USOO6359111B1 (12) United States Patent (10) Patent No.: US 6,359,111 B1 Meyer et al. (45) Date of Patent: Mar. 19, 2002

(54) OPIOID RECEPTOR TARGETING 5,352,680 A 10/1994 Portoghese et al...... 514/279 5,411,965 A 5/1995 Reid et al...... 514/279 (75) Inventors: Damon L. Meyer, Bellevue; Sudhakar 5,455,230 A 10/1995 Schiller ...... 514/18 Kasina. Mercer Island, both of WA 5,457,208 A 10/1995 Portoghese et al...... 546/35 (US) s s 5,464,841. A 11/1995 Portoghese et al...... 514/279 5,552.404 A 9/1996 Chang et al...... 514/255 (73) Assignee: NeoRX Corporation, Seattle, WA (US) 5,591.6025,578,725 A * 11/19961/1997 PortoghSee tal. alr ...... 435/69.1 546/35 5,622,946 A 4/1997 Sessler et al...... 514/185 (*) Notice: Subject to any disclaimer, the term of this 5,631,263 A 5/1997 Portoghese et al...... 514/279 patent is extended or adjusted under 35 5,658,908 A 8/1997 Chang et al...... 514/252 U.S.C. 154(b) by 0 days. 5,753,206 A 5/1998 McBride et al...... 424/1.69 5,772,981 A 6/1998 Govindan et al...... 424/1.49 (21) Appl. No.: 09/321,054 5,804,595 A * 9/1998 Portoghese et al...... 514/428 (22) Filed: May 27, 1999 FOREIGN PATENT DOCUMENTS Related U.S. Application Data wo WO 955. : go (60) prisional application No. 60/087.209, filed on May 28, WO 94/11021 * 5/1994 WO WO 98/04917 2/1998 (51) Int. Cl." ...... A61K 31/381; A61K 31/4355; OTHER PUBLICATIONS A61K 51/04; CO7D 489/00; CO7D 489/09 (52) U.S. Cl...... 530/302; 424/1.45; 424/9.1; American Radiolabeled Chemicals, Inc., Technical Data 424/9.44; 424/9.6; 514/2; 514/17, 514/250; Sheet, “ART549 Naltrindole, 5, 7–H).” Feb. 17, 1997. 514/259; 514/279; 514/281; 514/282; 530/345; 544/233; 544/283; 54.6/35; 54.6/39; 546/40; (List continued on next page.) 546/44; 546/45; 546/46; 534/10 (58) Field of Search 424/1.45, 9.1 fis, Examiner Jeffrey E. R. llectual ------n in is 74) Attorney, Agent, or Firm-Seed Intellectual Property 5143,5,6,7,85.250,250,276.2s.424/9.42, 9.44, 9.6; 530/302, 330, 345; Law Group PLLC 282, 562, 566, 616, 665; 54.6/34, 35, 39, (57) ABSTRACT 40, 44, 45, 46; 544/233,283; 534/10, 11, 12, 13, 14; 562/556, 565, 566; 564/154 A compound comprising a ligand portion which has binding affinity for an opioid receptor, and a therapeutically or (56) References Cited diagnostically effective group Selected from radionuclide chelating agents, fluorochromes, toxins, drugs, polyboron U.S. PATENT DOCUMENTS moieties, proteins, biological response modifiers, chemical 4231923 A 11/1980 Miller et al...... 260/112.5 R moieties capable of binding to other molecules of interest, 4,257,773. A 3/1981 Miller et al...... 23/230 B and radioisotopes Selected from therapeutically effective 4,263,255. A 4/1981 Miller et al...... 422/61 alpha and beta emitters, and diagnostically effective gamma 4,649.200 A 3/1987 Portoghese et al...... 546/26 emitters. The compound may be used in a method of treating 4,767,718 A * 8/1988 Meyers ...... 436/5O1 cancer, and in a method of imaging opioid receptors either 4,816,586 A 3/1989 Portoghese ...... 544/340 5,089,249 A 2/1992 Fritzberg et al...... 424/1.1 inside or outside of the central nervous System. 5,298.622 A 3/1994 Portoghese et al...... 546/15 5,332,818 A 7/1994 Nagase et al...... 546/37 17 Claims, 23 Drawing Sheets

US 6,359,111 B1 Page 2

OTHER PUBLICATIONS Luthra et al., “The Preparation of Carbon-11 Labelled Diprenorphine: a New Radioligand for the Study of the Campa et al., “Characterization of 8 Opioid Receptors in Opiate Receptor System In vivo,” J. Chem. Soc. Chem. Lung Cancer Using a Novel Nonpeptidic Ligand, Cancer Commun. 20: 1365–1456, 1995. Research 56: 1965-1701, 1996. Chang et al., “A Novel, Potent and Selective Nonpeptidic Olmsted et al., “A Remarkable Change of Opioid Receptor Delta Opioid Receptor Agonist BW373U86,” The Journal of Selectivity on the Attachment of a Peptidomimetic X Pharmacology And Experimental Therapeutics 267(1): Address Element to the 8 Antagonist, Natrindole: 5'- 852-857, 1993. N°-Alkylamidino)methyl)naltrindole Derivatives as a Channing et al., “Radiosynthesis of 'F3-Acetylcyclo Novel Class of K Opioid Receptor Antagonists,” J. Med. foxy: A High Affinity Opiate Antagonist,” Int. J. Appl. Chem. 36: 179-180, 1993. Radiat. Isot. 36(6): 429–433, 1985. Otte et al., “Yttrium-90-labelled somatostatin-analogue for Cohen et al., “F-18-Cyclofoxy PET Imaging In Man,” The cancer treatment,” The Lancet 351: 417-418, 1998. Journal of Nuclear Medicine 29(5): p. 796, Abstract No. 228, 1988. Portoghese et al., “ö Opioid Antagonist Activity and Binding Dannals et al. “Radiosynthetic of an Opiate Receptor Bind Studies of Regioisomeric Isothiocyanate Derivatives of Nal ing Radiotracer: 'CICarfentanil.” Int. J. Appl. Radiat. Isot. trindole: Evidence for 8 Receptor Subtypes,” J. Med. Chem. 36(4), 303-306, 1985. 35(2): 4086–4091, 1992. Dasher et al., “Electrophilic Opioid Ligands. Oxygen Teth Portoghese et al., “Application of the Message-Address ered C-Methylene-y-lactone, Acrylate, Isothiocyanate, and Concept in The Design of Highly Potent and Selective Epoxide Derivatives of 63-Naltrexol,” Journal of Medicinal Non-Peptide ö Opioid Receptor Antagonists,” Journal of Chemistry 35(13): 2374-2384, 1992. Medicinal Chemistry 31(2): 281-282, 1988. Frost et al., “Comparison Of C-11 Diprenorphine And C-11 Carfentanil Binding To Opiate Receptors. In Man By PET,” Portoghese et al., “Naltrindole, a highly Selective and potent The Journal of Nuclear Medicine 29(5): p. 796, Abstract No. non-peptide ö opioid receptor antagonist, European Jour 231, 1988. nal of Pharmacology 146:185-186, 1988. Frost et al., “Imaging Opiate Receptors in the Human Brain Portoghese et al., “Opioid Agonist and Antagonist Activities by Positron Tomography,” Journal of Computer Assisted of Morphindoles Related to Naltrindole,” J. Med. Chem. 35: Tomography 9(2): 231-236, 1985. 4325-4329, 1992. Frost et al., “Multi-Compartmental Analysis. Of C-11-Diprenorphine Binding To Opiate Receptors. In Man Portoghese, "Bivalent ligands and the message-address con By PET.” The Journal of Nuclear Medicine 29(5): p. 796, cept in the design of Selective opioid receptor antagonists,” Abstract No. 230, 1988. TiPs 10: 230-235, 1989. Jones et al., “Regional cerebral opioid receptor Studies with Schmidhammer et al., “Synthesis And Biological Evaluation 'Cldiprenorphine in normal volunteers,” Journal of Neu Of 14-Alkoxymorphinans. 14.' 14-Ethoxy-5-Methyl Sub roscience Methods 23: 121-129, 1988. stituted Indolomorphinans With 8 Opioid Receptor Selec Koolpe et al., “Opioid Agonists and Antagonists. tivity,” Bioorganic & Medicinal Chemistry Letters 7(2): 6-DeSoxy-6-Substituted Lactone, Epoxide, and Glycidate 151-156, 1997. Ester Derivatives of Naltrexone and Oxymorphone,” Jour Vogel et al., “Cyclic morphiceptin analogs: cyclization Stud nal of Medicinal Chemistry 28:949–957, 1985. Lever et al., “Synthesis. Of Carbon-11 Labeled Diprenor ies and opioid activities in vitro,” Int. J. Peptide & Protein phine: A Radioligand For Positron Emission Tomographic Res. 48: 495-502, 1996. Studies Of Opiate Receptors.” Tetrahedron Letters 28(35): Weerawarna et al., “Isothiocyanate-Substituted K-Selective 4015-4018, 1987. Opioid Receptor Ligands Derived from N-Methyl-N- Lever et al., “Synthesis Of N1'-(I'CMethyl)Naltrindole (1S)-1-phenyl-2-(1-pyrrolidinyl)ethylphenylaceta ('CMeNTI): A Radioligand For Positron Emission Tomo mide,” Journal of Medicinal Chemistry 37(18): 2856-2864, graphic Studies Of Delta Opioid Receptors,” Journal of 1994. Labelled Compounds and Radiopharmaceuticals XXXVI(2): 137-145, 1995. * cited by examiner U.S. Patent Mar. 19, 2002. Sheet 1 of 23 US 6,359,111 B1 (C H2)- NH, O N O

NH 2

(NHCH-NH. O

FIG. IA U.S. Patent Mar. 19, 2002. Sheet 2 of 23 US 6,359,111 B1 C (CH2)-NH

O OCH-CNH-(CH)-NH.

O A V R F F CHCH- NHCHCH-NH HC

-NH(CH)s-NH.

FIG. IB U.S. Patent Mar. 19, 2002. Sheet 3 of 23 US 6,359,111 B1

COOH

RO O N-(X)-(O)-CH, (UYN COOH COOH FIG. 2B

R R’ 1. S W NH i. U.S. Patent Mar. 19, 2002. Sheet 4 of 23 US 6,359,111 B1

U.S. Patent Mar. 19, 2002. Sheet 5 of 23 US 6,359,111 B1

U.S. Patent Mar. 19, 2002. Sheet 6 of 23 US 6,359,111 B1

H2N-OH, HCl ( EtOH

R= H, COCH3 N

U.S. Patent Mar. 19, 2002. Sheet 7 of 23 US 6,359,111 B1

HN-OH. HC1 / EtOH

U.S. Patent Mar. 19, 2002. Sheet 8 of 23 US 6,359,111 B1

U.S. Patent Mar. 19, 2002. Sheet 9 of 23 US 6,359,111 B1

8 FIG. 4B U.S. Patent Mar. 19, 2002. Sheet 10 of 23 US 6,359,111 B1

U.S. Patent Mar. 19, 2002. Sheet 11 of 23 US 6,359,111 B1

NHS/DCCATHF CH, U.S. Patent Mar. 19, 2002. Sheet 12 of 23 US 6,359,111 B1

------TFA/CHC1 e.

U.S. Patent Mar. 19, 2002. Sheet 13 of 23 US 6,359,111 B1

DMF/EtN--- U.S. Patent Mar. 19, 2002. Sheet 14 of 23 US 6,359,111 B1

DMF/EtN U.S. Patent Mar. 19, 2002. Sheet 15 of 23 US 6,359,111 B1

COH OH

-J

FIG. 8A U.S. Patent Mar. 19, 2002. Sheet 16 of 23 US 6,359,111 B1 A N OH

HO o O O r O, s O N

FIG. 8B U.S. Patent Mar. 19, 2002. Sheet 17 of 23 US 6,359,111 B1

Narayer.{X-N-coh “"“”N- K-No

Butoacy y-cotsu N S Z + re-- COBu COBu BOP, 3. EtiFN N OH K)-4)- C Butoacyl-cotsu

O --~k-H (VW (H2,2 Pd/C) 9. Cotsu CoatBu

Fig. 9A U.S. Patent Mar. 19, 2002. Sheet 18 of 23 US 6,359,111 B1

COH COH

Fig. 9B U.S. Patent Mar. 19, 2002. Sheet 19 of 23 US 6,359,111 B1

r NH r OH HN OH + n h, A- B2Br Ho o'wr of 'o' N KCO,mass OMF Yerazine Natrindcle N > OH N OH 1. NaH zC ow" 2 BCHCOMe BZO ow N O l HOC 5

Fig. 10 U.S. Patent Mar. 19, 2002. Sheet 20 of 23 US 6,359,111 B1

Nk N 7N PIOC OH O C t H. All Mary-uninocaprosta, 2-Hedren. Ro obd Roy ov BOP, EPN GA benzoic acid 1. COH

r OH r NaOH, MeOH OH Roy ov" wn RO O 13 orn-r-co r 3 - 3 BOP, EPN SH ButOC- - M-COBu Ro1 o' N N S H V-f 14 COatsu COBu 1. TFA

HOacy -coh o1 o' -- N N O COH CO2H

Fig. 11 U.S. Patent Mar. 19, 2002 Sheet 21 of 23 US 6,359,111 B1

SH 1. DCC, NHS N 2. H. PoC N-a-N-CO2HH O

FIG. 12 U.S. Patent Mar. 19, 2002. Sheet 22 of 23 US 6,359,111 B1

FIG. I.3 U.S. Patent Mar. 19, 2002. Sheet 23 of 23 US 6,359,111 B1

BzIOC Vr • )-1COHco SH Glutamate O-Benzyl ester HNOC alo? O Y de H -rco, 1. BOP, EtipN Hoc isoglutamine 2. BOP, 5. EtiFN O HOC 3. H. PdC 2 so 11'N, 4. FA ck-N-CBZ-Lysins

N

HO NN al N O COH N.

coh CO2H

Ho:c- N -COH K JC,NN D coh CO2H

HOCyryl-COH N N. R-(- (V-7) V1N O O doh CO2H SH & 2 XX HO2CV2'? ?v YOU -COH Hoy To Y R-O- SN-O-O O coh('\-v Coah

FIG. I.4 US 6,359,111 B1 1 2 OPOLD RECEPTOR TARGETING Chem. 36:179-180 (1993); Portoghese, J. Med. Chem. 35:4325-4329 (1992); Portoghese et al., J. Med. Chem. CROSS-REFERENCE TO RELATED 35:4086–4091 (1992); Portoghese, TiPS Reviews APPLICATION 10:230–235 (1989); Portoghese et al., J. Med. Chem. 31:281–282 (1988); Portoghese, Eur. J. Pharm 146:185-186 This application claims benefit of U.S. Provisional Patent (1988); Schmidhammer et al., Bioorganic & Med. Chem. Application No. 60/087,209, filed on May 28, 1998, which Let. 7:151-156 (1997); Vogel et al., Int. J. Peptide Protein application is incorporated herein by reference. Res. 48:495-502 (1996); Weerawarna et al., J. Med. Chem. TECHNICAL FIELD 37:2856–2864 (1994). 1O There is a need in the art for effective opioid receptor The present invention relates to therapeutic and diagnostic ligands which demonstrate Specificity to one or more chemical agents, and more particularly to compounds that intended opioid receptors, and can deliver functional agents can bind to an opioid receptor while being conjugated to a to the opioid receptor. The present invention fulfills this need therapeutically or diagnostically active group. and further provides other related advantages as disclosed 15 herein. BACKGROUND OF THE INVENTION Studies of the binding of various ligands in brain and SUMMARY OF THE INVENTION other tissues have revealed the presence of Several distinct In one aspect, the present invention provides a compound receptors that bind to opioid compounds. that includes a ligand portion (L) which has binding affinity Three major categories of opioid receptors in animals are for an opioid receptor, and a therapeutically or diagnosti recognized, and these are commonly designated as Ö (delta), cally effective group (X). The group (X) may be selected K (kappa) and u (mu). Several review articles are available, from, for example, radionuclide chelating agents, which describe the research which elucidated the presence fluorochromes, toxins, drugs, polyboron moieties, proteins, of these receptors, and the endogenous peptides and other biological response modifiers, chemical moieties capable of compounds with which they interact. See, e.g., Akil et al., 25 binding to other molecules of interest, and radioisotopes Annu. Rev. Neurosci. 7:223-255 (1984). Selected from therapeutically effective alpha and beta Three distinct families of peptides have been identified emitters, and diagnostically effective gamma emitters. that interact with opioid receptors: the enkephalins, the In another embodiment, the invention provides a thera endorphins, and the dynophins. Each family is derived from peutic method using a compound described above. In this a distinct precursor polypeptide, designated proenkephalin, embodiment, a therapeutically effective amount of a com pro-opiomelanocortin (POMC), and prodynorphin, respec pound of the invention is administered to a Subject in need tively. These peptides, and the precursors thereof, are gen thereof, Such that a compound of the invention binds to an erally found in Viva associated with cells that mediate pain, opioid receptor. For instance, the invention provides a however they may also be found associated with cells that 35 method of treating cancer comprising administering to a regulate the autonomic nervous System and neuroendrocri Subject in need thereof a therapeutically effective amount of nological functions, among others. a compound of the invention. The cancer may be Small cell There are also numerous non-peptidic compounds that lung cancer or other neuroendrocrine cancer. may serve as ligands for opioid receptors. Some compounds In another embodiment, the invention provides a diag interact non-specifically with all opioid receptor types, while 40 nostic method using a compound described above. In this other compounds demonstrate a higher affinity for one embodiment, a diagnostically effective amount of a com receptor type than other types. Moreover, a compound may pound of the invention is administered to a Subject in need interact with a receptor as an agonist, a partial agonist, or an thereof, Such that the compound binds to an opioid receptor. antagonist, at each receptor type. See, e.g., Martin, Phar The compound may be administered within or outside of the macol. Rev. 35:283-323 (1983). A discussion of many 45 central nervous System. In a related embodiment, the inven compounds, both peptidic and non-peptidic, which may tion provides for an in vitro diagnostic method using a Serve as ligands for opioid receptorS is found in Jaffe et al., compound described above. In this related embodiment, a Opioid Analgesics and Antagonists, Chapter 21 of Goodman diagnostically effective amount of a compound of the inven and Gilman's The Pharmacological Basis of Therapeutics, tion is contacted in vitro with an opioid receptor. 8" Ed. Ed. A. G. Gilman, T. N. Rall, A. S. Nies, P. Taylor. 50 These and related embodiments of the present invention Pergamon Press (1990). will be apparent upon reference to the following drawings Opioid receptor ligands have been implicated in the and detailed description. mediation of many important physiological responses, including pain/analgesia, appetite, gastric Secretion, epilep BRIEF DESCRIPTION OF THE DRAWINGS tic Seizures and other neurological disorders, renal function, 55 FIGS. 1A and 1B illustrate fluorochromes that may be cardiovascular responses, and traumatic paralysis, among conjugated to an opioid receptor ligand to prepare a diag others. Due to the physiological significance of opioid nostically active compound of the invention. receptor ligands, extensive research has been conducted into FIGS. 2A through 2H illustrate compound of the inven developing new ligands which may be used in various tion containing various L. groups, various X groupS and therapies. See, e.g., U.S. Pat. Nos. 5,658,908; 5,631,263; 60 5,578,725; 5,552,404, 5,464,841; 5,455,230; 5,411,965; various extender arms E disposed therebetween. 5,352,680; 5,332,818; 5,298,622; 4.816,586; 4,649,200. See FIGS. 3A and 3B illustrate synthetic methodology for also Campa et al., Cancer ReS. 56: 1965-1701 (1996); Chang preparing a compound of the present invention. et al., PCT Published Application No. WO93/15062; Chang FIGS. 4A and 4B illustrate synthetic methodology for et al., J. Pharm. Exp. Therap. 267,852-857 (1993); Dasher 65 preparing a compound of the present invention. et al., J. Med. Chem. 35:2374-2384 (1992); Koolpe et al., J. FIG. 5 illustrates Synthetic methodology for preparing a Med. Chem. 28:949–957 (1985); Olmsted et al., J. Med. compound of the present invention. US 6,359,111 B1 3 4 FIGS. 6A and 6B illustrate synthetic methodology for Sors thereof. Alternatively, the L portion of the compound preparing a compound of the present invention. may be non-peptidic. Suitable non-peptidic L portions FIGS. 7A and 7B illustrate synthetic methodology for include, without limitation, buprenorphine, butorphanol, preparing a compound of the present invention. fentanyl, meperidine, methadone, morphine, nalbuphine, FIGS. 8A and 8B each provide chemical structures for nal orphine, naloxone, naltrindole, pentazocine, four preferred compounds of the invention, having propoxyphene, Sufentanil, and derivatives thereof. The non naltrindole-type structures for the ligand binding portions of peptidic ligand may be a morphine analog, where Suitable the compounds, Separated by an extender arm to various morphine analogs include, without limitation, apomorphine, diagnostically or therapeutically effective groups. buprenorphine, butorphanol, codeine, dextrorphan, heroin, FIGS. 9A and 9B illustrate synthetic methodology for hydrocodone, hydromorphone, levallorphan, levorphanol, preparing a naltrindole derivative of the present invention. nalbuphine, nalmefene, thebaine, nalorphine, naloxone, FIG. 10 illustrates synthetic methodology for preparing a naltrexone, oxycodone, oxymorphone, and derivatives naltrindole derivative having a functional group to which an thereof. extender arm and/or a diagnostically or therapeutically 15 effective group may be added to the indole nitrogen of In other compounds of the invention, L has the formula naltrindole. FIG. 11 illustrates Synthetic methodology for preparing a naltrindole derivative having a functional group on the aromatic ring of the indole portion of the molecule, where the functional group is elaborated to an extender arm and then DOTA as a chelating agent. FIG. 12 illustrates synthetic methodology for adding a Succinimide group to a naltrindole derivative. FIG. 13 illustrates synthetic methodology for adding an 25 additional portion to an extender arm in a compound of the present invention. FIG. 14 illustrates synthetic methodology to add various extender arms to a naltrindole derivative, and then adding a where a preferred Stereochemistry is shown in the formula DOTA chelating group to the end of the extender arm to below: provide representative compounds of the present invention. DETAILED DESCRIPTION OF THE INVENTION 35 The present inventors have discovered compounds that contain both a ligand portion (L) having binding affinity for an opioid receptor, as well as a group (X) which imparts therapeutic or diagnostic efficacy to the compound. The group X may be, for example, any of a radionuclide chelat 40 w w ing agent, fluorochrome (fluorophore), toxin, drug, a poly O boron moiety, protein, a biological response modifier, a chemical moiety capable of binding to other molecules of interest, and a radioisotope Selected from therapeutically where G is selected from effective alpha and beta emitters, and diagnostically effec 45 tive gamma emitters. In the compounds of the invention, the ligand portion (L) may be primarily Specific for a 6 opioid receptor, a K opioid ? An N No receptor, or a u opioid receptor. Preferred compounds of the invention are primarily Specific for 6 opioid receptors. In the 50 fnA 'N ! fn{ E compounds of the invention, L may function primarily as an P w antagonist for a particular receptor, or it may function E E primarily as an agonist for a particular receptor. In other words, the compound may have affinity for only one receptor, or more than one receptor, and when it has affinity 55 for more than one receptor, it may have high affinity for all ? receptors, or may have high affinity for only one or two receptors. Also, the compound may function entirely as an agonist, or entirely as an antagonist, or may demonstrate E mixed agonist/antagonist efficacy for a particular receptor, 60 where that efficacy is independent of the compound's effi ? cacy at any other receptor. ?n- E AnO An S The L portion of the compound may be peptidic, i.e., formed of a plurality of peptide bonds, where the peptide N N ^N. bonds are preferably the condensation product (amides) of 65 As 2 A. A N E amino acids. Suitable peptidic ligand are enkephalins, E endorphins, dynorphins and peptidic physiological precur US 6,359,111 B1 S -continued ? A. N R5

