WO2003086476A1 - Technetium-labeled rotenone derivatives, and methods of use thereof - Google Patents

Abstract

One aspect of the present invention relates to Tc-99m-labeled rotenone derivatives. These derivatives are useful, for example, as myocardial blood flow imaging agents. Reported herein are the synthesis, radiolabeling, and testing of Tc-99m-labeled rotenone complexes as single photon emitting probes for improved delineation of myocardial perfusion. The Tc-99m-rotenone derivatives have unique properties. The instant invention also relates to technetium-99m-labeled rotenone derivatives which exhibit high uptake and retention in the myocardium in proportion to flow. Another aspect of the present invention relates to kits compring a Tc-99m-labeled rotenone derivative.

Description

Technetium-Labeled Rotenone Derivatives, and Methods of Use Thereof

Background ofthe Invention Coronary heart disease (CHD) is the leading cause of death in the United States, accounting for roughly 24% of all deaths. The cost of cardiovascular diseases in 1999 is estimated by the American Heart Association (AHA) at $286.5 billion. Myocardial perfusion scintigraphy is widely used in the evaluation of patients with known or suspected coronary artery disease (CAD). The extensive clinical use of stress myocardial perfusion imaging has resulted largely from its demonstrated improved diagnostic sensitivity and specificity for detection of CAD as compared with exercise electrocardiogram. However, there remains a general need for myocardial flow tracers with improved tracer kinetics. Zaret B and Beller GA, Wintergreen Panel Summaries J. Nuclear Cardiology (1999) 6:111. Although several tracers are currently available for perfusion imaging, all of these tracers suffer from one or more limitations which render them less than ideal agents for assessment of cardiac perfusion (i.e., limited extraction at high flow (Tc99m-sestamibi, Tl-201 Chloride) (Marshall, R.C., Leidholdt E.M. Jr., Zhang, D. Y„ Barnett, CA. "Technetium- 99m hexakis 2-methoxy-2-isobutyl isonitrile and thalliurn-201 extraction, washout, and retention at varying coronary flow rates in rabbit heart" Circulation (1990) 82: 998-1007), lack of ideal isotope (Tl-201 chloride), high liver extraction (Tc99m-teboroxime and Tc99m-sestamibi) (Marshall, R.C., Leidholdt E.M. Jr., Zhang, D. Y., Barnett, CA. "The effect of flow on technetium-99m-teboroxime (SQ30217) and mallium-201 extraction and retention in rabbit heart" J. Nucl. Med. (1991) 32: 1979-1988).

Hence, Technetium-99m myocardial perfusion tracers are needed with: improved extraction on first pass; better linearity with true blood flow; improved detection of myocardial viability; and reduced accumulation in non cardiac tissues. Identification of an agent that allows for improved noninvasive delineation of myocardial perfusion and which could be routinely prepared at most clinical institutions or purchased from a centralized nuclear pharmacy would be of considerable benefit in the diagnosis and treatment of heart disease. Flow tracers should be developed that enhance our ability to achieve absolute quantitation and permit better detection of the presence and extent of coronary disease. Nuclear cardiographic imaging is a sensitive and specific diagnostic procedure for the evaluation of patients with known or suspected coronary artery disease but this modality is limited by the quality of the myocardial imaging radiopharmaceutical used. Developing a more efficient myocardial blood flow agent could reduce the need to perform the expensive procedure of invasive exploratory coronary angiography.

Generally, radiopharmaceuticals may be used as diagnostic or therapeutic agents by virtue of the physical properties of their constituent radionuclides. Thus, their utility is not based on any pharmacologic action. Most clinically used drugs of this class are diagnostic agents incorporating a gamma-emitting nuclide which, because of physical or metabolic properties of its coordinated ligands, localizes in a specific organ after intravenous injection. The resultant images can reflect organ structure or function. These images are obtained by means of a gamma camera that detects the distribution of ionising radiation emitted by the radioactive molecules.

In radioimaging, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located using a gamma-radiation detecting camera (this process is often referred to as gamma scintigraphy). The imaged site is detectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alternatively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast).

Many of the procedures presently conducted in the field of nuclear medicine involve radiopharmaceuticals which provide diagnostic images of blood flow (perfusion) in the major organs and in tumors. The regional uptake of these radiopharmaceuticals within the organ of interest is proportional to flow; high flow regions will display the highest concentration of radiopharmaceutical, while regions of little or no flow have relatively low concentrations. Diagnostic images showing these regional differences are useful in identifying areas of poor perfusion, but do not provide metabolic information of the state of the tissue within the region of apparently low perfusion.

However, many radionuclides are less than ideal for routine clinical use. For example, the positron-emitting isotapes (such as ¹⁸F) are cyclotron-produced and shortlived, thus requiring that isotope production, radiochemical synthesis, and diagnostic imaging be performed at a single site or region. The costs of procedures based on positron- emitting isotopes are very high, and there are very few of these centers worldwide. While ¹²³I-radiopharmaceuticals may be used with widely-available gamma camera imaging systems, ¹²³I has a 13 hour half-life (which restricts the distribution of radiopharmaceuticals based on this isotope) and is expensive to produce. Nitroimidazoles labeled with ³H are not suitable for in vivo clinical imaging and can be used for basic research studies only.

A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency of detection, a radionuclide that emits gamma energy in the 100 to 200 keV range is preferred. To minimize the absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have a source ofthe radionuclide always available at the clinical site.

A variety of radionuclides are known to be useful for radioimaging, including Ga- 67, Tc-99m, In-Ill, 1-123, 1-125, Yb-169 and Re-186. The preferred radioisotope for medical imaging is Tc-99m. Its 140 keV gamma-photon is ideal for use with widely- available gamma cameras. It has a short (6 hour) half life, which is desirable when considering patient dosimetry. Tc-99m is readily available at relatively low cost through commercially-produced "Mo/Tc-99m generator systems. As a result, over 80% of all radionuclide imaging studies conducted worldwide utilize Tc-99m. See generally Reedijk J. "Medicinal Applications of heavy-metal compounds" Curr. Opin. Chem. Biol. (1999) 3(2): 236-240; and Horn, R. K., Katzenellenbogen, J. A. "Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results" Nuc. Med. and Biol. (1997) 24: 485-498. These advantages, coupled with the fact that Single Photon Emission Computed Tomography cameras are optimized for the 140 keV energy of Tc-99m, clearly demonstrate the superiority of Tc-99m-labeled imaging agents.

Recently, a new Tc(I) labeling system has been developed. Aberto, R., Schibli, R., Egli, A., Schubiger, A. P., Abram, U., Kaden, T. A. "A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [⁹⁹ⁿTc(OH₂)₃(CO)₃]⁺ from [^99mTcO₄]^" in Aqueous Solution and Its Reaction with a Bifunctional Ligand" J. Am. Chem. Soc. (1998) 120: 7987-7988; and Alberto, R., Schibli, R., Daniela, A., Schubiger, A. P., Abram, U., Abram, S., Kaden, T. A. "Application of technetium and rhenium carbonyl chemistry to nuclear medicine ~ Preparation of psfetJ^TcCLXCO^] from [NBu₄][TcO₄] and structure of [NEt₄][Tc₂(u-Cl)₃(CO)₆]; structures of the model complexes [NEt₄][Re₂(u- OEt)₂(u-OAc)(CO)₆] and [ReBr({-CH₂S(CH₂)₂Cl}₂(CO)₃]" Transition Met. Chem. (1997) 22: 597-601. This system takes advantage of the organometallic Tc(I) carbonyl chemistry. Importantly, the chemistry of [⁹⁹'ⁿTc(OH₂)₃(CO)₃]⁺ has been elucidated and simplified to the point where the methods are routine and offer a practical alternative to the currently employed Tc(V) chemistry. In contrast to the highly reactive Tc(V)-oxo cores, where the chemistry is sometimes unpredictable and includes labeling cleanup steps, the Tc(I) method offers an attractive labeling alternative. However, unlike the Tc(V)-oxo core, the Tc(I)(CO)₃ ⁺ core limits the number of possible coordination geometries available for Tc due to the presence of the three carbonyl groups. The facial arrangement of carbonyl ligands around the metal center also impose steric constraints on the binding possibilities ofthe remaining three sites.

Moreover, the [^99mTc(OH₂)₃(CO)₃]⁺ complex can be readily prepared in saline under 1 atm of carbon monoxide (CO). This water and air stable Tc(I) complex is a practical precursor to highly inert Tc(I) complexes, due in part to the d⁶ electron configuration of the metal center. As already pointed out, the preparation ofthe organometallic tris(aquo) ion is simple and straightforward, allowing for convenient manipulation and product formation. Substitution of the labile H₂O ligands has been shown to leave the Tc(CO)₃ ⁺ core intact. This stable core has the additional advantage of being smaller and less polar than the routinely employed Tc(V)-oxo systems. This characteristic could be advantageous in biologically relevant systems where the addition of the metal center effects the size, shape, and potentially the bioactivity ofthe compounds.

Although various chelators are currently employed in the binding of tecmetium, all of these tracers suffer from one or more disadvantages which render them less than ideal: HYNIC requires coligands; MAG3 may be only used with the Tc(V)-oxo species; EDTA DTPA is used primarily primarily with Tc(V)-oxo and its ability to retain label is poor. Hence, additional Technetium-99m chelators are needed. Novel radiolabeled chelators that display rapid, efficient labeling and demonstrate superior labeling retention for both Tc(N)-oxo and Tc(I)-tricarbonyl cores without the use of coligands are attractive candidates for clinical evaluation as potential chelators for biologically relevant molecules.

Rotenone

Rotenone has a high affinity for mitochondria. The myocardium is an organ rich in mitochrondria. Novel technetium radiolabeled rotenone analogs that display efficient myocardial uptake and adequate myocardial retention are attractive candidates for clmical evaluation of myocardial blood flow. Over the past six years several research groups have been involved in the development of radiolabeled analogs ofthe natural product rotenone as a marker of mitochondrial content and activity. Rotenone is a specific, high-affinity inhibitor of complex I (NADHubiquinone oxidoreductase), the proximal enzyme of the mitochondrial electron transport chain. Since rotenone inhibition defines the activity of complex I, defects in radiotracer binding can be expected to reflect functional changes in the enzyme, and hence, abnormalities ofthe mitochondrial energy metabolism.

