WO2011140376A1 - Controlled drug release from dendrimers - Google Patents
Controlled drug release from dendrimers Download PDFInfo
- Publication number
- WO2011140376A1 WO2011140376A1 PCT/US2011/035403 US2011035403W WO2011140376A1 WO 2011140376 A1 WO2011140376 A1 WO 2011140376A1 US 2011035403 W US2011035403 W US 2011035403W WO 2011140376 A1 WO2011140376 A1 WO 2011140376A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dendrimer
- optionally substituted
- drug
- solution
- lys
- Prior art date
Links
- 239000000412 dendrimer Substances 0.000 title claims abstract description 234
- 229920000736 dendritic polymer Polymers 0.000 title claims abstract description 224
- 238000013267 controlled drug release Methods 0.000 title description 2
- 239000003814 drug Substances 0.000 claims abstract description 191
- 229940079593 drug Drugs 0.000 claims abstract description 187
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 41
- 229940002612 prodrug Drugs 0.000 claims abstract description 35
- 239000000651 prodrug Substances 0.000 claims abstract description 35
- 238000007068 beta-elimination reaction Methods 0.000 claims abstract description 21
- 150000003384 small molecules Chemical class 0.000 claims abstract description 16
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 229920001223 polyethylene glycol Polymers 0.000 claims description 79
- 150000001875 compounds Chemical class 0.000 claims description 74
- 239000002202 Polyethylene glycol Substances 0.000 claims description 72
- 238000000034 method Methods 0.000 claims description 46
- 238000005859 coupling reaction Methods 0.000 claims description 40
- 125000003118 aryl group Chemical group 0.000 claims description 39
- 230000008878 coupling Effects 0.000 claims description 38
- 238000010168 coupling process Methods 0.000 claims description 38
- 125000001072 heteroaryl group Chemical group 0.000 claims description 38
- 125000001424 substituent group Chemical group 0.000 claims description 36
- 125000000217 alkyl group Chemical group 0.000 claims description 30
- 229910052760 oxygen Inorganic materials 0.000 claims description 27
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 25
- 125000004446 heteroarylalkyl group Chemical group 0.000 claims description 25
- 125000000524 functional group Chemical group 0.000 claims description 24
- 229920000729 poly(L-lysine) polymer Polymers 0.000 claims description 22
- 229920000962 poly(amidoamine) Polymers 0.000 claims description 21
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 19
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 14
- 229920000642 polymer Polymers 0.000 claims description 12
- 229910052717 sulfur Inorganic materials 0.000 claims description 12
- 229910052727 yttrium Inorganic materials 0.000 claims description 12
- 125000003107 substituted aryl group Chemical group 0.000 claims description 11
- 230000007246 mechanism Effects 0.000 claims description 10
- 230000001681 protective effect Effects 0.000 claims description 10
- 125000004946 alkenylalkyl group Chemical group 0.000 claims description 6
- 125000005038 alkynylalkyl group Chemical group 0.000 claims description 6
- 125000005017 substituted alkenyl group Chemical group 0.000 claims description 4
- 125000004426 substituted alkynyl group Chemical group 0.000 claims description 4
- 238000005304 joining Methods 0.000 claims description 2
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 92
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 11
- 238000013270 controlled release Methods 0.000 abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 263
- 239000000243 solution Substances 0.000 description 194
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 138
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 136
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 119
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 116
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 115
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 106
- 238000002360 preparation method Methods 0.000 description 92
- 229940093499 ethyl acetate Drugs 0.000 description 86
- 235000019439 ethyl acetate Nutrition 0.000 description 86
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 81
- 239000011347 resin Substances 0.000 description 75
- 229920005989 resin Polymers 0.000 description 75
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 69
- 239000000562 conjugate Substances 0.000 description 68
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 67
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 60
- 239000000047 product Substances 0.000 description 60
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 59
- -1 PEG-camptothecin ester Chemical class 0.000 description 59
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 58
- 239000000543 intermediate Substances 0.000 description 56
- 238000006243 chemical reaction Methods 0.000 description 55
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 53
- 239000007787 solid Substances 0.000 description 50
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 42
- 125000005647 linker group Chemical group 0.000 description 36
- 229940086542 triethylamine Drugs 0.000 description 33
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 32
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 28
- 230000015572 biosynthetic process Effects 0.000 description 28
- 239000011541 reaction mixture Substances 0.000 description 28
- 239000000725 suspension Substances 0.000 description 28
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 26
- 238000003786 synthesis reaction Methods 0.000 description 26
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 25
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 24
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 24
- 150000001412 amines Chemical class 0.000 description 24
- 230000002829 reductive effect Effects 0.000 description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 23
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 23
- 238000003756 stirring Methods 0.000 description 23
- 125000003277 amino group Chemical group 0.000 description 22
- 239000003921 oil Substances 0.000 description 22
- 235000019198 oils Nutrition 0.000 description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 21
- FZFAMSAMCHXGEF-UHFFFAOYSA-N chloro formate Chemical compound ClOC=O FZFAMSAMCHXGEF-UHFFFAOYSA-N 0.000 description 21
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 150000001345 alkine derivatives Chemical class 0.000 description 20
- 238000001914 filtration Methods 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 20
- 229920002521 macromolecule Polymers 0.000 description 20
- 239000000741 silica gel Substances 0.000 description 20
- 229910002027 silica gel Inorganic materials 0.000 description 20
- 239000012267 brine Substances 0.000 description 17
- 230000021615 conjugation Effects 0.000 description 17
- 239000000284 extract Substances 0.000 description 17
- 238000000746 purification Methods 0.000 description 17
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 17
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 16
- 235000019341 magnesium sulphate Nutrition 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 238000005160 1H NMR spectroscopy Methods 0.000 description 15
- 238000005406 washing Methods 0.000 description 15
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 14
- 150000001408 amides Chemical class 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 14
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 14
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 150000003573 thiols Chemical class 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 12
- 150000001540 azides Chemical class 0.000 description 12
- 239000002585 base Substances 0.000 description 12
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Natural products O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 12
- 239000010410 layer Substances 0.000 description 12
- 239000000178 monomer Substances 0.000 description 12
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 11
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 11
- 239000012043 crude product Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 150000002148 esters Chemical class 0.000 description 11
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 229910000029 sodium carbonate Inorganic materials 0.000 description 11
- 238000010532 solid phase synthesis reaction Methods 0.000 description 11
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- 125000000304 alkynyl group Chemical group 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 10
- ZCSHNCUQKCANBX-UHFFFAOYSA-N lithium diisopropylamide Chemical compound [Li+].CC(C)[N-]C(C)C ZCSHNCUQKCANBX-UHFFFAOYSA-N 0.000 description 10
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 description 10
- ZJIFDEVVTPEXDL-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) hydrogen carbonate Chemical compound OC(=O)ON1C(=O)CCC1=O ZJIFDEVVTPEXDL-UHFFFAOYSA-N 0.000 description 9
- WKGZJBVXZWCZQC-UHFFFAOYSA-N 1-(1-benzyltriazol-4-yl)-n,n-bis[(1-benzyltriazol-4-yl)methyl]methanamine Chemical compound C=1N(CC=2C=CC=CC=2)N=NC=1CN(CC=1N=NN(CC=2C=CC=CC=2)C=1)CC(N=N1)=CN1CC1=CC=CC=C1 WKGZJBVXZWCZQC-UHFFFAOYSA-N 0.000 description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 9
- 238000000502 dialysis Methods 0.000 description 9
- 239000000706 filtrate Substances 0.000 description 9
- 229960002949 fluorouracil Drugs 0.000 description 9
- 235000010378 sodium ascorbate Nutrition 0.000 description 9
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 9
- 229960005055 sodium ascorbate Drugs 0.000 description 9
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 239000012258 stirred mixture Substances 0.000 description 9
- 0 CC(C(*)=O)NC(C(CCCC**)NC(OC(C)(C)C)=O)=O Chemical compound CC(C(*)=O)NC(C(CCCC**)NC(OC(C)(C)C)=O)=O 0.000 description 8
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 8
- 108010039918 Polylysine Proteins 0.000 description 8
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 8
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 8
- 230000006320 pegylation Effects 0.000 description 8
- 229920000656 polylysine Polymers 0.000 description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 7
- 238000005481 NMR spectroscopy Methods 0.000 description 7
- 235000019647 acidic taste Nutrition 0.000 description 7
- 125000003342 alkenyl group Chemical group 0.000 description 7
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 7
- JUFFVKRROAPVBI-PVOYSMBESA-N chembl1210015 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N[C@H]1[C@@H]([C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@]3(O[C@@H](C[C@H](O)[C@H](O)CO)[C@H](NC(C)=O)[C@@H](O)C3)C(O)=O)O2)O)[C@@H](CO)O1)NC(C)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 JUFFVKRROAPVBI-PVOYSMBESA-N 0.000 description 7
- KOUDNRIDVHWUCH-UHFFFAOYSA-N chloromethylcarbamic acid Chemical compound OC(=O)NCCl KOUDNRIDVHWUCH-UHFFFAOYSA-N 0.000 description 7
- 238000002425 crystallisation Methods 0.000 description 7
- 230000008025 crystallization Effects 0.000 description 7
- 238000003379 elimination reaction Methods 0.000 description 7
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 7
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 7
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 238000010898 silica gel chromatography Methods 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 7
- 239000004472 Lysine Substances 0.000 description 6
- OFKKPUNNTZKBSR-VIFPVBQESA-N N(6)-(2,4-dinitrophenyl)-L-lysine Chemical compound OC(=O)[C@@H](N)CCCCNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O OFKKPUNNTZKBSR-VIFPVBQESA-N 0.000 description 6
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 6
- 235000019502 Orange oil Nutrition 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 125000002843 carboxylic acid group Chemical group 0.000 description 6
- 238000000354 decomposition reaction Methods 0.000 description 6
- RMBPEFMHABBEKP-UHFFFAOYSA-N fluorene Chemical compound C1=CC=C2C3=C[CH]C=CC3=CC2=C1 RMBPEFMHABBEKP-UHFFFAOYSA-N 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- 150000002576 ketones Chemical class 0.000 description 6
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 6
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 6
- NIHNNTQXNPWCJQ-UHFFFAOYSA-N o-biphenylenemethane Natural products C1=CC=C2CC3=CC=CC=C3C2=C1 NIHNNTQXNPWCJQ-UHFFFAOYSA-N 0.000 description 6
- 239000010502 orange oil Substances 0.000 description 6
- 239000012074 organic phase Substances 0.000 description 6
- PWXJULSLLONQHY-UHFFFAOYSA-N phenylcarbamic acid Chemical compound OC(=O)NC1=CC=CC=C1 PWXJULSLLONQHY-UHFFFAOYSA-N 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 239000002243 precursor Substances 0.000 description 6
- 238000004007 reversed phase HPLC Methods 0.000 description 6
- 239000012279 sodium borohydride Substances 0.000 description 6
- 229910000033 sodium borohydride Inorganic materials 0.000 description 6
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 150000003457 sulfones Chemical class 0.000 description 6
- 238000003828 vacuum filtration Methods 0.000 description 6
- 125000001917 2,4-dinitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C(=C1*)[N+]([O-])=O)[N+]([O-])=O 0.000 description 5
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 5
- 230000001588 bifunctional effect Effects 0.000 description 5
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 5
- 229940127093 camptothecin Drugs 0.000 description 5
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 5
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 5
- 239000012230 colorless oil Substances 0.000 description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 5
- 235000018417 cysteine Nutrition 0.000 description 5
- XCEBOJWFQSQZKR-UHFFFAOYSA-N dbco-nhs Chemical compound C1C2=CC=CC=C2C#CC2=CC=CC=C2N1C(=O)CCC(=O)ON1C(=O)CCC1=O XCEBOJWFQSQZKR-UHFFFAOYSA-N 0.000 description 5
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 238000001704 evaporation Methods 0.000 description 5
- 230000008020 evaporation Effects 0.000 description 5
- 229960001519 exenatide Drugs 0.000 description 5
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 5
- 210000002751 lymph Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000010647 peptide synthesis reaction Methods 0.000 description 5
- 230000009257 reactivity Effects 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- 229920006395 saturated elastomer Polymers 0.000 description 5
- HHVIBTZHLRERCL-UHFFFAOYSA-N sulfonyldimethane Chemical compound CS(C)(=O)=O HHVIBTZHLRERCL-UHFFFAOYSA-N 0.000 description 5
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 5
- 125000003396 thiol group Chemical group [H]S* 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- NIFAOMSJMGEFTQ-UHFFFAOYSA-N 4-methoxybenzenethiol Chemical class COC1=CC=C(S)C=C1 NIFAOMSJMGEFTQ-UHFFFAOYSA-N 0.000 description 4
- OQKYEMHWZYHWBL-UHFFFAOYSA-N 9h-fluoren-1-ylmethanol Chemical compound C1C2=CC=CC=C2C2=C1C(CO)=CC=C2 OQKYEMHWZYHWBL-UHFFFAOYSA-N 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- 102100036864 Electroneutral sodium bicarbonate exchanger 1 Human genes 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108010011459 Exenatide Proteins 0.000 description 4
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 4
- 108010087132 Sodium-Bicarbonate Symporters Proteins 0.000 description 4
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 4
- 239000000556 agonist Substances 0.000 description 4
- 125000003545 alkoxy group Chemical group 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 150000001718 carbodiimides Chemical class 0.000 description 4
- PVAWKBXCZQBVLC-UHFFFAOYSA-N carbonic acid;1-hydroxypyrrolidine-2,5-dione Chemical compound OC(O)=O.ON1C(=O)CCC1=O PVAWKBXCZQBVLC-UHFFFAOYSA-N 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000013058 crude material Substances 0.000 description 4
- 125000004093 cyano group Chemical group *C#N 0.000 description 4
- 125000004122 cyclic group Chemical group 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 125000005843 halogen group Chemical group 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 239000005457 ice water Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- YNESATAKKCNGOF-UHFFFAOYSA-N lithium bis(trimethylsilyl)amide Chemical compound [Li+].C[Si](C)(C)[N-][Si](C)(C)C YNESATAKKCNGOF-UHFFFAOYSA-N 0.000 description 4
- 229920001427 mPEG Polymers 0.000 description 4
- 125000000449 nitro group Chemical class [O-][N+](*)=O 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 description 4
- 125000006239 protecting group Chemical group 0.000 description 4
- 229960001153 serine Drugs 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 4
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 4
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 4
- BMJRTKDVFXYEFS-XIFFEERXSA-N (2s)-2,6-bis(9h-fluoren-9-ylmethoxycarbonylamino)hexanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(=O)O)CCCCNC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 BMJRTKDVFXYEFS-XIFFEERXSA-N 0.000 description 3
- HTFFMYRVHHNNBE-YFKPBYRVSA-N (2s)-2-amino-6-azidohexanoic acid Chemical compound OC(=O)[C@@H](N)CCCCN=[N+]=[N-] HTFFMYRVHHNNBE-YFKPBYRVSA-N 0.000 description 3
- QRZUPJILJVGUFF-UHFFFAOYSA-N 2,8-dibenzylcyclooctan-1-one Chemical compound C1CCCCC(CC=2C=CC=CC=2)C(=O)C1CC1=CC=CC=C1 QRZUPJILJVGUFF-UHFFFAOYSA-N 0.000 description 3
- NTQCWVJNMKBKFB-UHFFFAOYSA-N 2-(7-amino-2-oxochromen-4-yl)acetamide Chemical compound C1=C(N)C=CC2=C1OC(=O)C=C2CC(=O)N NTQCWVJNMKBKFB-UHFFFAOYSA-N 0.000 description 3
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 3
- WKGFWVPSZSRWKS-UHFFFAOYSA-N 2-amino-n-(3',6'-dihydroxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-yl)acetamide Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(NC(=O)CN)=CC=C21 WKGFWVPSZSRWKS-UHFFFAOYSA-N 0.000 description 3
- WHYHCPIPOSTZRU-UHFFFAOYSA-N 6-azidohexan-1-ol Chemical compound OCCCCCCN=[N+]=[N-] WHYHCPIPOSTZRU-UHFFFAOYSA-N 0.000 description 3
- ZSGRJAYPJLSJJT-UHFFFAOYSA-N 6-azidohexanal Chemical compound [N-]=[N+]=NCCCCCC=O ZSGRJAYPJLSJJT-UHFFFAOYSA-N 0.000 description 3
- 108090000317 Chymotrypsin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 3
- 108090000371 Esterases Proteins 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 3
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 229960002376 chymotrypsin Drugs 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 229960003067 cystine Drugs 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 150000002367 halogens Chemical class 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 3
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 210000004324 lymphatic system Anatomy 0.000 description 3
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 3
- ZMDGICHYDVYQDX-UHFFFAOYSA-N methylidenecarbamic acid Chemical compound OC(=O)N=C ZMDGICHYDVYQDX-UHFFFAOYSA-N 0.000 description 3
- SENLDUJVTGGYIH-UHFFFAOYSA-N n-(2-aminoethyl)-3-[[3-(2-aminoethylamino)-3-oxopropyl]-[2-[bis[3-(2-aminoethylamino)-3-oxopropyl]amino]ethyl]amino]propanamide Chemical compound NCCNC(=O)CCN(CCC(=O)NCCN)CCN(CCC(=O)NCCN)CCC(=O)NCCN SENLDUJVTGGYIH-UHFFFAOYSA-N 0.000 description 3
- 125000003854 p-chlorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1Cl 0.000 description 3
- 230000013878 renal filtration Effects 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 239000012465 retentate Substances 0.000 description 3
- 229940126586 small molecule drug Drugs 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 229910052721 tungsten Inorganic materials 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- JXHSXGOKAZHPKB-FQEVSTJZSA-N (2r)-2-amino-3-[(2-methoxyphenyl)-diphenylmethyl]sulfanylpropanoic acid Chemical compound COC1=CC=CC=C1C(SC[C@H](N)C(O)=O)(C=1C=CC=CC=1)C1=CC=CC=C1 JXHSXGOKAZHPKB-FQEVSTJZSA-N 0.000 description 2
- BUBGAUHBELNDEW-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylsulfanylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCSC)C(O)=O)C3=CC=CC=C3C2=C1 BUBGAUHBELNDEW-SFHVURJKSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- WUUVFJVVBLDQAM-UHFFFAOYSA-N 1-(6-azidohexanoyl)pyrrolidine-2,5-dione Chemical compound [N-]=[N+]=NCCCCCC(=O)N1C(=O)CCC1=O WUUVFJVVBLDQAM-UHFFFAOYSA-N 0.000 description 2
- KRRHTMTYAJRMKV-UHFFFAOYSA-N 1-(9h-fluoren-2-yl)-n-methylmethanamine Chemical compound C1=CC=C2C3=CC=C(CNC)C=C3CC2=C1 KRRHTMTYAJRMKV-UHFFFAOYSA-N 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- GZHPNIQBPGUSSX-UHFFFAOYSA-N 2-bromo-1-(3-nitrophenyl)ethanone Chemical compound [O-][N+](=O)C1=CC=CC(C(=O)CBr)=C1 GZHPNIQBPGUSSX-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- NHKMTYFIAPMWAJ-UHFFFAOYSA-N 6-azidohexyl carbonochloridate Chemical compound ClC(=O)OCCCCCCN=[N+]=[N-] NHKMTYFIAPMWAJ-UHFFFAOYSA-N 0.000 description 2
- IAIJAGVTYGSUGW-UHFFFAOYSA-N 9h-fluorene-2-carbonyl chloride Chemical compound C1=CC=C2C3=CC=C(C(=O)Cl)C=C3CC2=C1 IAIJAGVTYGSUGW-UHFFFAOYSA-N 0.000 description 2
- IBIDFEWDKNJSRD-UHFFFAOYSA-N 9h-fluorene-2-carboxylic acid Chemical compound C1=CC=C2C3=CC=C(C(=O)O)C=C3CC2=C1 IBIDFEWDKNJSRD-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 2
- 102400001282 Atrial natriuretic peptide Human genes 0.000 description 2
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 2
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical group NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 2
- 238000003512 Claisen condensation reaction Methods 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000007446 Glucagon-Like Peptide-1 Receptor Human genes 0.000 description 2
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 229940127024 acid based drug Drugs 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 125000002355 alkine group Chemical group 0.000 description 2
- 125000002947 alkylene group Chemical group 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 125000004391 aryl sulfonyl group Chemical group 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- UGUUDTWORXNLAK-UHFFFAOYSA-N azidoalcohol Chemical class ON=[N+]=[N-] UGUUDTWORXNLAK-UHFFFAOYSA-N 0.000 description 2
- ICCBZGUDUOMNOF-UHFFFAOYSA-N azidoamine Chemical compound NN=[N+]=[N-] ICCBZGUDUOMNOF-UHFFFAOYSA-N 0.000 description 2
- BLFLLBZGZJTVJG-UHFFFAOYSA-N benzocaine Chemical compound CCOC(=O)C1=CC=C(N)C=C1 BLFLLBZGZJTVJG-UHFFFAOYSA-N 0.000 description 2
- 125000001743 benzylic group Chemical group 0.000 description 2
- 239000007998 bicine buffer Substances 0.000 description 2
- 239000012455 biphasic mixture Substances 0.000 description 2
- XGIUDIMNNMKGDE-UHFFFAOYSA-N bis(trimethylsilyl)azanide Chemical compound C[Si](C)(C)[N-][Si](C)(C)C XGIUDIMNNMKGDE-UHFFFAOYSA-N 0.000 description 2
- 125000001246 bromo group Chemical group Br* 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000006352 cycloaddition reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- SGDINNZGYDHHKM-UHFFFAOYSA-N dilithium;trimethylsilylazanide Chemical compound [Li+].[Li+].C[Si](C)(C)[NH-].C[Si](C)(C)[NH-] SGDINNZGYDHHKM-UHFFFAOYSA-N 0.000 description 2
- KZTYYGOKRVBIMI-UHFFFAOYSA-N diphenyl sulfone Chemical group C=1C=CC=CC=1S(=O)(=O)C1=CC=CC=C1 KZTYYGOKRVBIMI-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 125000006575 electron-withdrawing group Chemical group 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 125000003983 fluorenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3CC12)* 0.000 description 2
- WBJINCZRORDGAQ-UHFFFAOYSA-N formic acid ethyl ester Natural products CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 2
- 239000012458 free base Substances 0.000 description 2
- 102000034238 globular proteins Human genes 0.000 description 2
- 108091005896 globular proteins Proteins 0.000 description 2
- 150000002374 hemiaminals Chemical class 0.000 description 2
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 2
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 125000002883 imidazolyl group Chemical group 0.000 description 2
- 150000002466 imines Chemical class 0.000 description 2
- 229960004768 irinotecan Drugs 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 239000012280 lithium aluminium hydride Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 2
- 125000004184 methoxymethyl group Chemical group [H]C([H])([H])OC([H])([H])* 0.000 description 2
- JCDWETOKTFWTHA-UHFFFAOYSA-N methylsulfonylbenzene Chemical compound CS(=O)(=O)C1=CC=CC=C1 JCDWETOKTFWTHA-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- AHVRPDDQUHKJEN-UHFFFAOYSA-N n-methyl-9h-fluorene-2-carboxamide Chemical compound C1=CC=C2C3=CC=C(C(=O)NC)C=C3CC2=C1 AHVRPDDQUHKJEN-UHFFFAOYSA-N 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 239000012038 nucleophile Substances 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- UXCJZSOZYIDKFL-QMMMGPOBSA-N prop-2-enyl (2s)-3-hydroxy-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@@H](CO)C(=O)OCC=C UXCJZSOZYIDKFL-QMMMGPOBSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 2
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 2
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 2
- 235000009518 sodium iodide Nutrition 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 229940124530 sulfonamide Drugs 0.000 description 2
- 150000003456 sulfonamides Chemical class 0.000 description 2
- 125000004434 sulfur atom Chemical group 0.000 description 2
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 2
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 150000003568 thioethers Chemical class 0.000 description 2
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- GZNAASVAJNXPPW-UHFFFAOYSA-M tin(4+) chloride dihydrate Chemical compound O.O.[Cl-].[Sn+4] GZNAASVAJNXPPW-UHFFFAOYSA-M 0.000 description 2
- FWPIDFUJEMBDLS-UHFFFAOYSA-L tin(II) chloride dihydrate Substances O.O.Cl[Sn]Cl FWPIDFUJEMBDLS-UHFFFAOYSA-L 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 229930195735 unsaturated hydrocarbon Natural products 0.000 description 2
- FBDOJYYTMIHHDH-OZBJMMHXSA-N (19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-2,4,6,8,10,14,20-heptaen-18-one Chemical compound CC[C@@]1(O)C(=O)OCC2=CN3Cc4cc5ccccc5nc4C3C=C12 FBDOJYYTMIHHDH-OZBJMMHXSA-N 0.000 description 1
- PQBYMIGEEFVPNG-UHFFFAOYSA-N (2,3,4,5,6-pentafluorophenyl) carbonochloridate Chemical compound FC1=C(F)C(F)=C(OC(Cl)=O)C(F)=C1F PQBYMIGEEFVPNG-UHFFFAOYSA-N 0.000 description 1
- UYABSLBMZAYXNN-UHFFFAOYSA-N (2,4-dinitrophenyl) carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(OC(Cl)=O)C([N+]([O-])=O)=C1 UYABSLBMZAYXNN-UHFFFAOYSA-N 0.000 description 1
- IQVLXQGNLCPZCL-ZDUSSCGKSA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2,6-bis[(2-methylpropan-2-yl)oxycarbonylamino]hexanoate Chemical compound CC(C)(C)OC(=O)NCCCC[C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(=O)CCC1=O IQVLXQGNLCPZCL-ZDUSSCGKSA-N 0.000 description 1
- IAMVVSHTWIUKAP-JTQLQIEISA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-6-[(2,2,2-trifluoroacetyl)amino]hexanoate Chemical compound FC(F)(F)C(=O)NCCCC[C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(=O)CCC1=O IAMVVSHTWIUKAP-JTQLQIEISA-N 0.000 description 1
- IYBNUJCDIAGXNX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,4-dinitroanilino)hexanoate Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC=C1NCCCCCC(=O)ON1C(=O)CCC1=O IYBNUJCDIAGXNX-UHFFFAOYSA-N 0.000 description 1
- AASBXERNXVFUEJ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) propanoate Chemical compound CCC(=O)ON1C(=O)CCC1=O AASBXERNXVFUEJ-UHFFFAOYSA-N 0.000 description 1
- MGHMWKZOLAAOTD-DEOSSOPVSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1h-indol-3-yl)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(=O)O)CC1=CNC2=CC=CC=C12 MGHMWKZOLAAOTD-DEOSSOPVSA-N 0.000 description 1
- CTYHQVFFQRDJSN-LHEWISCISA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[[(4-methoxyphenyl)-diphenylmethyl]amino]hexanoic acid Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)NCCCC[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 CTYHQVFFQRDJSN-LHEWISCISA-N 0.000 description 1
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 1
- DJGMNCKHNMRKFM-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pent-4-ynoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC#C)C(=O)O)C3=CC=CC=C3C2=C1 DJGMNCKHNMRKFM-SFHVURJKSA-N 0.000 description 1
- AMKHAJIFPHJYMH-ZETCQYMHSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]pent-4-ynoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC#C AMKHAJIFPHJYMH-ZETCQYMHSA-N 0.000 description 1
- AYMLQYFMYHISQO-QMMMGPOBSA-N (2s)-3-(1h-imidazol-3-ium-5-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CN=CN1 AYMLQYFMYHISQO-QMMMGPOBSA-N 0.000 description 1
- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical compound OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 description 1
- NXLNNXIXOYSCMB-UHFFFAOYSA-N (4-nitrophenyl) carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(OC(Cl)=O)C=C1 NXLNNXIXOYSCMB-UHFFFAOYSA-N 0.000 description 1
- MKDYODPQRXHJIA-UHFFFAOYSA-N (7-azido-1-cyanoheptan-2-yl) carbonochloridate Chemical compound ClC(=O)OC(CC#N)CCCCCN=[N+]=[N-] MKDYODPQRXHJIA-UHFFFAOYSA-N 0.000 description 1
- CPKDVQINHQPRPQ-UHFFFAOYSA-N (7-azido-1-methylsulfonylheptan-2-yl) carbonochloridate Chemical compound CS(=O)(=O)CC(OC(Cl)=O)CCCCCN=[N+]=[N-] CPKDVQINHQPRPQ-UHFFFAOYSA-N 0.000 description 1
- KGTPYDIBRWTWKI-UHFFFAOYSA-N (7-azido-1-morpholin-4-ylsulfonylheptan-2-yl) carbonochloridate Chemical compound [N-]=[N+]=NCCCCCC(OC(=O)Cl)CS(=O)(=O)N1CCOCC1 KGTPYDIBRWTWKI-UHFFFAOYSA-N 0.000 description 1
- ICGGBEVXGJRFBH-UHFFFAOYSA-N (8-azido-1-morpholin-4-yl-1-oxooctan-3-yl) carbonochloridate Chemical compound [N-]=[N+]=NCCCCCC(OC(=O)Cl)CC(=O)N1CCOCC1 ICGGBEVXGJRFBH-UHFFFAOYSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- OYWRDHBGMCXGFY-UHFFFAOYSA-N 1,2,3-triazinane Chemical compound C1CNNNC1 OYWRDHBGMCXGFY-UHFFFAOYSA-N 0.000 description 1
- 125000001399 1,2,3-triazolyl group Chemical group N1N=NC(=C1)* 0.000 description 1
- YRIZYWQGELRKNT-UHFFFAOYSA-N 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione Chemical compound ClN1C(=O)N(Cl)C(=O)N(Cl)C1=O YRIZYWQGELRKNT-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- XXJGBENTLXFVFI-UHFFFAOYSA-N 1-amino-methylene Chemical compound N[CH2] XXJGBENTLXFVFI-UHFFFAOYSA-N 0.000 description 1
- GODZNYBQGNSJJN-UHFFFAOYSA-N 1-aminoethane-1,2-diol Chemical compound NC(O)CO GODZNYBQGNSJJN-UHFFFAOYSA-N 0.000 description 1
- KAZUCVUGWMQGMC-UHFFFAOYSA-N 1-methoxy-4-methylsulfonylbenzene Chemical compound COC1=CC=C(S(C)(=O)=O)C=C1 KAZUCVUGWMQGMC-UHFFFAOYSA-N 0.000 description 1
- LLVWLCAZSOLOTF-UHFFFAOYSA-N 1-methyl-4-[1,4,4-tris(4-methylphenyl)buta-1,3-dienyl]benzene Chemical compound C1=CC(C)=CC=C1C(C=1C=CC(C)=CC=1)=CC=C(C=1C=CC(C)=CC=1)C1=CC=C(C)C=C1 LLVWLCAZSOLOTF-UHFFFAOYSA-N 0.000 description 1
- KYWXRBNOYGGPIZ-UHFFFAOYSA-N 1-morpholin-4-ylethanone Chemical compound CC(=O)N1CCOCC1 KYWXRBNOYGGPIZ-UHFFFAOYSA-N 0.000 description 1
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- QWENRTYMTSOGBR-UHFFFAOYSA-N 1H-1,2,3-Triazole Chemical compound C=1C=NNN=1 QWENRTYMTSOGBR-UHFFFAOYSA-N 0.000 description 1
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Chemical group C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 1
- XBNGYFFABRKICK-UHFFFAOYSA-N 2,3,4,5,6-pentafluorophenol Chemical compound OC1=C(F)C(F)=C(F)C(F)=C1F XBNGYFFABRKICK-UHFFFAOYSA-N 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- PQVYYVANSPZIKE-UHFFFAOYSA-N 2-(benzenesulfonyl)ethanol Chemical compound OCCS(=O)(=O)C1=CC=CC=C1 PQVYYVANSPZIKE-UHFFFAOYSA-N 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 1
- SQVIAVUSQAWMKL-UHFFFAOYSA-N 3-[2-(ethylamino)-1-hydroxyethyl]phenol Chemical compound CCNCC(O)C1=CC=CC(O)=C1 SQVIAVUSQAWMKL-UHFFFAOYSA-N 0.000 description 1
- WDFQBORIUYODSI-UHFFFAOYSA-N 4-bromoaniline Chemical compound NC1=CC=C(Br)C=C1 WDFQBORIUYODSI-UHFFFAOYSA-N 0.000 description 1
- 125000000590 4-methylphenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)C([H])([H])[H] 0.000 description 1
- GVZKQMUSVBGSIZ-UHFFFAOYSA-N 4-methylsulfonylmorpholine Chemical compound CS(=O)(=O)N1CCOCC1 GVZKQMUSVBGSIZ-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- DZNNFZGDBUXWMV-ZUWDIFAMSA-N 581079-18-7 Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(OC(=O)[C@H](C)NC(=O)COCCOCC(=O)N[C@@H](C)C(=O)O[C@@]3(CC)C5=C(C(N6CC7=CC8=CC=CC=C8N=C7C6=C5)=O)COC3=O)CC)C4=NC2=C1 DZNNFZGDBUXWMV-ZUWDIFAMSA-N 0.000 description 1
- JCORXJUUSVCJEP-UHFFFAOYSA-N 6-azidohexanoic acid Chemical compound OC(=O)CCCCCN=[N+]=[N-] JCORXJUUSVCJEP-UHFFFAOYSA-N 0.000 description 1
- JNTPTNNCGDAGEJ-UHFFFAOYSA-N 6-chlorohexan-1-ol Chemical compound OCCCCCCCl JNTPTNNCGDAGEJ-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- UQFCGBBNQQUTPM-UHFFFAOYSA-N 7-azido-1-methylsulfonylheptan-2-ol Chemical compound CS(=O)(=O)CC(O)CCCCCN=[N+]=[N-] UQFCGBBNQQUTPM-UHFFFAOYSA-N 0.000 description 1
- 102000055025 Adenosine deaminases Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 229930183010 Amphotericin Natural products 0.000 description 1
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 102000004452 Arginase Human genes 0.000 description 1
- 108700024123 Arginases Proteins 0.000 description 1
- OIXQINQYMGNCII-YRVFCXMDSA-N Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(O)C=C1 OIXQINQYMGNCII-YRVFCXMDSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 125000006847 BOC protecting group Chemical group 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- VMQMZMRVKUZKQL-UHFFFAOYSA-N Cu+ Chemical compound [Cu+] VMQMZMRVKUZKQL-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000005698 Diels-Alder reaction Methods 0.000 description 1
- 108010061435 Enalapril Proteins 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 description 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 1
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 1
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 1
- 241000270431 Heloderma suspectum Species 0.000 description 1
- 108091016366 Histone-lysine N-methyltransferase EHMT1 Proteins 0.000 description 1
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 1
- 238000006736 Huisgen cycloaddition reaction Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 229940122199 Insulin secretagogue Drugs 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 229910010084 LiAlH4 Inorganic materials 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 125000000520 N-substituted aminocarbonyl group Chemical group [*]NC(=O)* 0.000 description 1
- GDIHDSKOVXEJQQ-UHFFFAOYSA-N NC(O)=O.NC(O)=O.NC(O)=O.N Chemical compound NC(O)=O.NC(O)=O.NC(O)=O.N GDIHDSKOVXEJQQ-UHFFFAOYSA-N 0.000 description 1
- 229910020889 NaBH3 Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910020667 PBr3 Inorganic materials 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 1
- ADUKCCWBEDSMEB-NSHDSACASA-N Prenalterol Chemical compound CC(C)NC[C@H](O)COC1=CC=C(O)C=C1 ADUKCCWBEDSMEB-NSHDSACASA-N 0.000 description 1
- 102100040918 Pro-glucagon Human genes 0.000 description 1
- 108010050276 Protein Kinase C-alpha Proteins 0.000 description 1
- 102000015537 Protein Kinase C-alpha Human genes 0.000 description 1
- KIDHWZJUCRJVML-UHFFFAOYSA-N Putrescine Natural products NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 229940127180 SS1P Drugs 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- OUDSMILBAOGSQM-UHFFFAOYSA-N [6-azido-1-(9H-fluoren-9-yl)hexyl] carbonochloridate Chemical compound C1=CC=C2C(C(CCCCCN=[N+]=[N-])OC(=O)Cl)C3=CC=CC=C3C2=C1 OUDSMILBAOGSQM-UHFFFAOYSA-N 0.000 description 1
- SJGVXDBLSLSCKX-UHFFFAOYSA-N [7-azido-1-(4-methylphenyl)sulfonylheptan-2-yl] carbonochloridate Chemical compound CC1=CC=C(S(=O)(=O)CC(CCCCCN=[N+]=[N-])OC(Cl)=O)C=C1 SJGVXDBLSLSCKX-UHFFFAOYSA-N 0.000 description 1
- XXSDGNYHJMVPOP-UHFFFAOYSA-N [7-azido-1-(benzenesulfonyl)heptan-2-yl] carbonochloridate Chemical compound [N-]=[N+]=NCCCCCC(OC(=O)Cl)CS(=O)(=O)C1=CC=CC=C1 XXSDGNYHJMVPOP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000012382 advanced drug delivery Methods 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- 125000005910 alkyl carbonate group Chemical group 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 125000004414 alkyl thio group Chemical group 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- OFCNXPDARWKPPY-UHFFFAOYSA-N allopurinol Chemical compound OC1=NC=NC2=C1C=NN2 OFCNXPDARWKPPY-UHFFFAOYSA-N 0.000 description 1
- 229960003459 allopurinol Drugs 0.000 description 1
- BHELZAPQIKSEDF-UHFFFAOYSA-N allyl bromide Chemical compound BrCC=C BHELZAPQIKSEDF-UHFFFAOYSA-N 0.000 description 1
- RMRFFCXPLWYOOY-UHFFFAOYSA-N allyl radical Chemical group [CH2]C=C RMRFFCXPLWYOOY-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- VPGTYWUPXHFJDF-UHFFFAOYSA-N amino azido carbonate Chemical group NOC(=O)ON=[N+]=[N-] VPGTYWUPXHFJDF-UHFFFAOYSA-N 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 229940009444 amphotericin Drugs 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 125000002178 anthracenyl group Chemical group C1(=CC=CC2=CC3=CC=CC=C3C=C12)* 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940127003 anti-diabetic drug Drugs 0.000 description 1
- 230000000326 anti-hypercholesterolaemic effect Effects 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000043 antiallergic agent Substances 0.000 description 1
- 239000000924 antiasthmatic agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 125000002029 aromatic hydrocarbon group Chemical group 0.000 description 1
- 150000001543 aryl boronic acids Chemical class 0.000 description 1
- 125000005362 aryl sulfone group Chemical group 0.000 description 1
- 125000005361 aryl sulfoxide group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 125000005335 azido alkyl group Chemical group 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- PFYXSUNOLOJMDX-UHFFFAOYSA-N bis(2,5-dioxopyrrolidin-1-yl) carbonate Chemical compound O=C1CCC(=O)N1OC(=O)ON1C(=O)CCC1=O PFYXSUNOLOJMDX-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 229940084891 byetta Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 1
- 229960000830 captopril Drugs 0.000 description 1
- 150000001715 carbamic acids Chemical class 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000005587 carbonate group Chemical group 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 150000003857 carboxamides Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- AOGYCOYQMAVAFD-UHFFFAOYSA-N chlorocarbonic acid Chemical class OC(Cl)=O AOGYCOYQMAVAFD-UHFFFAOYSA-N 0.000 description 1
- CGLRXKACBSXHIU-UHFFFAOYSA-N chloromethyl carbamate Chemical compound NC(=O)OCCl CGLRXKACBSXHIU-UHFFFAOYSA-N 0.000 description 1
- 238000007265 chloromethylation reaction Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000012679 convergent method Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- ZPWOOKQUDFIEIX-UHFFFAOYSA-N cyclooctyne Chemical compound C1CCCC#CCC1 ZPWOOKQUDFIEIX-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 230000003544 deproteinization Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- AQEFLFZSWDEAIP-UHFFFAOYSA-N di-tert-butyl ether Chemical class CC(C)(C)OC(C)(C)C AQEFLFZSWDEAIP-UHFFFAOYSA-N 0.000 description 1
- 125000004663 dialkyl amino group Chemical group 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 150000001991 dicarboxylic acids Chemical class 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 125000001028 difluoromethyl group Chemical group [H]C(F)(F)* 0.000 description 1
- 108010067755 dinitrophenyl-bovine serum albumin Proteins 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- GBXSMTUPTTWBMN-XIRDDKMYSA-N enalapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)CC1=CC=CC=C1 GBXSMTUPTTWBMN-XIRDDKMYSA-N 0.000 description 1
- 229960000873 enalapril Drugs 0.000 description 1
- 229930013356 epothilone Natural products 0.000 description 1
- HESCAJZNRMSMJG-KKQRBIROSA-N epothilone A Chemical class C/C([C@@H]1C[C@@H]2O[C@@H]2CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 HESCAJZNRMSMJG-KKQRBIROSA-N 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- FCZCIXQGZOUIDN-UHFFFAOYSA-N ethyl 2-diethoxyphosphinothioyloxyacetate Chemical compound CCOC(=O)COP(=S)(OCC)OCC FCZCIXQGZOUIDN-UHFFFAOYSA-N 0.000 description 1
- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229960004695 etilefrine Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002039 glucoregulatory effect Effects 0.000 description 1
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- VPFMEXRVUOPYRG-UHFFFAOYSA-N hex-5-ynoic acid Chemical compound OC(=O)CCCC#C VPFMEXRVUOPYRG-UHFFFAOYSA-N 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- IKXNIQJDNKPPCH-UHFFFAOYSA-N hydron;prop-2-yn-1-amine;chloride Chemical compound [Cl-].[NH3+]CC#C IKXNIQJDNKPPCH-UHFFFAOYSA-N 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 125000003454 indenyl group Chemical group C1(C=CC2=CC=CC=C12)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 125000003010 ionic group Chemical group 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001786 isothiazolyl group Chemical group 0.000 description 1
- 125000000842 isoxazolyl group Chemical group 0.000 description 1
- 150000004715 keto acids Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000006138 lithiation reaction Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- NBTOZLQBSIZIKS-UHFFFAOYSA-N methoxide Chemical compound [O-]C NBTOZLQBSIZIKS-UHFFFAOYSA-N 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000006518 morpholino carbonyl group Chemical group [H]C1([H])OC([H])([H])C([H])([H])N(C(*)=O)C1([H])[H] 0.000 description 1
- MZAVPBQCWWIYEQ-UHFFFAOYSA-N n-(6-chloro-1,3-benzothiazol-2-yl)-1-benzothiophene-2-sulfonamide Chemical compound C1=CC=C2SC(S(=O)(=O)NC3=NC4=CC=C(C=C4S3)Cl)=CC2=C1 MZAVPBQCWWIYEQ-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- FBUKVWPVBMHYJY-UHFFFAOYSA-N noncarboxylic acid Natural products CCCCCCCCC(O)=O FBUKVWPVBMHYJY-UHFFFAOYSA-N 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- JQAHIGRMEIQAEL-UHFFFAOYSA-N oct-5-yn-1-ol Chemical compound CCC#CCCCCO JQAHIGRMEIQAEL-UHFFFAOYSA-N 0.000 description 1
- NWLSXMQRWBUIHI-UHFFFAOYSA-N oct-5-ynyl carbonochloridate Chemical compound CCC#CCCCCOC(Cl)=O NWLSXMQRWBUIHI-UHFFFAOYSA-N 0.000 description 1
- 238000010915 one-step procedure Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229960001639 penicillamine Drugs 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachloro-phenol Natural products OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 description 1
- 239000000863 peptide conjugate Substances 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- QIIPQYDSKRYMFG-UHFFFAOYSA-M phenyl carbonate Chemical class [O-]C(=O)OC1=CC=CC=C1 QIIPQYDSKRYMFG-UHFFFAOYSA-M 0.000 description 1
- AHWALFGBDFAJAI-UHFFFAOYSA-N phenyl carbonochloridate Chemical class ClC(=O)OC1=CC=CC=C1 AHWALFGBDFAJAI-UHFFFAOYSA-N 0.000 description 1
- 125000003170 phenylsulfonyl group Chemical group C1(=CC=CC=C1)S(=O)(=O)* 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- XYFCBTPGUUZFHI-UHFFFAOYSA-O phosphonium Chemical compound [PH4+] XYFCBTPGUUZFHI-UHFFFAOYSA-O 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- IPNPIHIZVLFAFP-UHFFFAOYSA-N phosphorus tribromide Chemical compound BrP(Br)Br IPNPIHIZVLFAFP-UHFFFAOYSA-N 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- RCIMBBZXSXFZBV-UHFFFAOYSA-N piromidic acid Chemical compound N1=C2N(CC)C=C(C(O)=O)C(=O)C2=CN=C1N1CCCC1 RCIMBBZXSXFZBV-UHFFFAOYSA-N 0.000 description 1
- 229960004444 piromidic acid Drugs 0.000 description 1
- 229960004358 prenalterol Drugs 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- PYNUOAIJIQGACY-UHFFFAOYSA-N propylazanium;chloride Chemical compound Cl.CCCN PYNUOAIJIQGACY-UHFFFAOYSA-N 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 150000003233 pyrroles Chemical class 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 125000005493 quinolyl group Chemical group 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 150000003333 secondary alcohols Chemical class 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 229960002135 sulfadimidine Drugs 0.000 description 1
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 description 1
- 125000005864 sulfonamidyl group Chemical group 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 229950009390 symclosene Drugs 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- UIYOVVYZPVVUMJ-UHFFFAOYSA-N tert-butyl carbamoyl carbonate Chemical compound CC(C)(C)OC(=O)OC(N)=O UIYOVVYZPVVUMJ-UHFFFAOYSA-N 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 125000005207 tetraalkylammonium group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000005000 thioaryl group Chemical group 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-M toluene-4-sulfonate Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-M 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 229960001082 trimethoprim Drugs 0.000 description 1
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 125000005500 uronium group Chemical group 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/641—Branched, dendritic or hypercomb peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2242—Atrial natriuretic factor complex: Atriopeptins, atrial natriuretic protein [ANP]; Cardionatrin, Cardiodilatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
- C08G83/002—Dendritic macromolecules
- C08G83/003—Dendrimers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
- C08G83/002—Dendritic macromolecules
- C08G83/003—Dendrimers
- C08G83/004—After treatment of dendrimers
Definitions
- the invention relates to delivery systems for drug molecules coupled to dendrimers. More specifically, the invention relates to compositions that provide for the release of multiple molecules of drug from protected dendrimeric macromolecules.