? ? A. nf -N F ).N NOO 57 R4 E E

R5 1O w sa F R4

N 1. 'N' ?' R4 S R5 E E and 15 ? where a further preferred compound has the formula shown w S R4 below, where R' is defined as above:

w A. Rs E

25

is a monocyclic, five- or six-membered, carbocyclic or heterocyclic, aromatic ring; 35 R" is selected from (C1-C5)alkyl, (C-C cycloalkyl) In another preferred compound of the invention, Lhas the C - Cs alkyl ene, (C-C 7 cycloalkenyl)- formula C-C alkylene, aryl, aralkyl, trans-(C-C)alkenyl, allyl and furan-2-ylC-Calkylene; 40 R is selected from H, OH and (C-C)alkyl-C(=O)0-; R is selected from H., (C-C-)alkyl and (C-C)alkyl-C (=O)-; and R" and Rare independently selected from a direct bond 45 to E, H, F, Cl, Br, NO, CN, COOR, NH, (C-C) alkyl, (C-C)alkoxy or together are a benzo ring fused RO tO 50 A. Another preferred compound of the invention has the formula O) s

55 with the proviso that one, but only one, of R" and R may be a direct bond to E; and 60 E is an extender arm, where E provides a stable chain of 2-60 atoms Selected from carbon, oxygen, nitrogen and Sulfur arm which covalently links together and dis tances the groupS L and X. 65 In a preferred compound of the invention, L has the formula wherein, independently at each occurrence, US 6,359,111 B1 7 8 G' is selected from O, S, NH and two hydrogens (when Ro is Selected from Calkyl and Coalkoxy, G' is two hydrogens, then each of the two carbon atoms to which G' is shown attached are themselves attached n is selected from 0 and 1; and to one of the two hydrogen atoms); G is selected from -CH=CH- and -CH-CH2-, 5 G is selected from -C-C- and S, where the ring including G' is aromatic; R is selected from H, C-C alkyl and hydroxyl protect ing groups including acetate; R" is selected from H, C-C alkyl and (C-C cycloalkyl) represents an aromatic or aliphatic ring. C-C alkyl, In compound of the invention there is a least one “X” R is selected from H, OH and a direct bond to E', such group, where the “X” group imparts diagnostic or therapeu that G' is -CH=CH- when R is OH: tic efficacy to the compound. Suitable “X” groups include, E" and E° each represent an extender arm (E); and without limitation, a radionuclide chelating group, fluoro either both of X" and X’ are radionuclide chelating groups 15 chrome (fluorophore), toxin, drug, a polyboron moiety, or one of X" and X is a radionuclide chelating group protein, a biological response modifier, a chemical moiety and the other of X" and X is hydrogen. capable of binding to other molecules of interest, and a Another preferred compound of the invention has the radioisotope Selected from therapeutically effective alpha formula and beta emitters, and diagnostically effective gamma emit 2O ters. Each of these types of “X” groups is described below. HC V A. Radionuclide Chelating Groups N In compounds of the invention, the radionuclide chelating group may be formed of two X groups which together 25 provide Sufficient chelating sites to bind a radionuclide. The two X groups are joined to the ligand portion of the compound at two sites, where extender arms may be present

R6O w" interposed between the ligand and the X groups, as shown w in the formula O El-X1. 3O El-X1 Another preferred compound of the invention has the formula Ligand--

H3C 35 V

N In compounds having X" and X groups, each of X" and X may have a structure selected from 40 A10 R6O El-X1. A-R11-A-R12 and N(CH2 A10-R13), 45 Another preferred compound of the invention has the formula wherein, A is an electron-donating moiety independently selected R8 at each occurrence from O, S, C(=O)NH and N(R'), R10 50 where R' is selected from hydrogen and pro-drug Substituents Selected from the group consisting of Prs C-C alkyl, -C(=O)(H, C-C alkyl or Ar), NH HN -C(=O)-(C-C alkylene)-N(independently H or C-C alkyl), -C(=O)C)-(H, C-C alkyl or Ar), SRo 55 -C(=O)CH(NH)(H, C-C alkyl or Ar), -(C- R, RoS R7 Calkylene)-C(=O)C)-(H, C-C alkyl or Ar), -(C-C alkylene)-OC(=O)-(H, C-C alkyl or wherein, independently at each occurrence, Ar), -(C-C alkylene)-N(independently H or R, is selected from -OH,-O-C(=O)-Calkyl, and 60 C-C alkyl), -(C-C alkylene)-NHC(=O)-Ar, -O-C alkyl, -(Calkylene)-CN, -(Calkylene)-NO, and Rs is selected from hydrogen and Calkyl, Ro is selected from hydrogen, -C(=O)-Calkyl, -C(=O)-phenyl; hemithioacetals, hemithioketals, -(C-Calkylene) and Sulfur protecting groups, Such that two Ro groups 65 may be joined together as a direct bond, thereby linking two Sulfur atoms as a disulfide linkage; US 6,359,111 B1 10 R" and R' each have a structure selected from

A10 -(C -case- \, R17 --it- and A. R'' and R' each have a structure selected from R18 R17 A10 R'' and R' are selected from H, C-C,hydrocarbyl, and CN, -NO, -NO, -C(OC-Calkyl)=NH, -itR18 N=C=O, -N=C=S, and –C(=O)CR' where R' is C-C hydrocarbyl; 15 R, R and R' are selected from H and protecting R'' and R' are selected from H., C.-Chydrocarbyl, groups for the A or A' moiety to which the R', R' CN, -NO, -NO, -C(OC-Calkyl)=NH, or R' is bonded; N=C=O, -N=C=S, and –C(=O)CR' where A” and R' may together form -N(CH-COOH); and R' is C-Chydrocarbyl, A' is selected from O and S. R, R and R' are selected from H and protecting In a preferred embodiment, the compound of the inven groups for the A or A' moiety to which the R', R' tion having X" and X groups has the formula or R' is bonded; A” and R' may together form -N(CH-COOH); and R20 R21 R21 A' is selected from O and S. 25 Alternatively, compounds of the invention may contain a (CH2) N-CH2- (CH, V-s-R. ligand portion (Ligand) conjugated at a single site to one X group, where the conjugation may be through an extender Ligand-- arm E, as represented by the formula Ligand-E-X. In preferred compounds of the formula Ligand-E-X, X cus - CH- (Ch.: As-Rs may be any of formulas (II), (III) or (IV), R20 R21 R21 (II) wherein R' and R'' are independently selected from hydrogen and methyl; 35 R’ is independently selected from hydrogen, acm, EOE, THP, C-Calkyl, acetate, benzoyl and Sulfur protect (III) ing groups, and A9-R14 each of m, n, p and q are Selected from 0 and 1 Such that 40 R?. YA when p+q=0 then m+n=0 or 1; and when p+q=1 then m+n=1; and when p+q=2 then m+n=1 or 2. k / k Alternatively, in compounds of the invention having X" R14-A9 and X groups, those groups together may form the cyclic (IV) Structure 45 R23 HOOC-CH V ?h-cool A. A. A. A. -CHCH NCHCH7 N >, N11 N11 N11 HOOC-CH CH-COOH

s 50 such that one methylene hydrogen of formula (IV) is wherein, replaced with a direct bond to E; A is an electron-donating moiety independently selected where, independently at each occurrence, at each occurrence from O, S, C(=O)NH and N(R'), 55 A is an electron-donating moiety selected from O, S, where R' is selected from hydrogen and pro-drug C(=O)NH and N(R'), where R' is selected from Substituents Selected from the group consisting of hydrogen and pro-drug Substituents Selected from C-C alkyl, -C(=O)(H, C-C alkyl or Ar), C-C alkyl, -C(=O)(H, C-C alkyl or Ar), -C(=O)-(C-C alkylene)-N(independently H or -C(=O)-(C-C alkylene)-N(independently H or C-C alkyl), -C(=O)C)-(H, C-C alkyl or Ar), 60 C-C alkyl), -C(=O)C)-(H, C-C alkyl or Ar), -C(=O)CH(NH2)(H, C-C alkyl or Ar), -C(=O)CH(NH2)(H, C-C alkyl or Ar), -(C-C alkylene)-(=O)C)-(H, C-C alkyl or Ar), -(C-C alkylene)-C(=O)C)-(H, C-C alkyl or -(C-C alkylene)-OC(=O)-(H, C-C alkyl or Ar), -(C-C alkylene)-OC(=O)-(H, C-C alkyl Ar), -(C-C alkylene)-N(independently H or or Ar), -(C-C alkylene)-N(independently H or C-C-alkyl), -(C-C alkylene)-NHC(=O)-Ar, 65 C-C alkyl), -(C-C alkylene)-NHC(=O)-Ar, -(C-C alkylene)-CN, -(C-C alkylene)-NO, -(C-C alkylene)-CN, -(C-C alkylene)-NO, and and US 6,359,111 B1 11 12 Other Suitable radionuclide chelating groupS may be represented by the following (“NS” or “NS”) formulae: (C1-Calkylene) / \, T R R R'' is selected from R S r f A10 X N N R - (-C) - and l R X A. wherein: 15 where R7 and R'' are selected from H, T is H or a Sulfur protecting group; C-C hydrocarbyl, -CN, -NO, -NO, each X independently represents H2 or O, C(OC-C alkyl)=NH, -N=C=O, -N=C=S, each R independently represents a Substituent Selected and -C(=O)CR' where R' is C-Chydrocarbyl; from the group consisting of hydrogen; alkyl, geminal R' is selected from H and protecting groups for A, dialkyl, a non-alkyl side chain of an amino acid other where A and R' may together form - N(CH than cysteine (alkyl side chains being covered when R COOH); is an alkyl group); and -(CH2)-Z, R’ is selected from H and CHCOOH: Z represents -COOH or -NH2, q is Selected from 0, 1, 2 and 3; and E represents one or more chemical bonds through which r is an integer of from 1 to about 4, and L is conjugated to X. 25 R" represents the position at which the radionuclide binding In preferred compounds of the formula Ligand-E-X, X group is joined, optionally through an extender arm, to the has a formula Selected from opioid ligand portion of the compound of the invention; and Y represents N or S. The Sulfur protecting group may be Selected from alkyl, COOH COOH aryl, acyl (preferably alkanoyl or benzoyl), thioacyl groups having 1 to about 7 carbons, and organothio groups having ls-N- 1 to about 10 carbons. For the R groups, the alkyl groups generally contain from 1 to 7 carbons, preferably from 1 to 4 carbons, and most 35 preferably represent methyl. In another preferred embodiment, the radionuclide chelat rN --> ing group has the following Structure: COOH COOH, W 40 X1 V X3 "\ y N C. (CH2) B N S-R2 Y \ NS Y N X2 A A' X4