Labeled rotenone studies have focused on brain and heart imaging (organs enriched with mitochondria) using tritium, carbon-11, fluorine-18, and iodine-125 isotopes. VanBrocklin HF, Enas JD, Hanrahan SM, Brennan K.M, O'Neil JP, Taylor SE. "[F- 18]Fluorodihydrorotenone: Synthesis and evaluation of a mitochondrial electron transport chain (ETC) complex I probe for PET" J. Nucl. Med. (1994) 35(5): 73P; Marshall, R.C, Powers-Risius, P., Reutter, B.W., Taylor, S.E., VanBrocklin, H.F., Huesman, R.H., Budinger, T.F. "Kinetic analysis of 1251-Iodorotenone as a deposited myocardial flow tracer: comparison with 99mTc-sestamibi" J. Nucl. Med. (2001) 42: 272-281; Blandini F and Greenamyre JT "Assay of [³H]Dihydrorotenone Binding to Complex I in Intact Human Platelets" Analytical Biochem.(1995) 230:16-19; Charalambous A, Manger TJ, Kilboum MR "Synthesis of (2-[^πC]Methoxy)rotenone, a Marker of Mitochondrial Complex I Activity" Nucl. Med. Biol.(1995) 22:65-69; O'Neil JP, Marshall RC, Powers-Risius P, NanBrocklin HF "[F-18]Fluororotenoids: Evaluation of Potential Myocardial Imaging Agents in an Isolated, Perfused Rabbit Heart Model" Poster #P1 presented to the 19th Annual Western Regional Meeting, Society of Nuclear Medicine, Monterey, CA. October 20-23, 1994; VanBrocklin HF, Enas JD, Hanrahan SM, O'Neil JP "Fluorine-18 Labeled Dihydrorotenone Analogs: Preparation and Evaluation of PET Mitochondrial Probes" Journal of Labelled Compounds and Radiopharmaceuticals,(1995) 37: 217-219; Kenski, DM; VanBrocklin, HF; O'Neil, JP. "Fluorine-18 Labeled Rotenone Analogs: Preparation and Evaluation of PET Mitochondrial Probes" J. Labelled Compd. Radiopharm.,(1999) 42, suppl. 1, S333-335. Studies on iodine-125 labeled rotenone in isolated blood perfused rabbit heart, a unique model for evaluating myocardial imaging agents, have demonstrated extraction superior to that of Tc-99m sestamibi (0.84 + 0.05 compared to 0.48 ± 0.10). Marshall, R.C., Powers-Risius, P., Reutter, B.W., Taylor, S.E., VanBrocklin, H.F., Huesman, R.H., Budinger, T.F. "Kinetic analysis of 1251-Iodorotenone as a deposited myocardial flow tracer: comparison with 99mTc-sestamibi" J. Nucl. Med. (2001) 42: 272- 281. It also was found to have greater net heart retention than that of Tc-99m sestamibi at 1 min (0.77 ± 0.08 vs. 0.41 + 0.11) and at 26 min (0.46 ± 0.13 vs. 0.27 ± 0.11) and better correlation with true flow.

Recently, an ¹⁸F-radiolabeled rotenone compound was developed and the ability to target the heart was demonstrated. VanBrocklin HF, Enas JD, Hanrahan SM, Brennan K.M, O'Neil JP, Taylor SE "[F-18]Fluorodihydrorotenone: Synthesis and evaluation of a mitochondrial electron transport chain (ETC) complex I probe for PET" J. Nucl. Med. (1994) 35(5): 73P. Subsequent studies with a ¹²⁵I-iodorotenone analog demonstrated superior extraction, retention, and uptake characteristics compared with that of ^99mTc- sestamibi in the in vitro rabbit heart. Marshall, R.C, Powers-Risius, P., Reutter, B.W., Taylor, S.E., VanBrocklin, H.F., Huesman, R.H., Budinger, T.F. "Kinetic analysis of 1251- Iodorotenone as a deposited myocardial flow tracer: comparison with 99mTc-sestamibi" J. Nucl. Med. (2001) 42: 272-281. The agent also demonstrated good linearity in relation to blood flow over a wide range of flow values.

Summary ofthe Invention One aspect of the present invention relates to a complex represented by A:

wherein

X represents independently for each occurrence O or S; Z represents a chelator comprising a radionuclide;

R represents independently for each occurrence H, lower alkyl, or halogen;

R' represents independently for each occurrence lower alkyl;

R" represents independently for each occurrence H, or lower alkyl; R³ represents independently for each occurrence H, or lower alkyl; and the stereochemical configuration at any stereocenter of a complex represented by A is R, S, or a mixture of these configurations.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)ammes, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides. In certain embodiments, the present mvention relates to a complex represented by A and the attendant definitions, wherem Z represents a chelator selected ftom the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino-carboxylic acids, and N_xS_y chelators, wherem x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators.

In certain embodiments, the present mvention relates to a complex represented by A and the attendant definitions, wherein said radionuclide is technetium.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R represents H.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R' represents methyl.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R" represents H. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R³ represents H.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA,

SHNH, and mercaptoacetyltripeptides.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmemyl)glycine, and N₂S₂ chelators.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides; and said radionuclide is technetium. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4; and said radionuclide is technetium.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators; and said radionuclide is technetium. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present mvention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherem x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherem X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis. Another aspect of the present invention relates to a composition, comprising a complex ofthe present invention; and a pharmaceutically acceptable excipient.

A third aspect of the present invention relates to a method of imaging a region in a patient, comprising the steps of: administering to a patient a diagnostically effective amount of an complex of the present invention; exposing a region of said patient to radiation; and obtaining an image of said region of said patient.

In certain embodiments, the present invention relates to the aforementioned method, wherein said region of said patient is the thorax.

In certain embodiments, the present invention relates to the aforementioned method, wherein said region of said patient is the heart.

A fourth aspect of the present invention relates to a kit, comprising a complex ofthe present invention in a container, and instructions for using said complex to image a region in a patient.

Detailed Description ofthe Invention

Herein, we report methods for the preparation of novel technetium rhenium- rotenone derivatives, analyses of their stability, kit formulations comprising them, and their use as tracers for cardiac imaging. The new imaging agents possess good first pass extraction, minimal washout and a high degree of uptake related to blood flow. These agents are significantly better than alternative tracers at measuring blood flow in the heart over a wide range of flow values. Further, our rotenone derivatives may be packaged into an easy to label kit, allowing clinicians to form readily stable Tc-imaging agents.

While there is evidence elucidateing important factors involved in the exchange of rotenone derivatives between blood and myocardium (RBC/ albumin binding, capillary permeability, sarcolemmal permeability, and cellular sequestration), the details of mechanism of exchange are still unclear. Marshall, R.C., Powers-Risius, P., Reutter, B.W., Taylor, S.E., VanBrocklin, H.F., Huesman, R.H., Budinger, T.F. "Kinetic analysis of 1251- Iodorotenone as a deposited myocardial flow tracer: comparison with 99mTc-sestamibi" J. Nucl. Med. (2001) 42: 272-281. We have discovered sites on the rotenone skeleton, including the 7' position, which has accommodated an iodine atom, that may also accommodate relatively large and bulky groups, such as metal chelates, without the loss of biological activity. In other words, we have discovered technetium-99m-labeled rotenone derivatives, and methods using them to measure flow blood flow. Technetium-99m was chosen as the radionuclide because of its desirable properties.

The radioisotope technetium-99m, with a 6 hour half-life, gamma energy of 140 keV (85% of the gamma photons emit at 140 keV), wide-spread availability, and low cost makes it a preferred choice of radionuclides for nuclear medicine imaging today. Reedijk J. "Medicinal Applications of heavy-metal compounds" Curr. Opin. Chem. Biol. (1999) 3(2): 236-240. These advantages, coupled with the fact that Single Photon Emission Computed Tomography (SPECT) cameras are optimized for the 140 keV energy of Tc-99m, clearly establish the superiority of Tc-99m-labeled imaging agents. The radioisotope is used in ~85% of all imaging agents currently employed. Horn, R. K., Katzenelleribogen, J. A. "Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results" Nuc. Med. and Biol. (1997) 24: 485-498. The novel rotenone derivatives utilize both N₂S₂ chelators (e.g., diaminodithiol

(DADT) and monoamine monoamide (MAMA)) and the picolinamine mono-acetic acid (PAMA) chelator as technetium chelating moieties. The DADT, MAMA and PAMA moieties were selected because they can chelate a wide range of technetium complexes, varying in metal oxidation states, size, and lipophilicity. Three series of novel rotenone analogs labeled with Tc-99m at the 7' position were prepared and their chemical structures identified by preparation of the corresponding rhenium analogs. The relationship in the Periodic Table between technetium and rhenium implies that Tc-99m radiopharmaceuticals can be designed by modeling analogous rhenium complexes. Rose, D. J., Maresca, K. P., Kettler, P.B., Chang, Y.D., Soghomomian, V., Chen, Q., Abrams, M.J., Larsen, S.K., Zubieta, J. "Synthesis and Characterization of Rhenium thiolate complexes" Inorganic Chem. (1996) 35: 3548-3558. Synthesis o the rhenium complexes allowed us a facile route to characterize structurally the complexes. The biodistribution of these tracers was studied in rats to evaluate their potential as myocardial perfusion tracers. The heart uptake characteristics of the Tc-99m labeled novel rotenone derivatives have beed compared. Uptake and retention was compared with Tc99m-sestamibi in a separate group of rats and with Tl-201 chloride in the same rats. The studies support the potential of this class of radiopharmaceutical for evaluating myocardial perfusion.

The recent advancement in the chemistry of Tc "metal cores" allowed us to utilize the new Tc(I) labeling system developed by Schubiger and coworkers. Alberto, R., Schibli, R., Egli, A., Schubiger, A. P., Abram, U., Kaden, T. A. "A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [⁹⁹ⁿTc(OH₂)₃(CO)₃]⁺ from [^99mTcO₄]^" in Aqueous Solution and Its Reaction with a Bifunctional Ligand" J. Am. Chem. Soc. (1998) 120: 7987-7988; and Alberto, R., Schibli, R., Daniela, A., Schubiger, A. P., Abram, U., Abram, S., Kaden, T. A. "Application of technetium and rhenium carbonyl chemistry to nuclear medicine. Preparation of [Net₄]₂[TcCl₃(CO)₃] from [NBu₄][TcO₄] and structure of [NEt₄][Tc₂(u-Cl)₃(CO)₆]; structures of the model complexes [NEt₄][Re₂(u-OEt)₂(u-OAc)(CO)₆] and [ReBr({- CH₂S(CH₂)₂Cl}₂(CO)₃]" Transition Met. Chem. (1997) 22: 597-601. This system takes advantage of the organometallic Tc(I) carbonyl chemistry. The chemistry of [^99mTc(OH₂)₃(CO)₃]⁺ has been elucidated and simplified to the point where the methods are routine and offer a practical alternative to the currently employed Tc(V) chemistry. In contrast to the highly reactive Tc(V)-oxo cores, where the chemistry is sometimes unpredictable and frequently requiring necessary cleanup steps, the Tc(I) method offers a distinct labeling alternative.

In the past, the organometallic Tc(I) complexes were extremely difficult to prepare and manipulate requiring the use of high temperatures and pressures. Today [⁹⁹ⁿ c(OH₂)₃(CO)₃]⁺ can be readily prepared in saline under 1 atm of carbon monoxide (CO) or without pressurized vials altogether utilizing the in situ CO producing boranocarbonate complex developed by Alberto and coworkers. Alberto, R., Wald, J., Ortner, K., Candreia, L., Pietzsch, H.-J. "Synthesis of derivatized cyclopentadienyl-tricarbonyl complexes of Tc- 99m in water with an in situ CO source" Journal of Labelled Compounds and Radiopharmaceuticals, (2001) 44: S54-S56. This water and air stable Tc(I) complex turns out to be a practical precursor to the formation of highly inert Tc(I) complexes, due in part to the formation ofthe d⁶ electron configuration of the metal center. The preparation ofthe organometallic aqua-ion is simple and straightforward, allowing for convenient manipulation and product formation. The easy substitution of the labile H₂O ligands has been demonstrated, leaving the Tc(CO)₃ ⁺ core intact. This stable core has the additional advantage of being smaller and less polar than the routinely employed Tc(V)-oxo systems. This could offer a big advantage in biologically relevant systems where the addition of the metal center effects the size, shape, and potentially the bioactivity ofthe compounds.

