Thioamide-containing compositions

Abstract

To provide thioamide-modified amino acids useful as albumin-targeting moieties, which offer tunable (and different) albumin binding and increased in vivo stability compared to the corresponding amide-modified compounds.SOLUTION: Provided is a composition comprising a thioamide represented by Formula (I). Therapeutic compounds incorporating these thioamide-linked albumin-targeting moieties are disclosed.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority of U.S. Provisional Patent Application 62 / 767,151, filed Nov. 14, 2018. The entire content of this related application is incorporated herein by reference.

[0002] The present invention generally relates to modified drugs, and more particularly to thioamide - modified drugs.

Background Art

[0003] Currently, several strategies are used to extend the lifespan of therapeutic drugs in serum, including altering the peptide sequence and secondary structure of peptide or polypeptide drugs to minimize protease activity. Another approach for extending the half - life of biomolecules is PEGylation. As yet another approach, an albumin - targeting moiety may be introduced to the drug.

Summary of the Invention

[0004] This specification provides thioamide - modified amino acids useful as albumin - targeting moieties. These compounds of this specification provide tunable (and different) albumin - binding properties and improved in - vivo stability compared to the corresponding amide - modified compounds.

[0005] In a first aspect, this specification discloses a composition containing a thioamide as disclosed below. In embodiments, it is useful, inter alia, as an albumin - targeting agent.

[0006] In a first aspect, this specification provides Formula (I):

Chemical Formula

[0007] In an embodiment, n is 2 or 3.

[0008] In an embodiment, the compound represented by formula (I) is (II):

Chemical formula

[0009] In an embodiment, n is 3.

[0010] In an embodiment, L 1 is X-Y-Z, where X is

Chemical formula

Chemical formula

[0011] In an embodiment, L 1 is Z, wherein: Z is C1-C 12 alkyl, wherein any of the methylene groups in the alkyl group may be replaced by NH or carbonyl.

[0012] In an embodiment, Z is

Chemical formula

[0013] In an embodiment, the chelating agent is

Chemical formula

[0014] In an embodiment, L 2 is -N(R 9 )-C1-C 12 alkyl-C(O)-. In an embodiment, L 2 is as follows.

[0015] In an embodiment, L 2 is -N(R 9 )-C4-C 30 alkylcycloalkyl-C(O)-C7-C 30 alkylaryl-C(O)-.

[0016] In an embodiment, L 2 is

Chemical formula

[0017] In an embodiment, L 2 is -N(R 9)-C7-C 30 Alkylaryl-C(O)NH-C7-C 30 Alkylaryl-C(O)NH-CH(CO2H)-C1-C 12 Alkyl-NHC(O)-C1-C 12 is alkyl-C(O)-, where C7-C 30 Alkylaryl may optionally be substituted by halo or hydroxyl.

[0018] In an embodiment, L 2 is

Chemical formula

[0019] In an embodiment, L 2 is

Chemical formula

[0020] In an embodiment, the compound represented by formula (I) is of formula (III):

Chemical formula

[0021] In an embodiment, n is 3.

[0022] In an embodiment, L 1 is X-Y-Z, where X is,

Chemical formula

[0023] In an embodiment, the chelating agent is

Chemical formula

[0024] In an embodiment, L 2 is

Chemical formula

[0025] In an embodiment, the compounds represented by formulas (II) and (III) are provided as therapeutic agents. In an embodiment, the compounds of the present invention include an albumin targeting moiety, a PMSA targeting moiety, and a drug moiety or a chelating agent moiety. In an embodiment, the 4-iodophenyl moiety is the albumin targeting moiety, urea (or a urea derivative) is the PMSA targeting moiety, and R 6 is the drug moiety or the chelating agent moiety. The PMSA targeting moiety, and the drug moiety or the chelating agent moiety may be bound to the albumin targeting group by a non-therapeutic linking moiety. In an embodiment, the linking moiety may consist of a PEG chain. In other embodiments, this linking moiety is a hybrid group of PEG and an alkyl group. In some embodiments, this linking moiety binds to a therapeutic agent that does not contain a PMSA-binding group. In an embodiment, the therapeutic agent binds to the albumin-binding group at the N-terminus of the lysine moiety of the albumin-binding group. In an embodiment, this linking group binds to an atom adjacent to the leaving group on the linking moiety via nucleophilic addition at the N-terminus of the lysine moiety of the albumin targeting group. In some embodiments, this leaving group is N-hydroxysuccinimide covalently bound to the carbonyl of the linking group.

[0026] In another aspect, the present specification provides a compound of formula (IV):

Chemical formula

[0027] Detailed Description The present specification discloses, inter alia, thioamide-containing compositions useful as albumin binders for imaging and therapeutic modalities.

[0028] In embodiments, the thioamide-containing compositions of the present invention bind to prostate-specific membrane antigen (PSMA) with high affinity and are similar to the PSMA-binding compounds disclosed in WO2018 / 098390 and WO2013 / 028664, the contents of which are hereby incorporated by reference in their entirety.

[0029] In embodiments, the compositions according to the present specification can be used in a method similar to the method taught for the thioamide-containing compounds disclosed in US2018 / 0066298, the contents of which are hereby incorporated by reference in their entirety.

[0030] In an embodiment, the present specification provides a modifying agent comprising lysine or ornithine and an albumin target group, wherein the lysine or ornithine is bound to the albumin target group by a thioamide moiety (i.e., a thioamide bond). In certain embodiments, the thioamide bond is more stable to hydrolysis in vivo compared to an amide bond. In certain embodiments, it is more stable to peptidase activity compared to an amide bond. In certain embodiments, the compounds of the present invention have higher in vivo stability than the corresponding compounds in which lysine or ornithine is bound to the albumin target group by an amide bond. One of ordinary skill in the art can bind a drug to the lysine or ornithine moiety using techniques known in the art.

[0031] Definitions A "therapeutically effective amount" of a compound or pharmaceutical composition means an amount effective to prevent, suppress, alleviate, or treat the symptoms of a particular disorder or disease.

[0032] "Pharmaceutically acceptable" indicates approval by a federal or state government regulatory authority or listing in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans.

[0033] "Carrier" means, for example, a diluent, adjuvant, preservative (e.g., thimerosal, benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, polysorbate 80), emulsifier, buffer (e.g., Tris HCL, acetate, phosphate), bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle, and the active substance of the present invention is administered together with these. A pharmaceutically acceptable carrier may be a sterile liquid such as water or oil, and examples include petroleum oil, animal oil, vegetable oil, synthetically derived oil such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or an aqueous saline solution and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly injectable solutions. The compositions of the present invention can be incorporated into particulate formulations of high molecular compounds such as polylactic acid, polyglycolic acid, or liposomes or micelles. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the components of the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention may be prepared, for example, in a liquid form or in a dry powder form (e.g., lyophilized powder form).Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins), 2000; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0034] "Enhanced binding" means a bond between at least two molecules, where at least one molecule changes from its native state, resulting in a higher binding affinity between the two molecules. For example, molecule A is not bound or weakly bound to molecule B, but when molecule A is modified, such as by introducing a non-natural amino acid with an affinity tag into molecule A (A'), molecule A' binds to molecule B with a higher affinity. In the methods and compositions of the present invention, molecule A’ is a polypeptide modified with a non-natural amino acid having an albumin-binding tag, such as Nε-(4-(4-iodophenyl)butanoyl)lysine, and molecule B is albumin, such as human serum albumin. Enhanced binding can be measured by various methods, such as measuring affinity by surface plasmon resonance or direct binding assays.