Y. i H Y S S Yi W N N1 H S \-coors. W

50 wherein: where W represents H or =O, R' is selected from H, COCH, EOE, THP, COPh, acm, C-Chydrocarbyl, and X and X are H or oxo but both are not oxo; sulfur protecting groups; and R is selected from H and X- and X are H or OXO, but both are not OXo, C-C hydrocarbyl, and One of X and X is bonded, either directly or indirectly (through an extender arm E), to the ligand portion (L) of a 55 W compound of the invention; "\ y N A is H, alkyl group of C or less, -CH-CH2-S-R N S-R2 or -CO-CH2-S-R, while in a preferred embodiment, when X or X is =0, then A is H; 60 A is H, alkyl group of C or less, -CHCH-S-R or -CO-CH2-S-R, while in a preferred / S-R2 embodiment, when X or X is =0, then A' is H; H Y is W (a) -CH-S-R, or H, when A is H or an alkyl 65 group of C or leSS and A' is H or an alkyl group of wherein R is selected from H., COCH, EOE, THP, COPh, C or less, or acm, C-Chydrocarbyl, and Sulfur protecting groups. (b) H; US 6,359,111 B1 13 14 Y is portion to provide an amide linkage between the two moi (a) -CH-S-R, or H, when A is H or an alkyl eties. Likewise, if the ligand portion of the molecule con group of C or leSS and A is H or an alkyl group of tains an amino group, then carboxylic acid-containing fluo C or less, while in a preferred embodiment, Y and rochromes may be condensed with the amino group to form Y are not both H, or a conjugate containing an amide group. Other methods known in the art of conjugating fluorochromes to chemical (b) H; moieties may be employed. R, R2, R., R., Rs, and R are independently Selected C. Toxin from Sulfur protecting groups, Several of the potent toxins useful within the present Q is H or a polar group to increase the hydrophilicity of invention consist of an A and a B chain. The A chain is the the compound; cytotoxic portion and the B chain is the receptor-binding n is 0 to 2; and portion of the intact toxin molecule (holotoxin). Preferred toxins in this regard include holotoxins, Such as abrin, ricin, Z is -(W) R', where W is -CH-, -CH-O-, modeccin, Pseudomonas eXotoxin A, Diphtheria toxin, per -CHCO-, or combination thereof, m is 0 to 5, and tussis toxin and Shiga toxin; and A chain or “A chain-like' R" is a chemically reactive group. In a preferred 15 molecules. embodiment, when Z is attached to the carbon desig D. Drug nated C. there is either no X or no Q at C. In another DrugS Suitable for use herein include conventional preferred embodiment, when Z is attached to the car chemo-therapeutics, Such as Vincristine, vinblastine, bon designated B there is either no X or no Q at B. In doxorubicin, bleomycin, methotrexate, 5-fluorouracil, another preferred embodiment, when X is =0 there is 6-thioguanine, cytarabine, cyclophosphamide and cis no Z at C. In another preferred embodiment, when X platinum, as well as other conventional chemotherapeutics is =0 there is no Z at B. as described in Cancer. Principles and Practice of R, R2, R,R,Rs and R are independently Selected from Oncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A. Sulfur protecting groups. Groups which may be used Rosenberg, J.B. Lippincott Co., Philadelphia, Pa., 1985, include any of the alkyl, acyl and aryl groups, disulfides 25 Chapter 14. A preferred drug within the present invention is and bunte salts known by those skilled in the art. a trichothecene. Other preferred drugs Suitable for use herein Preferred groups are those that result in the formation as a diagnostic or therapeutic active agent in the practice of of a thioacetal, hemithioacetal, thioester or acetami the present invention include experimental drugs as domethyl Substituent. Particularly preferred groups described in NCI Investigational Drugs, Pharmaceutical include p-anisylidine, acetonyl, tetrahydrylfuranyl, Data 1987, NIH Publication No. 88-2141, Revised Novem ethoxyethyl, tetrahydrylpyranyl, acetamidomethyl and ber 1987. derivatives thereof. A drug may be incorporated into a compound of the Preferred chelating groups of the invention include, with invention by any Suitable conjugation reaction, whereby a out limitation, DOTA, NS, NS, NS, MAMA, DTPA and reactive Site in the drug is reacted with a reactive Site in the porphyrins. Preferred compound of the invention, having 35 ligand portion of the compound. Optionally, an extender arm opioid receptor ligand (L) portions and radionuclide binding may be disposed intermediate the drug and the ligand arms are shown in FIGS. 2A through 2H, where R=H, CH, portion. or COCH; W=H or (=O); n=0–6; X=an extender arm E. Polyboron Moiety having 1-10 carbon atoms with 0-3 heteroatoms selected The compounds of the invention may incorporate one or from N, S, and O; and R=H, COCH, EOE, THP, COPh, 40 more boron atoms. Boron atoms are known to be activated acm, Me, etc. and Sulfur protecting groups known in the art. by external neutrons to generate a therapeutically-effective B. Fluorochromes Species. Therefore, a compound of the invention may incor Compounds of the present invention may contain a fluo porate boron atoms, and then be administered to a Subject in rochrome in addition to a ligand portion (L) having binding order that the compound associates with a particular opioid affinity for an opioid receptor. The fluorochrome, which may 45 ligand. Upon Such association, boron atoms are brought into also be referred to as a fluorophore, provides the compound proximity with the opioid receptor. The Subject is then of the invention with diagnostic efficacy. Thus, when a exposed to neutrons, which causes the boron atoms to be compound of the invention incorporates a fluorochrome, the converted into a therapeutically effective Species, and more compound may be contacted with tissue containing opioid specifically the 'B atoms of the polyboron moiety are receptor(s) to localize the fluorochrome at the receptor Site. 50 converted to an alpha particle and a Linucleus atom upon An advantage of this method of fluorescently marking thermal neutron bombardment. The alpha particles are par opioid receptor positive tissue over the use of fluorescently ticularly effective at killing nearby cells. labeled anti-receptor antibody is that NID Rh (for example) For discussion of this technology, See, e.g., Ranadive et al. recognizes only active functional receptor whereas antibod Nucl. Med. Biol. 20:663–668, 1993; Zamenhofetal. J. Natl ies could bind to non-functional or blocked receptor. The 55 Can. Inst. 84:1290–91, 1992; Barth et al. Cancer localized compound may then be detected by Standard 70:2995-3007, 1992; Kalend et al. Med. Phys. 18:662, techniques used in the art to detect fluorochromes. 1991; Barth et al. Cancer Res. 50:1061-70, 1990; Alam et al. Suitable fluorochromes which may be incorporated into a J. Med. Chem. 32:2326–30, 1989; Perks et al. Brit. J. Radiol. compound of the invention are shown in FIGS. 1A and 1B. 61:1115-26, 1988; Katanaka et al. Boron Neutron Capture These fluorochromes, and many others, may be obtained 60 Therapy For Tumors, pages 349-378, 1986 (Nishimura from commercial Sources. For instance, Molecular Probes Co.); Barth et al. Hybridoma 5(1):543–554, 1986; Golden (Corvallis Oreg.) sells many fluorochromes. A fluorochrome berg et al. P.N.A.S. USA81:560-563, 1984; Mizusawa et al. may be incorporated into a compound of the invention by a P.N.A.S. USA79:3011-3014, 1982; Ichihashi et al. J. Invest. Standard conjugation reaction. For instance, when the ligand Dermatol. 78:215-218, 1982; portion of the compound contains a carboxylic acid group, 65 Preferably, the polyboron moiety contains Several tens, any of the fluorochromes of FIGS. 1A and 1B which contain and more preferably hundreds of boron atoms. As the a reactive amino group, may be condensed with the ligand number of boronatoms in the polyboron moiety is increased, US 6,359,111 B1 15 16 the opportunity for thermal neutrons to impact a "B atom, Preferably, the beta-emitter is not tritium because tritium is and the opportunity for an impacted 'Batom to emit a lethal not a high energy beta emitter, and thus tritium is not alpha particle, increases. Thus, the polyboron moiety is effective in therapeutic methods according to the present preferably a boron-containing polymer. Suitable boron invention. The therapeutic radioisotope may be an alpha containing materials which may function as therapeutically emitter, such as Pb, Bi and At. Each of these radioisotopes effective chemical agents Suitable for binding to an opioid may be introduced into a compound of the present invention receptor ligand according to the present invention are dis using the “PIP” chemistry described herein. closed in the aforecited references. I. Pretargeting F. Protein AS used herein, includes proteins include polypeptides In a preferred embodiment of the invention, a compound and peptides, and may be an intact molecule, a fragment of the present invention is used in pretargeting methods. thereof, or multimers or aggregates of intact molecules Essentially, Such pretargeting methods are characterized by and/or fragments, and may occur in nature or be produced, an improved targeting ratio or increased absolute dose to the e.g., by Synthesis (including chemical and/or enzymatic) or target cell Sites in comparison to conventional cancer diag genetic engineering. Suitable examples of protein include, nosis or therapy. A general description of pretargeting meth without limitation, horseradish peroxidase, green fluorescent 15 ods may be found in U.S. Pat. Nos. 4,863,713, 5,578,287, protein and Streptavidin. and 5,630,996. Moreover, typical pretargeting approaches G. A Chemical Moiety Capable of Binding to Other Mol are Summarized below. ecules of Interest Pretargeting methods are of two general types: three-step The “X” group of a compound of the present invention pretargeting methods and two-step pretargeting methods. may be a chemical moiety that is capable of binding to other The three-step pretargeting protocol features administra molecules of interest. One Suitable example of a compound tion of an targeting moiety-ligand conjugate, which is of the invention having a chemical moiety capable of allowed to localize at a target Site and to dilute in the binding to a molecule of interest is shown below, where the circulation. This is followed by administration of an anti chemical moiety is capable of reacting with any primary or ligand which binds to the targeting moiety-ligand conjugate Secondary amine-containing compound: 25 and clears unbound targeting moiety-ligand conjugate from the blood, as well as binds to targeting moiety-ligand O O conjugate at the target Site. The complementary/anti complementary Set of molecules discussed above may func tion as the ligand/anti-ligand pair in the pretargeting method. --( Thus, the anti-ligand fulfills a dual function by clearing targeting moiety-ligand conjugate not bound to the target Site as well as attaches to the target Site to form a targeting moiety-ligandanti-ligand complex. Finally, a diagnostic or therapeutic active agent-ligand conjugate that exhibits rapid The “X” group which is a chemical moiety capable of 35 whole body clearance is administered. binding to other molecules of interest may be one member When the active agent-ligand conjugate in circulation of a complementary/anti-complementary Set of molecules comes into close proximity to the targeting moiety that demonstrate Specific binding, generally of relatively ligand:anti-ligand complex bound to the target Site, the high affinity. Exemplary complementary / anti anti-ligand portion of the complex binds to the ligand complementary pairs include Zinc finger protein/dsDNA 40 portion of the circulating active agent-ligand conjugate, thus fragment, enzyme/inhibitor, hapten/antibody, lectin/ producing a targeting moiety-ligand: anti-ligand ligand carbohydrate, ligand/receptor, and biotin/avidin or Strepta active agent “Sandwich' at the target Site. Furthermore, Vidin. AS used herein, the antibody present in a binding pair because the unbound diagnostic or therapeutic active agent may be an antibody fragment, preferably an antigen-binding is attached to a rapidly clearing ligand (rather than a slowly antibody fragment. 45 clearing targeting moiety, Such as antibody, antibody Biotin/avid in or streptavidin is a preferred fragment), this technique provides decreased non-target complementary/anti-complementayry pair. Thus, a preferred exposure to the active agent. compound of the present invention may include either biotin Alternatively, the two-step pretargeting methods elimi (which binds to either avidin or streptavidin) or avidin nate the Step of administering the above identified anti (which binds to biotin) or streptavidin (which also binds to 50 ligand. These “two-step” procedures feature targeting biotin). moiety-ligand or targeting moiety -anti-ligand H. A Therapeutically or Diagnostically Effective Radioiso administration, followed by the administration of active tope agent conjugated to the opposite member of the ligand/anti The compound of the invention may incorporate a thera ligand pair. peutically or diagnostically effective radioisotope. In one 55 AS an optional Step in the two-step pretargeting methods embodiment, the compound of the invention contains radio of the present invention, ligand or anti-ligand, designed nuclide binding arms, which can chelate with a therapeuti Specifically to provide a clearance function, is administered cally or diagnostically effective radioisotope. This embodi to facilitate the clearance of circulating targeting moiety ment of the invention is described else-where. However, in ligand or targeting moiety-anti-ligand. Thus, in the two-step another embodiment, the radioisotope is covalently incor 60 pretargeting approach, the clearing agent does not become porated into a compound of the invention. Diagnostically bound to the target cell population, either directly or through effective radioisotopes include gamma-emitterS Such as the previously administered target cell bound targeting indium ('In) and technectium ("Tc), and positron emit moiety-anti-ligand or targeting moiety-ligand conjugate. terS Such as carbon 11. Preferably, the radioisotope is a A targeting moiety in the pretargeting methods of the gamma-emitter rather than a positron emitter. 65 present invention has the functional property that it binds to For therapeutic purposes, the radioisotope may be a a defined target cell population, Such as tumor cells that beta-emitter, such as 'Y, Re, and 188Re, among others. contain opioid receptors. Thus, a ligand portion (L) of US 6,359,111 B1 17 18 compounds of the present invention provide the needed ring. The nature of the aromatic ring is not critical and may targeting moiety in the pretargeting approach. be mono-, bi-, tri-, or higher number of rings, but the Ligand/Anti-ligand pairs Suitable for use in the present monocyclic ring is preferred based on increased water invention include biotin/avidin or Streptavidin, haptens and Solubility. The aromatic rings may consist of all carbon epitopes/antibody, fragments or analogs thereof, including 5 atoms or may contain heteroatoms Such as nitrogen, oxygen, mimetics, lectins/carbohydrates, Zinc finger proteins/dsDNA or Sulfur. Inclusion of heteroaromatic ringS Such as fragments, enzyme inhibitors/enzymes, and analogs and pyridines, furans, or thiophenes can assist in increasing derivatives thereof. Preferred ligands and anti-ligands bind water Solubilities of the radioiodinated Small molecule con to each other with an affinity of at least about K210'M' jugates. The presence of the a heteroaromatic ring also aids or Ks 10M. Biotin/avidin or streptavidin is a preferred in elimination of water Soluble metabolites via Sulfoxide and ligand/anti-ligand pair. Sulfone formation in the case of thiophene ring System, In general Such pretargeting methods will preferably thereby decreasing toxicity to the host. Further substitution include the administration of a anti-ligand that provides a on the aromatic ring, exclusive of X and R, with polar clearance function. The clearance is probably attributable to Substituents Such as a nitro, Sulfonic acid, carboxylic acid, or croSS-linking and/or aggregation of conjugates that are cir 15 dialkyl amino group can also be used to enhance water culating in the blood, which leads to complex/aggregate solubility. Increased water solubility is desirable to give clearance by the recipient's RES (reticuloendothelial higher yields and less potential aggregation in the conjuga System). In one embodiment of the present invention, the tion reaction with targeting agent and to cause leSS pertur anti-ligand clearance of this type is preferably accomplished bation of the lipophilicity of the compound of the invention. with a multivalent molecule. However, a univalent molecule Other Substituents can be added to impart Some control of Sufficient size to be cleared by the RES on its own could against enzymatic degradation. also be employed. J. PIP Labeling The symbol R indicate any substituent that meets the “PIP” literally stands for p-iodophenyl, and more specifi following three requirements: First, the R substituent must cally refers to a labeling technique that employs a reagent not highly activate ring Artoward electrophilic Substitution. 25 In other words, R cannot be linked to an Arby a linkage that having the partial formula shown below, which may more increases the electron density of Ar on the order of the precisely be called a p-iodobenzyl group: increase produced by a hydroxy or amino Substitution. Second, R should be a short-chain Substituent So that uncon O jugated or cleaved radiohalogenated molecules can be rap idly removed by the kidneys. Thus, R may contain an alkyl or other spacer chain between the aryl linkage and the functional group for targeting agent conjugation, but Such a Spacer chain should preferably contain no more than 5, and most preferably no more than 3, Straight-chain carbon 35 atoms. Third, the R Substituent should bear a functional PIP labeling may be used to add a radioisotope (denoted group that is available for conjugation to the ligand portion “X”) to an aromatic or heteroaromatic ring (collectively (L) of a compound of the invention, under conjugation “Ar”) in compounds of the invention. The radioisotope may conditions, Such as acylation or amidation, that preserve the be an isotope of iodine, bromine, fluorine, or astatine. The biological activity of the opioid receptor ligand. Thus, R aromatic ring may contain one or more Substituents (denoted 40 should provide a functional group, Such as imide ester or “R”), which preferably do not activate Ar toward electro imidate ester, for covalent attachment of corresponding philic substitution on the order produced by hydroxy or functional groups (or conjugated attachment sites) of target amino Substitution of the ring, wherein Said bond or said ing agents, or carrier molecule Such as amino acid polymers Substituent has attached thereto a functional group Suitable that can in turn be conjugated to targeting agent molecule. for conjugation. 45 Suitable functional groups for the above-Stated purpose As utilized herein, the symbol *X indicates any radioiso include phenolic esters (e.g., para-nitrophenol), imide esters tope of: iodine, particularly I-123, I-125, and I-131; (e.g., Succinimide ester), imidate esters, anhydrides, bromine, particularly Br-75, Br-76 and Br-77; fluorine, acylsuccinimides, aldehydes, isothiocyanates, diazo, particularly F-18; and, astatine, particularly At-211. Pre amines, hydrazines, alkyl halides, maleimides, and other ferred radiohalogens X for diagnostic imaging purposes 50 groups that can be used to attach the molecule to a targeting include I-131 and most preferably I-123 for imaging with agent through a covalent bond. gamma cameras, and for positron tomographic imaging. Representative R Substituents include alkyl acids, amido F-18, Br-75, and Br-76. For clinical radiotherapy, preferred alkyl acid, nitrile, alkyl nitrile, amido alkyl nitrile, imide radiohalogens X include I-131, Br-77, and At-211. Pre ester, alkyl imide esters, amido alkyl imide ester, imidate ferred radiohalogens X for in vitro radioimmunoassay 55 ester, alkyl imidate esters, and amido alkyl imidate ester. purposes include I-125, and I-131. Pursuant to this invention Illustrative but nonlimiting example of radiohalogenated the radiohalogen X is preferably para- or meta-positioned small molecules of this invention include: N-succinimidyl on ring Ar relative to Substituent R in order to render the 3-(4-) iodophenyl)propionate; methyl-3-(4-'') radiohalogen leSS Susceptible to catabolism by dehalogenase iodophenyl)-propioimidate; N-succinimidyl-4- enzymes. 60 iodobenzoate; methyl-4-131) iodobe in Zimidate; The Symbol Ar indicates any aromatic or heteroaromatic N-succinimidyl-4- iodobenzamido-acetate or ring. Preferred rings Ar include benzene, pyridine, furan, N-succinimidyl-4-'liodohippurate; methyl-4-I''') and thiophene, the latter three because of the enhanced water io do be n zemio do ace timidate; and 4- ) solubility they convey. The attachment of the radiohalogen iodobenzamidoacetonitale. The Synthesis of radiohaloge to a carbon atom in an aromatic ring is preferred over 65 nated Small molecules Such as these is described in attachment to an alkyl carbon atom due to the increased European Patent Application 0 203 764 published Dec. 3, bond Strength of the carbon-halogen bond in the aromatic 1986. The radiolabeling procedures described therein gen US 6,359,111 B1 19 20 erally involve Substituting an organometallic group which is -continued a tri-alkyl stannane of the formula Sn(R), wherein R is a H lower alkyl group, preferably Sn(n-Bu) or SnMes, on a H 1CN haloaromatic compound. A radioisotope of a halogen then is Substituted for the organometallic group by halodemetaliza O St. Hi fl. tion. In addition, the following U.S. Patents, which generally Y-CH: Hics1O disclose methods for radiohalogenation of Small molecules /V Y for protein labeling, are incorporated herein by reference: HC S S U.S. Pat. Nos. 4885,153; 5,045.303; 5,213,787; and 5,609, 848. 2-Methyl tetrahydrofuranyl Tetrahydropyranyl (THP) H Sulfur Protecting Groups HC C The compounds of the invention may contain one or more protecting groups for one or more atoms. Suitable protecting 3 St. hi? y groups are Set forth in Greene, “Protective Groups in 15 HCS-O Hics-O Organic Chemistry”, John Wiley & Sons, New York N.Y. (1991). (Y, Yu, Sulfur atoms, particularly when present as part of a radionuclide binding group, may be protected in compounds Ethoxyethyl (EOE) 2-Methyl tetrahydropyranyl of the present invention. In one embodiment of the invention, the Sulfur protecting group, when taken together Advantages of using hemithioacetal Sulfur protecting with the Sulfur atom to be protected, is a hemithioacetal groups include the fact that a separate Step for removal of the group. Suitable hemithioacetals include, but are not limited Sulfur-protective groups is not necessary. The protecting to, those having the following formulae. groups are displaced from the compounds of the invention 25 during the radiolabeling Step, in what is believed to be -S-CH-O-CH metal-assisted acid cleavage; i.e., the protective groups are displaced in the presence of the radionuclide at an acidic pH, -S-CH-O-(CH)-OCH and the radionuclide is bound by the opioid receptor ligand of the invention. The radiolabeling procedure thus is and Simplified, which is especially advantageous when the com pounds of the invention are to be radiolabeled in a hospital -S-CH-O-CH-CH(CH). laboratory shortly before use. In addition, the basic pH conditions and harsh conditions Preferred hemithioacetals generally are of the following asSociated with certain known radiolabeling procedures, or formula, wherein the Sulfur atom is the Sulfur atom of the 35 procedures for removal of other Sulfur protective groups, are chelating portion of the compound of the invention. avoided. Because basic and/or harsh conditions are avoided, the compounds of the invention may incorporate (at Sites other than are desirably deprotected) protecting groups and OR20 other chemical functionality which should desirably survive R22-C-R21 the radiolabeling Step, but which would decompose under 40 basic and/or harsh conditions. Such base labile groups S include any group which may be destroyed, hydrolyzed, or otherwise adversely affected by exposure to basic pH. Cer colo tain protein conjugation groups, including activated esters, isothiocyanates, maleimides, and other Michael acceptors, wherein R is a lower alkyl group, preferably of from 2 to 45 among others, are relatively base labile. Thus, these group 5 carbon atoms, and R’ is H or a lower alkyl group, may be incorporated into compounds of the invention, and preferably of from 1 to 3 carbon atoms. Alternatively, R will retain their integrity throughout the radiolabeling pro and Rif may be taken together with the carbon atom and the cedure of the present invention. oxygen atom shown in the formula to define a nonaromatic Other preferred Sulfur atom protecting groups including ring, preferably comprising from 3 to 7 carbon atoms in 50 ACM and i-PrCO, as defined below: addition to the carbon and oxygen atoms shown in the formula. R' represents hydrogen or a lower alkyl group wherein the alkyl group preferably is of from 1 to 3 carbon atOmS. 55 i Examples of Such preferred hemithioacetals include, but CH2 =o are not limited to: H-N--CH, H3C CH3 Acetamidomethyl (ACM) -Pro H2 CH 60 OMSt. O 3 The acetamidomethyl group may be displaced from a Y-CH: YCH, compound of the invention during radiolabeling conducted /V at about 50 C. in a reaction mixture having a pH of about 3 to 6. The use of an acetamidomethyl group generally " 65 improves the water solubility of the compound of the Tetrahydrofuranyl Methoxymethyl (MOM) invention, which is desirable in therapeutic and diagnostic applications involving water-based living organisms. US 6,359,111 B1 21 22 Extender Arms are useful as cytotoxic agents for radiotherapy. Such emitters E is a group that functions as an “extender arm' and may include, without limitation, isotopes such as Scandium", be useful to distance the opioid binding site (L) from the scandium', scandium", copper' (Cu), copper'7 (Cu), therapeutically or diagnostically effective X group of the gallium', gallium, yttrium' ('Y), ruthenium’ (Ru), compound of the invention. Extender arms of the invention covalently link an X group to an L portion through a chain palladium', rhodium'", rhodium' ('Rh), palla of 2-60 atoms Selected from carbon, oxygen, nitrogen and dium (Pd), samarium, rhenium (Re), rhe Sulfur. The chain is “stable' in that it can exist at room nium' (188 Re), rhenium'', gold'('Au), gold' temperature without Spontaneously decomposing to con (Au), radium, lead (Pb), and lead (Pb). Of Stituent atoms or molecular fragments. However, in one these, Re, Re, and 'Y are preferred. embodiment of the invention, the chains are stable in terms of their thermodynamic stability, yet are unstable in that they The compounds of the invention may also be used to are metabolically degraded and cleaved by, for example, an chelate alpha radiation emitting materials such as bismuth'' enzyme. Thus, the extender arm may include Selectively ('Bi), positron emitters such as zirconium, fluorescent cleaveable linkerS Such as carboxylate esters, acetate esters, 15 members of the lanthanide Series of elements Such as imidate esters and carbamates, among others. Other groups terbium and europium and of the transition Series Such as which may be used include methylene (-CH-), methyl ruthenium, and paramagnetic materials. Such as gadolinium eneoxy (-CHO-), methylenecarbonyl (-CH-CO-), and iron. In addition the compounds of the invention are also or combinations thereof. The number, m, of groupS Such as these would be typically 0 to 30 and preferably 0 to 5. Suitable for binding numerous other metal ions which may Polyamides, as formed from, for example, condensation of be useful for a variety of purposes, including those in which amino acids, are a preferred extender arm. a catalytic property of the metalion is of utility. Iron, copper, Extender arms may incorporate side chains, e.g., amino Vanadium, rhodium, platinum, palladium and titanium are acid residue Side chains, which can impart desirable prop examples of metal ions useful in catalyzing a variety of erties to the molecule Such as enhanced receptor binding 25 organic reactions, Such as the cleavage of nucleic acids by affinity, increased hydrophobicity, or improved pharmaco the iron-catalyzed generation of hydroxyl free radicals. kinetics to allow enhanced vocalization. Synthesis of Opioid Receptor Ligands with X Groups Preferred radionuclides for use in conjunction with a Compounds of the invention are conveniently prepared by diagnostic kit are "Tc, Ru 'In and Pb and with a the chemical coupling of L portion of the molecule to an X therapeutic kit are Re, Re, 'Y, 7Cu, Rh, 'Au, group. An extender arm E may be disposed between the L 'Au and ’’Bi. and X portions of the molecule. To prepare molecules of the formula L-E-X, a ligand molecule and an E-X group may be Methods for preparing these isotopes are known. For Separately Synthesized, and then the two groups joined example, molybdenum/technetium generators for producing together. Alternatively, the ligand molecule may be Synthe 35 '"Te are commercially available. In addition, tungsten/ sized with an extender arm, i.e., L-E, and the X group may rhenium generators for producing Re are available. Pro be separately Synthesized, before combining the two groups cedures for producing Re include the procedures together to form L-E-X. The Examples set forth at the end described by Deutsch et al., (Nucl. Med. Biol. Vol. of this specification illustrate various Synthetic approaches 13:4:467-477, 1986) and Vanderheyden et al. (Inorganic to compounds of the invention. 40 Metal Species Chem. Vol. 24:1666–1673, 1985), and methods for produc The compounds of the invention described above having tion of Re have been described by Blachot et al. (Int'l J. radionuclide chelating groups may be chelated with one or of Applied Radiation and Isotopes Vol. 20:467-470, 1969) more metals or metalions, which are collectively referred to and by Klofutar et al. (J. of Radioanalytical Chem. Vol. herein as "metal Species', to provide useful therapeutic and 45 5:3–10, 1970). Production of 'Pd is described in Fawwaz diagnostic agents. For in vitro diagnostic purposes, fluores et al. (J. Nucl. Med. 25:796, 1984). Production of Pb and cent metal Species Such as Terbium and Europium may be 'Bi is described in Gansow et al. (Amer: Chem. Soc. Symp. preferred since their complexes would be chemically similar Ser. 241:215–217, 1984), and Kozah et al. (Proc. Nat'l Acad. to In and Y complexes but would be detectable by their Sci USA 83:474–478, 1986). fluorescence. Representative references in this regard 50 include Min Li and Paul R. Selvin, “Amino Reactive Forms Compounds of the Invention Chelated with Radionuclides of a Luminescent Diethylenetriaminepentaacetic Acid Che late of Terbium and Europium: Attachment to DNA and The present invention provides compounds having affin Energy Transfer Measurements,” Bioconi. Chem. 8:127-132 ity to one or more opioid receptors, where the compounds (1997); and Harri Takalo et al., “Synthesis of Europium (III) 55 also have radionuclide binding group for chelation with a Chelates Suitable for Labeling of Bioactive Molecules,” radionuclide. The chemical Structure of Such chelates, which Bioconj. Chem. 5:278-282 (1994). are also referred to herein as radiolabeled compounds, is For diagnostic purposes, preferred metal Species which essentially the Same as that of a compound of the invention may be chelated according to the invention include gamma with the exception that a metal is shown complexing with emitter isotopes which are useful for diagnostic Scintigra 60 four or more electron-donating atoms (i.e., S, O or N) of the phy. Such gamma emitters include, without limitation, binding groups (also referred to as binding arms). gallium", gallium, indium', and technetium'" (''"Tc). Indium'' with a half-life of 2.8 days, and technetium’" In one embodiment of the invention, a compound of the with a half-life of 6 hrs, are particularly useful gamma invention is chelated to a radionuclide So as to include the emitters. 