The non-polar precursor Tc(CO)₃ ⁺, with three tightly bound "innocent" carbonyls, provides three open coordination sites, allowing for a large degree of flexibility in the choice of ligands. In one embodiment of the present invention, the picolinamine mono- acetic acid (PAMA) ligand provides both oxygen and nitrogens as potential donor atoms. Recent work has demonstrated the high affinity of the Tc(CO)₃ ⁺ core for this chelator system. Alberto, R., Schibli, R., Egli, A., Schubiger, A. P., Abram, U., Pietzsch, H.-J., Johannsen B. "First Application of fac-[^99mTc(OH₂)₃(CO)₃]⁺ in Bioorganometallic Chemistry: Design, Structure, and in Vitro Affinity of a 5-HT_1A Receptor Ligand Labeled with ^99mTc" J. Am. Chem. Soc. (1999) 121 : 6076-6077; and Leirer, M., Knor, G., Vogler, A. "Electronic spectra of l,2-diiminetricarbonylrhenium(I)chloride complexes with imidazole derivatives as ligands" Inorg. Chim. Acta (1999) 288: 150-153.

One aspect of the present mvention relates to the design and synthesis of a series of rotenone derivatives, comprising a metal chelator substituent, which may be varied in terms of size and lipophility. Another aspect of the present invention relates to labeling the aforementioned rotenone derivatives with Tc-99m, using labeling methods based on both the Tc(V)-oxo core and Tc(I)(CO)₃L₃ core. The labeled rotenone analogues are characterized structurally by comparison to the corresponding Re(IV) and Re(I) derivatives.

Further, the present invention relates to assessment of the in vivo pharmacokinetic properties in rats of the Tc-99m labeled novel rotenone derivatives, including a comparison to the conesponding pharmacokinetic properties of Tl-201 chloride. Also, a control experiment compares the biodistribution in rats of Tc99m-sestamibi with that of the Tc-

99m labeled rotenone analogs ofthe present invention.

Early and accurate diagnosis of coronary artery disease is critical for the treatment, management and successful outcome of heart disease. To address this issue we have developed easy to label kit formulated ^99mTc-cardiac imaging agents. The technetium-99m labeled rotenone derivatives have also been assessed for their suitability for inclusion in a kit, including a determination of their stability as a function of time and concentration. The present invention also relates to an examination ofthe stability of these complexes in buffer at physiological pH, and in human plasma and serum components. Identification of an agent that allows for improved noninvasive delineation of myocardial perfusion and which could be routinely prepared at most clinical institutions or purchased from a centralized nuclear pharmacy would be of considerable benefit in the diagnosis and treatment of heart disease and constitute a significant diagnostic and commercial opportunity.

Biodistribution studies in rats have also been performed to deteπnine the metabolic fate of the new Tc-99m-rotenone derivatives, and to compare them with Tc99m-sestamibi and Tl-201 chloride in rats.

Optimum complexes to image myocardial blood flow are being developed, and mechanistic studies are being performed to characterize flow and biochemically related behaviors, and to develop a convenient kit formulation for the optimum Tc-agent.

Metal Chelator Groups

Chelators which bind to radionuclides are known in the art. See, e.g., M. Nicolini et al., eds., "Technetium and Rhenium in Chemistry and Nuclear Medicine," SGEditoriali, Padova (1995). In general, chelators used in the complexes of the present mvention are capable of binding to radionuclides, such as Tc(CO)₃ ⁺ or Tc(O)³⁺. In a preferred embodiment, a chelator moiety will be a tetradentate chelator, i.e., will be capable of four- point binding to a radionuclide. Exemplary tetradentate chelators include N₂S₂ and N₃S chelators, as described in, e.g., A. R. Fritzberg, et al., J. Nucl. Med. 23:592-598 (1982); S. Liu and D. S. Edwards, in M. Nicolini et al., eds., "Technetium and Rhenium in Chemistry and Nuclear Medicine," op. cit, pp. 383-393; and S. Vallabhajousula et al., J. Nucl. Med. 30:599-604 (1989). An N₂S₂ chelator can chelate a radionuclide through two nitrogen atoms (e.g., amido nitrogens of a peptide backbone) and two sulfur atoms (e.g., of a mercaptoacetyl moiety), while N₃S chelators can chelate to a radionuclide through three nitrogen atoms and one sulfur atom.

As defined herein, the term "N_xS_y chelating compound" includes bifunctional chelators that are capable of: (i) coordmately binding a radionuclide; and (ii) covalently attaching to an rotenone analog. Preferred N_xS_y chelating compounds have the N₂S₂ (generally described in U.S. Pat. Nos. 4,897,225 or 5,164,176 or 5,120,526), N₃S (generally described in U.S. Pat. No. 4,965,392), N₂S₃ (generally described in U.S. Pat. No. 4,988,496), N₂ S₄ (generally described in U.S. Pat. No. 4,988,496), N₃S₃ (generally described in U.S. Pat. No. 5,075,099) or N₄ (generally described in U.S. Pat. Nos. 4,963,688 and 5,227,474) cores. Preferred N_xS_y chelating compounds have N₂ S₂ and N₃ S cores. Exemplary N_xS_y chelating compounds are described in Fritzberg et al., Proc. Natl. Acad. Sci. USA 85:4024-29, 1988; in Weber et al., Bioconj. Chem. 1:431-37, 1990; and in the references cited therein.

The N₂S₂ chelating compounds include diamide, dimercaptide bifunctional chelators of the N_xS_y family capable of stably complexing a radionuclide through two nitrogen atoms and two sulfur atoms that are appropriately positioned. N₂S₂ chelating compounds are generally described in U.S. Pat. No. 4,897,225. The N₃S chelatmg compounds include triamide, mercaptide bifunctional chelators of the N_xS_y family capable of stably complexing a radionuclide through three nitrogen atoms and one sulfur atom that are appropriately positioned. Preferred N₃S chelating compounds are described in U.S. Pat. Nos. 4,965,392 and 5,091 ,514.

Other commonly used chelating agents include, for example, diethylene triamine pentaacetic acid (DTPA) and ethylene diamine tetracetic acid (EDTA). Other chelators appropriate to link a radionuclide to a compound in accordance with the present invention are described in standard texts such as Advanced Inorganic Chemistry, 4th edition, 1980, F. A. Cotton and G. Wilkinson, John Wiley & Sons. The most suitable metal chelating agent will vary with the metal to be chelated, e.g. depending on its particular coordination geometry. Chelators suitable specifically for linking ^99mTc to rotenone compounds in accordance with the present invention preferably present, as a metal coordinating configuration, a combination of four nitrogen and sulfur metal-coordinating atoms.

Further, proteins have been labeled with technetium-99m (^99mTc) using the hydrazino nicotinamide (SHNH) chelator (Abrams M. J., Juweid M., tenKate C. I., Schwartz D. A., Hauser M. M., Gaul F. E., Fuccello A. J., Rubin R. H., Strauss H. W., Fischman A. J, J. Nucl. Med., 31 : 2022-2028, 1990), and the label was found to be stable both in vitro and in vivo (Hnatowich D. J., Mardirossian G., Ruscowski M., Fogarasi M, Virzi F, Winnard P Jr., J. Nucl. Med., 34; 109-119, 1993). The SHNH chelator was initially used for oligonucleotides, however, transfer of label nonspecifically to proteins from oligonucleotides labeled in this manner was observed. The identical oligonucleotide; radiolabeled with In-I l l using the chelator diethylenetriamine-pentaacetic acid (DTPA) showed no tendency to bind to serum proteins under circumstances in which the ^99mTc- SHNH-labeled oligonucleotide was largely protein bound (Hnatowich D. J., Winnard P. Jr., Virzi F, Fogarasi M, Sano T, Smith C L, Cantor C R, Rusckowski M., J. Nucl. Med., 36: 2306-2314, 1995), Lastly, the N-[N-[N-[(benzoylthio)acetyl]glycyl]glycyl]glycine (MAG₃) chelator of

^99mTc was originally developed as an alternative to radiolabeled hippuran for renal function studies (Fritzberg A R., Kasina S., Eshima D., Johnson D. L., J. Nucl. Med., 27: 111-116; 1986). This succinimide ester mercapto-acetyl tripeptide is protected against disulfide-bond formation by a benzoyl group, which must be heated to 100 C for 10 min during labeling to remove the protecting group. This benzoyl-protected chelator has also been used to radiolabel antibodies with ^99mTc (Fritzberg A. R., Berninger R. W., Hadley S. W. et al., Pharmaceutical Res., 5: 325-334; 1988) and radioactive rhenium (Goldrosen M H., Biddle W C, Pancook S. Bakshi S., Vanderheyden J-L., Fritzberg A. R., Morgan A. C, Foon K. A., Cancer Res., 50: 7973-7978; 1990). However, past use of the chelator for protein labeling has been limited since the benzoyl protecting group requires extreme alkaline pH or boiling temperatures for sulfur deprotection. The MAG₃ chelator has also been used to label antibodies by post-conjugation methods through the use of an isophfhaloyl group for protection in place of the benzoyl group (Weber R. W., Boutin R. H., Nedelman M. A., Lister- James J., Dean R. D., Bioconjug. Chem 1:431-437, 1990).

Accordingly, prefened chelator moieties include amidothiols, including, e.g., mercaptoacetyltripeptides, such as, e.g., mercaptoacetyltriglycine (MAG₃), mercaptoacetyltriserine. Mercaptoacetyl-tripeptides can chelate radionuclides, such as Tc(CO)₃ ⁺ or Tc(O)³⁺, by coordination through the three amide nitrogens of the peptide backbone, and the terminal mercapto group. Other chelator moieties which may find use in the present invention include cyclams, porphyrins, crown ethers, azacrown ethers, and the like. Linking Moiety

In certain embodiments, the chelator is bonded directly to the methylene carbon of the isopropylidene group at the 2-position of the dihydrofuran nucleus of rotenone. In other embodiments, the chelator and the rotenone core of the complexes of the present invention are connected by a linker moiety, i.e., a covalent tether. Linking moieties provided by the invention are comprised of at least two linker functional groups each capable of covalently bonding to the rotenone core and the chelator. Such functional groups include but are not limited to primary and secondary amines, hydroxyl groups, carboxylic acid groups and thiol groups. In other words, for connection to the chelator, a rotenone analog may comprise a linker that serves to create a physical separation between the chelator and the rotenone. A linker may be an alkyl chain that is derivatized for coupling to the chelator. The spacer may comprise one or more amino acid residues.

To reiterate, the chelator may be linked to the rotenone core in a variety of ways. For example, if the chelator terminates in a carboxyl group or derivative thereof, the carboxyl group may be activated with carbodiimide and an alcohol to form an active ester or may already contain an active ester group that is reacted with an available amino group of a polypeptide or an amino sugar to form an amide bond. Alternatively, if the chelator terminates in an amino group, the amino group may be used to react with an aldehyde group, which may be derived by glycol cleavage of a sugar with periodate, to form a Schiff s base or cyclic imine or under conditions favoring reductive amination to form a secondary or tertiary amine or cyclic imine linkage.