- conjugates have been prepared wherein PEG is a releasable carrier of the drug or prodrug.
- the drug is attached by an ester or carbonate linkage that can be cleaved by esterase-catalyzed hydrolysis.
- ester or carbonate linkage that can be cleaved by esterase-catalyzed hydrolysis.
- Examples are PEG-camptothecin, PEG-SN38, PEG-irinotecan and PEG-docetaxel.
- Additional adaptations have been made to accommodate amine-containing drugs whereby a PEG moiety is connected by a cleavable ester to a self-immolating carbamate. This technology has been applied to peptides and proteins as well as to daunorubicin, amphotericin, Ara-C and other small molecules.
- PCT publication WO2009/158668 describes releasable drug conjugates to macromolecules wherein the rate of beta elimination is controlled by a trigger independent of the link to the macromolecule itself. This solves a problem left unsolved in the prior art.
- the release mechanism set forth in the '668 PCT publication has not been applied to instances where a multiplicity of drug molecules is coupled covalently, but releasably, to dendrimeric macromolecules. It is also limited to drugs that contain an amine functional group.
- this approach offers a means whereby the coupled drug is protected from hydrolysis by the presence of a protective polymer, such as PEG, on different sites at the surface or interstices of the solid support.
- a protective polymer such as PEG
- Dendrimers have been used as carriers for therapeutic compounds, either by entrapment of a drug in cavities within the dendrimer, or by covalently linking drug molecules to the surface. This is reviewed in Svenson, S., Eur J Pharm Biopharm (2009) 71 :445-462 and Cheng, Y., J Pharm. Set (2007) 97:123-143. Entrapment within dendrimer cavities is limited to small molecules, and covalent attachment approaches have thus far been limited to systems in which a small drug is hydrolytically or enzymatically cleaved from the dendrimer surface.
- Unmodified cationic dendrimers such as polyamidoamine dendrimer(PAMAM) or poly-L-lysine (PLL) have biocompatibility and toxicology shortcomings, for example, disruption of cell membranes and also have very short half-lives, typically ⁇ 20 min. Toxicity may be reduced by functionalizing the surface of the dendrimer with non-ionic or anionic groups (Kaminskas, L., et al, Mol Pharm (2007) 4:949-961).
- PAMAM dendrimers are not biodegradable and are retained in the liver and kidney, raising a concern - albeit unproven - of toxicity upon chronic dosing, though PLL dendrimers while retained in the liver and kidney, appear to be broken down to constituent monomers.
- PEGylation of PAMAM and PLL dendrimers neutralizes the surface positive charges and reduces or eliminates their propensity to lyse cells and cause acute toxicity. It has also been shown that the hydrophilic PEG moiety increases water solubility of guest drug molecules, and that PEGylated dendrimers effectively accumulate in tumor tissue via the enhanced permeability and retention and thus serve as targeting delivery vehicles for antitumor agents. PEGylated PLL shows almost complete (>90%) bioavailability when
- PEGylation of cationic dendrimers can decrease renal filtration and dramatically increase the half-life from minutes to several days.
- Composite results show that long half-lives may be achieved with PEGylated dendrimers of MW >40 kDa by varying either the number or size of the PEG chain. That is, the MW of the total dendrimer-PEG conjugate rather than the dendrimer or individual PEG chains dictates the extent of renal filtration.
- the size of PEGylated poly L-lysine dendrimer complexes can be specifically manipulated to dictate their pharmacokinetics, biodegradation and bioresorption behavior.
- camptothecin attached to the dendrimer surface of PLLi 6 (PEG5000) 8 (i.e., a PLL with 16 functional groups at the surface, 8 of which are occupied by PEG of 5000 molecular weight) via an ester linker was completely protected from serum esterases, whereas an analogous PEG-camptothecin ester hydrolyzed ⁇ 10-fold faster in serum than buffer.
- PEG5000 PLLi 6
- compositions of the invention overcome problems associated with coupling drugs to the conventional monovalent linear PEG carrier.
- the molecular weight of the PEG carrier In order to minimize kidney filtration, the molecular weight of the PEG carrier must be at least about 40,000 and the drug is limited therefore to about 1 ⁇ per 40 mg PEG. Thus, only very potent drugs can employ this system as a practical matter.
- Linear PEG's also provide only limited protection against enzymes that may modify and/or destroy the bound drug. Drugs bound to linear PEG may retain significant biological activity; while this is a requirement for a stably-modified drug, use of PEGylated-drug as a carrier for slow release of active free drug requires that the PEGylated form be substantially inactive due to the relatively higher dosages involved.
- the present invention permits increasing the drug payload and protects the drug against degrading enzymes as well as blocking access of the drug to its biological receptor. Like the technology described in the '668 publication, the activity of the drug is silenced until it is released, permitting administration of relatively large doses as depots.
- the invention provides controlled release forms of multiple molecules of drugs, such as growth factors and small molecule drugs, coupled covalently to sites on dendrimeric macromolecules that can serve as delivery systems to extend the half-life of such drugs or growth factors.
- a linker is covalently bound to multiple sites on the dendrimer, which linker is in turn coupled to an appropriate drug or prodrug.
- the drug or prodrug is then released at the desired rate through a beta elimination reaction at physiological pH.
- the drug sites on the dendrimer may be contained within a protective layer of polymer bound to adjacent sites on the solid support.
- the invention is directed to a multiplicity of substituents of the formula
- R 1 and R 2 are independently CN; N0 2 ;
- R 3 is H or optionally substituted alkyl
- heteroaryl or heteroarylalkyl each optionally substituted;
- R 4 is optionally substituted alkyl
- R and R may be joined to form a 3-8 member ring
- R 1 and R 2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
- each R 5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
- D is a residue of a drug or prodrug coupled through O, S, or N;
- Y is absent and X is O or S;
- Y is NBCH 2 and X is O;
- B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
- R , R , R or B is coupled to a dendrimeric macromolecule.
- Said dendrimer may optionally also be further coupled to a protective inert polymer, such as PEG.
- a protective inert polymer such as PEG.
- the invention is directed to a dendrimeric macromolecule coupled, optionally through an additional linker, to a multiplicity of substituents of the formula
- R and R is independently CN; N0 2 ;
- R is H or optionally substituted alkyl
- heteroaryl or heteroarylalkyl each optionally substituted; or OR or NR 2 wherein each R is independently H or optionally
- R 4 is optionally substituted alkyl
- heteroaryl or heteroarylalkyl each optionally substituted;
- R and R may be joined to form a 3-8 member ring
- R 1 and R 2 may be H, or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
- each R 5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
- D is a residue of a drug or prodrug coupled through O, S, or N;
- Y is absent and X is O or S;
- Y is NBCH 2 and X is O;
- B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
- the dendrimer may also optionally comprise a multiplicity of protective inert polymers, such as PEG.
- compositions of the invention thus offer prolonged blood circulation times, protection of drugs against hydrolases/proteases, high capacity, and inactivity against targets until released.
- the invention is directed to methods to prepare the compositions of the invention, and methods to employ them in medical/veterinary/physiological procedures. It also includes intermediates and precursors in the synthesis of formulas (1) and (2).
- the invention thus further includes "precursor" molecules of formula (3) in which L is a nucleofuge— i. e. , a leaving group that permits binding of a nucleophile.
- L is a nucleofuge—i. e. , a leaving group that permits binding of a nucleophile.
- the invention includes compounds with formulas identical to that of formula (1), wherein multiple substituents are coupled to a dendrimeric macromolecule through R , R , R , or B except that in lieu of the drug or prodrug, the nucleofuge, of formula (3), instead, is present, i.e., the dendrimer macromolecule has substituents of formula (3)
- R 1 , R 2 , R 5 , X, Y and m are as defined in formula (1) or (2);
- L is a nucleofuge for coupling the drug or prodrug to the remainder of the molecule.
- the dendrimer has not yet been coupled.
- formula (1) or (2) without coupling to the dendrimer may be used as an intermediate.
- Z is a drug or prodrug or a nucleofuge.
- the coupling of any of these intermediates to dendrimer may employ a "connector" - i. e. , one or more bifunctional organic molecules that connect the substituents on these formulas to the dendrimer.
- the invention includes compounds of the formula:
- M is a dendrimer
- D is a drug
- J is a joining moiety that releases D by a beta elimination mechanism, wherein m is at least 8, and may by larger - e.g., 16, 32 or more.
- Figure 1 shows a typical dendrimer structure that has a core with three functional groups.
- Figures 2A and 2B show a typical monomer for the production of a polyamidoamine dendrimer(PAMAM) and an early-stage phase of construction of said dendrimer, respectively.
- PAMAM polyamidoamine dendrimer
- Figure 3 is a diagram of a prior art description of a PEGylated polylysine wherein the PEGylation is contained within the dendrimer.
- Figure 4 is a diagram of a prior art description of a PEG protected PAMAM dendrimer showing relative dimensions of coupled drugs.
- Figure 5 is a graph showing the relationship of free energy to half-life for release of label from substituted ⁇ -elimination compounds.
- Figure 6 is a graph showing the free energy correlation between Hammett sigma values and rate of release of label by ⁇ -elimination.
- Figure 7 shows the time course of release of fluorescein from PEGylated polylysine dendrimer.
- Figure 8 shows the kinetics of release of fluorescein from coupling to a PEG-coated polylysine dendrimer.
- Figures 9a and 9b show, respectively, raw data showing concentration of various conjugates in plasma in a rat model system and the calculated release rate of the drug from the conjugate depending on the nature of the trigger. These data demonstrate the control over drug release rates using beta-elimination linkers.
- Figure 10 shows a comparison of in vivo and in vitro release rates of drugs as a function of the Hammett constants associated with the trigger.
- the present invention represents an improvement over standard PEGylation practices, and has additional advantages over the PEGylated releasable drug compositions such as those described in the above-referenced PCT publication WO2009/158668. Advantages include retaining a drug or prodrug in inactive form until released from the macromolecular carrier, a multiplicity of binding sites for the drug so that the drug dosage may be increased, thus permitting delivery of less potent drugs, and provision of protection against degrading enzymes or other inactivating conditions.
- compositions of the invention afford effective delivery of drugs to the lymphatic system. Because the compounds of the invention have molecular weights that are significantly higher than the molecular weight of the drug, they are capable of maintaining the drug in the lymphatic system when the compounds are administered subcutaneously. Compounds with molecular weights of 40,000 or more are effectively maintained in the lymphatic system. Further, because the lymph lacks esterases present in plasma that might release drugs from esterified linkages, the favorable pH of the lymph (which is identical to that of plasma) permits release of the active drug from the conjugate. Thus, the compounds of the invention effectively release drug into the lymph when delivery to the lymph is desired, as would be the case, for example, with respect to lymphomas.
- compositions of the invention comprise dendrimer macromolecules coupled to multiple copies of one or more drugs and optionally further coupled to protective hydrophilic polymers such as PEG.
- the dendrimer has a generation value of at least 4, where the core is assigned generation zero.
- Dendrimers are synthetic polymers characterized by repeated chain branchings emanating from a central core, giving rise to a fractal-like topology and a large number of chain endings. Dendrimers are composed of a core, one or more layers (or generations) of branched monomers, and a layer of end-groups that double each "generation" and terminate the various chains.
- Dendrons are wedge-shaped structural sub-components of a parent dendrimer. Each functional group of a core gives rise to a dendron; at higher generations, branches arising from a functional group can be viewed as small dendrons.
- Figure 2 shows a typical dendrimer structure.
- G refers to the number of layers in the dendrimer, and Z is the number of end groups on the dendrimer outer surface.
- the core is generation 0 (GO).
- a monomer directly attached to the core can be considered a 1st generation monomer (Gl); a monomer attached to a Gl monomer is a 2nd generation monomer (G2), etc.
- a variegated (i. e. , multi-functional) dendrimer has more than one type of functional group incorporated into at least one generational layer (Roberts, B. P., et al, New Journal of Chemistry (2008) 32: 1543-1555). Variegation of dendrimer end-groups allows detailed control over the chemical composition of the surface, while variegation of a more internal monomer provides control of its emanating branch(es) or an entire dendron.
- Exemplary dendrimers include dendrimeric forms of poly-L-lysine (PLL) and polyamidoamine (PAMAM).
- PLL poly-L-lysine
- PAMAM polyamidoamine
- Figure 3 A The structure of one embodiment that may form the core of a PAMAM dendrimer is shown in Figure 3 A and an alternative form is shown in Figure 3B where coupling has already occurred at two of four available amino groups.
- Dendrimers can be synthesized by divergent or convergent approaches
- a generation is grown by attaching monomers with two N-blocked functional groups (e.g., fBoc -Lys) to each core amino group, followed by blocking group removal to give a dendrimer with four amino end-groups.
- monomers with two N-blocked functional groups e.g., fBoc -Lys
- blocking group removal e.g., fBoc -Lys
- This can be repeated multiple times to form dendrimers with 8, 16, 32, etc., amino groups at the surface.
- steric congestion increases, surface groups become less accessible, and incomplete reactions may result in non-perfect dendrimers.
- dendrons are synthesized first and then coupled to a core molecule.
- the dendrons are typically constructed by the same strategies as for divergent growth, and once the dendrons have reached the desired generation they are coupled to a small core molecule.
- two PLL dendrons can be prepared as described above, and then connected to a Lys core to make a PLL dendrimer.
- the last coupling step is potentially difficult due to steric hindrance, but the large difference in size between the dendrimer and dendrons allows purification of the final product.
- Dendrimers have been extensively studied by a variety of physical, computational and experimental approaches (Caminade, A., et ah, Advanced drug delivery reviews (2005) 57:2130-2146).
- Table 1 shows how PAMAM and PLL dendrimer diameters grow with increasing generation.
- the high surface and relatively lower interior densities of larger dendrimers supports cavities with diameters ranging from 5 to 15 A that may be joined to channels connecting to the surface.
- the PEG molecules extend from the surface ⁇ 1 1 or 12 A per 1,000 MW to form a shell or "brush" around the dendrimer.
- ellipsoid dendrimers become almost spherical, and the PEG molecules more "stretched", thus increasing the PEG layer thickness.
- Each glycol unit of a PEG is 3.3 A, so a 5,000 kDa PEG (1 13 units) has a fully extended length of 377 A, considerably larger than the ⁇ 55 A PEG shell of the PEGylated dendrimer.
- R d dendrimer radius
- R total radius
- L thickness of PEG layer.
- compositions of the present invention suitable drugs are releasably coupled to multiple sites on a dendrimeric macromolecule through linkages which permit release of the drug or prodrug by ⁇ -elimination under physiological conditions.
- small molecules and many therapeutic peptides/proteins would fit within the dimensions/volume of a PEG 5000 shell, providing that a) suitable orientation of the guest molecule can be achieved, and b) that the PEG density is sterically accommodating.
- the PEG MW could be increased to 10,000 which, although not yet studied, should provide a larger shell, afford more protection, and accommodate the larger peptides/proteins.
- the drug conjugate of formula (1) or (2) is designed to control the pharmacokinetics of the drug or prodrug the residue of which when coupled to the remainder of the molecule is designated as "D".
- the mechanism whereby the drug or prodrug is released is shown below.
- the rate is controlled according to a pH dependent ⁇ -elimination mechanism.
- the groups R 1 and R are selected to provide the appropriate reactivity of the intervening proton in R -CH-R , thus providing control over the rate of drug or prodrug release.
- the properties of R 1 and R 2 may be modulated by the optional addition of electron-donating or electron-withdrawing substituents, for example, in aryl moieties contained therein.
- R 1 or R 2 can behave as the "trigger."
- the nature of the "trigger” controls the acidity of the intervening proton in R ⁇ CH- R 2 , which, when released, permits the electron pair thus freed to effect ⁇ -elimination.
- the half-life of the reaction may be in the range of that when Y is NBCH 2j depending on the nature of B. Half lives are very short for embodiments of B that are highly electronegative. Examples of the ability to control release rates of drugs or prodrugs from these linkers are given in the Examples below and shown in Figures 9 and 10.
- the mechanism of ⁇ -elimination release is shown below. taking place under conditions typical of those of biological systems, for example a pH of between 6 and 8 and a temperature of between 25 and 40°C, at a rate such that the half-life of the reaction is between 1 and 10,000 hours, or between 1 and 5,000 hours, or between 1 and 1 ,000 hours or between 1 and 100 hours or between 1 and 10 hours.
- this intermediate may decompose according to:
- B-N CH + H 2 0 ⁇ B-NH-CH 2 -OH (addition of water to the imine to form hemiaminal)
- reactions (l)-(4) shown above may be transient and therefore may not be detectable under physiological or other chemical reaction conditions.
- the degree to which the R 1 and/or R 2 8 roups activate the adjacent C-H bond may be expressed by the resulting acidity of the C-H bond; this acidity may in turn be expressed as the pK a of the C-H bond, wherein a lower pK a denotes a more acidic, more readily ionized C-H bond.
- Listings of approximate pKa values for various groups are common in the art, for example in Bordwell, F. G., "Equilibrium acidities in dimethyl sulfoxide solution," Accounts of Chemical Research (2002) 21 :456-463 (incorporated herein by reference).
- suitably activating groups include, but are not limited to, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted alkenes, optionally substituted alkynes, sulfones, sulfoxides, nitriles, ketones, esters, amides, and nitro groups.
- the R 1 and/or R 2 8 roups may be joined to form a cyclic structure, for example
- Substituents on the R 1 and/or R 2 g roups may optionally be added to provide further control over the acidity of the adjacent C-H, and thus the rate of the beta-elimination reaction.
- electron-withdrawing substituents will increase the rate of the beta-elimination reaction, while electron-donating substituents will decrease the rate of the beta-elimination reaction.
- the electronic effect of various substituents is well-known in the art, and may be expressed for example as linear free-energy (Hammett) relationships.
- aromatic systems for example substituted aryl, heteroaryl, arylketones, heteroarylketone, arylsulfone,
- An "electron-donating group” is substituent that will result in a decrease in the acidity of a benzylic hydrogen ion.
- suitable electron-donating substituents include but are not limited to, lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, and dialkylamino.
- Electrode-withdrawing groups result in an increase in the acidity of a benzylic hydrogen ion.
- Non-hydrogen electron-donating or electron-withdrawing substituents may be present in multiple positions on rings to which they are bound. While, for convenience, in most examples, only a single occurrence of a non-hydrogen substituent on a single ring is shown, multiple substituents may also be present and are within the scope of the invention. The substituents may be the same or different.
- B groups that reduce the reactivity of the carbamate N lone pair, for example via extended conjugation and/or electron-withdrawing ability, reduce the rate of competing decomposition by the El -elimination pathway.
- B is optionally substituted aryl, or optionally substituted heteroaryl.
- B is substituted aryl or substituted heteroaryl, substituted with groups having positive Hammett sigma constants (Table 3).
- B is phenyl or phenyl substituted with alkoxycarbonyl, CN, Br, N0 2 , sulfonamide or
- X is O and Y is NBC3 ⁇ 4, the NBC3 ⁇ 4 serves as an adaptor to provide the unstable carbamate thus permitting coupling to drugs that do not contain amino functional groups. It is estimated that only about 32% of the presently available small molecule drugs have primary or secondary amino groups available as functionalities. However, permitting the inclusion of groups with functional groups that are aliphatic primary or secondary alcohols permits about 45% of approved small molecule drugs to be included. Other functional groups that are acceptable to permit the constructs of the invention to contain the drug are sulfonamides, phenols, pyrroles, imides and thiols. Taken together, then, approximately 71% of currently approved drugs are amenable to inclusion in the constructs of the invention.
- B include a stabilizer, such as an aryl group, which prevents spontaneous cleavage or hydrolysis.
- the dendrimers may be coupled to Formulas 1, 2, or 3 through, additional
- the additional connectors are bifunctional organic compounds, such as DBCO- NHS. Many such connectors are commercially available, for example from Pierce Chemical Co, Rockford, IL. Various bifunctional connecters are well known in the art, including dicarboxylic acids or anhydrides, diamines, or heterobifunctional connecters. The selection of the connector will, of course, depend on the nature of the functional groups on the substituents on the dendrimer and on the intermediates corresponding to formulas (1) - (3).
- alkyl includes linear, branched, or cyclic saturated hydrocarbon groups of 1-8 carbons, or in some embodiments 1-6 or 1-4 carbon atoms.
- alkoxy includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.
- alkenyl includes non-aromatic unsaturated hydrocarbons with carbon- carbon double bonds.
- alkenyl (C 2 ) is meant a mono-, di-, tri-, or tetra-substituted carbon-carbon double bond of any geometric configuration.
- alkynyl includes non-aromatic unsaturated hydrocarbons with carbon- carbon triple bonds.
- alkynyl (C 2 ) is meant a mono- or di-substituted carbon- carbon triple bond.
- aryl includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl.
- heteroaryl includes aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.
- alkenyl, alkynyl, aryl or heteroaryl moieties may be coupled to the remainder of the molecule through an alkylene linkage.
- the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.
- halogen includes bromo, fluoro, chloro and iodo.
- a "nucleofuge” is a leaving group that takes with it the electron pair by which it is bonded.
- exemplary nucleofuges are halogen, OH, alkoxy, hydroxysuccinimide,
- each R is independently alkyl, aryl, or heteroaryl.
- protein and “peptide” are used interchangeably regardless of chain length, and these terms further include pseudopeptides which comprise linkages other than amide linkages, such as CH 2 NH 2 linkages as well as peptidomimetics.
- nucleic acids and “oligonucleotides” are also used interchangeably regardless of chain length.
- the nucleic acids or oligonucleotides may be single-chain or duplexed or may be DNA, RNA, or modified forms thereof with altered linkages, such as phosphodiesters, phosphoramidates, and the like.
- these terms also include those with side chains not found in nature in the case of proteins as well as pseudopeptide bonds and bases not found in nature in the case of nucleic acids as well as backbone modifications such as peptide nucleic acids.
- Small molecules in the context of drugs is a term well understood in the art, and is meant to include compounds other than proteins and nucleic acids that either are synthesized or are isolated from nature and in general do not resemble proteins or nucleic acids. Typically, they have molecular weights ⁇ 1,000, although there is no specific cutoff recognized.
- a wide variety of drugs may be included as the embodiment of D. Each of these drugs will be coupled through a nitrogen, oxygen or sulfur to the remainder of the molecule.
- suitable drugs include those for human or veterinary use including, but not limited to, antidiabetic drugs; growth promoters; antibacterials including aminoglycosides, penicillins, cephalosporins, macrolides and peptides, trimethoprim, piromidic acid, and sulfamethazine; analgesic and anti-inflammatory drugs, antiallergic and antiasthmatic drugs,
- drugs include alcohols such as paclitaxel and analogues, epothilones and analogues, camptothecin and analogues such as irinotecan, and nucleosides such as 5-fluorouracil and capecitabine.
- the drug is a peptide comprising a serine residue.
- the drug is a small molecule comprising an arylol group; examples of such drugs include sn-38, etilefrine, prenalterol, and estradiol.
- the drug is a peptide comprising a tyrosine residue.
- the drug may be a small molecule comprising a thiol group.
- examples of such drugs include penicillamine, captopril, and enalapril.
- the drug may be a small molecule comprising a thioaryl or thioheteroaryl group; examples of such drugs include 6-mercaptopurine.
- the drug is a nitrogen-containing heterocycle; examples include 5-fluorouracil and allopurinol.
- Other drugs are peptide, protein, and nucleic acid drugs. Examples of peptide drugs suitable for use in the invention include, e.g.
- GLP-1 glucagon-like peptide 1
- AMF atrial natriuretic factor
- protein drugs include immunotoxin SS1P, adenosine deaminase, arginase, and others.
- nucleic acid-based drugs include the sense strand and antisense strand of any gene from an animal, and particularly from a mammal.
- genes can be those that are already the subjects of antisense D As or RNAs, or small interfering R As that have been provided with the purpose of treating various diseases, for example genes for protein kinase C- alpha, BCL-2, ICAM-1, tumor necrosis factor alpha and the like.
- precursor refers to a dendrimeric macromolecule similar to formula (1), but wherein rather than linked to the drug or prodrug, the macromolecule is coupled to a nucleofuge for further binding to a drug or prodrug as in formula (3)
- R 1 , R 2 , R 5 , X, Y and m are as defined in formula (1) or (2);
- L is a nucleofuge
- the active form of the drug is directly released from the conjugates of the invention, in some cases, it is possible to release the active drug in the form of a prodrug thereof.
- a prodrug thereof On example of such a system is shown below:
- drug conjugates include conjugates both of drugs and prodrugs.
- any substituent may itself be optionally substituted
- the substitution on any ring system may be alkyl, alkenyl, alkynyl or an additional ring each optionally substituted.
- Optional substitutions on any substituent, including the above, include halo, nitro, cyano, OR, SR, NR 2 , OCOR, NRCOR, COOR, CONR 2 , SOR, S0 2 R, SONR 2 , S0 2 NR 2 , wherein each R is independently alkyl, alkenyl, alkynyl, aryl or heteroaryl.
- R and R together exert the most control over the release rate for the drug, though R 5 and m have some impact as well.
- one of R 1 and R 2 is hydrogen or is alkyl, arylalkyl or heteroarylalkyl and the other comprises one of the remaining embodiments set forth hereinabove.
- neither of R 1 and R 2 is hydrogen or is alkyl, arylalkyl or heteroarylalkyl.
- R 1 may be H and the other optionally substituted phenyl or both R 1 and R 2 may be optionally substituted phenyl.
- the substitutions on the phenyl rings may be at 1-5 positions but preferably 3 or less. If both R 1 and R 2 are optionally substituted phenyl, they need not be substituted identically, or may be identically substituted.
- Suitable substituents include methoxy, halo, nitro, cyano and the like for example as shown in Table 3 or the substitutions listed above.
- R 1 and R 2 is R 6 S-, R 6 S(0) -, or R 6 S(0) 2 - wherein R 6 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.
- R 6 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.
- the remaining member of R 1 and R 2 may then be, H, for example, or any of the alternative embodiments set forth above.
- R 1 and R 2 may be cyano and the other optionally selected from the permissible substituents set forth above, in particular phenyl optionally substituted at one or more positions, for example, with halo, CN, N0 2 , methoxy and the like.
- R 1 and R 2 is optionally substituted benzoyl and the other hydrogen or any of the other suitable choices, such as optionally substituted phenyl.
- R -CH- and forms thereof optionally substituted with electron- withdrawing and/or electron-donating groups as described above, wherein G is a bond; C 0; SO, S0 2 , CZ 2 , or CZ 2 CZ 2 wherein each Z independently is H or CI.
- Y is NBCH 2
- the substituents D or a leaving group in general coupled to the CH 2 element may be released by a competing mechanism designated El . This is illustrated in Figure 3 where the leaving group or drug/prodrug, represented in the alternative by Z is removed as shown followed by the release of
- Each R 5 is independently H, or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted.
- each R 5 is H.
- one of R 5 is H and the other is substituted alkyl or substituted phenyl, comprises an azidoalkyl group or is azido-(CH 2 ) 3- 6, monoalkylamino-(CH 2 )3 -6 , N 3 (CH 2 ) 3- 6 N(Me)C0(CH 2 ) 3-6 -, or -(CH 2 ) 3-6 -C0 2 H, or a protected variant thereof.
- one of R 5 is any one of the particular embodiments described above, further comprising a dendrimer or a functional group allowing for connection to a dendrimer, and the other R 5 is H.
- the B group may be alkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, each optionally substituted.
- the nature of the B group influences the stability of the N-methylene- carbamate toward decomposition via El -type elimination reactions.
- B is optionally substituted aryl, or optionally substituted heteroaryl.
- B is aryl or heteroaryl, each substituted with at least one group having a positive Hammett sigma constant.
- B is unsubstituted phenyl or phenyl substituted with
- B may also be phenyl substituted with an aminocarbonyl, such as morpholinocarbonyl, or a sulfonamidyl, or B may be propargyl, 4-ethoxycarbonylphenyl, propyl, 4-(N,N-diethylcarboxamido)phenyl, 4-morpholinocarbonylphenyl, or 4- morpholinosulfonylphenyl.
- B is any one of the particular embodiments described above, further comprising a dendrimer or a functional group allowing for connection to a dendrimer.
- Compounds of the invention either coupled to a dendrimer via one of R 1 , R 2 , R 5 , and B or R ⁇ and B comprises a functional group that allows for connection to a dendrimer.
- Suitable functional groups that allow for connection to a dendrimer comprise an alkyl or aryl
- R l , R ⁇ R ⁇ and B groups comprises a dendrimer or an alkyl or aryl group further substituted with one or more amino, azido, hydroxy, carboxylic acid, alkynyl, thiol, maleimido, or 1,3-dicarbonyl groups, or protected variants thereof.
- the compounds of formulas (1) or (2) are derived from precursors and intermediates where either the drug/prodrug or the dendrimer is added as the last step. Thus, in one pathway, a compound of the formula
- R , R , R or B are not yet coupled to the dendrimeric macromolecule can be used as an intermediate.
- Either the drug/prodrug or the dendrimer may be coupled first. If the dendrimer is coupled first, the novel compounds of formula (3) wherein a dendrimer is coupled to one of R 1 , R 2 , R 5 or B (if present) is formed. Alternatively, an intermediate containing the drug/prodrug can be first formed and then coupled to the dendrimer.
- one step in the synthesis is coupling the remainder of the molecule to the dendrimer; thus, intermediates are synthesized which contain functional groups in the appropriate R 1 , R 2 , R 5 or B substituents that permit such coupling.
- Methods for conjugation of the intermediates to the dendrimers are generally known in the art.
- an amide linkage is formed between an amino group and a carboxylic acid group; thus, a intermediate comprising an amino group can be conjugated to a dendrimer that contains or is modified to contain a carboxylic acid group, or a intermediate comprising a carboxylic acid group can be conjugated to a dendrimer comprising an amino group as the reactive group.
- the conjugation may be performed by reacting the intermediate and dendrimer in the presence of a condensing agent, for example a carbodiimide such as
- DCC dicyclohexylcarbodiimide
- EDCI l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
- a uronium reagent such as 0-benzotriazole-N,N,N',N'-tetramethyluronium-hexafiuorophosphate (HBTU)
- a phosphonium reagent such as benzotriazole-l-yl-oxy-tris- (dimethylamino)phosphonium hexafluorophosphate (BOP).
- the carboxylic acid group may be activated for conjugation in a prior step, for example by conversion to an acid chloride using thionyl chloride or oxalyl chloride, or to an active ester such as a pentafluorophenyl ester using a carbodiimide and pentafluorophenol or an N-hydroxysuccinimidyl ester using a carbodiimide and N-hydroxysuccinimide, and the resulting activated carboxylate may then be reacted with the amine in a second step.
- the amine and carboxylic acid groups may initially be present in protected form as required for stability and/or compatibility with additional chemical transformations, and deprotected prior to the conjugation step.
- Amine groups may be protected as carbamates, preferably tert- butoxycarbonyl ( l BOC), allyloxycarbonyl (Alloc), or other carbamate groups that may be removed under neutral-to-acidic conditions.
- Carboxylic acids may be protected as esters that may be removed under neutral-to-acidic conditions, such as tert-butyl ( l Bu), trityl (Ph 3 C), allyl (All), or methoxymethyl (MOM).
- a thioether linkage is formed between a thiol group and a maleimide group; thus, a intermediate comprising thiol group can be conjugated to a dendrimer comprising a maleimide group, or a intermediate comprising a maleimide group can be conjugated to a dendrimer that is modified, for example, by a bifunctional linker thiol group.
- the thiol group may initially be present in protected form as required for stability and/or compatibility with additional chemical transformations, and deprotected prior to the conjugation step. Suitable protecting groups include those that may be removed under neutral-to-acidic conditions, for example tert-butyl ethers ( l Bu) or trityl ethers.
- a 1 ,2,3-triazole linkage is formed between an alkyne and an azide group; thus, a intermediate comprising an alkyne group can be conjugated to a dendrimer modified to contain an azide group, or a intermediate comprising an azide group can be conjugated to a solid support modified to contain an alkyne group.
- the conjugation reactions may be performed under metal catalysis, typically using copper or ruthenium, or may be performed in the absence of catalyst using an activated alkyne such as a cyclo-octyne.
- Related cycloaddition methods known in the art may be employed, for example Diels- Alder
- an enamino-ketone linkage is formed between an amino group and a 1,3-dicarbonyl group; thus, a intermediate comprising an amino group can be conjugated to a dendrimer modified to contain a 1,3-dicarbonyl group, or a intermediate comprising a 1,3-dicarbonyl group can be conjugated to a solid support comprising an amine group.
- a intermediate comprising a 1,3-dicarbonyl group is reacted with an antibody such as m38C2 comprising a suitably reactive lysine ⁇ -amino group (Doppalapudi, et al, Bioorganic & Medicinal Chemistry Letters (2007) 17:501-506, incorporated herein by reference).
- the R 1 , R 2 , R 5 , or B groups in the intermediate independently may optionally be substituted by optionally protected amine, optionally protected carboxylic acid, optionally protected thiol, maleimide, alkyne, or azide groups to allow for conjugation with dendrimers.
- the R 1 , R 2 , R 5 , or B groups independently are substituted by dendrimers connected via, for example, carboxylic amide, thioether, or 1,2,3-triazole groups.
- coupling of the drug is illustrated below.
- coupling to dendrimer may or may not have already been conducted.
- the drug is coupled prior to coupling to dendrimers.
- R 1 , R 2 and R 5 are as above-defined or alternatively, one of R 1 , R 2 and R 5 is coupled to a dendrimer.