65 following structure, where any of the “R” groups may be For therapeutic purposes, the compounds according to the joined, directly or indirectly, to a ligand portion (L) of the invention may be chelated to beta radiation emitters which compound of the invention. US 6,359,111 B1 23 24 have been described in the prior art, See, e.g., U.S. Pat. NoS. R 5,227,474; 5,164, 176; 5,120,526; 5,112,953; 5,091,514; 5,075,099: 4,988,496; 4,965,392; and 4,963,688, as well as R S. N X references cited therein. In one embodiment of the present invention, compounds of the invention comprising acetamidomethyl and/or X N N R hemithioacetal Sulfur protective groups are radiolabeled with a metal radionuclide by reacting the compound with the radionuclide under conditions of acidic pH. It is believed R X that the acidic pH and the presence of the metal both R S. S R contribute to the displacement of the Sulfur protective groups from the chelating compound. The radionuclide is in chelatable form when reacted with the compound of the X N N X invention. 15 In the case of technetium and rhenium, being in “chelat able form' generally requires a reducing Step. A reducing R R1 agent will be employed to reduce the radionuclides (e.g., in the form of pertechnetate and perrhenate, respectively) to a In the above structure, M is a radionuclide ion, to which lower oxidation state at which chelation will occur. Many 1 or 2 oxygen atoms may be bonded (to provide monooxo Suitable reducing agents, and the use thereof, are known, and dioxo Species), or 1 nitrogen atom may be bonded (to see, e.g., U.S. Pat. Nos. 4,440,738; 4,434,151; and 4,652, provide a nitrido Species) especially if the metal is Tc or Re. 440. Such reducing agents include, but are not limited to, The radioisotopes '"Te and Re/Re are commercially Stannous ion (e.g., in the form of Stannous Salts. Such as available as pertechnetate (TcO) and perrhenate (ReO) Stannous chloride or Stannous fluoride), metallic tin, ferrous which are in their most stable +7 oxidation state. In order for 25 ion (e.g., in the form of ferrous Salts. Such as ferrous chloride, the donor atoms of the binding arms to coordinate with Tc ferrous Sulfate, or ferrous ascorbate) and many others. and Re, the pertechnetate and perrhenate have to be reduced Sodium pertechnetate (i.e., ''"Te0, which is in the +7 to lower oxidation States with reducing agents Such as oxidation level) or sodium perrhenate (i.e., ReO, stannous chloride (SnCl). The donor atom of the binding ReO) may be combined simultaneously with a reducing arms in compounds of the invention may utilize Tc and Re agent and a chelating compound of the invention in accor in their +5 oxidation State. dance with the radiolabeling method of the invention, to A variety of metal ions or complex ions may be employed form a chelate. as the radionuclide. These metals include, but are not limited In one preferred embodiment, the radionuclide is treated to, copper, e.g., "Cu and 'Cu; technetium, e.g., ''"Te; with a reducing agent and a complexing agent to form an rhenium, e.g., Re and Re; lead, e.g., -Pb and 'Pb: 35 intermediate complex (i.e., an “exchange complex”). This palladium, e.g. 'Pd; bismuth, e.g., 'Bi, and gold, e.g., embodiment is preferred when Tc or Re are the radionuclide. 'Au. The metal may be present as an ion, e.g., "Cu" and Complexing agents are compounds which bind the radio "Cu" (copper may end up in S-containing ligands as Cu") nuclide more weakly than do the compounds of the or as an oxidized form, e.g., ''"TcO", ReO" or invention, and may be weak chelators. Any of the Suitable ReO" when incorporated in the chelate compounds. 40 known complexing agents may be used, including but not The dashed lines in the formula presented for the chelate limited to gluconic acid, glucoheptonic acid, tartaric acid, compounds of the invention represent four coordinate cova methylene disphosphonate, glyceric acid, glycolic acid, lent bonds, between the metal radionuclide M and the Sulfur mannitol, oxalic acid, malonic acid, Succinic acid, bicine, and the three nitrogen atoms (an NS chelator) shown in the N,N'-bis(2-hydroxy ethyl) ethylene diamine, citric acid, formula on the left, and between the radionuclide M and the 45 ascorbic acid and gentisic acid. Tartaric acids gluconic acid two Sulfur and two nitrogen atoms (an NS chelator) shown or glucoheptonic acid are preferred as the Tc-complexing in the formula on the right. In either case, the metal agent and citric acid for rhenium. radionuclide is bound through relatively stable bonds in the When the radionuclide in the form of an exchange com chelated compounds of the invention. Also in the above pleX is reacted with a compound having radionuclide bind formulas, =X represents a carbonyl group or two hydrogen 50 ing arms according to the present invention, the radionuclide atoms. R and R/R' are organic groups. will transfer to the compound which binds the radionuclide Synthesis of Opioid Recepetor Ligands/Binding Arms more Strongly to form chelates of the invention. Heating Chelated with Radionuclides may be required to promote transfer of the radionuclide. The compounds of the present invention may be Radionuclides in the form of Such exchange complexes also radiolabeled, using conventional procedures, with any of a 55 are considered to be in “chelatable form” for the purposes of variety of radionuclide metals to form the corresponding the present invention. radionuclide metal chelates. AS Set forth above, these radio Chelates of Pb, Bi, and 'Pd may be prepared by nuclide metals include, but are not limited to, copper (e.g., combining the appropriate Salt of the radionuclide with a 7Cu and 'Cu); yttrium (Y), technetium (e.g., ''"Tc); Suitable compound of the invention at an appropriate pH, palladium (e.g., "Pd), indium (e.g., 'In), rhenium (e.g., 60 and incubating the reaction mixture at room temperature or Re and Re); lead (e.g. 'Pb); and bismuth (e.g., at higher temperatures. It is not necessary to treat the lead, '''Bi). The selection of suitable chemistry to achieve che bismuth, palladium, and copper radioisotopes with a reduc lation between a binding arm in a compound of the invention ing agent prior to chelation, as Such isotopes are already in and a radionuclide will depend, for example, on the identity an oxidation State Suitable for chelation (i.e., in chelatable of the radionuclide and the presence or absence of protecting 65 form). The specific radiolabeling reaction conditions may groups bound to the atoms which undergo the chelation with vary Somewhat according to the particular radionuclide and the radionuclide. Many aspects of the chemistry of chelation chelating compound involved. US 6,359,111 B1 25 26 The rhenium chelate may be formed from various routes. 25 C-100° C. for 10–30 min. The percent formation of For example, reducing perrhenate to rhenium (IV) hexachlo radiolabeled compound is determined by ITLC and a gra ride by employing hypophosphorous acid and concentrated dient HPLC system using a radiometric detector. HCl at 95 C. yields rhenium hexachloride. The rhenium The following general method may be used to radiolabel hexachloride may then be converted to rhenium dioxo opioid receptor ligands having protected Sulfur atoms in diethylenediamine chloride in 90% ethylenediamine at room their binding arms with Re: sodium perrhenate (3 mL, 15 temperature. At a basic pH in the presence of a compound mCi, produced from a W-188/Re-188 research scale of the invention having radionuclide binding arms, the generator) is added to a vial containing lyophilized mixture rhenium dioxo diethylenediamine chloride exchanges rap comprising citric acid, 75 mg, Stannous chloride, 0.75 mg, idly with, for example, a binding arm having the NS gentisic acid, 0.25 mg, and lactose, 100 mg. The Vial is configuration of chelating atoms. agitated gently to mix the contents, then incubated at room '"Te-radiolabeling of compound of the invention having temperature for 10 minutes to form a "Re-citrate interme radionuclide binding arms with N and/or S atoms in the diate exchange complex. To a separate Vial containing binding arms may be accompanied by the following Meth 0.50-1.0 mg of the opioid receptor ligand comprising a ods A or B. 15 hemithioacetate (ethoxyethyl) sulfur protecting group, 0.5 Method A: Stannous gluconate kits are prepared contain mL of isopropyl alcohol is added and the vial is agitated for ing 5.0 mg Sodium gluconate, 0.1 mg Stannous chloride, 2 min for complete dissolution of the ligand. Next, 0.3 mL 0.1-1.0 mg of an opioid receptor ligand of the invention with of this Solution is transferred to the Vial containing the N.S.-binding arms, and 1.0–5.0 mg of lactose. The pH of the Re-citrate complex prepared above. After gentle mixing, solution is maintained between 5 and 7.5 using hydrochloric the vial is incubated at 95 C-100° C. for 15–30 min. Then, acid, acetic acid or Sodium hydroxide. To the Stannous immediately transferred to a 0°C. ice bath for two minutes. gluconate kit is added 1.0 mL Sodium pertechnetate (Na The yields of Re-analog generally range between 99mTcO) in saline with a specific activity of 35-50 mCi/ 90-95% as measured by reverse phase C, HPLC analysis. mL. The vial is thoroughly mixed and incubated at 25 The following general method may be used to radiolabel C-100° C. for 15-30 minutes. The percent formation of 25 opioid receptor ligands having unprotected Sulfur atoms in '"Tc-chelate is determined by ITLC and HPLC using a their binding arms with Re (in general, a slightly higher radiometric detection System. pH is used when the Sulfur atoms are in unprotected form vS. Method B: Stannous tartrate kits are prepared in an when the Sulfur atoms are in protected form): Sodium evacuator vial under nitrogen to contain 0.5 mL of disodium perrhenate (3 mL, 15 mCi, produced from a W-188/Re-188 tartrate (10 mg/mL) and 0.1 mL stannous chloride (1.0 research Scale generator) is added to a vial containing mg/mL in ethanol). The pH of the solution is kept between lyophilized mixture comprising citric acid, 75 mg, Stannous 5 and 7.5 preferably 6.5. To this stannous tartrate solution is chloride, 0.75 mg, gentisic acid, 0.25 mg, and lactose, 100 added 1.0 mL of Sodium pertechnetate at a specific concen mg. The Vial is agitated gently to mix the contents, then tration of 35-50 mCi/mL in Saline. The reaction mixture is incubated at room temperature for 10 minutes to form a allowed to Stand at room temperature. In another evacuated 35 Re-citrate intermediate exchange complex. In another vial, 200 uL of sodium phosphate (0.5 M, pH 8.0 or 10.0) evacuated vial, 200-500 lull of sodium phosphate (0.5 M, pH and 1.0 mL of N opioid receptor ligand (1.0 mg/mL) are 8.0) and 1.0 mL of opioid receptor ligand (0.5-1.0 mg/mL) added successively. Then Tc-99m-Tartrate (50 mCi) is are added successively. Then Re-citrate (50 mCi) is added, and the vial is incubated at 25 C-100° C. for 10–30 added, and the vial is incubated at 25 C-100° C. for 10–30 min. The percent formation of radiolabeled N-opioid recep 40 min. The percent formation of radiolabeled compound is tor ligand is determined by ITLC and HPLC using a radio determined by ITLC and a gradient HPLC system using a metric detection System. radiometric detector. The following general method may be used to radiolabel Compositions Containing Diagnostically or Therapeuti opioid receptor ligands with radionuclide binding arms cally Effective Compounds having Opioid Receptor Affinity having protected Sulfur atoms in their binding arms with 45 and Radionuclide Chelates Thereof Re: a solution of 11.0 mg of citric acid 370 ug of gentisic The present invention provides a pharmaceutical or vet acid and 460 tug of Stannous chloride dihydrate in 100 till erinary composition (hereinafter, Simply referred to as a HO is adjusted to a final pH of 2. To this solution, 500 till pharmaceutical composition) containing a compound of the of ReO (1-50 mCi) is added and incubated for 1-2 invention having opioid receptor affinity as described above, minutes to form Re-citrate. Immediately following, 50 in admixture with a pharmaceutically acceptable carrier or hemithioacetal protected opioid receptor ligand (0.1 to 1.0 diluent. The invention further provides a pharmaceutical mg in 600 u isopropanol) is added. The reaction mixture is composition containing a chelate of a compound of the incubated at 90 C-100° C. for 15–30 min and then is invention having opioid receptor affinity as described above, brought to room temperature by rapid cooling in an ice bath. in admixture with a pharmaceutically acceptable carrier or The following general method may be used to radiolabel 55 diluent. opioid receptor ligands having unprotected Sulfur atoms in The pharmaceutical compositions of the present invention their binding arms with Re (in general, a slightly higher may be in any form which allows for the composition to be pH is used when the Sulfur atoms are in unprotected form vS. administered to a Subject. For example, the composition may when the Sulfur atoms are in protected form): a Solution of be in the form of a Solid, liquid or gas (aerosol). Typical 11.0 mg of citric acid 370 ug of gentisic acid and 460 ug of 60 routes of administration include, without limitation, oral, stannous chloride dihydrate in 0.1–1.0 mL HO is adjusted topical, parenteral, Sublingual, rectal, vaginal, and intrana to a final pH of 2. To this solution, 500 lull of ReO (1-50 Sal. The term parenteral as used herein includes Subcutane mCi) is added and incubated for 1-2 minutes to form ous injections, intravenous, intramuscular, intrasternal injec Re-citrate. In another evacuated vial, 200 uL of sodium tion or infusion techniques. Pharmaceutical composition of phosphate (0.5M, pH 8.0) and 1.0 mL of the opioid receptor 65 the invention are formulated So as to allow the active analog (0.5-1.0 mg/mL) are added Successively. Then ingredients contained therein to be bioavailable upon admin Re-citrate (50 mCi) is added, and the vial is incubated at istration of the composition to a Subject. Compositions that US 6,359,111 B1 27 28 will be administered to a subject take the form of one or tetraacetic acid; bufferS Such as acetates, citrates or phos more dosage units, where for example, a tablet may be a phates and agents for the adjustment of tonicity Such as Single dosage unit, and a container of opioid receptor ligand Sodium chloride or dextrose. Calcium (+2) may be added to of the invention in aerosol form may hold a plurality of the composition in instances where the chelate may have dosage units. Some undesirable toxicity. The parenteral preparation can be Materials used in preparing the pharmaceutical composi enclosed in ampoules, disposable Syringes or multiple dose tions should be pharmaceutically pure and non-toxic in the Vials made of glass or plastic. Physiological Saline is a amounts used. It will be evident to those of ordinary skill in preferred adjuvant. An injectable pharmaceutical composi the art that the optimal dosage of the active ingredient(s) in tion is preferably Sterile. the pharmaceutical composition will depend on a variety of A liquid composition intended for either parenteral or oral factors. Relevant factors include, without limitation, the type administration should contain an amount of the inventive of Subject (e.g., human), the particular form of the active compound or chelate Such that a Suitable dosage will be ingredient, the manner of administration and the composi obtained. Typically, this amount is at least 0.01% of a tion employed. compound of the invention in the composition, however the In general, the pharmaceutical composition includes a 15 actual amount of label that is effective may be less than compound of the invention having opioid receptor affinity or 0.01%. When intended for oral administration, this amount chelate thereofas described herein, in admixture with one or may be varied to be between 0.1 and about 70% of the more carriers. The carrier(s) may be particulate, So that the weight of the composition. Preferred oral compositions compositions are, for example, in tablet or powder form. The contain between about 4% and about 50% of the active carrier(s) may be liquid, with the compositions being, for opioid receptor ligand compound of the invention. Preferred example, an oral Syrup or injectable liquid. In addition, the compositions and preparations according to the present carrier(s) may be gaseous, So as to provide an aerosol invention are prepared So that a parenteral dosage unit composition useful in, e.g., inhalatory administration. contains between 0.01 to 1% by weight of active compound. When intended for oral administration, the composition is The pharmaceutical composition may be intended for preferably in either Solid or liquid form, where Semi-Solid, 25 topical administration, in which case the carrier may Suit Semi-liquid, Suspension and gel forms are included within ably comprise a Solution, emulsion, ointment or gel base. the forms considered herein as either Solid or liquid. The base, for example, may comprise one or more of the AS a Solid composition for oral administration, the com following: petrolatum, lanolin, polyethylene glycols, position may be formulated into a powder, granule, com beeswax, mineral oil, diluents Such as water and alcohol, and pressed tablet, pill, capsule, chewing gum, wafer or the like emulsifiers and Stabilizers. Thickening agents may be form. Such a Solid composition will typically contain one or present in a pharmaceutical composition for topical admin more inert diluents or edible carriers. In addition, one or istration. If intended for transdermal administration, the more of the following adjuvants may be present: binders composition may include a transdermal patch or iontophore Such as carboxymethylcellulose, ethyl cellulose, microcryS sis device. Topical formulations may contain a concentration talline cellulose, or gelatin; excipients Such as Starch, lactose 35 of the inventive compound of from about 0.1 to about 10% or dextrins, disintegrating agents Such as alginic acid, W/V (weight per unit volume). Sodium alginate, Primogel, corn Starch and the like, lubri The composition may be intended for rectal cants Such as magnesium Stearate or SteroteX, glidants Such administration, in the form, e.g., of a Suppository which will as colloidal Silicon dioxide, Sweetening agents Such as melt in the rectum and release the drug. The composition for Sucrose or Saccharin, a flavoring agent Such as peppermint, 40 rectal administration may contain an oleaginous base as a methyl Salicylate or orange flavoring, and a coloring agent. Suitable nonirritating excipient. Such bases include, without When the composition is in the form of a capsule, e.g., a limitation, lanolin, cocoa butter and polyethylene glycol. gelatin capsule, it may contain, in addition to materials of the The composition may include various materials which above type, a liquid carrier Such as polyethylene glycol or a modify the physical form of a Solid or liquid dosage unit. For fatty oil. 45 example, the composition may include materials that form a The composition may be in the form of a liquid, e.g., an coating shell around the active ingredients. The materials elixir, Syrup, Solution, emulsion or Suspension. The liquid which form the coating Shell are typically inert, and may be may be for oral administration or for delivery by injection, Selected from, for example, Sugar, shellac, and other enteric as two examples. When intended for oral administration, coating agents. Alternatively, the active ingredients may be preferred composition contain, in addition to the present 50 encased in a gelatin capsule. compounds, one or more of a Sweetening agent, The composition in Solid or liquid form may include an preservatives, dye/colorant and flavor enhancer. In a com agent which binds to the compound of the invention having position intended to be administered by injection, one or opioid receptor affinity or chelate thereof and thereby assists more of a Surfactant, preservative, wetting agent, dispersing in the delivery of the active components. Suitable agents agent, Suspending agent, buffer, Stabilizer and isotonic agent 55 which may act in this capacity include a monoclonal or may be included. polyclonal antibody, an antigen-binding fragment of a The liquid pharmaceutical compositions of the invention, monoclonal or polyclonal antibody, a protein or a liposome. whether they be Solutions, Suspensions or other like form, The pharmaceutical composition of the present invention may include one or more of the following adjuvants: Sterile may consist of gaseous dosage units, e.g., it may be in the diluents Such as water for injection, Saline Solution, prefer 60 form of an aeroSol. The term aerosol is used to denote a ably physiological Saline, Ringer's Solution, isotonic Sodium variety of Systems ranging from those of colloidal nature to chloride, fixed oils. Such as Synthetic mono or digylcerides Systems consisting of pressurized packages. Delivery may which may serve as the Solvent or Suspending medium, be by a liquefied or compressed gas or by a Suitable pump polyethylene glycols, glycerin, propylene glycol or other System which dispenses the active ingredients. AeroSols of Solvents, antibacterial agents Such as benzyl alcohol or 65 compounds of the invention may be delivered in Single methyl paraben; antioxidants Such as ascorbic acid or phase, bi-phasic, or tri-phasic Systems in order to deliver the Sodium bisulfite, chelating agents Such as ethylenediamine active ingredient(s). Delivery of the aerosol includes the US 6,359,111 B1 29 30 necessary container, activators, Valves, Subcontainers, Spac Methods of administering effective amounts of the inven ers and the like, which together may form a kit. Preferred tive compounds or chelates thereof include the administra aerosols may be determined by one skilled in the art, without tion of inhalation, oral or parenteral forms. Such dosage undue experimentation. forms include, but are not limited to, parenteral Solutions, Whether in Solid, liquid or gaseous form, the pharmaceu tablets, capsules, Sustained release implants and transdermal tical composition of the present invention may contain one delivery Systems, or inhalation dosage Systems employing or more known pharmacological agents useful in the thera dry powder inhalers or pressurized multi-dose inhalation peutic effect that is desired. The pharmaceutical compositions may be prepared by devices. The dosage amount and frequency are Selected to methodology well known in the pharmaceutical art. A com create an effective level of the agent without harmful effects. position intended to be administered by injection can be It will generally range from a dosage of about 0.1 to 100 prepared by combining the compound of the invention mg/Kg/day, and typically from about 0.1 to 10 mg/Kg/day having opioid receptor affinity or chelate thereof with water where administered orally or intravenously. Also, the dosage So as to form a Solution. A Surfactant may be added to range will be typically from about 0.01 to 1 mg/Kg/day facilitate the formation of a homogeneous Solution or SuS where administered intranasally or by inhalation. pension. Surfactants are compounds that non-covalently 15 Therapeutic Applications of Opioid Recepetor Ligands interact with the inventive compound/chelate So as to facili having Radionuclide Binding Arms Chelated with Radio tate dissolution or homogeneous Suspension of the inventive nuclides compound/chelate in the aqueous delivery System. Yet another embodiment of the invention provides meth The present invention provides that an effective amount of ods for using a compound of the invention having opioid a compound or composition of the present invention may be receptor affinity chelated with radionuclide as described used to detect, diagnose or treat diseases of cells having above for therapeutic purposes. The therapeutic method may opioid receptors. These cells are typically mammalian cells. be used for delivering a radionuclide to a target Site within Diseases that may be detected, diagnosed or treated accord a mammalian host. The method comprises the Step of ing to the present invention include certain dementia of the administering to a mammal a therapeutically effective dose brain. Thus, an imaging compound of the invention may be 25 of one of the radiolabeled compounds as described above. A injected or otherwise delivered into the central nervous therapeutically effective dose is generally from about 20 System, to identify and in Some instances quantitate opiate mCi to about 300 mCi. Preferred radionuclides are Re, receptors in the brain. Dementia of the brain include 90Y, 188Re, 67CU, 105Rh, 198Au, 199Au and 212Bi. A pre Schizophrenia, epilepsy, Alzheimer's disease, and abnor ferred mammal is man. malities due to narcotic drug addition. Compounds of the The chelates of the present invention may be administered invention having morphine-like ligand groups are particu to a mammalian host, normally by injection, intravenously, larly Suitable for monitoring changes in the brain due to intraarterially, peritoneally, intratumorally, or the like, narcotic drug addition. A preferred compound for this depending upon the particular site at which the radionuclide method contains a "Te atom. is desired. Generally, from about 1-30 mL will be injected A disease that may be detected or treated according to the 35 into a host, depending upon the size of the host, with about present invention is cancer, including the detection or treat 1-10 mCi/Kg of host. For human hosts, the therapeutic ment of neuroendocrine tumors. Adult neuroendocrine dosage will usually be about 70-700 mCi/70 Kg host, more tumors that may be detected or treated according to the usually about 25-35 mCi/70 Kg host of the diagnostic present invention include, without limitation, (listed as indo dosage. For lower mammals, e.g., mice, 1-50 uCi will be lent biology/aggressive biology) carcinoid tumors (many 40 used for biodistribution Studies, while up to or greater than primary sites)/Small cell lung cancer, atypical or poorly 0.5-2 mCi will be used for imaging studies. Multiple differentiated carcinoids (many primary Sites); islet cell injections may be use d in a therapeutic method, where the tumor (pancreas)/extrapulmonary Small cell carcinoma injections may be spaced, for example, about 1 week apart. (many primary sites); pheochromocytoma (adrenal)/ After administration of the radionuclide, depending upon its peripheral neuroepithelioma-neurons (usually in 45 purpose, the host may be treated in various ways for adolescents); medullary carcinoma (thyroid)/market cell detection of the radioactive emissions from the Site or Sites tumor (skin); and paraganglioma-neurons/neuroblastoma where the radionuclide Specifically binds. (adrenal). One or more neuroendocrine carcinomas may be Diagnostic Applications of Opioid Receptor Ligands hav found in the gastrointestinal tract, including without ing Radionuclide Binding Arms Chelated with Radionu limitation, carcinoid tumors, gastrinomas, insulinomas, 50 clides glucagonoma, Somatostatinoma, Vipomas, and pancreatic Yet another embodiment of the invention provides meth polypeptideoma. Leukemia and lymphomas are other cancer ods for using the radiolabeled compounds described above types that may be detected or treated according to the present for diagnostic purposes. The diagnostic method may be used invention. to detect the presence or absence of a target Site within a Neuroendrine tumors may be targeted with compounds of 55 mammalian host. The method comprises the Steps of admin the invention having naltrindole-like ligand portions. Opioid istering to a mammal a diagnostically effective dose of one receptors have been reported to be present on lung cancer of the radiolabeled compounds described above. This step is cells, to a greater extent than on other cell types of a host, followed by a step of detecting the biodistribution of the outside of the central nervous System (see, e.g., Campa et al. radionuclide in the mammal to deter mine the presence or Can. Res. 56: 1965-1701, 1996). A charged compound of the 60 absence of the target Site in the host. present invention that is administered Systemically should A diagnostically effective dose of a radiolabeled com not be able to cross the blood-brain barrier. DOTA pound is generally from about 5 to about 35 and typically derivatives of the invention are examples of Such charged from about 10 to about 30 mCi per 70 Kg body weight. The compounds of the present invention. Thus, a chemical of the precise dose for a radiolabeled compound is dependent upon invention which is toxic to cells should become localized at 65 the particular receptor which is being targeted because the lung cancer cells, and not adventitiously bind to opioid level of uptake of a radiolabeled compound into a tumor is receptor in the central nervous System. dependent upon the number of receptorS for the compound US 6,359,111 B1 31 32 and its affinity for the receptors. The precise dose further tive compound. When the radionuclide is to be reduced to a depends upon the particular route of administration, e.g., lower oxidation State (e.g., technetium and rhenium, as intravenous, intracompartmental, intraarterial or intratu discussed above), the kits may contain a reducing agent moral. It will be evident to one skilled in the art how to which is effective in reducing a particular metal determine the optimal effective dose for a particular radio radionuclide, which is to be chelated by the inventive labeled compound and a particular route of administration. compound, to an oxidation State at which an exchange Preferred radionuclides are '''In, Ru, ''"Tc and Pb. A complex of the radionuclide may be formed. In addition, a preferred mammal is man. The therapeutic method may be used for delivering a kit may additionally contain a complexing agent with which radionuclide to a target Site within a mammalian host. A the reduced radionuclide will form an exchange complex, preferred target Site is a lung, which may contain Small cell where this exchange complex is a useful intermediate in cancer. The method comprises the Step of administering to a forming the radiolabeled compound. mammal a therapeutically effective dose of one of the The kit components and instructions will be Somewhat radiolabeled compounds described above. A therapeutically different when the inventive compound is to be radiolabeled effective dose is generally from about 20 mCi to about 700 with a technetium isotope (i.e., a diagnostic kit) than when mCi. Preferred radionuclides are 'Y, Re, Re, 7Cu, 15 the inventive compound is to be radiolabeled with a yttrium, 'Rh, 'Au, 'Au 'Bi. A preferred mammal is man. indium, rhenium, lead, bismuth, palladium, or copper iso The chelates of the present invention may be administered tope (i.e., a therapeutic kit). The different components and to a mammalian host, normally by injection, intravenously, procedures are discussed in more detail below. In the fol intraarterially, peritoneally, intratumorally, or the like, lowing discussion, the term “Separate containers' is meant depending upon the particular site at which the radionuclide to include not only separate, individual containers (e.g., is desired. Generally, from about 1-30 ml, will be injected vials) but also physically separate compartments within the into a host, depending upon the size of the host, with about Same container. 