Synthesis of Rotenone Derivatives To achieve the goal of a technetium labeled rotenone analog, we will synthesize a series of novel pendant modified derivatives. A major concern when designing a chelated- Tc-99m labeled pharmaceutical is that the inclusion ofthe Tc-ligand in the carrier molecule should not drastically alter its biological behavior. To achieve this we will examine several of the pendant conjugation techniques as reviewed by Horn, Katzenellenbogen, et al. Horn, R. K., Katzenellenbogen, J. A. "Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results" Nuc. Med. and Biol. (1997) 24: 485-498. In these labeling approaches the chelated radionuclide is bound to the bio-molecule via a pendant chain distant to the receptor-binding site. Some advantages of this design include the ability to change the length and location of the pendant chain, as well as, the ability to change chelating moieties at the end.

Although various chelators are currently employed in the binding of tectnetium, all of these tracers suffer from one or more disadvantages which render them less than ideal: HYNIC requires coligands; MAG3 may be only used with the Tc(V)-oxo species; EDTA/DTPA is used primarily primarily with Tc(V)-oxo and its ability to retain label is poor. Hence, additional Technetium-99m chelators are needed. Novel radiolabeled chelators that display rapid, efficient labeling and demonstrate superior labeling retention for both Tc(V)-oxo and Tc(I)-tricarbonyl cores without the use of coligands are attractive candidates for clinical evaluation as potential chelators for biologically relevant molecules.

In certain embodiments, we have used two well-established chelate groups, N₂S₂ (diaminodithiol (DADT) and amideaminobisthiol (MAMA)) and the newer tricarbonyl aminomethyl pyridine (PAMA) system (Alberto, R., Schibli, R., Egli, A., Schubiger, A. P., Abram, U., Pietzsch, H.-J., Johannsen B. "First Application of fac-[^99mTc(OH₂)₃(CO)₃]⁺ in Bioorganometallic Chemistry: Design, Structure, and in Vitro Affinity of a 5-HT_1A Receptor Ligand Labeled with ^99mTc" J. Am. Chem. Soc. (1999) 121: 6076-6077) can produce stable technetium labeled molecules. Advantages of these systems are that they provide a robust, neutral charged core. The N₂S₂ chelators possess a formal 3- charge; therefore, upon addition of the metal-oxo (3+) core, the overall charge remains predictably neutral. Using the neutral metal chelate analogs has a number of advantages: a) there is no charge change and the labeled molecule is expected to retain the membrane diffusional properties of rotenone, b) these chelators have proven to be good ligands for binding Tc- 99m at room temperature in high radiochemical yields and purity, c) the ligands keep the metal in a thermodynamically stable +5 or +1 oxidation state, and d) the size of the Tc- 99m-chelate is similar to that of the phenyl group, which should not perturb the mitochondria binding system. Warren GL, Caldwell JH, Kremer PA, et al. "New iodinated phenyl fatty acids for imaging myocardial metabolism" J. Nucl. Med. (1986) 939-940. Two preferred derivatives include conjugation through one of the amines of the diaminodithiol (DADT) chelate (1) or the amine of the monoamine monoamide (MAMA)

(2).

A second embodiment, exemplified by analog 3, incorporates the technetium (I) tricarbonyl center capped with a coordinating picolinamine mono-acetic acid moiety (PAMA).

All compounds of the present invention are also synthesized with macroscopic quantities of rhenium for characterization by conventional spectral and elemental analyses, fast atom bombardment mass spectrometry, ^lH and ¹³C NMR spectrometry, and infrared analysis. Following purification, all non-radioactive compounds are analyzed for chemical purity by elemental analysis, thin-layer and high-pressure liquid chromatography. X-ray crystallography may also be performed on rhenium analogs. All radiolabeled agents are analyzed for radio-homogeneity by thin-layer and high-pressure liquid chromatography.

A synthetic pathway to a reactive rotenone substrate is shown in Scheme 1. VanBrocklin HF, Enas JD, Hamahan SM, O'Neil JP "Fluorine-18 Labeled Dihydrorotenone Analogs: Preparation and Evaluation of PET Mitochondrial Probes" Journal of Labelled Compounds and Radiopharmaceuticals,(1995) 37: 217-219. Rotenone is converted directly into the 7'-hydroxydihydrorotenols upon treatment with excess borane. Oxidation with Mn0₂ gives the conesponding 6'- epimers of 7' -hydroxy dihydrorotenone that are separable on normal phase HPLC. The epimers are then treated with methane sulfonyl chloride to form the corresponding mesylates.

The rotenone-MAMA chelator 2 has been prepared as shown below.

Likewise, the rotenone-picolinamine mono-acetic acid (PAMA) chelator 3 has been prepared as shown below.

Labeling rotenone analogs with Tc-99m using methods based on the Tc(V)-oxo and Tc(I)(CO)₃L₃ cores

TcCVVoxo core

Preparation of the Tc-99m-labeled rotenone derivatives is achieved by adding 10 mCi of TcO₄ ^" to a 0.9% saline solution of sodium gluceptate (200 mg/3 mL). After 20 minute incubation, 400 ul is added to a solution of 400 ul of sodium acetate (50 mM, pH 5.2) and the appropriate rotenone N₂S₂ derivative (50 ug). The mixture is heated at 80 °C for 30 min. The mixture is then extracted with ethyl acetate (3 x 1 mL), dried over sodium sulfate, and dried under N₂. Ηie residue is then re-dissolved in ethanol (400 ul) and purity checked via HPLC by a Vydac C18 (5 mm, 25 cm) column using methanol to elute the reaction products.

Tc(I)(COV- core

The Tc(I) carbonyl chemistry allows for the possibility of an alternative route to form stable ^99mTc-rotenone complexes. To explore this labeling method we place Na₂CO₃ (0.004 g, 0.038 mmol) and NaBH₄ (0.005 g, 0.13 mmol) in a vial. Next, the vial is sealed and flushed with CO for 10 min. To the vial is added 1 mL of Na ^99mTc0₄ ^" in saline. Finally the solution is heated to 75° C for 30 minutes. After cooling, 0.3ml of 1M PBS solution is added (pH 7.4), resulting in the stable formation of [^99mTc(OH₂)₃(CO)₃]⁺. This Tc(I) tricarbonyl species is then heated at 75° C for 30 minutes with the PAMA derivatized rotenone to form the ^99mTc-rotenone complex. A 'one pot' synthesis has also been performed where the PAMA derivatized rotenone is added to the vial with the Na₂CO₃, NaBH₄, and flushed with CO for 10 min. After the flush, 1 mL of Na ^99mTcO₄ ^" in saline is added. Finally the solution is heated to 75° C for 30 minutes. The reaction is then checked for purity via HPLC by a Vydac C18 (5 mm, 25 cm) column using methanol to elute the reaction products. Synthesis of rhenium analogs for structural characterization

The properties ofthe Group VII metals technetium and rhenium are very similar due to their periodic relationship. Therefore, the metals typically demonstrate similar reaction chemistry, for example, the thiol, nitrogen, phosphine and oxo-chemistry of these two metals. Likewise, penhenate and pertechnetate have very similar chemistries. Rose, D. J., Maresca, K. P., Nicholson, T., Davison, A., Jones, A. G., Babich, J._; Fischman, A., Graham, W., DeBord, J. R. D., Zubieta, J. "Synthesis and Characterization of Organohydrazine Complexes of Technetium, Rhenium, and Molybdenum with the {M(η l-HxNNR)(η2- HyNNR)} Core and Their Relationship to Radiolabeled Organohydrazine-Derivatized Chemotactic Peptides with Diagnostic Applications" Inorg. Chem. (1998) 37: 2701-2716. The similar reductions of the M(VII) oxo species by SnCl₂ allow for easy substitution ofthe nonradioactive rhenium as a model for the medicinally useful technetium-99m, which routinely uses tin-reduced ^99mTc. Synthesizing the rhenium-rotenone complexes provides a facile route to characterize structurally the products. The characterized products, in turn, lead to the development of new Tc-rotenone derivatives based on the presence or absence of a structural feature found in the rhenium data obtained. The periodic relationship between Tc and Re indicates that Tc-99m radiopharmaceuticals can be designed by modeling analogous rhenium complexes. Nicholson, T., Cook, J., Davison, A., Rose, D. J., Maresca K. P., Zubieta, J. A., Jones, A. G. "The synthesis and characterization of [MC1₃(N=NC₅H₄NH)(HN=NC₅H₄N)] from [MO₄]^" (where M = Re, Tc) organodiazenido, organodiazene-chelate complexes" Inorg. Chim. Acta (1996) 252: 421-426.

Re(V)-oxo core

The synthesis of the rhenium analogs follows the established chemistry of the N₂S₂ system in forming stable, neutral, rhenium-oxo complexes. Davison A, Jones AG, Orvig C, et al: A new class of oxotechnetium (5+) chelate complexes containing a TcON₂S₂ core. Inorg. Chem. (1981) 20: 1629-1631; Kung HF, Guo Y-Z, Mach RH, et al: New Tc-99 complexes based on N₂S₂ ligands. J. Nucl. Med.(1986) 27: 1051; Kung HF, Molnar M, Billings J, et al: Synthesis and biodistribution of neutral lipid-soluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. (1984) 25: 326-332; and Kung HF, Yu CC, Billings J, et al: Synthesis of new bis(aminoethanethiol) (BAT) derivatives: Possible ligands for ^99mTc brain imaging agents. J. Med. Chem.(1985) 28: 1280-1284. Our N₂S₂ system, with three easily removed protons forms a predictablable metal-complex with an overall net charge of zero. The synthesis of the Re(V) complexes is accomplished by reacting [TBA][ReOBr₄(OPPh₃)j with the appropriate rotenone ligand in the ratio of 1: 1.2 in 10 mL of methanol and three equivalents of NEt₃ as base. The reaction is then allowed to reflux for ^lA hour. After cooling the reaction products are purified using a small column using the method established by Spies and co-workers. Spies, H., Fietz, T., Glaser, M., Pietzsch, H.-J., Johannsen, B. In "Technetium and Rhenium in Chemistryand Nuclear Medicine 3", Nicollini, M., Bandoli, G., Mazzi, U., eds., Padova, Italy, (1995) 4, 243. Alternatively, the rhenium (V) starting material [ReOCl₃(PPh₃)₂] may be employed as the potential rhenium starting material. This versatile material has proven successful in the past in dealing with nitrogen and sulfur donor atoms. Maresca, K. P., Femia, F. J., Bonavia, G. H., Babich, J. W., Zubieta, J. "Cationic comples of the '3+1' oxorhenium-thiolate complexes" Inorganic Chemistry Acta (2000) 297: 98-105; and Maresca, K. P., Rose, D. J., Zubieta, J. "Synthesis and charaterization of a binuclear rhenium nitropyrazole" Inorganica Chimica Acta (1997) 260: 83-88. The synthesized rhenium-rotenone complexes are run through a chiral HPLC column for separation and purification purposes following the procedures described for the technetium complexes. The complexes are then analyzed by elemental analysis, infrared spectroscopy, mass spectroscopy, and NMR spectroscopy. Finally, the rhenium-rotenone complexes are crystallized.

