CN1761488A - Vitamin receptor binding drug delivery conjugates - Google Patents
Vitamin receptor binding drug delivery conjugates Download PDFInfo
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- CN1761488A CN1761488A CNA2004800076791A CN200480007679A CN1761488A CN 1761488 A CN1761488 A CN 1761488A CN A2004800076791 A CNA2004800076791 A CN A2004800076791A CN 200480007679 A CN200480007679 A CN 200480007679A CN 1761488 A CN1761488 A CN 1761488A
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Abstract
The invention describes a vitamin receptor binding drug delivery conjugate, and preparations therefor. The drug delivery conjugate consists of a vitamin receptor binding moiety, a bivalent linker (L), and a drug. The vitamin receptor binding moiety is a vitamin, or a vitamin receptor binding analog or a derivative thereof, and the drug includes analogs or derivatives thereof. The vitamin receptor binding moiety is covalently linked to the bivalent linker, and the drug, or the analog or the derivative thereof, is covalently linked to the bivalent linker, wherein the bivalent linker (L) comprises one or more spacer linkers, releasable linkers, and heteroatom linkers. Methods and pharmaceutical compositions for eliminating pathogenic cell populations using the drug delivery conjugate are also described.
Description
The cross reference of related application
35U.S.C. § 119 (e) according to the U.S.; the denomination of invention that the application applied on January 27th, 1 is 60/442 for the patent application number of " vitamin receptor binding drug delivery conjugates (Vitamin-ReceptorBinding Drug Delivery Conjugates) "; 845; the denomination of invention of application on August 1st, 2003 is 60/492 for the patent application number of " vitamin receptor binding drug delivery conjugates "; the denomination of invention of application on October 31st, 119 and 2003 is that the patent application number of " vitamin receptor binding drug delivery conjugates " is 60/516,188 priority.Whole disclosures of each of these applications all are attached to herein by reference.
Invention field
The present invention relates to be used for the compositions and the method for targeting drug delivery.More particularly, the present invention relates to be used for the treatment of the vitamin receptor binding drug delivery conjugates of pathogenic cell disease that colony causes, and correlation technique and pharmaceutical composition.
Background of invention
Immune system provides method for the foreign pathogens of identification and elimination tumor cell, other pathogenic cell and intrusion.Although immune system normally provides powerful protective wire, cancerous cell, other pathogenic cell or infectant are escaped host immune response and propagation or are continued to exist and follow that the host's is pathogenic in many cases.Having developed many chemotherapeutic and X-ray therapy eliminates as the tumor in duplicating.Yet many present available chemotherapeutic and radiotherapy scheme be toxic side effect all, influences the cell of normal host cell such as hemopoietic system because they not only destroy pathogenic cell.The toxic and side effects of these anticarcinogens highlights the demand to the new therapy of exploitation tool pathogenic cell colony's selectivity host toxicity minimizing simultaneously.
Researcher has been developed by making this class cell of cytotoxic compound targeting destroy the therapeutic scheme of pathogenic cell.Many schemes in these schemes are used the toxin of puting together with antibody, and described antibody with have only that described pathogenic cell just has or combined by the antigen of this pathogenic cell overexpression, thereby attempting to minimize is delivered to Normocellular toxin.Utilize the method, develop some immunotoxin of forming by following antibody: the antibody of specific antigen on the anti-pathogenic cell, be connected to for example antibody on ricin, Pseudomonas exotoxin, diphtheria toxin, diphtherotoxin and the tumor necrosis factor of toxin.These immunotoxin targeting have by the pathogenic cell of the specific antigen of described antibody recognition, for example tumor cell (Olsnes, S., Immunol.Today, 10, the 291-295 pages or leaves, 1989; Melby, E.L., Cancer Res., 53 (8), 1755-1760 page or leaf, 1993; Better, M.D., the PCT publication No. WO 91/07418 that on May 30th, 1991 announced).
The method of pathogenic cell colonies such as cancerous cell or exotic disease substance is among the another kind of targeting host, and the immunoreation that strengthens the anti-pathogenic cell of host is to avoid giving the needs of chemical compound, and described chemical compound also has independently host toxicity.The immunization therapy strategy of a report is to tumor cell surface with antibody such as genetic engineering poly antibodies, be illustrated in described cell surface with constant region with antibody, and therefore inducing tumor cell is killed (De Vita by various immune-mediated processes, V.T., Biologic Therapy of Cancer, second edition, Philadelphia, Lippincott, 1995; Soulillou, J.P., United States Patent (USP) 5,672,486).Yet these methods have been defined difficult complicated on the tumor specific antigen.
Summary of the invention
Attempting to develop to pathogenic cell tool specificity and during to effective therapy of normal cytotoxicity minimum, developing vitamin receptor binding drug delivery conjugates.The present invention is applicable to the pathogenic cell colony that causes various pathological changes in the host animal body.The pathogenic cell of available drug delivery conjugates of the present invention treatment comprises the cell of tumor cell, infectant such as antibacterial and virus, antibacterial or viral infection and anyly uniquely expresses, preferentially expresses or overexpression vitamin receptor or in conjunction with other type pathogenic cell of the receptor of vitamin D 3-analogies or derivant.
In one embodiment, provide vitamin receptor binding drug delivery conjugates.Described drug delivery conjugates comprises vitamin receptor bound fraction, bivalence joint and medicine." V " used herein be meant the vitamin receptor bound fraction and comprise vitamin and vitamin receptor in conjunction with analog or derivant, term " vitamin or its analog or derivant " is meant can be in conjunction with vitamin and the analog and the derivant of vitamin receptor." D " used herein is meant medicine and comprises its analog or derivant.Described vitamin or its analog or derivant are covalently bound to bivalence joint (L), and described medicine or its analog or derivant are also covalently bound to bivalence joint (L).Described bivalence joint (L) can comprise polylinker.For example, bivalence joint (L) can comprise one or more components, and it is selected from the spacer nipple (l that arranges by any order
sBut) releasing-joint (l
r) and hetero atom joint (l
H) and combination.
The drug delivery conjugates that this embodiment is described comprises:
V-L-D
V-(l
r)
c-D
V-(l
s)
a-D
V-(l
s)
a-(l
r)
c-D
V-(l
r)
c-(l
s)
a-D
V-(l
H)
b-(l
r)
c-D
V-(l
r)
c-(l
H)
b-D
V-(l
H)
d-(l
r)
c-(l
H)
e-D
V-(l
s)
a-(l
H)
b-(l
r)
c-D
V-(l
r)
c-(l
H)
b-(l
s)
a-D
V-(l
H)
d-(l
s)
a-(l
r)
c-(l
H)
e-D
V-(l
H)
d-(l
r)
c-(l
s)
a-(l
H)
e-D
V-(l
H)
d-(l
s)
a-(l
H)
b-(l
r)
c-(l
H)
e-D
V-(l
H)
d-(l
r)
c-(l
H)
b-(l
s)
a-(l
H)
e-D
V-(l
s)
a-(l
r)
c-(l
H)
b-D
V-[(l
s)
a-(l
H)
b]
d-(l
r)
c-(l
H)
e-D
Wherein a, b, c, d and e are 0,1,2,3 or 4 independently of one another, (l
a), (l
H) and (l
r) as defined herein, V is vitamin or its analog or derivant, D is medicine or its analog or derivant, and wherein bivalence joint L comprises one and perhaps manyly arranges and with (the l of any combination by any order
s), (l
H) and (l
r).Self-evident, the example of aforementioned bivalence joint L is intended to explanation, and the unrestricted (l that is contained in described bivalence joint
H), (l
s) and (l
r) assembling kind widely.
Self-evident, but each of described spacer nipple, hetero atom joint and releasing-joint all is a bivalence.Equally, but being connected between various spacer nipples defined herein, hetero atom joint and releasing-joint, but and being connected between various spacer nipple, hetero atom joint and releasing-joint and D and/or V, but can occur on any atom that is present in various spacer nipples, hetero atom joint and the releasing-joint, but and may not be at any obvious end of various spacer nipples, hetero atom joint and releasing-joint.For example, in illustrative embodiment, wherein the bivalence joint is:
That is, wherein bivalence joint L is-(l
H)-(l
s)
5-(l
r-l
H)
2-D, wherein respectively, (l
H) be nitrogen, (l
s)
5Be Ala-Glu-Lys-Asp-Asp, (l
r-l
H)
2For-(CH
2)
2-S-S-(CH
2)
2-O-C (O)-O-, described (l
r-l
H)
2Joint is connected to described (l
s)
5The middle part of joint.
In another embodiment, provide vitamin receptor binding drug delivery conjugates.Described drug delivery conjugates comprises vitamin receptor bound fraction, bivalence joint (L) and medicine, and bivalence joint (L) comprises one or more hetero atom joints (lH).Described vitamin receptor bound fraction is by the first hetero atom joint (l
H)
dCovalently bound to bivalence joint (L), medicine is by the second hetero atom joint (l
H)
eCovalently bound to bivalence joint (L).Bivalence joint (L) but also comprise one or more spacer nipples and releasing-joint, but wherein spacer nipple and releasing-joint can pass through the 3rd hetero atom joint (l
H)
bCovalently bound mutually.The drug delivery conjugates that this embodiment is described is as follows:
V-(l
H)
d-(l
s)
a-(l
H)
b-(l
r)
c-(l
H)
e-D
Wherein a, b, c, d and e are 0,1,2,3 or 4 independently of one another, (l
s), (l
H) and (l
r) and V and D are as defined herein, and wherein bivalence joint L comprises (l as described
s), (l
H) and (l
r).
In another embodiment, provide vitamin receptor binding drug delivery conjugates.Described drug delivery conjugates comprises vitamin receptor bound fraction, bivalence joint (L) and medicine, and bivalence joint (L) comprises a hetero atom joint (l
H).Described vitamin receptor bound fraction is vitamin or its analog or derivant, and medicine comprises its analog or derivant.Vitamin or its analog or derivant are covalently bound to bivalence joint (L), and medicine or its analog or derivant are covalently bound to bivalence joint (L).Bivalence joint (L) but also comprise spacer nipple and releasing-joint, but and spacer nipple and releasing-joint can be covalently bound mutually by the hetero atom joint.The drug delivery conjugates that this embodiment is described is as follows:
V-(l
s)
a-(l
H)
b-(l
r)
c-D
Wherein a, b and c are 0,1,2,3 or 4 independently of one another, (l
s), (l
H) and (l
r) and V and D are as defined herein, and wherein bivalence joint L comprises (l as described
s), (l
H) and (l
r).
The vitamin receptor binding drug delivery conjugates of general formula V-L-D is provided in another embodiment.In this embodiment, L is by one or more joint (l that arrange by any order
r)
c, (l
s)
a(l
H)
bAnd constitute (l wherein
rBut) be releasing-joint, (l
s) be spacer nipple, (l
H) be the hetero atom joint, a, b and c are 0,1,2,3 or 4 independently of one another, and V is vitamin or its analog or derivant, and D is medicine or its analog or derivant.Self-evident, but drug delivery conjugates described herein can comprise the bivalence joint with a more than spacer nipple releasing-joint or hetero atom joint.For example, but thought over and comprised two or more releasing-joints (l
r) the bivalence joint.In addition, but the configuration of this releasing-joint comprises the bivalence joint, but wherein releasing-joint is covalently bound mutually, but and wherein releasing-joint be spaced from each other by one or more hetero atom joints and/or spacer nipple.
In another embodiment, describe the vitamin receptor binding drug delivery conjugates of general formula V-L-D, wherein L comprises the (l that arranges by any order
s)
a(l
H)
bAnd the bivalence joint of combination, wherein (l
s)
a(l
H)
bAnd V and D are as defined herein.In this embodiment, the medicine in the drug delivery conjugates can be hapten, such as but not limited to fluorescein, dinitrophenyl etc.
In another embodiment, describe the vitamin receptor binding drug delivery conjugates of general formula V-L-D, wherein L comprises the (l that arranges by any order
s)
a, (l
H)
b(l
r)
cAnd the bivalence joint of combination, wherein (l
s)
a(l
H)
bAnd V and D are as defined herein, and at least one (l wherein
r) not disulphide.Self-evident, have a more than (l in this embodiment
r), promptly wherein c greater than 1 bivalence joint, but but except that another or other releasing-joint, also can comprise the disulphide releasing-joint.
Aspect of various vitamin receptor binding drug delivery conjugates as herein described, but the bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkoxy methyl oxygen base, and wherein said methyl is optional to be replaced by alkyl or substituted aryl.
On the other hand, but the bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkyl-carbonyl, and wherein said carbonyl and described medicine or its analog or derivant form the acyl group aziridine.
On the other hand, but the bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form the inferior cycloalkyloxy of 1-alkoxyl.
On the other hand, the bivalence joint comprises and is combined together to form alkylidene amino carbonyl (dicarboxyl arlydene) but spacer nipple, hetero atom joint and the releasing-joint of carboxylate.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form disulfide group alkyl-carbonyl hydrazides, wherein said hydrazides and described medicine or its analog or derivant form hydrazone.
On the other hand, but the bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkyl-carbonyl hydrazides, and wherein said hydrazides and described medicine or its analog or derivant form hydrazone.
On the other hand; the bivalence joint comprises and is combined together to form 3-sulfenyl alkyl sulphonyl alkyl (two replace silicyl) but hetero atom joint, spacer nipple, hetero atom joint, spacer nipple and the releasing-joint of oxygen base that wherein said two replace silicyls is replaced by alkyl or the optional aryl that replaces.
On the other hand, the bivalence joint comprises many spacer nipples that are selected from naturally occurring aminoacid and stereoisomer thereof.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkoxy carbonyl, wherein said carbonyl and described medicine or its analog or derivant form carbonic ester.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group aryl-alkoxy carbonyl, wherein said carbonyl and described medicine or its analog or derivant form carbonic ester, and described aryl is optional the replacement.
On the other hand, but but the bivalence joint comprises hetero atom joint, spacer nipple releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkoxyl alkoxyl alkylidene, wherein said alkylidene and described medicine or its analog or derivant form hydrazone, each alkyl is independently selected, and described oxygen base alkoxyl is optional by alkyl or the optional aryl replacement that replaces.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkoxy carbonyl hydrazides.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkyl amino, wherein said amino and described medicine or its analog or derivant form the bivinyl amide.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkyl amino, wherein said amino and described medicine or its analog or derivant form the bivinyl amide, and described alkyl is an ethyl.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkyl amino-carbonyl, wherein said carbonyl and described medicine or its analog or derivant form carbamate.
On the other hand, but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkyl amino-carbonyl, wherein said carbonyl and described medicine or its analog or derivant form carbamate, and described alkyl is an ethyl.
On the other hand; but but the bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group aryl-alkoxy carbonyl, and wherein said carbonyl and described medicine or its analog or derivant form carbamate or carbamoyl aziridine.
On the one hand, but releasing-joint, spacer nipple and hetero atom joint can arrange in such a way, make that the functional group that discharges is also referred to as anchimeric assistance cracking or fracture in fracture that chemically promotes other key or cracking after the bond fission in the bivalence joint.The illustrative embodiment of this bivalence joint or its part comprises the chemical compound with following formula:
Wherein X is a hetero atom, for example nitrogen, oxygen or sulfur, n is selected from 0,1,2 and 3 integer, R is hydrogen or substituent group, comprising can be by inducing or the substituent group of resonance stabilized positive charge by on aromatic ring, alkoxyl etc. for example, symbol (*) but expression forms the junction point of additional spacer nipple, hetero atom joint or the releasing-joint of bivalence joint, or medicine or its analog or derivant, or the junction point of described vitamin or its analog or derivant.Self-evident, other substituent group includes but not limited to be present in hydroxyl on aromatic ring, benzyl carbon, alkanoic acid or the methylene bridge, alkyl, alkoxyl, alkylthio group, halogen etc.Promote that cracking can comprise the mechanism that relates to benzene first intermediate, benzyne intermediate, lactone cyclisation, oxygen intermediate, β-elimination etc.Self-evident equally, but except that the cracking after the releasing-joint cracking, but the initial cracking of releasing-joint also can be by the facilitation of anchimeric assistance mechanism.
In another embodiment, provide the vitamin receptor binding drug delivery conjugates intermediate.Described intermediate comprises the vitamin receptor bound fraction, has the bivalence joint and the coupling group of first end and second end.Described vitamin receptor bound fraction is vitamin or its analog or derivant, and described coupling group comprises nucleopilic reagent, electrophilic reagent or its precursor.Described vitamin receptor bound fraction is covalently bound to described bivalence joint at first end of described bivalence joint, described coupling group is second terminal covalently bound to described bivalence joint described bivalence joint, but and described bivalence joint comprise one or more spacer nipple releasing-joints and the hetero atom joint and the combination thereof of arranging by any order.
In another embodiment, the vitamin receptor binding drug delivery conjugates intermediate is described.Described intermediate comprises bivalence joint, medicine or its analog or derivant and the coupling group with first end and second end.As described herein, but the bivalence joint comprises one or more components that are selected from spacer nipple releasing-joint and hetero atom joint.Described coupling group is covalently bound to described bivalence joint at first end of described bivalence joint, and described medicine or its analog or derivant are covalently bound to described bivalence joint at second end of described bivalence joint.In addition, described coupling group is nucleopilic reagent, electrophilic reagent or its precursor that can form covalent bond with the vitamin receptor bound fraction, and wherein said vitamin receptor bound fraction is vitamin or its analog or derivant.