- the compound of formula (A) is first activated for condensation by reaction with a suitable reagent, for example phosgene or triphosgene, optionally in the presence of N-hydroxysuccinimide; 1,1-carbonyldiimidazole;
- a suitable reagent for example phosgene or triphosgene, optionally in the presence of N-hydroxysuccinimide; 1,1-carbonyldiimidazole;
- the resulting compound comprises a carbamate linkage.
- the amino group that reacts with the intermediate may be a terminal alpha-amino group or the amino group of a side-chain, for example of a lysine, ornithine, or unnatural amino acid residue.
- the activating reagent may be a substituted phenyl chloroformate, for example, 4-nitrophenyl chloroformate, 2,4-dinitrophenyl chloroformate, or pentafluorophenyl chloroformate, resulting in formation of an intermediate substituted phenyl carbonate.
- the precursors wherein the drug is an oligonucleotide or nucleic acid may be prepared by chemical synthesis of the drug comprising a 5 '-terminal modification that allows for conjugation.
- the oligonucleotide may be chemically synthesized such that the 5'- terminal nucleotide unit, added at the last round of synthesis, comprises a phosphate group modified to contain an amino-alkyl group.
- the resulting amine-modified nucleic acid molecule is then conjugated to form a molecule of formula (3). See, for example, Zhao, et al,
- the intermediate formed b reaction with an amino acid is then employed in standard peptide synthesis: peptide synthesis
- the intermediate is attached during the synthesis of the peptide.
- the final step in the synthesis of the peptide by solid-phase peptide synthesis methods well-known in the art involves attachment of the N-terminal amino acid of the sequence of the peptide in protected form.
- the final step uses the N-terminal amino acid in a form using the intermediate as the protecting group, which is not removed.
- R is the side chain of an amino acid
- This embodiment is advantageous in that the position and stoichiometry of derivitization is completely controlled.
- the intermediate is a compound wherein Y is NBCH 2 .
- the nucleofuge coupled to CH 2 is similarly displaced by the drug or prodrug.
- the intermediate may or may not already be coupled to the dendrimeric macromolecule.
- Those intermediate compounds wherein m is 0 may be prepared by the addition of a carbanion R I R 2 CH " formed by reacting R 1 R 2 CH 2 with a strong base, for example butyllithium, NaH, lithium diisopropylamide, lithium bis(trimethylsilylamide), or similar, with a molecule to produce a compound of formula (A)
- a strong base for example butyllithium, NaH, lithium diisopropylamide, lithium bis(trimethylsilylamide), or similar
- Compounds wherein m is 1 and both R 5 are H may be prepared by addition of the carbanion derived by lithiation of R ! R 2 CH 2 , for example using a strong base such as NaH, butyllithium, lithium bis(trimethyl-silylamide), or similar, to an unsaturated compound such as methyl 3-(dimethyIamino)-acrylate to provide an intermediate ester, which may be reduced, either via one step or through multiple steps, to the corresponding unsaturated aldehyde:
- a strong base such as NaH, butyllithium, lithium bis(trimethyl-silylamide), or similar
- hexahydrotriazine This results in an intermediate where a leaving group coupled to the methylene is a halo group— i.e., Y-Cl, wherein Y is NBCH 2 .
- This intermediate can be converted to an intermediate with a drug comprising OH, SH or a heterocyclic nitrogen group under anhydrous conditions in the presence of mild base.
- Suitable bases include tertiary amines such as triethyl amine.
- the reaction mixture may optionally include Nal or a
- Suitable solvents include any inert anhydrous solvent. Attachment of Protective Polymers
- the dendrimers may also include protective polymer (the most common example would be PEG, but other hydrophilic polymers could also be used).
- pre-assembled units comprising a PEG, a releasable linker or drug conjugate or some combination of these units may be prepared.
- the pre-assembled units are then attached to the outer shell of the dendrimer, either in solution or while on a solid phase synthesis support.
- Such pre-assembled units may be constructed in a stepwise process starting from a trifunctional matrix molecule wherein each functionality may be selectively attached to a PEG, a releasable linker or drug conjugate, and to the dendrimer outer shell.
- Suitable functionalities include carboxylic acids, amines, maleimides, azides, thiols, and alkynes, which may be present in protected form.
- an amino acid comprising a carboxylic acid group and two
- differentially protected functional groups can be converted into such a pre-assembled unit by selective deprotection of one protected functional group, attachment of a PEG, then deprotection of the second protected functional group and attachment of the drug conjugate, then final attachment of the pre-assembled unit through the carboxylic acid to the dendrimer.
- azidonorleucine is reacted with an activated PEG molecule, for example a PEG N-hydroxysuccinimide carbonate, so as to produce N a -PEG-azidonorleucine.
- the N a -PEG- azidonorleucine is then either attached to the outer shell of the dendrimer through standard amide-forming reactions to provide a PEGylated dendrimer having an array of azide
- a protected cysteine for example S-(monomethoxytrityl)- cysteine
- an activated PEG molecule for example a PEG N-hydroxysuccinimide carbonate
- N a -PEG-S(mmt)-cysteine is attached to a dendrimer having outer shell amines using standard amide forming reactions, and the resulting dendrimer can be detritylated using mild acid and the resulting thiols reacted with a maleimide-linker or maleimide-drug conjugate.
- the N a -PEG-S(mmt)-cysteine can be reacted with an amine-linker or amine-drug conjugate using standard amide-forming reactions, and the complete pre-assembled unit can be detritylated using mild acid and coupled to a dendrimer having maieimide groups on the outer shell.
- the conjugates of the invention that are designed to release drugs at controllable rates are administered to subjects in a manner similar to medicaments in general.
- the subjects may be model systems such as mice, rats or rabbits or may be human patients or may be veterinary subjects such as companion animals, livestock, and avian subjects.
- the conjugates of the invention are typically administered by injection, in general by intravenous injection, but other dosage mechanisms are also within the scope of the invention such as oral administration, administration by suppository, transdermal or transmucosal administration and the like.
- the dosage levels will depend on the nature of the drug, the condition to be treated, the nature of the subject, and the judgment of the attending professional. The selection of appropriate release rates for a particular drug or protocol are also dependent on these factors.
- the use and administration of the compounds of the invention is within the skill of the practitioner.
- the conjugates of the invention are particularly useful and advantageous in treating diseases of the lymph system wherein subcutaneous injection is preferred.
- a series of model linker scaffolds having a range of functional groups as potential pKa modulators were designed, prepared and linked via carbamate bonds to N e -2,4-dinitrophenyl-L-lysine (N e -DNP-Lys) for evaluation of release rates; DNP-Lys is water soluble and is a strong chromophore to permit HPLC-UV analysis. Rates of release at pH 7.4 and/or 8.3 were determined.
- the DNP carbamates were prepared as follows. A suspension of N-DNP-L-Lys HC1 (35 mg, 0.1 mmol) in 600 ⁇ . of water was treated successively with 1.0 N NaOH (200 ⁇ ) and 1.0 M NaHC0 3 . A 0.1 M solution of the N-HS carbonate in acetonitrile (1.0 mL) was added to the stirred mixture to give a clear yellow solution. After 1 hr, the mixture was diluted with 10 mL water and loaded onto a Bond-ElutTM CI 8 extraction column (1 gm). The column was washed successively with water, 1% CF 3 C0 2 H water, water, and 50% MeOH/water.
- R x 4-chloro, H, 4-methyl, 4-methoxy, 2,4-dimethyl, and 2,4,6-trimethyl;
- R y -(CH 2 ) 3 C ⁇ CH, -(CH 2 ) 5 N 3 , -(CH 2 )2-maleimide
- stably conjugated Lys(DNP) was also prepared by click chemistry between Na-hexynoyl-Lys(DNP)-OH and 40 kDa-PEG-azide and administered to rats as a control.
- Competitive ELISA for DNP-Lys using DNP-BSA and an anti-DNP antibody conjugated to alkaline phosphatase is employed.
- Preparations 4-19 describe intermediates and compounds of formula (3), not yet linked to dendrimer, i.e., compounds wherein X is O and Y is NBCH 2 wherein the CH 2 group is coupled to a nucleofuge.
- the nucleofuge is replaced by a model system permitting monitoring release of cysteine coupled to 4-dinitrophenyl.
- N-hydroxysuccinimide carbonates the crude chloroformate is dissolved in anhydrous tetrahydrofuran (2 mL/mmol) and treated with pyridine (2 equivalents) and N-hydroxysuccinimide (4 equivalents) at ambient temperature for 30 minutes. The mixture is diluted with ethyl acetate, washed successively with 0.1 N HCl, water, and brine, then dried over MgS0 4 , filtered, and evaporated. The crude carbonates are purified by silica gel
- triethylamine (1 equivalent) is added to a mixture of the amine or aniline (1 equivalent) and the chloroformate (1 equivalent) in an inert anhydrous solvent, for example dichloromethane, tetrahydrofuran, or ethyl acetate. After stirring for 1 h at ambient temperature, the mixture is evaporated to dryness, and the residue is dissolved in ethyl acetate and washed successively with 1 N HCl, water, sat. aq. NaHC0 3 , and brine, then dried over MgS0 4 , filtered, and evaporated to provide the crude carbamate, which is purified as described above.
- an inert anhydrous solvent for example dichloromethane, tetrahydrofuran, or ethyl acetate.
- an alcohol is converted to a carbamate without isolation of the intermediate chloroformate.
- Pyridine (0.33 equivalent) is added dropwise to a vigorously stirred solution of the alcohol (1 equivalent) and triphosgene (0.33 equivalent) in anhydrous tetrahydrofuran (2 mL/mmol) cooled on ice. After 1 hr, the mixture is allowed to warm to ambient temperature and kept overnight. The mixture is cooled on ice, and the amine or aniline (2 equivalents) is added. The mixture is allowed to warm to ambient temperature and kept overnight. The mixture is then evaporated to dryness, and the residue is dissolved in ethyl acetate and washed successively with 1 N HC1, water, sat. aq. NaHC0 3 , and brine, then dried over MgS0 4 , filtered, and evaporated to provide the crude carbamate, which is purified as described above.
- a solution of N-chloromethyl carbamate in methanol is allowed to stand at ambient temperature for 1 h, then concentrated to dryness to provide the N-methoxymethyl carbamate.
- N-Alkoxymethyl Carbamates N-Phenoxymethyl Carbamates, N-Thiomethyl Carbamates, and N-Thiophenylmethyl Carbamates
- Triethylamine (0.7 mL) was added to a stirred mixture of 4-bromoaniline (0.85 g) and 9-fluorenylmethoxycarbonyl chloride (1.3 g) in 25 mL of dichloromethane. The mixture was stirred for 1 h at ambient temperature, then washed with 1 N HCl, water, sat. aq. NaHC0 3 , and brine. The organic solution was dried over MgS0 4 , filtered, and evaporated.
- Triethylamine (0.7 mL) was added to a stirred mixture of ethyl 4-aminobenzoate (0.85 g) and 9-fluorenylmethoxycarbonyl chloride (1.3 g) in 25 mL of dichloromethane. The mixture was stirred for 1 h at ambient temperature, then washed with 1 N HCl, water, sat. aq. NaHC0 3 , and brine. The organic solution was dried over MgS0 , filtered, and evaporated. Preparation 13
- Step 1 A solution of cystine bis(allyl ester) p-toluenesulfonate and
- Preparations 20-22 describe intermediates and compounds of formula (3) not yet linked to dendrimer, i.e., compounds wherein X is O and Y is NBCH 2 , wherein the methylene group is coupled to the OH group of a serine residue.
- Step 1 N-(tert-butoxycarbonyl)-L-serine allyl ester: To a stirred solution of allyl bromide (2.3 mL, 26.6 mmol) and tricaprymethylammonium chloride (4.00 g, 9.90 mmol) in CH 2 C1 2 (35 mL) was added a solution of N-(te /-butoxycarbonyl)-L-serine (1.03 g, 5.02 mmol) and NaHC(3 ⁇ 4 (0.43 g, 5.12 mmol) in water (16 mL). The biphasic reaction mixture was vigorously stirred at room temperature for 48 hours.
- Step 2 A solution of N-(tert-butoxycarbonyl)-L-serine allyl ester (0.175 g,
- Step 1 A solution of N-(6-(2,4-dinitrophenylamino)hexanoyl-L-serine allyl ester (0.050 g, 0.118 mmol), 0-(9-fluorenylmethyl) N-phenyl N-chloromethyl carbamate (0.043 g, 0.118 mmol) and triethylamine (16.1 mL, 0.116 mmol) in anhydrous CH 2 C1 2 (2 mL) was heated at reflux for 1 hour.
- Step 2 Tetrakis(triphenylphoshine)palladium(0) (0.002 g, 1.7 ⁇ ) was added to a stirred solution of the allyl ester from Step 1 (0.030 g, 40 ⁇ ) and phenylsilane (9.8 mL, 80 ⁇ ) in anhydrous tetrahydrofuran (0.5 mL). The reaction mixture was stirred at ambient temperature for 30 minutes and was then concentrated. Silica gel and CH 2 C1 2 were added and the mixture again concentrated and loaded onto a short silica gel column.
- Preparations 23 and 24 together describe constructs of the invention wherein Y is NBCH 2 and the linker for binding to dendrimer is coupled to R 1 .
- the construct includes the drug SN38 which provides a hydroxyl group for binding to the CH 2 of the adaptor.
- a solution of fluorene-2-carbonyl chloride (prepared from fluorene-2-carboxylic acid and oxalyl chloride) in THF is added to aqueous methylamine (2 molar equivalents) to prepare N-methyl fluorene-2-carboxamide.
- Reduction of the amide using LiAlH 4 in ether provides 2-((methylamino)methyl)fluorene.
- the amine is protected by reaction with di-tert-butyl dicarbonate to provide 2-((N- t BOC-N-methylamino)methyl)fluorene.
- the carbamate is dissolved in trifluoroacetic acid to remove the 'BOC protecting group. After evaporation to dryness, the resulting amine is dissolved in THF and treated with N-(6-azidohexanoyl)succinimide and triethylamine (2 equivalents) to provide
- Preparations 25-30 are prophetic examples showing the preparation of embodiments wherein Y is NBCH 2 and the linker is coupled through R 5 .
- N 3 (CH 2 ) n C0 2 R' (n 3-6) in the presence of a strong base, for example NaH, lithium bis(trimethylsilyl)amide (LiHMDS), or lithium diisopropylamide (LDA), provides a ketone which is reduced to the alcohol by reaction with a mild reductant, for example sodium borohydride in methanol. The resulting alcohol is then converted into the carbamate via the chloroformate, and then into the N-chloromethylcarbamate as described above.
- a strong base for example NaH, lithium bis(trimethylsilyl)amide (LiHMDS), or lithium diisopropylamide (LDA)
- LiHMDS lithium bis(trimethylsilyl)amide
- LDA lithium diisopropylamide
- the BOC group is removed from the carbamate by treatment with trifluoroacetic acid.
- the resulting amine is coupled with a macromolecule comprising a carboxylic acid using a condensing agent, for example a carbodiimide such as EDCI.
- An ethyl (2-phenylsulfonyl)acetate is deprotonated using excess NaH in THF and alkylated with N-(6-bromohexyl) ethyl carbamate.
- the product is reduced using lithium aluminum hydride in ether to provide the methylamino alcohol, which is N-protected as the BOC carbamate.
- the alcohol is converted to the chloroformate and thence into the carbamate and into the N-chloromethyl carbamate according to the previous procedures.
- a phenyl methylsulfone is deprotonated with NaH in tetrahydrofuran, then acylated with glutaric anhydride to provide a keto-acid.
- the resulting acid is protected as the tert-butyl ester, and the ketone is reduced using NaB3 ⁇ 4.
- the resulting alcohol is converted into the carbamate via the chloroformate, and thence to the N-chloromethyl carbamate as described above.
- Peptide synthesis is performed using standard methods for solid-phase peptide synthesis, using a serine, tyrosine, or cysteine in a suitably protected form such that the side chains of these residues may be selectively deblocked without deprotection of other residues.
- the partially deprotected peptide is reacted with an excess of intermediate of formula (3) which is not yet linked to dendrimer in the presence of a mild base. After washing the resin, the product peptide is deblocked and cleaved from the resin to provide the corresponding intermediate prior to dendrimer linkage wherein D is a peptide.
- CCK8 (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH 2 ) is synthesized on solid support using Rink resin using methods known in the art, for example as described in US Patent 4,769,445 (incorporated herein by reference).
- Commercial Fmoc-Phe-Rink amide- MBHA resin is pre-swollen in DMF for 30 min, then suspended and shaken in piperidine/DMF (1 :4 by volume, 50 ml) for 30 min at room temperature to remove the Fmoc group.
- the product is isolated by filtration and washed (3 X 50 ml each) with DCM, 5% N,N-diisopropylethylamine (DIEA) in DCM, and DCM to give the free base of Phe-Rink amide-MBHA-Resin.
- Fmoc- Asp(O l Bu)-OH (1.23 g, 3 mmol), DCC (0.62 g, 3 mmol), and HOBt (0.69 g, 4.5 mmol) are dissolved in 50 ml of 4:1 by volume DCM/DMF with stirring at 0° for 1 hour.
- Phe- Rink amide- MBHA resin (1 meq) is suspended in the filtered reaction mixture (precipitated DCU removed) and shaken for 2 to 15 hours at room temperature.
- the Fmoc-Asp-(O l Bu)-Phe- Rink amide- MBHA resin product is collected by filtration and washed with DCM.
- the Fmoc-Asp-(O'Bu)- Phe- Rink amide-MBHA resin is suspended and shaken in piperidine/DMF (1 :4 by volume, 50 ml) for 3 min at room temperature and then a second time for 7 min to remove the Fmoc group.
- the Fmoc-Met-Asp-(O t Bu)-Phe-Rink amide-MBHA resin product is collected by filtration and washed with DCM and DMF.
- the Fmoc-Met-Asp-(O l Bu)- Phe- Rink amide-MBHA resin is deprotected and coupled sequentially with Fmoc-Trp-OH (1.28 g, 3 mmol), Fmoc-Gly-OH (0.89 g, 3 mmol), Fmoc-Met-OH (1.12 g, 3 mmol), Fmoc-Tyr- OH (1.37 g, 3 mmol), and Boc-Asp(O f Bu)-OH (1.23 g, 3 mmol) to provide Boc-Asp(0 [ Bu)-Tyr- Met-Gly-Trp-Met-Asp(OtBu)-Phe-Rink amide-MBHA resin.
- Boc-Asp(0 [ Bu)-Tyr-Met- Gly-Trp-Met-Asp OTSu ⁇ Phe-Rink amide-MBHA resin is washed with DCM (3x50 ml), suspended and shaken in a mixture of 0-(9-fluorenylmethyl) N-phenyl N-chloromethylcar- bamate (10 equivalents) and triethylamine (1 equivalent) in DCM.
- the resin is isolated by filtration and washed (3 50 ml each) with DCM.
- cysteine-containing peptide is prepared by solid phase synthesis using the methods described above, incorporating an
- cysteine residue Prior to cleavage from the resin, the cysteine residue is selectively deblocked using (Ph 3 P) 4 Pd and phenylsilane in DCM, then reacted with a compound of formula (II) as described above. The peptide is finally deblocked, removed from the resin, and purified as described above.
- linker-drug compounds of formula (III) may be prepared from 5-fluorouracil and a compound of formula (II) analogously to the procedures used by Taylor and Sloane, "1-Alkylcarbonyloxymethyl Prodrugs of 5-Fluorouracil (5-FU):
- Examples 1-5 are prophetic examples describing how dendrimers are coupled to intermediates to obtain the constructs of the invention.
- Coupling reactions to dendrimers are monitored to insure completeness of reactions, typically using chromogenic or fluorogenic reactions.
- Acylation reactions use chromogenic leaving groups (e.g., p-nitro-phenyl (pNP) esters and carbonates) that can be continuously monitored.
- Free amines of the dendrimer are determined by the chromogenic reaction with TNPS.
- Alkynes and azides are determined by click reactions with fluorogenic azide and alkyne reagents, respectively.
- Thiols are determined by chromogenic reaction with DTNB.
- Modified dendrimers are analyzed by MS.
- chloroformate, HSE for attachment to alcohol (carbonate) and amine (carbamate) groups of drugs on one end, and functional groups for attachment to dendrimers on the other: maleimido for attachment to thiols, carboxyl for amino groups, alkyne for azides, and azides for alkynes are prepared.
- linkers used in this example are acid-stable and base-labile, so basic conditions
- acylation reactions are used for PEGylation of dendrimers, they are performed before attachment of drugs containing nucleophiles (e.g. , peptides) to avoid modifying the drug.
- free amines are coupled to activated mPEG, for example, PEG(5000)-pNP carbonate or
- PLL dendrimers in which half of the surface amines are free, and the other half attached to PEG [PLLi 6 (s-PEG5000) 8 (a-NH 2 ) 8 ].
- a carbonate is formed at the 20-OH by reaction with 5-octynol chloroformate (from 5-octynol and triphosgene/pyridine).
- the alkyne of the linker is attached to PLL 16 (PEG5000)g(azide) 8 containing 8 azide end-groups by CuAAC, providing a stable conjugate except for the carbonate moiety.
- the conjugate i. e.
- carbonate is studied (by HPLC) at pH 7.4 ⁇ serum over a long period, to demonstrate that the carbonate is stable to serum PEGylated dendrimers linked to CPT by a carbonate using one or more of our releasable linkers ⁇ e.g., phenyl-sulfone analogs, see Table 4).
- Kinetic studies are performed at pH 7.4 ⁇ serum (by HPLC) to determine the ⁇ -eliminative/release rate.
- the aforementioned conjugate provides a soluble particle that is ⁇ 6% weight CPT, almost 10-fold higher density than could be achieved with linear mono-methoxy PEG.
- Exendin-4 a 39-amino acid peptide from the Gila monster, is an agonist of the GLP-1 receptor, and thus an insulin secretagogue with glucoregulatory effects.
- Exendin sequence H?N-HGEGTFTSDLSKi ? QMEEE A VRLFIE WLK? 7 NGGP S S G APPPS-NH 2 (trypsin sites and amino terminus in red, a-helix underlined): Exendin-4 has a longer plasma lifetime than GLP-1 ( ⁇ 5 min), but a half-life of only 2.5 hrs. It is marketed for type 2 diabetes as Byetta ® .
- Exendin 4 consists of a 5-turn a-helix (Leu 10 to Asn 28 ; -27 A) with mobile N- and C-termini. It is relatively stable against plasma proteases in vitro (tj/ 2 -10 hr), and attachment to a PEG-dendrimer should increase stability even more. It has three potential trypsin cleavage sites: Lys 12 and/or Lys 2 7 are most susceptible, and Arg 20 is ⁇ 14-fold more resistant. PEGylation of the exendin N-terminus yields an inactive conjugate, whereas PEG-Lys 12 or PEG-Lys 27 are about as active agonists as the native peptide.
- a releasable linker is coupled to the a- or Lysi 2 amino groups of exendin, and each linker-exendin is coupled to a PEG5000 PLL dendrimer to give PLLi 6 (oc-PEG5000) 6 (s- exendin) 8 .
- Linear PEG-exendin controls are also prepared.
- Exendin is prepared by SPPS by Fmoc/tBu chemistry, using an orthogonal blocking group at the side chain of the Lys 12 (e.g., monomethoxytrityl, MMT).
- the blocking group at the intended site of reaction ( -amino Fmoc or Lysi 2 side chain MMT) is removed, and an HSE ester of an alkyne-containing or azide-containing releasable linker coupled to the free-amino group on-resin.
- Blocking groups and resin are removed (TFA), and the modified carbamoylated peptides purified by HPLC.
- the linker is attached to
- the in vitro rate of ⁇ -eliminative release and escape of the peptide from the dendrimer is determined at pH 7.4 ⁇ serum and 8.4 using SEC HPLC; the observed rate is a composite of the ⁇ -elimination and diffusion of the free peptide through PEG, but the latter should not contribute significantly.
- the PEG and PEG-dendrimer conjugates are tested as agonists of the GLP1 receptor in membrane or cell-based assays (e.g., RTN-m5f cells, ATCC as described (Young, A., et al, (2000) WO00/66629) pages 74-75) before and after ⁇ -eliminative release of native exendin.
- membrane or cell-based assays e.g., RTN-m5f cells, ATCC as described (Young, A., et al, (2000) WO00/66629) pages 74-75
- Conjugates are then treated with mild base to release native exendin that shows full activity as GLP1 agonists.
- conjugates are treated with trypsin and sera to determine accessibility to proteases; at various times, excess PMSF is added to quench serine proteases, peptides are released by mild base-catalyzed ⁇ -elimination and the remaining native peptide determined by HPLC.
- Examples 6-9 are working examples describing construction of a dendrimer which has as its core two lysine residues linked through diaminohexane and provided with azido groups for linkage to drug.
- Examples 10 and 1 1 are prophetic examples showing linkage of this intermediate both to PEG and to an alkynyl linker coupled to drug.
- Trifluoroacetic acid (5 mL) was added to a stirred solution of DAH[Lys] 2 [a-Boc] 2 [8- Boc] 2 (0.200 g; 0.259 mmol) in dichloromethane (5 mL). The solution was stirred at room temperature for 1 hour and was then concentrated on the roto-vap. The crude salt was dissolved in N,N-dimethylformamide (8 mL) and triethylarnine (0.58 mL; 4.16 mmol) added. To this solution was added Boc-Lys(Boc)-OSu (0.505 g; 1.14 mmol) and the reaction mixture stirred for 20 hours.
- Trifluoroacetic acid (4 mL) was added to a stirred suspension of DAH[Lys] 4 [a- Boc] 4 [s-Boc] 4 (0.139 g; 82.4 ⁇ ) in dichloromethane (4 mL) and the resulting solution stirred at ambient temperature for 1.5 hours.
- the solution was concentrated and the crude salt was taken up in N,N-dimethylformamide (4 mL) and triethylamine (368 ⁇ ; 2.7 mmol) added.
- a solution of Boc-L-azidonorleucine succinimidyl ester (0.268 g;
- Trifluoroacetic acid is added to a stirred suspension of DAH[Lys] 4 [BOC-ANL]8 in dichloromethane as described for other examples above and the resulting solution is stirred at ambient temperature for 1.5 hours.
- the solution is concentrated and the crude salt is taken up in ⁇ , ⁇ -dimethylformamide and triethylamine sufficient to neutralize the salts and allow subsequent coupling is added.
- mw 5000 monomethoxy- polyethylene glycol succinimidyl ester
- the reaction mixture is stirred at room temperature for 22 hours and is then dialyzed against methanol using a 10,000-mw cutoff dialysis membrane to remove small molecule reagents and byproducts.
- the dialysate is evaporated to dryness to provide the product.
- a solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS0 4 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys] 4 [NL] 8 [mPEG 5 ooo]8[N 3 ] 8 and 10 equivalents of alkyne-linker-drug in 1 :2
- Examples 12-16 result in compounds of the invention wherein 4 copies of the peptide drug exendin are coupled through linkers to the polylysine dendrimer that has been PEGylated.
- X is O and Y is absent.
- Rink-amide TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH 2 ) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques. After removal of the last FMOC group and washing the resin three times with dichloromethane to remove excess piperidine, the resin is treated with a solution of 3 equivalents of the alkynyl-Linker
- the mmt group is removed by treatment with dilute CF3CO2H in dichloromethane, and the resin is treated with a solution of 3 equivalents of the alkynyl-Linker succinimidyl carbonate in dichloromethane/DMF and 1.5 equivalent of N-methylmorpholine. After coupling is completed, the resin is washed to remove excess reagents.
- the linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol.
- the linker- exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1 % trifluoroacetic acid, and lyophilized.
- Step 1 2-Bromo-3'-nitroacetophenone (2.98 g, 12.2 mmol) was dissolved in acetonitrile (12 mL). Water (12 mL) then sodium hydrogen carbonate (2.04 g, 24.3 mmol) were added. The resulting biphasic mixture was vigorously stirred and 4-methoxythiophenol (1.5 mL, 12.2 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 1.5 hours. It was then diluted with water and extracted with ethyl acetate (x3).
- Step 2 To an ice-cooled stirred solution of the sulfide of Step 1 (2.75 g, 9.07 mmol) in ethyl acetate (75 mL) was added peracetic acid (5.8 mL of a 32 wt% solution in dilute acetic acid, 27.6 mmol) slowly over 10 minutes. The solution was stirred at ice-bath temperature for 10 minutes and then at ambient temperature for 2 hours. The suspension was dissolved by the addition of ethyl acetate (75 mL) and this solution was washed with 1 M sodium carbonate (x 2), water, 0.1 M sodium hydrosulfite (x 2), water, 1 M sodium carbonate and saturated sodium chloride.
- Step 3 Tin (II) chloride dihydrate (2.69 g, 1 1.9 mmol) was added to a stirred suspension of the sulfone of Step 2 (1.00 g, 2.98 mmol) in ethanol (30 mL). The reaction mixture was heated at gentle reflux for 30 minutes and the resulting yellow solution allowed to cool to room temperature. The solution was poured onto crushed ice and the pH adjusted to pH 8 with 1 M sodium carbonate. The suspension was equilibrated to room temperature and diluted to -200 mL volume with water.
- Step 4 To a stirred solution of 5-hexynoic acid (392 ⁇ , 3.46 mmol) and oxalyl chloride (351 ⁇ L, 4.15 mmol) in anhydrous dichloromethane (5 mL) was added 2 drops of anhydrous ⁇ , ⁇ -dimethylformamide resulting in gas evolution. The solution was stirred for 15 minutes after which time gas evolution had ceased and a further 2 drops of anhydrous ⁇ , ⁇ -dimethylformamide were added (no gas evolution). The solution was stirred for 10 minutes and was then concentrated on the rotary evaporator.
- Step 5 To a stirred suspension of the amide of Step 4 (0.484 g, 1.21 mmol) in methanol (12 mL) was added in portions over 5 minutes sodium borohydride (0.102 g,
- Step 6 Pyridine (156 ⁇ ,, 1.93 mmol) was added to a stirred solution of the alcohol of Step 5 (0.387 g, 0.964 mmol) and triphosgene (0.41 1 g, 1.39 mmol) in anhydrous
- Rink-amide TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH 2 ) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques. After removal of the last FMOC group and washing the resin three times with dichloromethane to remove excess piperidine, the resin is treated with a solution of 3 equivalents of the alkynyl-Linker
- linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol.
- the linker- exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1% trifluoroacetic acid, and lyophilized.
- Examples 17-20 describe synthesis of compounds of the invention wherein a drug is coupled to a polylysine dendrimer through a different linker, and wherein X is O and Y is absent.
- Trifluoroacetic acid (4 mL) is added to a stirred suspension of DAH[Lys] 4 [a- Boc] 4 [s-Boc] 4 (0.139 g; 82.4 ⁇ ) in dichloromethane (4 mL) and the resulting solution is stirred at ambient temperature for 1.5 hours.
- the solution is concentrated and the crude salt is taken up in N,N-dimethylformamide (4 mL) and triethylamine (368 ⁇ L; 2.7 mmol) added.
- a solution of Boc-L-propargylglycine succinimidyl ester (726 ⁇ ) in ⁇ , ⁇ -dimethyl-formamide is added to this solution.
- the reaction mixture is stirred at room temperature for 22 hours and is then added by pipette to a stirred ice-water (200 mL) solution.
- the resulting suspension is stirred for 30 minutes and is then collected by filtration.
- the solid is washed with water and dried. It is re-suspended in acetonitrile (2 mL) and stirred for 30 minutes. The solid is collected, washed with acetonitrile and dried to give the product.
- Trifluoroacetic acid is added to a stirred suspension of DAH[Lys] 4 [BOC-PG] 8 in dichloromethane as described for other examples above and the resulting solution is stirred at ambient temperature for 1.5 hours.
- the solution is concentrated and the crude salt is taken up in ⁇ , ⁇ -dimethylformamide and triethylamine sufficient to neutralize the salts and allow subsequent coupling is added.
- To this solution is added a molar excess of
- reaction mixture is stirred at room temperature for 22 hours and is then dialyzed against methanol using a 10,000-mw cutoff dialysis membrane to remove small molecule reagents and byproducts. The dialysate is evaporated to dryness to provide the product.
- a solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS0 4 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys]4p ⁇ L]s[mPEG 5 oooMAlkyne]8 and 10 equivalents of azido-linker-drug in 1 :2 water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-exendin. The dialysate is lyophilized to provide the product.
- DMSO dimethylsulfoxide
- Examples 21-23 describe coupling of exendin to the polylysine through still a different linker, wherein X is O and Y is absent.
- Step 1 2-Bromo-3'-nitroacetophenone (2.98 g, 12.2 mmol) was dissolved in acetonitrile (12 mL). Water (12 mL) then sodium hydrogen carbonate (2.04 g, 24.3 mmol) were added. The resulting biphasic mixture was vigorously stirred and 4-methoxythiophenol (1.5 mL, 12.2 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 1.5 hours. It was then diluted with water and extracted with ethyl acetate (x 3).
- Step 2 To an ice-cooled stirred solution of the sulfide of Step 1 (2.75 g, 9.07 mmol) in ethyl acetate (75 mL) was added peracetic acid (5.8 mL of a 32 wt% solution in dilute acetic acid, 27.6 mmol) slowly over 10 minutes. The solution was stirred at ice-bath temperature for 10 minutes and then at ambient temperature for 2 hours. The suspension was dissolved by the addition of ethyl acetate (75 mL) and this solution was washed with 1M sodium carbonate (x 2), water, 0.1 M sodium hydrosulfite (x 2), water, 1 M sodium carbonate and saturated sodium chloride.
- Step 3 Tin (II) chloride dihydrate (2.69 g, 1 1.9 mmol) was added to a stirred suspension of the sulfone of Step 2 (1.00 g, 2.98 mmol) in ethanol (30 mL). The reaction mixture was heated at gentle reflux for 30 minutes and the resulting yellow solution allowed to cool to room temperature. The solution was poured onto crushed ice and the pH adjusted to pH 8 with 1 M sodium carbonate. The suspension was equilibrated to room temperature and diluted to -200 mL volume with water.
- Step 4 To a stirred solution of 6-azidohexanoic acid and oxalyl chloride in anhydrous dichloromethane is added 2 drops of anhydrous ⁇ , ⁇ -dimethylformamide resulting in gas evolution. The solution is stirred for 15 minutes after which time gas evolution ceases and a further 2 drops of anhydrous ⁇ , ⁇ -dimethylformamide are added. The solution is stirred for 10 minutes and is then concentrated on the rotary evaporator. The crude acid chloride is dissolved in anhydrous dichloromethane and slowly added to a stirred suspension of the aniline of Step 3 and triethylamine in anhydrous dichloromethane.
- Step 5 To a stirred suspension of the amide of Step 4 in methanol is added in portions over 5 minutes sodium borohydride as described for the preparation of the alkynyl linkers above. The resulting solution is stirred for 35 minutes and then quenched by the addition of saturated ammonium chloride.
- Step 6 Pyridine is added to a stirred solution of the alcohol of Step 5 and triphosgene in anhydrous tetrahydrofuran as described for the preparation of the alkynyl linkers above. The resulting suspension is stirred for 30 minutes and is then filtered and the filtrate concentrated under reduced pressure. The crude chloroformate is taken up in anhydrous tetrahydrofuran and N-hydroxysuccinimide followed by pyridine is added. The reaction mixture is stirred at ambient temperature for 35 minutes and is then filtered and concentrated. The residue is dissolved in ethyl acetate and washed with water, 0.1 M hydrochloric acid, saturated sodium hydrogen carbonate, water and saturated sodium chloride. The organic solution is dried over magnesium sulfate and concentrated under reduced pressure to yield the crude product. Purification by silica gel column chromatography affords the succinimidyl carbonate.
- Rink amide-TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH 2 ) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques. After removal of the last FMOC group and washing the resin three times with dichloromethane to remove excess piperidine, the resin is treated with a solution of 3 equivalents of the azido-Linker succinimidyl carbonate of Example 21 in dichloromethane/DMF and 1.5 equivalents of N-methylmorpholine. After coupling is completed, the resin is washed to remove excess reagents.
- the linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol.
- the linker-exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1% trifluoroacetic acid, and lyophilized.
- a solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS0 4 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys] 4 [NL] 8 [mPEG 5 ooo]8[alkyne]8 and 10 equivalents of azido-linker-exendin in 1 :2 water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-exendin.
- DMSO dimethylsulfoxide
- dialysate is lyophilized to provide the product.
- Examples 24-26 describe the preparation of a polylysine dendrimer coupled through an azido linker to SN38 wherein X is O and Y is NBCH 2 .
- a solution of fluorene-2-carbonyl chloride (prepared from fluorene-2-carboxylic acid and oxalyl chloride) in THF is added to aqueous methylamine (2 molar equivalents) to prepare N-methyl fluorene-2-carboxamide.
- Reduction of the amide using L1AIH 4 in ether provides 2-((methylamino)methyl)fluorene.
- the amine is protected by reaction with di-tert-butyl dicarbonate to provide 2-((N- t BOC-N-methylamino)methyl)fluorene.
- the carbamate is dissolved in trifluoroacetic acid to remove the l BOC protecting group. After evaporation to dryness, the resulting amine is dissolved in THF and treated with N-(6-azidohexanoyl)succinimide and triethylamine (2 equivalents) to provide 0-(9-(2-((N-(6- azidohexanoyl)-N-methylamino)methyl)fluorenylmethyl) N-phenylcarbamate.
- a solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS0 4 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys] 4 [NL] 8 [mPEG 50 oo] 8 [alkyne] 8 and 10 equivalents of azido-linker-SN38 in 1 :2 water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-SN38. The dialysate is lyophilized to provide the product.
- This example supports dendrimer assembly via dendrons in Example 28, method B.
- Step 2 A solution of the N-(mPEG 5 ooo-oxycarbonyl)-L-azidonorleucine from Step 1 in dry acetonitrile is treated with N,N'-disuceinimidyl carbonate (1.2 equivalents) and
- Examples 28 and 29 describe preparation of dendrimers and coupling to alkynyl linker drugs, respectively.
- Step 1 H-Lys-Gly-Resin.
- TentaGel resin loaded with Fmoc-Glycine is deprotected by treatment with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
- the resin is treated with a DMF solution containing a 4-fold excess of HBTU-activated N a ,N E -bis(9-fluorenylmethoxycarbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH) and HOBt, and a 4-fold excess of ⁇ , ⁇ -diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
- Step 2 [H-Lys] 2 -Lys-Gly-Resin.
- the resin from Step 1 is treated with a DMF solution containing an 8-fold excess of HBTU-activated N a ,N e -bis(9- fluorenylmethoxycarbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH) and HOBt, and an 8-fold excess of N,N-diisopropylethyIamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
- Step 3 [[H-Lys] 2 -Lys] 2 -Lys-Gly-Resin.
- the resin from Step 2 is treated with a DMF solution containing a 4-fold excess of HBTU-activated N a ,N 8 -bis(9-fiuorenylmethoxycarbonyl)- L-lysine (Fmoc-Lys(Fmoc)-OH) and HOBt, and an 4-fold excess of N,N-diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
- Step 4 [[[ANL] 2 -Lys] 2 -Lys] 2 -Lys-Gly-Resin.
- the resin from Step 3 is treated with a DMF solution containing a 4-fold excess of Fmoc-L-azidonorleucine succinimidyl ester, and an 4-fold excess of ⁇ , ⁇ -diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
- Step 5 [[[mPEG 5000 -ANL] 2 -Lys] 2 -Lys] 2 -Lys-Gly-Resin.
- the resin from Step 4 is treated with a DMF solution containing a 4-fold excess of monomethoxypolyethylene glycol propionate succinimidyl ester (mw 5000), and an 4-fold excess of ⁇ , ⁇ -diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is washed 3x with dichloromethane and dried under vacuum.