Re(D(COL+ core

The Re(I)(CO)₃ ⁺ system follows similar reaction chemistry to that of the Tc-99m tricarbonyl core. The use of [NEt₄]₂[ReBr₃(CO)₃], as the starting material leads to easy formation of the_ αc-Re(CO)₃(L)₃ core. The [NEt₄]₂[ReBr₃(CO)₃] is readily derived from the

[ReBr(CO)₅]. The synthesis of the Re(I) complexes has been accomplished by reacting

[NEt₄]₂[ReBr₃(CO)₃] with the appropriate rotenone ligand in the ratio of 1: 1.2 in 10 mL of H₂O and three equivalents of NEt₃ as base. The reaction is allowed to heat at 80 °C for 4 hours. After cooling the reaction products are purified using a small column, using the method established by Alberto and coworkers. Alberto, R., Schibli, R., Egli, A., Schubiger, A. P., Abram, U., Kaden, T. A. "A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [^99mTc(OH₂)₃(CO)₃]⁺ from [^99mTcO₄]^" in Aqueous Solution and Its Reaction with a Bifunctional Ligand" J. Am. Chem. Soc. (1998) 120: 7987-7988. This versatile material has proven successful in the past for dealing with nitrogen and oxygen donor atoms. The synthesized rhenium-rotenone complexes are run through a HPLC column for separation and purification purposes following the procedures previously described for the technetium complexes. Next, the complexes are analyzed by: elemental analysis, infrared spectroscopy, mass spectroscopy, and NMR spectroscopy. Finally, the rhenium-rotenone complexes are crystallized.

In vivo rat studies to evaluate the ^99mTc-rotenone complexes for heart uptake

Vertebrate animals in this research project are used to investigate the biodistribution and pharmacokinetics of new technetium-rotenone complexes and determine their uptake in the heart. Rats (Sprague Dawley, male, at 80-100 grams each) are used for the whole body biodistribution studies. Three compounds are evaluated in this series with five time points 5, 15, 30, 60, and 120 minutes with five animals per time point. This many animals are needed to provide accurate statistics in the clearance rate measurements and to account for intraspecies variation.

Stability Assessment

We have already developed the proposed stability tests for ^99mTc-labeled complexes. The stability of the radiolabeled compounds in solution and in plasma is determined as a function of time and solution conditions such as pH and solvents. Specifically, after radiolabeling and isolation, the product is allowed to sit at room temperature for 48 hours, after which HPLC analysis is performed to check for degree of label retention, as well as potential product degradation. We then analyze for the reformation of TcO₄ ^" and the presence of the reduced species Tc0₂. To assist in predicting the in vivo label stability, we perform ligand challenges. Specifically, we incubate the product with a competing biological ligand, such as cysteine, albumin, and transferrin, testing the stability of the radiolabel via HPLC analysis. Finally we test the product in plasma as a function of time and pH. Development of a convenient kit formulation for the agent with the highest heart specificity and selectivity

The widespread availability of Tc-99m from a generator has made it the most frequently used nuclide in nuclear medicine. The advantage of a Tc-99m labeled rotenone complex is that it allows for the possible development of a convenient "kit" formulation, thereby permitting more accessibility of the diagnostic agent. Based on our previous results, we believe that the ^99mTc-rotenone complexes can be synthesized in a "one-step" procedure. The appropriate coligand, as well as the conect stoichiometric amounts of reducing agents and coligands are being developed. Both the Tc(V)-oxo system and the Tc(I)-carbonyl system possess the potential to be routes to ^99mTc-rotenone kits. By selecting the N₂S₂ and PAMA as chelates for the ^99mTc we have chosen stable cores with saturated metal binding sites, limiting the number of possible side-products. The potential to prepare the compound into a kit lies in the chelates' ability to bind rapidly and securely to the metal.

Kits The present invention also includes radiopharmaceutical kits containing the labeled compounds ofthe present invention. Such kits may contain the labeled compounds in sterile lyophilized form, and may include a sterile container of a radiopharmaceutically acceptable reconstitution liquid. Suitable reconstitution liquids are disclosed in Remington's Pharmaceutical Sciences and The United States Pharmacopia~The National Formulary, cited above. Such kits may alternatively contain a sterile container of a composition of the radiolabeled compounds of the invention. Such kits may also include, if desired, other conventional kit components, such as, for example, one or more carriers, one or more additional vials for mixing. Instructions, either as inserts or labels, indicating quantities of the labeled compounds of the invention and carrier, guidelines for mixing these components, and protocols for administration may also be included in the kit. Sterilization of the containers and any materials included in the kit and lyophilization (also referred to as freeze-drying) of the labeled compounds of the invention may be carried out using conventional sterilization and lyophilization methodologies known to those skilled in the art. Another aspect of the present invention is diagnostic kits for the preparation of radiopharmaceuticals. Diagnostic kits of the present invention comprise one or more vials containing the sterile, non-pyrogenic, formulation comprised of a predetermined amount of a rotenone analog, a reducing agent, and optionally a solubilization aid or other components such as transfer ligands, buffers, lyophilization aids, stabilization aids, and bacteriostats. The inclusion of one or more optional components in the formulation will frequently improve the ease of synthesis ofthe radiopharmaceutical by the practising end user, the ease of manufacturing the kit, the shelf-life of the kit, or the stability and shelf-life of the radiopharmaceutical. The improvement achieved by the inclusion of an optional component in the formulation must be weighed against the added complexity of the formulation and added cost to manufacture the kit. The one or more vials that contain all or part of the formulation can independently be in the form of a sterile solution or a lyophilized solid. Solubilization aids useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monoloeate, polysorbates, poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers (Pluronics) and lecithin. Prefened solubilizing aids are polyethylene glycol, and Pluronics.

Buffers useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to phosphate, citrate, sulfosalicylate, and acetate. A more complete list can be found in the United States Pharmacopeia. Lyophilization aids useful in the preparation diagnostic kits useful for the preparation of radiopharmaceuticals include but are not limited to mannitol, lactose, sorbitol, dextran, Ficoll, and polyvinylpyrrolidine(PVP).

Stabilization aids useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, and inositol.

Bacteriostats useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to benzyl alcohol, benzalkonium chloride, chlorobutanol, and methyl, propyl or butyl paraben. A component in a diagnostic kit can also serve more than one function. A reducing agent can also serve as a stabilization aid, a buffer can also serve as a transfer ligand, a lyophilization aid can also serve as a transfer, ancillary or co-ligand and so forth. The predetermined amounts of each component in the formulation are determined by a variety of considerations that are in some cases specific for that component and in other cases dependent on the amount of another component or the presence and amount of an optional component. In general, the minimal amount of each component is used that will give the desired effect of the formulation. The desired effect of the formulation is that the practicing end user can synthesize the radiopharmaceutical and have a high degree of certainty that the radiopharmaceutical can be safely injected into a patient and will provide diagnostic information about the disease state of that patient.

The diagnostic kits of the present invention also contain written instructions for the practicing end user to follow to synthesize the radiopharmaceuticals. These instructions may be affixed to one or more of the vials or to the container in which the vial or vials are packaged for shipping or may be a separate insert, termed the package insert.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The term "chelator", as used herein, refers to a moiety that is capable of binding a radionuclide, preferably through non-covalent interactions, e.g., through ionic interactions. Chelator moieties suitable for use in the compositions and methods of the invention are preferably capable of binding to a radionuclide with a high affinity, e.g., a binding affinity sufficiently high to permit binding of a radionuclide, preferably under physiological conditions, e.g., in vivo.

The term "linking group" as used herein refers to a chemical group that serves to couple the rotenone analog to the chelator while not adversely affecting either the targeting function of the rotenone analog or the metal binding function of the chelator. Suitable linking groups include alkyl chains; alkyl chains optionally substituted with one or more substituents and in which one or more carbon atoms are optionally replaced with nitrogen, oxygen or sulfur atoms. Other suitable linking groups include those having the formula A¹- A²-A³, wherein A¹ and A³ are independently selected from N, O and S; and A² includes alkyl optionally substituted with one or more substituents, and in which one or more carbon atoms are optionally replaced with nitrogen, oxygen or sulfur atoms; aryl optionally substituted with one or more substituents; and heteroaryl optionally substituted with one or more substituents. Still other suitable linking groups include amino acids and amino acid chains functionalized with one or more reactive groups for coupling to the rotenone analog and/or chelator.

The term "diagnostic kit," as used herein, comprises a collection of components, termed the fonnulation, in one or more vials which are used by the practising end user in a clinical or pharmacy setting to synthesize the radiopharmaceutical. The kit provides all the requisite components to synthesize and use the radiopharmaceutical except those that are commonly available to the practising end user, such as water or saline for injection, a solution of the radionuclide, equipment for heating the kit during the synthesis of the radiopharmaceutical if required, equipment necessary for administering the radiopharmaceutical to the patient such as syringes and shielding, and imaging equipment.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term "electron- withdrawing group" is recognized in the art, and denotes the tendency of a substitaent to attract valence electrons from neighboring atoms, i.e., the substitaent is electronegative with respect to neighboring atoms. A quantification of the level of electron- withdrawing capability is given by the Hammett sigma (σ) constant. This well known constant is described in many references, for instance, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 edition) pp. 251-259. The Hammett constant values are generally negative for electron donating groups (σ[P] = - 0.66 for NH2) and positive for electron withdrawing groups (σ[P] = 0.78 for a nitro group), σ[P] indicating para substitution. Exemplary electron-withdrawing groups include nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the like. Exemplary electron- donating groups include amino, methoxy, and the like. The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In prefened embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. Likewise, prefened cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Prefened alkyl groups are lower alkyls. In prefened embodiments, a substitaent designated herein as alkyl is a lower alkyl.

The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "aryl" as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pynole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structare may also be refened to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. The terms ortho, meta and para apply to 1,2-, 1,3- and 1 ,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ort/.o-dimethylbenzene are synonymous.

The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, azetidine, azepine, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pynole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pynolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pynolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, - CF3, -CN, or the like.

The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom ofthe ring is carbon.

As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -CI, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-.

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:

R ' R 10

/ ¹⁰ 1 +

— N. _or — N- R_l0

\ Rα

Rc wherein R9, Ri g and R'10 each independently represent a group permitted by the rules of valence.

The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein Rg is as defined above, and R'i ]_ represents a hydrogen, an alkyl, an alkenyl or -(CH2)_m-R8, where m and Rg are as defined above.

The term "amido" is art recognized as an amino-substitated carbonyl and includes a moiety that can be represented by the general formula:

O

/ ^R o wherein R9, RI Q are as defined above. Prefened embodiments of the amide will not include imides which may be unstable.

The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In prefened embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2)_m-R8, wherein m and Rg are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.

The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and Rj j represents a hydrogen, an alkyl, an alkenyl, -(CH2) - or a pharmaceutically acceptable salt, R'n represents a hydrogen, an alkyl, an alkenyl or -(CH2)_m-Rg, where m and Rg are as defined above. Where X is an oxygen and R\ or R'n is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R\ \ is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when RJ is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'ι i is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and Rj _j or R' \ \ is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and R_{j j} is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and Rj 1 ' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and Ri \ is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and R^ \ is hydrogen, the above formula represents an "aldehyde" group.

The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substitaent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl, -0-(CH2)m"R8» where m and Rg are described above.

The term "sulfonate" is art recognized and includes a moiety that can be represented by the general formula:

in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, /j-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, /?-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, j->-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.

The term "sulfate" is art recognized and includes a moiety that can be represented by the general formula:

O

II O— S— 0R₄ι

II ⁴¹ o in which R4 is as defined above.