In the illustrative embodiment of another vitamin receptor binding drug delivery conjugates intermediate as herein described, described coupling group is Michael (Michael) receptor, described bivalence joint comprise (O) NHN=that has formula-C ,-NHC (O) NHN=or-CH
2C (O) but the releasing-joint of NHN=.Aspect an illustrative of vitamin receptor binding drug delivery conjugates intermediate described herein, described coupling group and described bivalence joint are combined together to form the chemical compound with following formula:
Or its protected derivant, wherein D is for illustrating medicine or its analog or the derivant that forms hydrazone as this paper; N is an integer, for example 1,2,3 or 4.Aspect the illustrative of another vitamin receptor binding drug delivery conjugates intermediate as herein described, vitamin or its analog or derivant comprise the alkyl hydrosulfide nucleopilic reagent.
In the illustrative embodiment of another vitamin receptor binding drug delivery conjugates intermediate as herein described, described coupling group is a hetero atom, for example nitrogen, oxygen or sulfur, bivalence joint comprise covalently bound vitamin or its analog or derivant one or more hetero atom joints and the one or more spacer nipple on the described coupling group.One illustrative aspect, vitamin receptor binding drug delivery conjugates intermediate described herein comprises the chemical compound with following formula:
Or its protected derivant, wherein X is oxygen, nitrogen or sulfur, m is an integer, and for example 1,2 or 3, wherein V, l
sAnd l
HAs defined herein.
Another illustrative aspect, vitamin receptor binding drug delivery conjugates intermediate described herein comprises the chemical compound with following formula:
Or its protected derivant, wherein X is nitrogen or sulfur, wherein V and l
sAs defined herein.
Another illustrative aspect, vitamin receptor binding drug delivery conjugates intermediate described herein comprises the chemical compound with following formula:
Or its protected derivant, wherein Y is hydrogen or substituent group, is electron-withdrawing substituent illustrative, includes but not limited to nitro, cyano group, halogen, alkyl sulphonyl, carboxylic acid derivates etc., wherein V and l
sAs defined herein.
In the illustrative embodiment of another vitamin receptor binding drug delivery conjugates intermediate as herein described, described coupling group is a michael acceptor, and the bivalence joint comprises covalent bond vitamin or its analog or derivant one or more hetero atom joints and the one or more spacer nipple on the described coupling group.Aspect the illustrative of a vitamin receptor binding drug delivery conjugates intermediate as herein described, described coupling group and bivalence joint are combined together to form the chemical compound with following formula:
Or its protected derivant, wherein X is oxygen, nitrogen or sulfur, m and n are the independent integer of selecting, and for example 1,2 or 3, wherein V, l
sAnd l
HAs defined herein.Aspect the illustrative of a vitamin receptor binding drug delivery conjugates intermediate as herein described, described medicine or its analog or derivant comprise the alkyl hydrosulfide nucleopilic reagent.
Aspect the illustrative of another vitamin receptor binding drug delivery conjugates intermediate as herein described, intermediate comprises the chemical compound with following formula:
Or its protected derivant; wherein V is vitamin or its analog or derivant; AA is the aminoacid that illustrative is selected from naturally occurring aminoacid or its stereoisomer; X is nitrogen, oxygen or sulfur; Y is hydrogen or substituent group; be electron-withdrawing substituent illustrative, include but not limited to nitro, cyano group, halogen, alkyl sulphonyl, carboxylic acid derivates etc.N and m are the independent integer of selecting, for example 1,2 or 3, and p is that integer is as 1,2,3,4 or 5.AA also can be any other aminoacid, for example any aminoacid with following general formula:
-N(R)-(CR′R″)
q-C(O)-
Wherein R is hydrogen, alkyl, acyl group or suitable nitrogen-protecting group, R ' and R " is hydrogen or substituent group, independently selects when wherein each occurs at every turn that q is that integer is as 1,2,3,4 or 5.Illustrative ground; R ' and/or R " independently meet but be not limited to hydrogen or be present in side chain on the naturally occurring aminoacid; for example methyl, benzyl, hydroxymethyl, sulfenyl methyl, carboxyl, carboxyl methyl, guanidine radicals propyl group etc., and the derivant of derivant and protection.Above-mentioned formula comprises all stereoisomerism variants.For example, aminoacid can be selected from agedoite, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithuric acid, threonine etc.Aspect the illustrative of a vitamin receptor binding drug delivery conjugates intermediate as herein described, described medicine or its analog or derivant comprise the alkyl hydrosulfide nucleopilic reagent.
In another embodiment, describe preparation and had the chemical compound of following formula or the method for its protected derivant:
Wherein L is for containing (l
r)
c, (l
s)
a(l
H)
bAnd the joint of combination; D is for forming medicine or its analog or the derivant of hydrazone, wherein (l
r)
c, (l
s)
a(l
H)
bAnd V as defined herein, and described method comprises the steps:
(a) make chemical compound or its protected derivant with following formula:
With the chemical compound with following formula or its protected derivatives reaction:
Generate the sulfenyl succinimide derivatives; With
(b) form the hydazone derivative of medicine or its analog or derivant with the sulfenyl succinimide derivatives.
In another embodiment, describe preparation and had the method for the chemical compound of following formula:
Wherein L is for comprising (l
r)
c, (l
s)
a(l
H)
bAnd the joint of combination; Wherein D is for forming medicine or its analog or the derivant of hydrazone, (l
r)
c, (l
s)
a(l
H)
bAnd V as defined herein, and described method comprises the steps:
Make chemical compound or its protected derivant with following formula:
With the chemical compound with following formula:
Or its protected derivatives reaction.
In another embodiment, pharmaceutical composition has been described.Described pharmaceutical composition comprises drug delivery conjugates of the present invention and medicine acceptable carrier thereof.
In another embodiment, the method that a kind of elimination has the interior pathogenic cell colony of host animal body of pathogenic cell colony has been described, the member of wherein said pathogenic cell colony have can and vitamin or the binding site of its analog or derivant, and wherein said binding site is by the unique expression of described pathogenic cell, overexpression or preferential the expression, and described method comprises the step that gives described host drug delivery conjugates of the present invention or its pharmaceutical composition.
The accompanying drawing summary
Fig. 1 shows the inhibition of EC112 (embodiment 9c) to the M109 tumor growth.
Fig. 2 shows the influence of EC112 (embodiment 9c) to the weight of animals.
Fig. 3 shows the inhibition of EC105 (embodiment 10a) to the M109 tumor growth.
Fig. 4 shows the influence of EC105 (embodiment 10a) to the weight of animals.
Fig. 5 shows that EC105 (embodiment 10a) lacks the inhibition to the 4T1 tumor growth.
Fig. 6 shows the inhibition of EC145 (embodiment 16b) to the M109 tumor growth.
Fig. 7 shows the inhibition of EC140 (embodiment 17a) to the M109 tumor growth.
Fig. 8 shows the inhibition of EC136 (embodiment 10b) to the L1210 tumor growth.
Fig. 9-16 shows EC135, EC136, EC137, EC138, EC140, EC145, EC158 and EC159 (being respectively embodiment 17b, 10b, 16a, 10c, 17a, 16b, 14e and 15) the synthetic inhibition of pair cell DNA.
Detailed Description Of The Invention
The present invention relates to vitamin receptor binding drug delivery conjugates, it contains the vitamin receptor knot Close part, divalence joint (L) and medicine, wherein said vitamin receptor bound fraction and described Medicine is separately optional to be connected on the described divalence joint (L) by the hetero atom joint. Divalence joint (L) Comprise one or more spacer nipple, hetero atom joints of arranging by any order and can discharge (namely Cleavable) joint and combination thereof.
Term used herein " but releasing-joint " refers to comprise that at least one is at physiological condition (for example pH labile bond, sour labile bond, oxidation instability or enzyme are or not the key that can rupture down Stable keys) joint. Self-evident, this physiological condition that causes bond fission comprises and occurring in Under the physiological pH, or dissolve for example its pH nuclear lower than kytoplasm pH of organelle as compartment The result of endosome and the standard chemical hydrolysis that takes place.
Self-evident, as described herein, but the cleavable key can connect two in the releasing-joint Adjacent atom, but and/or described releasing-joint appoint one or both ends connect other joint or V and/or D. The feelings of two adjacent atoms in but this cleavable key connects releasing-joint Under the condition, after this bond fission, but described releasing-joint fragments into two or more fragments. Or The person, but this cleavable key releasing-joint and another part for example the hetero atom joint, But every joint, another releasing-joint, medicine or its analog or derivative or vitamin or its In the situation between analog or the derivative, after this bond fission, but releasing-joint is from other one Separate in the branch.
The unstability of cleavable key can be adjusted, for example by the cleavable key position or Near it replacement changes, and for example comprises the α branch branching of adjoining the cleavable disulfide bond, increases tool Substituting group hydrophobicity on the silicon in the part of hydrolyzable silicon oxygen bond, homologization forms hydrolyzable section Divide the alkoxyl of ketal or acetal etc.
According to the present invention, described vitamin receptor binding drug delivery conjugates can be used for treatment with the place Having pathogenic cell colony among the master is the disease of feature, wherein member's tool of pathogenic cell colony Vitamin or its analog or the derivative binding site that can reach are arranged, wherein said binding site By the unique expression of described pathogenic cell, overexpression or preferential the expression. Described pathogenic cell The selective elimination partly connected by the vitamin of described vitamin receptor binding drug delivery conjugates To vitamin receptor, transport protein or other specific binding vitamin or its analog or spread out Biological surface exists on the albumen and mediates, and described surface exist albumen by described cause a disease thin The unique expression of born of the same parents, overexpression or preferential the expression. The unique expression of described pathogenic cell, excessive The surface of expressing or preferentially expressing exists albumen to provide the method for selective elimination pathogenic cell In non-pathogenic cell, do not exist or acceptor that concentration is lower.
For example, the surface expression vitamin receptor such as high-affinity folacin receptor is on tumour cell Overexpression. Ovary, mammary gland, colon, lung, nose, pharynx and the brain epithelioma table that all is in the news Reach high-level folacin receptor. In fact, known 90% the human ovarian's tumour of surpassing is with Gao Shui This acceptor of flat expression. Therefore, it is thin that drug delivery conjugates of the present invention can be used for treating various tumours Born of the same parents' type, and other pathogenic cell type such as infectant, described cell type precedence table Reach vitamin receptor, and therefore have the surface can and vitamin or vitamin D 3-analogies or spread out The bioconjugation position.
Except vitamin as herein described, self-evident, other part can with described herein or The medicine of considering or joint coupling form part-joint-drug conjugate, and this conjugate can Medicine is passed to required target facilitation. Except described vitamin and analog and derivative, this A little other parts can be used for forming the drug delivery conjugates that can be attached on the target cell. General next Say that any part of cell surface receptor can be puted together as being prepared into joint-medicine effectively The target part of thing. Illustrative other part that this paper considers comprises from library screening to be identified The peptide part that goes out, tumor cell specific peptide, tumor cell specific is fit (aptamer), swollen Oncocyte specific carbohydrate, tumor cell specific monoclonal antibody or polyclonal antibody, anti-(for example anti-EphA2 or other are transferred cancer to the Fab of body or scFv (being the strand variable region) fragment The Fab fragment of antibody cell specific expression or the albumen that metastasis cancer cell is exclusive), From the organic molecule of combinatorial libraries, growth factor (such as EGF, FGF, insulin and pancreas islet Plain like growth factor, and homeopeptide), somatostatin and analog thereof, turn to the iron egg In vain, PLC, cholate, selectin, steroid hormone, contain the Arg-Gly-Asp peptide, Retinoids, various half lactadherin, δ-opioid receptor part, pancreozymin A acceptor Part, angiotensins AT1 or AT2 receptor-specific part, peroxisome proliferation Thing activated receptor λ part, beta-Lactam antibiotic for example penicillin, organic molecule (comprise anti-Microbial medicine and other molecule, described molecular specificity is in conjunction with preferentially being expressed in tumour cell Or the acceptor on the infection biological body), antimicrobial agents and other is according to acceptor or other cell The crystal structure design of surface protein adapts to the medicine of concrete receptors bind pocket, in tumour The preferential tumour antigen of expressing of cell surface or the fragment of other molecule or all these molecules. The example of tumour specific antigen that can be used as the binding site of ligand-immunogen conjugates comprises Liver is joined protein family member's extracellular epi-position, for example EphA2. In normal cell, EphA2 Expression is limited in cell-cell contact, but in the metastatic tumour cell, EphA2 is distributed in Whole cell surface. Therefore, the EphA2 on the transitional cell can approach to be attached to be conjugated in and exempt from On the Fab fragments on the epidemic focus, described albumen is kept off the Fab that is attached on the normal cell On the fragment, produce metastasis cancer cell ligands specific-immunogen conjugates.
The present invention has also considered the purposes of the combination of part-joint-drug conjugate, with maximization The pathogenic cell that target will be eliminated.
The illustrative drug delivery conjugates is as follows:
V-L-D
V-(l
r)
c-D
V-(l
s)
a-D
V-(l
s)
a-(l
r)
c-D
V-(l
r)
c-(l
s)
a-D
V-(l
H)
b-(l
r)
c-D
V-(l
r)
c-(l
H)
b-D
V-(l
H)
d-(l
r)
c-(l
H)
e-D
V-(l
s)
a-(l
H)
b-(l
r)
c-D
V-(l
r)
c-(l
H)
b-(l
s)
a-D
V-(l
H)
d-(l
s)
a-(l
r)
c-(l
H)
e-D
V-(l
H)
d-(l
r)
c-(l
s)
a-(l
H)
e-D
V-(l
H)
d-(l
s)
a-(l
H)
b-(l
r)
c-(l
H)
e-D
V-(l
H)
d-(l
r)
c-(l
H)
b-(l
s)
a-(l
H)
e-D
V-(l
s)
a-(l
r)
c-(l
H)
b-D
V-[(l
s)
a-(l
H)
b]
d-(l
r)
c-(l
H)
e-D
Wherein a, b, c, d and e are 0,1,2,3 or 4 independently of one another, (ls) be spacer nipple, (lH) be the hetero atom joint, (lrBut) be releasing-joint, V is vitamin or its analog or derivative, D is medicine or its analog or derivative, and wherein divalence joint L comprises a perhaps (l of many any arrangements and any combinations)、(l
H) and (lr). Self-evident, aforementioned The example of divalence joint L is intended to explanation, and the unrestricted (l that is contained in described divalence jointH)、(l
s) and (lr) widely assembling kind.
In the embodiment of a drug delivery conjugates V-L-D, wherein V is vitamin folic acid; L is not the ethylenediamine of following formula:
In the embodiment of another drug delivery conjugates V-L-D, wherein V is vitamin folic acid, D is Mitomycin C C; L is not the L-Cys-(S-sulfenyl ethyl) of following formula:
L is not the L-Asp-L-Arg-L-Asp-L-Cys-(S-sulfenyl ethyl) of following formula:
L is not the L-Arg-L-Cys-(S-sulfenyl ethyl)-L-Ala-L-Gly-OH of following formula:
Also considered drug delivery conjugates of the present invention, wherein vitamin or its analog or derive But thing is connected on the releasing-joint, and the latter is connected on the medicine by spacer nipple again. And, Described medicine and described vitamin or its analog or derivative can be connected to separately the interval and connect On the head, but wherein said spacer nipple interconnects by releasing-joint. In addition, described medicine Thing and described vitamin or its analog or derivative, but can be connected to separately on the releasing-joint, But wherein said releasing-joint interconnects by spacer nipple. The hetero atom joint can place appoints Between what two joint, or between any joint and vitamin or its analog or the derivative, Or between any joint and medicine or its analog or the derivative. Other can also to have considered all Arrangement and the combination of energy.
In one embodiment, the invention provides vitamin receptor binding drug delivery conjugates. Described drug delivery conjugates is by vitamin receptor bound fraction, divalence joint (L) and ingredients. Described vitamin receptor bound fraction, for can be attached on the vitamin receptor vitamin or Its analog or derivative, described medicine comprise its analog with pharmaceutically active or derive Thing. Described vitamin or its analog or derivative, covalently bound to described divalence joint (L) On, medicine or its analog or derivative, also covalently bound to described divalence joint (L). Divalence joint (L) comprises one or more spacer nipples of arranging by any order, can discharge and connect Head and hetero atom joint and combination thereof. For example, described hetero atom joint can be nitrogen, described can Releasing-joint and described hetero atom joint can be combined together to form bilvalent radical, described bilvalent radical Comprise alkylidene aziridine-1-base, alkylidene carbonyl aziridine-1-base, carbonylic alkyl aziridine-1-base, alkylidene sulfonic group aziridine-1-base, sulfonic group alkyl aziridine-1-base, sulfonyl Alkyl aziridine-1-base or alkylidene sulfonyl aziridine-1-base, wherein each described release The optional quilt of joint as undefined substituent X2Replace. Perhaps, the hetero atom joint can be nitrogen, Oxygen, sulphur and formula-(NHR1NHR
2)-、-SO-、-(SO
2)-and-N (R3) O-, wherein R1、R
2And R3Independently be selected from separately hydrogen, alkyl, aryl, aryl alkyl, substituted aryl, replacement virtue Alkyl, heteroaryl, substituted heteroaryl and alkoxyalkyl. In another embodiment, institute State the hetero atom joint and can be oxygen, described spacer nipple can be optional quilt as undefined substituting group X1The 1-alkylidene succinimide that replaces-3-base, but described releasing-joint can be methylene, 1-alkane Oxygen base alkylidene, 1-alkoxyl cycloalkylidene, 1-alkoxyl alkylidene carbonyl, 1-alkoxyl Asia Naphthene base carbonyl, but wherein each described releasing-joint is chosen quilt as undefined substituent X 2 wantonly Replace, but and wherein said spacer nipple and described releasing-joint be connected to separately hetero atom Form succinimide-1-base alkyl acetal or ketal on the joint.