- Step 6 The dendrimer is cleaved from the resin by treatment with CF 3 C0 2 H for 4 hours, followed by removal of the resin by filtration and evaporation to provide the dendrimer product.
- Steps 1-3 are as in Method A above.
- Step 4. [[[mPEG 50 o 0 -ANL] 2 -Lys] 2 -Lys] 2 -Lys-Gly-Resin.
- the resin from Step 3 is treated with a DMF solution containing a 4-fold excess of N-(mPEG 5 ooo-oxycarbonyl)-L- azidonorleucine succinimidyl ester, and an 4-fold excess of ⁇ , ⁇ -diisopropylethylamine is added. Coupling proceeds for 4 hours.
- Step 5 The dendrimer is cleaved from the resin by treatment with CF 3 C0 2 H for 4 hours, followed by removal of the resin by filtration and evaporation to provide the dendrimer product.
- DMSO dimethylsulfoxide
- the conjugation mixture is shaken overnight, and the resin is filtered and washed 3x with DMSO, 3x with water, 3x with methanol, and 3x with dichloromethane and dried under vacuum.
- the dendrimer is cleaved from the resin by treatment with CF 3 C0 2 H for 4 hours, followed by removal of the resin by filtration and evaporation to provide the crude product, which is dialyzed against water to remove small- molecule contaminants.
- the dialysate is lyophilized to provide the product.
- fluorescein as model drug i.e., compounds of the formula
- 6-azido-l-hexanol a mixture of 6-chloro-l-hexanol (25 g, 183 mmol) and sodium azide (32.5 g, 500 mmol) in 200 mL of water was heated at reflux for 20 h, then cooled to ambient temperature and extracted 3x with ethyl acetate. The combined extracts were washed with brine, dried over MgS0 4 , filtered, and concentrated to yield the product as a pale yellow oil (28.3 g).
- R a in R a CH 3 contains the trigger present in the final product, R 1
- Compounds prepared according to this method include:
- R-CH 3 4-acetylmorpholine
- R-CH 3 " fluorene
- the crude HS carbonate was first chromatographed on silica gel using a gradient of ethyl acetate in hexane, followed by crystallization. All compounds were crystalline with the exception of that obtained from 1- (methanesulfonyl)-7-azido-2-heptanol.
- Compounds prepared according to this method include:
- Example 35 600 mg, 0.88 mmol
- dichloromethane 5 mL
- the resulting mixture was allowed to stir at room temperature for 1.5 h.
- the mixture was then concentrated under reduced pressure to give 1310 mg of residue estimated to comprise 890 mg of free trifluoroacetic acid.
- This material was dissolved in methanol (25 mL) and treated with 24 mL of methanol-washed Dowex ® (Dow Chemical Co.) MonosphereTM 550A for 0.5 h.
- the resin was removed by filtration, and the filtrate was concentrated to dryness to give the product (420 mg) as a viscous clear oil.
- Example 37
- the reaction mixture was dialyzed once against 50% methanol + 0.5% v/v acetic acid using a Spectra Por ® (Spectrum Laboratories) 2 membrane (12-14 kDa-cutoff), then three times against methanol + 0.2% v/v acetic acid.
- the dialysis mixture was concentrated to dryness to give the product as a dark orange-yellow solid.
- the reaction mixture was then diluted with 200 mL of ethyl acetate, washed with 0.5 N NaOH (200 mL in small portions), then with 0.1 N HC1 (200 mL in small portions), then with water, and finally brine.
- the extract was dried over MgS0 4 and concentrated to dryness to give the product (205 mg) as a white solid.
- Examples 47-56 describe preparation of fluorescein as model drug coupled to PEGylated
- Boc-Lys(Boc)-DAB-DNP (0.500 g; 0.858 mmol) was dissolved in anhydrous dichloromethane (5 mL) and trifluoroacetic acid (5 mL) was added. The resulting solution was stirred at room temperature for 2 hours and was concentrated. Ethyl acetate was added and the mixture was concentrated. The brown oil thus obtained was dissolved in anhydrous DMF (5 mL) and triethylamine (598 ⁇ ; 4.29 mmol) added.
- the product was eluted with 1 : 1 water/acetonitrile + 0.1% TFA and the product containing fractions pooled and diluted with methanol. This solution was treated with methanol-washed Dowex ® (Dow Chemical Co.) 55 OA MonosphereTM resin for 1 h and was then filtered and concentrated to give the product as an orange glassy solid (0.022 g).
- Trifluoroacetic acid (0.5 mL) was added to a solution of [Boc-Lys(mPEG 5 kD)]s- [Lys] 4 [Lys] 2 Lys-DAB-DNP (0.1 g) in 0.5 mL of dichloromethane. After 6 h, the mixture was concentrated to give a yellow oil (0.271 g). This was dissolved in methanol and treated with 10 mL of methanol-washed Dowex ® (Dow Chemical Co.) 550 A MonosphereTM resin for 1 h and was then filtered and concentrated to give the product as an pale yellow solid (68 mg). Analysis by UV absorbance indicated a total of 0.752 ⁇ of DNP.
- reaction solution was diluted with water (2 mL) and was dialysed (MWCO 12-14K) against 10 mM sodium acetate, pH 5, (250 mL) for 22 hours in the dark.
- the buffer was replaced with methanol (250 mL) and the dialysis continued for 7 hours with a change of solvent after 4 hours.
- the retentate was concentrated on the roto-vap.
- Compounds include those wherein R 1 is phenylsulfonyl, 4-chlorophenylsulfonyl and morpholinosulfonyl.
- Rates of release of the drug from the conjugates of the invention can readily be determined by methods known in the art including chromatographic methods, such as HPLC. Where, for example, a fluorescent marker is used as a model system for the drug, the
- fluorescence attributable to freed fluorescent compound is readily determined as compared to fluorescence emitted by the conjugate.
- the in vivo release of drug from the conjugates of the invention may be measured by determining the pharmacokinetics of the conjugates as compared with the pharmacokinetics of a non-releasable conjugate of the same size. . Such data are preferably obtained in rats as compared to mice as they exhibit more favorable clearance rates for the high molecular weight conjugates of the invention.
- the conjugates are administered to a model subject such as a rat, for example, by intravenous administration, and blood samples are periodically taken and plasma isolated. The level of conjugate in the plasma as a function of time is then determined. This may be done by chromatographic separation (for example, HPLC analysis after deproteinization coupled to UV, fluorescence, or mass spectrometric detection), or in appropriate cases by a direct assay such as ELISA, bioactivity, or fluorescence.
- chromatographic separation for example, HPLC analysis after deproteinization coupled to UV, fluorescence, or mass spectrometric detection
- a direct assay such as ELISA, bioactivity, or fluorescence.
- macromolecular conjugates adhere to a one-compartment model, conjugates of the invention can disappear from the plasma by one of two mechanisms: release of drug from the conjugate, and clearance of the intact conjugate (e.g., by renal filtration).
- the rate of loss of a releasable conjugate from the plasma is thus the sum of the rates of loss by release of the drug and by clearance of the conjugate.
- the rate of loss of a non-releasable conjugate is just the rate of clearance of the conjugate from the plasma, since no drug is released.
- the rate of drug release from a conjugate of the invention can be calculated as the difference in rates of loss of the releasable conjugate from that of a corresponding non-releasable conjugate.
- Figure 10 shows that the variation of the rate constant as a function of the nature of the trigger in vitro and in vivo follow the same pattern which correlates as expected with the Hammett constants associated with the trigger.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Endocrinology (AREA)
- Zoology (AREA)
- Cardiology (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to compositions that comprise dendrimers useful in medical and veterinary applications that provide controlled release of drugs, such as peptides, nucleic acids and small molecules. The drugs are covalently coupled to the dendrimer through a linkage that releases the drug or a prodrug through controlled beta elimination.
Description
CONTROLLED DRUG RELEASE FROM DENDRIMERS
Cross-Reference to Related Application
[0001] This application claims priority from U.S. provisional application 61/331,749 filed 5 May 2010. The contents of this document are incorporated herein by reference.
Technical Field
[0002] The invention relates to delivery systems for drug molecules coupled to dendrimers. More specifically, the invention relates to compositions that provide for the release of multiple molecules of drug from protected dendrimeric macromolecules.
Background Art
[0003] There is a plethora of approaches to arranging for controlled release of drugs or growth factors useful in medicine. For example, compositions and methods have been described for controlled release of drugs covalently coupled to polyethylene glycol (PEG). As opposed to a drug bound irreversibly to PEG in order to enhance its half-life and diminish its
immunogenicity, conjugates have been prepared wherein PEG is a releasable carrier of the drug or prodrug. Typically, the drug is attached by an ester or carbonate linkage that can be cleaved by esterase-catalyzed hydrolysis. The adjustment of release rates in these cases is, however, difficult. Examples are PEG-camptothecin, PEG-SN38, PEG-irinotecan and PEG-docetaxel. Additional adaptations have been made to accommodate amine-containing drugs whereby a PEG moiety is connected by a cleavable ester to a self-immolating carbamate. This technology has been applied to peptides and proteins as well as to daunorubicin, amphotericin, Ara-C and other small molecules.
[0004] Another system has been developed at the Weizmann Institute wherein PEG or other macromolecule is attached to beta elimination linkers such as fluorenyl methoxycarbonyl (Fmoc) or its 2-sulfo derivative (Fms). These are described in U.S. patent 7,585,837. However, rate-of-release control remains a problem.
[0005] PCT publication WO2009/158668 describes releasable drug conjugates to macromolecules wherein the rate of beta elimination is controlled by a trigger independent of the link to the macromolecule itself. This solves a problem left unsolved in the prior art.
[0006] The release mechanism set forth in the '668 PCT publication has not been applied to instances where a multiplicity of drug molecules is coupled covalently, but releasably, to dendrimeric macromolecules. It is also limited to drugs that contain an amine functional group. In addition to providing a controllable rate of release of more than one drug molecule from the dendrimer itself, this approach offers a means whereby the coupled drug is protected from hydrolysis by the presence of a protective polymer, such as PEG, on different sites at the surface or interstices of the solid support.
[0007] Dendrimers have been used as carriers for therapeutic compounds, either by entrapment of a drug in cavities within the dendrimer, or by covalently linking drug molecules to the surface. This is reviewed in Svenson, S., Eur J Pharm Biopharm (2009) 71 :445-462 and Cheng, Y., J Pharm. Set (2007) 97:123-143. Entrapment within dendrimer cavities is limited to small molecules, and covalent attachment approaches have thus far been limited to systems in which a small drug is hydrolytically or enzymatically cleaved from the dendrimer surface. Unmodified cationic dendrimers such as polyamidoamine dendrimer(PAMAM) or poly-L-lysine (PLL) have biocompatibility and toxicology shortcomings, for example, disruption of cell membranes and also have very short half-lives, typically <20 min. Toxicity may be reduced by functionalizing the surface of the dendrimer with non-ionic or anionic groups (Kaminskas, L., et al, Mol Pharm (2007) 4:949-961). PAMAM dendrimers are not biodegradable and are retained in the liver and kidney, raising a concern - albeit unproven - of toxicity upon chronic dosing, though PLL dendrimers while retained in the liver and kidney, appear to be broken down to constituent monomers.
[0008] It has been shown that PEGylation of PAMAM and PLL dendrimers neutralizes the surface positive charges and reduces or eliminates their propensity to lyse cells and cause acute toxicity. It has also been shown that the hydrophilic PEG moiety increases water solubility of guest drug molecules, and that PEGylated dendrimers effectively accumulate in tumor tissue via the enhanced permeability and retention and thus serve as targeting delivery vehicles for antitumor agents. PEGylated PLL shows almost complete (>90%) bioavailability when
administered subcutaneously, providing downstream benefit in terms of patient compliance. Most importantly, PEGylation of cationic dendrimers can decrease renal filtration and dramatically increase the half-life from minutes to several days. Composite results show that long half-lives may be achieved with PEGylated dendrimers of MW >40 kDa by varying either the number or size of the PEG chain. That is, the MW of the total dendrimer-PEG conjugate
rather than the dendrimer or individual PEG chains dictates the extent of renal filtration. The size of PEGylated poly L-lysine dendrimer complexes can be specifically manipulated to dictate their pharmacokinetics, biodegradation and bioresorption behavior.
[0009] In particular, camptothecin attached to the dendrimer surface of PLLi6(PEG5000)8 (i.e., a PLL with 16 functional groups at the surface, 8 of which are occupied by PEG of 5000 molecular weight) via an ester linker was completely protected from serum esterases, whereas an analogous PEG-camptothecin ester hydrolyzed ~ 10-fold faster in serum than buffer. A tetra- peptide chymotrypsin substrate attached to the dendrimer end groups
was protected against chymotrypsin hydrolysis (kcat/KM 0.1 uMl s"1) compared to the peptide- dendrimer without PEG (~5-fold), or to the free peptide (~8-fold), or to the peptide attached to PEG (~12-fold). IFN-a-2b attached to the core of a 4-armed PEGylated-dendrimer showed ~ 10-fold lower cytotoxicity and anti-viral activity, as well as trypsin resistance and prolonged serum half-life compared to the free cytokine. Thus, it appears that molecules covalently bound to a dendrimer core and immersed within a PEG layer are protected against hydrolytic enzymes.
[0010] The compositions of the invention overcome problems associated with coupling drugs to the conventional monovalent linear PEG carrier. In order to minimize kidney filtration, the molecular weight of the PEG carrier must be at least about 40,000 and the drug is limited therefore to about 1 μιηοΐβ per 40 mg PEG. Thus, only very potent drugs can employ this system as a practical matter. Linear PEG's also provide only limited protection against enzymes that may modify and/or destroy the bound drug. Drugs bound to linear PEG may retain significant biological activity; while this is a requirement for a stably-modified drug, use of PEGylated-drug as a carrier for slow release of active free drug requires that the PEGylated form be substantially inactive due to the relatively higher dosages involved. The present invention permits increasing the drug payload and protects the drug against degrading enzymes as well as blocking access of the drug to its biological receptor. Like the technology described in the '668 publication, the activity of the drug is silenced until it is released, permitting administration of relatively large doses as depots.
* The rate of diffusion of chymotrypsin into the PEG shell is kcat/KM; so the results indicate that proteins of at least this size (25 kDa) should as well escape out of the PEG layer.
Disclosure of the Invention
[0011] The invention provides controlled release forms of multiple molecules of drugs, such as growth factors and small molecule drugs, coupled covalently to sites on dendrimeric macromolecules that can serve as delivery systems to extend the half-life of such drugs or growth factors. A linker is covalently bound to multiple sites on the dendrimer, which linker is in turn coupled to an appropriate drug or prodrug. The drug or prodrug is then released at the desired rate through a beta elimination reaction at physiological pH. In an additional embodiment, the drug sites on the dendrimer may be contained within a protective layer of polymer bound to adjacent sites on the solid support.
[0012] Thus, in one aspect, the invention is directed to a multiplicity of substituents of the formula
= 0-1
least one, or both R1 and R2 is independently CN; N02;
optionally substituted aryl;
optionally substituted heteroaryl;
optionally substituted alkenyl;
optionally substituted alkynyl;
COR3 or SOR3 or S02R3 wherein
R3 is H or optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted;
heteroaryl or heteroarylalkyl, each optionally substituted; or
OR or NR2 wherein each R is independently H or optionally
substituted alkyl;
SR4 wherein
R4 is optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted; or
heteroaryl or heteroarylalkyl, each optionally substituted;
J
wherein R and R may be joined to form a 3-8 member ring; and
wherein one and only one of R1 and R2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
D is a residue of a drug or prodrug coupled through O, S, or N;
Y is absent and X is O or S; or
Y is NBCH2 and X is O;
wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
1 9 ^
wherein one of R , R , R or B is coupled to a dendrimeric macromolecule.
[0013] In some embodiments, said dendrimeric macromolecule has a G of at least 2, 3 or 4, wherein G is the number of generations included in the dendrimer, where the core of the dendrimer is assigned G = 0. G values of 3 or 4 are preferred when Y is absent.
[0014] Said dendrimer may optionally also be further coupled to a protective inert polymer, such as PEG.
[0015] Alternatively phrased, the invention is directed to a dendrimeric macromolecule coupled, optionally through an additional linker, to a multiplicity of substituents of the formula
wherein the variables are defined as above— specifically
m = 0-l
1 9
at least one or both of R and R is independently CN; N02;
optionally substituted aryl;
optionally substituted heteroaryl;
optionally substituted alkenyl;
optionally substituted alkynyl;
COR3 or SOR3 or S02R3 wherein
R is H or optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted;
heteroaryl or heteroarylalkyl, each optionally substituted; or
OR or NR2 wherein each R is independently H or optionally
substituted alkyl;
SR4 wherein
R4 is optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted; or
heteroaryl or heteroarylalkyl, each optionally substituted;
1 2
wherein R and R may be joined to form a 3-8 member ring; and
wherein one and only one of R1 and R2 may be H, or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
D is a residue of a drug or prodrug coupled through O, S, or N;
Y is absent and X is O or S; or
Y is NBCH2 and X is O;
wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
1 2 5
wherein said coupling is through any of R , R , R or B.
[0016] The dendrimer may also optionally comprise a multiplicity of protective inert polymers, such as PEG.
[0017] The compositions of the invention thus offer prolonged blood circulation times, protection of drugs against hydrolases/proteases, high capacity, and inactivity against targets until released.
[0018] In other aspects, the invention is directed to methods to prepare the compositions of the invention, and methods to employ them in medical/veterinary/physiological procedures. It also includes intermediates and precursors in the synthesis of formulas (1) and (2).
[0019] The invention thus further includes "precursor" molecules of formula (3) in which L is a nucleofuge— i. e. , a leaving group that permits binding of a nucleophile. Thus, the invention includes compounds with formulas identical to that of formula (1), wherein multiple substituents are coupled to a dendrimeric macromolecule through R , R , R , or B except that in lieu of the drug or prodrug, the nucleofuge, of formula (3), instead, is present, i.e., the dendrimer macromolecule has substituents of formula (3)
wherein R1, R2, R5, X, Y and m are as defined in formula (1) or (2); and
wherein L is a nucleofuge for coupling the drug or prodrug to the remainder of the molecule.
[0020] In some embodiments of formula (3), the dendrimer has not yet been coupled.
Similarly, formula (1) or (2) without coupling to the dendrimer may be used as an intermediate. These intermediates can be summarized in formula (5):
R1 R5 O
R2— C (CH=CH)m— C— X— C— Y Z (5)
H R5
where Z is a drug or prodrug or a nucleofuge.
[0021] The coupling of any of these intermediates to dendrimer may employ a "connector" - i. e. , one or more bifunctional organic molecules that connect the substituents on these formulas to the dendrimer.
[0022] More generally, the invention includes compounds of the formula:
M-(J-D)m
wherein M is a dendrimer, D is a drug and J is a joining moiety that releases D by a beta elimination mechanism, wherein m is at least 8, and may by larger - e.g., 16, 32 or more.
Intermediate values for m are also included.
Brief Description of the Drawings
[0023] Figure 1 shows a typical dendrimer structure that has a core with three functional groups.
[0024] Figures 2A and 2B show a typical monomer for the production of a polyamidoamine dendrimer(PAMAM) and an early-stage phase of construction of said dendrimer, respectively.
[0025] Figure 3 is a diagram of a prior art description of a PEGylated polylysine wherein the PEGylation is contained within the dendrimer.
[0026] Figure 4 is a diagram of a prior art description of a PEG protected PAMAM dendrimer showing relative dimensions of coupled drugs.
[0027] Figure 5 is a graph showing the relationship of free energy to half-life for release of label from substituted β-elimination compounds.
[0028] Figure 6 is a graph showing the free energy correlation between Hammett sigma values and rate of release of label by β-elimination.
[0029] Figure 7 shows the time course of release of fluorescein from PEGylated polylysine dendrimer.
[0030] Figure 8 shows the kinetics of release of fluorescein from coupling to a PEG-coated polylysine dendrimer.
[0031] Figures 9a and 9b show, respectively, raw data showing concentration of various conjugates in plasma in a rat model system and the calculated release rate of the drug from the conjugate depending on the nature of the trigger. These data demonstrate the control over drug release rates using beta-elimination linkers.
[0032] Figure 10 shows a comparison of in vivo and in vitro release rates of drugs as a function of the Hammett constants associated with the trigger.
Modes of Carrying Out the Invention
[0033] The present invention represents an improvement over standard PEGylation practices, and has additional advantages over the PEGylated releasable drug compositions such as those described in the above-referenced PCT publication WO2009/158668. Advantages include retaining a drug or prodrug in inactive form until released from the macromolecular carrier, a multiplicity of binding sites for the drug so that the drug dosage may be increased, thus permitting delivery of less potent drugs, and provision of protection against degrading enzymes or other inactivating conditions.
[0034] Another advantage of the compositions of the invention is that they afford effective delivery of drugs to the lymphatic system. Because the compounds of the invention have molecular weights that are significantly higher than the molecular weight of the drug, they are capable of maintaining the drug in the lymphatic system when the compounds are administered subcutaneously. Compounds with molecular weights of 40,000 or more are effectively maintained in the lymphatic system. Further, because the lymph lacks esterases present in plasma that might release drugs from esterified linkages, the favorable pH of the lymph (which is identical to that of plasma) permits release of the active drug from the conjugate. Thus, the
compounds of the invention effectively release drug into the lymph when delivery to the lymph is desired, as would be the case, for example, with respect to lymphomas.
[0035] The compositions of the invention comprise dendrimer macromolecules coupled to multiple copies of one or more drugs and optionally further coupled to protective hydrophilic polymers such as PEG. In one embodiment, the dendrimer has a generation value of at least 4, where the core is assigned generation zero.
[0036] Dendrimers are synthetic polymers characterized by repeated chain branchings emanating from a central core, giving rise to a fractal-like topology and a large number of chain endings. Dendrimers are composed of a core, one or more layers (or generations) of branched monomers, and a layer of end-groups that double each "generation" and terminate the various chains.
[0037] Dendrons are wedge-shaped structural sub-components of a parent dendrimer. Each functional group of a core gives rise to a dendron; at higher generations, branches arising from a functional group can be viewed as small dendrons. Figure 2 shows a typical dendrimer structure.
[0038] Generation, G, refers to the number of layers in the dendrimer, and Z is the number of end groups on the dendrimer outer surface. As used herein, the core is generation 0 (GO). A monomer directly attached to the core can be considered a 1st generation monomer (Gl); a monomer attached to a Gl monomer is a 2nd generation monomer (G2), etc. In this system of numbering Z=2(G+1). Thus, for a dendrimer where the core has 2 functional groups,
for GO Z =2 (2(0+I)), for Gl Z=4 (2(1+I), etc.†
[0039] A variegated (i. e. , multi-functional) dendrimer has more than one type of functional group incorporated into at least one generational layer (Roberts, B. P., et al, New Journal of Chemistry (2008) 32: 1543-1555). Variegation of dendrimer end-groups allows detailed control over the chemical composition of the surface, while variegation of a more internal monomer provides control of its emanating branch(es) or an entire dendron.
[0040] Exemplary dendrimers include dendrimeric forms of poly-L-lysine (PLL) and polyamidoamine (PAMAM). The structure of one embodiment that may form the core of a PAMAM dendrimer is shown in Figure 3 A and an alternative form is shown in Figure 3B where coupling has already occurred at two of four available amino groups.
† Some workers refer to the layer wherein a monomer is coupled to the core as GO, especially for polyamidoamine (PAMAM) dendrimers; thus, Z=2(G+"2 and the designated G is one number lower than used here (i. e., Gl here is GO in this alternative designation).
Synthesis of Dendrimers
[0041] Four and eight-branched (Z=4 and 8) Fmoc-PLL-resin (Applied Biosystems) and t-Boc-PLL-benzhydryl amides (Aldrich) are commercially available, as are a large number of functionalized PEG derivatives (e.g., on the World Wide Web at creativepegworks.com).
[0042] Dendrimers can be synthesized by divergent or convergent approaches
(Carlmark, A., et ah, Chem Soc Rev (2009) 38:352-362). Divergent synthesis assembles the molecule from the core, extending radially in each generation to the periphery, whereas convergent methods start at the surface and pre-synthesized units (dendrons) are attached together. In some methods, a hybrid synthesis strategy is employed, for example a strategy wherein initial synthesis is divergent, and final synthesis employs attachment of more elaborate dendron units. Synthesis can be either in solution or on a solid support.
[0043] In an example of divergent dendrimer growth, starting from an initiator core containing two amino groups (e.g., Lys), a generation is grown by attaching monomers with two N-blocked functional groups (e.g., fBoc -Lys) to each core amino group, followed by blocking group removal to give a dendrimer with four amino end-groups. This can be repeated multiple times to form dendrimers with 8, 16, 32, etc., amino groups at the surface. As generations increase, steric congestion increases, surface groups become less accessible, and incomplete reactions may result in non-perfect dendrimers.
[0044] In convergent synthesis, individual dendrons are synthesized first and then coupled to a core molecule. The dendrons are typically constructed by the same strategies as for divergent growth, and once the dendrons have reached the desired generation they are coupled to a small core molecule. Thus, two PLL dendrons can be prepared as described above, and then connected to a Lys core to make a PLL dendrimer. The last coupling step is potentially difficult due to steric hindrance, but the large difference in size between the dendrimer and dendrons allows purification of the final product.
Characteristics of Dendrimers
[0045] Dendrimers have been extensively studied by a variety of physical, computational and experimental approaches (Caminade, A., et ah, Advanced drug delivery reviews (2005) 57:2130-2146).
[0046] For example, Table 1 shows how PAMAM and PLL dendrimer diameters grow with increasing generation. With increasing generations, dendrimers develop through a continuum of molecular shapes ranging from open, extended structures to ellipsoids, to closed globular
spheroids. Since the number of ends increase exponentially with generation, and surface area increases with the square, steric crowding of the branches at high generations results in a crowded surface; after about G5 (Z=64) there is a decrease in accessibility of the ends and thus their reactivity. The high surface and relatively lower interior densities of larger dendrimers supports cavities with diameters ranging from 5 to 15 A that may be joined to channels connecting to the surface.
Table 1 Some properties of PAMAM and PLL dendrimers
Synthesis of PEGylated Dendrimers
[0047] Cationic dendrimers up to 4 or 5 generations (Z=32 or 64) have been efficiently PEGylated on their surfaces ( aminskas, L., Mol Pharm (2008) 5:449-463; Fox, M. E., et al, Mol Pharm (2009) 6:1562-1572; and Hedden R. C, et al, Macromolecules (2003)
36: 1829-1835). Using variegated dendrimers (Roberts, B. P., supra), it is also possible to control the degree {i.e., density) and specific sites of PEGylation or drug conjugation of dendrimers. For example, using a residue containing two different {e.g., Glu, Cys) or orthogonally protected {e.g., a-Fmoc-s-tBoc-Lys) functional groups at the last generation, selective deblocking allows a drug to be attached to one group and PEG to the other; hence, a drug:PEG of 1 : 1. It is also possible to attach a moiety {e.g. , drug) to one functional group and another biiunctional monomer to the other; this terminates branching from one site, but allows
continuation at the other; here, PEGylation of surface groups would provide a drug:PEG of 1 :2. Numerous variations of structures are feasible using variegated dendrimers.
[0048] Table 2 summarizes the dimensions (i.e. , outer radii of the dendrimer and PEG- dendrimer, thickness and volume of the PEG layer) of G3 to G5 PAMAM dendrimers conjugated to PEG 550 and 5000 with two different grafting densities (n=Z and Z/2). In general, the PEG molecules extend from the surface ~1 1 or 12 A per 1,000 MW to form a shell or "brush" around the dendrimer. With increasing PEG density, ellipsoid dendrimers become almost spherical, and the PEG molecules more "stretched", thus increasing the PEG layer thickness. Each glycol unit of a PEG is 3.3 A, so a 5,000 kDa PEG (1 13 units) has a fully extended length of 377 A, considerably larger than the ~55 A PEG shell of the PEGylated dendrimer.
Table 2 Dimensions of PEG-PAMAM constructs *
* Rd=dendrimer radius, R=total radius, L=thickness of PEG layer.
[0049] In the compositions of the present invention, suitable drugs are releasably coupled to multiple sites on a dendrimeric macromolecule through linkages which permit release of the drug or prodrug by β-elimination under physiological conditions. A depiction of the prior art (lacking the β-elimination release system) shown in Figure 4 provides a conceptual
understanding of the type of delivery system contemplated by the invention.
[0050] Additional PEGylated polylysine dendrimers useful in the invention have been described, recently, by Choi, J. S., et ah, Bioconjugate Chem. (1999) 10:62-65. In these dendrimers, as shown in Figure 4, the polyethylene glycol polymer is internal to the dendrimer and is essentially at the GO level.
[0051] Figure 5 depicts the reported diameters of a G4 PAMAM dendrimer (Z=32; r~2.5 A) in which surface residues are attached to PEG 5000 to give a -55 A shell thickness (Lee, H., et al, J. Phys. Chem. (2009) 113:13202-13207). Superimposed within the PEG shell are scaled drawings of a typical small molecule (ca. ΙθΑ), a 20 residue a-helix (~6-turns, 34 x 15 A, 30 nm3; e.g. , type B 1 gCPR peptide ligand), a -20 kDa 4-helix bundle (v=l 9.5 nm3), a 6 kDa globular protein (d=26 A, v=78 nm3) and 20 kDa globular protein (d=40 A, v=260 nm3). Thus, small molecules and many therapeutic peptides/proteins would fit within the dimensions/volume of a PEG 5000 shell, providing that a) suitable orientation of the guest molecule can be achieved, and b) that the PEG density is sterically accommodating. The PEG MW could be increased to 10,000 which, although not yet studied, should provide a larger shell, afford more protection, and accommodate the larger peptides/proteins.
Nature of the Drug Conjugate
[0052] The drug conjugate of formula (1) or (2) is designed to control the pharmacokinetics of the drug or prodrug the residue of which when coupled to the remainder of the molecule is designated as "D". The mechanism whereby the drug or prodrug is released is shown below. The rate is controlled according to a pH dependent β-elimination mechanism. The groups R1 and R are selected to provide the appropriate reactivity of the intervening proton in R -CH-R , thus providing control over the rate of drug or prodrug release. The properties of R1 and R2 may be modulated by the optional addition of electron-donating or electron-withdrawing substituents, for example, in aryl moieties contained therein.
[0053] In other words, either R1 or R2, or R1 and R2 in combination, can behave as the "trigger." The nature of the "trigger" controls the acidity of the intervening proton in R^CH- R2, which, when released, permits the electron pair thus freed to effect β-elimination. For compounds wherein Y is absent; the half-life of the reaction may be in the range of that when Y is NBCH2j depending on the nature of B. Half lives are very short for embodiments of B that are highly electronegative. Examples of the ability to control release rates of drugs or prodrugs from these linkers are given in the Examples below and shown in Figures 9 and 10.
[0054] The mechanism of β-elimination release is shown below.
taking place under conditions typical of those of biological systems, for example a pH of between 6 and 8 and a temperature of between 25 and 40°C, at a rate such that the half-life of the reaction is between 1 and 10,000 hours, or between 1 and 5,000 hours, or between 1 and 1 ,000 hours or between 1 and 100 hours or between 1 and 10 hours. The product carbamic acids are typically highly unstable, and further decompose to release C02 (or COS) and DH when Y is absent, and C02, DH as well as B-NH2 and H2C=0 when Y is NBCH2.
[0055] In further detail, when X is O and Y is NBCH2, the initial product of the
β elimination is HX— (C=0)N(B)— CH2D. In one mechanism, this intermediate may decompose according to:
(1) HX-(C=0)-N(B)-CH2-D→ COX + B-NH-CH2-D (an unstable
formaldehyde aminal)
(2) B-NH-CH2-D→ B-N=CH + DH (imine formation)
(3) B-N=CH + H20→ B-NH-CH2-OH (addition of water to the imine to form hemiaminal)
(4) B— NH2 + 0=CH2 (breakdown of hemiaminal to formaldehyde + amine.) The various intermediates of the beta-elimination and subsequent decomposition
reactions (l)-(4) shown above may be transient and therefore may not be detectable under physiological or other chemical reaction conditions.
[0056] When X is O or S and Y is absent, the drug itself has an amino group and the elimination leads directly to a carbamate which decomposes to drug and C02 or COS.
[0057] The degree to which the R1 and/or R2 8roups activate the adjacent C-H bond may be expressed by the resulting acidity of the C-H bond; this acidity may in turn be expressed as the pKa of the C-H bond, wherein a lower pKa denotes a more acidic, more readily ionized C-H bond. Listings of approximate pKa values for various groups are common in the art, for example in Bordwell, F. G., "Equilibrium acidities in dimethyl sulfoxide solution," Accounts of Chemical Research (2002) 21 :456-463 (incorporated herein by reference). Examples of suitably activating groups include, but are not limited to, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted alkenes, optionally substituted alkynes, sulfones, sulfoxides, nitriles, ketones, esters, amides, and nitro groups. The R1 and/or R2 8roups may be joined to form a cyclic structure, for example
and substituted forms thereof.
[0058] Substituents on the R1 and/or R2 groups may optionally be added to provide further control over the acidity of the adjacent C-H, and thus the rate of the beta-elimination reaction. In general, electron-withdrawing substituents will increase the rate of the beta-elimination reaction, while electron-donating substituents will decrease the rate of the beta-elimination reaction. The electronic effect of various substituents is well-known in the art, and may be expressed for example as linear free-energy (Hammett) relationships. For aromatic systems, for example substituted aryl, heteroaryl, arylketones, heteroarylketone, arylsulfone,
heteroarylsulfone, and arylsulfoxide, and heteroarylsulfoxide groups, the electronic effects of substituents are described by Hammett sigma parameters, with a positive sigma value denoting electron- withdrawing (rate-accelerating relative to H) and a negative sigma value denoting electron-donating (rate-retarding relative to H) effects. Table 3 provides a listing of Hammett sigma constants for various substituents.
[0059] An "electron-donating group" is substituent that will result in a decrease in the acidity of a benzylic hydrogen ion. Examples of suitable electron-donating substituents, include but are not limited to, lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, and dialkylamino. "Electron-withdrawing groups" result in an increase in the acidity of a benzylic hydrogen ion. Examples of suitable electron-withdrawing substituents include, but are not limited to, halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C(=0)-R, wherein R is H, lower alkyl, lower alkoxy, or amino, or SOR or S02R, where R is lower alkyl, aryl, or heteroaryl. Non-hydrogen electron-donating or electron-withdrawing substituents may be present in multiple positions on rings to which they are bound. While, for convenience, in most examples, only a single occurrence of a non-hydrogen substituent on a single ring is shown, multiple substituents may also be present and are within the scope of the invention. The substituents may be the same or different.
[0060] The foregoing is something of an oversimplification, because in some cases, whether a substituent is electron-withdrawing or electron-donating depends on its position in an aromatic ring. This is reflected in the following table of linear free energy (Hammett) relationships, where a positive sigma value denotes electron-withdrawing effect and a negative sigma value
indicates an electron-donating effect. As shown in the table, for example, OMe is electron- withdrawing when present in the meta position but electron-donating in the para (or ortho) position.
Table 3 Selected Hammett Sigma Constants for Aromatic Substituents
Substituent a(meta) a(para) Substituent a(meta) a(para)
H 0 0 F +0.34 +0.06
CH3 -0.07 -0.17 CI +0.37 +0.23
CH3CH2 -0.07 -0.15 Br +0.39 +0.23
Me2CH -0.05 -0.15 I +0.35 +0.18
Me3C -0.1 -0.2 SH +0.25 +0.15
Me3Si -0.04 -0.07 MeS +0.15 0
NH2 -0.16 -0.66 C1CH2 +0.1 1 +0.12
Me2N -0.15 -0.83 CF3 +0.43 +0.54
OH +0.12 -0.37 CN +0.56 +0.66
OMe +0.12 -0.27 CHO +0.35 +0.42
OCH2CH3 +0.10 -0.24 CH3C=0 +0.38 +0.50
AcNH +0.07 -0.15 C02H +0.37 +0.45
Ph +0.06 -0.01 NO +0.62 +0.91
CH2-CH +0.05 -0.02 N02 +0.71 +0.78
HC(=0)NH +0.19 0 Me3N+ +0.88 +0.82
[0061] When Y is NBCH2, the nature of the B group influences the stability of the N-methylene-carbamate toward decomposition via El -type elimination reactions.
[0062] El proceeds as shown:
hydrolysis
+ H2CO
[0063] B groups that reduce the reactivity of the carbamate N lone pair, for example via extended conjugation and/or electron-withdrawing ability, reduce the rate of competing decomposition by the El -elimination pathway. In preferred embodiments of the invention, B is optionally substituted aryl, or optionally substituted heteroaryl. In one embodiment of the invention, B is substituted aryl or substituted heteroaryl, substituted with groups having positive Hammett sigma constants (Table 3). In one specific embodiment of the invention, for example, B is phenyl or phenyl substituted with alkoxycarbonyl, CN, Br, N02, sulfonamide or
carboxamide.
[0064] The embodiment wherein X is O and Y is NBC¾, the NBC¾ serves as an adaptor to provide the unstable carbamate thus permitting coupling to drugs that do not contain amino functional groups. It is estimated that only about 32% of the presently available small molecule drugs have primary or secondary amino groups available as functionalities. However, permitting the inclusion of groups with functional groups that are aliphatic primary or secondary alcohols permits about 45% of approved small molecule drugs to be included. Other functional groups that are acceptable to permit the constructs of the invention to contain the drug are sulfonamides, phenols, pyrroles, imides and thiols. Taken together, then, approximately 71% of currently approved drugs are amenable to inclusion in the constructs of the invention.
[0065] In embodiments where Y is NBCH2, it is preferable that B include a stabilizer, such as an aryl group, which prevents spontaneous cleavage or hydrolysis.
Some Definitions
[0066] The dendrimers may be coupled to Formulas 1, 2, or 3 through, additional
"connectors". The additional connectors are bifunctional organic compounds, such as DBCO- NHS. Many such connectors are commercially available, for example from Pierce Chemical Co, Rockford, IL. Various bifunctional connecters are well known in the art, including dicarboxylic acids or anhydrides, diamines, or heterobifunctional connecters. The selection of the connector will, of course, depend on the nature of the functional groups on the substituents on the dendrimer and on the intermediates corresponding to formulas (1) - (3).
[0067] The term "alkyl" includes linear, branched, or cyclic saturated hydrocarbon groups of 1-8 carbons, or in some embodiments 1-6 or 1-4 carbon atoms.
[0068] The term "alkoxy" includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.
[0069] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons with carbon- carbon double bonds. By the term "alkenyl (C2)" is meant a mono-, di-, tri-, or tetra-substituted carbon-carbon double bond of any geometric configuration.
[0070] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons with carbon- carbon triple bonds. By the term "alkynyl (C2)" is meant a mono- or di-substituted carbon- carbon triple bond.
[0071] The term "aryl" includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term
"heteroaryl" includes aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.
[0072] In some instances, alkenyl, alkynyl, aryl or heteroaryl moieties may be coupled to the remainder of the molecule through an alkylene linkage. Under those circumstances, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.
[0073] The term "halogen" includes bromo, fluoro, chloro and iodo.
[0075] A "nucleofuge" is a leaving group that takes with it the electron pair by which it is bonded. Exemplary nucleofuges are halogen, OH, alkoxy, hydroxysuccinimide,
paratoluenesulfonate, alkylsulfonate, and R2S+, wherein each R is independently alkyl, aryl, or heteroaryl.
[0076] The terms "protein" and "peptide" are used interchangeably regardless of chain length, and these terms further include pseudopeptides which comprise linkages other than amide linkages, such as CH2NH2 linkages as well as peptidomimetics.