[0035] The terms "isolated", "purified", and "biologically pure" mean a substance that is substantially or essentially free of components that are normally present in its native state.

[0036] The term "subject" means a mammal. Thus, the subject means, for example, a dog, a cat, a horse, a cow, a pig, a guinea pig, etc. Preferably, the subject is a human. When the subject is a human, in this specification, the subject may be referred to as a patient.

[0037] The various methodologies of the present invention include steps that involve comparing values, levels, features, characteristics, properties, etc. with a "suitable control" that is interchangeably referred to herein as an "appropriate control". The "suitable control" or "appropriate control" is a control or standard well known to those skilled in the art that is useful for the purpose of comparison. In one embodiment, the "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. that is determined prior to performing the methodology, as described herein.

[0038] As used herein, "non-natural" amino acids mean amino acids that do not exist in nature (novel synthetic amino acids), or amino acids that exist in nature but do not naturally exist within proteins (natural but non-proteinogenic amino acids).

[0039] The term "thioyl" means a divalent chemical functional group that has conventionally been shown as a carbon atom with a double bond to a sulfur atom.

[0040] As used herein, "drug" means a pharmaceutical formulation containing at least one pharmaceutically active compound. In one embodiment, the pharmaceutically active compound has a molecular weight up to 1500 Da and / or is a peptide, protein, polysaccharide, vaccine, DNA, RNA, enzyme, antibody or fragment thereof, hormone or oligonucleotide, or a mixture of these pharmaceutically active compounds.

[0041] Compounds containing a thioamide bond In one aspect, the present specification discloses compounds comprising an albumin targeting moiety. An “albumin targeting moiety”, “albumin targeting molecule” or “albumin targeting tag” is a small molecule incorporated into a second molecule, such as a polypeptide, which directs the second molecule to associate with albumin, preferably in vivo. Such association involves binding interactions between albumin and the albumin targeting tag.

[0042] As used herein, the term “drug” means a pharmaceutical formulation containing at least one pharmaceutically active compound. In one embodiment, the pharmaceutically active compound has a molecular weight up to 1500 Da and / or it is a peptide, protein, polysaccharide, vaccine, DNA, RNA, enzyme, antibody or fragment thereof, hormone or oligonucleotide, or a mixture of these pharmaceutically active compounds.

[0043] The drug can include, for example, an anti-inflammatory drug as disclosed in the embodiments, which is an anti-inflammatory substance, for example, the anti-inflammatory substance disclosed in USSN12 / 351,417. As used herein, the "anti-inflammatory therapeutic agent" means a compound for treating an inflammatory disease or related symptoms. Anti-inflammatory therapeutic agents include non-steroidal anti-inflammatory drugs (NSAIDs; for example, aspirin, ibuprofen, naproxen, methyl salicylate, diflunisal, indomethacin, sulindac, diclofenac, ketoprofen, ketorolac, carprofen, fenoprofen, mefenamic acid, piroxicam, meloxicam, methotrexate, celecoxib, valdecoxib, parecoxib, etoricoxib and nimesulide), corticosteroids (for example, prednisone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, tramcinolone and fluticasone), rapamycin, rho-kinase inhibitors, viral CC-chemokine inhibitors (vCCIs), glucocorticoids, steroids, beta-agonists, anticholinergic agents, methylxanthines, sulfasalazine, dapsone, psoralen, proteins, peptides, DMARDs, glucocorticoids, methotrexate, sulfasalazine, chloroquine, gold, gold salts, copper, copper salts, penicillamine, D-penicillamine, cyclosporine, lipoxin, degradation products and protective substances, but are not limited thereto.

[0044] In certain embodiments, the anti-inflammatory therapeutic agent is selected from the group consisting of proteins, peptides, NSAIDs, DMARDs, glucocorticoids, methotrexate, sulfasalazine, chloroquine, gold, gold salts, copper, copper salts, penicillamine, D-penicillamine, cyclosporine and dexamethasone. The anti-inflammatory therapeutic agent can also be The Pharmacological Basis of Therapeutics, 10 thIt is provided in Gilman et al., eds., McGraw-Hill Press (2001) and Remington's Pharmaceutical Science's, 18th ed. Easton: Mack Publishing Co. (1990).

[0045] Other drugs include, for example, analgesics, anti - hair loss drugs, anti - angina drugs, antibacterial substances, antidepressants, antifungal drugs, antihypertensive drugs, anticancer drugs, antipyretics, antipsychotics, anxiolytics, bronchodilators, glucocorticoids, immunosuppressants, acetylsalicylic acid, alpha - atrial natriuretic peptide, arginine vasopressin, atropine, augmerosen, atorvastatin, avastin, calcitonin, chlorhexidine, chorionic gonadotropin, adrenocorticotropic hormone, desmopressin, epibatidine, arvictus, exenatide, herceptin, humira, humulin, ketoconazole, lanreotide, lutropin alpha, metoprolol, minoxidil, nesiritide, octreotide, paclitaxel, paracetamol, pegaptanib, recombinant follicle - stimulating hormone, recombinant growth factor, remicade, rituxan, sermorelin, somatotropin, taxane derivative, taxol, teriparatide acetate, thyrotropin, triclosan, urofollitropin, zolea, actinomycin D, albendazole, aldosterone, alprazolam, amiodarone, amitriptyline, amprenavir, asimadoline, atorvastatin, bunitrolol, buspirone, camptothecin, carbamazepine, carvedilol, celiprolol, cyclosporin A, cimetidine, clotrimazole, colchicine, cortisone, daunorubicin, debrisoquine, diazepam, digitoxin, digoxin, diltiazem, docetaxel, domperidone, doxorubicin, efavirenz, epirubicin, erythromycin, ergotamine, estradiol, estradiol glucuronide, erlotinib, etoposide, phenytoin, fentanyl, felodipine, phenothiazine, fexofenadine, fluoroquinolone, fluorouracil, FK - 506, gentamicin, griseofulvin, imatinib, indinavir, itraconazole, ivermectin, ketoconazole, kemperol, levofloxacin, lidocaine, loperamide, losartan, lovastatin, mebendazole, methylprednisolone, methotrexate, mibefradil, midazolam, nisoldipine, morphine, nelfinavir, nicardipine, nitrendipine, nifedipine, ondansetron, paclitaxel, pentazocine, pradicantel, prednisolone, prednisone,Quercetin, quinidine, ranitidine, rapamycin, rifabutin, rifampicin, ritonavir, saquinavir, sirolimus, sulfamethizole, tacrolimus, tamoxifen, talinolol, teniposide, terfenadine, tetracycline, topotecan, triamcinolone, valspodar, verapamil, vinblastine, vincristine, vindesine, zopiclone, herbicide, insecticide, fungicide, anti-aging product, anti-acne product, facial care product, pigmented cosmetic, cosmetic, personal care product, sunscreen / sun care product, tooth cleaner, toothpaste or mouthwash product, shampoo product, fragrance, hair product, food additive, essential oil, Mentha piperita oil, Thyme oil, cinnamon oil, eugenol, lemon oil, curcumin, folic acid, 4-aminobenzoic acid, niacin or vitamin B3, pantothenic acid or vitamin B5, thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, ascorbic acid, pteroylpolyglutamic acid, folinic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-coumaric acid, caffeic acid, vitamins A, D, E, K and their derivatives, phospholipids, carotenoids, fatty acids, omega-3 fatty acids, cod liver oil, linolenic acid, amino acids, phytosterols, phytostanols, polyphenols, chlorhexidine, bovine serum albumin and mixtures thereof, but not limited thereto.,