Described spacer nipple is selected from carbonyl; thiocarbonyl; alkylidene; cycloalkylidene; the alkylidene cycloalkyl; the alkylidene carbonyl; the cycloalkylidene carbonyl; the carbonylic alkyl carbonyl; 1-alkylidene succinimide-3-base; 1-(carbonylic alkyl) succinimide-3-base; the alkylidene sulfonic group; the sulfonyl alkyl; alkylidene sulfonic group alkyl; alkylidene sulfonyl alkyl; carbonyl tetrahydrochysene-2H-pyranose; the carbonyl tetrahydrofuran base; 1-(carbonyl tetrahydrochysene-2H-pyranose) succinimide-3-base and 1-(carbonyl tetrahydrofuran base) succinimide-3-base, the wherein optional quilt of each described spacer nipple as undefined substituent X1Replace. In this embodiment, described hetero atom joint can be nitrogen, Described spacer nipple can be alkylidene carbonyl, cycloalkylidene carbonyl, carbonylic alkyl carbonyl, 1-(carbonyl The base alkyl) succinimide-3-base, wherein each spacer nipple quilt as undefined substituent X1The optional replacement,, described spacer nipple is connected on the described nitrogen and forms acid amides. Perhaps, described assorted The atom joint can be sulphur, and described spacer nipple can be alkylidene and cycloalkylidene, wherein each Described spacer nipple is optional by carboxyl substituted, and described spacer nipple is connected on the described sulphur and forms Mercaptan. In another embodiment, described hetero atom joint can be sulphur, described spacer nipple Can be 1-alkylidene succinimide-3-base and 1-(carbonylic alkyl) succinimide-3-base, institute Stating spacer nipple is connected to and forms succinimide-3-base mercaptan on the described sulphur.
In the alternative of above-mentioned embodiment; described hetero atom joint can be nitrogen; but described releasing-joint and described hetero atom joint can be combined together to form bilvalent radical; described bilvalent radical comprises alkylidene aziridine-1-base, carbonylic alkyl aziridine-1-base, sulfonic group alkyl aziridine-1-base or sulfonyl alkyl aziridine-1-base, but wherein each described releasing-joint is chosen quilt as undefined substituent X wantonly2Replace. In this alternative embodiment, described spacer nipple can Be carbonyl, thiocarbonyl, alkylidene carbonyl, cycloalkylidene carbonyl, carbonylic alkyl carbonyl, 1-(carbonyl The base alkyl) succinimide-3-base, the wherein optional quilt of each described spacer nipple as undefined Substituent X1Replace, but and wherein said spacer nipple be connected to shape on the described releasing-joint Become the aziridine acid amides.
Described substituent X1Can be alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R4-carbonyl, R5-carbonylic alkyl, R6-acylamino-and R7-amidoalkyl, wherein R4And R5Independently be selected from separately amino acid, amino acid derivativges and peptide, and R wherein6And R7Independently be selected from separately amino acid, amino acid derivativges and peptide. In this embodiment, described hetero atom joint can be nitrogen, described substituent X1With described assorted The atom joint can be combined together to form heterocycle with the spacer nipple that is connected them.
But described releasing-joint can be methylene; 1-alkoxyl alkylidene; 1-alkoxyl cycloalkylidene; 1-alkoxyl alkylidene carbonyl; 1-alkoxyl cycloalkylidene carbonyl; the carbonyl aryl carbonyl; carbonyl (carboxyl aryl) carbonyl; carbonyl (two carboxyl aryl) carbonyl; halo alkylidene carbonyl; alkylidene (dialkyl group silicyl); alkylidene (alkylaryl silicyl); alkylidene (diaryl silicyl); (dialkyl group silicyl) aryl; (alkylaryl silicyl) aryl; (diaryl silicyl) aryl; oxygen base ketonic oxygen base; oxygen base ketonic oxygen base alkyl; sulfonyl oxygen base; oxygen base sulfonyl alkyl; the imino group alkylidene; the carbonyl alkylen group imino group; the imino group cycloalkylidene; carbonyl cycloalkylidene imino group; the alkylidene sulfenyl; alkylidene aryl sulfenyl and carbonylic alkyl sulfenyl, but wherein each described releasing-joint is optional by one or more as undefined substituent X2Replace.
In the above-described embodiment, described hetero atom joint can be oxygen, but described releasing-joint can be methylene, 1-alkoxyl alkylidene, 1-alkoxyl cycloalkylidene, 1-alkoxyl alkylidene carbonyl and 1-alkoxyl cycloalkylidene carbonyl, but wherein each described releasing-joint is chosen quilt as undefined substituent X wantonly2Replace, but and described releasing-joint be connected on the described oxygen and form Acetal or ketal. Perhaps, described hetero atom joint can be oxygen, but described releasing-joint can be Methylene, the aryl that wherein said methylene is optionally substituted replaces, but described releasing-joint Be connected to and form acetal or ketal on the oxygen. In addition, described hetero atom joint can be oxygen, and is described But releasing-joint can be the sulfonyl alkyl, but described releasing-joint is connected to and forms alkyl on the oxygen Sulphonic acid ester.
But in the embodiment of another above-mentioned releasing-joint embodiment, described hetero atom joint can be nitrogen, but described releasing-joint can be imino group alkylidene, carbonyl alkylen group imino group, imino group cycloalkylidene and carbonyl cycloalkylidene imino group, but wherein each described releasing-joint is chosen quilt as undefined substituent X wantonly2Replace, but described releasing-joint is connected on the nitrogen and forms Hydrazone. In alternate configuration, described hydrazone can be used carboxylic acid derivates, orthoformate derivative or ammonia But base formoxyl derivative acidylate forms various acylhydrazone releasing-joints.
Perhaps, described hetero atom joint can be oxygen, but described releasing-joint can be alkylidene (dialkyl group silicyl), alkylidene (alkylaryl silicyl), alkylidene (diaryl silicyl), (dialkyl group silicyl) aryl, (alkylaryl silicyl) aryl and (diaryl silicyl) aryl, but wherein each described releasing-joint is chosen quilt as undefined substituent X wantonly2Replace, but described releasing-joint is connected to and forms silanol on the oxygen.
But in above-mentioned releasing-joint embodiment, described medicine can comprise nitrogen-atoms, and is described The hetero atom joint can be nitrogen, but described releasing-joint can be carbonyl aryl carbonyl, carbonyl (carboxyl Aryl) carbonyl, carbonyl (two carboxyl aryl) carbonyl, but that described releasing-joint can be connected to is assorted former Form acid amides on the sub-nitrogen, also be connected on the described medicine nitrogen and form acid amides.
But in above-mentioned releasing-joint embodiment, described medicine can comprise oxygen atom, and is described The hetero atom joint can be nitrogen, but described releasing-joint can be carbonyl aryl carbonyl, carbonyl (carboxyl Aryl) carbonyl, carbonyl (two carboxyl aryl) carbonyl, but that described releasing-joint can be connected to is described Form acid amides on the hetero atom joint nitrogen, also can be connected on the described medicine oxygen and form ester.
Described substituent X2Can be alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R4-carbonyl, R5-carbonylic alkyl, R6-acylamino-and R7-amidoalkyl, wherein R4And R5Independently be selected from separately amino acid, amino acid derivativges and peptide, and R wherein6And R7Independently be selected from separately amino acid, amino acid derivativges and peptide. In this embodiment, described hetero atom joint can be nitrogen, described substituent X2With described assorted The atom joint can be combined together to form heterocycle with the spacer nipple that is connected them.
Described heterocycle can be pyrrolidines, piperidines, oxazolidine, isoxazole alkyl, thiazolidine, different Thiazolidine, pyrrolidones, piperidones, oxazolidone, isoxazole alkyl ketone, thiazolidone, Isothiazolidine ketone and succinimide.
Described medicine can be mitomycin, E09 or mitomycin analogs, in this embodiment, but described releasing-joint is selected from carbonylic alkyl sulfenyl, carbonyl tetrahydrochysene-2H-pyranose, carbonyl tetrahydrofuran base, 1-(carbonyl tetrahydrochysene-2H-pyranose) succinimide-3-base and 1-(carbonyl tetrahydrofuran base) succinimide-3-base, but wherein each described releasing-joint is optional by a substituent X2Replace, the aziridine of wherein said mitomycin is connected to described releasing Put and form the acyl group aziridine on the joint.
Described medicine can comprise nitrogen-atoms, but described releasing-joint can be the optional basic X that is substituted2The halo alkylidene carbonyl that replaces, but described releasing-joint is connected on the described medicine nitrogen and forms Acid amides.
Described medicine can comprise oxygen atom, but described releasing-joint can be the optional basic X that is substituted2The halo alkylidene carbonyl that replaces, but described releasing-joint is connected on the described medicine oxygen and forms Ester.
Described medicine can comprise two key nitrogen-atoms, and in this embodiment, described release connects Head can be alkylidene carbonylamino and 1-(alkylidene carbonylamino) succinimide-3-base, and is described But releasing-joint can be connected on the described medicine nitrogen and form hydrazone.
Described medicine can comprise sulphur atom, and in this embodiment, but described releasing-joint can Be alkylidene sulfenyl and carbonylic alkyl sulfenyl, but described releasing-joint can be connected to described medicine Form disulphide on the sulphur.
Vitamin can be the folic acid that comprises nitrogen-atoms, in this embodiment, described spacer nipple can be alkylidene carbonyl, cycloalkylidene carbonyl, carbonylic alkyl carbonyl, 1-alkylidene succinimide-3-base, 1-(carbonylic alkyl) succinimide-3-base, and wherein each described spacer nipple is chosen wantonly and is substituted basic X1Replace, described spacer nipple be connected to form on the folic acid nitrogen imidodicarbonic diamide or Alkylamide. In this embodiment, described substituent X1Can be alkyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, carboxyl, carboxyalkyl, guanidine alkylation, R4-carbonyl, R5-carbonylic alkyl, R6-acylamino-and R7-amidoalkyl, wherein R4And R5Independently be selected from separately amino acid, amino acid derivativges and peptide, and R wherein6And R7Independently be selected from separately amino acid, amino acid derivativges and peptide.
Term used herein " alkyl " refers to choose wantonly the unit price Linear Carbon atomic link of branch, Such as methyl, ethyl, propyl group, 3-methyl amyl etc.
Term used herein " cycloalkyl " refers to that wherein the unit price carbon of part formation ring is former Subchain, such as cyclopropyl, cyclohexyl, 3-ethyl cyclopenta etc.
Term used herein " alkylidene " refers to choose wantonly the divalence Linear Carbon atom of branch Chain, such as methylene, ethylidene, propylidene, 3-methyl pentylidene etc.
Term used herein " cycloalkylidene " refers to that wherein a part forms the dicovalent carbon that encircles Atomic link for example encircles the third-1,1-, two bases, ring the third-1,2-two bases, hexamethylene-Isosorbide-5-Nitrae-two base, 3-ethyl Ring penta-1,2-two bases, 1-methylene hexamethylene-4-base etc.
Term used herein " heterocycle " refers to unit price carbon and hetero atom chain, and is wherein said assorted Atom is selected from nitrogen, oxygen and sulphur, forms ring comprising at least one heteroatomic part, Such as aziridine, pyrrolidines, oxazolidine, 3-methoxyl group pyrrolidines, 3-methyl piperazine etc.
The alkane of this paper definition that term used herein " alkoxyl " refers to be combined with end group oxygen Base, such as methoxyl group, ethyoxyl, propoxyl group, 3-methyl amoxy etc.
Term " halogen " or " halo " refer to fluorine, chlorine, bromine and iodine.
Term used herein " aryl " refers to aromatic monocyclic or many rings of carbon atom, for example Phenyl, naphthyl etc.
Term used herein " heteroaryl " refers to carbon atom and at least one heteroatomic virtue Family's monocycle or many rings, described hetero atom is selected from nitrogen, oxygen and sulphur, for example pyridine radicals, pyrimidine radicals, Indyl, benzoxazolyl etc.
Term used herein " substituted aryl " or " substituted heteroaryl " refer to by one or A plurality of selected substituting groups are halogen, hydroxyl, amino, alkyl or dialkyl amido, alkane for example Aryl or heteroaryl that oxygen base, alkyl sulphonyl, cyano group, nitro etc. replace.
Term used herein " imino group alkylidene " refers to contain the alkylidene of this paper definition and the bilvalent radical of nitrogen-atoms, the end group carbon atom bivalent of wherein said alkylidene is bonded on the nitrogen-atoms, for example formula-(CH)=N-,-(CH2)
2(CH)＝N-、-CH
2C (Me)=N-etc.
Term used herein " amino acid " typically refers to the aminoalkyl carboxylate, wherein institute It is optional by alkyl, hydroxy alkyl, mercaptoalkyl, aminoalkyl, carboxyalkyl etc. to state alkyl Replace, comprise and group corresponding to natural amino acid, for example serine, cysteine, Methionine, aspartic acid, glutamic acid etc.
Term used herein " aryl alkyl " refers to that the alkylidene that is defined by this paper replaces The aryl of this paper definition, such as benzyl, phenethyl, Alpha-Methyl benzyl etc.
Should be appreciated that above-mentioned term generation chemistry capable of being combined relevant group, for example " alkoxyl Alkyl " refer to methoxy, ethoxyethyl group etc., " halogenated alkoxy alkyl " refers to Trifluoromethoxy ethyl, 1,2-two fluoro-2-chloro second-1-base oxygen base propyl group etc.
Term used herein " amino acid derivativges " refers generally to the aminoalkyl carboxylate, its In amino or carboxylic acid ester groups separately optional replaced by alkyl, carboxyalkyl, alkyl amino etc. or Optional protected; Middle divalent alkyl fragment is optional by alkyl, hydroxy alkyl, sulfydryl alkane The replacements such as base, aminoalkyl, carboxyalkyl, comprise with naturally occurring amino acid in exist Group corresponding to side chain, for example serine, cysteine, methionine, aspartic acid, The group that exists in the glutamic acid etc.
Term used herein " peptide " refers to that generally a series of mutual covalency of amido link that pass through connect The amino acid that connects and amino acid analogue and derivative.
But described releasing-joint comprises at least one key that can rupture (pH for example under physiological condition Labile bond, sour labile bond, oxidation instability or enzyme labile bond). One or more can The cracking key can be present in one or two ends of cleavable joint inside and/or cleavable joint. Do not say And explain, the unstability of described cleavable key can by can promote or this key of facilitation disconnected The functional group of splitting or fragment are included in the divalence joint L and adjust, and are also referred to as anchimeric assistance. In addition, self-evident, extra functional group or fragment can be included in the divalence joint L, should The divalence joint can promote or the extra fragments of the described vitamin receptor of facilitation discharges described Bound drug conjugate after the bond fission of joint.
But the bond cleavage solution mechanism of illustrative releasing-joint comprises following oxygen and promotes cracking:
Wherein Z is vitamin or its analog or derivative, or medicine or its analog or spread out Biology, or vitamin or the drug moiety of respectively doing for oneself and being connected with the other parts of divalence joint, But for example comprise one or more spacer nipples, hetero atom joint and/or other releasing-joint Medicine or vitamin part. In this embodiment, the acid catalyzed elimination of carbamate Cause CO2Release and nitrogen moiety be connected on the Z, and can be by water or any other road The cationic formation of benzyl that easy this alkali is caught.
But another is illustrative to be connected on the releasing-joint or to comprise wherein bond cleavage solution mechanism, but forming section divalence joint L comprises that following β eliminates and bivinyl β elimination mechanism:
Wherein X is nucleopilic reagent, GSH, glutathione or biological reductant etc., Z or Z ' Be vitamin or its analog or derivative, or medicine or its analog or derivative, or be The vitamin or the drug moiety that are connected with the other parts of divalence joint. Self-evident, described The bond cleavage solution also can be eliminated by the acid catalysis of carbamate moiety and take place, and can pass through by upper State the aryl of the β sulphur in the example or stabilisation that disulphide provides by anchimeric assistance. At these In the modification of embodiment, but described releasing-joint is carbamate moiety.
But another illustrative mechanism relates to releasing-joint, spacer nipple and hetero atom joint with this The rearrangement of the mode of kind: after the bond cleavage solution in the divalence joint, the functional group of release chemistry promotes it The fracture of its key or cracking are also referred to as anchimeric assistance cracking or fracture. This divalence joint or The illustrative embodiment of its part comprises the compound with following formula:
Wherein X is hetero atom, for example nitrogen, oxygen or sulphur, and n is selected from 0,1,2 and 3 Integer, R is hydrogen or substituting group, comprising can be steady by the resonance of inducing or pass through on aromatic ring Decide the substituting group of positive charge, such as alkoxyl etc., and Z or Z ' are that vitamin or its are similar Thing or derivative, or medicine or its analog or derivative, or be and other of divalence joint Vitamin or drug moiety that part connects. Self-evident, other substituting group comprises but does not limit In hydroxyl, alkyl, alkoxyl, alkylthio group, halogen etc. can be present in aromatic ring, benzyl carbon, On carbamate nitrogen, alkanoic acid or the methylene bridge. Promoting cracking to comprise relates in the benzene first The mechanism of mesosome, benzyne intermediate, lactone cyclisation, oxygen intermediate, β-elimination etc. Equally, But except the cracking after the releasing-joint cracking, but the initial cracking of releasing-joint also can lead to Cross the facilitation of anchimeric assistance mechanism.
In this embodiment, can be the cracking of the hydroxy alkanoic acid facilitation methylene bridge of ring-type, Its mode is by for example oxonium ion, but and bond cleavage solution or the key of the described releasing-joint of facilitation Fragmentation after the cracking. Perhaps, the acid catalyzed oxonium ion of methylene bridge promotes that cracking can Start the fragmentation cascade reaction of this illustrative joint or its fragment. Perhaps, carbamate But the acid catalyzed hydrolysis facilitation β that can be the hydroxy alkanoic acid of ring-type eliminate, and by for example oxygen The cracking of ion facilitation methylene bridge. Self-evident, other describes metabolism, life at this paper Bond fission under the rational faculty or the cell condition or the chemism of cracking can start this fragmentation level Connection.