[0077] The terms "nucleic acids" and "oligonucleotides" are also used interchangeably regardless of chain length. The nucleic acids or oligonucleotides may be single-chain or duplexed or may be DNA, RNA, or modified forms thereof with altered linkages, such as
phosphodiesters, phosphoramidates, and the like. For both the proteins and nucleic acids useful as drugs in the invention, these terms also include those with side chains not found in nature in the case of proteins as well as pseudopeptide bonds and bases not found in nature in the case of nucleic acids as well as backbone modifications such as peptide nucleic acids.
[0078] Small molecules in the context of drugs is a term well understood in the art, and is meant to include compounds other than proteins and nucleic acids that either are synthesized or are isolated from nature and in general do not resemble proteins or nucleic acids. Typically, they have molecular weights <1,000, although there is no specific cutoff recognized.
Nevertheless, the term is well understood in the fields of pharmacology and medicine.
[0079] A wide variety of drugs may be included as the embodiment of D. Each of these drugs will be coupled through a nitrogen, oxygen or sulfur to the remainder of the molecule. Examples of suitable drugs include those for human or veterinary use including, but not limited to, antidiabetic drugs; growth promoters; antibacterials including aminoglycosides, penicillins, cephalosporins, macrolides and peptides, trimethoprim, piromidic acid, and sulfamethazine; analgesic and anti-inflammatory drugs, antiallergic and antiasthmatic drugs,
antihypercholesterolemic drugs, beta-adrenergic blockers and antihypertensive drugs, antineoplastic drugs, and antiviral drugs. As explained above, the inclusion of the "adaptor" NBC¾ as Y permits drugs other than those bearing primary or secondary amines to be included.
[0080] Further examples of such drugs include alcohols such as paclitaxel and analogues, epothilones and analogues, camptothecin and analogues such as irinotecan, and nucleosides such as 5-fluorouracil and capecitabine. In another embodiment, the drug is a peptide comprising a serine residue. In another embodiment, the drug is a small molecule comprising an arylol group; examples of such drugs include sn-38, etilefrine, prenalterol, and estradiol. In another embodiment, the drug is a peptide comprising a tyrosine residue. If coupling is through S, the drug may be a small molecule comprising a thiol group. Examples of such drugs include penicillamine, captopril, and enalapril. The drug may be a small molecule comprising a thioaryl or thioheteroaryl group; examples of such drugs include 6-mercaptopurine. In another embodiment, the drug is a nitrogen-containing heterocycle; examples include 5-fluorouracil and allopurinol.
[0081] Other drugs are peptide, protein, and nucleic acid drugs. Examples of peptide drugs suitable for use in the invention include, e.g. , glucagon-like peptide 1 (GLP-1), atrial natriuretic factor (ANF), and many others. Examples of protein drugs include immunotoxin SS1P, adenosine deaminase, arginase, and others.
[0082] Examples of nucleic acid-based drugs include the sense strand and antisense strand of any gene from an animal, and particularly from a mammal. Such genes can be those that are already the subjects of antisense D As or RNAs, or small interfering R As that have been provided with the purpose of treating various diseases, for example genes for protein kinase C- alpha, BCL-2, ICAM-1, tumor necrosis factor alpha and the like.
[0083] The term "precursor" refers to a dendrimeric macromolecule similar to formula (1), but wherein rather than linked to the drug or prodrug, the macromolecule is coupled to a nucleofuge for further binding to a drug or prodrug as in formula (3)
wherein R1, R2, R5, X, Y and m are as defined in formula (1) or (2); and
wherein L is a nucleofuge.
[0084] While typically, the active form of the drug is directly released from the conjugates of the invention, in some cases, it is possible to release the active drug in the form of a prodrug thereof. On example of such a system is shown below:
wherein Q = O or NH, D' is the active form of a drug,
M = usual aryl substitutions.
[0085] To avoid misunderstanding, the "drug conjugates" described herein include conjugates both of drugs and prodrugs.
Exemplary Substituents
[0086] Because the substituents R1, R2, R5, and X are shared by all of the compounds of formulas (l)-(3) and any intermediates in their preparation, the various embodiments of these substituents as presented in the alternative set forth below in connection with the compounds of formula (1) or (2) may be extrapolated to precursors and intermediates thereto.
[0087] When any substituent may itself be optionally substituted, the substitution on any ring system may be alkyl, alkenyl, alkynyl or an additional ring each optionally substituted. Optional substitutions on any substituent, including the above, include halo, nitro, cyano, OR, SR, NR2, OCOR, NRCOR, COOR, CONR2, SOR, S02R, SONR2, S02NR2, wherein each R is independently alkyl, alkenyl, alkynyl, aryl or heteroaryl.
[0088] As noted above, in the compounds of the invention, R and R together exert the most control over the release rate for the drug, though R5 and m have some impact as well. In some instances, one of R1 and R2 is hydrogen or is alkyl, arylalkyl or heteroarylalkyl and the other comprises one of the remaining embodiments set forth hereinabove. In other instances, neither of R1 and R2 is hydrogen or is alkyl, arylalkyl or heteroarylalkyl.
[0089] For example, one of R1 may be H and the other optionally substituted phenyl or both R1 and R2 may be optionally substituted phenyl. The substitutions on the phenyl rings may be at 1-5 positions but preferably 3 or less. If both R1 and R2 are optionally substituted phenyl, they need not be substituted identically, or may be identically substituted. Suitable substituents include methoxy, halo, nitro, cyano and the like for example as shown in Table 3 or the substitutions listed above.
[0090] In other embodiments, one or both of R1 and R2 is R6S-, R6S(0) -, or R6S(0)2- wherein R6 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl. The remaining member of R1 and R2 may then be, H, for example, or any of the alternative embodiments set forth above.
[0091] In other instances, one or both of R1 and R2 may be cyano and the other optionally selected from the permissible substituents set forth above, in particular phenyl optionally substituted at one or more positions, for example, with halo, CN, N02, methoxy and the like.
[0092] In another set of instances, one or both of R1 and R2 is optionally substituted benzoyl and the other hydrogen or any of the other suitable choices, such as optionally substituted phenyl.
I 2
[0093] When R and R are joined to form cyclic structures, this includes groups wherein the
1
R -CH-
and forms thereof optionally substituted with electron- withdrawing and/or electron-donating groups as described above, wherein G is a bond; C=0; SO, S02, CZ2, or CZ2CZ2 wherein each Z independently is H or CI. In embodiments wherein Y is NBCH2, the substituents D or a leaving group in general coupled to the CH2 element may be released by a competing mechanism designated El . This is illustrated in Figure 3 where the leaving group or drug/prodrug, represented in the alternative by Z is removed as shown followed by the release of
formaldehyde. This is a slower, competing reaction which is not controlled appreciably by Ph, and thus results in a lack of control of the rate of release. The level of competition by this El reaction is controlled by the nature of B which influences the stability of the N-methylene- carbamate by decomposition via El elimination. B groups that reduce the reactivity of the carbamate nitrogen loan pair reduce the rate of the El elimination pathway. These groups provide this control through extended conjugation and/or electron withdrawing ability and thus reduce the rate of El . Thus, heteroaryl or aryl substituents are preferred especially those substituted with groups having positive Hammett sigma constants. For example, one embodiment is that wherein B is phenyl or phenyl substituted with alkoxycarbonyl, CN, N02 or Br.
[0094] Each R5 is independently H, or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted. In certain embodiments, each R5 is H. In other embodiments, one of R5 is H and the other is substituted alkyl or substituted phenyl, comprises an azidoalkyl group or is azido-(CH2)3-6, monoalkylamino-(CH2)3-6, N3(CH2)3- 6N(Me)C0(CH2)3-6-, or -(CH2)3-6-C02H, or a protected variant thereof. In additional embodiments, one of R5 is any one of the particular embodiments described above, further
comprising a dendrimer or a functional group allowing for connection to a dendrimer, and the other R5 is H.
[0095] The B group may be alkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, each optionally substituted. The nature of the B group influences the stability of the N-methylene- carbamate toward decomposition via El -type elimination reactions. B groups that reduce the reactivity of the carbamate N lone pair, for example via extended conjugation and/or electron- withdrawing ability, reduce the rate of competing decomposition by the El -elimination pathway. In preferred embodiments of the invention, B is optionally substituted aryl, or optionally substituted heteroaryl. In one embodiment of the invention, B is aryl or heteroaryl, each substituted with at least one group having a positive Hammett sigma constant. In one specific embodiment of the invention, B is unsubstituted phenyl or phenyl substituted with
alkoxycarbonyl, CN, N02, or Br, B may also be phenyl substituted with an aminocarbonyl, such as morpholinocarbonyl, or a sulfonamidyl, or B may be propargyl, 4-ethoxycarbonylphenyl, propyl, 4-(N,N-diethylcarboxamido)phenyl, 4-morpholinocarbonylphenyl, or 4- morpholinosulfonylphenyl. In additional embodiments, B is any one of the particular embodiments described above, further comprising a dendrimer or a functional group allowing for connection to a dendrimer.
[0096] Compounds of the invention either coupled to a dendrimer via one of R1, R2, R5, and B or R^^ and B comprises a functional group that allows for connection to a dendrimer. Suitable functional groups that allow for connection to a dendrimer comprise an alkyl or aryl
1 2 5 group, further substituted with a reactive chemical moiety. Thus, at least one of the Rl, R\ R\ and B groups comprises a dendrimer or an alkyl or aryl group further substituted with one or more amino, azido, hydroxy, carboxylic acid, alkynyl, thiol, maleimido, or 1,3-dicarbonyl groups, or protected variants thereof.
Synthesis of the Compounds of Formulas 1 or 2
[0097] The compounds of formulas (1) or (2) are derived from precursors and intermediates where either the drug/prodrug or the dendrimer is added as the last step. Thus, in one pathway, a compound of the formula
wherein R , R , R or B (if present) are not yet coupled to the dendrimeric macromolecule can be used as an intermediate. Either the drug/prodrug or the dendrimer may be coupled first. If the dendrimer is coupled first, the novel compounds of formula (3) wherein a dendrimer is coupled to one of R1, R2, R5 or B (if present) is formed. Alternatively, an intermediate containing the drug/prodrug can be first formed and then coupled to the dendrimer.
[0098] Thus, one step in the synthesis is coupling the remainder of the molecule to the dendrimer; thus, intermediates are synthesized which contain functional groups in the appropriate R1, R2, R5 or B substituents that permit such coupling.
[0099] Methods for conjugation of the intermediates to the dendrimers are generally known in the art. In one method, an amide linkage is formed between an amino group and a carboxylic acid group; thus, a intermediate comprising an amino group can be conjugated to a dendrimer that contains or is modified to contain a carboxylic acid group, or a intermediate comprising a carboxylic acid group can be conjugated to a dendrimer comprising an amino group as the reactive group. The conjugation may be performed by reacting the intermediate and dendrimer in the presence of a condensing agent, for example a carbodiimide such as
dicyclohexylcarbodiimide (DCC) or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), a uronium reagent such as 0-benzotriazole-N,N,N',N'-tetramethyluronium-hexafiuorophosphate (HBTU), or a phosphonium reagent such as benzotriazole-l-yl-oxy-tris- (dimethylamino)phosphonium hexafluorophosphate (BOP).
[0100] Alternately, the carboxylic acid group may be activated for conjugation in a prior step, for example by conversion to an acid chloride using thionyl chloride or oxalyl chloride, or to an active ester such as a pentafluorophenyl ester using a carbodiimide and pentafluorophenol or an N-hydroxysuccinimidyl ester using a carbodiimide and N-hydroxysuccinimide, and the resulting activated carboxylate may then be reacted with the amine in a second step. The amine and carboxylic acid groups may initially be present in protected form as required for stability and/or compatibility with additional chemical transformations, and deprotected prior to the conjugation step. Amine groups may be protected as carbamates, preferably tert- butoxycarbonyl (lBOC), allyloxycarbonyl (Alloc), or other carbamate groups that may be removed under neutral-to-acidic conditions. Carboxylic acids may be protected as esters that may be removed under neutral-to-acidic conditions, such as tert-butyl (lBu), trityl (Ph3C), allyl (All), or methoxymethyl (MOM).
[0101] In a second method, a thioether linkage is formed between a thiol group and a maleimide group; thus, a intermediate comprising thiol group can be conjugated to a dendrimer comprising a maleimide group, or a intermediate comprising a maleimide group can be conjugated to a dendrimer that is modified, for example, by a bifunctional linker thiol group. The thiol group may initially be present in protected form as required for stability and/or compatibility with additional chemical transformations, and deprotected prior to the conjugation step. Suitable protecting groups include those that may be removed under neutral-to-acidic conditions, for example tert-butyl ethers (lBu) or trityl ethers.
[0102] In a third method, a 1 ,2,3-triazole linkage is formed between an alkyne and an azide group; thus, a intermediate comprising an alkyne group can be conjugated to a dendrimer modified to contain an azide group, or a intermediate comprising an azide group can be conjugated to a solid support modified to contain an alkyne group. The conjugation reactions may be performed under metal catalysis, typically using copper or ruthenium, or may be performed in the absence of catalyst using an activated alkyne such as a cyclo-octyne. Related cycloaddition methods known in the art may be employed, for example Diels- Alder
cycloadditions between a 1,3-diene and a dienophile.
[0103] In a fourth method, an enamino-ketone linkage is formed between an amino group and a 1,3-dicarbonyl group; thus, a intermediate comprising an amino group can be conjugated to a dendrimer modified to contain a 1,3-dicarbonyl group, or a intermediate comprising a 1,3-dicarbonyl group can be conjugated to a solid support comprising an amine group. In one embodiment, a intermediate comprising a 1,3-dicarbonyl group is reacted with an antibody such as m38C2 comprising a suitably reactive lysine ε-amino group (Doppalapudi, et al, Bioorganic & Medicinal Chemistry Letters (2007) 17:501-506, incorporated herein by reference).
[0104] Thus, the R1, R2, R5, or B groups in the intermediate independently may optionally be substituted by optionally protected amine, optionally protected carboxylic acid, optionally protected thiol, maleimide, alkyne, or azide groups to allow for conjugation with dendrimers. Once conjugated, the R1, R2, R5, or B groups independently are substituted by dendrimers connected via, for example, carboxylic amide, thioether, or 1,2,3-triazole groups.
Coupling of Drug/Prodrug
[0105] For conjugates where Y is absent, coupling of the drug is illustrated below. In formula (A), coupling to dendrimer may or may not have already been conducted. Typically, the drug is coupled prior to coupling to dendrimers. Thus, R1, R2 and R5 are as above-defined or alternatively, one of R1, R2 and R5 is coupled to a dendrimer.
with a drug or prodrug molecule D. The compound of formula (A) is first activated for condensation by reaction with a suitable reagent, for example phosgene or triphosgene, optionally in the presence of N-hydroxysuccinimide; 1,1-carbonyldiimidazole;
1,1-carbonylditriazole; Ν,Ν-disuccinimidyl carbonate, or similar reagents for the conversion of a compound of formula (A) into an activated compound, wherein W = F, CI, imidazolyl, triazolyl, or O-succinimidyl, and then coupled to the drug.
(A) (A*) (3)
[0106] For example, reaction of a compound of formula (A) wherein x = O with triphi and N-hydroxysuccinimide yields a compound wherein x = O and W = O-succinimidyl:
[0107] Compounds wherein x = O and W = O-succinimidyl are particularly preferred when the drug or prodrug molecule to be conjugated has an amino group. In this case, the resulting compound comprises a carbamate linkage. For cases wherein the drug or prodrug is a peptide or protein, the amino group that reacts with the intermediate may be a terminal alpha-amino group
or the amino group of a side-chain, for example of a lysine, ornithine, or unnatural amino acid residue.
[0108] Alternatively, the activating reagent may be a substituted phenyl chloroformate, for example, 4-nitrophenyl chloroformate, 2,4-dinitrophenyl chloroformate, or pentafluorophenyl chloroformate, resulting in formation of an intermediate substituted phenyl carbonate.
[0109] Intermediates wherein x = O and W = F or CI are particularly preferred when the drug or prodrug molecule to be conjugated has no amino group, but instead has a hydroxy group, for example when the drug or prodrug is a peptide or protein from a side-chain tyrosine, serine, or threonine residue, or when the drug or prodrug is nucleic acid-based such as a deoxynucleic acid or ribonucleic acid, or a small molecule.
[0110] The precursors wherein the drug is an oligonucleotide or nucleic acid may be prepared by chemical synthesis of the drug comprising a 5 '-terminal modification that allows for conjugation. For example, the oligonucleotide may be chemically synthesized such that the 5'- terminal nucleotide unit, added at the last round of synthesis, comprises a phosphate group modified to contain an amino-alkyl group. The resulting amine-modified nucleic acid molecule is then conjugated to form a molecule of formula (3). See, for example, Zhao, et al,
Bioconjugate Chemistry (2005) 16(4):758-766.
[0111] In the case of peptide-, protein-, or nucleic acid-based drugs, multiple reactive groups may be present leading to multiple reactions. The extent of this multiple reaction may be controlled using standard conditions known in the art, for example by varying the reaction temperature, concentrations, and stoichiometries in order to obtain the desired reaction product.
[0112] In one embodiment of the invention, where the drug is a peptide, the intermediate formed b reaction with an amino acid is then employed in standard peptide synthesis: peptide synthesis
[0113] In another method, the intermediate is attached during the synthesis of the peptide. For example, the final step in the synthesis of the peptide by solid-phase peptide synthesis methods well-known in the art involves attachment of the N-terminal amino acid of the
sequence of the peptide in protected form. The final step uses the N-terminal amino acid in a form using the intermediate as the protecting group, which is not removed.
cleavage
wherein R is the side chain of an amino acid.
[0114] This embodiment is advantageous in that the position and stoichiometry of derivitization is completely controlled.
[0115] Similar reactions are employed where the intermediate is a compound wherein Y is NBCH2. The nucleofuge coupled to CH2 is similarly displaced by the drug or prodrug. In this case, as well, the intermediate may or may not already be coupled to the dendrimeric macromolecule.
Preparation of Intermediate Compounds
[0116] Those intermediate compounds wherein m is 0 may be prepared by the addition of a carbanion RIR2CH" formed by reacting R1R2CH2 with a strong base, for example butyllithium, NaH, lithium diisopropylamide, lithium bis(trimethylsilylamide), or similar, with a molecule to produce a compound of formula (A)
[0117] Alternatively, compounds of formula (A) wherein x = O and one R5 is H may be
1 2
prepared by a two-step process. In the first step, the addition of a carbanion R R CH" formed by reacting R!R2CH2 with a strong base, with an ester R5-C(=0)OR*, wherein R* is lower alkyl, produces an intermediate ketone R,R2CH-CR5=0, which may in the second step be reacted with
a suitable reducing agent, for example NaBH4 or NaBH3CN, to provide the compound of formula (A) wherein X=0, and one R5 is H.
[0118] For example, when R'R2CH2 is fluorene, this is reacted with a strong base, for example, to form a fluorenyl carbanion, which is then reacted with R5 2-CO, the reaction is as follows:
[0119] Corresponding compounds wherein x is S may be similarly prepared using the appropriate analogue R5 2-C=S, or may alternatively be prepared by subsequent chemical transformation of formula (A) where x is O using methods known in the art, for example activation of the alcohol group in (A), for example by conversion to a bromide using PBr3 or Ph3PBr2, or by conversion to the tosylate or triflate, and displacement by a suitable nucleophilic group such as thiourea or thiosulfate. In one embodiment, thiosulfate is used to form an intermediate that is hydrolyzed by acid treatment to form the thiol.
[0120] Compounds wherein m is 1 and both R5 are H may be prepared by addition of the carbanion derived by lithiation of R!R2CH2, for example using a strong base such as NaH, butyllithium, lithium bis(trimethyl-silylamide), or similar, to an unsaturated compound such as methyl 3-(dimethyIamino)-acrylate to provide an intermediate ester, which may be reduced, either via one step or through multiple steps, to the corresponding unsaturated aldehyde:
[0121] Reaction of the unsaturated aldehyde with a substituted or unsubstituted arylboronic acid, aryl-B(OH)2, in the presence of a palladium catalyst, for example as described in Org. Letts. (2005) 7:4153-5, provides a compound wherein one R5 is substituted aryl, one R5 is H, and X = O.
[0122] Alternatively, reaction of the unsaturated aldehyde with an alkylborane according to the method of Soderquist provides compounds wherein x = O, one R5 is H and the other is CH2CH=CH2 or CH2CCH. See Burgos, C. H., et al., J. Am. Chem. Soc. (2005) 127:8044.
[0123] The compounds of formula (A) may then be derivatized to the drug. In these intermediates and the drug conjugate, all of the embodiments which correspond to the many
1 ^ illustrated forms of formulas (1) and (2), and specifically embodiments of R , R and R are retained.
[0124] In instances where Y is NBC¾, an additional intermediate is prepared from the compound of formula (A), for example, by activating the compound of formula (A) where x is O to a chloroformate as described above and then further reacting this compound with
hexahydrotriazine. This results in an intermediate where a leaving group coupled to the methylene is a halo group— i.e., Y-Cl, wherein Y is NBCH2. This intermediate can be converted to an intermediate with a drug comprising OH, SH or a heterocyclic nitrogen group under anhydrous conditions in the presence of mild base. Suitable bases include tertiary amines such as triethyl amine. The reaction mixture may optionally include Nal or a
tetraalkylammonium iodide to accelerate the reaction. Suitable solvents include any inert anhydrous solvent.
Attachment of Protective Polymers
[0125] The dendrimers may also include protective polymer (the most common example would be PEG, but other hydrophilic polymers could also be used).
[0126] In one approach, only a portion of the reactive sites on the derivatized dendrimer are provided with drug conjugate or polymer by controlling the stoichiometry of the coupling reaction and the remaining sites are then coupled to the other component.
[0127] Alternatively, pre-assembled units comprising a PEG, a releasable linker or drug conjugate or some combination of these units may be prepared. The pre-assembled units are then attached to the outer shell of the dendrimer, either in solution or while on a solid phase synthesis support. Such pre-assembled units may be constructed in a stepwise process starting from a trifunctional matrix molecule wherein each functionality may be selectively attached to a PEG, a releasable linker or drug conjugate, and to the dendrimer outer shell. Suitable functionalities include carboxylic acids, amines, maleimides, azides, thiols, and alkynes, which may be present in protected form.
[0128] For example, an amino acid comprising a carboxylic acid group and two
differentially protected functional groups can be converted into such a pre-assembled unit by selective deprotection of one protected functional group, attachment of a PEG, then deprotection of the second protected functional group and attachment of the drug conjugate, then final attachment of the pre-assembled unit through the carboxylic acid to the dendrimer. In one example, azidonorleucine is reacted with an activated PEG molecule, for example a PEG N-hydroxysuccinimide carbonate, so as to produce Na-PEG-azidonorleucine. The Na-PEG- azidonorleucine is then either attached to the outer shell of the dendrimer through standard amide-forming reactions to provide a PEGylated dendrimer having an array of azide
functionalities on the outer shell that can be subsequently coupled with alkynyl-linkers or alkynyl drug conjugate, or is first reacted with an alkynyl-linker or alkynyl-drug conjugate under Cu(I) catalysis to provide the complete pre-assembled unit, which is then attached to a dendrimer having amine groups on the outer shell using standard amide-forming reactions.
azidonorleucine N -PEG-azidonorleucine
^[linker]— Drug 1. [Dendrimer]-NH2
HBTU
Cu(l)
2. alkynyl-linker-drug Cu(l)
Na-PEG-azidonorleucine
[0129] In another example, a protected cysteine, for example S-(monomethoxytrityl)- cysteine, is reacted with an activated PEG molecule, for example a PEG N-hydroxysuccinimide carbonate, so as to produce Na-PEG-S(mmt)-cysteine. This can be attached to a dendrimer having outer shell amines using standard amide forming reactions, and the resulting dendrimer can be detritylated using mild acid and the resulting thiols reacted with a maleimide-linker or maleimide-drug conjugate. Alternatively, the Na-PEG-S(mmt)-cysteine can be reacted with an amine-linker or amine-drug conjugate using standard amide-forming reactions, and the complete pre-assembled unit can be detritylated using mild acid and coupled to a dendrimer having maieimide groups on the outer shell.
S-(monomethoxytrityl)cysteine
Na-PEG-azidonorleucine
1. H2N-linker-drug
HBTU 1. [Dendrimer]-NH2
2. aleimide-[dendrimer] HBTU
2. CF3C02H
3. Maleimide-linker-Drug
Administration and Use
[0130] The conjugates of the invention that are designed to release drugs at controllable rates are administered to subjects in a manner similar to medicaments in general. The subjects may be model systems such as mice, rats or rabbits or may be human patients or may be veterinary subjects such as companion animals, livestock, and avian subjects. The conjugates of the invention are typically administered by injection, in general by intravenous injection, but other dosage mechanisms are also within the scope of the invention such as oral administration, administration by suppository, transdermal or transmucosal administration and the like. The dosage levels will depend on the nature of the drug, the condition to be treated, the nature of the subject, and the judgment of the attending professional. The selection of appropriate release rates for a particular drug or protocol are also dependent on these factors. Thus, the use and administration of the compounds of the invention is within the skill of the practitioner. Further, as noted above, the conjugates of the invention are particularly useful and advantageous in treating diseases of the lymph system wherein subcutaneous injection is preferred.
[0131] The following examples are intended to illustrate but not to limit the invention.
Preparation 1
Release Rate Determination— Phenyl Sulfones
[0132] A series of model linker scaffolds having a range of functional groups as potential pKa modulators (substituted aromatics, ketones, nitriles, sulfones) were designed, prepared and linked via carbamate bonds to Ne-2,4-dinitrophenyl-L-lysine (Ne-DNP-Lys) for evaluation of release rates; DNP-Lys is water soluble and is a strong chromophore to permit HPLC-UV analysis. Rates of release at pH 7.4 and/or 8.3 were determined.
2 3
[0133] Starting alcohols, obtained commercially or prepared by standard methods, were converted into N-hydroxysuccinimide (HS) carbonates either using a one-step procedure with disuccinimidyl carbonate (Manoharan, J. Org. Chem. (1999) 64:6468-6472) or by a two-step procedure wherein the alcohol is first converted into the chloroformate using
triphosgene/pyridine and then to the carbonate by treatment with N-HS (Tsubery, H., et al, J. Biol. Chem. (2004) 279:38118-38124).
[0134] The DNP carbamates were prepared as follows. A suspension of N-DNP-L-Lys HC1 (35 mg, 0.1 mmol) in 600 μΐ. of water was treated successively with 1.0 N NaOH (200 μί) and 1.0 M NaHC03. A 0.1 M solution of the N-HS carbonate in acetonitrile (1.0 mL) was added to the stirred mixture to give a clear yellow solution. After 1 hr, the mixture was diluted with 10 mL water and loaded onto a Bond-Elut™ CI 8 extraction column (1 gm). The column was washed successively with water, 1% CF3C02H water, water, and 50% MeOH/water. The product was eluted with MeOH, then evaporated to give the product as a yellow glass. Kinetic analyses were performed by HPLC (CI 8; linear MeOH/water + 0.5% HO Ac gradient) using a UV/vis monitor. The areas under the DNP and starting material peaks were integrated to determine extent of reaction.
[0135] The t 2 values of β-eliminative cleavage of DNP-Lys carbamates at pH 7.4 and/or 8.3 are shown in Table 4.
Table 4
[0136] The half- lives for cleavage span 2 hr to >10 days. That cleavage was generated by β-eliminative reactions was evidenced by the different half-lives, and determination that 0-benzyl-N-(Ne-2,4-DNP-Lys) carbamate (which cannot undergo O-alkyl scission) showed less than 0.25% cleavage after 5 days at 37°C and pH 7.4 (t1 2 >3 yrs ). 0-benzyl-N-(Ne-2,4-DNP- Lys) carbamate undergoes no detectable hydrolysis in 50% human serum after 1 week at 37°C. This demonstrates the stability of carbamates to serum hydrolases. In general, compared to C-H, a) electron withdrawing groups at R1 increase the rate; b) alkyl groups at R3 increase the rate; and c) aryl moieties at R decrease the rate.
[0137] A good linear free energy relationship, shown in Figure 5, was observed for the substituted (phenylsulfonyl)ethyl linkers, allowing estimation of release rates for other substituted linkers in this series based on SAR using Hammett sigma parameters, for the substituents on the phenyl group as shown. Thus, substituents can be selected to provide either slower (e.g. , 4-OMe, σρ = -0.27; 4-OH, σρ = -0.37; 4-Me2N, σρ = -0.83) or intermediate release rates (e.g. , 4-F, σρ = +0.06; 4-CI, σρ - +0.23; 3-Br, am = +0.39; 4-CF3, σρ = +0.54).
Preparation 2
Release Rate Determination— Effect of R5
[0138] From the studies in Preparation 1, the phenyl sulfone moieties at R1 appeared to provide rates (t]/2 ~2 to 72 hr) spanning a range suitable for use in our conjugates. These were converted into bifunctional linkers containing a N-HS carbonate for attachment to amine- containing molecules and an acylated 3-aminophenyl moiety at R5 for attachment to PEG, or to dendrimers. In particular, linkers having the general structure shown below were prepared.
Rx = 4-chloro, H, 4-methyl, 4-methoxy, 2,4-dimethyl, and 2,4,6-trimethyl;
Ry = -(CH2)3C≡CH, -(CH2)5N3, -(CH2)2-maleimide
[0139] The N-HS carbonate linkers with Ry = -(CH2)3C≡ CH were attached to Ne-DNP-Lys, and the rates of Lys(DNP) release were measured in 0.1 M HEPES, pH 7.40 at 25°C or 37°C using HPLC. All compounds gave good first-order kinetics, with tj/2 16 to 120 hours (Table 5) and a temperature coefficient Q12 of 5.7 ±0.1.
Table 5 Rates of H-Lys(DNP)-OH release from compounds (Ry = -(CH2)3C≡ CH)
No. R1 ti/2, hr
25°C 37°C 25°C 37°C
1 4-C1 0.0074 0.0434 94 16
2 H 0.004 0.0236 170 30
3 2,4-Me2 0.0021 0.012* 330 57
4 4-Me 0.0018 0.0104* 380 67
5 4-OMe 0.0013 0.0074 530 94
6 2,4,6-Me3 0.001 0.0057 690 120
* extrapolated from data at 25°C.
[0140] A good correlation between release rate and Hammett sigma constants was also observed for the mono-substituted compounds is shown in Figure 6.
Preparation 3
Effect of Coupling to a Macromolecule
[0141] The linker with Rx = 4-methoxy, Ry = -(CH2)3C≡CH coupled to Ne-DNP-Lys was conjugated with 40 kDa PEG-azide using copper-catalyzed Huisgen cycloaddition. Examination of the release of H-Lys(DNP)-OH indicated that the rate of release from the macromolecular conjugate (k = 0.0059 h"1, t = 118 hrs) was similar to that of the unconjugated linker
(ti 2 = 94 hr).
[0142] Preliminary results of determination of effects of human sera on the rate of release from PEG-conjugates suggest there may be a uniform 3-fold rate enhancement of cleavage. The conjugate of 40 kDa PEG with this compound was administered to rats to determine
pharmacokinetics; stably conjugated Lys(DNP) was also prepared by click chemistry between Na-hexynoyl-Lys(DNP)-OH and 40 kDa-PEG-azide and administered to rats as a control. Competitive ELISA for DNP-Lys using DNP-BSA and an anti-DNP antibody conjugated to alkaline phosphatase is employed.
[0143] Preparations 4-19 describe intermediates and compounds of formula (3), not yet linked to dendrimer, i.e., compounds wherein X is O and Y is NBCH2 wherein the CH2 group is coupled to a nucleofuge. In Preparation 19, the nucleofuge is replaced by a model system permitting monitoring release of cysteine coupled to 4-dinitrophenyl.
Preparation 4
General Preparation of Chloroformates and N-Hydroxysuccinimide Carbonates
[0144] Pyridine (0.33 equivalent) is added dropwise to a vigorously stirred solution of the alcohol (1 equivalent) and triphosgene (0.33 equivalent) in anhydrous tetrahydrofuran
(2 mL/mmol) cooled on ice. After 1 hr, the mixture is allowed to warm to ambient temperature and kept overnight. The mix is then filtered and concentrated under vacuum on a rotary evaporator. The resulting crude chloroformate is used without further purification.
[0145] To prepare N-hydroxysuccinimide carbonates, the crude chloroformate is dissolved in anhydrous tetrahydrofuran (2 mL/mmol) and treated with pyridine (2 equivalents) and N-hydroxysuccinimide (4 equivalents) at ambient temperature for 30 minutes. The mixture is diluted with ethyl acetate, washed successively with 0.1 N HCl, water, and brine, then dried over MgS04, filtered, and evaporated. The crude carbonates are purified by silica gel
chromatography (ethyl acetate/hexane).
Preparation 5
General Preparation of Carbamates
[0146] A solution of the chloroformate (1 equivalent) in acetone (2 mL/mmol) is added dropwise to a vigorously stirred mixture of the amine or aniline (1 equivalent) and NaHC03 (2 equivalents) in water (2 mL/mmol). After 30 minutes, carbamates which precipitate as solids are collected by vacuum filtration, washed with water, and dried; carbamates which separate as oils are extracted with ethyl acetate. The extract is dried over MgS04, filtered, and evaporated to provide the crude carbamate. In either case, the crude carbamate is further purified by column chromatography (Si02) or by crystallization.
[0147] Alternatively, triethylamine (1 equivalent) is added to a mixture of the amine or aniline (1 equivalent) and the chloroformate (1 equivalent) in an inert anhydrous solvent, for example dichloromethane, tetrahydrofuran, or ethyl acetate. After stirring for 1 h at ambient temperature, the mixture is evaporated to dryness, and the residue is dissolved in ethyl acetate and washed successively with 1 N HCl, water, sat. aq. NaHC03, and brine, then dried over MgS04, filtered, and evaporated to provide the crude carbamate, which is purified as described above.
[0148] Alternatively, an alcohol is converted to a carbamate without isolation of the intermediate chloroformate. Pyridine (0.33 equivalent) is added dropwise to a vigorously stirred solution of the alcohol (1 equivalent) and triphosgene (0.33 equivalent) in anhydrous tetrahydrofuran (2 mL/mmol) cooled on ice. After 1 hr, the mixture is allowed to warm to ambient temperature and kept overnight. The mixture is cooled on ice, and the amine or aniline (2 equivalents) is added. The mixture is allowed to warm to ambient temperature and kept overnight. The mixture is then evaporated to dryness, and the residue is dissolved in ethyl acetate and washed successively with 1 N HC1, water, sat. aq. NaHC03, and brine, then dried over MgS04, filtered, and evaporated to provide the crude carbamate, which is purified as described above.
Preparation 6
N-Chloromethylation of Carbamates
[0149] A mixture of the carbamate (1 equivalent) and paraformaldehyde (3 equivalents of formaldehyde) in 1 :1 tetrahydrofuran/chlorotrimethylsilane (1 mL/mmol) in a sealed screw-cap vial is heated at 55°C until a clear solution is obtained. The mixture is concentrated under vacuum on a rotary evaporator, and the residue is dissolved in ethyl acetate, filtered, and concentrated again to provide the crude N-chloromethyl carbamate.
Preparation 7
N-Methoxymethyl Carbamates
[0150] A solution of N-chloromethyl carbamate in methanol is allowed to stand at ambient temperature for 1 h, then concentrated to dryness to provide the N-methoxymethyl carbamate.
Preparation 8
N-Alkoxymethyl Carbamates, N-Phenoxymethyl Carbamates, N-Thiomethyl Carbamates, and N-Thiophenylmethyl Carbamates
[0151] A solution of the alcohol, phenol, thiol, or thiophenol (1 equivalent) and the
N-chloromethylcarbamate (1 equivalent) in an inert anhydrous solvent, for example
tetrahydrofuran, dichloromethane, or ethyl acetate, is treated dropwise with triethylamine (1 equivalent). After 1 hour, the mixture is evaporated to dryness. The crude product is purified by silica gel chromatography.
Preparation 9
0-(9-FluorenylmethvD-N-Phenyl Carbamate
[0152] A solution of 9-fluorenylmethoxycarbonyl chloride (2.6 g) in 20 mL of acetone was added slowly to a stirred mixture of aniline (0.93 g) and NaHC03 (2.5 g) in 20 mL of water. After 1 hour, the solid precipitate was collected by vacuum filtration, washed with water, and air dried. Crystallization from ethyl acetate provided the product.
Preparation 10
0-(9-Fluorenylmethyl)-N-Propargyl Carbamate
[0153] A solution of 9-fluorenylmethoxycarbonyl chloride (2.6 g) in 20 mL of acetone was added slowly to a stirred mixture of propargylamine hydrochloride (0.91 g) and NaHC03 (2.5 g) in 20 mL of water. After 1 hour, the solid precipitate was collected by vacuum filtration, washed with water, and air dried. Crystallization from ethyl acetate/hexane provided the product.
Preparation 11
0-(9-FluorenylmethyD N-(4-Bromophenyl Carbamate
[0154] Triethylamine (0.7 mL) was added to a stirred mixture of 4-bromoaniline (0.85 g) and 9-fluorenylmethoxycarbonyl chloride (1.3 g) in 25 mL of dichloromethane. The mixture was stirred for 1 h at ambient temperature, then washed with 1 N HCl, water, sat. aq. NaHC03, and brine. The organic solution was dried over MgS04, filtered, and evaporated.
Preparation 12
0-(9-Fluorenylmethyl N-(4-(Ethoxycarbonyl)Phenyl) Carbamate
[0155] Triethylamine (0.7 mL) was added to a stirred mixture of ethyl 4-aminobenzoate (0.85 g) and 9-fluorenylmethoxycarbonyl chloride (1.3 g) in 25 mL of dichloromethane. The mixture was stirred for 1 h at ambient temperature, then washed with 1 N HCl, water, sat. aq. NaHC03, and brine. The organic solution was dried over MgS0 , filtered, and evaporated.
Preparation 13
0-(9-Fluorenylmethyl)-N-Propyl Carbamate
[0156] A solution of 9-fluorenylmethoxycarbonyl chloride (2.6 g) in 20 mL of acetone was added slowly to a stirred mixture of propylamine hydrochloride (0.91 g) and NaHC03 (2.5 g) in 20 mL of water. After 1 hour, the solid precipitate was collected by vacuum filtration, washed with water, and air dried. Crystallization from ethyl acetate/hexane provided the product.
Preparation 14
O-Ethyl N-Phenyl Carbamate
[0157] A solution of ethyl chloroformate (1.1 g) in 20 mL of acetone was added slowly to a stirred mixture of aniline (0.93 g) and NaHC03 (2.5 g) in 20 mL of water. After 1 hour, the mixture was extracted with ethyl acetate. The extract was dried over MgS04, filtered, and evaporated to provide the crude product as an oil that slowly crystallized upon standing.
Crystallization from ethyl acetate/hexane provided the product.
Preparation 15
[0158] Pyridine (0.86 mL) was added to a solution of 2-(phenylsulfonyl)ethanol (1.0 g) and triphosgene (2.2 g) in anhydrous tetrahydrofuran (10 mL). After stirring for 30 min, the mixture was filtered and evaporated to an oil under vacuum to provide 0.93 g of the crude chloroformate. The chloroformate was redissolved in 10 mL of acetone and added to a mixture of aniline (0.37 g) and NaHC03 (0.42 g) in 10 mL of water. Ethyl acetate (5 mL) was added, and after 1 hour, the organic phase was collected, dried over MgS04, filtered, and evaporated to provide the 1.1 g of the crude product as an orange oil. Crystallized from 1 : 1 ethyl acetate/hexane.
Preparation 16
Q-(9-Fluorenylmethyl N-Phenyl N-Chloromethyl Carbamate
[0159] A suspension of 0-(9-fiuorenylmethyl) N-phenyl carbamate (1.58 g),
paraformaldehyde (0.25 g), chlorotrimethylsilane (5 mL) and tetrahydrofuran (5 mL) was heated at 55°C in a sealed vial for 20 h. The resulting clear solution was concentrated to an oil using a rotary evaporator. The residue was dissolved in ethyl acetate and re-concentrated, resulting in crystallization. The crystals were suspended in 2:1 hexane/ethyl acetate, collected, and dried to provide 1.45 g of the N-chloromethyl carbamate.