1.0-10 mCi/Kg of host. For human hosts, the therapeutic In accordance with one embodiment of the invention, a dosage will usually be about 70-700 mCi/70 Kg host, more diagnostic kit comprises the following reagents (in Separate usually about 25-35 mCi/70 Kg host of the diagnostic 25 containers unless otherwise noted), presented in the general dosage. For lower mammals, e.g., mice, 1-50 uCi will be order of use. used for biodistribution Studies, while up to or greater than 1. A reducing agent effective in reducing pertechnetate 0.5-2.0 mCi will be used for imaging studies. After admin ('"TcO, which is in the +7 oxidation level) to a lower istration of the radionuclide, depending upon its purpose, the oxidation State at a neutral to acidic pH So that a technetium host may be treated in various ways for detection of the eXchange complex can be formed. Many Suitable reducing radioactive emissions from the Site or Sites where the agents are known in the art, including but not limited to radionuclide Specifically binds. The total dose can be given Stannous ion, (e.g., in the form of Stannous Salts such as as a Single injection or can be split into Several multiple Stannous chloride or Stannous fluoride), metallic tin, forma injections. midine Sulfinic acid, ferric chloride, ferrous Sulfate, ferrous Kits Containing Opioid Receptor Ligands having Radio 35 ascorbate, and alkali salts of borohydride. Preferred reduc nuclide Binding Arms Chelated With Radionuclides, and ing agents are Stannous Salts. Preparation and Uses Thereof 2. A complexing agent with which the reduced '"Te will In another embodiment of the invention, an opioid recep form an exchange complex, thus protecting the "Tc from tor ligand having radionuclide binding arms as described hydrolysis. In order to achieve efficient transfer or exchange above may be included in a kit for producing a metal 40 of the '"Tc from this complex to the inventive compound, Species-chelated compound of the invention (a "radiolabeled the complexing agent advantageously binds the radionuclide compound”) for radiopharmaceutical use. Reagents useful in more weakly than the compound will. Complexing agents reactions to radiolabel the compound with a radionuclide which may be used include, but are not limited to, gluconic may be included. Such kits also may comprise a means for acid, glucoheptonic acid, methylene diphosphonate, glyceric purifying the radiolabeled compound away from the reaction 45 acid, glycolic acid, mannitol, Oxalic acid, malonic acid, mixture in which it was formed, as well as Specific instruc succinic acid, bicine, N,N'-bis(2-hydroxyethyl) ethylene tions for producing the radiolabeled compound using the kit diamine, citric acid, ascorbic acid and gentisic acid. Good components. results are obtained using gluconic acid or glucoheptonic Such kits generally will be used in hospitals, clinics or acid as the Tc-complexing agent (or “exchange agent” in other medical facilities. Since Such facilities generally have 50 these cases), as they efficiently transfer the "Te to the ready acceSS on a daily basis to radionuclides, including inventive compound at a pH at which the active ester is isotopes of technetium, and Since isotopes of rhenium, stable. yttrium, lead, bismuth, palladium, and copper may be pre 3. A compound of the present invention, having an opioid pared as described above, inclusion of the radionuclide in receptor ligand Site and radionuclide binding arms, as the kit is optional. Exclusion of the radionuclide permits 55 described above. Storage of the kit, whereas kits containing the radionuclide 4. Additional reagents for use in the radiolabeling are (either as a separate component or as the radiolabeled optional components of the kits. Examples of Such addi compound) would have to be used within a narrow time tional reagents include, without limitation, buffers, alcohols, frame (depending on the half-life of the particular isotope); acidifying Solutions, and other Such reagents as are more otherwise, radioactive decay of the radioisotope would 60 fully described herein. Such additional reagents are gener diminish the effectiveness of the diagnostic or therapeutic ally available in medical facilities and thus are optional technique for which the radiolabeled compound is used. For components of the kit because they can be readily obtained Re and 'Y, on-site radiolabeling would avoid radiolytic by people using the kits. However, these reagents preferably degradation of the labeled compound due to the beta particle are included in the kit to ensure that reagents of Sufficient emission. 65 purity and Sterility are used, Since the resulting radiolabeled The kits may be diagnostic or therapeutic kits, depending compounds may be administered to mammals, including on which radionuclide is used for chelating with the inven humans, for medical purposes. US 6,359,111 B1 33 34 5. Optionally, a container of a compound of the invention, produce low yields, as well as generating an excessive which may be administered in non-radiolabeled form to a amount of radioactivity for administration to a single human or mammal, is included in the kit. This inventive patient. When about 75 to 100 mCi of TcO are to be added, compound is reactive with essentially the same target Sites the Stannous gluconate complex preferably is formed from (i.e., opioid receptor(s)) as the radiolabeled compound and about 3 to about 10 mg of sodium gluconate and about 0.075 reduces binding of the radiolabeled compound to croSS to about 0.250 mg of stannous chloride dihydrate; preferably reactive binding sites on non-target tissues. The two com from about 4 to about 6 mg of Sodium gluconate and about pounds may be the Same, or the compound to be radiola 0.075 to about 0.125 mg of stannous chloride dihydrate. beled may, for example, be somewhat different from the Using the reagents described above, Sodium pertechnetate compound which is to be administered in non-radiolabeled is combined with the reducing agent and complexing agent. form. The latter inventive compound is administered as an When the Sodium pertechnetate is added to Stannous unlabeled specific blocker (prior to administration of the gluconate, the radionuclide is effectively reduced to a lower radiolabeled compound) in an amount effective in improving oxidation State and complexed with gluconate to form an diagnostic imaging of the desired target sites (e.g., tumors as eXchange complex. The Stannous gluconate and pertechne described above). tate may be combined in various ways. In one embodiment 6. Optionally, the kit may also include a container of a of the invention, Sterile water is added to a vial containing opioid receptor ligand which does not bind through specific 15 a Stannous gluconate preparation in dry Solid form. A portion mechanisms to Sites within the human or mammal to which of the resulting solution is combined with about 0.75 mL the radiolabeled compound is to be administered. This sodium pertechnetate (about 75 to 100 mCi). In another opioid receptor compound is administered as an "irrelevant” embodiment of the invention, Sodium pertechnetate (about 1 compound (prior to administration of the radiolabeled mL) is added directly to a lyophilized preparation compris compound) in an amount effective in decreasing nonspecific ing Stannous gluconate, gentisic acid as a Stabilizer, and uptake of certain radiolabeled compounds, as described lactose as a filler compound. In either case, the reaction above. mixture is incubated at about 25 C. to about 50 C., In one embodiment of the invention, a radiolabeled com preferably at about 25 C. to about 3 C. for a minimum of pound may be produced using a kit as described above, 10 minutes. Incubation for 10 minutes generally gives according to the following general procedure. The procedure 25 Sufficient yields of the desired technetium exchange com is preferably conducted under Sterile conditions. In this plex (e.g., technetium gluconate) while minimizing the particular embodiment of the invention, the kit includes formation of insoluble technetium dioxide, which may reagents in amounts Suitable for preparation of an amount of increase with increased incubation time. radiolabeled compound Suitable for injection into one An opioid receptor ligand having radionuclide binding human for diagnostic purposes. arms of the invention is added to an organic Solvent which An aqueous Solution containing a reducing agent and a is effective in dissolving the ligand and effective for the complexing agent (each as described above) is prepared. The exchange reaction that follows. Suitable solvents should be Solution may be prepared, for example, by combining stan nontoxic in mammals and inert toward the reactants in the nous chloride dihydrate (which includes the Stannous ion reaction mixture. Organic Solvents which may be used reducing agent) and Sodium gluconate (a complexing agent) include, without limitation, acetonitrile, ethyl acetate, and to form a Stannous gluconate complex. This Stannous glu 35 methylethyl ketone. When the radiolabeled compound is to conate complex may be provided in a single container in the be injected into humans, however, Suitable organic Solvents kit. In one embodiment of the invention, the Stannous include, but are not limited to, alcohols Such as ethanol, gluconate complex is provided in the kit in dry Solid form. butanol, tert-butyl alcohol and propanol, and polar aprotic Optionally, one or more Stabilizer compounds may be solvents such as DMSO and dimethylformamide (DMF). added to the Stannous gluconate complex. Many Such sta 40 The choice of Solvent may vary according to the particular bilizer compounds are known in the art, and Some are opioid receptor compound of the invention is to be included discussed in connection with the therapeutic kits below. For in the kit. For example, when the opioid receptor compound example, gentisic acid may be added to a container of the includes a tetrafluorophenyl ester group, ethanol may react Stannous gluconate complex to stabilize (minimize oxida with the ester in a transesterification reaction, producing tion of) the Stannous ion reducing agent, and the resulting 45 ethyl ester as a by-product. A preferred organic Solvent is mixture may be provided in the kit in dry solid form or as isopropyl alcohol. The concentration of the organic Solvent a lyophilized preparation. A filler compound advantageously in the following Tc-labeling eXchange reaction mixture is added prior to lyophilization, as described for the thera should be between about 10% and about 30%, preferably peutic kit below. For example, lactose may be added as a between about 15% and about 25%. filler compound in an amount effective to facilitate lyo 50 The Solution comprising the opioid receptor compound in philization. The amounts of Stannous chloride and Sodium the organic solvent is then acidified to a pH of about 2.0 to gluconate preferably are not So large as to have adverse about 5.0, preferably 2.8 to 3.3. At these acidic pH effects on the desired reactions and product. For example, an conditions, the formation of insoluble TcO will be mini excessively large amount of free Sodium gluconate may mized. Also, in this pH range, any hemithioacetal or thio Slow the transchelation Step and require addition of exces acetal Sulfur-protecting groups present in the opioid receptor Sive amounts of buffer necessary to raise the pH in Subse 55 compound will be displaced by a metal-assisted acid cleav quent Steps, and the reaction mixtures would then be unde age during the technetium labeling eXchange reaction to Sirably dilute. An acceptable ratio of Stannous chloride form the corresponding technetium chelate compound. Also, dihydrate to Sodium gluconate (by weight) is from about hydrolysis of ester groups on the opioid receptor compound 1:10 to about 1:100, preferably from about 1:25 to about is minimized under acidic conditions when compared to 1:70, most preferably about 1:41.6. 60 basic conditions. Suitable acids are added in amounts Suf The amount of "Te added may vary. When the diag ficient to displace the Sulfur-protective groups, if present, in nostic kit is designed for preparation of a radiolabeled the presence of the metal radionuclide (i.e., in amounts compound to be injected into a Single human patient, the Sufficient to adjust the reaction mixture to the above amount of pertechnetate to be added to the following reac described pH values range). Suitable acids include but are tion mixture may be from about 50 to about 200 mCi, 65 not limited to, phosphoric acid, Sulfuric acid, nitric acid, preferably from about 75 to about 100 mCi of the radionu glacial acetic acid, hydrochloric acid and combinations clide. Greater amounts may interfere with the reaction and thereof. Also included are Solutions including Such acids and US 6,359,111 B1 35 36 buffers (e.g., acetate and phosphate buffers). A Solution components of the kit because they can be readily obtained including glacial acetic acid and 0.2N HCl at a ratio of 2:14 by people using the kits. However, these reagents preferably is a typical acid Solution in the present invention. are included in the kit to ensure that reagents of Sufficient The acidified Solution of opioid receptor compound is purity and Sterility are used, Since the resulting radiolabeled combined with the previously prepared technetium compounds may be administered to mammals, including eXchange complex Solution, to form the corresponding humans, for medical purposes. radiolabeled compound. Typically, about 100 ug to about 5. Optionally, a container of opioid receptor ligand of the 150 lug, preferably about 135 ug of opioid receptor com invention, which may be administered in non-radiolabeled pound is combined with the Tc-gluconate complex prepared form to a human or mammal, is included in the kit. This from the 75 to about 100 mCi of technetium as described inventive ligand is reactive with essentially the same target above. The reaction mixture is heated to between about 50 Sites as the radiolabeled compound and reduces binding of C. and 100° C. for a time ranging from about 5 minutes to the radiolabeled compound to cross-reactive binding sites on about 45 minutes. Typically, the desired radiolabeled com non-target tissues. The two compounds may be the same, or pound may be produced by heating at about 75 C. for about the compound to be radiolabeled may, for example, be 15-2 minutes. Heating the reaction mixture accelerates the somewhat different from the compound which is to be eXchange reaction to form the chelate between the opioid 15 administered in non-radiolabeled form. The latter opioid receptor compound and the radionuclide. Upon completion receptor ligand of the invention is administered as an of the reaction, the mixture is transferred immediately to a unlabeled specific blocker (prior to administration of the O C. ice bath for a minimum of 2 minutes to stop the radiolabeled compound) in an amount effective in improving reaction quickly and at this temperature the radiopharma diagnostic imaging of the desired target sites (e.g., tumors as ceutical is stable. For practical purposes, the radiopharma described above). ceutical can be stored at room temperature for Several hours. 6. Optionally, the kit may also include a container of an An aqueous Solution including a buffer may then be added opioid receptor ligand of the invention which does not bind to the reaction mixture in order to reduce the concentration through Specific mechanisms to Sites within the human or of the organic Solvent(s) and to adjust the pH as desired. mammal to which the radiolabeled compound is to be Suitable buffers include nontoxic buffers which are inert administered. This opioid receptor ligand of the invention is toward the reactants, Such as, but not limited to, Sodium 25 administered as an "irrelevant” compound (prior to admin phosphate buffer and sodium bicarbonate buffer, preferably istration of the radiolabeled compound) in an amount effec at a concentration of about 1.0 M and a pH of about 10. tive in decreasing nonspecific uptake of certain radiolabeled Buffers such as TRIS must be used cautiously because the compounds, as described above. free amine groups of TRIS are reactive with ester groups that In one embodiment of the invention, an opioid receptor may be present as part of the opioid receptor ligand of the ligand of the invention radiolabeled with either Re or invention. Sufficient buffer is added to reduce the organic "Re may be prepared using such a kit, according to the Solvent concentration to an amount ranging from about 10% following general procedure. The procedure is conducted to about 15%, preferably from about 7.5% to about 12.5% under Sterile conditions. (on a weight basis, based on the entire weight of reaction Perrhenate (the ReO form of the Re or Re isotope) product Solution). 35 is reacted with reducing agent and a complexing agent. For In accordance with another embodiment of the invention, instance, citric acid (a complexing agent) may be combined a therapeutic kit includes the following reagents. with Stannous chloride (a reducing agent) in a single con 1. A reducing agent effective in reducing ReO, which is tainer (in which a Stannous citrate complex is believed to in the +7 oxidation level, to a lower oxidation State at a form) and the perrhenate may then be added thereto. neutral to acidic pH So that a rhenium exchange complex can The amounts of Stannous chloride and citric acid added be formed. Many Suitable reducing agents for this purpose 40 should not be So large as to have adverse affects on the are known in the art, including but not limited to Stannous desired reactions. For example, an excessively large amount ion (e.g., in the form of Stannous Salts. Such as Stannous of free citric acid may lower the pH to a level which makes chloride or Stannous fluoride), metallic tin, formamidine addition of large quantities of buffer necessary to raise the Sulfinic acid, ferric chloride, ferrous Sulfate, ferrous pH in Subsequent Steps, and the reaction mixtures would be ascorbate, and alkali salts of borohydride. Preferred reduc 45 undesirably dilute. An acceptable ratio of Stannous chloride ing agents are Stannous Salts. to citric acid (by weight) generally ranges from about 1:10 2. A complexing agent with which the reduced Re will to about 1:500, preferably from about 1:20 to about 1:200, form an exchange complex, thus protecting the Re from most preferably about 1:100. hydrolysis. In order to achieve efficient transfer or exchange One or more Stabilizer compounds may be added to the of the Re from this complex to the opioid receptor ligand of 50 Stannous citrate complex. Many Such Stabilizer compounds the invention, the complexing agent advantageously binds are known. See, e.g., U.S. Pat. Nos. 4,440,738 and 4,510, the radionuclide more weakly than the ligand will. Com 125. Advantageously, gentisic acid is added to the Stannous plexing agents which may be used include, but are not citrate to Stabilize (e.g., to prevent oxidation of) the Stannous limited to, methylene diphosphonate, glyceric acid, glycolic ion. The Stabilizer is added to a Solution including the acid, mannitol, Oxalic acid, malonic acid, Succinic acid, Stannous chloride reducing agent (and the complexing bicine, N,N'-bis(2-hydroxyethyl) ethylene acid, succinic 55 agent) in an amount effective to Stabilize the Stannous ion So diamine, citric acid, ascorbic acid, gentisic acid, tartaric that the shelf life (stability) of the stannous ion is increased. acid, and gluconic acid. A referred complexing agent is citric The solution may be lyophilized and provided in the kit as acid, which may be used to form a Re-complexing gent a lyophilized powder. complex (or “exchange agent” in these cases). When the stannous citrate solution is to be lyophilized, a 3. A opioid receptor ligand of the invention as described 60 “filler compound” may be added to the solution to provide above. bulk or mass and to aid in the lyophilization process. Lactose 4. Additional reagents for use in the radiolabeling are is a Suitable filler compound. optional components of the kits. Examples of Such addi In one particular embodiment of the invention, an aque tional reagents include, without limitation, buffers, alcohols, ous Solution of Stannous citrate may be prepared by com acidifying Solutions, and other Such reagents as are more 65 bining about 75 mg citric acid with about 750 tug stannous fully described herein. Such additional reagents are gener chloride. About 250 ug gentisic acid may then be added. ally available in medical facilities and thus are optional Typically, as the amount of gentisic acid decreases, the US 6,359,111 B1 37 38 Stabilizing effect was not as efficient, whereas when the from about 100 ug to about 200 lug of opioid receptor ligand amount of gentisic acid increases there is a negative affect on of the invention per mL of reaction mixture. yields. About 100 mg lactose (a preferred amount) may then To accomplish chelation of the opioid receptor ligand of be added to the preparation, although about 20 mg is the invention, the reaction mixture may be heated to a typically adequate. The final Solution (about 2 mL volume) temperature between about 50° C. and 100° C. for a time of may then be lyophilized. from about 5 to about 45 minutes. Typically, heating at about Perrhenate may then be added to the stannous citrate 75 C. for about 10 minutes is sufficient. Upon completion preparation. Perrhenate may be introduced into the prepa of the reaction, the mixture may be transferred immediately ration as an aqueous Solution of the Sodium salt (e.g., eluted to a 0° C. ice bath for a minimum of 2 minutes in order to from a rhenium generator) or as an aqueous Solution of the stop the reaction and is stable at 0°C. to 25 C. for several tetrabutylammonium ion pair. Either way, perrhenate is hours. incubated with a Solution that includes a reducing agent and The following examples are provided for purposes of a complexing agent. The reaction mixture is incubated at a illustration, not limitation. temperature ranging from about 25 C. to about 50 C., EXAMPLES preferably at about 25° C. to 37° C., for a minimum of 10 RPHPLC conditions for Examples 1 and 2 are as follows: minutes. Incubation for 10 minutes generally gives Sufficient 15 Hewlett Packard 1090 HPLC with diode array detection; yields of the desired rhenium exchange complex (e.g., analytical reverse phase C18 column eluted at 1.0 mL/min rhenium-citrate), while minimizing the formation of with a mixture of Solvents A and B, where A=0.1% trifluo insoluble rhenium dioxide. roacetic acid (TFA) in water, B=90% acetonitrile (MeCN) An opioid receptor ligand of the invention, which may containing 0.1% TFA. RPHPLC conditions for Examples contain thioacetal or hemithioacetal Sulfur-protecting groups 3–5 and 8-20 are as follows: Hewlett Packard 1090 HPLC in the radionuclide binding arms, may then be dissolved in with diode array detection; analytical reverse phase C18 an organic Solvent which is both effective in dissolving the column eluted at 1.0 mL/min with a mixture of Solvents. A ligand and is Suitable for the exchange reaction that follows. and B, where A=5% MeCN, 0.1% TFA, B=95% MeCN, Suitable solvents should be non-toxic in mammals and inert 0.1% TFA. Each gradient is preceded by 4 minutes under toward the reactants in the reaction mixture. Organic Sol initial solvent conditions and followed by a wash period at vents which may be used include, without limitation, 25 higher %B (usually 100%B) and re-equilibration to starting acetonitrile, ethyl acetate, and methyl ethyl ketone. When conditions. the radiolabeled compound is to be injected into humans, Unless otherwise Stated, chemicals and reagents are however, Suitable organic Solvents include but are not lim obtained from chemical Supply houses known in the art, for ited to alcohols Such as ethanol, butanol, tert-butyl alcohol, example, Aldrich, Milwaukee, Wis. As used herein, ret time and propanol and polar aprotic Solvents Such as DMSO and represents retention time; con HCl represents concentrated dimethylformamide (DMF). The choice of solvent may vary hydrochloric acid; h represents hours, NID represents nal according to the particular opioid receptor ligand of the trindole, TLC represents thin layer chromatography; PSI invention included in the kit. For example, when the ligand represents pounds per Square inch; EtOAC represents ethyl includes a tetrafluorophenyl ester group, ethanol may react acetate; min represents minutes; with the ester in a transesterification reaction, producing 35 ethyl ester by-products which are undesirably lipophilic. A /- COMe preferred organic Solvent is isopropyl alcohol. N The Solution which includes the opioid receptor ligand of V the invention in the organic Solvent may be combined with NH2 the rhenium exchange complex Solution prepared above, to NPNCH form the corresponding rhenium-chelated opioid receptor 40 Example 1 ligand. Typically, the reaction is advantageously conducted at a pH of from about 1.5 to about 5.0, preferably from about N-phenyl-N-carboxymethyl hydrazine (NPNCH), 1.7 to about 2.0. At these acidic pH conditions, the formation CAS Registry No. 4408-70-2 of insoluble ReO will be minimized; and as explained Nitrosation of N-phenyl glycine (NPG). (Procedure from above, hemithioacetal and thioacetal Sulfur-protecting 45 Organic Synthesis II pg. 460) groups (if present) will be displaced by a metal-assisted acid NPG (Aldrich; 1.51 g, 10 mmol) is dissolved in 1.45 mL cleavage during the rhenium labeling eXchange reaction to con HCl, then diluted with 2 mL H2O and cooled on ice. form the corresponding rhenium chelate compound. Also, NaNO (0.70 g, 10 mmol) is dissolved in 2 mL HO and hydrolysis of ester groups on the opioid receptor ligand added over 10 minutes to the cooled NPG solution. The compound of the invention is minimized under acidic con 50 precipitate which forms is filtered. Product is analyzed by ditions when compared to basic conditions. If adjustment of isocratic RPHPLC with 30%B: NPG ret time=3.7 min, the pH of the reaction mixture is necessary, Suitable acids 2=240, 290 nm, product ret time=6.2 min, 2=270 nm. may be added in amounts Sufficient to displace the Sulfur Reduction of N-nitroso, N-phenylglycine protective groups in the presence of the metal radionuclide The precipitate as prepared above. is Suspended in 3 mL (i.e., in amounts Sufficient to adjust the reaction mixture to HOAc and added in 30 fractions to 2.0 g Zn dust suspended the above-described pH values range). Suitable acids 55 in 3 mL HO. Analysis of the reaction mixture by RPHPLC include, but are not limited to, phosphoric acid, Sulfuric acid, (30% B) reveals disappearance of the starting material, and nitric acid, glacial acetic acid, hydrochloric acid, and com appearance of two products, one identical by retention time binations thereof. Also included are Solutions comprising and UV spectrum with NPG, the other a new compound with Such acids and buffers (e.g., acetate and phosphate buffers). ret time=2.8 min and 2–274 nm. The reaction mixture is The amount of opioid receptor ligand of the invention 60 filtered and the Solids washed with water. The filtrate is reacted with the Re-citrate intermediate may vary according evaporated to dryneSS under reduced pressure, then applied to the reaction Volume, which in turn varies according to the to a C18 flash column equilibrated in 10% MeCN, 0.1% Volume in which perrhenate was added in an earlier Step TFA, and eluted with a stepwise gradient of 10, 20, 30, and (e.g., perrhenate may be added as an eluent from the 50% MeCN, all containing 0.1% TFA. Product containing generator or may first be concentrated). In one embodiment 65 fractions are identified by HPLC, combined and evaporated of the invention, the concentration of chelating compound in under reduced pressure. Product is analyzed by H NMR in the reaction mixture (in which the chelate is formed) ranges dDMF: 6 (ppm)=7.25 (t, 2H), 7.1 (d. 2H), 6.8 (t, 1H), 4.4 US 6,359,111 B1 39 40 (S, 2H), and by low resolution electron impact mass spec Example 3 troscopy (MS): molecular ion=166.1. 1'-N-carboxymethyl-3-O-benzyl naltrindole (BZl CMNID) 3-O-benzyl natrindole (BZl-NID) was prepared according to “Synthesis of N1'-(''Climethyl)naltrindole (''C MeNTI): "A Radioligand for Positron Emission Tomo graphic Studies of Delta Opioid Receptors,” J. Labeled Compa. Radiopharm. 36:137–45 (1995). Bzl-NID (300.6 mg, 0.6 mmol) is taken up in 1 mL dry DMF and added to a stirred suspension of NaH (60% dispersion in mineral oil; 126.9 mg, ~3 mmol) in 3 mL dry DMF. The suspension is allowed to stir 20 min, then methyl bromoacetate (Aldrich, 71 mL, 0.75 mmol) is added dropwise. The reaction is followed by RPHPLC using a 14 min gradient from 20 to 70% B. Immediately after completion of addition of the CO2H 15 methyl bromoacetate, RPHPLC reveals 2 new peaks with CMNID retention times of 17.5 and 19.2 min, respectively. After 30 to 60 min only the 17.5 min peak remains. The reaction is Example 2 quenched by the addition of 2 mL of a 4:1 MeOH:AcOH N1'-carboxymethyl naltrindole. (CMNID) mixture. The quenched reaction mixture is evaporated under reduced pressure, taken up in 5 mL MeOH, which is then The following procedure was adapted from “ö Opioid saturated with water, and the mixture applied to a C18 flash Antagonist Activity and Binding Studies of Regioisomeric column equilibrated in 50% MeOH. The column is eluted Isothiocyanate Derivatives of Naltrindole: Evidence for 8 with a step gradient of 50, 60, 70,80, and 100% MeOH, with Receptor Subtypes”, J. Med. Chem. 35:4086–91 (1992). the product eluting at the end of the 70% step. Product NPNCH (51 mg, 0.31 mmol) and naltrexone hydrochlo 25 containing fractions are evaporated to dryneSS under reduced ride (Sigma, 100.3 g, 0.26 mmol) are stirred overnight in 3 preSSure. mL methanol (MeOH). The resulting mixture is evaporated "H NMR (CDOD): comparison with "H NMR of Bzl to dryneSS under reduced pressure, then taken up in 5 mL NID shows the appearance of 2 new asymmetric doublets, con HCl and placed on an oil bath heated to 150° C. under 1H each, at 8=4.8 and 4.92 ppm, respectively, attributable to a water cooled condenser. After 1 hour the heat is turned off the methylene protons of the carboxymethyl moiety. ESI and the mixture left overnight. RPHPLC reveals a new peak MS: expected M+H (CHNO+H)=563. with ret time 7.1 min (30% B) and a UV spectrum indis That the product of Example 3 was the free acid rather tinguishable from authentic naltrindole (provided by Dr. than the methyl ester is Surprising. The late eluting peak that Wendell Nelson, Univ. of Washington). appears early in the reaction and then disappears has been The reaction mixture is filtered and the Solid applied to a shown to be the expected ester. While the conversion of C18 flash column equilibrated in 25% MeCN containing 35 methyl ester to acid under the reaction conditions is not 0.1% TFA. The column is eluted with a step gradient of 25, understood, the Spectral data are clear and the product 30, and 35% MeCN, all containing 0.1% TFA. Fractions performs as expected in Subsequent reactions. containing the putative product are identified by RPHPLC, pooled, and evaporated to dryneSS under reduced pressure. The product is analyzed by "H NMR in CDOD and by low 40 resolution FAB MS. Comparison of the H NMR spectrum with that of naltrindole in CDCls, naltrindole in CDOD, and naltrexone hydrochloride in CDOD reveals that the product spectrum is attributable to the protonated form (i.e., TFA salt) of the desired product. Signals at Ö=5.20 and 5.05 ppm (distorted doublets, 1H each), not found in any of the 45 other spectra, are attributable to the prochiral methylene protons of the carboxymethyl group. FAB MS gives the expected M-H peak at 473.