Drug delivery conjugates as herein described can be by the synthetic method preparation of this area approval. The institute The selection of hetero atom joint is depended in the selection of stating synthetic method, and described functional group is present in institute But state on spacer nipple and the described releasing-joint. Generally speaking, relative keys forms the reaction description In Richard C.Larock, " Comprehensive Organic Transformations, a guide To functional group preparations, " VCH Publishers, Inc.New York (1989) and Theodora E.Greene ﹠ Peter G.M.Wuts, " Protective Groups Ion Organic Synthesis, " second edition, John Wiley ﹠ Sons, Inc.New York (1991), the disclosure of described document is incorporated herein by reference.
The formation of conventional acid amides and ester
For example, wherein said hetero atom joint is nitrogen-atoms, is present in spacer nipple and maybe can releases The functional end-group of putting on the joint is carbonyl, and shown in scheme 1, required amide groups can lead to Coupling reaction or the acylation reaction of crossing corresponding carboxylic acid or derivative obtain, and wherein L is suitably choosing The leaving group of selecting is halogen, trifluoro-methanesulfonyl oxy (triflate), phenyl-pentafluoride oxygen base, three for example Methyl silicane oxygen base, succinimide-N-oxygen base etc., and amine.
Scheme 1
Coupling reagent comprise DCC, EDC, RRDQ, CGI, HBTU, TBTU, HOBT/DCC, HOBT/EDC, BOP-Cl, PyBOP, PyBroP etc. Perhaps, mother Body acid can be converted to the carbonyl derivative of activation, for example acyl chlorides, N-hydroxy-succinamide Base ester, pentafluorophenyl group ester etc. Become the acid amides reaction also can be in alkali such as triethylamine, diisopropyl second Amine, N carry out under the existence such as N-dimethyl-4-aminopyridine. Suitable one-tenth acid amides described herein Solvent comprises CH2Cl
2、CHCl
3, THF, DMF, DMSO, acetonitrile, EtOAc etc. Illustratively, described acid amides can be-15 ℃ to about 80 ℃ approximately in scope, or about 0 ℃ extremely about 45 Prepare under ℃ temperature. Acid amides can be by for example nitrogenous aziridine ring, carbohydrate and alpha-halogenated carboxylic acids preparation. The illustrative carboxylic acid derivates that is applicable to into acid amides comprises having following formula:
Deng compound, wherein n is that integer is such as 1,2,3 or 4.
Equally, wherein said hetero atom joint is oxygen atom, is present in spacer nipple and maybe can releases The functional end-group of putting on the joint is carbonyl, and required ester group can or be derived by corresponding carboxylic acid Thing obtains with the coupling reaction of alcohol.
Coupling reagent comprises DCC, EDC, CDI, BOP, PyBOP, the different propylene of chloro-carbonic acid, EEDQ, DEAD, PPh3Deng. Solvent comprises CH2Cl
2、CHCl
3, THF, DMF, DMSO, acetonitrile, EtOAc etc. Alkali comprises triethylamine, diisopropyl-ethamine and N, and N-two Methyl-4-aminopyridine. Perhaps, parent acid can be converted to the carbonyl derivative of activation, example Such as acyl chlorides, N-hydroxy-succinamide base ester, pentafluorophenyl group ester etc.
The formation of conventional ketal and acetal
In addition, wherein said hetero atom joint is oxygen atom, is present in spacer nipple and maybe can releases The functional end-group of putting on the joint is the 1-alkoxyalkyl, shown in scheme 2, and required acetal Or ketal group can form the reaction preparation by ketal and the acetal of correspondent alcohol and enol ether. Scheme 2
R
1=H or alkyl
R
2=alkyl
Solvent comprises ethanol, CH
2Cl
2, CHCl
3, THF, ether, DMF, DMSO, acetonitrile, EtOAc etc.The formation usable acid catalyst of this acetal and ketal is finished.Wherein said hetero atom joint comprises two oxygen atoms, but described releasing-joint is for choosing wantonly by radicals X as herein described
2The methylene that replaces, shown in scheme 3, required symmetrical acetal or ketal group can form reaction by acetal and ketal by corresponding pure and mild aldehydes or ketones and form.
Scheme 3
Perhaps, wherein the aryl that is optionally substituted of methylene replaces, and required acetal or ketal can prepare step by step, and wherein L be the leaving group of suitably selection such as halogen, trifluoroacetyl oxygen base, trifluoro-methanesulfonyl oxy etc., shown in scheme 4.Scheme 4 described methods are conventional preparation, generally follow R.R.Schmidt etc., Chem.Rev., and 2000,100, the step that 4423-42 summarized, the disclosure of described document is attached to herein by reference.
Scheme 4
The aryl alkyl ethers of gained produces the intermediate oxonium ion with processing such as oxidant such as DDQ, then handles to produce acetal or ketal with another kind of alcohol.
The formation of conventional butanimide
In addition, wherein said hetero atom joint is as nitrogen-atoms, oxygen atom or sulphur atom, but the functional group that is present on spacer nipple or the releasing-joint is a succinimide derivatives, shown in scheme 5, the carbon-heteroatom bond of gained can form by the Michael addition of corresponding amine, alcohol or mercaptan and maleimide derivant, and wherein X is the hetero atom joint.
Scheme 5
The solvent that forms Michael addition comprises THF, EtOAc, CH
2Cl
2, DMF, DMSO, H
2O etc.The alkali of the available adding equimolar amounts of the formation of this Michael addition chemical compound such as triethylamine, H ü nig alkali or finish by the pH to 6.0-7.4 that regulates aqueous solution.Self-evident, when described hetero atom joint is oxygen or nitrogen-atoms, the scalable reaction condition comes the facilitation Michael addition, activates maleimide for example by employing higher reaction temperatures, adding catalyst, stronger solvent such as DMF, the DMSO etc. of use polarity, and with silylation reagent.
The formation of conventional siloxy
In addition, wherein said hetero atom joint is an oxygen atom, but the functional group that is present on spacer nipple or the releasing-joint is the silicyl derivant, shown in scheme 6, required siloxy can form by making corresponding silicyl derivant and alcohol reaction, and wherein L be the leaving group of suitably selection such as halogen, trifluoroacetyl oxygen base, trifluoro-methanesulfonyl oxy etc.
The silicyl derivant comprises suitably functionalized silicyl derivant, for example vinylsulfonyl alkyl diaryl, or diaryl, or alkylaryl silicyl chlorination thing.Available β-chloro ethylsulfonyl alkyl precursor substitutes the vinylsulfonyl alkyl.Any proton inertia and anhydrous solvent and any nitrogenous base can be used as reaction medium.Temperature range in this transforms can change between-78 ℃ to 80 ℃.
The formation of conventional hydrazone
In addition, wherein said hetero atom joint is a nitrogen-atoms, but the functional group that is present on spacer nipple or the releasing-joint is an imino derivative, and shown in scheme 7 reaction equations (1) and (2), required hydrazone group can form by corresponding aldehydes or ketones and hydrazine or hydrazide derivatives are reacted respectively.
Available solvent comprises THF, EtOAc, CH
2Cl
2, CHCl
3, CCl
4, DMF, DMSO, MeOH etc.The temperature range of using in this transforms can be between 0 ℃ to 80 ℃.Available any acidic catalyst such as mineral acid, H
3CCOOH, F
3CCOOH, p-TsOHH
2O, p-methyl benzenesulfonic acid pyridine etc.With regard to acylhydrazone in the reaction equation (2), described acylhydrazone can prepare by beginning the acidylate hydrazine with suitable carboxylic acid or derivant, such as above-mentioned scheme 1 general introduction, make hydrazides and corresponding aldehydes or ketones react the formation acylhydrazone subsequently.Perhaps, described hydrazone functional group can begin to form by making hydrazine and corresponding aldehydes or ketones reaction.The hydrazone of gained can be subsequently with suitable carboxylic acid or derivant acidylate, such as above-mentioned scheme 1 general introduction.
The formation of conventional disulphide
In addition; wherein said hetero atom joint is a sulphur atom; but the functional group that is present on the releasing-joint is the alkylidene thiol derivative; shown in scheme 8, required disulphide group can form with the reaction of alkylidene thiol derivative by making corresponding alkyl or aryl sulphonyl-mercapto alkyl derivative or corresponding heteroaryl disulfide group alkyl derivative such as pyridine-2-base disulfide group alkyl derivative etc.
Available solvent is THF, EtOAc, CH
2Cl
2, CHCl
3, CCl
4, DMF, DMSO etc.The temperature range of using in this transforms can change between 0 ℃ to 80 ℃.Required alkyl or aryl sulphonyl-mercapto alkyl derivative can prepare with the scheme of this area approval; also can be according to Ranasinghe and Fuchs; Synth.Commun.18 (3), the method preparation of 227-32 (1988), the disclosure of described document is attached to herein by reference.The preparation method of other asymmetric dialkyl disulphides is based on asymmetric heteroaryl-alkyl disulfide for example 2-sulfenyl pyridine radicals, 3-nitro-2-sulfenyl pyridine radicals and the similar transthiolation of disulphide and alkyl hydrosulfide, as WO88/01622, european patent application 0116208A1 and United States Patent (USP) 4,691,024 is described, and the disclosure of described document is attached to herein by reference.
The formation of conventional carbonic ester
In addition, wherein said hetero atom joint is an oxygen atom, but the functional group that is present on spacer nipple or the releasing-joint is the alkoxy carbonyl derivant, shown in scheme 9, required carbonate group can form by chemical compound and the activatory alkoxy carbonyl derivatives reaction that corresponding hydroxyl is replaced, and wherein L is suitable leaving group.
Available solvent is THF, EtOAc, CH
2Cl
2, CHCl
3, CCl
4, DMF, DMSO etc.The temperature range of using in this transforms can change between 0 ℃ to 80 ℃.Any base catalyst for example inorganic base, amine alkali, polymer can be used for the described reaction of facilitation in conjunction with alkali etc.
The formation of conventional semicarbazones
In addition, wherein said hetero atom joint is a nitrogen-atoms, but the functional group that is present on spacer nipple or the releasing-joint is an imino derivative, but the functional group that is present on other spacer nipple or other releasing-joint is alkyl amino or aromatic yl aminocarbonyl derivant, shown in scheme 10, required semicarbazones base can react by corresponding aldehydes or ketones and semicarbazide derivative and form.
Available solvent is THF, EtOAc, CH
2Cl
2, CHCl
3, CCl
4, DMF, DMSO, MeOH etc.The temperature range of using in this transforms can change between 0 ℃ to 80 ℃.Available any acidic catalyst such as mineral acid, H
3CCOOH, F
3CCOOH, p-TsOHH
2O, p-methyl benzenesulfonic acid pyridine etc.In addition, forming aspect the semicarbazones, described hydrazone functional group can begin to form by making hydrazine and corresponding aldehydes or ketones reaction.The hydrazone of gained can be subsequently with isocyanates or carbamoyl derivatives carbamyl halogen acidylate for example, obtain semicarbazones.Perhaps, corresponding semicarbazides can form semicarbazides by making for example carbamyl halogen reaction of hydrazine and isocyanates or carbamoyl derivatives.Semicarbazides can form semicarbazones with corresponding aldehydes or ketones reaction subsequently.
The formation of conventional sulphonic acid ester
In addition; wherein said hetero atom joint is an oxygen atom; but the functional group that is present on spacer nipple or the releasing-joint is a sulfonyl-derivatives; shown in scheme 11; required sulfonate group can form by the chemical compound that corresponding hydroxyl is replaced and the reaction of activatory sulfonyl-derivatives, and wherein L is suitable leaving group such as halogen etc.
Available solvent is THF, EtOAc, CH
2Cl
2, CHCl
3, CCl
4Deng.The temperature range of using in this transforms can change between 0 ℃ to 80 ℃.Any base catalyst for example inorganic base, amine alkali, polymer can be used for the described reaction of facilitation in conjunction with alkali etc.
The formation of conventional folic acid-peptide
Contain folic acid peptidyl fragment Pte-Glu-(AA)
n-NH (CHR
2) CO
2The continuation method that H (3) supports by polymer with standard method Fmoc-strategy for example, goes up at acid-sensitive sense Fmoc-AA-Wang resin (1) and to prepare, shown in scheme 12.
Scheme 12
(a) 20% piperidines/DMF; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF; (c) Fmoc-Glu (O-t-Bu)-OH, PyBop, DIPEA, DMF; (d) 1.N
10(TFA)-Pte-OH; PyBop, DIPEA, DMSO; (e) TFAA, (CH
2SH)
2, i-Pr
3SiH; (f) NH
4OH, pH10.3.
In the illustrative embodiment of methods described herein, R
1Be Fmoc, R
2Be the amino acid side chain of required due care, DIPEA is a diisopropylethylamine.The available standards coupling method, for example PyBOP and other described herein or means known in the art wherein are used as activating reagent to guarantee effective coupling to the coupling agent illustrative.The Fmoc protecting group is removed under standard conditions after each coupling step, and described standard conditions are for example used piperidines, tetrabutylammonium processing such as (TBAF).Shown in scheme 12, the aminoacid member of available due care, for example Fmoc-Glu-OtBu, N
10-TFA-Pte-OH etc., and be representative with Fmoc-AA-OH in the step (b).Therefore, AA is meant the amino acid starting material of any due care.Self-evident, term amino acid used herein is meant anyly have amine and the carboxylic acid functional of being separated by one or more carbon, and comprises naturally occurring α and beta amino acids, and these amino acid whose derivant and analog.Usually, have the protected aminoacid of side chain such as protected serine, threonine, cysteine, aspartate etc. and also can be used for the synthetic of folic acid-peptide as herein described.In addition, γ, δ or longer homologous amino acid, also can be used as raw material be included in folic acid-peptide described herein synthetic in.In addition, have the amino acid analogue or the branched structure of homology side chain, as nor-leucine, 2-amino-3-methylpentanoic acid, Beta-methyl threonine, Beta-methyl cysteine, β, Beta-Dimethylcysteine etc. also can be used as raw material be included in folic acid-peptide described herein synthetic in.
It is inferior that the coupling sequence (step (a) and (b)) that relates to Fmoc-AA-OH is carried out " n ", supports peptide 2 with the preparation solid, and wherein n is integer and can be 0 to about 100.After the last coupling step, remove remaining Fmoc base (step (a)), described peptide then is coupled to (step (c)) on the glutamate derivatives, deprotection, and be coupled on the pteroic acid of TFA-protection (step (d)).Subsequently, described peptide downcuts (step (e)) by with trifluoroacetic acid, dithioglycol and triisopropyl silane treated from polymer support.These reaction conditions cause t-Bu, t-Boc and Trt protecting group to be removed simultaneously, and described protecting group can form the amino acid side chain of part due care.Described TFA protecting group is by (step (f) is removed, and obtains containing folic acid peptidyl fragment 3 with alkali treatment.
But spacer nipple and releasing-joint, and described hetero atom joint can make up by different way.Illustrate, described joint interconnects by the hetero atom joint, for example alkylidene--amino--and alkylidene carbonyl, alkylidene--sulfenyl--carbonylic alkyl butanimide-3-base etc., as shown in the formula further described, wherein integer x and y are 1,2,3,4 or 5:
The illustrative embodiment of another joint described herein, but comprise releasing-joint, and this joint comes cracking by the chemism that relates to β-elimination under condition described herein.On the one hand, but this releasing-joint comprises β-sulfenyl, beta-hydroxy and beta-amino substituted carboxylic acid and derivant thereof, for example ester, amide, carbonic ester, carbamate and urea.On the other hand, but this releasing-joint comprises 2-and 4-sulfenyl aryl ester, carbamate and carbonic ester.
In addition, being connected of vitamin or medicine and hetero atom joint can be made by being present in the medicine that changes into the hetero atom joint or the reactive functional groups on the vitamin, for example aklavine (aclamycin) ketone changes into corresponding hydrazone, folic acid changes into corresponding amide etc., as shown in the formula illustrated:
Bivalence joint (L) but comprise and one or morely be selected from spacer nipple releasing-joint, hetero atom joint and with the components of any order combination.For example, but considered spacer nipple releasing-joint and the hetero atom joint and the combination thereof of table 1 and table 2 illustrated.The catalogue of these joints is not comprehensive, only is illustrative and shall not be construed as restriction to invention described herein.Be positioned at preceding asterisk of said structure and the asterisk that is shown in table 1 and table 2, but be equal to the illustrative junction point of additional space joint releasing-joint or hetero atom joint, or be the medicine of described vitamin receptor binding drug delivery conjugates or the illustrative junction point of vitamin component.Self-evident, but bivalence joint L comprises one or more spacer nipple releasing-joints and hetero atom joint, those joints that comprise table 1 and table 2 illustrated, but and this spacer nipple releasing-joint and hetero atom joint can any order combination to form bivalence joint L.
Table 1.The joint of design, and the combination of some spacer nipple and hetero atom joint
Table 2.The joint of design, but and the combination of some releasing-joint and hetero atom joint
Drug delivery conjugates of the present invention also can be by intermediate preparation.The chemical compound that can prepare in one embodiment, following formula:
V-L-Z
1
Z wherein
1Electrophilic reagent, nucleopilic reagent or precursor for the connection that is applicable to facilitation medicine or its analog or derivant.
On the one hand, Z
1Can be leaving group, its connection that can make medicine is undertaken by being present in hetero atoms such as nucleophilic residues on medicine or its analog or the derivant such as nitrogen.