Preparation 17
Q-(9-Fluorenylmethyl) N-Phenyl N-Methoxymethyl Carbamate
[0160] Prepared by dissolving 0-(9-fluorenylmethyl) N-phenyl N-chloromethyl carbamate (Preparation 14) in methanol. 1H-NMR (d6-DMSO) δ 7.86 (2H, d, J = 7 Hz), 7.42-7.22 (m, 9H), 7.14 (m, 2H), 4.83 (2H, br s), 4.47 (2H, d, J = 6 Hz), 4.18 (1H, m), 3.11 (3H, br s).
Preparation 18
N-(2,4-Dinitrophenyl)Cysteine Allyl Ester
[0161] Step 1. A solution of cystine bis(allyl ester) p-toluenesulfonate and
2,4-dinitrofluorobenzene in THF was treated with triethylamine for 24 hrs. The mixture was diluted with ethyl acetate, washed successively with 1 N HC1, water, sat. aq. NaHC03, and brine, then dried over MgS04, filtered, and evaporated to provide bis(N-DNP)-cystine bis(allyl ester).
[0162] Step 2. A solution of bis(N-DNP)-cystine bis(allyl ester) (326 mg) in THF (2 mL) was treated with a solution of dithiothreitol (115 mg) in water (1 mL), followed by addition of 1 M NaHC03 (50 μί). The bright yellow mixture immediately turned dark. After 15 minutes, the mixture was diluted with 10 mL of water and acidified with 1 N HCl (50 μί), then extracted with ethyl acetate. The extract was washed with water and brine. The yellow solution was over MgS04, filtered, and evaporated to provide crude N-DNP-cysteine allyl ester. The crude material was dissolved in dichloromethane and filtered through 5 mL of silica gel using 1 : 1 ethyl acetate/hexane to elute the bright yellow product (371 mg).
Preparation 19
S-nSf-(9-Fluorenylmethoxycarbonyl-N-Phenylamino)Methyl N-(2,4-Dinitrophenyl)-Cysteine
[0163] A solution of N-(DNP)-cysteine allyl ester of Preparation 18 (82 mg) and
0-(9-fluorenylmethyl) N-phenyl N-chloromethyl carbamate of Preparation 16 (91 mg) in dichloromethane (1 mL) was treated with triethylamine (35 μί) for 1 hour, then filtered through silica gel using 1 : 1 ethyl acetate/hexane and concentrated to dryness. The product was purified by silica gel chromatography.
[0164] A solution of the allyl ester, phenylsilane (75 μΤ), and
tetrakis(triphenylphosphine)palladium (15 mg) in THF (2.5 mL) was stirred at ambient temperature for 10 minutes, then evaporated to dryness. The residue was dissolved in dichloromethane and loaded onto a 5 mL column of silica gel, which was eluted sequentially with 1 :4 ethyl acetate/hexane, ethyl acetate, and 0.5% acetic acid/ethyl acetate. Fractions containing product were combined and evaporated.
[0165] 1H-NMR (d6-DMSO): d 13.7 (1H, br s), 9.01 (1H, d, J = 7 Hz), 8.85 (1H, d,
J = 3 Hz), 8.25 (1H, dd, J = 3, 9 Hz), 7.82 (1H, d, J = 7), 7.40-7.25 (m, 7H), 7.25-7.15 (m, 3H), 7.11 (m, 2H), 4.96 (m, 1H), 4.81 (s, 2H), 4.30 (m, 2H), 4.08 (m, 1H), 3.18 (m, 2H).
[0166] Preparations 20-22 describe intermediates and compounds of formula (3) not yet linked to dendrimer, i.e., compounds wherein X is O and Y is NBCH2, wherein the methylene group is coupled to the OH group of a serine residue.
Preparation 20
N-(6-(2,4-Dinitrophenylamino)Hexanoyl-L-Serine Allyl Ester
[0167] Step 1. N-(tert-butoxycarbonyl)-L-serine allyl ester: To a stirred solution of allyl bromide (2.3 mL, 26.6 mmol) and tricaprymethylammonium chloride (4.00 g, 9.90 mmol) in CH2C12 (35 mL) was added a solution of N-(te /-butoxycarbonyl)-L-serine (1.03 g, 5.02 mmol) and NaHC(¾ (0.43 g, 5.12 mmol) in water (16 mL). The biphasic reaction mixture was vigorously stirred at room temperature for 48 hours. It was diluted with water (50 mL) and extracted with CH2C12 (3 x 50 mL). The combined organic extracts were dried over MgS04, filtered, and concentrated under reduced pressure to yield a colorless oil (5.95 g). Purification using a Thomson Instruments Single Step 80 g silica gel cartridge eluting with 60%
hexanes/40% ethyl acetate produced LR2-1 (1.01 g, 82%) as a colorless oil. ]H NMR
(DMSO- d) δ 1.37 (9H, s), 3.63 (2H, m), 4.00 (2H, m), 4.53 (2H, m), 4.89 (1H, t, J = 6.2 Hz), 5.18 (1H, dd, J = 1.4 Hz, J = 10.6 Hz), 5.30 (1H, dd, J = 1.6 Hz, J = 17.1 Hz), 5.84 (1H, m), 6.98 (1H, d, J = 8.2 Hz).
[0168] Step 2. A solution of N-(tert-butoxycarbonyl)-L-serine allyl ester (0.175 g,
0.731 mmol) in 4 M hydrogen chloride/dioxane (2 mL) was stirred at ambient temperature for 40 minutes. The reaction mixture was concentrated on a rotary evaporator and the crude HCl salt was taken up in anhydrous tetrahydrofuran (3 mL). To this solution was added N-succinimidyl 6-(2,4-dinitroanilino)hexanoate (0.288 g, 0.791 mmol) and triethylamine (102 mL, 0.731 mmol). The reaction mixture was stirred at room temperature for 30 minutes and the solvent was evaporated. The residue was partitioned between ethyl acetate and water and the phases were separated. The organic phase was washed with saturated NaHC03 and saturated NaCl. It was dried over MgS04, filtered, and concentrated under reduced pressure to yield the crude product (0.293 g) as a yellow oil. Purification using a Thomson Instruments Single Step 12 g silica gel cartridge eluting with 50% hexanes/50% ethyl acetate followed by ethyl acetate gave the product
(0.222 g, 72%) as a yellow oil. 1H NMR (DMSO- tf) δ 1.32 (2H, m), 1.52-1.64 (4H, m), 2.15 (2H, t, J = 7.0 Hz), 3.44 (2H, m), 3.59 (1H, m), 3.66 (1H, m), 4.33 (1H, m), 4.55 (2H, m), 5.02 (1H, t, J = 5.5 Hz), 5.17 (1H, m), 5.28 (1H, m), 5.83 (1H, m), 7.21 (1H, d, J = 9.5 Hz), 8.12 (1H, d, J = 7.9 Hz), 8.23 (1H, dd, J = 2.5 Hz, J = 9.4 Hz), 8.85 (2H, m).
Preparation 21
0-(N-((9-Fluorenylrnethoxy Carbonyl -N-Phenyl Aminomethvn N-(6-(2.4-
[0169] Step 1. A solution of N-(6-(2,4-dinitrophenylamino)hexanoyl-L-serine allyl ester (0.050 g, 0.118 mmol), 0-(9-fluorenylmethyl) N-phenyl N-chloromethyl carbamate (0.043 g, 0.118 mmol) and triethylamine (16.1 mL, 0.116 mmol) in anhydrous CH2C12 (2 mL) was heated at reflux for 1 hour. Further aliquots of 0-(9-fluorenylmethyl) N-phenyl N-chloromethyl carbamate (0.043 g, 0.118 mmol) and triethylamine (16.1 mL, 0.116 mmol) were added and reflux maintained for 1 hour. The solution was cooled to room temperature, diluted with CH2C12, washed with saturated NaCl solution, dried over MgS04, filtered, and concentrated under reduced pressure. The crude material (0.145 g) was purified using a Thomson Instruments Single Step 12 g silica gel cartridge eluting with 50% hexanes/50% ethyl acetate followed by 30% hexanes/70% ethyl acetate to furnish the intermediate allyl ester (0.030 g, 33%) as a yellow oil. 1H NMR (DMSO- d) δ 1.31 (2H, m), 1.52-1.63 (4H, m), 2.15 (2H, t, J = 7.3 Hz), 3.41 (2H, m), 3.43-3.70 (2H, br. m), 4.15 (1H, br, m), 4.43-4.54 (5H, br. m), 4.87 (2H, br. m), 5.14 (1H, m), 5.25 (1H, m), 5.79 (1H, m), 7.12-7.38 (12, m), 7.82 (2H, d, J = 7.4 Hz), 8.21 (1H, dd, J = 2.5 Hz), J = 9.5 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.84 (2H, m).
[0170] Step 2. Tetrakis(triphenylphoshine)palladium(0) (0.002 g, 1.7 μηιοΐ) was added to a stirred solution of the allyl ester from Step 1 (0.030 g, 40 μηιοΐ) and phenylsilane (9.8 mL, 80 μηιοΐ) in anhydrous tetrahydrofuran (0.5 mL). The reaction mixture was stirred at ambient temperature for 30 minutes and was then concentrated. Silica gel and CH2C12 were added and the mixture again concentrated and loaded onto a short silica gel column. The column was
eluted with 30% hexanes/70% ethyl acetate followed by ethyl acetate and finally ethyl acetate containing 0.5% acetic acid to generate the carboxylic acid (0.024 g, 86%) as a yellow oil. Ή NMR (DMSO-i d) δ 1.31 (2H, m), 1.51-1.62 (4H, m), 2.14 (2H, t, J = 7.3 Hz), 3.40 (2H, m), 3.45-3.80 (2H, br. m), 4.14 (1H, br. m), 4.41 (3H, br. m), 4.87 (2H, br. m), 7.16-7.30 (12H, m),
7.82 (2H, d, J = 7.6 Hz), 8.08 (1H, d, J = 8.1 Hz), 8.20 (1H, dd, J = 2.7 Hz, J = 9. 6 Hz),
8.83 (2H, m).
Preparation 22
0-(N-Ethoxycarbonyl-N-Phenyl) Aminomethyl) N-(6-(Z4- Dinitrophenylamino Hexanoyl)-Serine
[0171] Prepared in two steps as described in Preparation 21 above but utilizing O-ethyl N-phenyl N-chloromethyl carbamate as the alkylating agent in step 1. 1H NMR (DMSO-i 6) δ 1.13 (3H, t, J = 7.0 Hz), 1.31 (2H, m), 1.51-1.63 (4H, m), 2.14 (2H, t, J = 7.3 Hz), 3.42 (2H, q, J = 6.7 Hz), 3.68 (1H, dd, J = 4.2 Hz, J = 9.7 Hz), 3.79 (1H, dd, J = 5.7 Hz, J = 9.7 Hz), 4.07 (2H, q, J = 7.1 Hz), 4.42 (1H, m), 4.94 (1H, d, J = 11.0 Hz), 5.01 (1H, d, J = 11.0 Hz), 7.19-7.37 (6H, m), 8.10 (1H, d, J = 8.2 Hz), 8.23 (1H, dd, J = 2.7 Hz, J = 9.7 Hz), 8.84 (2H, m), 12.75 (1H, br. s).
[0172] Preparations 23 and 24 together describe constructs of the invention wherein Y is NBCH2 and the linker for binding to dendrimer is coupled to R1. The construct includes the drug SN38 which provides a hydroxyl group for binding to the CH2 of the adaptor.
Preparation 23
Q-((9-(2-(N-(6-Azidohexanoyl N-Methyl Aminomethyl)Fluorenyl)Methyl)
N-Phenyl N-Chloromethyl Carbamate
[0173] A solution of fluorene-2-carbonyl chloride (prepared from fluorene-2-carboxylic acid and oxalyl chloride) in THF is added to aqueous methylamine (2 molar equivalents) to prepare N-methyl fluorene-2-carboxamide. Reduction of the amide using LiAlH4 in ether provides 2-((methylamino)methyl)fluorene. The amine is protected by reaction with di-tert-butyl dicarbonate to provide 2-((N-tBOC-N-methylamino)methyl)fluorene.
[0174] A solution of the 2-((N-tBOC-N-methylamino)methyl)fluorene in anhydrous tetrahydrofuran (THF) is cooled to -78°C, then treated with a solution of lithium
bis(trimethylsilyl)amide in THF (1.2 molar equivalents). After 1 hr, ethyl formate is added and the mixture is allowed to warm to ambient temperature. The mixture is diluted with ethyl acetate and washed successively with 0.1 N HC1, water, saturated aqueous NaHC03, and brine, then dried over MgS04, filtered, and evaporated to provide the 2-((N-lBOC-N- methylamino)methyl)-fluorene-9-carboxaldehyde. This compound is dissolved in methanol and treated with NaB¾ to provide 9-(2-((N-tBOC-N-methylamino)methyl)fluorenylmethanol.
[0175] The 9-(2-((N-tBOC-N-methylamino)methyl)fluorenylmethanol is dissolved in THF and treated with triphosgene and pyridine according to the general procedure of Preparation 4 to provide the chloroformate. The chloroformate is reacted with aniline according to the method of
Preparation 5 to provide 0-(9-(2-((N-tBOC-N-methylamino)methyl)fluorenylmethyl)
N-phenylcarbamate .
[0176] The carbamate is dissolved in trifluoroacetic acid to remove the 'BOC protecting group. After evaporation to dryness, the resulting amine is dissolved in THF and treated with N-(6-azidohexanoyl)succinimide and triethylamine (2 equivalents) to provide
0-(9-(2-((N-(6-azidohexanoyl)-N-methylamino)methyl)fluorenylmethyl) N-phenylcarbamate.
[0177] Reaction of 0-(9-(2-((N-(6-azidohexanoyl)-N-methylamino)methyl)fluorenylmethyl) N-phenylcarbamate. with paraformaldehyde in 1 :1 THF/chlorotrimethylsilane provides the product N-chloromethyl carbamate.
Preparation 24
Linker-Drug Compound with SN-38
[0178] A solution of the N-chloromethylcarbamate of Preparation 23 (1 equivalent), SN-38 (1 equivalent), and sodium iodide (10 equivalents) in anhydrous acetone is treated with triethylamine (1 equivalent). The product is purified by silica gel chromatography.
[0179] Preparations 25-30 are prophetic examples showing the preparation of embodiments wherein Y is NBCH2 and the linker is coupled through R5.
Preparation 25
General Scheme for Preparation of Azidoalkyl-Linkers
[0180] Claisen condensation of R-CH2-Trigger with an co-azidoalkanoate ester
N3(CH2)nC02R' (n = 3-6) in the presence of a strong base, for example NaH, lithium bis(trimethylsilyl)amide (LiHMDS), or lithium diisopropylamide (LDA), provides a ketone which is reduced to the alcohol by reaction with a mild reductant, for example sodium borohydride in methanol. The resulting alcohol is then converted into the carbamate via the chloroformate, and then into the N-chloromethylcarbamate as described above.
Preparation 26
General Scheme for Preparation of BOC-Protected Amine Linkers
R
[0181] Claisen condensation of R-CH2-Trigger with an co-((N-tert-butoxycarbonyl N-alkyl)amino)alkanoate ester (n = 3-6) in the presence of a strong base, for example NaH, lithium bis(trimethylsilyl)amide (LiHMDS), or lithium diisopropylamide (LDA), provides a ketone which is reduced to the alcohol by reaction with a mild reductant, for example sodium borohydride in methanol. The resulting alcohol is then converted into the carbamate using amine B-NH2 as described in Preparation 5. The carbamate is converted into the
N-chloromethylcarbamate as described in Preparation 6.
[0182] After coupling with a molecule comprising an alcohol, thiol, phenol, or thiophenol group, the BOC group is removed from the carbamate by treatment with trifluoroacetic acid. The resulting amine is coupled with a macromolecule comprising a carboxylic acid using a condensing agent, for example a carbodiimide such as EDCI.
Preparation 27
Alternate Scheme for Preparation of Azidoalkyl-Linkers
CI
[0183] The BOC group is removed from the intermediate BOC-protected carbamate of Preparation 25 by treatment with trifluoroacetic acid, and reaction of the resulting amine with an co-azidoalkanoate N-hydroxysuccinimide ester (n = 3-6) provides the azidoamide. This is converted into the N-chloromethylcarbamate as described in Preparation 26.
Preparation 28
Preparation of a Sulfonyl-Triggered Amine Linker
[0184] An ethyl (2-phenylsulfonyl)acetate is deprotonated using excess NaH in THF and alkylated with N-(6-bromohexyl) ethyl carbamate. The product is reduced using lithium aluminum hydride in ether to provide the methylamino alcohol, which is N-protected as the BOC carbamate. The alcohol is converted to the chloroformate and thence into the carbamate and into the N-chloromethyl carbamate according to the previous procedures.
Preparation 29
Preparation of a Sulfonyl-Triggered Azide Linker
[0185] The BOC group is removed from the intermediate BOC-protected carbamate of Preparation 25 by treatment with trifluoroacetic acid, and reaction of the resulting amine with an co-azidoalkanoate N-hydroxysuccinimide ester (n = 3-6) provides the azidoamide. This is converted into the N-chloromethylcarbamate as described in Preparation 27.
Preparation 30
Synthesis of a Sulfonyl- Activated Acid Linker
[0186] A phenyl methylsulfone is deprotonated with NaH in tetrahydrofuran, then acylated with glutaric anhydride to provide a keto-acid. The resulting acid is protected as the tert-butyl ester, and the ketone is reduced using NaB¾. The resulting alcohol is converted into the carbamate via the chloroformate, and thence to the N-chloromethyl carbamate as described above.
Preparation 31
Synthesis of Linked Peptides
[0187] Peptide synthesis is performed using standard methods for solid-phase peptide synthesis, using a serine, tyrosine, or cysteine in a suitably protected form such that the side chains of these residues may be selectively deblocked without deprotection of other residues. The partially deprotected peptide is reacted with an excess of intermediate of formula (3) which is not yet linked to dendrimer in the presence of a mild base. After washing the resin, the product peptide is deblocked and cleaved from the resin to provide the corresponding intermediate prior to dendrimer linkage wherein D is a peptide.
[0188] As one example, CCK8 (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2) is synthesized on solid support using Rink resin using methods known in the art, for example as described in US Patent 4,769,445 (incorporated herein by reference). Commercial Fmoc-Phe-Rink amide- MBHA resin is pre-swollen in DMF for 30 min, then suspended and shaken in piperidine/DMF (1 :4 by volume, 50 ml) for 30 min at room temperature to remove the Fmoc group. The product is isolated by filtration and washed (3X50 ml each) with DCM, 5% N,N-diisopropylethylamine (DIEA) in DCM, and DCM to give the free base of Phe-Rink amide-MBHA-Resin. Fmoc- Asp(OlBu)-OH (1.23 g, 3 mmol), DCC (0.62 g, 3 mmol), and HOBt (0.69 g, 4.5 mmol) are dissolved in 50 ml of 4:1 by volume DCM/DMF with stirring at 0° for 1 hour. Phe- Rink amide- MBHA resin (1 meq) is suspended in the filtered reaction mixture (precipitated DCU removed) and shaken for 2 to 15 hours at room temperature. The Fmoc-Asp-(OlBu)-Phe- Rink amide- MBHA resin product is collected by filtration and washed with DCM. The Fmoc-Asp-(O'Bu)- Phe- Rink amide-MBHA resin is suspended and shaken in piperidine/DMF (1 :4 by volume, 50 ml) for 3 min at room temperature and then a second time for 7 min to remove the Fmoc group. The product is isolated by filtration and washed (3x50 ml each) with DMF and DCM to give the free base of Asp^O'Bu^Phe-Rink amide-MBHA resin. Fmoc-Met-OH (1.12 g, 3 mmol), DCC (0.62 g, 3 mmol), and HOBt (0.69 g, 4.5 mmol) are dissolved in 50 ml of 4:1 by volume DCM/DMF with stirring at 0° for 1 hour. Asp-(OlBu)-Phe-Rink amide-MBHA resin (1 meq) is suspended in the filtered reaction mixture (precipitated DCU removed) and shaken for 2 to 15 hours at room temperature. The Fmoc-Met-Asp-(OtBu)-Phe-Rink amide-MBHA resin product is collected by filtration and washed with DCM and DMF. The Fmoc-Met-Asp-(OlBu)- Phe- Rink amide-MBHA resin is deprotected and coupled sequentially with Fmoc-Trp-OH (1.28 g, 3 mmol), Fmoc-Gly-OH (0.89 g, 3 mmol), Fmoc-Met-OH (1.12 g, 3 mmol), Fmoc-Tyr- OH (1.37 g, 3 mmol), and Boc-Asp(OfBu)-OH (1.23 g, 3 mmol) to provide Boc-Asp(0[Bu)-Tyr- Met-Gly-Trp-Met-Asp(OtBu)-Phe-Rink amide-MBHA resin. The Boc-Asp(0[Bu)-Tyr-Met- Gly-Trp-Met-Asp OTSu^Phe-Rink amide-MBHA resin is washed with DCM (3x50 ml), suspended and shaken in a mixture of 0-(9-fluorenylmethyl) N-phenyl N-chloromethylcar- bamate (10 equivalents) and triethylamine (1 equivalent) in DCM. The resin is isolated by filtration and washed (3 50 ml each) with DCM. The resulting Boc-Asp(OlBu)-Tyr(OX)-Met- Gly-Trp-Met-Asp(OtBu)-Phe-Rink amide-MBHA resin is cleaved from the resin and deblocked by shaking with a mixture of 8% phenol, 5% thioanisole, 5% water, and 3%
3,6-dioxa-l,8-octanedithiol in trifluoroacetic acid (10 mL/g resin) for 4 hours. The resin is
removed by filtration, and the peptide is precipitated by addition of 10 volumes of ether. The crude peptide is purified by reversed-phase HPLC.
[0189] In another example, a cysteine-containing peptide is prepared by solid phase synthesis using the methods described above, incorporating an
S-(allyloxycarbonylaminomethyl)-cysteine [Cys(allocam)] or S-(N-[2,3,5,6-tetrafluoro-4-(N'- piperidino)phenyl]-N-allyloxycarbonyl-amino)cysteine [Cys(fnam)] residue. Prior to cleavage from the resin, the cysteine residue is selectively deblocked using (Ph3P)4Pd and phenylsilane in DCM, then reacted with a compound of formula (II) as described above. The peptide is finally deblocked, removed from the resin, and purified as described above.
Preparation 32
Linker-Drug Compounds of 5-Fluorouracil
[0190] As an example of preparing compounds of the invention where D is the residue of a drug coupled through a heterocyclic N, linker-drug compounds of formula (III) may be prepared from 5-fluorouracil and a compound of formula (II) analogously to the procedures used by Taylor and Sloane, "1-Alkylcarbonyloxymethyl Prodrugs of 5-Fluorouracil (5-FU):
Synthesis,Physicochemical Properties, and Topical Delivery of 5-FU", J Pharmaceutical Sci. 87(1): 15-20 (1998), and by Roberts and Sloane, "Synthesis of 3-Alkylcarbonyl-oxymethyl Derivatives of 5-Fluorouracil", J. Heterocyclic Chem. 39: 905-910 (each incorporated herein by reference). Thus, a suspension of a compound of formula (II) wherein L is CI (1 mmol) and Nal (1.3 mmol) in dry acetonitrile (1 mL) is stirred in the dark for 24 h, then filtered to afford a solution of the compound of formula (II) wherein L is I. The filtrate is allowed to react with a mixture of l-(allyloxycarbonyl-oxymethyl)-5-fluorouracil [Liu, Fullwood, and Rimmer, "Synthesis of Allyloxycarbonylmethyl-5-fluorouracil and copolymerizations with
N-vinylpyrrolidinone", J Materials Chem. 10: 1771-7, 2000] (0.8 mmol) and
l,8-bis(dimethylamino)naphthalene at ambient temperature. After 6 h, the mixture is diluted with ether, stirred for 1 h, and filtered. The filtrate is concentrated to provide the crude protected product, which is treated with a mixture of tetrakis(triphenylphosphine)-palladium(0) and phenylsilane in anhydrous THF for 1 h to remove the allyloxycarbonylmethyl protecting group. The mixture is evaporated, and the residue is purified by silica gel chromatography to provide the linker-drug compound of formula (III).
[0191] Examples 1-5 are prophetic examples describing how dendrimers are coupled to intermediates to obtain the constructs of the invention.
Example 1
Coupling to Dendrimers
[0192] Coupling reactions to dendrimers are monitored to insure completeness of reactions, typically using chromogenic or fluorogenic reactions. Acylation reactions use chromogenic leaving groups (e.g., p-nitro-phenyl (pNP) esters and carbonates) that can be continuously monitored. Free amines of the dendrimer are determined by the chromogenic reaction with TNPS. Alkynes and azides are determined by click reactions with fluorogenic azide and alkyne reagents, respectively. Thiols are determined by chromogenic reaction with DTNB. Modified dendrimers are analyzed by MS.
[0193] Sets of "releasable" bi-functional linkers with an activated group (e.g.,
chloroformate, HSE) for attachment to alcohol (carbonate) and amine (carbamate) groups of drugs on one end, and functional groups for attachment to dendrimers on the other: maleimido for attachment to thiols, carboxyl for amino groups, alkyne for azides, and azides for alkynes are prepared.
[0194] The linkers used in this example are acid-stable and base-labile, so basic conditions
(e.g., for blocking group removal) cannot be used subsequent to attachment of the linker-drug moiety. If acylation reactions are used for PEGylation of dendrimers, they are performed before attachment of drugs containing nucleophiles (e.g. , peptides) to avoid modifying the drug.
[0195] PLL dendrimer (Z=8) is coupled with commercially available oc-BOC-s-Alloc-Lys or a-BOC-s-trifluoroacetyl-Lys (Sigma-Aldrich) or with a-BOC-e-(trifluoroacetyl)-Lys, providing surface-variegated dendrimers with Z=16 using a minor modification of a method to prepare 50% PEGylated PLL dendrimers (Kaminskas, L. M., et al, J. Pharm Sci (2009) 98:3871-3875). After removal of the ε- Alloc group (Pd(PPh3)4) or the trifluoroacetyl group (methoxide), free amines are coupled to activated mPEG, for example, PEG(5000)-pNP carbonate or
mPEG-succinimidyl succinate. The tBOC groups are removed (25% TFA/DCM) to provide PLL dendrimers in which half of the surface amines are free, and the other half attached to PEG
[PLLi6(s-PEG5000)8(a-NH2)8]. Free surface amines of the PEGylated dendrimers are reacted with an activated carboxylic acid containing a functional group (e.g., R'CO-pNP, where R'=alkyne, azide, thiol, etc.) for subsequent coupling to an appropriate functionalized linker- drug (X-Drug) to give PLLi6(s-PEG5000)8(cc-Drug)8.
Example 2
β-Eliminative Release of Molecules from PEGylated Dendrimers
[0196] Several releasable linker-carbamate-DNP analogs shown in Tables 4 and 5 are attached to PLLi6(a-PEG5000)6(azide)8 (PEG5000)8 by CuAAC. Rates of DNP release at pH 7.4 ± serum are determined by HPLC for comparison to those determined for the linkers themselves.
Example 3
Protection of Peptides Against Proteases
[0197] The fluorogenic 7-amino-4-carbamoylmethylcoumarin (ACC) amide of a consensus trypsin-type sequence, N-Ac-Cys-PheSerArg-ACC (-16 A fully extended) is prepared
(Harris, J. L., et al, Proc. Natl. Acad. Set USA (2000) 97:7754-7759), and reacted at the thiol with bi-functional reagents that crosslink thiol and amino groups, and that differ in lengths separating the reactant groups. (Pierce offers a series of such reagents with spacer arm lengths ranging incrementally from 4.4 A to >50 A.) The derivatized peptides are attached to the 8 free amino groups of the intermediate PEGylated dendrimers described in Example 1 via the NHS group. Two linkers such that the cleavage site of the fully extended tetra-peptide is -20 and 40 A from the dendrimer into the 50 A PEG 5000 layer are used. Kinetics of hydrolysis catalyzed by trypsin (MW 24 kDa; d~40 A) and the larger tissue plasminogen activator (MW -70 kDa; d~60 A) of the conjugates and free peptide controls is monitored by fluorogenic release of ACC. Analogous experiments using a PEG 10,000 dendrimer - presumably having a deeper PEG-layer - are than tested to determine if protection against proteases can be increased with increased PEG size.
Example 4
Protection of Alkyl Carbonate Moieties in Linkers Attached to PEGylated Dendrimers
[0198] Within the 7517 small molecule pharmaceuticals in the CMC database, 23% have aliphatic hydroxyl moieties, and a suitable linker is provided for incorporating these into the invention conjugates. It has been reported that esters of the 20-OH of camptothecin (CPT) attached to the core of a PEGylated PLL dendrimer completely are stable towards serum for up to 60 hrs, whereas corresponding PEG-conjugates hydrolyze in a few hours. If carbonates within a PEGylated dendrimers are likewise protected from esterases, our releasable linkers would allow predictable, controlled release of hydroxyl-containing small molecules from PEGylated dendrimers.
[0199] Using CPT as a model hydroxyl-containing drug, a carbonate is formed at the 20-OH by reaction with 5-octynol chloroformate (from 5-octynol and triphosgene/pyridine). The alkyne of the linker is attached to PLL16(PEG5000)g(azide)8 containing 8 azide end-groups by CuAAC, providing a stable conjugate except for the carbonate moiety. The conjugate (i. e. , carbonate) is studied (by HPLC) at pH 7.4 ± serum over a long period, to demonstrate that the carbonate is stable to serum PEGylated dendrimers linked to CPT by a carbonate using one or more of our releasable linkers {e.g., phenyl-sulfone analogs, see Table 4). Kinetic studies are performed at pH 7.4 ± serum (by HPLC) to determine the β-eliminative/release rate. The aforementioned conjugate provides a soluble particle that is ~6% weight CPT, almost 10-fold higher density than could be achieved with linear mono-methoxy PEG.
Example 5
PEGylated Dendrimer-Peptide Conjugates
[0200] Exendin-4, a 39-amino acid peptide from the Gila monster, is an agonist of the GLP-1 receptor, and thus an insulin secretagogue with glucoregulatory effects.
[0201] Exendin sequence: H?N-HGEGTFTSDLSKi ? QMEEE A VRLFIE WLK? 7NGGP S S G APPPS-NH2 (trypsin sites and amino terminus in red, a-helix underlined): Exendin-4 has a longer plasma lifetime than GLP-1 (~5 min), but a half-life of only 2.5 hrs. It is marketed for type 2 diabetes as Byetta®.
[0202] Exendin 4 consists of a 5-turn a-helix (Leu10 to Asn28; -27 A) with mobile N- and C-termini. It is relatively stable against plasma proteases in vitro (tj/2 -10 hr), and attachment to a PEG-dendrimer should increase stability even more. It has three potential trypsin cleavage
sites: Lys12 and/or Lys27 are most susceptible, and Arg20 is ~14-fold more resistant. PEGylation of the exendin N-terminus yields an inactive conjugate, whereas PEG-Lys12 or PEG-Lys27 are about as active agonists as the native peptide.
[0203] A releasable linker is coupled to the a- or Lysi2 amino groups of exendin, and each linker-exendin is coupled to a PEG5000 PLL dendrimer to give PLLi6(oc-PEG5000)6(s- exendin)8. Linear PEG-exendin controls are also prepared.
[0204] Exendin is prepared by SPPS by Fmoc/tBu chemistry, using an orthogonal blocking group at the side chain of the Lys12 (e.g., monomethoxytrityl, MMT). The blocking group at the intended site of reaction ( -amino Fmoc or Lysi2 side chain MMT) is removed, and an HSE ester of an alkyne-containing or azide-containing releasable linker coupled to the free-amino group on-resin. Blocking groups and resin are removed (TFA), and the modified carbamoylated peptides purified by HPLC. The linker is attached to
a) the dendrimer shell of PLL-PEG 5000 containing 8 PEGs and 8 azide or alkyne end groups, and
b) a control azide- or alkyne-modified linear PEG 5000 by CuAAC.
[0205] The in vitro rate of β-eliminative release and escape of the peptide from the dendrimer is determined at pH 7.4 ± serum and 8.4 using SEC HPLC; the observed rate is a composite of the β -elimination and diffusion of the free peptide through PEG, but the latter should not contribute significantly.
[0206] The PEG and PEG-dendrimer conjugates are tested as agonists of the GLP1 receptor in membrane or cell-based assays (e.g., RTN-m5f cells, ATCC as described (Young, A., et al, (2000) WO00/66629) pages 74-75) before and after β-eliminative release of native exendin.
[0207] Conjugates are then treated with mild base to release native exendin that shows full activity as GLP1 agonists.
[0208] The conjugates are treated with trypsin and sera to determine accessibility to proteases; at various times, excess PMSF is added to quench serine proteases, peptides are released by mild base-catalyzed β-elimination and the remaining native peptide determined by HPLC.
[0209] Examples 6-9 are working examples describing construction of a dendrimer which has as its core two lysine residues linked through diaminohexane and provided with azido groups for linkage to drug. Examples 10 and 1 1 are prophetic examples showing linkage of this intermediate both to PEG and to an alkynyl linker coupled to drug.
Example 6
DAHrLvsl?ra-Bocl2rs-Boc1z
[0210] To a stirred solution of Na,N8-bis(tert-butoxycarbonyl)-L-lysine succinimidyl ester (1.91 g; 4.31 mmol) in N,N-dimethylformamide (5 mL) was added a solution of
1 ,6-diaminohexane (0.200 g; 1.72 mmol) in N,N-dimethylformamide (5 mL) over 5 minutes. The reaction mixture was stirred at ambient temperature for 20 hours and was then diluted with water (100 mL). The aqueous phase was extracted 3x with ethyl acetate and the combined organic extracts were washed with water, saturated sodium hydrogen carbonate solution and brine. The organic phase was dried over magnesium sulfate and concentrated to give a colorless oil (1.93 g). Purification using a Thomson Instruments Single Step 40 g silica gel cartridge eluting with 1 : 1 ethyl acetate/hexanes followed by 70:30 ethyl acetate/hexanes furnished
DAH[Lys]2[a-Boc]2[s-Boc]2 (1.15 g ; 86%) as a white solid. 1H NMR (DMSO-rfd) 1.17-1.52 (50H, br. m, 2.85 (4H, br. m), 2.96 (4H, m), 3.77 (2H, m), 6.70 (4H, m), 7.69 (2H, m).
Example 7
DAHiLvsyg-Bocldjs
[0211] Trifluoroacetic acid (5 mL) was added to a stirred solution of DAH[Lys]2[a-Boc]2[8- Boc]2 (0.200 g; 0.259 mmol) in dichloromethane (5 mL). The solution was stirred at room temperature for 1 hour and was then concentrated on the roto-vap. The crude salt was dissolved in N,N-dimethylformamide (8 mL) and triethylarnine (0.58 mL; 4.16 mmol) added. To this solution was added Boc-Lys(Boc)-OSu (0.505 g; 1.14 mmol) and the reaction mixture stirred for 20 hours. The solution was added to ice-water with stirring (500 mL) by pipette and the resulting fine suspension stirred for 35 minutes. The solid was collected by filtration, washed with water and dried. It was then re-suspended in acetonitrile (2 mL) and stirred for 1 hour. The solid was collected, washed with acetonitrile and dried to give DAH[Lys]4[oc-Boc]4[s-Boc] (0.337 g; 77%) as a white solid. 1H NMR (DMSO-c¾) 1.21 (116H, Br. m), 2.82-3.06 (8H, br. m), 3.79 (4H, br. m), 4.17 (2H, br. m), 6.67-6.91 (8H, m), 7.66-7.81 (6H, m).
Example 8
Boc-L-Azidonorleucine succinimidyl ester (BOC-ANL-OSu
[0212] To a stirred solution of N-(tert-butoxycarbonyl)-L-azidonorleucine (0.505 g;
1.85 mmol) and triethylamine (284 xL; 2.04 mmol) in tetrahydrofuran (10 mL) was added Ν,Ν'-disuccinimidyl carbonate (0.523 g; 2.04 mmol). The reaction mixture was stirred at ambient temperature for 1.5 hours and was then concentrated on the roto-vap. The residue was taken up in ethyl acetate and washed with water, saturated sodium hydrogen carbonate solution and saturated sodium chloride solution. The organic phase was dried over magnesium sulfate and concentrated to give a colorless oil (0.744 g). Purification by column chromatography using a Thomson Instruments Single Step 40 g silica gel cartridge eluting with hexanes followed by 1 : 1 ethyl acetate/hexanes produced the product (0.536 g; 79%) as a colorless oil. Ή NMR (DMSO-i¾) 1.39 (9H, s), 1.46 (4H, m), 1.74 (2H, m), 2.80 (4H, br s), 3.30 (2H, m overlaps solvent), 4.31 (1H, m), 7.61 (1H, d, J = 7.8 Hz). Fmoc-L-azidonorleucine succinimidyl ester is prepared similarly starting from Fmoc-L-azidonorleucine.
Example 9
PAH I vsl 4 ΓΒ OC- ANLIg
DAH[Lys]4[a-Boc]4[6-Boc]
1. CF3COzH, CH2CI2
2. BOC-ANL-OSu, Et3N, DMF
DAH[Lys]4[BOC-ANL]8
[0213] Trifluoroacetic acid (4 mL) was added to a stirred suspension of DAH[Lys]4[a- Boc]4[s-Boc]4 (0.139 g; 82.4 μτηοϊ) in dichloromethane (4 mL) and the resulting solution stirred at ambient temperature for 1.5 hours. The solution was concentrated and the crude salt was taken up in N,N-dimethylformamide (4 mL) and triethylamine (368 μί; 2.7 mmol) added. To this solution was added a solution of Boc-L-azidonorleucine succinimidyl ester (0.268 g;
726 μιηοΐ) in Ν,Ν-dimethyl-formamide. The reaction mixture was stirred at room temperature for 22 hours and was then added by pipette to a stirred ice-water (200 mL) solution. The resulting suspension was stirred for 30 minutes and was then collected by filtration. The solid was washed with water and dried. It was re-suspended in acetonitrile (2 mL) and stirred for
30 minutes. The solid was collected, washed with acetonitrile and dried to give DAH[Lys]4[BOC-ANL]8 (0.181 g; 75%) as a white solid.
Example 10
DAHfLvsl.f LlgrmPEGjnnnlgf al.
DAH[Lys]4[BOC-ANL]8
1. CF3C02H, CH2CI2
2. mPEG(5000)-NHS, Et3N, D F
DAH[Lys]4[NL]8[mPEG50oo]8[N3]8
[0214] Trifluoroacetic acid is added to a stirred suspension of DAH[Lys]4[BOC-ANL]8 in dichloromethane as described for other examples above and the resulting solution is stirred at ambient temperature for 1.5 hours. The solution is concentrated and the crude salt is taken up in Ν,Ν-dimethylformamide and triethylamine sufficient to neutralize the salts and allow subsequent coupling is added. To this solution is added a molar excess of monomethoxy-
polyethylene glycol succinimidyl ester (mw 5000) as a solution in Ν,Ν-dimethylformamide. The reaction mixture is stirred at room temperature for 22 hours and is then dialyzed against methanol using a 10,000-mw cutoff dialysis membrane to remove small molecule reagents and byproducts. The dialysate is evaporated to dryness to provide the product.
Example 1 1
Conjugation of Alkvnyl-Linker-Drug with DAH[Lys]4(NL]s[mPEGsnon]g[N ]s
DAH[Lys]4[NL]8[mPEG50oo [N3 χ o
-Linker— O Drug
CuS04, TBTA, sodium ascorbate
DMSO/H20
[0215] A solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS04 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys]4[NL]8[mPEG5ooo]8[N3]8 and 10 equivalents of alkyne-linker-drug in 1 :2
water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-drug. The dialysate is lyophilized to provide the product.