50

55 Example 4 Methyl-N-(1'-N-(3-O-benzyl)-naltrindolyl)-6- acetamidocaproate (BZl-NID-ACM) 60 Bzl-CMNID (302.2 mg, 0.538 mmol) and methyl-6- a mino capro ate hydrochloride (prepared from 6-aminocaproic acid (Aldrich) in methanolic HCl) are dis solved in 6 mL dry DMF, followed by BOP (Benzotriazol 1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophos 65 phate; Chem-Impex Intl., Wood Dale Ill., 290.4 mg., 0.65 mmol) and ethyldiisopropyl amine (Aldrich; 0.935 mL, 5.5 mmol). The reaction is followed by PRHPLC, using a US 6,359,111 B1 41 42 gradient form 40 to 100% B in 18 min, and by TLC using Silica eluted with 1:1 ethyl acetate (EtOAc):hexane (hex) -continued containing 2% triethylamine. Analysis at early timepoints shows the disappearance of Bzl-CMNID and appearance of C# 8 °C (ppm) & H (ppm) a new peak (RPHPLC ret time=10.2 min), which disappears 5 2. ' 17462 or 169.02 5.9 with the appearance of a product peak (RPHPLC ret time= 3." 39.28 3.1 4" 29.17 1.0-1.4 13.9 min). When starting material and intermediate peaks 5" 26.22 1.0-1.4 are consumed, the reaction mixture is evaporated to dryneSS 6" 24.61 1.0-1.4 under reduced preSSure and applied, as a concentrated Solu 7. 34.05 2.05 tion in CHCl, to a Silica flash column equilibrated in 3:1 8" EtOAc:hex containing 2% triethylamine. The column is 9" 51.69 3.55 eluted with a stepwise gradient of 150 mL 3:1, 100 mL 2:1, and 200 mL 1:1 EtOc:hex all containing 2% triethylamine. FAB MS gives the expected M+H ion (CHNO+H) Product containing fractions are identified by TLC, pooled, 690. and evaporated under reduced preSSure. Reaction yield is 15

88%. 'C and H NMR spectra assignments (tentative) are shown below:

