Conjugates comprising self-immolative groups and methods related thereto

1. A ligand-drug conjugate having a structure represented by formula I

wherein:

G is

A is a ligand;

B is an active agent;

W is —C(O)NR′—, —C(O)O—, —S(O) 2 NR′—, —P(O)R″NR′—, —S(O)NR′—, or —PO 2 NR′—, in each case wherein the C(O), S, or P is directly bound to the phenyl ring;

R′ and R″ are each independently hydrogen, (C 1 -C 8 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, mono- or di-(C 1 -C 8 )alkylamino, (C 3 -C 20 )heteroaryl, or (C 6 -C 20 )aryl;

each Z independently is hydrogen, (C 1 -C 8 )alkyl, or an electron-withdrawing;

n is 1 to 3;

m is 0 or 1;

L is a linker that covalently links A to W, wherein the linker comprises an alkylene having 1 to 100 carbon atoms and further comprises a hydrophilic amino acid, and wherein the nitrogen of W is bonded to the hydrophilic amino acid;

R 1 and R 2 are each independently hydrogen, (C 1 -C 8 )alkyl, or (C 3 -C 8 )cycloalkyl, or R 1 and R 2 taken together with the carbon atom to which they are attached form a (C 3 -C 8 )cycloalkyl ring;

n 10 is 1-12;

R 3 is hydrogen or a carboxyl-protecting group; and

each R 4 is independently hydrogen or a hydroxyl-protecting group.

2. The conjugate of claim 1 , wherein R 3 and each R 4 are hydrogen.

3. The conjugate of claim 1 , wherein W is —C(O)NR′—, wherein the C(O) is bonded to the phenyl ring and NR′ is bonded to L.

4. The conjugate of claim 1 , wherein the alkylene comprises either:

i) the alkylene comprises at least one unsaturated bond;

ii) the alkylene comprises at least one heteroarylene;

iii) A carbon atom of the alkylene is replaced by one or more heteroatoms selected from nitrogen (N), oxygen (O), and sulfur (S); or

iv) the alkylene is substituted with one or more alkyls having 1 to 20 carbon atoms.

5. The conjugate of claim 1 , wherein W represents —C(O)NR′— and the nitrogen of W is a nitrogen atom of a hydrophilic amino acid.

6. The conjugate of claim 5 , wherein the hydrophilic amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

7. The conjugate of claim 1 , wherein the linker is covalently bound to the ligand by a thioether bond, and the thioether bond comprises a sulfur atom of a cysteine of the ligand.

8. The conjugate of claim 7 , wherein the linker comprises at least one isoprenyl unit.

9. The conjugate of claim 8 , wherein the linker comprises an oxime and the at least one isoprenyl unit covalently links the oxime to the ligand.

10. The conjugate of claim 1 , wherein the linker comprises a connection unit represented by —(CH 2 ) r (V(CH 2 ) p ) q —, —((CH 2 ) p V) q —, —(CH 2 ) r (V(CH 2 ) p ) q Y—, —((CH 2 ) p V) q (CH 2 ) r —, —Y(((CH 2 ) p V) q — or —(CH 2 ) r (V(CH 2 ) p ) q YCH 2 —

wherein:

r is an integer from 0 to 10;

p is an integer from 1 to 10;

q is an integer from 1 to 20;

V and Y are each independently a single bond, —O—, —S—, —NR 21 —, —C(O)NR 22 —, —NR 23 C(O)—, —NR 24 SO 2 —, or —SO 2 NR 25 —; and

R 21 to R 25 are each independently hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkyl(C 6 -C 20 )aryl or (C 1 -C 6 )alkyl(C 3 -C 20 )heteroaryl.

11. The conjugate of claim 10 , wherein q is an integer from 4 to 20.

12. The conjugate of claim 10 , wherein r is 2.

13. The conjugate of claim 10 , wherein p is 2.

14. The conjugate of claim 10 , wherein V and Y are each independently —O—.

15. The conjugate of claim 1 , wherein the linker comprises a binding unit formed by a 1,3-dipolar cycloaddition reaction, hetero-Diels-Alder reaction, nucleophilic substitution reaction, non-aldol type carbonyl reaction, addition to carbon-carbon multiple bond, oxidation reaction, or click reaction.