[0046] In an embodiment, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triaza-cyclo-nonane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or diethylenetriaminepentaacetic acid (DTPA).

[0047] The selection of drugs targeting albumin can be determined by those skilled in the art. In the case of polypeptide therapeutic agents, their function may be to bind to serum albumin, for example, to increase the half-life of the therapeutic polypeptide in serum. These embodiments relate to albumin targeting of polypeptides. General examples of therapeutic polypeptides include, but are not limited to, antibodies, chimeric antibodies, monoclonal antibodies, single-chain antibodies, Fab, Fab', F(ab')2, Fv, and scF, Fc fusions, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Other examples of therapeutic peptides include salmon calcitonin; β-interferon; interferon; veraglucerase-; taliglucerase-; glucarpidase (e.g., for methotrexate toxicity treatment); elosulfase- (e.g., for Morquio syndrome treatment); aldesleukin; anakinra; insulin lispro; uricase (e.g., for gouty tophus treatment); paricalcitol.

[0048] The drug-containing thioamide composition can be administered by any desired suitable means. For in vivo delivery (i.e., delivery to a subject having arthritis or other inflammatory diseases), a delivery system that is biocompatible, preferably biodegradable and non-immunogenic, is desirable. Further, it is desirable to provide a therapeutically effective amount of the compound in a physiologically acceptable carrier. Injection into an individual can be performed subcutaneously, intravenously, intramuscularly, intraperitoneally, intra-articularly, or, for example, directly into a local area. In vivo delivery can also be performed using syrups, elixirs, liquids, tablets, pills, sustained-release capsules, aerosols, transdermal patches, injections, infusions, ointments, etc.

Examples

[0049] The following examples are provided for illustrative purposes and are not intended to limit the present disclosure in any way. Those skilled in the art will readily recognize various non-essential parameters that can be modified to obtain substantially the same results.

[0050] Example 1: Synthesis of a thioamide-containing composition The thioamide-containing composition is prepared using the following synthetic scheme: [Chemical formula]

[0051] Synthesis of 3-(4-iodophenyl)propanoic acid: [Chemical formula]

[0052] To a mixture of 3-phenylpropanoic acid (20.0 g, 133.18 mmol), H5IO6 (6.18 g, 26.68 mmol), iodine (14.54 g, 57.3 mmol), and 10 M H2SO4 (5.0 mL), water (36 mL) and acetic acid (166 mL) were added, and the mixture was heated at 70 °C for 19 hours. The reaction mixture was cooled and evaporated to dryness. The residue was dissolved in EtOAc (300 mL), washed with Na2S2O3 (2 × 200 mL) and brine (2 × 200 mL), dried over Na2SO4, filtered, and evaporated to leave a yellow solid. The crude product was precipitated from EtOAc / hexane at 0 °C to give the title compound as a pale yellow solid (15.0 g, 42%). 1 H NMR (400 MHz, CDCl3) δ 10.7 (s(br), 1 H), 7.63 (d, J = 8.2 Hz, 2 H), 6.99 (d, J = 8.2 Hz, 2 H), 2.92 (t, J = 7.6 Hz, 2 H), 2.68 (t, J = 7.6 Hz, 2 H).

[0053] Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(4-iodophenyl)propanoate: [Chemical formula]

[0054] Under a nitrogen atmosphere, 3-(4-iodophenyl)propanoic acid (9.40 g, 34 mmol) was dissolved in dichloromethane (100 mL), and N-hydroxysuccinimide (6.0 g, 51.1 mmol, 1.5 eq) was added. After cooling the mixture to 0 °C, a dichloromethane solution of dicyclohexylcarbodiimide (DCC, 10.55 g, 51.1 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 6 hours, filtered, and the filtrate was evaporated to dryness. The residue was purified by silica gel column chromatography using straight (CH2Cl2 / methanol). The appropriate fractions were evaporated to give the title compound (10.98 g, 86.5%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (d, J = 8.2 Hz, 2 H), 7.14 (d, J = 8.2 Hz, 2 H), 3.01 (t, J = 6.84 Hz, 2 H), 2.91 (d, J = 6.96 Hz, 2 H), 2.81 (s, 4 H).

[0055] N 2 Synthesis of N-(tert-butoxycarbonyl)-N6-(4-(4'-iodophenyl)propanoyl)-L-lysine: [Chemical formula]

[0056] Under a purified nitrogen atmosphere, 2,5-dioxopyrrolidin-1-yl-3-(4-iodophenyl)propanoate (2 g, 5.36 mmol, 1.06 eq) was dissolved in 20 mL, and N 2It was treated with (tert-butoxycarbonyl)-L-lysine (1.35 g, 5.04 mmol). The mixture was cooled to 0 °C and DIPEA (0.88 ml, 5.04 mmol) was added dropwise. After 5 hours, the solvent was removed at room temperature and the title compound was isolated by extraction with basic followed by pH 3.5 water and CH2Cl2. The final organic extracts were combined, dried over Na2SO4, filtered, and evaporated to leave a colorless solid (80.0%). 1 H NMR (400 MHz, CD3CN) δ 7.65 (d, J = 8.3 Hz, 2 H), 7.04 (d, J = 8.3 Hz, 2 H), 6.36 (t, 1 H, NH), 5.62 (d, J = 7.1 Hz, 1 H, NH), 4.02 (dd, 1 H), 3.11 (m, 2 H), 2.85 (t, J = 7.44 Hz, 2 H), 2.39 (t, J = 7.80 Hz, 2 H), 1.43(s,9H).