[0216] Examples 12-16 result in compounds of the invention wherein 4 copies of the peptide drug exendin are coupled through linkers to the polylysine dendrimer that has been PEGylated. In these embodiments, X is O and Y is absent.
Example 12
Alkynyl-Linker - Exendin (N-terminally linked)
[0217] Rink-amide TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH2) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques. After removal of the last FMOC group and washing the resin three times with dichloromethane to remove excess piperidine, the resin is treated with a solution of 3 equivalents of the alkynyl-Linker
succinimidyl carbonate in dichloromethane/DMF and 1.5 equivalent of N-methylmorpholine. After coupling is completed, the resin is washed to remove excess reagents. The linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol. The linker-exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1% trifluoroacetic acid, and lyophilized.
Example 13
Alkynyl-Linker - Exendin (Lys -linked)
[0218] Rink-amide TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH2) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques, with the exception that Lys12 is introduced as Fmoc-Lys(mmt) (mmt = monomethoxytrityl) and the final coupling is performed using BOC-His in place of Fmoc-His. The mmt group is removed by treatment with
dilute CF3CO2H in dichloromethane, and the resin is treated with a solution of 3 equivalents of the alkynyl-Linker succinimidyl carbonate in dichloromethane/DMF and 1.5 equivalent of N-methylmorpholine. After coupling is completed, the resin is washed to remove excess reagents. The linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol. The linker- exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1 % trifluoroacetic acid, and lyophilized.
Example 14
Synthesis of an Example Alkynyl Linker Succinimidyl Carbonate
[0219] While the following procedure is given in detail for the 4-methoxyphenylsulfonyl trigger, other alkynyl-linker succinimidyl carbonates comprising arylsulfonyl triggers may be prepared by substitution of 4-methoxythiophenol in Step 1 below with other substituted thiophenols.
[0220] Step 1. 2-Bromo-3'-nitroacetophenone (2.98 g, 12.2 mmol) was dissolved in acetonitrile (12 mL). Water (12 mL) then sodium hydrogen carbonate (2.04 g, 24.3 mmol) were added. The resulting biphasic mixture was vigorously stirred and 4-methoxythiophenol (1.5 mL, 12.2 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 1.5 hours. It was then diluted with water and extracted with ethyl acetate (x3). The combined organic extracts were washed with 1M sodium carbonate and saturated sodium chloride, they were then dried over magnesium sulfate and concentrated under reduced pressure to give an orange oil (3.85 g) which solidified on standing. To the crude material was added ethyl acetate/hexanes (1 :4, 20 mL) and the mixture stirred overnight. The solid was collected, washed with ethyl acetate/hexanes (1 :4) and dried to yield the sulfide (2.75 g, 74%) as a pale orange solid. 1H NMR (DMSO-<¾) 63 733 Ds, 4.53 (2H, s), 6.86 (2H, d, J = 8.4 Hz), 7.28 (2H, d, J = 8.4 Hz), 7.80 (1H, t, J = 8.0 Hz), 8.36 (1H, d, J = 7.8 Hz), 8.45 (1H, m), 8.62 (1H, t,
J = 1.8 Hz).
[0221] Step 2. To an ice-cooled stirred solution of the sulfide of Step 1 (2.75 g, 9.07 mmol) in ethyl acetate (75 mL) was added peracetic acid (5.8 mL of a 32 wt% solution in dilute acetic acid, 27.6 mmol) slowly over 10 minutes. The solution was stirred at ice-bath temperature for 10 minutes and then at ambient temperature for 2 hours. The suspension was dissolved by the addition of ethyl acetate (75 mL) and this solution was washed with 1 M sodium carbonate (x 2), water, 0.1 M sodium hydrosulfite (x 2), water, 1 M sodium carbonate and saturated sodium chloride. It was then dried over magnesium sulfate and concentrated under reduced pressure to furnish the sulfone as a white solid (3.04 g, 100%). Ή NMR (DMSO-<¾) 53.843 Ds, 5.41 (2H, s), 7.09 (2H, d, J = 8.8 Hz), 7.72 (2H, d, J = 8.7 Hz), 7.80 (1H, t, J - 7.9 Hz), 8.35 (1H, m), 8.47 (1H, ddd, J = 0.9 Hz, J = 2.3 Hz, J = 8.2 Hz), 8.62 (1H, t, J = 1.9 Hz).
[0222] Step 3. Tin (II) chloride dihydrate (2.69 g, 1 1.9 mmol) was added to a stirred suspension of the sulfone of Step 2 (1.00 g, 2.98 mmol) in ethanol (30 mL). The reaction mixture was heated at gentle reflux for 30 minutes and the resulting yellow solution allowed to cool to room temperature. The solution was poured onto crushed ice and the pH adjusted to pH 8 with 1 M sodium carbonate. The suspension was equilibrated to room temperature and diluted to -200 mL volume with water. It was then extracted with ethyl acetate (x 3) and the combined organic layers were washed with saturated sodium chloride, dried over magnesium sulfate and concentrated under reduced pressure to produce the aniline (0.813 g, 89%) as a yellow solid. Ή NMR (DMSO-dtf) 53.823 Ds, 5.08 (2H, s), 5.36 (2H, br. s), 6.81 (1H, dt, J = 1.9 Hz, J = 7.2 Hz), 7.04-7.15 (5H, m), 7.79 (2H, d, J = 8.8 Hz).
[0223] Step 4. To a stirred solution of 5-hexynoic acid (392 μί, 3.46 mmol) and oxalyl chloride (351 μL, 4.15 mmol) in anhydrous dichloromethane (5 mL) was added 2 drops of anhydrous Ν,Ν-dimethylformamide resulting in gas evolution. The solution was stirred for 15 minutes after which time gas evolution had ceased and a further 2 drops of anhydrous Ν,Ν-dimethylformamide were added (no gas evolution). The solution was stirred for 10 minutes and was then concentrated on the rotary evaporator. The crude acid chloride was dissolved in anhydrous dichloromethane (5 mL) and slowly added to a stirred suspension of the aniline of Step 3 (0.813 g, 2.66 mmol) and triethylamine (1.1 mL, 8.07 mmol) in anhydrous
dichloromethane (20 mL). The resulting solution was stirred at room temperature for 2 hours and was then diluted with ethyl acetate. The solution was washed with water, 1 M sodium carbonate, water and saturated sodium chloride. It was dried over magnesium sulfate and concentrated under reduced pressure to give the crude amide as a brown oil (1.28 g).
Purification utilizing a Thomson Instruments Single Step 40 g silica gel cartridge and eluting with 100% hexanes followed by 50%> ethyl acetate/50%) hexanes gave the amide (0.484 g, 46%») as a white solid. Ή NMR (DMSO- 6) δί□□□□□, 2.21 (2H, td, J = 2.7 Hz, J = 7.1 Hz), 2.41 (2H, t, J = 7.5 Hz), 2.82 (1H, t, J = 2.7 Hz), 3.84 (3H, s), 5.18 (2H, s), 7.11 (2H, d, J = 9.2 Hz), 7.41 (1H, t, J = 8.1 Hz), 7.65 (1H, d, J = 8.3 Hz), 7.79 (2H, d, J = 9.2 Hz), 7.84 (1H, d,
J = 7.8 Hz), 8.09 (1H, t, J = 1.8 Hz), 10.15 (1H, s).
[0224] Step 5. To a stirred suspension of the amide of Step 4 (0.484 g, 1.21 mmol) in methanol (12 mL) was added in portions over 5 minutes sodium borohydride (0.102 g,
2.70 mmol). The resulting solution was stirred for 35 minutes and then quenched by the addition of saturated ammonium chloride. It was then concentrated on the rotary evaporator, diluted with water and extracted with ethyl acetate (x 3). The organic extracts were combined, washed with saturated sodium chloride and dried over magnesium sulfate. The solution was concentrated to afford the alcohol (0.438 g, 90%) as white foam. 1H NMR (DMSO-J6) 61.71 (2H, m), 2.19 (2H, td, J = 2.7 Hz, J = 7.0 Hz), 2.37 (2H, t, J = 7.4 Hz), 2.80 (1H, t, J = 2.7 Hz), 3.37 (1H, dd, J = 3.0 Hz, J = 14.6 Hz), 3.56 (1H, dd, J = 8.8 Hz, J = 14.5 Hz), 4.88 (1H, m), 5.59 (1H, d, J = 4.3 Hz), 6.93 (1H, d, J = 7.7 Hz), 7.09 (2H, d, J = 8.7 Hz), 7.17 (1H, t, J = 7.8 Hz), 7.45 (1H, d, J = 8.1 Hz), 7.49 (1H, s), 7.78 (2H, d, J = 8.8 Hz).
[0225] Step 6. Pyridine (156 μΐ,, 1.93 mmol) was added to a stirred solution of the alcohol of Step 5 (0.387 g, 0.964 mmol) and triphosgene (0.41 1 g, 1.39 mmol) in anhydrous
tetrahydrofuran (10 mL). The resulting suspension was stirred for 30 minutes and was then filtered and the filtrate concentrated under reduced pressure. The crude chloroformate was taken up in anhydrous tetrahydrofuran (10 mL) and N-hydroxysuccinimide (0.598 g, 5.20 mmol) followed by pyridine (249 μί, 3.09 mmol) added. The reaction mixture was stirred at ambient temperature for 35 minutes and was then filtered and concentrated. The residue was dissolved in ethyl acetate and washed with water, 0.1 M hydrochloric acid, saturated sodium hydrogen carbonate, water and saturated sodium chloride. The organic solution was dried over magnesium sulfate and concentrated under reduced pressure to yield the crude product (0.469 g) as a pale yellow oil. Purification by silica gel column chromatography eluting with 100% hexanes followed by 50% ethyl acetate/50% hexanes produced the succinimidyl carbonate (0.296 g, 52%) as a pale yellow oil. !H NMR (DMSO-d5) 1.73 (2H, m), 2.19 (2H, td), J = 2.8 Hz, J = 7.1 Hz), 2.39 (2H, t, J = 7.4 Hz), 2.77 (4H, s), 2.80 (1H, t, J = 2.6 Hz), 3.86 (3H, s), 3.89 (1H, dd, J - 3.2 Hz, J = 15.1 Hz), 4.30 (1H, dd, J = 9.7 Hz, J = 15.1 Hz), 5.87 (1H, dd,
J = 3.2 Hz, J - 9.7 Hz), 7.07 (1H, d, J = 8.4 Hz), 7.11 (2H, d, J = 8.9 Hz), 7.28 (1H, t, J = 8.0 Hz, 7.55 (1H, d, J = 8.2 Hz), 7.61 (1H, s), 7.82 (2H, d, J = 8.9 Hz.
Example 15
Preparation of an Example Alkynyl-Linker-Exendin-4 Compound
[0226] Rink-amide TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH2) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques. After removal of the last FMOC group and washing the resin three times with dichloromethane to remove excess piperidine, the resin is treated with a solution of 3 equivalents of the alkynyl-Linker
succinimidyl carbonate of Example 14 in dichloromethane/DMF and 1.5 equivalents of
N-methylmorpholine. After coupling is completed, the resin is washed to remove excess reagents. The linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol. The linker- exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1% trifluoroacetic acid, and lyophilized.
Example 16
DAH[Lys]4[ L]8[mPEG5ooo]8[N3]8
O
X
Linker—O NH-Exendin-OH
CuS04, TBTA, sodium ascorbate DMSO/H20
O^ EGsoooOMe
[0227] A solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS04 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated azido dendrimer DAH[Lys]4[NL]8[mPEG5oo0]8[N3]8 and 10 equivalents of alkynyl-linker-exendin in
1 :2 water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water using a 10,000-mw cutoff dialysis membrane until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-exendin. The dialysate is lyophilized to provide the product.
[0228] Examples 17-20 describe synthesis of compounds of the invention wherein a drug is coupled to a polylysine dendrimer through a different linker, and wherein X is O and Y is absent.
Example 17
Boc-L-Propargylglycine Succinimidyl Ester (BOC-PG-OSu)
[0229] To a stirred solution of N-(tert-butoxycarbonyl)-L-propargyl glycine (1.8 mmol) and triethylamine (2.0 mmol) in tetrahydrofuran (10 niL) is added Ν,Ν'-disuccinimidyl carbonate (2.0 mmol). The reaction mixture is stirred at ambient temperature for 1.5 hours and is then concentrated on the roto-vap. The residue is taken up in ethyl acetate and washed with water, saturated sodium hydrogen carbonate solution and saturated sodium chloride solution. The organic phase is dried over magnesium sulfate and concentrated. Purification by silica gel chromatography provides the product. Fmoc-L-propargylglycine succinimidyl ester is prepared similarly, starting from Fmoc-L-propargylglycine.
Example 18
DAHrLvsMBOC-PGIg
DAH[Lys]4[a-Boc]4[s-Boc]4
1. CF3C02H, CH2CI2
2. BOC-PG-OSu, Et3N, DMF
DAH[Lys]4[BOC-PG]8
[0230] Trifluoroacetic acid (4 mL) is added to a stirred suspension of DAH[Lys]4[a- Boc]4[s-Boc]4 (0.139 g; 82.4 μηιοΐ) in dichloromethane (4 mL) and the resulting solution is stirred at ambient temperature for 1.5 hours. The solution is concentrated and the crude salt is taken up in N,N-dimethylformamide (4 mL) and triethylamine (368 μL; 2.7 mmol) added. To this solution is added a solution of Boc-L-propargylglycine succinimidyl ester (726 μιηοΐ) in Ν,Ν-dimethyl-formamide. The reaction mixture is stirred at room temperature for 22 hours and is then added by pipette to a stirred ice-water (200 mL) solution. The resulting suspension is stirred for 30 minutes and is then collected by filtration. The solid is washed with water and
dried. It is re-suspended in acetonitrile (2 mL) and stirred for 30 minutes. The solid is collected, washed with acetonitrile and dried to give the product.
Example 19
DAHrLvsl4f L1srmPEGsmnlgrAlkvnels
DAH[Lys]4[BOC-PG]8
1. CF3C02H, CH2CI2
2. mPEG(5000)-NHS, Et3N,
DAH[Lys]4[NL]8[mPEG500o]8[alkyne]8
[0231] Trifluoroacetic acid is added to a stirred suspension of DAH[Lys]4[BOC-PG]8 in dichloromethane as described for other examples above and the resulting solution is stirred at ambient temperature for 1.5 hours. The solution is concentrated and the crude salt is taken up in Ν,Ν-dimethylformamide and triethylamine sufficient to neutralize the salts and allow
subsequent coupling is added. To this solution is added a molar excess of
monomethoxypolyethylene glycol succinimidyl ester (mw 5000) as a solution in
Ν,Ν-dimethylformamide. The reaction mixture is stirred at room temperature for 22 hours and is then dialyzed against methanol using a 10,000-mw cutoff dialysis membrane to remove small molecule reagents and byproducts. The dialysate is evaporated to dryness to provide the product.
Example 20
DAHfLysMNLfefmPEGsoooMalkynek
N3-Linker-Drug
sodium ascorbate
CuS04l TBTA
DMSO/H20
DAH[Lys]4[NL]8[mPEG5ooo]8[linker-drug]8
[0232] A solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS04 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys]4p^L]s[mPEG5oooMAlkyne]8 and 10 equivalents of azido-linker-drug in 1 :2
water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-exendin. The dialysate is lyophilized to provide the product.
[0233] Examples 21-23 describe coupling of exendin to the polylysine through still a different linker, wherein X is O and Y is absent.
Example 21
Synthesis of an Example Azido-Linker Succinimidyl Carbonate
[0234] While the following procedure is given in detail for the 4-methoxyphenylsulfonyl trigger, other azido-linker succinimidyl carbonates comprising arylsulfonyl triggers may be prepared by substitution of 4-methoxythiophenol in Step 1 below with other substituted thiophenols.
[0235] Step 1. 2-Bromo-3'-nitroacetophenone (2.98 g, 12.2 mmol) was dissolved in acetonitrile (12 mL). Water (12 mL) then sodium hydrogen carbonate (2.04 g, 24.3 mmol) were added. The resulting biphasic mixture was vigorously stirred and 4-methoxythiophenol (1.5 mL, 12.2 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 1.5 hours. It was then diluted with water and extracted with ethyl acetate (x 3). The combined organic extracts were washed with 1M sodium carbonate and saturated sodium chloride, they were then dried over magnesium sulfate and concentrated under reduced pressure to give an orange oil (3.85 g) which solidified on standing. To the crude material was added ethyl acetate/hexanes (1 :4, 20 mL) and the mixture stirred overnight. The solid was collected, washed with ethyl acetate/hexanes (1 :4) and dried to yield the sulfide (2.75 g, 74%) as a pale orange solid. 1H NMR (DMSO- d) 53.733 Ds, 4.53 (2H, s), 6.86 (2H, d, J = 8.4 Hz), 7.28 (2H, d, J = 8.4 Hz), 7.80 (1H, t, J = 8.0 Hz), 8.36 (1H, d, J = 7.8 Hz), 8.45 (1H, m), 8.62 (1H, t,
J = 1.8 Hz).
[0236] Step 2. To an ice-cooled stirred solution of the sulfide of Step 1 (2.75 g, 9.07 mmol) in ethyl acetate (75 mL) was added peracetic acid (5.8 mL of a 32 wt% solution in dilute acetic acid, 27.6 mmol) slowly over 10 minutes. The solution was stirred at ice-bath temperature for 10 minutes and then at ambient temperature for 2 hours. The suspension was dissolved by the addition of ethyl acetate (75 mL) and this solution was washed with 1M sodium carbonate (x 2), water, 0.1 M sodium hydrosulfite (x 2), water, 1 M sodium carbonate and saturated sodium chloride. It was then dried over magnesium sulfate and concentrated under reduced pressure to furnish the sulfone as a white solid (3.04 g, 100%). 1H NMR (OMSO-d6 53.843 Ds, 5.41 (2H, s), 7.09 (2H, d, J = 8.8 Hz), 7.72 (2H, d, J = 8.7 Hz), 7.80 (1H, t, J = 7.9 Hz), 8.35 (1H, m), 8.47 (1H, ddd, J = 0.9 Hz, J = 2.3 Hz, J - 8.2 Hz), 8.62 (1H, t, J = 1.9 Hz).
[0237] Step 3. Tin (II) chloride dihydrate (2.69 g, 1 1.9 mmol) was added to a stirred suspension of the sulfone of Step 2 (1.00 g, 2.98 mmol) in ethanol (30 mL). The reaction mixture was heated at gentle reflux for 30 minutes and the resulting yellow solution allowed to cool to room temperature. The solution was poured onto crushed ice and the pH adjusted to pH 8 with 1 M sodium carbonate. The suspension was equilibrated to room temperature and diluted to -200 mL volume with water. It was then extracted with ethyl acetate (x 3) and the combined organic layers were washed with saturated sodium chloride, dried over magnesium sulfate and concentrated under reduced pressure to produce the aniline (0.813g, 89%) as a yellow solid. 1H NMR (DMSO-i/<5) 53.823 Ds, 5.08 (2H, s), 5.36 (2H, br. s), 6.81 (1H, dt, J = 1.9 Hz, J - 7.2 Hz), 7.04-7.15 (5H, m), 7.79 (2H, d, J = 8.8 Hz).
[0238] Step 4. To a stirred solution of 6-azidohexanoic acid and oxalyl chloride in anhydrous dichloromethane is added 2 drops of anhydrous Ν,Ν-dimethylformamide resulting in gas evolution. The solution is stirred for 15 minutes after which time gas evolution ceases and a further 2 drops of anhydrous Ν,Ν-dimethylformamide are added. The solution is stirred for 10 minutes and is then concentrated on the rotary evaporator. The crude acid chloride is dissolved in anhydrous dichloromethane and slowly added to a stirred suspension of the aniline of Step 3 and triethylamine in anhydrous dichloromethane. The resulting solution is stirred at room temperature for 2 hours and is then diluted with ethyl acetate. The solution is washed with water, 1 M sodium carbonate, water and saturated sodium chloride. It is dried over magnesium sulfate and concentrated under reduced pressure to give the crude amide. Purification utilizing a silica gel column provides the amide.
[0239] Step 5. To a stirred suspension of the amide of Step 4 in methanol is added in portions over 5 minutes sodium borohydride as described for the preparation of the alkynyl linkers above. The resulting solution is stirred for 35 minutes and then quenched by the addition of saturated ammonium chloride. It is then concentrated on the rotary evaporator, diluted with water and extracted with ethyl acetate (x 3). The organic extracts are combined, washed with saturated sodium chloride and dried over magnesium sulfate. The solution is concentrated to afford the alcohol.
[0240] Step 6. Pyridine is added to a stirred solution of the alcohol of Step 5 and triphosgene in anhydrous tetrahydrofuran as described for the preparation of the alkynyl linkers above. The resulting suspension is stirred for 30 minutes and is then filtered and the filtrate concentrated under reduced pressure. The crude chloroformate is taken up in anhydrous tetrahydrofuran and N-hydroxysuccinimide followed by pyridine is added. The reaction mixture is stirred at ambient temperature for 35 minutes and is then filtered and concentrated. The residue is dissolved in ethyl acetate and washed with water, 0.1 M hydrochloric acid, saturated sodium hydrogen carbonate, water and saturated sodium chloride. The organic solution is dried over magnesium sulfate and concentrated under reduced pressure to yield the crude product. Purification by silica gel column chromatography affords the succinimidyl carbonate.
Example 22
Preparation of an Example Azido-Linker-Exendin-4 Compound
[0241] Rink amide-TentaGel resin containing the exendin-4 sequence (HGEGTFTSDLSKQ MEEEAV-RLFIEWLKNGGPSSGAPPPS-NH2) in protected form is prepared by solid-phase synthesis using standard FMOC/t-Bu solid-phase synthesis techniques. After removal of the last FMOC group and washing the resin three times with dichloromethane to remove excess piperidine, the resin is treated with a solution of 3 equivalents of the azido-Linker succinimidyl carbonate of Example 21 in dichloromethane/DMF and 1.5 equivalents of N-methylmorpholine.
After coupling is completed, the resin is washed to remove excess reagents. The linker-exendin is removed from the resin and deblocked by treatment with a cocktail of trifluoroacetic acid, phenol, thioanisole, and 3,6-dioxa-l,8-octanedithiol. The linker-exendin is then purified by reversed-phase HPLC using a gradient of acetonitrile and water containing 0.1% trifluoroacetic acid, and lyophilized.
Example 23
Conjugation of Azido-Linker-Exendin with DAH[ ys]4|^L]s[mPEGsnnn
[0242] A solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS04 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys]4[NL]8[mPEG5ooo]8[alkyne]8 and 10 equivalents of azido-linker-exendin in 1 :2 water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-exendin. The dialysate is lyophilized to provide the product.
[0243] Examples 24-26 describe the preparation of a polylysine dendrimer coupled through an azido linker to SN38 wherein X is O and Y is NBCH2.
Example 24
Preparation of an Azido-linker chloromethyl carbamate
Q-(T9-(2-(N-f6-azidohexanoyl) N-methyl)aminomethyl fluorenyl)methyl) N-phenyl N-chloromethyl carbamate
[0244] A solution of fluorene-2-carbonyl chloride (prepared from fluorene-2-carboxylic acid and oxalyl chloride) in THF is added to aqueous methylamine (2 molar equivalents) to prepare N-methyl fluorene-2-carboxamide. Reduction of the amide using L1AIH4 in ether provides 2-((methylamino)methyl)fluorene. The amine is protected by reaction with di-tert-butyl dicarbonate to provide 2-((N-tBOC-N-methylamino)methyl)fluorene.
[0245] A solution of the 2-((N-tBOC-N-methylamino)methyl)fluorene in anhydrous tetrahydrofuran (THF) is cooled to -78°C, then treated with a solution of lithium
bis(trimethylsilyl)amide in THF (1.2 molar equivalents). After 1 hr, ethyl formate is added and the mixture is allowed to warm to ambient temperature. The mixture is diluted with ethyl acetate and washed successively with 0.1 N HCl, water, saturated aqueous NaHC03, and brine, then dried over MgS04, filtered, and evaporated to provide the 2-((N-*BOC-N-
methylamino)methyl)-fluorene-9-carboxaldehyde. This compound is dissolved in methanol and treated with NaBH4 to provide 9-(2-((N-tBOC-N-methylamino)methyl)fluorenylmethanol.
[0246] The 9-(2-((N-tBOC-N-methylamino)methyl)fluorenylmethanol is dissolved in THF and treated with triphosgene and pyridine according to the general procedure of Example 2 to provide the chloroformate. The chloroformate is reacted with aniline according to the method of Example 3 to provide 0-(9-(2-((N-tBOC-N-methylamino)methyl)fluorenylmethyl)
N-phenylcarbamate.
[0247] The carbamate is dissolved in trifluoroacetic acid to remove the lBOC protecting group. After evaporation to dryness, the resulting amine is dissolved in THF and treated with N-(6-azidohexanoyl)succinimide and triethylamine (2 equivalents) to provide 0-(9-(2-((N-(6- azidohexanoyl)-N-methylamino)methyl)fluorenylmethyl) N-phenylcarbamate.
[0248] Reaction of 0-(9-(2-((N-(6-azidohexanoyl)-N-methylamino)methyl)fluorenylmethyl) N-phenylcarbamate with paraformaldehyde in 1 :1 THF/chlorotrimethylsilane provides the product N-chloromethyl carbamate.
Example 25
Azido-Linker-Drug Compound with SN-38
[0249] A suspension of the N-chloromethylcarbamate of Example 24 (2 equivalents) and sodium iodide (20 equivalents) in anhydrous acetone is allowed to stir overnight protected from light. The mixture is filtered and evaporated, and the residue is redissolved in anhydrous acetonitrile and added to a solution of SN-38 (1 equivalent) and triethylamine (1 equivalent). After stirring overnight protected from light, the mixture is evaporated to dryness. The residue
is redissolved in ethyl acetate, washed with sat. aq. NH4C1, water, and brine, then dried over magnesium sulfate, filtered, and evaporated. The product is purified by silica gel
chromatography.
Example 26
[0250] A solution of sodium ascorbate (1.5 M in water) is added to a blue mixture of 0.1 M CuS04 in water and 50 mM TBTA in dimethylsulfoxide (DMSO), and the resulting colorless solution is immediately added to a solution of 1 equivalent of the PEGylated alkyne dendrimer DAH[Lys]4[NL]8[mPEG50oo]8[alkyne]8 and 10 equivalents of azido-linker-SN38 in 1 :2 water/DMSO. The mixture is allowed to react overnight, and is then dialyzed against water until HPLC analysis reveals complete removal of the uncoupled alkynyl-linker-SN38. The dialysate is lyophilized to provide the product.
Example 27
N-fmPEG^jon-Qxycarbonyn-L-Azidonorleucine Succinimidyl Ester
[0251] This example supports dendrimer assembly via dendrons in Example 28, method B.
[0252] Step 1. A solution of commercial monomethoxypolyethylene glycol succinimidyl carbonate (mw = 5,000) in acetonitrile (1 equivalent) is added to a solution of L-azidonorleucine (1 equivalent) in 0.1 M aqueous NaHC03. After stirring at ambient temperature for 2 hrs, the mixture is acidified with CF3C02H and evaporated to dryness to provide N-(mPEG5ooo- oxycarbonyl)-L-azidonorleucine. The crude product is purified by precipitation from THF by addition of methyl tert-butyl ether.
[0253] Step 2. A solution of the N-(mPEG5ooo-oxycarbonyl)-L-azidonorleucine from Step 1 in dry acetonitrile is treated with N,N'-disuceinimidyl carbonate (1.2 equivalents) and
4-(dimethylamino)pyridine (0.1 equivalent) until gas evolution ceases and a clear solution is obtained. Methyl tert-butyl ether is added to precipitate the PEGylated product, which is collected by vacuum filtration and dried.
[0254] Examples 28 and 29 describe preparation of dendrimers and coupling to alkynyl linker drugs, respectively.
Example 28
Solid-phase Synthesis of Dendrimers
Gly[Lys]7[NL]8[mPEG50oo]8[N3]8
Method A. Linear synthesis
[0255] Step 1. H-Lys-Gly-Resin. TentaGel resin loaded with Fmoc-Glycine is deprotected by treatment with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine. The resin is treated with a DMF solution containing a 4-fold excess of HBTU-activated Na,NE-bis(9-fluorenylmethoxycarbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH) and HOBt, and a 4-fold excess of Ν,Ν-diisopropylethylamine is added. Coupling proceeds for
4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
[0256] Step 2. [H-Lys]2-Lys-Gly-Resin. The resin from Step 1 is treated with a DMF solution containing an 8-fold excess of HBTU-activated Na,Ne-bis(9- fluorenylmethoxycarbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH) and HOBt, and an 8-fold excess of N,N-diisopropylethyIamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
[0257] Step 3. [[H-Lys]2-Lys]2-Lys-Gly-Resin. The resin from Step 2 is treated with a DMF solution containing a 4-fold excess of HBTU-activated Na,N8-bis(9-fiuorenylmethoxycarbonyl)- L-lysine (Fmoc-Lys(Fmoc)-OH) and HOBt, and an 4-fold excess of N,N-diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
[0258] Step 4. [[[ANL]2-Lys]2-Lys]2-Lys-Gly-Resin. The resin from Step 3 is treated with a DMF solution containing a 4-fold excess of Fmoc-L-azidonorleucine succinimidyl ester, and an 4-fold excess of Ν,Ν-diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is treated with 20% piperidine in DMF, followed by washing the resin 3x with DMF to remove excess piperidine.
[0259] Step 5. [[[mPEG5000-ANL]2-Lys]2-Lys]2-Lys-Gly-Resin. The resin from Step 4 is treated with a DMF solution containing a 4-fold excess of monomethoxypolyethylene glycol propionate succinimidyl ester (mw 5000), and an 4-fold excess of Ν,Ν-diisopropylethylamine is added. Coupling proceeds for 4 hours. After washing the resin 3x with DMF, the resin is washed 3x with dichloromethane and dried under vacuum.
[0260] Step 6. The dendrimer is cleaved from the resin by treatment with CF3C02H for 4 hours, followed by removal of the resin by filtration and evaporation to provide the dendrimer product.
Method B. Semi-convergent synthesis.
[0261] Steps 1-3 are as in Method A above.
[0262] Step 4. [[[mPEG50o0-ANL]2-Lys]2-Lys]2-Lys-Gly-Resin. The resin from Step 3 is treated with a DMF solution containing a 4-fold excess of N-(mPEG5ooo-oxycarbonyl)-L- azidonorleucine succinimidyl ester, and an 4-fold excess of Ν,Ν-diisopropylethylamine is added. Coupling proceeds for 4 hours.
[0263] Step 5. The dendrimer is cleaved from the resin by treatment with CF3C02H for 4 hours, followed by removal of the resin by filtration and evaporation to provide the dendrimer product.
Example 29
Solid-phase Conjugation of Alkvnyl-Linker-Drugs and Dendrimers
Gly [Lys] 7 [NL]8 [mPEG5ooo]8 [triazolyl-linker-exendin] g
[0264] A [[[mPEG5ooo-ANL]2-Lys]2-Lys]2-Lys-Gly-Resin from Example 28 (containing 8 equivalents of azido groups) and an alkynyl-linker-exendin compound (for example, that from Example 15 above) (10 equivalents) in 2:1 DMSO/water is treated with a fresh catalyst mixture prepared by addition of sodium ascorbate (1.5 M in water) to a mixture of 0.1 M CuS04 in water and 50 mM TBTA in dimethylsulfoxide (DMSO). The conjugation mixture is shaken overnight, and the resin is filtered and washed 3x with DMSO, 3x with water, 3x with methanol, and 3x with dichloromethane and dried under vacuum. The dendrimer is cleaved from the resin by treatment with CF3C02H for 4 hours, followed by removal of the resin by filtration and evaporation to provide the crude product, which is dialyzed against water to remove small- molecule contaminants. The dialysate is lyophilized to provide the product.
Examples 30-34 describe preparation of Formula d) without linkage to dendrimer using
H Linker for coupling to dendrimer
wherein D = fluorescein as a model for drug
Example 30
Preparation of 6-Azidohexanal
[0265] (1) 6-azido-l-hexanol: a mixture of 6-chloro-l-hexanol (25 g, 183 mmol) and sodium azide (32.5 g, 500 mmol) in 200 mL of water was heated at reflux for 20 h, then cooled to ambient temperature and extracted 3x with ethyl acetate. The combined extracts were washed with brine, dried over MgS04, filtered, and concentrated to yield the product as a pale yellow oil (28.3 g).
[0266] (2) 6-azidohexanal: Solid trichloroisocyanuric acid (4.3 g) was added in small portions to a vigorously stirred mixture of 6-azido-l-hexanol (7.15 g) and sodium bicarbonate (5.0 g) in dichloromethane (100 mL) and water (10 mL). The mixture was stirred for an additional 30 minutes after addition, then filtered through a pad of Celite™. The organic phase was separated and washed successively with sat. aq. NaHC03 and brine, then dried over MgS04, filtered, and concentrated to provide the product (5.8 g), which was used without further purification.
Example 31
†H R*
Preparation of Azidoalcohols of the Formula N3(CH2)n CH CH
[0267] In these reactions, Ra in RaCH3 contains the trigger present in the final product, R1
i.e., Ra = R2— C
H
[0268] A 1.6 M solution of n-butyllithium (3.1 mL, 5.0 mmol) in hexane was added dropwise to a stirred solution of Ra— CH3 (5.0 mmol) in anhydrous tetrahydrofuran (THF) (15 mL) cooled to -78°C. After addition, the cooling bath was removed and the mixture was allowed to warm slowly to 0°C over approximately 30 min. The mixture was then cooled back to -78°C, and 6-azidohexanal from Example 30 (5.5 mmol) was added. After stirring for 15 minutes, the cooling bath was removed and the mixture was allowed to warm. At the point where the mixture became clear, 5 mL of saturated aq. NH4C1 was added and the mixture was
allowed to continue warming to ambient temperature. The mixture was diluted with ethyl acetate and washed successively with water and brine, and then dried over MgS04, filtered, and evaporated to provide the crude product as an oil. Chromatography on silica gel using a gradient of ethyl acetate in hexane provided the purified products.
[0269] Compounds prepared according to this method include:
l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptanol
(Ra-CH3 = CF3 methyl sulfone);
l-(4-chlorophenylsulfonyl)-7-azido-2-heptanol (R-CH3 = 4-chlorophenyl
methyl sulfone);
l-(phenylsulfonyl)-7-azido-2-heptanol (R-C¾ = phenyl methyl sulfone);
l-(4-methylphenylsulfonyl)-7-azido-2-heptanol (R-CH3 = 4-methylphenyl
methyl sulfone);
l-(4-methoxyphenylsulfonyl)-7-azido-2-heptanol (R-CH3 = 4-methoxyphenyl methyl sulfone);
l-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptanol (R-CH3 - 2,4,6-trimethylphenyl methyl sulfone);
l-(morpholinosulfonyl)-7-azido-2-heptanol (R-CH3 = 4-(methylsulfonyl)-morpholine; l-(methanesulfonyl)-7-azido-2-heptanol (R-CH3 = dimethyl sulfone);
l-cyano-7-azido-2-heptanol (R-CH3 = acetonitrile);
l-(morpholinocarbonyl)-7-azido-2-heptanol (R-CH3 = 4-acetylmorpholine); and l-(9-fluorenyl)-6-azido-l-hexanol ("R-CH3" = fluorene).
Example 32
Preparation of Azido-Linker Chloroformates
[0270] Pyridine (160 μΐ,) was added dropwise to a stirred solution of the azidoalcohol of Example 31 (1.0 mmol) and triphosgene (500 mg) in 15 mL of anhydrous THF. The resulting suspension was stirred for 10 minutes, then filtered and concentrated to provide the crude chloroformate as an oil.
[0271] Compounds prepared according to this method include:
l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptyl chloroformate
l-(4-chlorophenylsulfonyl)-7-azido-2-heptyl chloroformate;
1 -(phenylsulfonyl)-7-azido-2-heptyl chloroformate;
1 -(4-methylphenylsulfonyl)-7-azido-2-heptyl chloroformate;
1 -(4-methoxyphenyIsulfonyl)-7-azido-2-heptyl chloroformate;
l-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptyl chloroformate;
1 -(morpholinosulfonyl)-7-azido-2-heptyl chloroformate;
1 -(methanesulfonyl)-7-azido-2-heptyl chloroformate;
1 -cyano-7-azido-2-heptyl chloroformate;
1 -(morpholinocarbonyl)-7-azido-2-heptyl chloroformate; and
1 -(9-fluorenyl)-6-azido- 1 -hexyl chloroformate.
[0272] Also prepared according to this method was 6-azidohexyl chloroformate, starting from 6-azidohexanol.
Example 33
Preparation of Azido-Linker Hvdroxysuccimidyl (HS) Carbonates
[0273] A solution of the chloroformate from Example 32 in 15 mL of dry THF was treated successively with N-hydroxysuccinimide (350 mg) and pyridine (250 μΕ) for 10 minutes. The mixture was then concentrated, and the residue was redissolved in ethyl acetate. After washing with 0.1 N HC1, water, sat. NaHC03, water, and brine, the solution was dried over MgS04, filtered, and evaporated. In some cases, the HS carbonate spontaneously crystallized, and was recrystallized from ethyl acetate/hexane. In other cases, the crude HS carbonate was first chromatographed on silica gel using a gradient of ethyl acetate in hexane, followed by crystallization. All compounds were crystalline with the exception of that obtained from 1- (methanesulfonyl)-7-azido-2-heptanol.
[0274] Compounds prepared according to this method include:
0-[l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate;
0-[l-(4-chlorophenylsulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate;
O- [ 1 -(phenylsulfonyl)-7-azido-2-heptyl] -O ' -succinimidyl carbonate;
0-[l-(4-methylphenylsulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate;
0-[ 1 -(4-methoxyphenylsulfonyl)-7-azido-2-heptyl] -O ' -succinimidyl carbonate;
0-[l-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate; O- [ 1 -(morpholinosulfonyl)-7-azido-2-heptyl] -0 ' -succinimidyl carbonate;
0-[l-(methanesulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate;
0-[l -cyano-7-azido-2-heptyl]-0'-succinimidyl carbonate;
0-[l -(morpholinocarbonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate; and
0-[l -(9-fluorenyl)-6-azido- 1 -hexyl]-0'-succinimidyl carbonate.
[0275] Also prepared according to this method was 0-[6-azidohexyl]-0'-succinimidyl carbonate, starting from 6-azidohexyl chloroformate.
Example 34
Preparation of Linked Fluoresceins of the Formula
[0276] A solution of 25 mM azide-linker-HS of Example 33 in DMSO (100 μΓ) was added to a 10 mg/mL solution of 5-(aminoacetamido)fluorescein (Invitrogen) in DMSO (1 15 μί). After 1 h at ambient temperature, the mixture was analyzed by reversed-phase HPLC, indicating complete consumption of azide-linker-HS and formation of a single linked fluorescein product. The solutions were used without purification.
Examples 35-42 describe the preparation of PEGylated dendrimer,
Example 35
Preparation of Boc-Lvs Boc)-HEGA
[0277] A solution of 20-azido-3, 6,9, 12, 15,18-hexaoxaicosan-l -amine (362 mg, 1.0 mmol) in
THF (5 mL) was added to a solution of Na,Ne-bis(te^butoxycarbonyl)-L-lysine 4-nitrophenyl ester {Boc-Lys(Boc)-OPNP} (580 mg, 1.2 mmol) in THF (5 mL) followed by Et3N (288 μΐ,, 209 mg, 2.1 mmol). The resulting mixture was allowed to stir for 2 h, then 1 N NaOH (3 mL) was added. Stirring was continued for 1.5 h, then the THF was removed under vacuum. The resulting suspension was extracted with ethyl acetate. The extract was washed with 0.5 N NaOH and water, then dried over MgS04, filtered and concentrated to dryness to give the product (610 mg) as a pale yellow oil.