19

25

BZ-NIDAC

Example 5 35 N-(1'-N-(3-O-benzyl)-naltrindolyl)-6- acetamidocaproic acid (Bzl-NID-AC) Bzl-NIDACM (315.5 mg, 0.46 mmol) is dissolved in 3 8 °C (ppm) & H (ppm) 40 mL MeOH. 1.0 mL 1.0 N NaOH is added. After -20 1. 117.43 6.60 minutes, 1 mL MeOH is added to aid in solubility, and the 2 119.61 6.7O milky white solution is left stirring overnight. 1 NHCl is 3 unassigned 4 unassigned added until the Solution is acidic, then the Solution is 5 8437 5.70 evaporated to a white Solid under reduced pressure. The 6 unassigned 45 residue is taken up in MeOH and mechanically Separated 7 unassigned from precipitated NaCl. Evaporation of the MeOH results in 8 24.37 2.8, 3.15 9 62.42 3.40 a residue with exceSS weight, indicating that Some Salt 1O 29.17 2.6, 2.9 remains in the product. RPHPLC using an 18 min gradient 11 unassigned from 40 to 100% B shows a single new peak ret time=11.1 12 unassigned 50 min. Hand 'C NMR spectra (CDOD) are similar to those 13 48.36 14 72.84 of Bzl-NIDACM, but with peaks shifted due to protonation 15 32.O2 1.8, 2.4 of the basic tertiary amine by the carboxyl group, and 16 43.84 2.3, 2.75 missing the Signals assigned to the methyl ester. ESI MS 17 59.81 2.45 gives the expected M+H ion (CHNO+H) at 676.3. 18 9.58 O.9 55 19 3.93f4.24 0.2fO.6 2O 3.93f4.24 0.2fO.6 COtBu 2 unassigned 3 unassigned l / \-COIBu 4 119.83 7.5 N N 5 120.62f124.04 7.1 60 6" 120.62f124.04 7.1 7. 110.18 7.3 D 1" 48.36 4.8, 4.9 ON 2. 1428O r N /N-cob 3".7" 127.32 or 128.95 7.3 4",6" 127.32 or 128.95 7.3 COtBu 5" 128.45 7.3 65 NBtBDOTA 1". 72.18 5.0, 5.1 US 6,359,111 B1 43 Example 6

2-(4-nitrobenzyl)-1,4,7,10-tetraazadodecane-N1, N4N7.N10-tetra-t-butylacetate (NBtBDOTA) To 2-(4-nitrobenzyl)-1,4,7,10-tetraazadodecane, (208.4 mg, 0.5 mmol) prepared as in U.S. Pat. No. 5,541,287, in 6 mL dry DMF and KCO (691 mg, 5 mmol) is added t-butyl bromoacetate (Aldrich, 488 mg, 370 mL, 2.5 mmol). The reaction mixture is heated at 50° C. under nitrogen and with a condenser. After 4 hours RPHPLC shows only two peaks in the ratio 93:7. RPHPLC system: 40% A/60% B to 100%B in 15 min; to 50% B/50% C in 5 min; to 20% B/80% C in 15 5 min, where A=0.1% TFA; B=60% MeCN/40% HO with 0.1% TFA; C=100% MeOH; elution rate=1.0 mL/min. The reaction is stopped, filtered, and the filtrate evaporated to BZ-NIDAC-BABDOTA dryneSS under reduced pressure. The crude product is applied to a Silica flash column equilibrated in 6% MeOH in Example 8 CHCl, then eluted with 1 L equilibration buffer followed by 0.5 L 7.5% MeOH in CHC1. Product containing frac (Bzl-NIDAC-BAB DOTA) ABtBDOTA (157.7 mg, 0.215 mmol), Bzl-NIDAC (96.7 tions are identified by TLC and HPLC, pooled, and evapo 25 mg, 0.143 mmol), and BOP (97.5 mg, 215 mmol) are rated to dryneSS under reduced preSSure. Integration of the combined in an oven dried reaction vessel, then diluted with 2.5 mL dry DMF followed by 250 mL ethyl-diisopropyl "H NMR spectrum (CDC1) shows the correct ratio of amine and Stirred magnetically. The reaction is followed by aromatic protons to t-butyl ester protons (4:36). High reso RPHPLC using a gradient from 40 to 100% B in 18 minutes: lution mass spectroScopy gives the expected value, 764.4825 the BZl-NIDAC peak is quickly consumed, resulting in a new (intermediate) peak with a ret time of ~14 min; the for the M+H ion (CHNO+H; calc. 764,4810). intermediate peak and the tEABDOTA peak disappear more Slowly, and a new peak appears with a ret time of ~1.6 min COtBu and a new UV spectrum. After stirring overnight, RPHPLC indicates that the reaction is complete with a yield>90%. l W V -COBu 35 The reaction mixture is evaporated under reduced preSSure N to a yellow oil which is taken up in MeCN/HO/0.1% TFA and applied to a C18 flash column equilibrated in 50% MeCN:HO containing 0.1% TFA. The column is eluted HN N N with a step gradient consisting of 100 mL portions of 50, 60, r N / N-CO.Bu 70, 80, and 90% MeCN, each containing 0.1%TFA. Product 40 containing fractions, identified by TLC, are pooled, neutral COtBu ized with Saturated NaHCO, evaporated to dryness under ABtBDOTA reduced pressure, and transferred to a tared vial. The final weight of product exceeds the Starting materials, Suggesting that some salt accompanies the product. The "H NMR and 45 C (CDC1) spectra are quite complex, but include features Example 7 associated with each of the reactants. ESI MS gives the expected M+H ion (CHN.O+H) of 1391.8. 2-(4-aminobenzyl)-1,4,7,10-tetraazadodecane-N1, N4,N7.N10-tetra-t-butylacetate (ABtBDOTA) 50 A solution of NBtBDOTA (280 mg, 0.366 mg) in 14 ml, MeOH is added to a Parrbottle containing 56 mg 10% Pd/C. D N. N N The bottle is charged with 50 psi Hi in a Parr hydrogenation COtBu apparatus. After 3 h TLC (Silica eluted with 7.5% MeOH in 55 CHCl) shows that the reaction is complete. The mixture is filtered. through a glass fiber membrane and the filtrate evaporated to dryneSS under reduced pressure. The residue is taken up in CHCl2, and evaporated to dryneSS again to 60 O remove residual MeOH. Reaction yield is quantitative. The "H NMR spectrum shows the expected shift in the aromatic H proton Signals expected for the nitro to amine conversion. r High resolution mass spectroScopy gives the expected value, 65 NIDAC-BABDOTA 734.5037 for the M+H ion (CH, NOs+H; calc. 734.5068). US 6,359,111 B1 45 46 Example 9 fivefold molar excess of the metal solution. The solution pH is checked and adjusted to 5 if necessary, then the Solution (NIDACBABDOTA) is heated to 80 C. in a closed tube for 30 min. The reaction The Bzl-NIDACBABDOTA of Example 8 is taken up in mixture is analyzed by RPHPLC using a gradient from 20 to 4 mL MeOH and placed in a flask containing 21.7 mg of 5 40% B in 18 min. NIDDOTA elutes at 13.7 min. The metal 10% Palladium on charcoal. The suspension is stirred under loaded chelates elute as two peaks, ret time 11.3 and 12.7 hydrogen gas overnight, then 23.7 mg additional Pd/C is min for In-NIDDOTA, and 10.8 and 12.9 min for added and the reaction continues over a Second night. The Y-NIDDOTA. The UV spectra of the metal loaded and metal reaction mixture is filtered through glass fiber filter paper, free compounds are nearly identical. The metal loaded evaporated to dryneSS under reduced pressure, taken up in compounds can be separated from each other and from 40% MeCN with 0.1% TFA and applied to a C18 flash excess metal by preparative RPHPLC using conditions column equilibrated in 4% MeCN/0.1% TFA. Column is derived from the analytical Separation conditions described eluted with a step gradient consisting of 40, 50, 55, and 60% above. Low resolution ESI mass spectra of the two Y MeCN, each containing 0.1% TFA. Product elutes as a 1. s loaded peaks are identical, with the M+H ions of 1163, slightly yellow band. which is the M+H ion for the chelate complex in which one of the carboxyls is protonated rather than bound to the CO2H CO2H yttrium (YC, HNO+H). The existence of two forms of M"NIDDOTA complex is explained by diastereomers that result from chelation of the metal on either side of the

macrocylic ring relative to the benzyl moiety, which is attached at a chiral center. N N

HOC-1 N-CO2H 25

~s.NH NIDDOTA1 35 Example 10 (NIDDOTA1) 40 The NIDACBABDOTA of Example 10 is taken up in 1 mL TFA. The reaction is followed by RPHPLC using a 30 min gradient from 0 to 100% B. After 1 hour the reaction shows consumption of Starting material and 4 new peaks. Example 12 After overnight reaction the reaction mixture shows a single 45 peak with ret time 18.5 min, (vs. 28 min for starting material) and an unchanged UV spectrum. The reaction mixture is evaporated to dryneSS under reduced pressure N-(1'-N-naltrindolyl)-6-acetamidocaproic acid then applied in equilibration solvent to a C18 flash column (NID-AC) equilibrated in 0.2% HC1. The column is eluted with a step 50 gradient consisting of 200 mL 0.2% HCl, 100 mL 10%, 100 mL 20%, 200 mL 30% MeCN, each containing 0.2% HC1. The product elutes in a Single yellow band. The product Bzl-NIDAC (Example 5; 92 mg) is added to a stirred flask fractions are identified by HPLC, pooled and evaporated containing 11.4 mg 10% Pd/C in 5 mL MeOH Hydrogen is under reduced pressure. The residue is taken up in Sterile 55 water, transferred to a tared Vial, and evaporated under introduced from a balloon reservoir and the Suspension left reduced pressure. Dry wit=42.4 mg. ESIMS of the desired stirring overnight. The reaction is monitored by RPHPLC product gives the expected M-H ion (C7H7NO+H) of (gradient: 20 to 70% B in 14 min). After overnight reaction 1077.4. RPHPLC shows a single peak eluting earlier than starting material (12 min vs. 19 min). The reaction mixture is filtered Example 11 60 through a glass fiber membrane, evaporated under reduced pressure, taken up in MeCN and re-evaporated. The H NMR Y* or In NIDDOTA1 spectrum (CDOD) indicates the desired product (benzyl An In" solution (~10 mM) is prepared by dissolving Signals absent), but also Suggests that the material is impure. InSO in 2 N. NH4OAc, pH 5; a Y" solution (-10 mM) is 65 Re-analysis by RPHPLC indicates that some degradation prepared by dissolving Y(NO) in 2 N. NH4OAc, pH 5. An has occurred during Workup, yielding two contaminants aliquot of NIDDOTA solution (6 mM) is mixed with a comprising ~20% of the total. US 6,359,111 B1 47 48

HO

CO2H

~" NIDF

Example 13 DMF. After 30 min tetramethylrhodamine cadaverine (Rh) (Molecular Probes, Eugene Oreg., cat. #A1318, 10 mg, (NID-F1) 25 0.019 mmol) is dissolved in 1 mL dry DMF and the solution NIDAC (used as prepared in Example 12; 15 mg, 0.025 added to the NIDAC/BOP reaction mixture. The reaction is mmol) and BOP (12.2 mg, 0.027 mmol) and ethyldiisopro followed by RPHPLC using a gradient from 20 to 70% B in pylamine (43.5 mL, 0.25 mmol) are combined in a pre-dried 14 minutes, monitored at 540 nm (background set at 450 vessel in 0.5 mL dry DMF. After 20 min fluorescein glyci nm). The rhodamine reagent is a mixture of isomers with the namide (Molecular Probes, Eugene Oreg., #A1363; 10 mg, carboxamide either as drawn or at the position indicated by 0.025mmol) is added. The reaction is monitored by the arrow (Molecular Probes catalog), which appear at 9 and RPHPLC using a gradient from 30 to 60%B in 18 min and 11 min. New peaks containing the rhodamine chromophore monitoring at 450 nm. The major product is purified by appear very slowly at 15.5 and 16 min. The reaction is preparative RPHPLC using a preparative column with the 35 allowed to proceed 11 dayS. Same gradient as for analytical monitoring, but run at 3 The main product peaks are purified by preparative mL/min over 1 hr. Product fraction is neutralized with RPHPLC, neutralized with minimum saturated NaHCO, minimum saturated NaHCO then rotovaped dry. evaporated to dryneSS under reduced pressure, taken up in

MeN O NMe2 2 O CO2H

NDRh

Example 14 MeOH for transfer to tared vessels and weighed. Yield of isomer A is 3 mg, isomer B is 4 mg. RPHPLC analysis of the (NID-Rh) isolated products indicates that they are pure with respect to NIDAC as isolated in Example 12 (13.6 mg, 0.023 65 UV/vis.-active materials. FAB MS of the isolated product mmol), BOP (9.6 mg, 0.022 mmol) and ethyldiiso fraction gives the expected molecular ion (CH7N, O, proplyamine (33 mL, 0.19 mmol) are combined in 2 mL dry 1082). US 6,359,111 B1 49 SO pylamine (0.75 mL, 4.4 mmol). The reaction is monitored by RPHPLC using a 14 min gradient from 20 to 70% B. Reaction is allowed to continue overnight, after which NID7AC is consumed and two new peaks are seen in a ratio of ~6:1. The reaction mixture is quenched by the addition of 1 mL of water, then stirred 10 min. The mixture is then evaporated under reduced pressure before being taken up in EtOAc/HO. The aqueous phase is made basic by addition of dilute NaOH and the EtOAc phase washed 2x with dilute acqueous NaOH. The EtOAc phase is then extracted with 0.5 M citric acid and the acid Solution washed 3x with EtOAc. The citric acid Solution is made basic by addition of Satu rated NaHCO and finally NaOH while being extracted with CO2H EtOAc. The EtOAc solution was evaporated to dryness NID7A under reduced preSSure. The residue was applied in equili bration solvent to a Silica flash column equilibrated in 1% 15 MeOH, 2% EtN/CHCl and eluted with 500 mL of equili Example 15 bration solvent. Product containing fractions were identified by UV absorbance and shown by TLC to be a single spot. 7"carboxy naltrindole (NID7A) Fractions were pooled, washed with water, then evaporated to dryness under reduced pressure. The "H NMR spectrum Method adapted from Portoghese et al., “7'-Substituted shows the expected changes from the spectrum of NID7A: Amino Acid Conjugates of Naltrindole. Hydrophilic Groups multi-proton multiplets at Ö=1.4, 1.7 and 2.3 ppm and a 3 as Determinants of Selective Antagonism of d- Opioid proton singlet at 3.7 ppm. The C NMR spectrum is Receptor-Mediated Antinociception in Mice,” J. Med. assignable by referring to the spectrum for BZl-NIDACM Chem. 38:402-7, 1995. (Example 4). ESIMS gives the expected nominal mass for Naltrexone hydrochloride (Sigma; 1.0508 g, 2.78 mmol) the M+H ion (CHNO+H)=586. and 2-hydrazinobenzoic acid (Aldrich; 0.6312 g, 3.33 25 mmol) are mixed in a flask. Acetic acid (25 mL) is added and the reaction flask is capped with a water cooled condenser and heated to reflux in an oil bath. The reaction is followed by RPHPLC and stopped after refluxing overnight. The major new peak elutes at 13.6 min in a 14 min gradient from 20 to 70%B and shows a 2 shifted from that of naltrin dole (280 nm) to about 312 nm. The reaction mixture is evaporated to dryness under reduced pressure, then taken up in 20% MeCN with 0.1% TFA and applied to a C18 flash column equilibrated in the Same Solvent. The column is 35 eluted with a step gradient consisting of 250 mL each of 20, 30, and 40% MeCN each containing 0.1% TFA. The main reaction product precipitates as a white powder from the NID7AC main product containing fractions, and is filtered and trans ferred to a tared vial. Isolation of residual product in the 40 fraction filtrates yields little additional product. Total reac Example 17 (NIDAC) tion yield is 33%. The H NMR spectrum (CDOD) shows NID7ACM produced in Example 16 is taken up in 8 mL Signals characteristic of the desired product: C5 methine, 5.7 MeOH (dissolution difficult) and treated with 1 mL 1 N ppm, doublets at 7.4 and 7.8 for C4' and C6', and a triplet at NaOH. After stirring overnight, RPHPLC shows the reaction 6.9 ppm for C5". Low resolution ESIMS gives the expected 45 to be about 70% complete. Additional 1 N. NaOH (0.5 mL) M+H ion (CHNO+H) at 459. is added and the mixture Stirred overnight again. The Solu tion is then neutralized with minimum 1 M HCl, then evaporated under reduced pressure. Evaporation is stopped when a white precipitate coats the flask and a Small aqueous Volume remains. The liquid is manually removed and the 50 product remains. "H NMR (CDOD) indicates that the product has the desired structure. ESIMS gives the expected nominal mass for the M+H ion (CH7NO+H)=572.

55

NID7ACM 60 Example 16 (NID7ACM) NTD7A (199.6 mg, 0.44 mmol), BOP (229.3 mg, 0.48 65 NID7ACNHS mmol), and ethyl-6-aminocaproate (101.7 mg, 0.55 mmol) are mixed in 6.5 mL dry DMF, followed by ethyl diisopro US 6,359,111 B1 S1 52 Example 18 4% triethylamine. Product containing fractions are identified by TLC and RPHPLC, and are pooled and evaporated under (NID7AC-NHS) reduced pressure. Residual triethylamine is removed by NID7AC (200 mg, 0.35 mmol), N-hydroxysuccinimide repeatedly adding MeCN to the residue and re-evaporating (NHS; 60 mg, 0.52 mmol) and dicyclohexylcarbodiimide 5 until triethylamine signals are no longer visible in the "H (DCC; 108 mg, 0.52 mmol) are mixed in 3 mL dry DMF. NMR spectrum.