On the other hand, Z
1Can be nucleopilic reagent, hetero atom such as nitrogen for example, it can replace the leaving group that is present on medicine or its analog or the derivant, for example carboxylic acid derivates such as acyl chlorides.
On the other hand, Z
1Can be precursor, for example can make the nitro of nucleophilic nitrogen, or can make the ester of close electric acyl chlorides by continuous hydrolysis and chlorination through reduction reaction.Self-evident, Z
1Can be the hetero atom joint.
In another embodiment, described drug delivery conjugates can be by following intermediate preparation:
Z
2-L-D
Z wherein
2Electrophilic reagent, nucleopilic reagent or precursor for the connection that is applicable to facilitation vitamin or its analog or derivant.
On the one hand, Z
1Can be leaving group, its connection that can make vitamin is undertaken by being present in hetero atoms such as nucleophilic residues on vitamin or its analog or the derivant such as nitrogen.
On the other hand, Z
1Can be nucleopilic reagent, hetero atom such as nitrogen for example, it can replace the leaving group that is present on vitamin or its analog or the derivant, for example carboxylic acid derivates such as acyl chlorides.
On the other hand, Z
1Can be precursor, for example can make the nitro of nucleophilic nitrogen, or can make the ester of close electric acyl chlorides by continuous hydrolysis and chlorination through reduction reaction.Self-evident, Z
2Can be the hetero atom joint.
In another embodiment, bivalence joint (L) can synthesize respectively, in subsequent step, be connected to then on vitamin and the medicine, for example by preparing the midbody compound of following formula:
Z
1-L-Z
2
Z wherein
1And Z
2Independent separately selection the, and as above definition.
Drug delivery conjugates of the present invention is used applicable to people's clinical medicine and veterinary.Therefore, contain described pathogenic cell colony and can be the people with the host animal of described vitamin receptor binding drug delivery conjugates treatment, or under veterinary's applicable cases, can be laboratory animal, agriculture and animal husbandry domestic animal, domestic animal or wild animal.The host animal that the present invention can be applicable to includes but not limited to the people, laboratory animal such as rodent (for example mice, rat, hamster etc.), rabbit, monkey, chimpanzee, performing animal such as Canis familiaris L., cat and rabbit, agriculture and animal husbandry animal such as cattle, horse, pig, sheep, goat, and the wild animal of stable breeding such as Bears, panda, lion, tiger, leopard, resemble, zebra, giraffe, gorilla, dolphin and whale.
The present invention is applicable to the pathogenic cell colony that causes various pathological changes in these host animal bodies.According to the present invention " pathogenic cell " be meant cancerous cell, infectant such as antibacterial and virus, antibacterial or viral infection cell, can cause that the activated macrophage of morbid state and any other unique express, preferentially express or overexpression vitamin receptor or in conjunction with the pathogenic cell of the receptor of vitamin D 3-analogies or derivant.Pathogenic cell also can comprise any disease that it causes, with vitamin receptor binding drug delivery conjugates treatment of the present invention, the cell that causes described disease symptoms to alleviate.For example, described pathogenic cell can be that tool is pathogenic in some cases, but the pathogenic host cell of tool not in other cases, for example causes the immune system cell of graft versus host disease.
Therefore, pathogenic cell colony can be the tumorigenesis cancer cell population of (comprising benign tumor and malignant tumor), also can be tumorigenesis not.Described cancer cell population can spontaneous generation or because sudden change in being present in the host animal system genitale or the process the somatic mutation produce, or it can induce generation by chemistry, virus or radiation.The present invention can be used for the cancer of treatment such as cancer, sarcoma, lymphoma, Hodgkin (Hodgekin ' s disease), melanoma, mesothelioma, Burkitt lymphoma (Burkitt ' s lymphoma), nasopharyngeal carcinoma, leukemia and myeloma.Described cancer cell population can include but not limited to oral cancer, thyroid carcinoma, endocrine adenocarcinoma, skin carcinoma, gastric cancer, the esophageal carcinoma, laryngeal carcinoma, cancer of pancreas, colon cancer, bladder cancer, osteocarcinoma, ovarian cancer, cervical cancer, uterus carcinoma, breast carcinoma, carcinoma of testis, carcinoma of prostate, rectal cancer, renal carcinoma, hepatocarcinoma and pulmonary carcinoma.
In pathogenic cell colony is in the embodiment of cancer cell population, and the effect of conjugate administration is served as reasons and reduced or eliminated tumor mass or suppress the therapeutic response that tumor cell proliferation is measured.With regard to tumor, eliminate the elimination can be the primary tumor cell, for shifted or be elimination at cell from the primary tumor separation process.With described vitamin receptor binding drug delivery conjugates prophylactic treatment, to prevent that tumor from by any Therapeutic Method, comprising that ocal resection, radiotherapy, chemotherapy or biotherapy recur after removing, and also think over according to the present invention.Described prophylactic treatment can be with the treatment of initial stage of drug delivery conjugates, for example with the treatment of multiple dose scheme every day, and/or can be initial stage treatment back additional treatment or the serial therapy after several days or several months at interval.Therefore, the elimination of the pathogenic cell colony that handles according to the present invention comprises minimizing pathogenic cell quantity, suppresses the treatment that pathogenic cell is bred, prevented the prophylactic treatment of pathogenic cell recurrence or causes the pathogenic cell that disease symptoms alleviates.
Under the situation that cancerous cell will be eliminated, but method coupling ocal resection of the present invention, radiotherapy, chemotherapy or biotherapy, for example other immunotherapy includes but not limited to monoclonal antibody therapy, the adoptive transfer with immunosuppressant treatment, immune effector cell, usefulness hemopoietic growth factor, cytokine and vaccine therapy.
The present invention also is applicable to the pathogenic cell colony that causes various infectious disease.For example, the present invention is applicable to such as antibacterial, comprises the pathogenic cell colony zymic fungus, virus, virus infected cell, mycoplasma and the parasite.The infectious organisms of available drug delivery conjugates treatment of the present invention is any infectious organisms that causes this area approval of animal morbidity, comprises the organism such as Gram-negative or gram-positive cocci or bacillus.For example, Bacillus proteus (Proteus sp.), Cray Bai Shi bacillus (Klebsiella sp.), Providence (Providencia sp.), yersinia (Yersinia sp.), Erwinia (Erwiniasp.), enterobacteria (Enterobacter sp.), Salmonella (Salmonella sp.), Serratieae (Serratia sp.), gas bacillus (Aerobacter sp.), Escherichia (Escherichia sp.), pseudomonas (Pseudomonas sp.), shigella (Shigella sp.), vibrio (Vibrio sp.), Aeromonas (Aeromonas sp.), Campylobacter (Campylobacter sp.), streptococcus (Streptococcus sp.), staphylococcus (Staphylococcus sp.), lactobacillus (Lactobacillussp.), micrococcus luteus (Micrococcus sp.), mora gram Salmonella (Moraxella sp.), bacillus cereus (Bacillus sp.), clostridium (Clostridium sp.), corynebacterium (Corynebacterium sp.), Erichsen bacterium (Eberthella sp.), micrococcus luteus (Micrococcussp.), mycobacteria (Mycobacterium sp.), Neisseria (Neisseria sp.), haemophilus (Haemophilus sp.), bacteroid (Bacteroides sp.), listeria spp (Listeriasp.), Erysipelothrix (Erysipelothrix sp.), acinetobacter calcoaceticus (Acinetobacter sp.), brucella (Brucella sp.), pasteurella (Pasteurella sp.), vibrio (Vibrio sp.), Flavobacterium (Flavobacterium sp.), Fusobacterium (Fusobacterium sp.), streptobacillus (Streptobacillus sp.), calymmatobacterium granulomatis (Calymmatobacterium sp.), legionella (Legionella sp.), treponema (Treponema sp.), burgdorferi (Borrelia sp.), leptospira (Leptospira sp.), actinomycetes (Actinomyces sp.), Nocard's bacillus (Nocardia sp.), rickettsia (Rickettsia sp.) and any other cause the antibacterial of the disease of available drug delivery conjugates treatment of the present invention among the host.
What is interesting is especially, the antibacterial of tool antibiotic resistance such as antibiotic resistance streptococcus and staphylococcus, or to antibiotic sensitive but cause with the recurrent infection of antibiotic therapy so that finally develop into the antibacterial of drug resistance organism.To antibiotic sensitive but cause with the recurrent infection of antibiotic therapy so that finally develop into the antibacterial of drug resistance organism, available drug delivery conjugates of the present invention is treated under the non-existent situation of antibiotic, or with the antibiotic coupling lower than the dosage that gives the patient under the normal condition, to avoid the development of these antibiotics resistance bacterial strains.
Virus, for example DNA and RNA viruses also can be treated according to the present invention.This viroid includes but not limited to DNA viruses such as human papillomavirus (papilloma viruses), parvovirus (parvoviruses), adenovirus (adenoviruses), herpesvirus (herpesviruses) and vaccinia virus (vaccina viruses), and RNA viruses such as arenavirus (arenaviruses), coronavirus (coronaviruses), rhinovirus (rhinoviruses), respiratory syncytial virus (respiratory syncytial viruses), influenza virus (influenza viruses), picorna virus (picornaviruses), paramyxovirus (paramyxoviruses), reovirus (reoviruses), retrovirus (retroviruses), slow virus (lentiviruses) and rhabdovirus (rhabdoviruses).
The present invention also is applicable to any fungus, comprises that yeast (yeast), mycoplasma (mycoplasmasp.), parasite (parasite) or other cause the infectious organisms of Animal diseases.The example of the fungus of available the inventive method and combination treatment comprises as mould growth or is the fungus of yeast sample, for example comprises the fungus that causes such as following disease: tinea, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, mucormycosis, chromoblastomycosis, dermatomycosis, Protothecosis, fusaridiosis, pityriasis, mycetoma, Brazilian blastomycosis, phaeohyphomycosis, pseudallescheriasis, sporotrichosis, trichosporosis, lung sac insect infection and candidiasis.
The present invention also can be used to treat parasitic infection, include but not limited to the infection that following organism causes: cestode (tapeworms), for example cestode (Taenia sp.), Hymenolepis (Hymenolepsis sp.), diphyllobothrium (Diphyllobothrium sp.) and echinococcus (Echinococcus sp.); Trematodiasis (flukes), for example fasciloopsis (Fasciolopsis sp.), heterophyid (Heterophyes sp.), after grow trematodiasis (Metagonimus sp.), a testis trematodiasis (Clonorchis), sheet trematodiasis (Fasciola sp.), paragonimus (Paragonimus sp.) and schistosomicide (Schitosoma sp.); Nematicide (roundworms), for example pinworm (Enterobius sp.), whipworm (Trichuris sp.), ascarid (Ascaris sp.), ancylostome (Ancylostoma sp.), plate mouth nematode (Necator sp.), quasi-colubriformis (Strongyloides sp.), trichinella (Trichinellasp.), Wuchereria (Wuchereria sp.), cloth Shandong filaricide (Brugia sp.), Loa loa (Loasp.), filaria volvulus (Onchocerca sp.) and imperial nematicide (Dracunculus sp.); Ameba (ameba), for example Na Shi worm (Naegleria sp.) and Acanthamoeba (Acanthamoeba sp.); And protozoon (protozoans), for example plasmodium (Plasmodium sp.), trypanosomicide (Trypanosomasp.), leishmania (Leishmania sp.), toxoplasma (Toxoplasma sp.), inner amoeba (Entamoeba sp.), giardia lamblia (Giardia sp.), isospora (Isospora sp.), Cryptosporidium (Cryptosporidium sp.) and intestinal born of the same parents worm (Enterocytozoon sp.).
The related pathogenic cell of drug delivery conjugates of the present invention also can be the cell that comprises endogenous pathogens, the cell of virus, mycoplasma, parasite or bacterial infection for example, and condition is that these cells are preferentially expressed vitamin receptor.
In one embodiment, vitamin receptor binding drug delivery conjugates can by in conjunction with vitamin part to vitamin receptor, transport protein or other specificity in conjunction with vitamin and preferentially exist on the albumen on the surface of expressing on the pathogenic cell, internalization is in the targeting pathogenic cell.For example, this internalization can take place by receptor-mediated endocytosis.If but drug delivery conjugates contains releasing-joint, then the vitamin part can be separated in born of the same parents with medicine, and medicine can work to its born of the same parents' internal target.
In an alternative embodiment, the vitamin of drug delivery conjugates part can be attached on the pathogenic cell, is combined closely in medicine and pathogenic cell surface.But described medicine can be subsequently cracking by releasing-joint be released.For example, if but described releasing-joint is a disulfide group, then described medicine can be discharged by protein disulphideisomerase.Described medicine can be absorbed by described vitamin receptor binding drug delivery conjugates combination pathogenic cell thereon subsequently, or described medicine can be close to the pathogenic cell picked-up of this cell by another.Perhaps, described medicine can be discharged by the protein disulphideisomerase of cell interior, but wherein said releasing-joint is a disulfide group.Described medicine also can discharge by hydrolysis mechanism, and acid-catalyzed hydrolysis is for example eliminated as described in mechanism as above some β, or is discharged by oxonium ion or lactone ion mechanism of production by the anchimeric assistance cracking.But the mechanism that medicine discharges is depended in the selection of one or more releasing-joints from conjugate.Self-evident, this selection can be defined in advance by the service condition of drug conjugate.
But do not comprise in the embodiment of releasing-joint at another described joint, described drug delivery conjugates vitamin part can be attached on the pathogenic cell, makes medicine place the pathogenic cell of pathogenic cell surface will attack by other molecular orientation that can be attached on the medicine.Perhaps, in this embodiment, described drug delivery conjugates can by in conjunction with internalization in targeted cells, and described vitamin part and described medicine can keep that cell is interior to be connected, wherein said medicine is brought into play its effect and is not needed partly to separate from described vitamin.
In yet another embodiment, or with the combination of above-mentioned embodiment, described vitamin receptor binding drug delivery conjugates can the mechanism of vitamin receptor work in the cell by relying on.For example, described drug delivery conjugates can be attached to and be present in the serum or be attached on the solubility vitamin receptor on the serum albumin, cause the circulation of described conjugate not prolong, and cause described conjugate not improve with respect to puting together medicine at the activity of pathogenic cell colony with respect to puting together medicine.
In another embodiment of the present invention, provide the vitamin receptor binding drug delivery conjugates of general formula V-L-D.L is selected from (l
s)
a, (l
H)
bAnd combination, wherein (l
s)
a, (l
H)
bWith V as defined herein, D is medicine such as immunogen.Described immunogen can be hapten, for example fluorescein, dinitrophenyl etc.In this embodiment, described vitamin receptor binding drug delivery conjugates is attached to described pathogenic cell surface, and with immunogen " labelling " cell, triggers the immunoreation at the pathogenic cell colony of labelling thus.In passive immunity, give host's antibody, or be present in the host system inbornly or, be attached on the immunogen, trigger endogenous immune response from the antibody that acquired immunity gets.Be attached to cell-, cause the cytotoxicity of complement-mediated, cell-mediated cytotoxicity, antibody opsonification and the phagocytosis of antibody-dependence, the transfer cell death of receptor clustering signal or dormancy or any body fluid or the cell immune response that other is stimulated to cell-binding partner-immunogen conjugates by antibodies of antibody induction in conjunction with the antibody on vitamin-immunogen conjugates.Can be need not under the situation of previous antibody opsonification by the immunocyte Direct Recognition in immunogen, directly causing death of pathogenic cell can be taken place.The more detailed description of this embodiment is seen U.S. Patent application 09/822,379, and this patent is attached to herein by reference.Self-evident, medicine is in the modification of immunogenic some this embodiment therein, but the bivalence joint also can comprise releasing-joint, as mentioned above, the vitamin receptor binding drug delivery conjugates of general formula V-L-D for example, wherein L is selected from (l
s)
a, (l
H)
b, (l
r)
cAnd combination, wherein (l
s) be spacer nipple, (l
H) be the hetero atom joint, (l
rBut) be releasing-joint, V is vitamin or its analog or derivant, a, b and c are integer.
Vitamin receptor binding drug delivery conjugates described herein comprises vitamin receptor bound fraction, bivalence joint (L), medicine and optional described vitamin receptor bound fraction and the described medicine hetero atom joint to the bivalence joint (L) that is connected.Bivalence joint (L) can comprise spacer nipple, can discharge (being cleavable) joint and hetero atom joint or its combination.
Vitamin receptor binding drug delivery conjugates described herein can be by very a large amount of vitamin or receptors bind vitamin D 3-analogies/derivant, joint and medication preparation.Drug delivery conjugates of the present invention can the intravital pathogenic cell of selectivity targeting host animal colony, and reason is to be applicable to that the bonded vitamin receptor of vitamin preferentially expresses on pathogenic cell.Illustrative vitamin partly comprises carnitine, inositol, thioctic acid, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., ascorbic acid, nicotinic acid, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B
12, and fat-soluble A, D, E and K.These vitamin and receptors bind analog and derivant constitute the targeting entity, described entity can be with described medicine by bivalence joint (L) coupling to form vitamin receptor binding drug delivery conjugates described herein.Therefore, term " vitamin " comprises vitamin D 3-analogies and/or derivant (for example being the pteroic acid of folic acid derivatives, biotin analog biological example born of the same parents element, biotinylsulfoxide, oxybiotin and other biotin receptor binding compounds etc.).Self-evident, according to the present invention, vitamin D 3-analogies or derivant can refer to mix heteroatomic vitamin, and vitamin D 3-analogies or derivant are covalently bound to bivalence joint (L) by described hetero atom joint.