[0057] Methyl N 2 -(tert-butoxycarbonyl)-N 6 Synthesis of N-(tert-butoxycarbonyl)-N-(3-(4-iodophenyl)propanoyl)-L-lysinate:

Chemical formula

[0058] N 2 -(tert-butoxycarbonyl)-N 6To a solution of (3-(4-iodophenyl)propanoyl)-L-lysine (0.76 g, 1.506 mmol) in dichloromethane (10 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.28 g, 1.506 mmol). The mixture was cooled to 0 °C, 4-dimethylaminopyridine (DMAP) (0.02 g, 0.1506 mmol, 0.1 eq) was added, and then dry MeOH (0.13 mL, 3.012 mmol, 2 eq) was added. The reaction mixture was stirred overnight at room temperature, the solvent was evaporated, and the residue was purified by silica gel column chromatography (eluent = 10% methanol in dichloromethane). The appropriate fractions were evaporated to isolate the title compound as a colorless solid (0.52 g, 66.6%). 1 H NMR (400 MHz, CD3CN) δ 7.65 (d, J = 8.28 Hz, 2 H), 7.04 (d, J = 8.28 Hz, 2 H), 6.34 (t, 1 H, NH), 5.65 (d, J = 7.04 Hz, 1 H, NH), 4.06 (dd, 1 H), 3.68 (s, 3H), 3.10 (m, 2 H), 2.85 (t, J = 7.48 Hz, 2 H), 2.85 (t, J = 7.72 Hz, 2 H), 1.72 (m, 1 H), 1.62 (m, 1 H), 1.42 (s, 9 H) 1.30 (m, 2 H).

[0059] Methyl N 2 -(tert-butoxycarbonyl)-N 6 Synthesis of Methyl N

Chemical formula

[0060] Under an argon atmosphere, methyl N 2 -(tert-butoxycarbonyl)-N 6-(3-(4-Iodophenyl)propanethioyl)-L-lysinate (0.49 g, 0.94 mmol) and Lawesson's reagent (0.095 g, 0.24 mmol) were dissolved in 20 mL of dry toluene and subsequently stirred at 70 °C overnight. The solvent was evaporated and the residue was purified by silica gel column chromatography (MeOH gradient 0 - 10% in CH2Cl2). Fractions were selected, evaporated, and the product was dried under dynamic vacuum. Yield: 40% 1 H NMR (400 MHz, CD3CN) δ 8.29 (S, 1 H), 7.65 (d, J = 8.12 Hz, 2 H), 7.05 (d, J = 8.16 Hz, 2 H), 5.57 (d, 1 H, NH), 4.09 (m, 1H), 3.69 (s, 3 H), 3.51 (q, J = 6.08, 2H), 3.01 (t, J = 7.60, Hz, 2 H), 2.84 (t, J = 7.64, Hz, 2 H), 1.74 (m, 1 H), 1.62 (m, 1 H), 1.52 (m, 2 H), 1.42 (s, 9 H), 1.31 (m, 2 H).

[0061] N 2 -(tert-Butoxycarbonyl)-N 6 -Configuration of (3-(4-iodophenyl)propanethioyl)-L-lysine:

Chemical Structure

[0062] Methyl N 2 -(tert-Butoxycarbonyl)-N 6-(3-(4-Iodophenyl)propanethioyl)-L-lysinate (0.40 g, 0.74 mmol) was dissolved in i-PrOH (13 mL) and added to a mixture of NaOH (0.051 g, 0.89 mmol) and CaCl2 (1.47 g, 13.3 mmol) in H2O (5 mL). The mixture was stirred at room temperature for 4 hours, neutralized with 1 M AcOH, and the excess i-PrOH was evaporated. Saturated aqueous NaCl was added to the residue and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over Na2SO4, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl2, 0 - 10% gradient of methanol). Fractions were selected and evaporated to isolate the title compound (0.38 g, 100%) as a yellow solid. 1 1H NMR (400 MHz, MeOD) δ 7.61 (d, J = 8.28 Hz, 2 H), 7.03 (d, J = 8.28 Hz, 2 H), 4.00 (m, 1 H), 3.53 (t, J = 7.16 Hz, 2 H), 3.01 (t, J = 7.2 Hz, 2H), 2.85 (t, J = 8.00 Hz, 2 H), 1.82 (m, 1 H), 1.66 (m, 1 H), 1.57 (m, 2 H), 1.46 (s, 9 H), 1.37 (q, J = 7.68 Hz, 2 H).

[0063] N 6 Synthesis of N-(3-(4-Iodophenyl)propanethioyl)-L-lysine, Trifluoroacetate Salt:

Chemical Structure

[0064] N 2 - (tert-Butoxycarbonyl)-N 6 -(3-(4-Iodophenyl)propanethioyl)-L-lysine was dissolved in a solution of trifluoroacetic acid (2 mL) in CH2Cl2. The solution was stirred at room temperature for 5 hours and the solvent was evaporated. The residue was further purified for use. 1 1H NMR (400 MHz, MeOD) δ 7.61 (d, J = 8.28 Hz, 2 H), 7.03 (d, J = 8.28 Hz, 2 H), 3.96 (t, J = 6.4 Hz, 1 H), 3.57 (td, J = 7.2 Hz, J = 2.2 Hz, 2 H), 3.04 (t, J = 8.60 Hz, 2 H), 2.86 (t, J = 7.92 Hz, 2 H), 1.95 (m, 2 H), 1.61 (m, 2 H), 1.40 (m, 2 H).

[0065] Example 2: Alternative synthesis of a thioamide-containing composition An alternative scheme for synthesizing the thioamide-containing composition is shown below:

Chemical formula

[0066] Synthesis of 4-(4-iodophenyl)butanoic acid:

Chemical formula

[0067] A mixture of 4-phenylbutanoic acid (20.0 g, 121.8 mmol), H5IO6 (5.56 g, 24.4 mmol), iodine (13.30 g, 52.4 mmol), 10 M H2SO4 (5.0 mL), water (36 mL), and acetic acid (166 mL) was heated at 70 °C for 19 h. The reaction mixture was cooled and the solvent was concentrated under reduced pressure. The residue was dissolved in EtOAc (300 mL) and washed with Na2S2O3 (2 × 200 mL) and brine (2 × 200 mL). The organic phase was separated, dried over Na2SO4, filtered, and evaporated to leave a yellow solid. The crude product was precipitated from EtOAc / hexane at 0 °C to give the product as a pale yellow solid (15.0 g, 42%). 11H NMR (400 MHz, CDCl3) δ 11.0 (s (broad), 1 H), 7.61 (d, J = 8.4 Hz, 2 H), 6.95 (d, J = 8.0 Hz, 2 H), 2.63 (t, J = 7.6 Hz, 2 H), 2.38 (t, J = 7.6 Hz, 2 H), 1.95 (A2B2, t, 2 H).