16. The conjugate of claim 15 , wherein the binding unit is represented by Formula A, B, C, or D:

wherein:

L 1 is a single bond or alkylene having 1 to 30 carbon atoms;

R 11 is hydrogen or alkyl having 1 to 10 carbon atoms; and

L 2 is alkylene having 1 to 30 carbon atoms.

17. The conjugate of claim 1 , comprising the structure:

wherein A represents the ligand; B represents the active agent; and n is 1 to 20.

18. The conjugate of claim 1 , wherein the ligand is an antibody.

19. The conjugate of claim 1 , wherein the ligand is a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab′, Fab′-SH, F(ab′) 2 , Fv, single chain Fv (“scFv”), diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, or fusion protein comprising the antigen-binding portion of an antibody.

20. The conjugate of claim 1 , wherein the ligand is selected from muromonab-CD3 abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab, efalizumab, tositumomab, bevacizumab, natalizumab, ranibizumab, eculizumab, rilonacept, certolizumab, romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept, belimumab, atacicept, an anti-CD20 antibody, canakinumab, tocilizumab, atlizumab, mepolizumab, pertuzumab, tremelimumab, ticilimumab, ipilimumab, IDEC-114, inotuzumab, aflibercept, teplizumab, otelixizumab, catumaxomab, the anti-EpCAM antibody IGN101, adecatumomab, oregovomab, dinutuximab, girentuximab, denosumab, bapineuzumab, motavizumab, efumgumab, raxibacumab, an anti-CD20 antibody, LY2469298, and veltuzumab.

21. The conjugate of claim 1 , wherein the ligand is an antibody and the antibody does not specifically bind CD19 or EGFR.

22. The conjugate of claim 1 , wherein the active agent is a chemotherapeutic agent or a toxin.

23. The conjugate of claim 1 , wherein the active agent is selected from:

(a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, antrmycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates or acids of any of the foregoing;

(b) monokine, a lympokine, a traditional polypeptide hormone, parathyroid hormone, thyroxine, relaxin, prorelaxin, a glycoprotein hormone, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-a, tumor necrosis factor-B, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, an osteoinductive factor, an interferon, interferon-α, interferon-β, interferon-γ, a colony stimulating factor (“CSF”), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, an interleukin (“IL”), IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, a tumor necrosis factor, TNF-α, TNF-β, a polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing;

(c) diphtheria toxin, botulium toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, O-amanitin, a derivative of amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, a dolastatin analog, cryptophycin, camptothecin, a derivative or metabolite of camptothecin, rhizoxin, a rhizoxin derivative, CC-1065, a CC-1065 analogue or derivative, duocarmycin, an enediyne antibiotic, esperamicin, epothilone, azonafide, aplidine, a toxoid, or a combination of any of the foregoing;

(d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, a stimulating agent, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;

(e) a radioactive label, 32 P, 35 S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule with a sequence complementary to a target, or a combination of any of the foregoing;

(f) an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, and an anti-parasitic agent, or a combination of any of the foregoing;

(g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene;

(h) 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;

(i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

(j) an aromatase inhibitor;

(k) a protein kinase inhibitor;

(l) a lipid kinase inhibitor;

(m) an antisense oligonucleotide;

(n) a ribozyme;

(o) a vaccine; and

(p) an anti-angiogenic agent.

24. The conjugate of claim 1 , wherein the conjugate comprises a structure selected from:

and

the wavy line wavy line represents a bond to the rest of L.

25. A pharmaceutical composition comprising the conjugate of claim 1 and a pharmaceutically acceptable excipient.

26. A method of treating cancer in a subject, comprising administering the conjugate of claim 1 to the subject.

27. A method for making the conjugate of claim 1 , comprising reacting a biomolecule with a prodrug, wherein:

the biomolecule comprises a ligand and a ketone or aldehyde;

the prodrug comprises an alkoxyamine; and

the reaction produces an oxime, thereby covalently linking the ligand to the prodrug.

28. A method for making the conjugate of claim 1 , comprising isoprenylating a ligand, wherein:

the ligand comprises an amino acid motif that is recognized by an isoprenoid transferase;

isoprenylating the ligand comprises incubating the ligand with an isoprenoid transferase and an isoprenoid transferase substrate; and

the substrate comprises the active agent.