The reaction is followed by RPHPLC using a 14 min Example 20 gradient from 20 to 70% B. After stirring overnight the reaction mixture is filtered, the filtrate evaporated to dryness (NID7-biotin) and applied to a Silica column equilibrated in CH2Cl2 NID7'-NH. (123 mg, 0.20 mmol) is stirred with biotina containing 2% triethylamine, then eluted with a step gradi 35 midocaproate N-hydroxySuccinimide ester (Sigma; 100 mg, ent consisting of 1, 2, 5, 10% isopropanol in CHCl, each 0.22 mmol) in 4 mL dry DMF. The reaction is monitored by containing 2% triethylamine. Product containing fractions RPHPLC using a gradient from 40 to 100% B in 18 min. A are identified by TLC and RPHPLC, and are evaporated to new peak appears eluting much later than NID7'-NH2 but dryneSS under reduced pressure. with the same UV absorbance. The product is purified by 40 flash chromatography on Silica using MeOH in CHCl containing 4% triethylamine. Example 21 45 NID-streptavidin NID7AC-NHS (5 mg, 7.5 mmol) is dissolved in 2.0 mL dry DMSO. A solution of streptavidin (SA) is prepared at 10 mg/mL in 50 mmol sodium borate buffer, pH 8.0. 50 To 2.0 mL of the SA solution (0.38 mmol SA) is added 0.2 mL of the NID7AC-NHS Solution. The reaction is left on a Shaker for 4 hours at room temperature. The conjugate is then concentrated in a centrifugal concentration device and separated from residual NID components by elution through Example 19 55 a desalting column. The conjugate is analyzed by UV spectroscopy and by ESI MS. (NID7-NH) Example 22 NID7AC-NHS (200 mg, 0.3 mmol) is dissolved in 4 mL Delta-Opioid Receptor Binding of NIDDOTA1 and dry DMF containing ethylene diamine (0.40 mL, 6 mmol). 60 The reaction is stirred at room temperature and monitored by NID Rh RPHPLC using a gradient from 20 to 70%B in 14 min. After The assay determines the concentration of test compound Stirring overnight the reaction is complete. It is evaporated capable of inhibiting the specific binding of H NID to the to dryneSS under reduced pressure and purified on a Silica plasma membrane of an intact cell. To do So, 1 to 4 Serial flash column equilibrated in 1% MeOH in CHCl contain 65 dilutions of test compounds and naltrindole (NID, Sigma) ing 4% triethylamine, then eluted with a step gradient are prepared to generate Solutions with concentrations rang consisting of 2, 3, 5, 10% MeOH in CHCl, each containing ing from 10 to 10' M in 50 mM Tris buffer, pH 7.5. H US 6,359,111 B1 S3 S4 NID (American Radiolabeled Chemicals, St. Louis, Mo., as tertiary butyl esters (compound 5) is reacted with the keto specific activity 49 dpm/10" mol) is prepared in Tris at a analog, compound 1. The reaction mixture is heated under concentration of 10 M. Cultured cells of the reflux in toluene using molecular Sieves to absorb water or neruablastoma/glioblastoma hybrid cell line NG108-15 Dean's Stork apparatus for azeotropic distillation of water (American Type Culture Collection) are briefly trypsinized, formed in the reaction mixture. The progreSS of the reaction washed with media, and resuspended at 6x10 cells/mL. 20 is monitored by TLC. After completion of the reaction, tuL of test compound or NID solution and 20 uL of H NID Solvent from the reaction mixture is removed under reduced are added to each of 96 wells of a Multiscreen assay system pressure and dried. The crude product is purified by florisil (Millipore) MAFB. 160 lull of cell suspension in Dulbecco's Silica gel column chromatography using ethyl acetate/ MEM--HAT with 10%. Fetal Bovine Serum are added. The ligroine as eluting Solvents. Fractions containing the product reactions are allowed to equilibrate 2 hr at 37 C., vacuum are combined and Solvent removed under reduced pressure filtered, washed 2x with 200 till ice cold Tris buffer and air and dried to yield. Compound 6 is provided in moderate dried. The filters are punched out of the plate into scintil yield. lation Vials and mixed on a shaker overnight before count Compound 6 is then catalytically reduced using 10% Ing. 15 Data is analyzed by the Prism Software package Palladium over activated carbon in ethanol Solvent. The (GraphPad Software, Inc., San Diego, Calif.) using a non reduction of imine to an amine is carried out in a Parr linear curve fit routine for one site competitive binding. The hydrogenation apparatus at 50-60 PSI (pounds per Square Software generates plots of bound counts VS. log molar inch). The reaction mixture is filtered. Solvent from the concentration of inhibitor and determines values for Log filtrate is removed under reduced preSSure and the residue is ECso (and associated uncertainty) that best fit the data. The dried under high vacuum to provide the crude product 7. values in Table 1 show that NID-DOTA is only about 3-fold The crude product 7 is used in next reaction without lower in affinity than NID and NID-Rh is only about 9-fold further purification. The tertiary butyl esters of the carboxy lower in affinity than NID. lic acid functional group protecting groups are removed in 50% trifluoroacetic acid-methylene chloride reagent. The 25 TABLE 1. reaction mixture is stirred at room temperature overnight. Solvent from the reaction mixture is removed under reduced Test # Compound Log EC (tS.E.) ECs (M) Test/Control pressure and dried to yield compound 8 (see FIG. 4B) in good yield. 1. ND -8.4 + 0.4 4.2 x 10 1. ND-DOTA -7.9 O2 1.2 x 10 2.8 2 ND -8.8 + 0.1 1.8 x 10 Example 26 2 ND-Rh -7.8 O1 1.6 x 10 8.9 As described in FIG. 5, compound 1 is reacted with compound 9. The hydrazide derivative compound 9 (in Example 23 molar excess) is stirred with compound 1 dissolved in 35 ethanol Solvent. The progreSS of the reaction is monitored by As described in FIG. 3A, one equivalent of keto com TLC. After completion of the reaction, solvent is removed pound 1 is reacted with 1.2-2.0 equivalents of hydroxy under reduced pressure and the residue is dried under high lamine hydrochloride in ethanol. The reaction mixture is Vacuum to give crude compound 10 in good yield. The crude heated under reflux to form the oXime, compound 2 in good product is purified by florisil Silica gel column chromatog yield. The oXime, compound 2 is reacted with compound 3 raphy using methanol/ligroin or methanol/EtOAC (EtOAc in 0.2 molar excess in DMF solvent using 5.0 molar excess 40 represents ethyl acetate) as elution Solvents. Fractions con of triethylamine. The reaction mixture is stirred at room taining the product are combined and Solvent removed under temperature (r.t.). The progress of the reaction is monitored reduced pressure, with the residue dried under high Vacuum by TLC. The crude product is purified by silica gel chro to yield compound 10. matography using ethylacetate initially and finally with 45 The hydrazone derivative, compound 10 is reduced using methanol/EtOAC as eluting Solvents. Fractions containing 10% Palladium on activated carbon. Compound 10 is taken the product are combined and the Solvent removed under into a hydrogenation bottle, ethanol is added followed by reduced pressure and dried under high Vacuum to yield 10% Pd/C. The catalytic reduction is carried out using a Parr compound 4 in good yield. hydrogenation apparatus using hydrogen at 50-60 PSI for 24 Example 24 50 hours. The reaction mixture is filtered and the solvent In analogy with Example 24 and as illustrated in FIG. 3B, removed under reduced pressure with the residue being Oxime, compound 2 is reacted with compound 18 in its dried under high vacuum to yield compound 11 in good chloride, mesylate or tosylate form. In FIG. 3B, R' repre yield. sents any of chloride, -O-SO-CH or -O-SO Example 27 Ar-CH. 0.2 excess equivalents of compound 18 are 55 reacted in anhydrous DMF solvent using 5.0 equivalents of As described in FIGS. 6A and 6B, compound 12 in DMF triethylamine at room temperature. The progreSS of the is reacted with 1.2 equivalents of Succinic anhydride and 5.0 reaction is monitored by TLC. After completion of the equivalents of triethylamine. The hemisuccinic acid reaction, the crude product is purified by Silica gel column derivative, compound 13 is purified by Silica gel column chromatography using ethyl acetate and MeOH/CH2Cl as 60 chromatography. The hemisuccinic acid is then esterified eluting Solvents. Fractions containing the product are pooled with N-hydroxysuccinimide in anhydrous THF using dicy and the Solvent removed under reduced pressure and dried to clohexylcarbodiimide as a coupling reagent. The yield compound 19 as an oxime ether in good yield. hemisuccinyl-NHS ester, compound 14 is then reacted with t-butyl ester analog of p-aminobenzyl DOTA, compound 5. Example 25 65 The reaction mixture is stirred in anhydrous DMF and 5.0 As described in FIGS. 4A and 4B, excess of equivalents of anhydrous triethylamine at room temperature. p-aminobenzyl DOTA having its carboxylic acids protected The progress of the reaction is monitored by TLC. Solvent US 6,359,111 B1 SS S6 from the reaction mixture is removed under reduced pres Sure and dried under high vacuum. The crude product is purified on a Silica gel column chromatography using 15% CHOH/CH2Cl as elution solvent. The fractions containing the product are pooled and the Solvent removed under reduced pressure, with the residue being dried under high Vacuum to yield compound 15 in good yield. The compound 15 is then stirred in 50% TFA/CHCl solvent overnight at room temperature. Solvent from the reaction mixture is removed and the crude product is recrystallized from ethanol to give compound 16 in moderate yield. Example 28 15 As described in FIG. 7A, compound 12 is reacted with where G is selected from compound 3. The reaction mixture is dissolved in anhydrous DMF solvent. To the magnetically stirred reaction mixture, 5.0 equivalents of anhydrous tri-ethylamine is added and continued Stirring at room temperature. The progreSS of the reaction is monitored by TLC. After completion of the ? As N ?o reaction, Solvent from the reaction mixture is removed under ?n ^? ! ?n 1. E high vacuum. The crude product is purified on a Silica gel P column using 15% methanol in methylene chloride as an E E elution Solvent. The fractions containing the product are combined and the Solvent removed under reduced preSSure 25 with the residue being dried under high vacuum to yield An A. A -E compound 17 in good yield. N ?' ?' Example 29 E As described in FIG. 7B, compound 12 is reacted with compound 20 in its chloride, meSylate or tosylate form. Thus, R' in FIG. 7B may be chloride, -O-SO-CH, or ?n-F E AnOF ?n's -O-SO-Ar-CH. The reaction mixture is dissolved in 35 anhydrous DMF solvent. To the stirred solution, 5.0 equiva lents of anhydrous triethylamine is added and Stirred at room An 4N ^. Aa N E temperature. The progreSS of the reaction is monitored by E TLC. Solvent from the reaction mixture is removed under high vacuum. The crude product is purified on a Silica gel 40 column using 15% methanol in methylene chloride as an & ^ -Ni. elution Solvent. The fractions containing the product are ? ? i 1 s combined and the Solvent removed under reduced pressure, An -N A. A. N N s7 with the residue being dried under high vacuum to yield N w N compound 21 in good yield. 45 E E All publications and patent applications mentioned in this R5 Specification are herein incorporated by reference to the f sas R4 Same extent as if each individual publication or patent SS application was specifically and individually incorporated 1. by reference. 50 As R4 As S Rs From the foregoing it will be appreciated that, although specific embodiments of the invention have been described E E and herein for purposes of illustration, various modifications may be made without deviating from the Spirit and Scope of 55 A. S R4 the invention. Accordingly, the invention is not limited w except as by the appended claims. & What is claimed is: ? R5 1. A compound comprising a ligand portion (L) which has E binding affinity for an opioid receptor, and a therapeutically 60 or diagnostically effective group (X) selected from radionu clide chelating agents, fluorochromes, toxins, drugs, poly boron moieties, proteins, biological response modifiers, chemical moieties capable of binding to other molecules, and radioisotopes Selected from therapeutically effective 65 alpha and beta emitters, and diagnostically effective gamma emitters, wherein L has the formula US 6,359,111 B1 57 58 is a monocyclic, five- or six-membered, carbocyclic or heterocyclic, aromatic ring, R" is selected from (C1-C5)alkyl, (C-C cycloalkyl) C1-C5 alkylene, (C5-C,cycloalkenyl)C1-C5 alkylene, s aryl, aralkyl, trans-(C-C)alkenyl, allyl and furan-2- y1C1-C5alkylene; R’ is selected from H, OH and (C-C)alkyl-C(=O)C)-; R is selected from H., (C-C-)alkyl and (C-C)alkyl-C 10 (=O)-; and R" and Rare independently selected from a direct bond to E, H, F, Cl, Br, NO, CN, COOR, NH, (C-C) wherein, alkyl, (C-C)alkoxy or together are a benzo ring fused is R is selected from H, C-C alkyl and hydroxyl protect tO ing groups, E" represents an extender arm (E); and A. X" is a radionuclide chelating group. (O) 2O 5. A compound comprising a ligand portion (L) which has s binding affinity for an opioid receptor, the compound having the Structure

with the proviso that one, but only one, of R" and R may 25 be a direct bond to E; and

E is an extender arm, where E provides a stable chain of 2–60 atoms selected from carbon, oxygen, nitrogen and so Sulfur which covalently links together and distances the groupS L and X. 2. A compound of claim 1 wherein L has the formula

35 wherein, R is selected from H, C-C-alkyl and hydroxyl protect ing groups,

E" represents an extender arm (E); and X is a radionuclide chelating group. 40 6. A compound comprising a ligand portion (L) which has binding affinity for an opioid receptor, and a therapeutically or diagnostically effective group (X) selected from radionu clide chelating agents, fluorochromes, toxins, drugs, poly 45 boron moieties, proteins, biological response modifiers, chemical moieties capable of binding to other molecules, and radioisotopes Selected from therapeutically effective alpha and beta emitters, and diagnostically effective gamma emitters, the compound having the formula 3. A compound of claim 1 wherein L has the formula 50 R8 R10 rx 55 NH HN

SRo R, RoS R7

wherein, independently at each occurrence, R, is selected from -OH,-O-C(=O)-Calkyl, and -O-C alkyl, 4. A compound comprising a ligand portion (L) which has 65 Rs is Selected from hydrogen and Calkyl, binding affinity for an opioid receptor, the compound having Ro is selected from hydrogen, -C(=O)-Calkyl, the Structure -C(=O)-phenyl; hemithioacetals, hemithioketals, US 6,359,111 B1 59 60 and Sulfur protecting groups, Such that two Ro groups R' has a structure selected from may be joined together as a direct bond, thereby linking two Sulfur atoms as a disulfide linkage; A10 Ro is selected from Calkyl and Calkoxy; R17 n is selected from 0 and 1; and and -itR18 R'' and R' are selected from H., C.-Chydrocarbyl, CN, -NO, -NO, -C(OC-Calkyl)=NH, N=C=O, -N=C=S, and –C(=O)CR' where R' is C-C hydrocarbyl; represents a 6-membered carbocyclic aromatic or aliphatic R'' and Rare selected from Hand protecting groups for ring. 15 the A or A9 moiety to which the R' or R' is bonded; 7. A compound comprising a ligand portion (L) which has A” and R' may together form -N(CH-COOH); and binding affinity for an opioid receptor, and a therapeutically A' is selected from O and S. or diagnostically effective group (X) selected from radionu 9. The compound of claim 7 having the formula clide chelating agents, fluorochromes, toxins, drugs, poly boron moieties, proteins, biological response modifiers, R20 R21 R21 chemical moieties capable of binding to other molecules, (CH)n-N-CH-(CH3) - V-s-R. and radioisotopes Selected from therapeutically effective Ligand alpha and beta emitters, and diagnostically effective gamma emitters, wherein the ligand portion (Ligand) is conjugated 25 to two X groups X and X, through two extender arms E" (CH2) R20-at-cut R21As R21 and Ef, as represented by the formula wherein R'' and R'' are independently selected from El-X1 hydrogen and methyl; R’ is independently selected from hydrogen, acm, EOE, re THP, C-Calkyl, acetate, benzoyl and Sulfur protect ing groups, and 35 each of m, n, p and q are Selected from 0 and 1 Such that 8. The compound of claim 7 wherein X" and X together when p+q=0 then m+n=0 or 1; and when p+q=1 then bind one radionuclide and each has a structure Selected from m+n=1; and when p+q=2 then m+n=1 or 2. 10. The compound of claim 7 wherein X" and X together A10 form the cyclic Structure 40 A-R11-A-R12 and sci-( A. A. A. A. A10-R13), >, N11 N1 N11

s wherein, 45 A is an electron-donating moiety independently selected wherein, A is an electron-donating moiety independently selected at each occurrence from O, S, C(=O)NH and N(R'), at each occurrence from O, S, C(=O)NH and N(R'), where R' is selected from hydrogen and pro-drug where R' is selected from hydrogen and pro-drug Substituents Selected from the group consisting of 50 Substituents Selected from the group consisting of C-C alkyl, -C(=O)(H, C-C alkyl or Ar), C-C alkyl, -C(=O)(H, C-C alkyl or Ar), -C(=O)-(C-C alkylene)-N(independently H or -C(=O)-(C-C alkylene)-N(independently H or C-C alkyl), -C(=O)C)-(H, C-C alkyl or Ar), C-C alkyl), -C(=O)C)-(H, C-C alkyl or Ar), -C(=O)CH(NH2)(H, C-C alkyl or Ar), 55 -C(=O)CH(NH2)(H, C-C alkyl or Ar), -(C-C alkylene)-C(=O)C)-(H, C-C alkyl or Ar), -(C-C alkylene)-C(=O)C)-(H, C-C alkyl or Ar), -(C-C alkylene)-OC(=O)-(H, C-C alkyl or Ar), -(C-C alkylene)-OC(=O)-(H, C-C alkyl or -(C-C alkylene)-N(independently H or C-C, Ar), -(C-C-alkylene)-N(independently H or alkyl), -(C-C alkylene)-NHC(=O)-Ar, -(C. C-C-alkyl), -(C-C alkylene)-NHC(=O)-Ar, 3alkylene)-CN, -(Calkylene)-NO, and 60 -(C-C alkylene)-CN, -(C-C alkylene)-NO, and -(C1-C3alkylene) ( \, -(C -case- \, 65 US 6,359,111 B1 61 62 R' has a structure selected from A10 (C1-C3alkylene) / \, R17 ---- and A. k R'' is selected from A10 R'' and R' are selected from H, C-C,hydrocarbyl, R17 CN, -NO, -NO, -C(OC-Calkyl)=NH, N=C=O, -N=C=S, and –C(=O)CR' where -it- and R' is C-C hydrocarbyl; 15 R18 A” and R' may together form -N(CH-COOH); and A" is selected from O and S. 11. A compound comprising a ligand portion (L) which where R7 and R are selected from H, has binding affinity for an opioid receptor, and a therapeu C-C hydrocarbyl, -CN, -NO, -NO, tically or diagnostically effective group (X) selected from C(OC-Calkyl)=NH, -N=C=O, -N=C=S, radionuclide chelating agents, fluorochromes, toxins, drugs, and –C(=O)CR' where R' is C-C hydrocarbyl; polyboron moieties, proteins, biological response modifiers, A' is selected from O and S; chemical moieties capable of binding to other molecules, and radioisotopes Selected from therapeutically effective R" is selected from H and protecting groups for A, alpha and beta emitters, and diagnostically effective gamma 25 where A and R' may together form -N(CH emitters, wherein the ligand portion (Ligand) is conjugated COOH); at one site to an X group through an extender arm E, as R’ is selected from H and CHCOOH: represented by the formula Ligand-E-X, wherein X is rep resented by any of formulas (II), (III) or (IV), q is Selected from 0, 1, 2 and 3; and E represents an extender arm through which L is conju (II) gated to X. 12. A compound of claim 11 wherein X has the formula

35 COOH COOH (III) A9-R 4 ls - N'- R14 NA9 40

k / l 4 R 14-A9 (IV) rN --> COOH COOH. R23 45 HOOC-CH V H-cool /N-CHCH-A-NCHCHHN q \ 13. A compound of claim 11 wherein X has the formula HOOC-CH CH-COOH W 50 Such that one methylene hydrogen of formula (IV) is F. y \ replaced with a direct bond to E; N S-R2 where, independently at each occurrence, A is an electron-donating moiety selected from O, S, 55 C(=O)NH and N(R'), where R' is selected from hydrogen and pro-drug Substituents Selected from C-C alkyl, -C(=O)(H, C-C alkyl or Ar), -C(=O)-(C-C alkylene)-N(independently H or C-C alkyl), -C(=O)C)-(H, C-C alkyl or Ar), 60 -C(=O)CH(NH2)(H, C-C alkyl or Ar), -(C-C alkylene)-C(=O)C)-(H, C-C alkyl or Ar), -(C-C alkylene)-OC(=O)-(H, C-C alkyl or Ar), -(C-C alkylene)-N(independently H or where W represents H or =O, R' is selected from H, C-Calkyl), -(C-C alkylene)-NHC(=O)-Ar, 65 COCH, EOE, THP, COPh, acm, C-Chydrocarbyl, and -(C-C-alkylene)-CN, -(C-C alkylene)-NO, Sulfur protecting groups; and R is selected from H and and C-C hydrocarbyl. US 6,359,111 B1 63 64 14. A compound of claim 11 wherein X has the formula =N-(O, NH, or direct bond)-E-(phenylene or direct bond-(CH), ; W -(O, S, or Se)-E-; H na N S-R2 E O R6 O -(CH)n-C-NH(CH3). CH-(CH3), C-: 1 N S-R2 H W -(CH2) -- N-(cis-N-(| )—cil- ; and O O where W represents H or =O, and R' is selected from H, COCH, EOE, THP, COPh, acm, C-Chydrocarbyl, and 15 Sulfur protecting groups. 15. A compound comprising a ligand portion (L) which o (Chi- (NH- citri-n-( )—cil has binding affinity for an opioid receptor, and a therapeu O O tically or diagnostically effective group (X) selected from radionuclide chelating agents, fluorochromes, toxins, drugs, polyboron moieties, proteins, biological response modifiers, chemical moieties capable of binding to other molecules, where m is selected from 0, 1, 2, 3, 4 and 5; n is selected and radioisotopes Selected from therapeutically effective from 1, 2, 3, 4 and 5; p is selected from 0, 1, 2, 3 and alpha and beta emitters, and diagnostically effective gamma 4, r is an integer of from 1 to about 4, and emitters, wherein L is conjugated to an X group through an 25 extender arm (E), where E provides a stable chain of 2–60 E is a 1-10 carbon moiety having 0–3 heteroatoms as atoms Selected from carbon, oxygen, nitrogen and Sulfur ethers or ester; which covalently links together and distances the groupS L R is selected from H, C-C alkyl and hydroxyl protect and X, and E has a formula Selected from ing groups, and wherein X is NY and Y is selected from H., (C-C)alkyl O and a direct bond to E. 16. The compound of claim 4 wherein R is acetate o ch)-( protecting group. NH (CH2) 17. The compound of claim 5 wherein R is acetate 35 protecting group. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,359,111 B1 Page 1 of 2 DATED : March 19, 2002 INVENTOR(S) : Damon L. Meyer et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 57 Line 47, the following formula:

should be corrected to read --

Column 61 Line 7, the following formula:

-(-),18 R and should be corrected to read UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,359,111 B1 Page 2 of 2 DATED : March 19, 2002 INVENTOR(S) : Damon L. Meyer et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 63 Line37, the following formula:

=N-(NHor NHCO)-(CH)-

should be corrected to read

=N-(NHor NHCO)-(CH)-. s

Signed and Sealed this Twelfth Day of November, 2002

Attest.

JAMES E ROGAN Attesting Officer Director of the United States Patent and Trademark Office UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,359,111 B1 Page 1 of 2 DATED : March 19, 2002 INVENTOR(S) : Damon L. Meyer et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 9 Line 5, the following formula: ti,R7 y, A10 x and

should read -- UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,359,111 B1 Page 2 of 2 DATED : March 19, 2002 INVENTOR(S) : Damon L. Meyer et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 11 Line 10, the following formula:

-(- s 1-4

should read -- -(- s 1-4

Signed and Sealed this Twenty-seventh Day of May, 2003

JAMES E ROGAN Director of the United States Patent and Trademark Office