[0068] Synthesis of N-(2-amino-5-nitrophenyl)-4-(4-iodophenyl)butanamide:

Chemical Structure

[0069] Under a nitrogen atmosphere, a solution of 4-(4-iodophenyl)butanoic acid (11.60 g, 40 mmol) in THF (200 mL) was cooled to -20 °C and sequentially treated with N-methylmorpholine (NMM) (8.8 mL, 80 mmol, 2.0 eq) and isobutyl chloroformate (5.2 mL, 40 mmol, 1.0 eq). The reaction mixture was stirred for 30 minutes, a solution of 4-nitro-1,2-phenylenediamine (6.12 g, 40 mmol, 1.0 equiv) in THF (100 mL) was added, and the mixture was maintained at -20 °C for an additional 1.5 hours and then at 23 °C for 15 hours. The mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in EtOAc (300 mL) and washed with aqueous solutions of 1 M NaH2PO4 (2 × 100 mL), saturated brine (2 × 100 mL), saturated NaHCO3 (2 × 100 mL), and saturated NaCl (2 × 100 mL). The organic phase was separated, dried over Na2SO4, and evaporated to dryness. The crude product was sonicated in EtOAc to form a solid. The EtOAc was decanted, and the remaining solid was filtered and dried under reduced pressure to obtain the title compound (10.77 g, 63%) as a yellowish-brown solid. 11H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1 H), 8.25 (d, J = 2.4 Hz, 1 H), 7.83 (dd, J = 8.8, 2.4 Hz, 1 H), 7.64 (d, J = 8.0 Hz, 2 H), 7.06 (d, J = 8.0 Hz, 2 H), 6.75 (d, J = 8.8 Hz, 1 H), 6.44 (s (broad), 2 H), 2.60 (t, J = 7.2 Hz, 2 H), 2.35 (t, J = 7.2 Hz, 2 H), 1.89 (A2B2, t, 2 H).

[0070] Synthesis of N-(2-Amino-5-nitrophenyl)-4-(4-iodophenyl)butanethioamide:

Chemical Structure

[0071] Under a nitrogen stream at 23 °C, P2S5 (4.44 g, 20 mmol, 1.0 eq) was added to a suspension of Na2CO3 (1.08 g, 10 mmol, 0.5 eq) in THF (200 mL). After 1 hour, the mixture was cooled to 0 °C and then introduced into a solution of N-(2-amino-5-nitrophenyl)-4-(4-iodophenyl)butanamide (8.50 g, 20 mmol) in THF (100 mL). The mixture was stirred and kept at 0 °C for 2 hours and then at 23 °C for an additional 1 hour. The solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (200 mL), washed with 5% aqueous NaHCO3 (2 × 100 mL), and the aqueous phase was extracted once with EtOAc (100 mL). The organic phases were combined, dried over Na2SO4, filtered, and evaporated to dryness. The residue was sonicated in EtOAc, and the remaining solid was filtered and dried under reduced pressure to obtain the title compound (6.89 g, 76%) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 11.0 (s (broad), 1 H), 7.95 (d, J = 2.4 Hz, 1 H), 7.92 (dd, J = 8.8, 2.4 Hz, 1 H), 7.65 (d, J = 8.0 Hz, 2 H), 7.07 (d, J = 8.0 Hz, 2 H), 6.78 (d, J = 8.8 Hz, 1 H), 6.50 (s (broad), 2 H), 2.76 (t, J = 7.6 Hz, 2 H), 2.64 (t, J = 7.6 Hz, 2 H), 2.08 (A2B2, t, 2 H).

[0072] Synthesis of 4-(4-Iodophenyl)-1-(6-nitro-1H-benzo[d][1,2,3]triazol-1-yl)butane-1-thione:

Chemical Structure

[0073] A solution of N-(2-Amino-5-nitrophenyl)-4-(4-iodophenyl)butanethioamide (5.68 g, 12.8 mmol) in 95% glacial acetic acid (300 mL) was cooled to 0 °C. NaNO2 (1.32 g, 19.2 mmol, 1.5 eq) was added to the stirred mixture over 20 minutes. After 30 minutes, the product was precipitated, filtered, washed with water, and the filtrate was extracted with EtOAc (2 × 150 mL). The organic phases were combined and washed successively with H2O (3 × 100 mL), saturated NaHCO3 (2 × 100 mL), and brine (2 × 100 mL). The organic phase was separated, dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The remaining solid was sonicated with a small amount of EtOAc (5 mL). The EtOAc was decanted, and the remaining solid was filtered. The remaining product (yellow solid, 3.23 g, 56%) was dried under reduced pressure. 11H NMR (400 MHz, CDCl3) δ 9.71 (d, J = 1.6 Hz, 1 H), 8.45 (dd, J = 8.8, 2.0 Hz, 1 H), 8.31 (d, J = 8.8 Hz, 1 H), 7.59 (d, J = 8.0 Hz, 2 H), 6.98 (d, J = 8.0 Hz, 2 H), 3.80 (t, J = 7.6 Hz, 2 H), 2.79 (t, J = 7.6 Hz, 2 H), 2.33 (A2B2, t, 2 H).

[0074] N 2 -(tert-Butoxycarbonyl)-N 6 - (4-(4-Iodophenyl)butanoyl)-L-lysine synthesis:

Chem.

[0075] To a cooled solution (0 °C) of the thioacylating reagent (5 mmol, 2.26 g) in 75 mL of THF was added dropwise over 1 hour a solution of Boc-Lys-OH (5 mmol, 1.23 g) and triethylamine in 15 mL of THF and 3.0 mL of H2O. After the addition was complete, the mixture was subsequently stirred overnight at room temperature. The mixture was extracted with EtOAc, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / EtOAc, 1 / 1, followed by 100% MeOH) to give the title compound (1.18 g) in 44% yield.

[0076] N 6 - (4-(4-Iodophenyl)butanoyl)-L-lysine trifluoroacetate synthesis:

Chem.

[0077] N 2 -(tert-Butoxycarbonyl)-N 6To a solution of (4-(4-iodophenyl)butanethioyl)-L-lysine (1.05 g, 2 mmol) in dry CH2Cl2 (2 mL) was added TFA (2.0 mL), and the reaction mixture was stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the remaining solid was dried under dynamic vacuum overnight. The residue was dissolved in ethyl acetate (10.0 mL) and precipitated over 12 hours. The precipitate was filtered and dried in vacuo to give the title amino acid (0.97 g, 91%) as a gray solid. 1 H NMR (400 MHz, MeOH-d4) δ 7.61 (d, J = 8.0 Hz, 2 H), 7.00 (d, J = 8.0 Hz, 2 H), 3.97 (t, J = 6.4 Hz, 1 H), 3.63 (t, J = 7.6 Hz, 2 H), 2.67-2.52 (m, 3 H), 2.11-1.85 (m, 4 H), 1.70 (A2B2, t, 2 H), 1.63-1.40 (m, 3 H).

[0078] Example 3: Butyl N 6 -(3-(4-Iodophenyl)propanethioyl)-L-lysinate synthesis

Chemical formula

[0079] Synthesis of tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinate:

Chemical formula

[0080] (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-lysine (3 g, 8.14 mmol) was dissolved in tert-butyl acetate (42 ml) and cooled to -10 °C. Perchloric acid (1.05 ml, 12.21 mmol, 1.5 eq) was added dropwise. After 4 hours, 200 mL of EtOAc and 100 mL of deionized water were added, and the mixture was fractionated with a separating funnel. The organic phases were combined, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The product was purified by silica gel column chromatography using 100% EtOAc as the eluent. The fractions of the product were evaporated to give a pale yellow, flowable powder. 1 1H NMR (400 MHz, CD3Cl) δ 7.71 (d, J = 7.5 Hz, 2 H), 7.58 (d, J = 7.9 Hz, 2 H), 7.35 (t, 2 H, J = 7.4 Hz,), 7.28 (d, J = 5.8 Hz, 2 H), 5.52 (d, J = 7.96 Hz, 1 H), 4.32 (t, 1H), 4.37 (t, 1H), 3.12 (t, 2H), 1.41 (s, 9 H).