Illustrative vitamin partly comprises folic acid, biotin, riboflavin, thiamine, vitamin B
12With the receptors bind analog and the derivant of these vitamin molecules, and other relevant vitamin receptor binding molecule.The embodiment of illustrative vitamin D 3-analogies and/or derivant comprises folacin and derivant such as folinic acid, pteroylglutamic acid, and folacin receptor is in conjunction with pteridine such as tetrahydrochysene pterin, dihydrofoilic acid, tetrahydrofolic acid and denitrogenation thereof and two denitrogenation analog.Term " denitrogenation " and " two denitrogenations " analog are meant to have the analog that substitutes this area approval of one or two nitrogen-atoms in naturally occurring folic acid structure or its analog or the derivant with carbon atom.For example, described denitrogenation analog comprises 1-denitrogenation, 3-denitrogenation, 5-denitrogenation, 8-denitrogenation and 10-denitrogenation folacin.Described two denitrogenation analog comprise, for example 1, and 5-two denitrogenations, 5,10-two denitrogenations, 8,10-two denitrogenations and 5,8-two denitrogenation folacins.Other is as the folic acid of the complex that forms part of the present invention, for folacin receptor in conjunction with analog aminopterin, methotrexate (methotrexate), N
10-methopterin, 2-deaminizating-hydroxyl folic acid, denitrogenation analog for example 1-denitrogenation methotrexate or 3-denitrogenation methotrexate and 3 ', 5 '-two chloro-4-amino-4-'-deoxy-ns
10-methyl pteroylglutamic acid (dichioromethotrexate).Aforementioned folacin and/or derivant are commonly referred to " folic acid ", reflect their abilities in conjunction with folacin receptor, and this class part is effective to improving transmembrane transport when puting together with exogenous molecules, for example through folate-mediated endocytosis as herein described.Other can be attached on the folacin receptor and the suitable ligand that starts receptor-mediated complex endocytosis transhipment comprises the anti-idiotype antibody of anti-folacin receptor.With the compound exogenous molecule of the anti-idiotype antibody of anti-folacin receptor, be used to trigger the transmembrane transport of complex of the present invention.
The embodiment of illustrative vitamin D 3-analogies and/or derivant also comprises the analog and the derivant of biotin, biological example born of the same parents element, biotinylsulfoxide, oxybiotin and other biotin receptor binding compounds etc.Self-evident, the analog and the derivant of other vitamin described herein are also considered at this paper.In one embodiment, the vitamin that can be used for drug delivery conjugates described herein comprises the vitamin that is attached on the vitamin receptor, described vitamin receptor specificity is expressed on activated macrophage, the folacin receptor in conjunction with folic acid or its analog or derivant for example described herein.
The binding site of vitamin can comprise the receptor of any vitamin molecules or derivatives thereof or analog, and this position can be attached on the receptor by specificity, and wherein said receptor or other albumen are by the unique expression of pathogenic cell colony, overexpression or preferential the expression.There is albumen by the unique expression of pathogenic cell, overexpression or preferential surface of expressing, typically is and is not present in or is present in receptor on the non-pathogenic cell, provide selectivity to eliminate the method for pathogenic cell with low concentration.Described vitamin receptor binding drug delivery conjugates can high-affinity be attached on the receptor on cancerous cell or other type pathogenic cell.Described high-affinity is in conjunction with being that described vitamin part is inherent, and perhaps binding affinity also can be used for strengthening by the making of vitamin (being analog or derivant) of chemical modification.
Described medicine can be anyly can be regulated or change the molecule of cell function in other mode, comprises pharmaceutical active compounds.Suitable molecule can include but not limited to: peptide, oligopeptide, oppositely inversion oligopeptide, protein, wherein at least one non-peptide bond replaces protein analogue, apoprotein, glycoprotein, enzyme, coenzyme, enzyme inhibitor, aminoacid and derivant thereof, receptor and other memebrane protein of peptide bond; Antigen and antibody thereof; Hapten and antibody thereof; Hormone, lipid, phospholipid, liposome; Toxin; Antibiotic; Analgesic; Bronchodilator; Beta-receptor blockader; Antimicrobial drug; Antihypertensive; Cardiovascular drug comprises antiarrhythmics, cardiac glycoside, anti-anginal drug and vasodilator; Central nervous system's medicine comprises central nervous excitation agent, psychotropic thing, antimanic drugs and tranquilizer; Antiviral agents; Antihistaminic; Anticarcinogen comprises chemotherapeutic; The tranquillizer; Antidepressants; The H-2 receptor antagonist; Anticonvulsant; Antinauseant; Prostaglandin and prostaglandin analogue; Muscle relaxant drug; The antibiotic medicine; Analeptic; Decongestant drug; Bendectin; Diuretic; Anti-spasmodics; Antiasthmatics; Antiparkinsonian drug; Expectorant; Cough medicine; Mucolytic; With mineral and nourishing additive agent.
In addition, described medicine can be any medicine known in the art, and described medicine is cytotoxicity, improve the tumor permeability, suppress tumor cell proliferation, promote apoptosis, reduce anti-apoptosis activity in the target cell, be used for the treatment of disease that infectant causes, strengthen at the endogenous immunoreation of pathogenic cell or be applicable to the medicine of the disease that treatment is caused by any kind pathogenic cell.Be applicable to that medicine of the present invention comprises adrenocortical hormone and corticosteroid, alkylating agent, antiandrogen, antiestrogen, androgen, aklavine and aklavine derivant, estrogen, antimetabolite such as cytosine arabinoside, purine analogue, pyrimidine analogue and methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, taxol, paclitaxel, paclitaxel derivant, Docetaxel _, cyclophosphamide, daunomycin, rhizomycin, the T2 toxin, plant alkaloid, prednisone, hydroxyurea, teniposide, mitomycin, dish suberite lactone, the microtubule inhibitor, Macrolide antineoplastic agent (epothilones), tubulysin, cyclopropyl benzo [e] indolone, open loop-cyclopropyl benzo [e] indolone, O-Ac-open loop-cyclopropyl benzo [e] indolone, bleomycin and any other antibiotic, chlormethine, nitroso ureas, vincristine, vinblastine and analog thereof and derivant are for example taken off acetyl vinblastine one hydrazides, colchicine, colchicine derivative, different colchicine, muscoril, the trityl cysteine, halichondrin B (HalicondrinB), dolastatin is dolastatin 10 for example, amanitin is α-amanitin for example, camptothecine, Irinotecan and other camptothecin derivative, geldanamycin and geldanamycin derivant, estramustine, nocodazole, MAP4, Demecolcine, inflammatory factor and proinflammatory factor, the medicine or the toxin of peptide and peptide mimics signal transduction inhibitor and the approval of any this area.Other comprises the Antimicrobe compound of penicillin, cephalosporin, vancomycin, erythromycin, clindamycin, rifampicin, chloromycetin, aminoglycoside antibiotics, gentamycin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin and any other this area approval applicable to medicine of the present invention.
In one embodiment, being used for medicine of the present invention kept stable 4 hours at serum at least.In another embodiment, the IC of medicine
50In the nanomole scope, in another embodiment, medicine is water miscible.If medicine is not water miscible, bivalence joint (L) can be by derivatization to increase water solublity.Term " medicine " also refers to any said medicine analog or derivant, includes but not limited to for example dolastatin 10, the amanitin derivant of α-amanitin, camptothecine and Irinotecan and other camptothecine and Irinotecan for example of dolastatin.Self-evident, according to the present invention, drug analogue or derivant can refer to mix heteroatomic medicine, and described drug analogue or derivant are covalently bound to bivalence joint (L) by described hetero atom joint.
Vitamin receptor binding drug delivery conjugates of the present invention can comprise vitamin receptor bound fraction, bivalence joint (L), medicine and optional described vitamin receptor bound fraction and the described medicine hetero atom joint to the bivalence joint (L) that is connected.Self-evident, according to the present invention, vitamin D 3-analogies or derivant can refer to mix heteroatomic vitamin, and described vitamin D 3-analogies or derivant are covalently bound to bivalence joint (L) by described hetero atom joint.Therefore, described vitamin can be covalently bound to bivalence joint (L) by described hetero atom joint, and perhaps vitamin D 3-analogies or derivant (promptly being mixed with hetero atom) can directly be connected on the bivalence joint (L).Equally, for the drug analogue or the derivant of medicine of the present invention and drug analogue or derivant can refer to mix heteroatomic medicine, described drug analogue or derivant are covalently bound to bivalence joint (L) by described hetero atom joint.Therefore, described medicine can be covalently bound to bivalence joint (L) by described hetero atom joint, and perhaps drug analogue or derivant (promptly being mixed with hetero atom) can directly be connected on the bivalence joint (L).Bivalence joint (L) can comprise spacer nipple, can discharge (being cleavable) joint, but and connects in the conjugate contain these two kinds of joints the hetero atom joint to the releasing-joint of joint at interval.
Therefore, according to the present invention, bivalence joint (L) can comprise the method that connects vitamin and medicine, for example connects by hetero atom joint (being spacerarm or bridging molecules), or closes bivalence joint (L) on vitamin or drug analogue or derivant by direct covalent bonds.Each method of attachment should not hinder vitamin or vitamin receptor bound derivative or analog to be attached on the vitamin receptor on the cell membrane, so that the inventive method can be operated.
Usually, can any way used according to the invention to form the conjugate between conjugate, bivalence joint (L) and described medicine or its analog or derivant between bivalence joint (L) and described vitamin or its analog or derivant, comprise any conjugate that interleaves the hetero atom joint.Similarly, but can use the conjugate method between the described spacer nipple of formation, described releasing-joint and the described hetero atom joint of any this area approval to form bivalence joint (L).Described conjugate can by any of these molecule directly put together formation, for example by hydrogen bond, ionic bond or covalent bond.For example, covalent bond can exist by amido link, ester bond, disulfide bond or the imido key that forms between acidic group, aldehyde radical, hydroxyl, amino, sulfydryl or hydrazo-.
But spacer nipple and/or releasing-joint (being the cleavable joint) can be any bio-compatible joint.For example, the cleavable joint can be under reduction in being present in cell or on the cell or the oxidizing condition and be easy to cracked joint, can be sour instability or the unstable joint of alkali the responsive joint of pH or can be by biochemistry or cracked joint of metabolic process such as the unstable joint of enzyme.Typically, but spacer nipple and/or releasing-joint comprise about 1 to about 30 carbon atoms, more typically, about 2 to about 20 carbon atoms.Usually use the low-molecular-weight joint (be molecular weight approximate about 30 to about 300 molecule).The precursor of this class joint usually selects to have nucleophilic or parent's electricity, or the two functional group, optional with the protected form that has cracked protecting group easily promoting their application in intermediate forms is synthetic.
The present invention also relates to comprise the pharmaceutical composition of the vitamin receptor binding drug delivery conjugates of effective dose, when with one or more dosed administration, effectively eliminate the intravital pathogenic cell of host animal colony.The preferred parenteral of described drug delivery conjugates gives host animal, for example gives in Intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous or the sheath.Perhaps, when described drug delivery conjugates gives host animal, can pass through other medical effective way such as oral, and can use any effective dose and suitable therapeutic dosage forms, comprise slow release formulation.
Isotonic saline solution, 5% glucose or the acceptable liquid-carrier of other well-known medicine that the example of parenteral dosage form comprises activating agent be the aqueous solution of liquid alcohol, dihydroxylic alcohols, ester and amide for example.Parenteral dosage form of the present invention can be for comprising the reconstruction lyophilized form of doses drug delivery conjugates.Aspect of this embodiment, can give more any slow release formulations known in the art, for example, be described in United States Patent (USP) 4,713,249; 5,266,333; With 5,417,982 biodegradable carbonic ester substrate, its disclosed content is attached to herein by reference, perhaps, can use deceleration pump (for example osmotic pumps).
At least a additional set compound for the treatment of the factor that comprises can give the host with the said method coupling or as the adjuvant of said method, and the elimination with the drug delivery conjugates that strengthens pathogenic cell colony perhaps can give more than a kind of additional procedures factor.The described treatment factor is optional from stimulating endogenous immunoreactive chemical compound, chemotherapeutic or another can replenish the treatment factor of the drug delivery conjugates effectiveness that is given.Except giving the above-mentioned conjugate of host, method of the present invention can stimulate endogenous immunoreactive chemical compound or compositions (for example cytokine) to implement by giving the host, and described chemical compound or compositions include but not limited to cytokine or immune cell growth factor such as interleukin-11-18, stem cell factor, basic FGF, EGF, G-CSF, GM-CSF, FLK-2 part, HILDA, MIP-1 α, TGF-α, TGF-β, M-CSF, IFN-α, IFN-β, IFN-γ, solubility CD23, LIF and combination thereof.
Can use the treatment of these factors effectively to make up.For example, in one embodiment, for example, the scope of measuring in the scheme of multiple dose every day is about 0.1MIU/m
2/ agent/sky is to about 15MIU/m
2The IL-2 of the treatment effective dose in/agent/sky and the scope of measuring in the scheme of multiple dose every day are about 0.1MIU/m
2/ agent/sky is to about 7.5MIU/m
2The IFN-α of the treatment effective dose in/agent/sky can be used to eliminate, reduce or offsets the intravital pathogenic cell of the host animal that contains pathogenic cell (MIU=hundred million international units with described drug delivery conjugates; m
2=approximate ordinary people's body surface area).IL-12 and IFN-α are with above-mentioned treatment effective dose in another embodiment, and as interleukin and interferon combination, in yet another embodiment, IL-15 and IFN-α are with above-mentioned treatment effective dose, as interleukin and interferon combination.In an alternative embodiment, IL-2, IFN-α or IFN-γ and GM-CSF are used in combination with above-mentioned treatment effective dose.The present invention also considers any other effective cell combinations of factors, comprises the combination of other interleukin and interferon and colony stimulating factor.
For example, itself be Cytotoxic or can be used to increase the chemotherapeutic of tumor permeability, also be fit in the methods of the invention unite use with drug delivery conjugates.This based chemotherapy medicated bag is drawn together adrenocortical hormone and corticosteroid, alkylating agent, antiandrogen, antiestrogen, androgen, aklavine and aklavine derivant, estrogen, antimetabolite such as cytosine arabinoside, purine analogue, pyrimidine analogue and methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, taxol, paclitaxel, paclitaxel derivant, Docetaxel _, cyclophosphamide, daunomycin, rhizomycin, the T2 toxin, plant alkaloid, prednisone, hydroxyurea, teniposide, mitomycin, dish suberite lactone, the microtubule inhibitor, the Macrolide antineoplastic agent, tubulysin, cyclopropyl benzo [e] indolone, open loop-cyclopropyl benzo [e] indolone, O-Ac-open loop-cyclopropyl benzo [e] indolone, bleomycin and any other antibiotic, chlormethine, nitroso ureas, vincristine, vinblastine and analog thereof and derivant are for example taken off acetyl vinblastine one hydrazides, colchicine, colchicine derivative, different colchicine, muscoril, the trityl cysteine, halichondrin B, dolastatin is dolastatin 10 for example, amanitin is α-amanitin for example, camptothecine, Irinotecan and other camptothecin derivative, geldanamycin and geldanamycin derivant, estramustine, nocodazole, MAP4, Demecolcine, inflammatory factor and proinflammatory factor, the medicine or the toxin of peptide and peptide mimics signal transduction inhibitor and the approval of any this area.Other comprises the Antimicrobe compound of penicillin, cephalosporin, vancomycin, erythromycin, clindamycin, rifampicin, chloromycetin, aminoglycoside antibiotics, gentamycin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, maytansine and analog thereof and derivant, gemcitabine and any other this area approval applicable to medicine of the present invention.
The treatment factor can be before described vitamin receptor binding drug delivery conjugates, give host animal afterwards or simultaneously, and the described treatment factor part same combination that can contain described drug delivery conjugates gives or be different from part the compositions administration of described drug delivery conjugates.Any this class contains the therapeutic combination of the treatment factor for the treatment of effective dose, can be used for the present invention.
In addition, can use more than one type drug delivery conjugates.For example, host animal can be in the co-administered scheme, and with having different vitamin, but the conjugate of same medicine is treated (folic acid-mitomycin conjugate and vitamin B for example
12-mitomycin conjugate).In other embodiment, host animal can be treated with containing the conjugate that identical vitamin is connected with different pharmaceutical, or treats with the conjugate that different vitamin are connected with different pharmaceutical.For example, host animal can be treated with folic acid-mitomycin and folic acid-cisplatin conjugate, perhaps uses folic acid-mitomycin and vitamin B
12-cisplatin conjugate is treated.In addition, can use the drug delivery conjugates that has identical or different vitamin, or comprise as the multivitamin of identical drug delivery conjugates ingredient and the drug delivery conjugates of multiple medicine.
The drug delivery conjugates of single daily dose can be according to for example coupling probability of radiotherapy and the significantly change of molecular weight, its route of administration and the tissue distribution of host's condition, the disease that will treat, conjugate and other Therapeutic Method.The effective dose that gives patient depends on patient's surface area, body weight and the doctor diagnosis to the patient disease.The effective dose mobility scale for about 1ng/kg for example to about 1mg/kg, about 1 μ g/kg to about 500 μ g/kg with about 1 μ g/kg about 100 μ g/kg extremely.
Any effective therapeutic regimen that gives drug delivery conjugates can use.For example, described drug delivery conjugates can single dose give maybe can separate and with every day multiple dose give.In addition, staggered therapeutic regimen, for example, the selective therapy that can be used as treatment every day on every Mondays by three days, and in order to define purpose of the present invention, these interruptions or staggered therapeutic regimens every day is considered to be equal to the treatment of every day and within the scope of the present invention.In one embodiment of the invention, the host treats to eliminate pathogenic cell colony with the described drug delivery conjugates of multiple injection.In one embodiment, the host is by multiple injection (preferred about 2 times be up to about 50 times) drug delivery conjugates, for example, and 12-72 hour time period or 48-72 hour time period.Drug delivery conjugates of extra injection can be after initial injection several days or some months gave in the time period, and the palindromia that causes of described extra injection prevention pathogenic cell.