The term "sulfonylamino" is art recognized and includes a moiety that can be represented by the general formula:

O

II N— S-R

^I o

R

The term "sulfamoyl" is art-recognized and includes a moiety that can be represented by the general formula:

The term "sulfonyl", as used herein, refers to a moiety that can be represented by the general formula:

O

II S — R44

II o in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term "sulfoxido" as used herein, refers to a moiety that can be represented by the general formula: 0

II

— S-R₄₄

in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A "selenoalkyl" refers to an alkyl group having a substitated seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -Se-(CH2)_m-R7, m and R7 being defined above.

Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n, etc., when it occurs more than once in any structare, is intended to be independent of its definition elsewhere in the same structare.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substitaent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

As used herein, the term "substitated" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substitaents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substitaents of organic compounds. Illustrative substitaents include, for example, those described herein above. The permissible substitaents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substitaents of organic compounds.

The phrase "protecting group" as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2^nd ed.; Wiley: New York, 1991). Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and tr-wω-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substitaent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, it may be isolated using chiral chromatography methods, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., functioning as analgesics), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in binding to opioid receptors. In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Compounds ofthe Invention

In certain embodiments, a complex ofthe present invention is represented by A:

wherein

X represents independently for each occurrence O or S;

Z represents a chelator comprising a radionuclide;

R represents independently for each occurrence H, lower alkyl, or halogen;

R' represents independently for each occurrence lower alkyl;

R" represents independently for each occurrence H, or lower alkyl;

R³ represents independently for each occurrence H, or lower alkyl; and

^• the stereochemical configuration at any stereocenter of a complex represented by A is R, S, or a mixture of these configurations.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O.

In certain embodiments, the present mvention relates to a complex represented by A and the attendant definitions, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino-carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein Z represents a chelator selected from the group consisting ofbis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, andN₂S₂ chelators.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein said radionuclide is technetium.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R represents H.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R' represents methyl.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R" represents H. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R³ represents H.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein the stereochemical relationship between the two instances of R³ is cis. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids,

N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides; and said radionuclide is technetium.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4; and said radionuclide is technetium.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators; and said radionuclide is technetium. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis. In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a complex represented by A and the attendant definitions, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

In certain embodiments, the present invention relates to a formulation, comprising a rotenone complex represented by A and the attendant definitions; and a pharmaceutically acceptable excipient.

The rotenone complexes of the present invention may be used as radiographic imaging agents. The rotenone complexes of the present invention are prepared by reacting a rotenone-chelator compound with a radionuclide containing solution under radionuclide complex forming reaction conditions. In particular, if a technetium agent is desired, the reaction is carried out with a pertechnetate solution under technetium 99 m complex forming reaction conditions. The solvent may then be removed by any appropriate means, such as evaporation. The rotenone complexes are then prepared for administration to the patient by dissolution or suspension in a pharmaceutically acceptable vehicle.

The present invention also relates to imaging agents containing a radionuclide complex as described above, in an amount sufficient for imaging, together with a pharmaceutically acceptable radiological vehicle. The radiological vehicle should be suitable for injection or aspiration, such as human serum albumin; aqueous buffer solutions, e.g tris(hydromethyl) aminomethane (and its salts), phosphate, citrate, bicarbonate, etc; sterile water; physiological saline; and balanced ionic solutions containing chloride and or dicarbonate salts or normal blood plasma cations such as calcium, potassium, sodium, and magnesium.

The concentration of the imaging agent according to the present invention in the radiological vehicle should be sufficient to provide satisfactory imaging, for example, when using an aqueous solution, the dosage is about 1.0 to 50 millicuries. The imaging agent should be administered so as to remain in the patient for about 1 to 3 hours, although both longer and shorter time periods are acceptable. Therefore, convenient ampules containing 1 to 10 mL of aqueous solution may be prepared.

Imaging may be carried out in any workable manner, for example by injecting a sufficient amount of the imaging composition to provide adequate imaging and then scanning with a suitable machine, such as a gamma camera. In certain embodiments, the present invention relates to a method of imaging a region in a patient, comprising the steps of: administering to a patient a diagnostically effective amount of a rotenone complex of the present invention comprising a radionuclide; exposing a region of said patient to radiation; and obtaining an image of said region of said patient. In certain embodiments of the method of imaging a region in a patient, said region of said patient is the thorax. In certain embodiments of the method of imaging a region in a patient, said region of said patient is the heart.

Pharmaceutical Formulations In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the complexes described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absoφtion, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion ofthe body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

Formulations of the present invention may be based in part on liposomes. Liposomes consist of a phospholipid bilayer which forms a shell around an aqueous core.

Methods for preparing liposomes for administration to a patient are known to those skilled in the art; for example, U.S. Pat. No. 4,798,734 describes methods for encapsulation of biological materials in liposomes. The biological material is dissolved in a aqueous solution, and the appropriate phospholipids and lipids are added, along with surfactants if required. The material is then dialyzed or sonicated, as necessary. A review of known methods is presented by G. Gregoriadis, Chapter 14 ("Liposomes"), in Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).

Formulations of the present invention may be based in part on polymeric microparticles. Microspheres formed of polymers or proteins are also well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract, as described in U.S. Pat. Nos. 4,906,474, 4,925,673, and 3,625,214, for example. There are a number of well-known methods, including solvent evaporation and coacervation phase separation, for preparing microspheres. Biqerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, as described, for example, by Mathiowitz et al., J. Appl. Polymer Sci. 35, 755-774(1988), and P. Deasy, in Microencapsulation and Related Drug Processes, pp. 61-193, (Dekker, 1984), the teachings of which are incorporated herein. The selection of a method depends on the drug properties and choice of polymer, as well as the size, external morphology, and degree of crystallinity desired, as discussed, for example, by Benita et al., J. Pharm. Sci. 73, 1721-1724 (1984), Jalil and Nixon, J. Microencapsulation, 7, 297-325(1990), and Mathiowitz et al., Scanning Microscopy 4, 329-340(1990), the teachings of which are incorporated herein. In solvent evaporation, described, for example, in Mathiowitz et al., (1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent. The drug, either in soluble or particulate form, is added to the polymer solution and the mixture is suspended in an aqueous phase containing a surface active agent such as poly (vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres. Microspheres of various sizes (1-1000 microns) and morphologies may be obtained by this method, which is useful for non-labile polymers.

Coacervation/phase separation techniques have been used to encapsulate both solid and liquid core materials with various polymer coatings. U.S. Pat. Nos. 2,730,456, 2,730,457, and 2,800,457 to Green and Schleichter, describe gelatin and gelatin-acacia (gum arabic) coating systems, for example. Simple coacervation employs a single colloid (e.g. gelatin in water) and involves the removal of the associated water from around the dispersed colloid by agents with a higher affinity for water, such as alcohols and salts. Complex coacervation employs more than one colloid, and the separation proceeds mainly by charge neutralization of the colloids carrying opposite charges rather than by dehydration. Coacervation may also be induced using nonaqueous vehicles, as described in Nakano et al., Int. J. Pharm, 4, 29-298(1980), for example. Hydrogel microspheres made of gel-type polymers such as alginate or polyphosphazenes or other dicarboxylic polymers can be prepared by dissolving the polymer in an aqueous solution, suspending the material to be incorporated into the mixture, and extruding the polymer mixture through a microdroplet forming device, equipped with a nitrogen gas jet. The resulting microspheres fall into a slowly stirring, ionic hardening bath, as illustrated, for example, by Salib, et al., Pharmazeutische Industrie 40- 11 A, 1230(1978), the teachings of which are incorporated herein. The advantage of this system is the ability to further modify the surface ofthe microspheres by coating them with polycationic polymers (such as polylysine) after fabrication, as described, for example, by Lim et al, J. Pharm Sci. 70, 351-354(1981). The microsphere particle size depends upon the extruder size as well as the polymer and gas flow rates.

Examples of polymers that can be used include polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt, polypropylene, polyethylenes including poly(ethylene glycol), poly(ethylene oxide), and poly (ethylene terephthalate), and polystyrene.

Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophihc proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

A diluent used in a composition of the present invention can be one or more compounds which are capable of densifying the active principle to give the desired mass. The preferred diluents are mineral phosphates such as calcium phosphates; sugars such as hydrated or anhydrous lactose, or mannitol; and cellulose or cellulose derivatives, for example microcrystalline cellulose, starch, corn starch or pregelatinized starch. Very particularly preferred diluents are lactose monohydrate, mannitol, microcrystalline cellulose and corn starch, used by themselves or in a mixtare, for example a mixtare of lactose monohydrate and corn starch or a mixtare of lactose monohydrate, corn starch and microcrystalline cellulose.

A binder employed in a composition of the present invention can be one or more compounds which are capable of densifying a compound of formula (I), converting it to coarser and denser particles with better flow properties. The preferred binders are alginic acid or sodium alginate; cellulose and cellulose derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose or methyl cellulose, gelatin; acrylic acid polymers; and povidone, for example povidone K-30; hydroxypropyl methyl cellulose and povidone K-30 are very particularly preferred binders.

A disintegrating agent employed in a composition of the present invention can be one or more compounds which facilitate the disintegration of the prepared formulation when it is placed in an aqueous medium. The preferred disintegrating agents are cellulose or cellulose derivatives such as sodium carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose, micro-crystalline cellulose, cellulose powder, crospovidone; pregelatinized starch, sodium starch glyconate, sodium carboxymethyl starch, or starch. Crospovidone, crosslinked sodium carboxymethyl cellulose and sodium carboxymethyl starch are preferred disintegrating agents.

An antiadhesive employed in a composition of the present invention can be one or more compounds which are capable of reducing the sticky character ofthe formulation, for example of preventing adhesion to metal surfaces. The preferred antiadhesives are compounds containing silicon, for example silica or talcum. A flow promoter employed in a composition of the present invention can be one or more compounds which are capable of facilitating the flow ofthe prepared formulation. The preferred flow promoters are compounds containing silicon, for example anhydrous colloidal silica or precipitated silica.

A lubricant employed in a composition of the present invention can be one or more compounds which are capable of preventing the problems associated with the preparation of dry forms, such as the sticking and/or seizing problems which occur in the machines during compression or filling. The preferred lubricants are fatty acids or fatty acid derivatives such as calcium stearate, glyceryl monostearate, glyceryl palmitostearate, magnesium stearate, sodium laurylsulfate, sodium stearylfumarate, zinc stearate or stearic acid; hydrogenated vegetable oils, for example hydrogenated castor oil; polyalkylene glycols or polyethylene glycol; sodium benzoate; or talcum. Magnesium stearate or sodium stearylfumarate is preferred according to the present invention.

A color employed in a formulation of the present invention can be one or more compounds which are capable of imparting the desired color to the prepared formulation. Ηie addition of a color can serve for example to differentiate between formulations containing different doses of active principle. The preferred colors are iron oxides.

As set out above, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palrnitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. Hie term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The fonnulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent. In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle fonning agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound ofthe present invention. Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by unifonnly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant

(for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixtare ofthe powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain 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, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstitated hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood ofthe intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absoφtion of the dmg then depends upon its rate of dissolution which, in tarn, may depend upon crystal size and crystalline form. Alternatively, delayed absoφtion of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature ofthe particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention will be that amount ofthe compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the subject compounds, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, or oral cavity; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

The term "treatment" is intended to encompass also prophylaxis, therapy and cure.