[0081] tert-Butyl N 2 -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N 6 -(3-(4-iodophenyl)propanethioyl)-L-lysinate synthesis:

Chemical Structure

[0082] Under a dry nitrogen atmosphere, a solution of 3-(4-iodophenyl)-1-(6-nitro-1H-benzo[d][1,2,3]triazol-1-yl)propane-1-thione (4.7 mmol, 2.05 g) in 50 mL of THF was treated with tert-butyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinate (2.06 g, 4.7 mmol). The mixture was cooled to 0 °C and DIPEA (4.2 mmol, 0.7 mL) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The mixture was neutralized with 1 M HCl and transferred to a separatory funnel containing EtOAc and deionized water. The organic fraction was isolated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH in CH2Cl2) to give the title compound in 70% yield. 1 1H NMR (400 MHz, CDCl 3 ) δ 7.78 (d, J = 7.56 Hz, 2 H), 7.57 (d, J = 7.4 Hz, 2H), 7.50 (d, J = 7.96 Hz, 2 H), 7.44 (t, J = 8.16 Hz, 2 H), 7.33 (t, J = 7.52 Hz, 2 H), 6.89 (d, J = 7.96 Hz, 2 H), 5.48 (d, J = 7.92 Hz, 1 H), 4.41 (m, 1 H), 4.33 (t, J = 7.32 Hz,1 H), 4.23 (m, 2 H), 3.56 (m, 2 H), 3.04 (m, 2 H), 2.81 (t, J = 7.52 Hz, 2 H), 1.82 (m, 1 H), 1.66 (m, 2 H), 1.50 (s, 9H), 1.38 (m, 2 H).

[0083] tert-Butyl N 6 -(3-(4-iodophenyl)propanethioyl)-L-lysinate synthesis:

Chemical formula

[0084] Under an N2 atmosphere, tert-butyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 -(3-(4-iodophenyl)propanethioyl)-L-lysinate (1.43 mmol, 1.0 g) in 15 mL of CH2Cl2 was added to a 10% solution of piperidine in CH2Cl2 (about 40 eq). (TLC during the process showed that the reaction was completed in the first 4 hours), and the mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (50% EtOAc in hexane). The product fractions were evaporated to give an off-white powder in 86% yield (0.51 g). 1 1H NMR (400 MHz, CDCl3) δ 7.66 (s (broad), 1 H), 7.61 (d, J = 8.24 Hz, 2H), 6.98 (d, J = 8.24 Hz, 2 H), 3.58 (m, 2 H), 3.28 (dd, J = 7.94, 5.08 Hz,1 H), 3.09 (t, J = 7.32 Hz, 2 H), 2.87 (t, J = 7.32 Hz, 2 H), 1.72 (m, 1 H), 1.58 (t, J = 7.20 Hz, 2 H), 1.48 (s, 9 H), 1.38 (m, 2 H).

[0085] Example 4: Further synthesis of a thioamide-containing composition

Chemical Structure

[0086] Synthesis of N-(2-amino-5-nitrophenyl)--(4-iodophenyl)propanamide:

Chemical Structure

[0087] Under an N2 atmosphere at 20 °C, carbonyl diimidazole (CDI, 8.8 mL, 80 mmol, 2.0 eq) was added to a THF solution (200 mL) of 3-(4-iodophenyl)propanoic acid (16.44 g, 40 mmol). Isobutyl chloroformate (5.2 mL, 40 mmol, 1.0 eq) was added dropwise, and the reaction mixture was stirred for 30 minutes. A solution of 4-nitro-1,2-phenylenediamine (6.12 g, 40 mmol, 1.0 eq) in THF (100 mL) was added, and the mixture was stirred at -20 °C for 1.5 hours and then at 23 °C for 15 hours. The mixture was filtered and evaporated to dryness. The residue was dissolved in EtOAc (300 mL) and washed with aqueous solutions of 1 M NaH2PO4 (2 × 100 mL), saturated brine (2 × 100 mL), saturated NaHCO3 (2 × 100 mL), and saturated NaCl (2 × 100 mL), dried over Na2SO4, and evaporated to dryness. The crude product was sonicated in EtOAc until it solidified. The EtOAc was decanted, the remaining solid was filtered, evaporated under reduced pressure, and the title compound (10.77 g, 63%) was obtained as a yellowish-brown solid. 1 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1 H), 8.20 (d, J = 2.6 Hz, 1 H), 7.84 (dd, J = 7.8, 2.6 Hz, 1 H), 7.64 (d, J = 8.2 Hz, 2 H), 7.10 (d, J = 8.2 Hz, 2 H), 6.75 (d, J = 9.04 Hz, 1 H), 6.43 (s (br), 2 H), 2.88 (t, J = 7.3 Hz, 2 H), 2.66 (t, J = 8.04 Hz, 2 H).

[0088] Synthesis of N-(2-amino-5-nitrophenyl)-3-(4-iodophenyl)propanethioamide:

Chemical formula

[0089] Under a stream of N2, at 23 °C, P2S5 (4.44 g, 20 mmol, 1.0 eq) was added to a suspension of Na2CO3 (1.08 g, 10 mmol, 0.5 eq) in THF (200 mL). After 1 hour, the mixture was cooled to 0 °C, and N-(2-amino-5-nitrophenyl)-3-(4-iodophenyl)propanamide (8.22 g, 20 mmol) in THF (100 mL) was added dropwise. The mixture was stirred at 0 °C for 2 hours and then at 23 °C for 1 hour. The solvent was evaporated, and the residue was dissolved in EtOAc (200 mL) and washed with 5% aqueous NaHCO3 (2 × 100 mL). The aqueous phase was extracted with additional EtOAc (100 mL), the organic phases were combined, dried over Na2SO4, filtered, and evaporated. The crude product was sonicated in EtOAc until solidified. The EtOAc was decanted, and the remaining solid was filtered and dried under reduced pressure to obtain the title compound (6.89 g, 76%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 11.0 (s (broad), 1 H), 7.84 (dd, J = 9.08, J = 2.4 Hz, 1 H), 7.84 (d, J = 2.64 Hz, 1 H), 7.67 (d, J = 8.24 Hz, 2 H), 7.12 (d, J = 8.28, 2 H), 6.77 (d, J = 9.12 Hz, 1 H), 6.43 (s (broad), 2 H), 3.04 (m, 2 H), 3.06 (m, 2 H).