In one embodiment, vitamin or its analog or derivant can be used for drug delivery conjugates of the present invention, comprise being attached to the conjugate of specificity on the receptor of expressing on the activated macrophage that described receptor is for example in conjunction with the folacin receptor of folic acid or its analog or derivant.For example, the conjugate of connection folic acid can be used for killing activated macrophage that causes host disease or the activity that suppresses described activated macrophage.This macrophage targeting conjugate when the patient of the disease of suffering from activated macrophage mediation, causes to concentrate and in conjunction with puting together medicine to kill activated macrophage or to suppress macrophage function in the activated macrophage colony.The elimination of activated macrophage colony, minimizing or inactivation cause the stopping or reducing of pathogeneticing characteristic of the activated macrophage mediation that will treat disease.Known exemplary disease by the activated macrophage mediation comprises rheumatoid arthritis, ulcerative colitis, Crohn disease, psoriasis, osteomyelitis, multiple sclerosis, atherosclerosis, pulmonary fibrosis, sarcoidosis, Sjogren's syndrome disease, organ-graft refection (GVHD) and chronic inflammatory disease.The administration of described drug delivery conjugates lasts till when described disease symptoms reduces or eliminates usually.
Be used to kill activated macrophage or suppress the activated macrophage function drug delivery conjugates can for example Intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous be mixed in animal or the patient who suffers from described disease with pharmaceutically acceptable carrier by parenteral.Perhaps, described drug delivery conjugates gives by other medical science usability methods, effective dose can standard or slow release formulation give.Described Therapeutic Method can use separately or with other treatment of generally acknowledging by the disease mediated Therapeutic Method coupling of activated macrophage.
Following drug delivery conjugates is illustrative drug delivery conjugates, thinks that they are in the scope of the invention described herein.These drug delivery conjugates can be used the methods described herein preparation except that this area approval scheme according to the present invention.
In addition, following drug delivery conjugates is illustrative drug delivery conjugates also, thinks that they are in the scope of the invention described herein.Appended synthetic method is illustrative method, can be used for preparing drug delivery conjugates described herein.
Add 1,2,4 of 1.0eg. in the acetonitrile solution of Diacetoxysciroenol (diacetoxyscirpenol (DAS)), the 5-PMDA adds the H ü nig alkali of 1eg. then.Under argon and room temperature, stirred reaction mixture 1.5 hours.If also have some DAS unreacteds, added the dianhydride of 0.2eg. and continuous stirring again 1 hour.The anhydrous DMSO solution that adds the pteroyl hydrazine (according to J.Am.Chem.Soc., 1997,119,10004 preparations, its disclosure is attached to herein by reference) of 1.2eg. adds the H ü nig alkali of 1.0eg. then.Stirred reaction mixture 1 hour, and in ether, precipitate.Gained precipitation reuse preparation HPLC purification.
Summarize as embodiment 10a, make the reaction of Boc-hydrazides and succinic anhydride, " amino-two-indyl-open loop-CBI reacts in the presence of EDC for products therefrom and condensing agent 5.After the NHS ester activation, the fragments of peptides Pte-γ-Glu-Asp-Arg-Asp-Dap-OH that forms that removes and have an acyl group hydrazone of free levulic acid of Boc provides the reaction gametophyte.The continuation method that this fragments of peptides is supported by polymer begins to prepare with Fmoc-Dap (Boc)-Wang resin with the Fmoc strategy, such as scheme 12 general introduction.
The reaction of Fmoc-hydrazides and 3-(2-pyridine radicals disulfide group) propanoic acid obtains Fmoc-hydrazide group-[3-(2-pyridine radicals disulfide group) propionic ester].With mitomycin c derivative (ametycin, N-(CH
2)
2SH) reaction produces the derivant that contains the disulphide ametycin.After the activation of successive NHS ester, provide the reaction gametophyte with the fragments of peptides Pte-γ-Glu-Dap-OH that forms that removes and have an acyl group hydrazone of levulic acid of the Fmoc of standard scheme, its can such as scheme 12 general introduction prepare.
Following illustrative example embodiment is not expected, shall not be construed as restrictive.For example, in being present in the chemical compound of this paper, the amino acid whose spatial chemistry that is used to form joint can be chosen wantonly and be selected from natural L configuration or D configuration.Each embodiment is by NMR, MS and/or UV spectral characterization, and/or represented by HPLC; Suitably write down selected characteristic signal.
Embodiment 1
According to P.Fuchs etc., J.Am.Chem.Soc., 1997,119,10004 methods of describing prepare diethylenetriamines folic acid, γ-amide (DETA-folic acid), the disclosure of the document is attached to herein by reference.This chemical compound (100mg) is dissolved among the 0.1N HCl of 2ml.Gained solution joins K
2PtCl
4In the 1ml 0.1N HCl agitating solution (158mg).Added 3ml DMSO and continuous stirring 3 days, filtering solution, filtrate is precipitated in acetonitrile, obtains the 170mg yellow powder;
MS(MALDI)1249.92，1286.27；
1H?NMR(D
2O)δ1.05(t，1H)，2.3(t，2H)，3.1(t，2H)，4.45(m，1H)，6.65(d，2H)，7.5(d，2H)，8.65(s，1H).
Embodiment 2a
N
10-trifluoroacetyl group protection contain folic acid peptidyl fragment N
10The continuation method that-TFA-Pte-Glu-Glu-Lys-OH supports by polymer is with the preparation of Fmoc-strategy, such as scheme 12 general introduction.On acid-sensitive sense Fmoc-Lys (Boc)-Wang resin, synthesize.PyBop is used as activator to guarantee the amino acid whose effective coupling with low equivalent.Under standard conditions (DMF of 20% piperidines), remove the Fmoc protecting group after each coupling step.Fmoc-Glu-OtBu and N
10-TFA-Pte-OH is as protected aminoacid member.After last installation step,, peptide is downcut from polymer support by with trifluoroacetic acid, ethane two mercaptan and triisopropyl silane treated.When also causing t-Bu and t-Boc protecting group, this reaction removes.Thick peptide preparation HPLC purification obtains N
10The tfa salt of-TFA-Pte-γ Glu-γ Glu-Lys-OH.The 2ml DMSO solution of 81mg (0.1mmol) gained peptide is with 15 μ L (0.11mmol) Et
3The Mitomycin A of N and 35mg (0.1mmol) is handled.Mitomycin A can be by ametycin according to M.Matsui, Y.Yamada, K.Uzu, and T.Hirata, J.Antibiot.21,189-198 (1968) and D.Vias, D.Benign, R.Partyka, and T.Doyle, J.Org.Chem.51, the described method preparation of 4307-4309 (1986), the disclosure of the document is attached to herein by reference.Reactant mixture at room temperature stirs 48h and lyophilizing and removes and desolvate.Except as otherwise noted, the evaporation of all solvents is all carried out under reduced pressure.Finally, the trifluoroacetyl group protecting group is separated in ammonium hydroxide aqueous solution (pH=10.0), and in acetonitrile precipitated product, obtain the yellow solid of the described conjugate of 102mg;
1H?NMR(D
2O)δ2.45(q，1H)，2.95(m，2H)，3.35(dd，1H)，3.5(d，1H)，6.5(d，2H)，7.55(d，2H)，8.55(s，1H).
Embodiment 2b
N
10-trifluoroacetyl group protection contain folic acid peptidyl fragment N
10The continuation method that-TFA-Pte-Glu-Cys-OH supports by polymer prepares with the Fmoc-strategy, as the description among 12 general introductions of scheme and the embodiment 2a.Make the reaction of cystamine and Mitomycin A (referring to Matsui etc., J.Antibiot.21,189-198 (1968); Vias etc., J.Org.Chem.51,4307-4309 (1986)) obtain containing the disulphide of mitomycin c derivative with free end amino, the acyl group hydrazides reaction of carbonyl and maleimide derivative is carried out in itself and levulic acid coupling then immediately.The michael acceptor of gained and N
10-TFA-Pte-Glu-Cys-OH reaction after the trifluoroacetyl group protecting group is removed with ammonium hydroxide aqueous solution (pH=10.0), by acetonitrile precipitation, obtains final conjugate;
MS(MALDI)1059.04，1148.44，1225.32，1300.8；
1H?NMR(D
2O)δ1.8(d，2H)，1.9(s，1H)，2.3(q，1h)，2.45(q，1H)，2.9(t，1H)，3.35(dd，1H)，4.45(s，1H)，4.5(dd，1H)，6.65(d，2H)，7.55(d，2H)，8.6(s，1H).
Embodiment 3
The intermediate succinimido of T-2 toxin is begun to synthesize by commercially available N-(2-ethoxy) maleimide to the inferior benzyl acetal of methoxyl group.Under the situation that silver oxide (I) (2eg.) exists as the weak base in the dichloromethane, its hydroxyl with the methoxyl group xylylene dichlorides is reacted.Crude product is purification on silicagel column.Containing in the presence of the T-2 toxin (1eg.) of OH-, use 1.5eg.2,3-two chloro-5,6-dicyano-benzoquinone (DDQ) oxidation processes gained to mehtoxybenzyl ether, to mehtoxybenzyl carbon middle bodily form formula, obtain the inferior benzyl acetal of required methoxyl group through stable.
Other reaction gametophyte, Pte-γ-Glu-Arg-Asp-Cys-OH, the continuation method of supporting by polymer prepares with the Fmoc-strategy.On the H-Cys of acid-sensitive sense (4-methoxyl group trityl)-2-chlorine trityl-resin, synthesize.PyBop is used as activator to guarantee the amino acid whose effective coupling with low equivalent.Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Glu-OtBu) and N
10-TFA-Pte-OH is as protected aminoacid member.Under standard conditions (DMF of 20% piperidines), remove the Fmoc protecting group after each coupling step.After last installation step,, peptide is downcut from polymer support by with trifluoroacetic acid, ethane two mercaptan and triisopropyl silane treated.When also causing t-Bu and t-Boc protecting group, this reaction removes.Thick peptide preparation HPLC purification obtains N
10-TFA-Pte-γ-Glu-Arg-Asp-Cys-OH.The trifluoroacetyl group protecting group is separated in ammonium hydroxide aqueous solution (pH=10.0).
At last, target molecule is to the inferior benzyl acetal of methoxyl group-constraint folic acid-drug conjugate, by aqueous buffer solution (pH=7.0) and the equimolar acetonitrile solution preparation that contains the acetal of maleimide T-2 toxin that mixes the gained peptide under argon.Stir under the room temperature after 1 hour, final conjugate preparation HPLC purification behind the collected flow point of lyophilizing, obtains yellow powder;
MS(m+H)
+1541.3；?
1H?NMR(DMSO-d
6)δ0.1(s，1H)，0.55(d，2H)，0.9(dd，3H)，1.65(s，1H)，2.0(d，1H)，3.75(d，2H)，5.25(d，1H)，6.65(d，2H)，6.9(d，2H)，7.3(t，2H)，7.65(d，2H)，8.65(s，1H).
Embodiment 4a
Embodiment 4b
Embodiment 4c
The chemical compound of embodiment 4a, 4b and 4c, according to the universal method preparation of describing among the embodiment 3, just the acyl group aziridine prepares by the acidylate (referring to scheme 1) of Mitomycin A with suitable commercially available N-(alkanoic acid) maleimide.
Embodiment 5
NaHCO at 2.2eg.
3Be alkali, acetonitrile/water (1/1) is under the condition of solvent, the Fmoc-OSu reaction of anti--4-aminocyclohexanol hydrochlorate and equimolar amounts; obtain the amino alcohol of N-Fmoc-protection; use the amino ketones that Swern condition (Synthesis, 1981,165) oxidation obtains corresponding N-Fmoc-protection.The trifluoroacetic acid ketalization of 4 normal original acid A esters and catalytic amount obtains the amino ketal of the N-Fmoc-protection of quantitative yield.With the trimethyl silyl trifluoromethayl sulfonic acid ester and 2,4 of equimolar amounts, 6-three-tert-butyl group-pyridine is handled this ketal, obtains product 4-Fmoc-aminocyclohexyl enol ether.At next step, in the presence of the trifluoroacetic acid of molecular sieve (3_) and catalytic amount, medicine, T-2 toxin are handled with four times of excessive enol ethers.Asymmetric mixing ketal purification on silica gel of gained.The Fmoc protecting group removes by the piperidines with resin-bonded DMF.In the presence of the H of 1.1eg. ü nig alkali, the maleimide acetic acid of free amino and 1.1eg.-NHS-ester reaction.The T-2 toxin contain the maleimide ketal through silica gel purification.
The fragments of peptides that contains folic acid, Pte-γ-Glu-β-Dap-Asp-Cys-OH, the continuation method of supporting by polymer is with the preparation of Fmoc-strategy, such as scheme 12 general introduction.On the Wang resin of the acid-sensitive sense that is loaded with Fmoc-L-Cys (Trt)-OH, synthesize.PyBop is used as activator to guarantee the amino acid whose effective coupling with low equivalent.Under standard conditions (DMF of 20% piperidines), remove the Fmoc protecting group after each coupling step.Fmoc-Asp (OtBu)-OH, Boc-Dap (Fmoc)-OH, Fmoc-Glu-OtBu and N
10-TFA-Pte-OH is as the aminoacid member of protection.After last installation step,, peptide is downcut from polymer support by with trifluoroacetic acid, ethane two mercaptan and triisopropyl silane treated.When also causing t-Bu and t-Boc and trityl-protecting group, this reaction removes.Finally, the trifluoroacetyl base section is separated in ammonium hydroxide aqueous solution and is obtained the required peptide that contains mercaptan.Thick peptide preparation HPLC purification.
At last, target molecule acetal-constraint folic acid-drug conjugate prepares by the aqueous buffer solution (pH=7.0) of mixing gained peptide and the acetonitrile solution that contains the maleimide acetal of equimolar T-2 toxin under argon.Stir under the room temperature after 1 hour, final conjugate preparation HPLC purification behind the collected flow point of lyophilizing, obtains yellow powder; ESMS (m-H)
-1474.5, (m+H)
+1476.2, (m+Na)
+1498.3.
According to (J.Am.Chem.Soc., 1997,119,10004 such as P.Fuchs; Synthetic referring to the described chemical compound 52 of for example document) method of describing prepares ethylenediamine folic acid, γ-amide (EDA-folic acid), and the disclosure of the document is attached to herein by reference.EDA-folic acid (600mg) is suspended among the anhydrous DMSO of 5ml.After 4 hours, the solution of gained is cooled to 20 ℃ in 60 ℃ of stirrings, adds 1,2,4 of 4eg., 5-PMDA (BTCA anhydride).After 5 minutes, in the well-beaten anhydrous acetonitrile of reactant mixture impouring.The precipitation of gained is separated BTCA (single acid anhydride)-EDA-folic acid that obtains 657mg by centrifugal.
In the anhydrous DMSO solution of well-beaten daunomycin, add solid BTCA (single acid anhydride)-EDA-folic acid (1.5eg.).Behind the restir 14 hours, 50% daunomycin is unreacted (HPLC) still, so add BTCA (single acid anhydride)-EDA-folic acid of 1.5eg..Behind the restir 4 hours, all daunomycin react completely.In HPLC figure, observe two new peaks, its retention time is close, represents two regional isomers (regioisomers) of final conjugate.Gained crude product post precipitation in acetonitrile separates, and uses the reversed-phase HPLC purification then.The structure of product and ES MS (m-H)
-1227.1 it is consistent.
In the presence of argon,, in the 4ml anhydrous methylene chloride solution of well-beaten 250mg (0.25mmol) paclitaxel and 130 μ L (0.73mmol) H ü nig alkali, slowly add 85 μ L (0.8mmol) Alloc-Cl in 0 ℃.Continuous stirring is 12 hours again, and product separates with the standard abstraction technique.This 2 '-alloc-paclitaxel white powder just need not to be further purified and can be directly used in next step.
In this step, 108mg (0.117mmol) 2 '-alloc-paclitaxel is dissolved in the 1.0ml anhydrous acetonitrile.In the presence of argon, while stirring with 1,2,4 of 25mg (0.117mmol), 5-PMDA (BTCA acid anhydride) and 21 μ l (0.120mmol) H ü nig alkali join in this solution.Continue to stir 2.5 hours.In independent reaction bulb, 52mg EDA-folic acid is in 60 ℃ of stirrings, up to all substances dissolvings (about 60 minutes).After being cooled to room temperature, reactant mixture joins in this solution before this, and continuous stirring is 2 hours again.Reactant mixture is added drop-wise in well-beaten acetonitrile/ether (20: 80).Centrifugalize goes out yellow mercury oxide, is further purified by preparation HPLC.Product structure and 1D and 2D (COSY)
1The H-NMR spectrum; ES MS (m+H)
+1555.5 it is consistent.
In the presence of argon, the mixture of 1.0eg. aklavine, 2.0eg. hydrazide group-[3-(2-pyridine radicals disulfide group) propionic ester] (SPDP-hydrazone) and a little p-methyl benzenesulfonic acid pyridine under agitation, is dissolved in the absolute methanol.Reactant mixture at room temperature stirred 8 hours.Solvent evaporation is extremely done.Residue is through with the pretreated silicagel column purification of the triethylamine in 1.5% chloroform/methanol (90: 10).The aklavine acylhydrazone of gained is dissolved in the acetonitrile of minimum.In the presence of argon, in gained solution, slowly add the Pte-γ-Glu-Cys-OH (water-soluble and transfer to pH=6.8) of equimolar amounts.Method is similar described in the preparation of Pte-γ-Glu-Cys-OH and the embodiment 2a, and is summarized in the scheme 12.The exchange reaction of disulphide took place in 10 minutes.Reactant mixture is slowly joined in the excessive acetonitrile, and separate the centrifugal back of gained precipitation.Precipitation is resuspended in the acetonitrile once more, stirs after 15 minutes centrifugalize.After high vacuum dry is spent the night, final conjugate enough pure (HPLC); ES MS (m+H)
+1474.1.