The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.

The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incoφorating the premix into the complete ration. Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and complete rations can be prepared and administered are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and B books, Corvallis, Ore., U.S.A., 1977).

Combinatorial Libraries

The subject complexes readily lend themselves to the creation of combinatorial libraries for the screening of pharmaceutical, agrochemical or other biological or medically- related activity or material-related qualities. A combinatorial library for the puφoses of the present invention is a mixture of chemically related compounds which may be screened together for a desired property; said libraries may be in solution or covalently linked to a solid support. The preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for the appropriate biological, pharmaceutical, agrochemical or physical property may be done by conventional methods.

Diversity in a library can be created at a variety of different levels. For instance, the substrate aryl groups used in a combinatorial approach can be diverse in terms of the core aryl moiety, e.g., a variegation in terms of the ring structare, and/or can be varied with respect to the other substituents.

A variety of techniques are available in the art for generating combinatorial libraries of small organic molecules. See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Patents 5,359,115 and 5,362,899: the Ellman U.S. Patent 5,288,514: the Still et al. PCT publication WO 94/08051; Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCT publication WO93/20242). Accordingly, a variety of libraries on the order of about 16 to 1,000,000 or more diversomers can be synthesized and screened for a particular activity or property. In an exemplary embodiment, a library of substituted diversomers can be synthesized using the subject reactions adapted to the techniques described in the Still et al. PCT publication WO 94/08051, e.g., being linked to a polymer bead by a hydrolyzable or photolyzable group, e.g., located at one of the positions of substrate. According to the Still et al. technique, the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead. In one embodiment, which is particularly suitable for discovering enzyme inhibitors, the beads can be dispersed on the surface of a permeable membrane, and the diversomers released from the beads by lysis of the bead linker. The diversomer from each bead will diffuse across the membrane to an assay zone, where it will interact with an enzyme assay. Detailed descriptions of a number of combinatorial methodologies are provided below.

A) Direct Characterization A growing trend in the field of combinatorial chemistry is to exploit the sensitivity of techniques such as mass spectrometry (MS), e.g., which can be used to characterize sub- femtomolar amounts of a compound, and to directly determine the chemical constitution of a compound selected from a combinatorial library. For instance, where the library is provided on an insoluble support matrix, discrete populations of compounds can be first released from the support and characterized by MS. In other embodiments, as part of the MS sample preparation technique, such MS techniques as MALDI can be used to release a compound from the matrix, particularly where a labile bond is used originally to tether the compound to the matrix. For instance, a bead selected from a library can be irradiated in a MALDI step in order to release the diversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin library format. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS 81:3998-4002) introduced a method for generating compound libraries by a parallel synthesis on polyacrylic acid-grated polyethylene pins arrayed in the microtifre plate format. The Geysen technique can be used to synthesize and screen thousands of compounds per week using the multipin method, and the tethered compounds may be reused in many assays. Appropriate linker moieties can also been appended to the pins so that the compounds may be cleaved from the supports after synthesis for assessment of purity and further evaluation (c.f, Bray et al. (1990) Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem 197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can be provided on a set of beads utilizing the strategy of divide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135; and U.S. Patents 4,631,211; 5,440,016; 5,480,971). Briefly, as the name implies, at each synthesis step where degeneracy is introduced into the library, the beads are divided into separate groups equal to the number of different substitaents to be added at a particular position in the library, the different substitaents coupled in separate reactions, and the beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carried out using an analogous approach to the so-called "tea bag" method first developed by Houghten, where compound synthesis occurs on resin sealed inside porous polypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Substituents are coupled to the compound- bearing resins by placing the bags in appropriate reaction solutions, while all common steps such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end ofthe synthesis, each bag contains a single compound. D) Combinatorial Libraries by Light-Directed, Spatially Addressable Parallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compound is given by its locations on a synthesis substrate is termed a spatially-addressable synthesis. In one embodiment, the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations on a solid support (Dower et al. (1991) Annu Rep Med Chem 26:271-280; Fodor, S.P.A. (1991) Science 251:767; Pirrung et al. (1992) U.S. Patent No. 5,143,854; Jacobs et al. (1994) Trends Biotechnol 12:19-26). The spatial resolution of photolithography affords miniaturization. This technique can be carried out through the use protection/deprotection reactions with photolabile protecting groups. The key points of this technology are illustrated in Gallop et al. (1994) J Med Chem

37:1233-1251. A synthesis substrate is prepared for coupling through the covalent attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers. Light is used to selectively activate a specified region of the synthesis support for coupling. Removal of the photolabile protecting groups by light (deprotection) results in activation of selected areas. After activation, the first of a set of amino acid analogs, each bearing a photolabile protecting group on the amino terminus, is exposed to the entire surface. Coupling only occurs in regions that were addressed by light in the preceding step. The reaction is stopped, the plates washed, and the substrate is again illuminated through a second mask, activating a different region for reaction with a second protected building block. The pattern of masks and the sequence of reactants define the products and their locations. Since this process utilizes photolithography techniques, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be addressed with appropriate resolution. The position of each compound is precisely known; hence, its interactions with other molecules can be directly assessed.

In a light-directed chemical synthesis, the products depend on the pattern of illumination and on the order of addition of reactants. By varying the lithographic patterns, many different sets of test compounds can be synthesized simultaneously; this characteristic leads to the generation of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compound library provided with an encoded tagging system. A recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using tags that uniquely encode the reaction steps a given bead has undergone and, by inference, the structure it carries. Conceptually, this approach mimics phage display libraries, where activity derives from expressed peptides, but the structures of the active peptides are deduced from the corresponding genomic DNA sequence. The first encoding of synthetic combinatorial libraries employed DNA as the code. A variety of other forms of encoding have been reported, including encoding with sequenceable bio-oligomers (e.g., oligonucleotides and peptides), and binary encoding with additional non-sequenceable tags.

1) Tagging with sequenceable bio-oligomers

The principle of using oligonucleotides to encode combinatorial synthetic libraries was described in 1992 (Brenner et al. (1992) PNAS 89:5381-5383), and an example of such a library appeared the following year (Needles et al. (1993) PNAS

90:10700-10704). A combinatorial library of nominally 7^ (= 823,543) peptides composed of all combinations of Arg, Gin, Phe, Lys, Val, D-Val and Thr (three-letter amino acid code), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), was prepared by a series of alternating rounds of peptide and oligonucleotide synthesis on solid support. In this work, the amine linking functionality on the bead was specifically differentiated toward peptide or oligonucleotide synthesis by simultaneously preincubating the beads with reagents that generate protected OH groups for oligonucleotide synthesis and protected H2 groups for peptide synthesis (here, in a ratio of 1:20). When complete, the tags each consisted of 69-mers, 14 units of which carried the code. The bead-bound library was incubated with a fiuorescently labeled antibody, and beads containing bound antibody that fluoresced strongly were harvested by fluorescence- activated cell sorting (FACS). The DNA tags were amplified by PCR and sequenced, and the predicted peptides were synthesized. Following such techniques, compound libraries can be derived for use in the subject method, where the oligonucleotide sequence ofthe tag identifies the sequential combinatorial reactions that a particular bead underwent, and therefore provides the identity of the compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive tag analysis. Even so, the method requires careful choice of orthogonal sets of protecting groups required for alternating co-synthesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the choice of reagents and conditions that can be employed for the synthesis of non-oligomeric libraries. In preferred embodiments, the libraries employ linkers permitting selective detachment ofthe test compound library member for assay.

Peptides have also been employed as tagging molecules for combinatorial libraries. Two exemplary approaches are described in the art, both of which employ branched linkers to solid phase upon which coding and ligand strands are alternately elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531). orthogonality in synthesis is achieved by employing acid-labile protection for the coding strand and base-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170). branched linkers are employed so that the coding unit and the test compound can both be attached to the same functional group on the resin. In one embodiment, a cleavable linker can be placed between the branch point and the bead so that cleavage releases a molecule containing both code and the compound (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894). In another embodiment, the cleavable linker can be placed so that the test compound can be selectively separated from the bead, leaving the code behind. This last construct is particularly valuable because it permits screening of the test compound without potential interference of the coding groups. Examples in the art of independent cleavage and sequencing of peptide library members and their corresponding tags has confirmed that the tags can accurately predict the peptide structure. 2) Non-sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a set of non-sequencable electrophone tagging molecules that are used as a binary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tags are haloaromatic alkyl ethers that are detectable as their trirnethylsilyl ethers at less than femtomolar levels by electron capture gas chromatography (ECGC). Variations in the length of the alkyl chain, as well as the nature and position of the aromatic halide substitaents, permit the synthesis of at least 40 such tags, which in principle can encode 2^0 (e.g., upwards of 10^) different molecules. In the original report (Ohlmeyer et al., supra) the tags were bound to about 1% of the available amine groups of a peptide library via a photocleavable o-nitrobenzyl linker. This approach is convenient when preparing combinatorial libraries of peptide-like or other amine-containing molecules. A more versatile system has, however, been developed that permits encoding of essentially any combinatorial library. Here, the compound would be attached to the solid support via the photocleavable linker and the tag is attached through a catechol ether linker via carbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem 59:4723-4724). This orthogonal attachment strategy permits the selective detachment of library members for assay in solution and subsequent decoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binary encoding with the electrophone tags attached to amine groups, attaching these tags directly to the bead matrix provides far greater versatility in the structures that can be prepared in encoded combinatorial libraries. Attached in this way, the tags and their linker are nearly as unreactive as the bead matrix itself. Two binary-encoded combinatorial libraries have been reported where the electrophone tags are attached directly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031) and provide guidance for generating the subject compound library. Both libraries were constructed using an orthogonal attachment strategy in which the library member was linked to the solid support by a photolabile linker and the tags were attached through a linker cleavable only by vigorous oxidation. Because the library members can be repetitively partially photoeluted from the solid support, library members can be utilized in multiple assays. Successive photoelution also permits a very high throughput iterative screening strategy: first, multiple beads are placed in 96-well microtiter plates; second, compounds are partially detached and transferred to assay plates; third, a metal binding assay identifies the active wells; fourth, the corresponding beads are rearrayed singly into new microtiter plates; fifth, single active compounds are identified; and sixth, the structures are decoded.

Exemplification The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for puφoses of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1

Rotenone Methyl Enol Ether

Rotenone (25 g, 0.063 mol) was placed in a 100 mL flask equipped with a stir bar. The white solid was dissolved in 500 mL of methanol. Added to the solution was trimethyl orthoformate (8.31 mL, 0.076 mol) via syringe. Next, p-toluenesulfonic acid (catalytic amount) was added. The solution was refluxed for 16 hrs, with additional 0.1 g of catalyst added after 8 hrs. The solution was then allowed to cool to room temperature. Neutralization of the reaction was accomplished by addition of potassium carbonate. The solution was then vacuumed down to residue. The residue was passed through a silca column using 1% methanol / methylene chloride yielding 3.83 g, 15% yield. 'H NMR (CDC1₃) 300 MHz: 1.40 (m, H), 1.78 (d, 3H), 2.95 (m, H), 3.25 (m, H), 3.73 (s, 3H), 3.86 (s, 3H), 3.90 (s, 3H), 4.15 (t, H), 4.52 (q, H), 4.93 (s, H), 5.10 (s, H), 5.21 (m, H), 5.32 (m, H), 6.44 (s, H), 6.46 (dd, H), 7.15 (dd, H), 7.92 (s, H).