[0090] Synthesis of 3-(4-iodophenyl)-1-(6-nitro-1H-benzo[d][1,2,3]triazol-1-yl)propane-1-thione:

Chemical Structure

[0091] N-(2-Amino-5-nitrophenyl)-3-(4-iodophenyl)propanethioamide (5.46 g, 12.8 mmol) was warmed to 40 °C in 95% glacial acetic acid, diluted with 5% water (300 mL), and then cooled to 0 °C. NaNO2 (1.32 g, 19.2 mmol, 1.5 eq) was added portionwise to the stirred solution over 20 minutes. After 30 minutes, the precipitated product was filtered, washed with water, and the filtrate was extracted with EtOAc (2 × 150 mL). The organic phases were combined and washed successively with H2O (3 × 100 mL), saturated NaHCO3 (2 × 100 mL), and brine (2 × 100 mL). The organic phase was separated, dried over Na2SO4, filtered, and evaporated. The solid thus obtained was sonicated in a small amount of EtOAc (5 mL), the mixture was filtered, and dried under dynamic vacuum to give the product as a yellow solid (3.23 g, 56%). 1 1H NMR (400 MHz, CDCl3) δ 9.73 (d, J = 1.96 Hz, 1 H), 8.47 (dd, J = 8.9, 2.08 Hz, 1 H), 8.33 (d, J = 8.9 Hz, 1 H), 7.64 (d, J = 8.32 Hz, 2 H), 7.06 (d, J = 8.28 Hz, 2 H), 4.08 (t, J = 7.64 Hz, 2 H), 3.28 (t, J = 7.92 Hz, 2 H).

[0092] Synthesis of N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N6-(3-(4-iodophenyl)propanethioyl)-D-lysine:

Chemical Structure

[0093] A solution of 3-(4-iodophenyl)-1-(6-nitro-1H-benzo[d][1,2,3]triazol-1-yl)propane-1-thione (0.46 mmol, 200.0 mg) in 4 mL of THF was added to Fmoc-L-Lys (0.46 mmol, 169.5 mg). After cooling the mixture to 0 °C, DIPEA (0.46 mmol, 81 μL) was added dropwise. After stirring the mixture at room temperature for 12 hours, it was neutralized with 1 M HCl and diluted with deionized water. The mixture was extracted with EtOAc, dried over Na2SO4, filtered, and evaporated. The residue was purified by chromatography (10% MeOH in CH2Cl2) to give the title compound in 20% yield. 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1 H), 7.88 (d, J = 7.48 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2 H), 7.60 (d, J = 8.16 Hz, 2 H), 7.40 (t, J = 7.36 Hz, 2 H), 7.32 (t, J = 7.44 Hz, 2 H), 7.02 (d, J = 8.12 Hz, 2 H), 6.90 (s, 1 H), 4.29 (m, 1 H), 4.21 (m, 2 H), 3.77 (m, 1 H), 2.92 (t, J = 8.12 Hz, 2 H), 2.77 (t, J = 8.16 Hz, 2 H), 2.92 (t, J = 8.12 Hz, 2 H), 1.70 (m, 1 H), 1.58 (m, 1 H), 1.48 (m, 2 H), 1.27 (m, 2 H).

[0094] Example 5: Suitable lysine derivatives

Chem.

[0095] Example 6: Stability against hydrolysis of thioamides

Chem.

[0096] Example 7: General procedure for formation of bioconjugates characterized by thioamide-albumin binding groups

Chem.

[0097] Under a dry nitrogen atmosphere, in a flame-dried flask, tert-butyl N 6 -(3-(4-iodophenyl)propanethioyl)-L-lysinate (0.200 g, 0.420 mmol, 1 eq) was dissolved in 2 mL of dry methylene chloride and set to stir. After adding anhydrous triethylamine (5.90 μL, 0.042 mmol, 0.1 eq) with a syringe, Fmoc-PEG8-NHS ester (0.383 g, 0.504 mmol, 1.2 eq) was dissolved in 1 mL of dry methylene chloride. The mixture was stirred at room temperature for 4 hours, then the solvent was evaporated, leaving a pale yellow syrup. The residue was purified by silica gel flash column chromatography (10% MeOH / DCM) to give the title compound as a pale yellow syrup (0.188 g, 40%).

[0098] tert-butyl N 2 -(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatricontan-31-yl)-N 6 -(3-(4-iodophenyl)propanethioyl)-L-lysinate synthesis:

Chem.

[0099] tert-butyl N 2 -(1-(9H-fluoren-9-yl)-3-thioxo-2,7,10-trioxa-4-azatridecan-13-yl)-N 6-(3-(4-Iodophenyl)propanethioyl)-L-lysinate (0.188 g, 0.168 mmol) was dissolved in a 10% piperidine solution in dry methylene chloride (5 mL). The mixture was stirred at room temperature for 4 hours, then the solvent was removed, leaving a pale yellow powder. The residue was purified by silica gel flash column chromatography (5% MeOH / DCM) to give a pale yellow syrup (0.133 g, 88%).

[0100] Synthesis of tert-butyl N2-((S)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12-diazanonatriacontan-39-oil)-N6-(3-(4-iodophenyl)propanethioyl)-L-lysinate:

Chemical Structure

[0101] Under a dry nitrogen atmosphere, in a flame-dried flask, tert-butyl N 2 -(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azapentatriacontan-31-oil)-N 6 -(3-(4-iodophenyl)propanethioyl)-L-lysinate (0.133 g, 0.147 mmol, 1 eq) was dissolved in 3 mL of dry methylene chloride. To the stirred solution, dry triethylamine (20.50 μL, 0.147 mmol, 1 eq) was added, and then a solution of Boc-Lys(Boc)-OSu (71.71 mg, 0.161 mmol, 1.1 eq) in 3 mL of dry methylene chloride was added. The mixture was stirred at room temperature for 4 hours, then the solvent was removed under reduced pressure, leaving a yellow solid. The residue was purified by silica gel flash column chromatography (5% MeOH / DCM) to give a yellow syrup (0.139 g, 70%).

[0102] Synthesis of tert-butyl N2-((S)-10-amino-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12-diazahentriacontan-39-oil)-N6-(3-(4-iodophenyl)propanoyl)-L-lysinate: [Chemical formula]

[0103] tert-Butyl N2-((S)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12-diazahentriacontan-39-oil)-N6-(3-(4-iodophenyl)propanoyl)-L-lysinate (0.139 g, 0.103 mmol) was treated with a solution of piperidine (10%) in 5 mL of dry methylene chloride. The mixture was stirred at room temperature for 4 hours, then the solvent was removed under reduced pressure, leaving a pale yellow powder. The residue was purified by silica gel flash column chromatography (5% MeOH / DCM) to give a pale yellow syrup (0.093 g, 80%).

[0104] Synthesis of N6-(3-(4-iodophenyl)propanoyl)-N2-(17-oxo-21-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azaheneicosanoyl)-L-lysine: [Chemical formula]

[0105] (S)-1-Carboxy-5-(3-(4-iodophenyl)propanethioamide)penta-1-aminium trifluoroacetate salt (0.100 g, 0.187 mmol, 1 eq) was dissolved in 1 mL of DMF (dimethylformamide), and triethylamine (52.20 μL, 0.374 mmol, 2 eq) was added. When triethylammonium trifluoroacetate began to precipitate, a potassium carbonate buffer (pH = 8) solution was added dropwise to form a homogeneous solution. Subsequently, NHS-PEG4-biotin (0.121 g, 0.205 mmol, 1.1 eq) was dissolved in 1 mL of DMF, added to the solution, and the mixture was stirred for 8 hours. Deionized water (2 mL) and methylene chloride (5 mL) were added, and the organic phase was washed with water (3 x 5 mL) to remove DMF. The organic phase was evaporated, and the colorless powdery residue was purified by silica gel flash column chromatography (5% MeOH / DCM) to obtain an off-white powder (0.100 g, 60%).