Example 36
Preparation of H-Lys-HEGA
[0278] Trifluoroacetic acid (5 mL) was added to a solution of Boc-Lys(Boc)-HEGA
(Example 35, 600 mg, 0.88 mmol) in dichloromethane (5 mL). The resulting mixture was allowed to stir at room temperature for 1.5 h. The mixture was then concentrated under reduced pressure to give 1310 mg of residue estimated to comprise 890 mg of free trifluoroacetic acid. This material was dissolved in methanol (25 mL) and treated with 24 mL of methanol-washed Dowex® (Dow Chemical Co.) Monosphere™ 550A for 0.5 h. The resin was removed by filtration, and the filtrate was concentrated to dryness to give the product (420 mg) as a viscous clear oil.
Example 37
Preparation of [Boc-LysfBoc^Lys-HEGA
[0279] To a solution of H-Lys-HEGA (Example 36, 400 mg, 0.836 mmol) in THF (6 mL) was added Boc-Lys(Boc)-OPNP (938 mg, 2.0 mmol) followed by Et3N (466 μί, 338 mg, 3.3 mmol). The resulting mixture was allowed to stir for 6.5 h, then 1 N NaOH was added (4 mL). Stirring was continued for 4 h. The resulting suspension was diluted with ethyl acetate (100 mL). The extract was washed with 0.5 N NaOH, then with 0.1 N HC1, then water, then dried over MgS04 and concentrated to dryness to give the product (722 mg, 76%) as sticky white solid.
Example 38
Preparation of ["Lys^Lys-HEGA
[0280] Trifluoroacetic acid (3 mL) was added to a solution of [Boc-Lys(Boc)]2Lys-HEGA (Example 37, 499 mg, 0.44 mmol) in dichloromethane (3 mL). The resulting mixture was allowed to stir at room temperature for 3.5 h. The mixture was then concentrated under reduced pressure to give 1 120 mg of residue estimated to comprise -800 mg of free trifluoroacetic acid.
This material was dissolved in methanol (20 mL) and treated with 20 mL of methanol washed Dowex® (Dow Chemical Co.) Monosphere™ 550A for 20 min. The resin was removed by filtration, and the filtrate was concentrated to dryness to give the product (323 mg) as a pale yellow oil.
Example 39
Preparation of [Boc-LysfBoc^JLvs^Lys-HEGA
[0281] To a solution of [Lys]2Lys-HEGA (Example 38, 300 mg, 0.41 mmol) in DMF (10 mL) was added Boc-Lys(Boc)-OPNP (916 mg, 2.0 mmol, 4.9 equiv) followed by Et3N (455 μΐ,, 330 mg, 3.3 mmol). The resulting mixture was allowed to stir for 23 h, then 1 N NaOH was added (4 mL). Stirring was continued for 3 h. The resulting suspension was diluted with ethyl acetate (150 mL). The extract was washed with 0.5 N NaOH, then with 0.1 N HC1,
then water, then brine, then dried over MgS04 , filtered and concentrated to dryness to give the product (730 mg, 87%) as white solid.
Example 40
[0282] Trifluoroacetic acid (4 mL) was added to a solution of [Boc-Lys(Boc)]4[Lys]2Lys- HEGA (Example 39, 200 mg) in dichloromethane (4 mL). The resulting mixture was allowed to stir at room temperature for 1.5 h. The mixture was then concentrated under reduced pressure to give 786 mg of residue estimated to comprise -664 mg of free trifluoroacetic acid. This material was dissolved in methanol (30 mL) and treated with 20 mL of methanol washed Dowex® (Dow Chemical Co.) Monosphere™ 550A (strong base anion exchange resin) for
30 min. The resin was removed by filtration, and the filtrate was concentrated to dryness to give the product (120 mg) as a sticky white solid.
Example 41
fLysl4fLyslzJLvs-HEG-rmPEG4n»j1
[0283] A 1.8 mM solution of [Lys]4[Lys]2Lys-HEGA (Example 40) in methanol (1.1 mL, 2 μπιοΐ) was mixed with a 1.2 mM solution of linear mPEG40kD-DBCO (DBCO is a linking group, see Example 58) in methanol (0.825 mL, 1 μηιοΓ). The resulting mixture was allowed to stir for 6 h, dialyzed against methanol (Spectra/Por® (Spectrum Laboratories) 2 membrane;
12-14 kDa-cutoff), and concentrated to dryness to give the PEGylated dendrimer (39 mg) as a white solid.
Example 42
Preparation of pBCO-Lvsq^BCO^fLvsl^Lvs-HEG-fmPEGdn jl
[0284] A 9.1 mM solution of DBCO-NHS (Click Chemistry Tools, 1660 μΐ, 15.1 μιηοΐ) was added to a solution of [Lys]4[Lys]2Lys-HEG-[mPEG40kD] (Example 41 , 39 mg, 0.946 μηιοΐ) in THF (1.9 mL). After stirring for 24 h the reaction mixture was diluted with methanol (4 mL), and dialyzed twice against methanol using a Spectra Por® (Spectrum Laboratories) 2 membrane (12-14 kDa-cutoff). The mixture was filtered through a 0.2 μη filter to remove precipitated materials and further dialyzed twice against 50% methanol followed by methanol. Some precipitate formed during the 50% methanol dialysis and redissolved during the methanol dialysis. The solution was concentrated to dryness to give the product (30.1 mg).
Example 43
Preparation of Fluorescein-Dendrimer-PEG con ufiates
R1
wherein Ra is R2— C wherein R1 = phenyl-S02CH2; R1 = (4-chlorophenyl)-S02CH2
H
= mo holino-S02CH2 , and R is H.
[0285] A mixture of acetic acid in methanol (5% v/v, 0.83 mL), a 0.81 mM methanol solution of [DBCO-Lys(DBCO)]4[Lys]2Lys-HEG-[mPEG40kD] (Example 42, 0.170 mL, 0.17 μπιοι), and a 11.7 mM solution of the linked fluorescein azide of Example 34 in DMSO (0.140 mL, 1.64 μπιοΐ) was allowed to sit at ambient temperature for 70 h. The presence of excess azide was verified by size exclusion HPLC. The reaction mixture was dialyzed once against 50% methanol + 0.5% v/v acetic acid using a Spectra Por® (Spectrum Laboratories) 2 membrane (12-14 kDa-cutoff), then three times against methanol + 0.2% v/v acetic acid. The dialysis mixture was concentrated to dryness to give the product as a dark orange-yellow solid.
[0286] Compounds prepared according to this method thus include PEGylated dendrimers
R1
wherein Ra is R2— C wherein R1 = phenyl-S02CH2; R1 = (4-chlorophenyl)-S02CH2, and
H
1 9
R = morpholino-S02CH2 , and R is H. Thus the product is of the formula
wherein D is modeled by fluorescein
Example 44
Fluorescein Release from Fluorescein-Dendrimer-PEG conjugates
[0287] The fluorescein-dendrimer-PEG conjugates of Example 43 were dissolved in 0.1 M bicine, pH 8.5, 37°C to a concentration of ~20 μΜ total fluorescein. Aliquots were periodically removed and analyzed by HPLC to determine released 5-(aminoacetamido)fluorescein. Results are shown in Figure 7.
Examples 45-46 describe synthesis of a more complex dendrimer.
Example 45
Preparation of [Boc-Lys(Tjoc [Lys] rLys1?Lys-HEGA
[0288] To a suspension of [Ly s] 4 [Ly s] 2Ly s-HEGA (Example 40, 75 mg, 0.060 mmol) in 1 : 1 DMF:DMSO (12 mL) was added Et3N (0.133 mL, 96.6 mg, 0.955 mmol) followed by Boc- Lys(Boc)-OPNP (270 mg, 0.578 mmol). The resulting mixture was allowed to stir for 25 h, during which time all of the starting material became soluble. Next 1 N NaOH (2 mL) was added to the reaction mixture for 3 h. The reaction mixture was then diluted with 200 mL of ethyl acetate, washed with 0.5 N NaOH (200 mL in small portions), then with 0.1 N HC1 (200 mL in small portions), then with water, and finally brine. The extract was dried over MgS04 and concentrated to dryness to give the product (205 mg) as a white solid.
Example 46
[0289] Trifluoroacetic acid (8 mL) was added to a solution of protected dendrimer [Boc- Lys(Boc)]8[Lys]4[Lys]2Lys-HEGA (Example 45, 98 mg, 0.025 mmol) in DCM (8 mL). The resulting mixture was allowed to stir for 2 h at room temperature then concentrated under vacuum to give 360 mg of residue. The residue was dissolved in methanol (15 mL) and treated methanol-washed Dowex® (Dow Chemical Co.) Monosphere™ 550A (10 mL) for 20 minutes. The resin was removed by filtration and the filtrate concentrated to provide the product.
Examples 47-56 describe preparation of fluorescein as model drug coupled to PEGylated
dendrimers.
Example 47
Preparation of N-ffert-butoxycarbonyiVN' -(2,4-dinitrophenyl)- 1 ,4-butanediamine
[0290] To a stirred solution of N-Boc-l,4-butanediamine (0.400 g; 2.12 mmol) and 1-fluoro- 2,4-dinitrobenzene (280 xL; 2.23 mmol) in anhydrous tetrahydrofuran (THF) (10 mL) was added triethylamine (886 ί; 6.36 mmol). The reaction solution was stirred at ambient temperature for 17 hours and was then concentrated. The residue was dissolved in ethyl acetate and washed with water, saturated NaHC03 and saturated NaCl. The organic solution was dried
over MgS04 and concentrated to give a yellow oil (1.12 g). Purification using a Thomson Instruments Single StEP™ silica gel column (25 g) and eluting with 20% ethylacetate/80% hexanes followed by 50% ethyl acetate/50%) hexanes produced the product as a yellow oil (0.751 g, 100%). 1H NMR (d6-DMSO) 51.36 (9H, s), 1.44 (2H, m) 1.58 (2H, m), 2.93 (2H, m), 3.46 (2H, m), 6.82 (1H, t, J = 5.6 Hz), 7.23 (1H, d, J = 9.5 Hz), 8.27 (1H, dd, J = 2.7 Hz, J = 9.6 Hz), 8.85 (2H, m).
Example 48
Preparation of Boc-Lys(Boc)-DAB-DNP
[0291] The Boc protected amine (0.751 g; 2.12 mmol) was dissolved in anhydrous dichloromethane (10 mL) and trifluoroacetic acid (10 mL) was added. The resulting solution was stirred at room temperature for 2 hours and was concentrated. Ethyl acetate was added and the mixture was concentrated. The brown solid thus obtained was dissolved in anhydrous Ν,Ν-dimethylformamide (DMF, 4 mL) and triethylamine (740 ί; 5.31 mmol) was added. To this solution was added a solution of Boc-Lys(Boc)-OPNP (1.19 g; 2.55 mmol) in anhydrous DMF (4 mL) and the solution stirred at room temperature for 18 hours. It was then poured into water (100 mL) and extracted with ethyl acetate (3x). The combined extracts were washed with water (2x) and saturated NaCl and were dried over MgS04 and concentrated to give an orange oil (2.00 g). Purification using a Thomson Instruments Single StEP™ silica gel column (25 g) and eluting with 50% ethylacetate/50% hexanes followed by 70% ethyl acetate/30%) hexanes produced Boc-Lys(Boc)-DAB-DNP as a yellow solid (0.985 g, 79%). 1H NMR (DMSO) 51.34-1.61 (28H, m), 2.82 (2H, m), 3.07 (2H, m), 3.47 (2H, m), 3.77 (1H, m), 6.74 (2H, m), 7.23 (1H, d, J = 9.7 Hz), 7.78 (1H, t, J = 6.0 Hz), 8.24 (1H, dd, J - 2.8 Hz, J = 9.5 Hz),
8.85 (2H, m).
Example 49
[0292] Boc-Lys(Boc)-DAB-DNP (0.500 g; 0.858 mmol) was dissolved in anhydrous dichloromethane (5 mL) and trifluoroacetic acid (5 mL) was added. The resulting solution was stirred at room temperature for 2 hours and was concentrated. Ethyl acetate was added and the mixture was concentrated. The brown oil thus obtained was dissolved in anhydrous DMF (5 mL) and triethylamine (598 μΕ; 4.29 mmol) added. To this solution was added a solution of Boc-Lys(Boc)-OPNP (0.963 g; 4.29 mmol) in anhydrous DMF (5 mL) and the solution stirred at room temperature for 18 hours. Additional triethylamine (188 μΕ; 1.35 mmol) was added and the reaction stirred for a further 3 hours. It was then slowly added to ice-water (250 mL) and extracted with ethyl acetate (3x). The combined extracts were washed with water (2x) and saturated NaCl and were dried over MgS04 and concentrated to give an orange oil (0.844 g). Purification using a Thomson Instruments Single StEP™ silica gel column (25 g) and eluting with 50% ethylacetate/50% hexanes followed by 100% ethyl acetate then 2% methanol/98% ethyl acetate and finally 5% methanol/95%) ethyl acetate produced [Boc-Lys(Boc)]2Lys-DAB- DNP as a yellow solid (0.367 g, 42%).
Example 50
Preparation of l¾oc-Lvs(BocYUrLvsl7Lvs-DAB-DNP
[0293] [Boc-Lys(Boc)]2Lys-DAB-DNP (0.670 g; 0.645 mmol) was dissolved in anhydrous dichloromethane (7 mL) and trifluoroacetic acid (7 mL) added. The resulting solution was stirred at room temperature for 2 hours and was concentrated on the roto-vap. Ethyl acetate was added and the mixture was concentrated. The brown oil thus obtained was dissolved in anhydrous DMF (7 mL) and triethylamine (1150 μΕ; 8.25 mmol) added. To this solution was added a solution of Boc-Lys(Boc)-OPNP (1.45 g; 3.10 mmol) in anhydrous DMF (7 mL). A further aliquot of triethylamine (400 μί; 2.87 mmol) was added and the solution stirred at room temperature for 18 hours, at which time HPLC analysis showed the reaction to be complete. The reaction mixture was added to water (160 mL) and extracted with ethyl acetate (3x). The combined extracts were washed with water (2x), 1M Na2C03, and saturated NaCl and were dried over MgS04 and concentrated to give a yellow solid (1.68 g). Purification using a Thomson Instruments Single StEP™ silica gel column (40 g) and eluting 100% ethyl acetate then 5% methanol/95% ethyl acetate and finally 7.5% methanol/92.5% ethyl acetate produced [Boc-Lys(Boc)]4[Lys]2Lys-DAB-DNP as a yellow solid (0.600 g, 48%). 1H NMR (DMSO-i¾) 1.15-1.59 (1 17H, m, Calc 118H), 2.73-2.98 (17H, m, Cole. 18H), 3.48 (2H, rn), 3.81 (4H, m), 3.99 (3H, m), 6.66 (2H, d, J = 8.2 Hz), 6.73 (4H, br. s), 6.89 (2H, d, J = 7.0 Hz), 7.22 (1H, d, J = 9.8 Hz), 7.65-7.7.95( 7H, m), 8.23 (1H, dd, J = 2.7 Hz, J = 9.5 Hz), 8.84 (2H, m). MS (ESI+) found 977.12 (M+2H)/2+, data deconvoluted to give mw 1952.24; calc for
C92H162N18027 1951.19.
Example 51
[0294] A solution of [Boc-Lys(Boc)]4[Lys]2Lys-DAB-DNP (Example 50; 0.2 g, 0.1 mmol) in trifluoroacetic acid (2 mL) was stirred at room temperature for 6.5 hours. The solution was concentrated and then ethyl acetate was added and the solution, again, concentrated. The residue (0.505 g) was dissolved in methanol (15 mL) and methanol washed Dowex® (Dow Chemical Co.) 550A Monosphere™ resin (-10 mL) was added. The mixture was stirred gently for 1 hour and then filtered. The resin was washed with methanol and the filtrate concentrated to give [Lys]4[Lys]2Lys-DAB-DNP as a dark orange oil (0.092 g).
Example 52
Preparation of rBoc-Lvs(Tfa^[Lvs]4rLvs1?Lvs-DAB-DNP
[0295] A solution of Boc-Lys(Tfa)-OSu (0.366 g, 0.83 mmol) in DMF (1 mL) was added to a solution of [Lys] [Lys]2Lys-DAB-DNP (Example 51 ; 0.10 g, 86.6 μηιοΓ) and triethylamine (97 μΐ,, 696 μηιοΐ) in DMF (3 mL). The solution was stirred at ambient temperature for 1.5 hours and was added slowly to water (25 mL) with stirring. The solid was collected by filtration and washed with water and acetonitrile. The solid was re-suspended in acetonitrile and the mixture stirred for 1 hour. The solid was collected by filtration, washed with acetonitrile and dried to give the product as a yellow solid (0.161 g; 50%).
Example 53
Preparation of rBoc-LvslgfLvs Lys Lys-DAB-DNP
[0296] The dendrimer [Boc-Lys(Tfa)]8[Lys]4[Lys]2Lys-DAB-DNP (Example 52; 0.025 g; 6.67 μιηοι) was dissolved in methanol (1 mL) and 1 M sodium hydroxide (160 μί; 160 mmol) was added. The reaction was kept at 37°C for 1.5 hours and was then diluted with water (2 mL). This solution was loaded onto a 1-g Bond-Elut™ (Varian) C1 solid phase extraction column and the column was washed with water and water + 0.1% TFA. The product was eluted with 1 : 1 water/acetonitrile + 0.1% TFA and the product containing fractions pooled and diluted with methanol. This solution was treated with methanol-washed Dowex® (Dow Chemical Co.) 55 OA Monosphere™ resin for 1 h and was then filtered and concentrated to give the product as an orange glassy solid (0.022 g).
Example 54
Preparation of PEG-Coated Dendrimer
rBoc-LvsimPEG^lsfLvs^fLvs^Lvs-DAB-DNP
[0297] A solution of [Boc-Lys]8[Lys]4[Lys]2Lys-DAB-DNP (Example 53; 0.020 g;
7.4 μη οι) and triethylamine (16.5 μί; 118 μηιοΓ) in Ν,Ν-dimethylformamide (1.5 mL) was treated with 5-kDa monomethoxy-polyethylene glycol succinimidyl carboxymethyl ester (JenKem technology, 0.591 g; 118 μηαοΐ) and the solution stirred at ambient temperature for 22 hours. The reaction mixture was diluted with water (10 mL) and dialysed (MWCO
12-14 kDa) against water (1 L) for 24 hours with a change of solvent after 6 hours. The water
was replaced with methanol (1 L) and the dialysis continued for a further 6 hours. The retentate was concentrated and the crude product was taken up in tetrahydrofuran (2.5 mL). This solution was added slowly to a stirred solution of methyl tert-butyl ether (25 mL). After 1 hour the precipitated solid was collected by filtration, washed with methyl tert-butyl ether and dried to give the PEG-coated dendrimer as a yellow solid (0.463 g).
Example 55
Preparation of DBCO-activated PEG-Coated Dendrimer
[DBCO-Lvs(mPEG^1g|Lvsl4[Lvs1?Lvs-DAB-D P
[0298] Trifluoroacetic acid (0.5 mL) was added to a solution of [Boc-Lys(mPEG5kD)]s- [Lys]4[Lys]2Lys-DAB-DNP (0.1 g) in 0.5 mL of dichloromethane. After 6 h, the mixture was concentrated to give a yellow oil (0.271 g). This was dissolved in methanol and treated with 10 mL of methanol-washed Dowex® (Dow Chemical Co.) 550 A Monosphere™ resin for 1 h and was then filtered and concentrated to give the product as an pale yellow solid (68 mg). Analysis by UV absorbance indicated a total of 0.752 μηιοΐ of DNP.
[0299] To a stirred solution of DAB[DNP][Lys]g[a-Boc]8[8-NHC(0)PEG5K]8 (0.752 μιηοΐ) in acetonitrile (1 mL) was added triethylamine (2 μί; 14.7 μηιοΐ) and DBCO-NHS (0.006 g; 12.5 μηιοΐ) and the solution stirred at ambient temperature for 6.5 hours. To the reaction solution was then added 10 mL of 10 mM taurine in HEPES pH 7.0 and the solution stirred for 17 hours. This solution was dialysed (MWCO 12-14K) against water (1L) for 7.5 hours with a
change of solvent after 4.5 hours. The water was replaced with methanol (1L) and the dialysis continued for 17 hours. The retentate was concentrated and the crude product was taken up in tetrahydrofuran (1.5 mL) and slowly added to a stirred solution of methyl t-butyl ether (15 mL) After 1 hour the solid was collected, washed with methyl t-butyl ether and dried to give the product (0.057 g) as a pale yellow solid.
Example 56
Preparation of Fluorescein-Conjugated to PEG-Coated Dendrimer
[0300] To 500 μΐ, of a 0.2 mM solution of [DBCO-Lys(mPEG5kD)MLys]4[Lys]2Lys-DAB- DNP in tetrahydrofuran was added 103 μΐ, of the 11.7 mM linked fluorescein azide solution in DMSO prepared in Example 34. The reaction solution was incubated in the dark for 17 hours at which time HPLC analysis indicated complete consumption of the fluorescein azide. A further
50 μΐ aliquot of the linked fluorescein azide was added and the reaction incubated in the dark for 24 hours. HPLC analysis now showed residual fluorescein azide. The reaction solution was diluted with water (2 mL) and was dialysed (MWCO 12-14K) against 10 mM sodium acetate, pH 5, (250 mL) for 22 hours in the dark. The buffer was replaced with methanol (250 mL) and the dialysis continued for 7 hours with a change of solvent after 4 hours. The retentate was concentrated on the roto-vap. Compounds include those wherein R1 is phenylsulfonyl, 4-chlorophenylsulfonyl and morpholinosulfonyl.
Example 57
Release of Fluorescein from PEG-Coated Dendrimer Conjugates
[0301] The fluorescein-dendrimer-PEG conjugates of Example 56 were dissolved in 0.1 M bicine, pH 8.5, 37°C to a concentration of -20 μΜ total fluorescein. Aliquots were periodically removed and analyzed by HPLC to determine released 5-(aminoacetamido)fluorescein. Results are shown in Figure 8.
Example 58
Preparation of mPEG4nkn-DBCO
[0302] A solution of 40-kDa monomethoxy polyethylene glycol amine hydrochloride (Jen em Technology, 2 g, 50 μιηοΐ), triethylamine (20 μί), and 6-aza-5,9-dioxo-9-(l,2- didehydrodibenzo[6,/jazocin-5(6H)-yi)nonanoic acid succinimidyl ester ("DBCO-NHS", Click Chemistry Tools, Macon, GA) (50 mg, 100 μηιοΐ) in 25 mL of THF was stirred for 24 h at
ambient temperature. The product was precipitated by addition of the reaction mixture to 100 mL of methyl tert-butyl ether (MTBE). The precipitate was collected by vacuum filtration, then redissolved in THF and the precipitation in MTBE was repeated to provide 1.98 g of product free of residual DBCO-NHS as determined by HPLC analysis.
Example 59
Kinetics of Release
[0303] Rates of release of the drug from the conjugates of the invention can readily be determined by methods known in the art including chromatographic methods, such as HPLC. Where, for example, a fluorescent marker is used as a model system for the drug, the
fluorescence attributable to freed fluorescent compound is readily determined as compared to fluorescence emitted by the conjugate.
[0304] The in vivo release of drug from the conjugates of the invention may be measured by determining the pharmacokinetics of the conjugates as compared with the pharmacokinetics of a non-releasable conjugate of the same size. . Such data are preferably obtained in rats as compared to mice as they exhibit more favorable clearance rates for the high molecular weight conjugates of the invention.
[0305] In more detail, the conjugates are administered to a model subject such as a rat, for example, by intravenous administration, and blood samples are periodically taken and plasma isolated. The level of conjugate in the plasma as a function of time is then determined. This may be done by chromatographic separation (for example, HPLC analysis after deproteinization coupled to UV, fluorescence, or mass spectrometric detection), or in appropriate cases by a direct assay such as ELISA, bioactivity, or fluorescence. As noted above, macromolecular conjugates adhere to a one-compartment model, conjugates of the invention can disappear from the plasma by one of two mechanisms: release of drug from the conjugate, and clearance of the intact conjugate (e.g., by renal filtration). The rate of loss of a releasable conjugate from the plasma is thus the sum of the rates of loss by release of the drug and by clearance of the conjugate. In contrast, the rate of loss of a non-releasable conjugate is just the rate of clearance of the conjugate from the plasma, since no drug is released. Thus, the rate of drug release from a conjugate of the invention can be calculated as the difference in rates of loss of the releasable conjugate from that of a corresponding non-releasable conjugate. This may be done by directly taking the difference in rates or this can be calculated from the slope of a plot of In (R/N) versus
time, where R is the concentration of releasable conjugate and N is the concentration of non- releasable conjugate, as shown in Figures 9a and 9b. As shown in panel a, the raw data simply show the logarithm (In) of the concentration of various conjugates and of a stable conjugate as a function of time. Panel b shows the difference in release rates of various releasable conjugates which are obtained by the calculation described above. The stable conjugate, of course, shows zero release rate whereas release rates of drug from various embodiments of the trigger for release in sample conjugates are shown.
[0306] Figure 10 shows that the variation of the rate constant as a function of the nature of the trigger in vitro and in vivo follow the same pattern which correlates as expected with the Hammett constants associated with the trigger.
Claims
1. A dendrimer coupled to a multiplicity of substituents of the formula
m = 0-l
at least one or both R1 and R2 is independently CN; N02;
optionally substituted aryl;
optionally substituted heteroaryl;
optionally substituted alkenyl;
optionally substituted alkynyl;
COR3 or SOR3 or S02R3 wherein
R is H or optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted;
heteroaryl or heteroarylalkyl, each optionally substituted; or OR or NR2 wherein each R is independently H or optionally
substituted alkyl;
SR4 wherein
R4 is optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted; or
heteroaryl or heteroarylalkyl, each optionally substituted;
wherein R1 and R2 may be joined to form a 3-8 member ring; and
wherein one and only one ofR1 and R may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
D is a residue of a drug or prodrug coupled through O, S, or N;
Y is absent and X is O or S; or
Y is NBCH2 and X is O; wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
wherein said coupling to the dendrimer is through any of R', R2, R5 or B.
2. The dendrimer of claim 1 wherein when X is O or S and Y is absent said
1 9 ^
dendrimer is coupled to R , R , R or B through one or more connecter residues.
3. The dendrimer of claim 1 wherein said dendrimer has a minimum G of 3 wherein G is the number of generations included in the dendrimer where the core of the dendrimer is assigned G = 0.
4. The dendrimer of claim 1 wherein the dendrimer is poly L-lysine (PLL) or polyamidoamine (PAMAM).
5. The dendrimer of any of claims 1-4 which further comprises a multiplicity of coupled inert protective polymers.
6. The dendrimer of claim 5 wherein the protective polymer is polyethylene glycol (PEG).
7. The dendrimer of any of claims 1-4 wherein the drug is a peptide, a nucleic acid or a small molecule.
8. The dendrimer of any of claims 1-4 wherein one of R1 and R is CN, or
1 9
wherein at least of one R and R comprises phenyl or phenylene.
1 9
9. The dendrimer of any of claims 1-4 wherein one of R and R is S02R and the other is H.
10. The dendrimer of any of claims 1-4 wherein m is 0.
I l l
11. A dendrimer coupled to a multiplicity of substituents of the formula
m = 0-l
at least one, or both R and R is independently CN; N02;
optionally substituted aryl;
optionally substituted heteroaryl;
optionally substituted alkenyl;
optionally substituted alkynyl;
COR3 or SOR3 or S02R3 wherein
R3 is H or optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted;
heteroaryl or heteroarylalkyl, each optionally substituted; or OR or NR2 wherein each R is independently H or optionally
substituted alkyl;
SR4 wherein
R4 is optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted; or
heteroaryl or heteroarylalkyl, each optionally substituted;
wherein R1 and R2 may be joined to form a 3-8 member ring; and
wherein one and only one ofR1 and R2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted;
Y is absent and X is O or S; or
Y is NBCH2 and X is O;
wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
L is a nucleofuge,
wherein said coupling to the dendrimer is through any of R 1 , R2 , R 5 or B.
12. The dendrimer of claim 1 1 wherein when X is O or S and Y is absent said dendrimer is coupled to R1, R2, R5 or B through one or more connecter residues.
13. The dendrimer of claim 1 1 wherein when X is O or S and Y is absent said dendrimer has a minimum G of 3 wherein G is the number of generations included in the dendrimer where the core of the dendrimer is assigned G = 0.
14. The dendrimer of claim 1 1 wherein the dendrimer is poly L-lysine (PLL) or polyamidoamine (PAMAM).
15. The dendrimer of any of claims 1-14 which is further coupled to a protective inert polymer.
16. The dendrimer of claim 15 wherein the protective polymer is polyethylene glycol (PEG).
17. A method to prepare the dendrimer of claim 1 which method comprises
(a) reacting a compound of the formula
wherein m, R1, R2, R5, X, Y, m and D are as defined in claim 1 ; and
wherein one of R1, R2, R5 and B comprises a functional group or connecter that couples formula (4) to a dendrimer
with a dendrimer under conditions whereby said dendrimer is coupled to
said compound, or
(b) reacting the dendrimer of claim 11 with a drug or prodrug under conditions whereby said drug or prodrug is coupled to said dendrimer.
18. A compound of the formula
M-(J-D)m
wherein M is a dendrimer, D is a drug and J is a joining moiety that releases D by a beta elimination mechanism, wherein m is an integer of at least 8.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11778373.8A EP2571496A4 (en) | 2010-05-05 | 2011-05-05 | Controlled drug release from dendrimers |
US13/696,301 US8703907B2 (en) | 2010-05-05 | 2011-05-05 | Controlled drug release from dendrimers |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US33174910P | 2010-05-05 | 2010-05-05 | |
US61/331,749 | 2010-05-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011140376A1 true WO2011140376A1 (en) | 2011-11-10 |
Family
ID=44904086
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/035403 WO2011140376A1 (en) | 2010-05-05 | 2011-05-05 | Controlled drug release from dendrimers |
Country Status (3)
Country | Link |
---|---|
US (1) | US8703907B2 (en) |
EP (1) | EP2571496A4 (en) |
WO (1) | WO2011140376A1 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011140392A1 (en) | 2010-05-05 | 2011-11-10 | Prolynx Llc | Controlled drug release from solid supports |
WO2013036847A1 (en) | 2011-09-07 | 2013-03-14 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
WO2015051307A1 (en) * | 2013-10-04 | 2015-04-09 | Prolynx Llc | Slow-release conjugates of sn-38 |
WO2015067791A1 (en) | 2013-11-11 | 2015-05-14 | Ascendis Pharma Relaxin Division A/S | Relaxin prodrugs |
US20150352246A1 (en) * | 2013-01-22 | 2015-12-10 | Prolynx Llc | Sealants having controlled degration |
WO2017068051A1 (en) * | 2015-10-21 | 2017-04-27 | Lek Pharmaceuticals D.D. | Peg-based dendron and process for producing the same |
EP3152248A4 (en) * | 2014-06-06 | 2017-11-29 | Starpharma Pty Ltd | Dendrimer-drug conjugates |
US11118016B2 (en) | 2018-07-19 | 2021-09-14 | Starpharma Pty Ltd. | Therapeutic dendrimer |
EP3958888A4 (en) * | 2019-04-26 | 2023-07-12 | Prolynx LLC | Slow-release cytokine conjugates |
US11730836B2 (en) | 2018-01-12 | 2023-08-22 | Prolynx Llc | Synergistic cancer treatment |
EP4037715A4 (en) * | 2019-09-30 | 2023-12-13 | OD Therapeutics Limited | Protein-macromolecule conjugates and methods of use thereof |
US12071517B2 (en) | 2018-07-19 | 2024-08-27 | Starpharma Pty Ltd. | Therapeutic dendrimer |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK2729179T3 (en) | 2011-06-06 | 2020-12-21 | Starpharma Pty Ltd | MACROMOLECULES |
JP2016534064A (en) | 2013-10-22 | 2016-11-04 | プロリンクス エルエルシー | Conjugates of somatostatin and its analogues |
CA2960592A1 (en) * | 2014-09-09 | 2016-03-17 | Ramot At Tel-Aviv University Ltd. | Agrochemical delivery system based on enzyme- or ph- responsive amphiphilic peg-dendron hybrids |
US10869939B2 (en) | 2015-08-03 | 2020-12-22 | Ramot At Tel-Aviv University Ltd. | Delivery system in micellar form having modular spectral response based on enzyme-responsive amphiphilic PEG-dendron hybrid polymers |
SG11201807832VA (en) | 2016-03-16 | 2018-10-30 | Prolynx Llc | Extended release conjugates of exenatide analogs |
JOP20190191A1 (en) | 2017-02-22 | 2019-08-08 | Astrazeneca Ab | Therapeutic dendrimers |
KR20220054368A (en) * | 2019-08-28 | 2022-05-02 | 프로린크스 엘엘시 | Conjugated inhibitors of DNA damage responses |
CN110755639A (en) * | 2019-11-13 | 2020-02-07 | 浙江大学 | Polyethylene glycol-dendritic polylysine/anhydride-cisplatin compound and preparation method and application thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080206183A1 (en) * | 2005-04-28 | 2008-08-28 | Central National De La Recherche Scientifique | Method of Preparing Grafted Polylysine Dendrimers |
WO2009158668A1 (en) * | 2008-06-26 | 2009-12-30 | Prolynx Llc | Prodrugs and drug-macromolecule conjugates having controlled drug release rates |
US20100003316A1 (en) * | 2007-08-06 | 2010-01-07 | Duke University | Methods and systems for treating cell proliferation disorders using plasmonics enhanced photospectral therapy (pepst) and exciton-plasmon enhanced phototherapy (epep) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2512499T3 (en) | 2003-04-08 | 2014-10-24 | Yeda Research And Development Co., Ltd. | Pegylated Reversible Drugs |
-
2011
- 2011-05-05 WO PCT/US2011/035403 patent/WO2011140376A1/en active Application Filing
- 2011-05-05 EP EP11778373.8A patent/EP2571496A4/en not_active Withdrawn
- 2011-05-05 US US13/696,301 patent/US8703907B2/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080206183A1 (en) * | 2005-04-28 | 2008-08-28 | Central National De La Recherche Scientifique | Method of Preparing Grafted Polylysine Dendrimers |
US20100003316A1 (en) * | 2007-08-06 | 2010-01-07 | Duke University | Methods and systems for treating cell proliferation disorders using plasmonics enhanced photospectral therapy (pepst) and exciton-plasmon enhanced phototherapy (epep) |
WO2009158668A1 (en) * | 2008-06-26 | 2009-12-30 | Prolynx Llc | Prodrugs and drug-macromolecule conjugates having controlled drug release rates |
Non-Patent Citations (2)
Title |
---|
BHADRA ET AL.: "PEGylated-poly-I-lysine dendrimers for delivery of Chloroquine phosphate", PROCEEDINGS OF THE 2004 INTEMATIONAL CONFERENCE ON MEMS, NANO AND SMART SYSTEMS (ICMENS '04), XP010836228 * |
See also references of EP2571496A4 * |
Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011140392A1 (en) | 2010-05-05 | 2011-11-10 | Prolynx Llc | Controlled drug release from solid supports |
US11179470B2 (en) | 2011-09-07 | 2021-11-23 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
EP2755691A4 (en) * | 2011-09-07 | 2016-05-25 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
US10398779B2 (en) | 2011-09-07 | 2019-09-03 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
US11454861B2 (en) | 2011-09-07 | 2022-09-27 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
US11181803B2 (en) | 2011-09-07 | 2021-11-23 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
US9649385B2 (en) | 2011-09-07 | 2017-05-16 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
WO2013036847A1 (en) | 2011-09-07 | 2013-03-14 | Prolynx Llc | Hydrogels with biodegradable crosslinking |
US20150352246A1 (en) * | 2013-01-22 | 2015-12-10 | Prolynx Llc | Sealants having controlled degration |
JP2016531895A (en) * | 2013-10-04 | 2016-10-13 | プロリンクス エルエルシー | SN-38 sustained release conjugate |
WO2015051307A1 (en) * | 2013-10-04 | 2015-04-09 | Prolynx Llc | Slow-release conjugates of sn-38 |
US10342792B2 (en) | 2013-10-04 | 2019-07-09 | Prolynx Llc | Slow-release conjugates of SN-38 |
WO2015067791A1 (en) | 2013-11-11 | 2015-05-14 | Ascendis Pharma Relaxin Division A/S | Relaxin prodrugs |
EP3152248A4 (en) * | 2014-06-06 | 2017-11-29 | Starpharma Pty Ltd | Dendrimer-drug conjugates |
US10730999B2 (en) | 2014-06-06 | 2020-08-04 | Starpharma Pty Ltd | Dendrimer-drug conjugates |
WO2017068051A1 (en) * | 2015-10-21 | 2017-04-27 | Lek Pharmaceuticals D.D. | Peg-based dendron and process for producing the same |
US11730836B2 (en) | 2018-01-12 | 2023-08-22 | Prolynx Llc | Synergistic cancer treatment |
US11118016B2 (en) | 2018-07-19 | 2021-09-14 | Starpharma Pty Ltd. | Therapeutic dendrimer |
US11970583B2 (en) | 2018-07-19 | 2024-04-30 | Starpharma Pty Ltd. | Therapeutic dendrimer |
US12071517B2 (en) | 2018-07-19 | 2024-08-27 | Starpharma Pty Ltd. | Therapeutic dendrimer |
EP3958888A4 (en) * | 2019-04-26 | 2023-07-12 | Prolynx LLC | Slow-release cytokine conjugates |
EP4037715A4 (en) * | 2019-09-30 | 2023-12-13 | OD Therapeutics Limited | Protein-macromolecule conjugates and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
EP2571496A1 (en) | 2013-03-27 |
US8703907B2 (en) | 2014-04-22 |
EP2571496A4 (en) | 2016-03-30 |
US20130123461A1 (en) | 2013-05-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8703907B2 (en) | Controlled drug release from dendrimers | |
JP6363125B2 (en) | Sustained release from macromolecular conjugates | |
US20210283264A1 (en) | Polymeric Prodrugs with a Self-Immolative Linker | |
US20220280654A1 (en) | Improved conjugation linkers | |
JP4824404B2 (en) | Releasable polymer conjugates based on aliphatic biodegradable linkers | |
CA2517459C (en) | Heterobifunctional polymeric bioconjugates | |
US8946405B2 (en) | Controlled release from solid supports | |
US20120114742A1 (en) | Polymer Conjugates with Decreased Antigenicity, Methods of Preparation and Uses Thereof | |
MXPA05001716A (en) | Releasable polymeric conjugates based on aliphatic biodegradable linkers. | |
JP2002508400A (en) | Polymeric prodrugs of amino and hydroxyl containing bioactive agents | |
JP2015172078A (en) | Prodrug and drug-macromolecule conjugate having controlled drug release rate | |
KR20220074897A (en) | Protein-macromolecule conjugates and methods of use thereof | |
EP3458103A1 (en) | Conjugates and conjugating reagents | |
CN102036687A (en) | Conjugates of a cholinesterase moiety and a polymer | |
KR20120090961A (en) | Oligomer-calcimimetic conjugates and related compounds | |
AU2011213827B2 (en) | Polymeric prodrug with a self-immolative linker | |
WO2003074089A1 (en) | Conjugates of the c domain of human gelatinase a and polyethylene glycol, methods of purification and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11778373 Country of ref document: EP Kind code of ref document: A1 |
|
REEP | Request for entry into the european phase |
Ref document number: 2011778373 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011778373 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13696301 Country of ref document: US |