Example 2 Hydroboration of Rotenone Methyl Enol Ether

Rotenone methyl enol ether (1.51 g, 3.69 mmol) was placed in a 250 mL flask equipped with a stirrer under nitrogen. The yellow solid was dissolved in 9-BBN (0.5 M solution in THF) (29.6 mL, 0.0148 mol). The mixtare was refluxed for 5 hrs. After cooling to room temperatare 6 mL of ethanol were added, followed by 2 mL of 6 N NaOH, and finally 4 mL of 30% H₂0₂. Hie additions were all performed dropwise resulting in a cloudy mixture which was then heated at 50 C for 1 hr. The solution was cooled, extracted with ether, washed with a solution of saturated sodium carbonate, and dried over sodium sulfate. The solution was then vacuumed down to residue. The residue was passed through a silca column using 1% methanol / methylene chloride yielding 0.660 g, 42% yield. Η NMR (CDC1₃) 300 MHz: 1.03 (m, 3H), 1.25 (m, H), 1.60 (m, H), 2.10 (m, H), 2.95 (m, H), 3.20 (m, H), 3.49 (s, H), 3.73 (s, 3H), 3.86 (s, 3H), 3.90 (s, 3H), 4.15 (t, H), 4.52 (q, H), 4.71 (m, H), 5.02 (m, H), 5.35 (m, H), 6.42 (dd, H), 6.44 (s, H), 7.14 (dd, H), 7.92 (s, H).

The hydroxymethyl rotenone derivative (0.568 g, 1.33 mmol) was placed in a 100 mL flask equipped with a stirrer under nitrogen. The flask containing the yellow solid was placed in an ice-bath. The solid was dissolved in 20 mL of methylene chloride, followed by addition of triethylamine (0.185 mL, 2.66 mmol). Finally, p-toluenesulfonyl chloride (0.508 g, 2.66 mmol) was added to the reaction mixture. The solution was allowed to stir at room temperatare for 4 hrs. The solution was then vacuumed down to residue. The residue was passed tlirough a silca column using 0.5 % methanol / methylene chloride yielding 0.376 g, 49% yield. Η NMR (CDC1₃) 300 MHz: 1.00 (m, 3H), 1.20 (m, H), 1.54 (s, H), 2.17 (m, H), 2.45 (s, 3H), 2.87 (m, H), 3.13 (m, H), 3.48 (s, H), 3.71 (s, 3H), 3.86 (s, 3H), 3.89 (s, 3H), 4.08 (m, H), 4.16 (m, H), 4.49 (m, H), 4.59 (m, H), 5.30 (m, H), 6.33 (m, H), 6.43 (s, H), 7.10 (d, H), 7.34 (d, 2H), 7.79 (m, 2H), 7.91 (s, H).

Example 4

The tosylated rotenone derivative (0.350 g, 0.60 mmol) was placed in a 100 mL flask equipped with a stirrer under nitrogen. The solid was dissolved in 5 mL of DMF, followed by addition of potassium carbonate (0.10 g, 0.72 mmol). Finally, the ethyl ester of picolineamine mono-acetic acid (PAMA) was added (0.138 g, 0.664 mmol) was added to the reaction mixture. The solution was heated at 105 C for 4 hrs. The solution was then vacuumed down to residue. The residue was passed through a HPLC silca gel column using 0-10% methanol/methylene chloride as the solvents, yielding 0.066 g, 18% yield. Η NMR (CDCL, 300 MHz) δ 1.00 (m, 3H), 1.26 (m, 3H), 1.55 (m, 2H), 2.15 (m, 2H), 2.38 (m, H), 2.85 (m, 2H), 3.21 (m, 2H), 3.48 (s, H), 3.72 (s, 3H), 3.86 (s, 3H), 3.89 (s, 3H), 4.20 (m, 2H), 4.52 (m, H), 4.70 (m, H), 4.75 (m, H), 4.85 (m, H), 5.30 (m, H), 6.42 (m, H), 6.43 (m, 2H), 7.12 (m, H), 7.30 (m, H), 7.79 (m, H), 7.91 (s, H), 8.55 (d, H).

Example 5

The procedure outlined in Example 4 was used with a mesylated rotenone derivative, yielding 0.006 g, 21% yield. Η NMR (CDC1₃) 300 MHz: 0.95 (m, 3H) 1.21 (m, 3H), 1.35 (m, H), 1.60 (m, H), 2.05 ( , 2H), 2.19 (m, H), 2.95 (m, H), 3.20 (m, H), 3.45 (s, 2H), 3.49 (s, H), 3.52 (s, 2H), 3.73 (s, 3H), 3.83 (s, 3H), 3.90 (s, 3H), 4.15 (t, H), 4.54 (q, H), 4.80 (m, H), 4.92 (m, H), 5.35 (s, H), 6.44 (s, H), 6.72 (d, H), 7.04 (dd, H), 7.18 (m, H), 7.31 (m, H), 7.54 (d, H), 7.67 (m, H) 7.98 (s, H), 8.43 (d, H). Example 6

The procedure outlined in Example 4 was used with a mesylated rotenone derivative and protected N,N'-bis(PMB-S-ethyl)glycine amide, yielding 0.010 g ofthe crude product.

Example 7

Rotenol-dipyridinemethylamine

Dipyridinemethylamine (DPMA) (0.0035 g, 0.0018 mmol) and o-Tos-Rotenol (0.01 g, 0.0018 mmol) were mixed in a 100 mL pressure tube in 2 mL of DMF under nitrogen. Potassium carbonate (0.05 g) and triethylamine (0.3 mL) were added to the solution. The mixtare was heated at 130° C for 3 hrs. The reaction mixture was vacuumed down to residue. The residue was purified through a pad of silica gel using methanol-methylene chloride to provide the product in 51 % yield. Η NMR (CDC1₃): 1.2 (m), 1.58 (s), 3.21 (m), 3.48 (m), 3.52 (m), 3.61 (m), 3.78 (s), 3.90 (m), 3.95 (d), 5.15 (s), 6.30(d), 6.48 (s), 6.70 (m), 6.85 (s), 7.20 (s), 7.25 (m), 7.35 (d), 7.52 (dd), 7.79 (d), 8.01 (d).

Example 8

Re(CO)₃(η₃-(rotenol-dipyridinemethylamine) The synthesis of the Re(I) tricarbonyl complex was accomplished by reacting [NEt₄]₂[ReBr₃(CO)₃] with the rotenol-DPMA in the ratio of 1:1.2 in 2.5 mL of methanol. The reaction mixture was heated at 120 °C for 6 hours. After cooling, the reaction product was purified using a small silica column using 95% methylene chloride 5% methanol. LCMS analysis using a C18 column with water and acetonitrile demonstrated multiple peaks, but near the retention time of the ^99mTc-labeled product demonstrated the molecular weight 864, which corresponds to the rhenium complex.

Example 9

Tc(CO)₃(η₃-(rotenol-dipyridinemethylamine) [^99mTc(CO)₃(H₂0)₃]⁺was heated with rotenol-DPMA in 0.5 mL (lmg / mL) of methanol at 100° C for 60 minutes. Purity, analyzed via C18 HPLC, showed >68% RCP. The product eluted with methanol at 20.5 minutes. The HPLC analysis was performed using a Supelco C 18 column, 25 cm x 4.6 mm column (5μm pore size), equipped with 2 cm guard using solvent A = 0.05 M triethylammonium phosphate buffer pH 2.5 and solvent B = methanol. The method employed was a gradient 5-95% B, 1 mL/ minute for 30 minutes. The gradient ramped from 5-95 from 3-20 minutes. In challenge experiments the HPLC purified product demonstrated no degradation in either 10 mM Cysteine or Histidine in PBS pH 7.2 at 37° C for 18 hrs.

Example 10 Animal Studies — Tc(CO)₃(η₃-frotenol-dipyridinemethylamine)

The biodistribution of Tc-99m-rotenol-DMPA was investigated in male rats (Sprague Dawley, n = 3/timepoint; ~180 gms). The complex was injected via the tail vein in 10% ethanol in saline (10 μCi / 100 μl). Animals were sacrificed at 5, 15, 30, and 60 minutes p.i. The results are shown below in Table 1.

Table 1

Biodistribution of Tc99m-Rotenol-DPMA Complex (expressed as Average %ID/g)

Incorporation By Reference

All ofthe patents and publications cited herein are hereby incoφorated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A complex represented by A:

wherein X represents independently for each occurrence O or S;

Z represents a chelator comprising a radionuclide;

R represents independently for each occurrence H, lower alkyl, or halogen;

R' represents independently for each occurrence lower alkyl;

R" represents independently for each occurrence H, or lower alkyl; R³ represents independently for each occurrence H, or lower alkyl; and the stereochemical configuration at any stereocenter of a complex represented by A is R, S, or a mixture of these configurations.

2. The complex of claim 1, wherein X represents O.

3. The complex of claim 1, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides .

4. The complex of claim 1, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino-carboxylic acids, andN_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4.

5. The complex of claim 1, wherein Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, andN₂S₂ chelators.

6. The complex of claim 1 , wherein said radionuclide is technetium.

7. The complex of claim 1 , wherein R represents H.

8. The complex of claim 1 , wherem R' represents methyl.

9. The complex of claim 1 , wherein R" represents H.

10. The complex of claim 1 , wherein R³ represents H.

11. The complex of claim 1, wherein the stereochemical relationship between the two instances of R³ is cis.

12. The complex of claim 1, wherein R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

13. The complex of claim 1, wherein R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

14. The complex of claim 1, wherein X represents 0; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino- carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides.

15. The complex of claim 1, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2- amino-carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4.

16. The complex of claim 1, wherein X represents O; and Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2- pyridylmethyl)glycine, and N₂S₂ chelators.

17. The complex of claim 1, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides; and said radionuclide is technetium,

18. The complex of claim 1, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4; and said radionuclide is technetium.

19. The complex of claim 1, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators; and said radionuclide is technetium.

20. The complex of claim 1, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-pyridyl-2-amino-carboxylic acids, N_xS_y chelators, wherein x is 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4, DTPA, EDTA, SHNH, and mercaptoacetyltripeptides; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

21. The complex of claim 1, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amines, N-(2-pyridylmethyl)-2-amino- carboxylic acids, and N_xS_y chelators, wherein x is 1, 2, 3, or 4, y is 0, 1, 2, or 3, and the sum of x and y is 4; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

22. The complex of claim 1, wherein X represents O; Z represents a chelator selected from the group consisting of bis(2-pyridylmethyl)amine, N-(2-pyridylmethyl)glycine, and N₂S₂ chelators; said radionuclide is technetium; R represents H; R' represents methyl; R" represents H; R³ represents H; and the stereochemical relationship between the two instances of R³ is cis.

23. A composition, comprising a complex according to any of claims 1-22; and a pharmaceutically acceptable excipient.

24. A method of imaging a region in a patient, comprising the steps of: administering to a patient a diagnostically effective amount of a complex of claim 1; exposing a region of said patient to radiation; and obtaining an image of said region of said patient.

25. The method of claim 24, wherein said region of said patient is the thorax.

26. The method of claim 24, wherein said region of said patient is the heart.

27. A kit, comprising a complex of claim 1 in a container, and instructions for using said compound to image a region in a patient.