[0106] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is understood that any concepts or elements of the invention that are shown separately for convenience are also included in the invention, in combination, as necessary. Such equivalents are intended to be encompassed by the claims.

[0107] The contents of the patents and literature cited herein are incorporated by reference in their entirety.

Claims

1. Formula (I): 【Chemical 1】 [wherein, R 1 is H, C 1 -C 6 alkyl or a protecting group; R 2 is H, C 1 -C 6 alkyl or a protecting group; R 3 is H, C 1 -C 6 alkyl or a protecting group; X is a therapeutic agent; and n is 0, 1, 2, 3, 4 or 5]] A compound represented by the formula or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein n is 2 or 3.

3. The compound represented by formula (I) is a compound represented by formula (II): [Chemical Formula 2] [wherein, R 1 is H, C 1 -C 6 alkyl or a protecting group; R 2 is H, C 1 -C 6 alkyl or a protecting group; R 3 is H, C 1 -C 6 alkyl or a protecting group; L 1 is a natural amino acid, unnatural amino acid or (X) q -(Y) r -(Z) s wherein X is C 1 -C 30 alkyl, Y is C 10 -C 30 heteroaromatic, and Z is C 1 -C 12 alkyl, wherein any of the methylene groups in the alkyl group of L 1 may be replaced by -O-, NH or carbonyl; R 4 is H, C 1 -C 6 alkyl or a protecting group; R 5 is H, C 1 -C 6 alkyl or a protecting group; R 6 is a therapeutic agent or a chelating agent; R 7 is H, C 1 -C 6 alkyl or a protecting group; R 8 is H, C 1 -C 6 alkyl or a protecting group; L 2 is a linking, -N(R 9 )-C 1 -C 12 alkyl-C(O)-, -N(R 9 )-C 4 -C 30 alkylcycloalkyl-C(O)-C 7 -C 30 alkylaryl-C(O)-, or -N(R 9 )-C 7 -C 30 alkylaryl-C(O)NH-C 7 -C 30 alkylaryl-C(O)NH-CH(CO 2 H)-C 1 -C 12 alkyl-NHC(O)-C 1 -C 12 alkyl-C(O)-, where the C 7 -C 30 alkylaryl may optionally be substituted by halo or hydroxyl; n is 0, 1, 2, 3, 4 or 5; m is 0, 1, 2, 3, 4 or 5; p is 0, 1, 2, 3, 4 or 5; q is 0 or 1; r is 0 or 1; and s is 0 or 1]] The compound according to claim 1, which is a compound represented by the formula or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, wherein n is 3.

5. L 1 is X-Y-Z, where X is [Chemical Formula 3] ; Y is 【Chemical Formula 4】 ; Z is C 1 -C 12 alkyl, wherein any of the methylene groups in the alkyl group may be replaced by NH or carbonyl, the compound according to claim 3.

6. L 1 is Z, where Z is C 1 -C 12 alkyl, wherein any of the methylene groups in the alkyl group may be replaced by NH or carbonyl, the compound according to claim 3.

7. Z is 【Chemical Formula 5】 The compound according to claim 6.

8. The chelating agent is 【Chemical Formula 6】 The compound according to claim 1.

9. L 2 is -N(R 9 )-C 1 -C 12 alkyl-C(O)-, the compound according to claim 1.

10. L 2 is 【Chemical Formula 7】 The compound according to claim 9.

11. L 2 is -N(R 9 )-C 4 -C 30 alkylcycloalkyl - C(O)-C 7 -C 30 alkylaryl - C(O)-, the compound according to claim 1.

12. L 2 is 【Chemical 8】 The compound according to claim 12.

13. L 2 is -N(R 9 )-C 7 -C 30 alkylaryl -C(O)NH-C 7 -C 30 alkylaryl -C(O)NH-CH(CO 2 H)-C 1 -C 12 alkyl -NHC(O)-C 1 -C 12 alkyl -C(O)-, where C 7 -C 30 alkylaryl is the compound according to claim 1, which may optionally be substituted by halo or hydroxyl.

14. L 2 is [Chemical Formula 9] The compound according to claim 13.

15. L 2 is 【Chemical 10】 The compound according to claim 13.

16. The compound represented by formula (I) is a compound represented by formula (III): 【Chemical 11】 [wherein, R 1 is H, C 1 -C 6 alkyl or a protecting group; R 2 is H, C 1 -C 6 alkyl or a protecting group; R 3 is H, C 1 -C 6 alkyl or a protecting group; L 1 is a natural amino acid, an unnatural amino acid or (X) q -(Y) r -(Z) s wherein X is C 1 -C 20 alkyl, Y is C 10 -C 30 aryl, and Z is C 1 -C 12 alkyl, wherein any of the methylene groups in the alkyl group of L 1 may be replaced by -O-, NH, carbonyl or thiocarbonyl; R 4 is H, C 1 -C 6 alkyl or a protecting group; R 5 is H, C 1 -C 6 alkyl or a protecting group; R 6 is a therapeutic agent or a chelating agent; R 7 is H, C 1 -C 6 alkyl or a protecting group; R 8 is H, C 1 -C 6 alkyl or a protecting group; R 9 is H, C 1 -C 6 alkyl or a protecting group; R 10 is H, C 1 -C 6 alkyl or a protecting group; L 2 is C 1 -C 30 alkyl-C 3 -C 18 heteroaryl-C 6 -C 18 aryl, wherein any of the methylene groups in the alkyl group may be replaced by -O-; n is 0, 1, 2, 3, 4 or 5; m is 0, 1, 2, 3, 4 or 5; p is 0, 1, 2, 3, 4 or 5; q is 0 or 1; r is 0 or 1; and s is 0 or 1]] The compound according to claim 1, which is a compound represented by the formula or a pharmaceutically acceptable salt thereof.

17. The compound according to claim 16, wherein n is 3.

18. L 1 is X-Y-Z, where X is 【Chemical 12】 ; Y is C 10 -C 30 is aryl; and Z is C 1 -C 12 alkyl, wherein any of the methylene groups in the alkyl group may be replaced by NH or carbonyl, the compound according to claim 16.

19. The chelating agent is 【Chemical 13】 The compound according to claim 16.

20. L 2 is 【Chemical 14】 The compound according to claim 16.

21. Formula (IV): 【Chemical Formula 15】 [wherein, R 1 is H, C 1 -C 6 alkyl or a protecting group; R 2 is H, C 1 -C 6 alkyl or a protecting group; and n is 0, 1, 2, 3, 4 or 5]] A compound represented by the formula or a pharmaceutically acceptable salt thereof.

22. The compound according to claim 1, wherein n is 2 or 3.

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