1.0eg. the mixture of aklavine and 1.2eg. β-maleimide base propanoic acid-TFA in the presence of argon, through stirring and dissolving in absolute methanol.Reactant mixture at room temperature stirred 1 hour.Solvent evaporation is extremely done.Residue passes through with the pretreated silicagel column of triethylamine in 1.5% chloroform/methanol (90: 10).In the presence of argon, in independent flask, fragments of peptides Pte-γ-Glu-γ-Glu-Cys-OH is water-soluble and transfer to pH=6.8.The preparation of Pte-γ-Glu-γ-Glu-Cys-OH is similar to method described in the embodiment 2a, and is summarized in the scheme 12.In the yellow solution of gained, slowly add the maleimide base-hydrazone that is dissolved in the aklavine in the minimum ethanol.Reactant mixture stirred 1 hour in the presence of argon.Remove methanol, residue is used HPLC purification, lyophilizing then on preparative column; ES MS (m+H)
+1722.3.
Embodiment 9b
Embodiment 9c
The chemical compound of embodiment 9b and 9c is by doxorubicin (14-Hydroxydaunomycin) derivant, according to the method preparation that is summarized in embodiment 9a.
Embodiment 10a
Efficient cytotoxicity medicine 5 " (N-Boc) amino analog; promptly pair-indyl-open loop-1; 2; 9,9a-tetrahydrochysene cyclopropane also [c] benzo [e] indole-4-ketone (two-indyl-open loop-CBI) according to D.Boger etc., J.Org.Chem.; 1992; 57,2873 prepare after at first reporting the modification slightly of method, and the disclosure of the document is attached to herein by reference.
Fragments of peptides Pte-γ-Glu-Asp-Arg-Asp-Cys-OH, the continuation method Fmoc-strategy by polymer is supported prepares on the H-Cys of acid-sensitive sense (4-methoxyl group trityl)-2-chlorine trityl-resin.Such as scheme 12 general introduction.PyBop is used as activator to guarantee the amino acid whose effective coupling with low equivalent.Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Glu-OtBu) and N
10-TFA-Pte-OH is as the aminoacid member of protection.Under standard conditions (DMF of 20% piperidines), remove the Fmoc protecting group after each coupling step.After last installation step,, peptide is downcut from polymer support by with trifluoroacetic acid, ethane two mercaptan and triisopropyl silane treated.When also causing t-Bu and t-Boc protecting group, this reaction removes.Thick peptide preparation HPLC purification obtains N
10-TFA-Pte-γ-Glu-Asp-Arg-Asp-Cys-OH.The trifluoroacetyl group protecting group is separated in ammonium hydroxide aqueous solution (PH=10.0).
Lev-Val-OH prepares with standard scheme, and this scheme is included in EDC and H ü nig alkali exists down, makes hydrochloric acid valine methyl ester and levulic acid (levolinic acid) condensation, uses this methyl-esterified compound of Lithium hydrate and water hydrolysis then.
The final assembling of compound conjugate starts from by 5 " (N-Boc) the last removal of amino-two-indyl-open loop-CBI N-Boc base, and in the presence of EDC, the coupling of the carboxyl functional group of free amine group and Lev-Val-OH.Acylhydrazone forms by acetyl propyl group (levolinic) part and finishes with the β-reaction of maleimide base propanoic acid TFA in oxolane of 1.2eg..Behind chromatography purification (silica gel, THF/ hexane=1/1), above-mentioned product is dissolved among the DMSO.In the presence of argon, in this solution, add Pte-γ-Glu-Asp-Arg-Asp-Cys-OH of 0.9eg. and reactant mixture was stirred 18 hours.Solvent is removed by lyophilizing, residue HPLC purification.
Embodiment 10b
Embodiment 10c
The chemical compound of embodiment 10c and 10c is by " (N-Boc) amino-two-indyl-open loop-CBI derivant is according to the method preparation that is summarized among the embodiment 10a.
In the presence of potassium carbonate, the S-alkylation of 2 mercapto ethanol is finished with allyl bromide, bromoallylene.Hydroxyl in the pi-allyl beta-hydroxy ethyl sulfur of gained is changed into chlorine by thionyl chloride.With hydrogen peroxide this product of oxidation (J.Am.Chem.Soc., 1950,72,59, the disclosure of the document is attached to herein by reference) in the presence of acetic acid and acetic anhydride, obtain pi-allyl β-chloroethyl sulfone.The reaction of this product and chloro dimethyl silane in the presence of the chlordene network platinum hydracid (IV) of catalytic amount, after the high temperature distillation simultaneously, obtains 3-(β-chloroethyl sulfonyl) propyl-dimethyl silyl chloride.When using the 1eg. pyridine as alkali, the hydroxyl of this chlorosilane silanization high cell toxicity chemical compound rhizomycin.Handle β-chloroethyl sulfone part in this molecule with excessive triethylamine, cause the β-elimination of hydrogen chloride smoothly, simultaneously each self-forming vinyl sulfone.
Other reaction gametophyte, i.e. fragments of peptides Pte-γ-Glu-Arg-Asp-Cys-OH, the continuation method of supporting by polymer is with the preparation of Fmoc-scheme, such as embodiment 2a and scheme 12 general introduction.
The final assembling of compound conjugate, by Michael addition reaction, the vinyl sulfone that the mercapto of fragments of peptides is connected the silicon joint on the rhizomycin is partly gone up and is realized.The reaction medium of this conversion is 50: 50 acetonitrile/water (pH=7.2).Stir after 24 hours under the room temperature, final conjugate carries out separating behind the HPLC on preparation type post; ES MS (m+H)
+1631.6; (m-H)
-1629.6.
Embodiment 12
The silicon of this rhizomycin-constraint conjugate, synthetic with embodiment 11 described schemes, just substitute Chlorodimethyl silane with commercially available chloro methylbenzene methyl-monosilane.
Embodiment 13
Contain the general preparation of cysteine disulfide bond chemical compound
According to Ranasinghe and Fuchs, Synth.Commun.18 (3), the thiosulfonates 4 (1 equivalent) of the method for 227-32 (1988) (disclosure of the document is attached to herein by reference) preparation, react with medicine, drug analogue or medicaments derivative 5 (1 equivalent), the methanol solution of preparation medicine thiosulfonates 6 is shown in scheme 13.R is an alkyl or aryl, and L is suitable leaving group, for example halogen, pentafluoro-benzyl etc., n is the integer of 1-4, X is-O-,-NH-,-C (O) O-or-C (O) NH-.Conversion can be by using TLC (silica gel; CHCl
3/ MeOH=9/1) observe every kind of consumption of raw materials to monitor easily.
Scheme 13
Contain folic acid peptidyl fragment Pte-Glu-(AA)
nThe continuation method Fmoc-strategy that-Cys-OH (9) supports by polymer is gone up preparation at acid-sensitive sense Fmoc-Cys (Trt)-Wang resin (7), shown in scheme 14.R
1Be Fmoc, R
2Be trityl, DIPEA is a diisopropylethylamine.PyBop is used as activator to guarantee effective coupling.The Fmoc protecting group is removed under standard conditions after each coupling step.Use the aminoacid member of due care, as Fmoc-Glu-OtBu, N
10-TFA-Pte-OH etc. shown in scheme 14, and represent with Fmoc-AA-OH in the step (b).Therefore, AA is meant the amino acid starting material of any due care.Coupling program (step (a) ﹠amp that relates to Fmoc-AA-OH; (b)) carry out " n " inferior, with the peptide 8 that the preparation solid is supported, wherein n is an integer, and can equal 0 to about 100.After the last coupling step, remove remaining Fmoc base, the gained peptide is coupled to (step (c)) on the glutamate derivatives subsequently, deprotection, and be coupled on the pteroic acid of TFA-protection (step (d)).Subsequently, the gained peptide is handled with trifluoroacetic acid, ethane two mercaptan and tri isopropyl silane, downcuts (step (e)) from polymer support.When causing t-Bu, t-Boc and Trt protecting group, remove these reaction conditions.The TFA protecting group obtains containing folic acid and contains Cys peptidyl fragment 9 by removing with alkali treatment (step (f)).
(a) 20% piperidines/DMF; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF; (c) Fmoc-Glu (O-t-Bu)-OH, PyBop, DIPEA, DMF; (d) 1.N
10(TFA)-Pte-OH; PyBop, DIPEA, DMSO; (e) TFAA, (CH
2SH)
2, i-Pr
3SiH; (f) NH
4OH, pH10.3.
(0.04M uses 0.1N NaHCO at deionized water
3Transfer pH to 7) in, in the presence of argon, about 30 minutes, form disulfide bond by making folic acid derivatives 9 (0.9-0.95 equivalent) and 6 reactions of medicine thiosulfonates, with the preparation drug conjugate.Behind the vacuum evaporation methanol, conjugate can be used preparation HPLC (Prep Novapak HR C18 19 * 300mM post; Mobile phase (A)-1.0mM phosphate buffer, pH=6; Organic facies (B)-acetonitrile; Condition-gradient 99%A and 1%B to 50%A and 50%B, in 30 minutes, the purification of flow velocity=15ml/min).
Embodiment 14a
1H?NMR(DMSO-d
6)δ4.7(d，1H)，4.95(t，1H)，6.7(d，4H)，6.9(t，1H)，7.95(d，2H)，8.1(d，2H)，8.2(m，1H)，8.3(s，1H)，8.4(s，1H)，8.7(s，1H)，10.2(s，1H)，11.8(d，2H).
Embodiment 14b
ES?MS(m-H)
-1436.4，(m+H)
+1438.3.
Embodiment 14c
1H?NMR(DMSO-d
6/D
2O)81.0(s，1H)，1.1(s，1H)，1.6(s，1H)，1.8(s，1H)，2.1(s，1H)，2.25(s，3H)，2.65(dd，2H)，3.7(d，1H)，4.4(t，1H)，4.55(q，2H)，4.6(d，2H)，4.95(d，1H)，5.9(t，1H)，6.15(s，1H)，6.6(d，2H)，7.85(d，2H)，7.95(d，2H)，8.6(s，1H)，8.95(d，1H).
Embodiment 14d
1HNMR(DMSO-d
6/D
2O)δ1.0(s，1H)，1.1(s，1H)，1.65(s，1H)，2.1(s，1H)，2.25(s，3H)，2.6(dd，2H)，3.25(dd，1H)，3.6(t，2H)，3.7(d，1H)，4.4(t，1H)，4.6(d，1H)，4.95(d，1H)，5.9(t，1H)，6.2(s，1H)，6.6(d，2H)，7.7(t，1H)，7.9(d，2H)，7.95(d，2H)，8.6(s，1H)，9.1(d，2H).
Embodiment 14e
1H?NMR(DMSO-d
6/D
2O)δ10.85(d，2H)，1.05(d，2H)，1.2(d，2H)，1.7(d，2H)，3.95(d，1H)，4.05(dd，1H)，5.4(dd，1H)，5.7(dd，1H)，6.65(d，2H)，7.6(d，2H)，7.95(s，1H)，8.65(s，1H).
Embodiment 14f
ES?MS(m+H)
+1487.23；
1H?NMR(DMSO-d
6/D
2O)δ0.9(t，2H)，1.3(t，2H)，2.15(t，2H)，3.2(dd，1H)，4.0(t，1H)，4.15(q，1H)，5.3(s，2H)，5.5(s，2H)，6.6(d，2H)，7.0(s，1H)，7.4(m，2H)，7.55(d，2H)，8.0(d，2H)，8.6(s，1H).
Embodiment 14a, 14b, 14c, 14d, 14e and 14f are prepared by following universal method.In the presence of argon, to well-beaten corresponding tool-OH base drug solution (1 normal anhydrous CH
2Cl
2Or anhydrous THF) in, adding 6-(trifluoromethyl) benzotriazole base 2-(2 '-pyridine radicals disulfide group ethyl carbonate ester (1.3 equivalent) and N, N-dimethyl aminopyridine (1.5 equivalent).Reactant mixture stirs 3h, and pyridine radicals disulfide group-deutero-medicine separates (＞65%, to each embodiment) by silica gel chromatography.Corresponding peptidyl fragment (0.5 equivalent) according to the universal method preparation of summarizing in the scheme 12 is dissolved among the DMSO.In the clear yellow solution of gained, add the deutero-medicine of pyridine radicals disulfide group.After 30 minutes, react completely gained conjugate HPLC purification.With regard to embodiment 14e, peptidyl fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH is earlier water-soluble, again pH value of solution is transferred to 2.5 with 0.1N HCl, makes peptidyl fragment precipitation.Gained peptidyl fragment is by centrifugal collection, drying, and is dissolved in and is used among the DMSO subsequently and the deutero-drug reaction of pyridine radicals disulfide group.
Intermediate 4-(the 2-pyridine radicals disulfide group) benzyl carbonate of SN 38 (10-hydroxyl-7-ethyl-camptothecin) is according to P.Senter etc., J.Org.Chem.1990,55,2875 described method preparations, the disclosure of the document is attached to herein by reference.Peptidyl fragment Pte-Glu-Asp-Arg-Asp-Cys-OH is dissolved in DMSO, and adds the deutero-medicine of pyridine radicals-disulfide group in the clear yellow solution of gained.After 30 minutes, react completely gained conjugate HPLC purification;
ES?MS(m+H)
+1425.38；
1H?NMR(DMSO-d
6/D
2O)δ0.9(t)，1.15(t)，3.9(t)，4.0(t)，4.25(t)，5.1(m)，5.2(s)，5.4(s)，6.55(d)，7.25(d)，7.35(d)，7.5(d)，7.9(d)，8.55(s).
Embodiment 16a
Embodiment 16b
The chemical compound of embodiment 16a and 16b is by peptidyl fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH preparation, and this peptidyl fragment is according to scheme 12 described universal method preparations.This peptidyl fragment produces folic acid conjugate embodiment 16a to the Michael addition of the maleimide radical derivative of open loop-CBI-pair-indole.Gained peptidyl fragment is also reacted with thiosulfonates or pyridine radicals disulfide group-activatory vinblastine, forms embodiment 16b.Maleimide radical derivative and the thiosulfonates and the pyridine radicals disulfide group-activatory vinblastine intermediate of open loop-CBI-pair-indole are used for the methods described herein preparation of other embodiment.
Embodiment 17a
Taking off acetyl vinblastine one hydrazides (1 equivalent) (referring to Barnett etc., J.Med.Chem., 1978,21,88, the disclosure of the document is attached to herein by reference) handles with 1 equivalent trifluoroacetic acid in new distillatory THF.Stir after 10 minutes, gained solution is handled with 1.05 normal N-(4-acetylbenzene methyl) maleimide.Acylhydrazone is formed in 45 minutes and finishes, evaporating solvent.The peptidyl fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH (0.85 equivalent) that the universal method of summarizing according to scheme 12 prepares, soluble in water, pH transfers to 2.5 with 0.1NHCl, makes the peptide precipitation.Gained peptidyl fragment is by centrifugal collection, drying, and is dissolved among the DMSO.The Michael-adduct that in the clear yellow solution of gained, adds H ü nig alkali (15 equivalent) and acylhydrazone.Behind the 1h, final conjugate HPLC purification.
Embodiment 17b
Embodiment 17c
Embodiment 17b and 17c are according to the described method of embodiment 17a, with the hydrazide derivatives preparation of corresponding peptidyl fragment and CBI.
The universal method preparation that the chemical compound of embodiment 18-41 is summarized according to embodiment 13.Embodiment 18-41 is by Electrospray Mass Spectrometry (ES MS) and comprise 1D and other spectral technique sign of 2D NMR and UV.
ES?MS(m+H)
+1071.9，(m+Na)
+1093.9；
1H?NMR(D
2O)δ2.6(t，4H)，2.7(t，4H)，4.15(s，2H)，5.45(s，2H)，7.75(d，2H)，8.15(d，2H)，8.9(s，1H).
Embodiment 19
UV(nm)233(max)，255，280；
1H?NMR(D
2O，NaOD，CD
3CN)δ1.15(d，3H)，2.3(s，3H)，3.6(s，1H)，3.85(s，3H)，4.9(s，1H)，5.3(s，1H)，6.5(d，2H)，7.3(m，1H)，7.5(d，2H)，7.65(d，2H)，8.4(s，1H).
ES?MS(m-H)
-935.6，(m+H)
+937.4，(m+Na)
+959.5.
Embodiment 21
1H?NMR(D
2O，NaOD，CD
3CN)δ0.1(s，1H)，1.1(s，3H)，1.2(s，3H)，1.75(s，3H)，1.9(s，3H)，2.05(s，3H)，2.35(s，3H)，3.3(dd，2H)，3.8(d，1H)，4.3(q，2H)，4.9(d，1H)，5.1(d，1H)，5.4(q，1H)，5.55(d，1H)，5.65(d，1H)，6.1(t，1H)，6.35(s，1H)，6.9(d，2H)，7.9(d，2H)，8.15(d，2H)，8.7(s，1H).
Embodiment 22
Embodiment 23
ES?MS(m-H)
-1136.5.
Embodiment 24
ES?MS(m-H)
-1136.3，(m+H)
+1138.0.
ES?MS(m+H)
+1382.3，(m+Na)
+1405.4.
Embodiment 26
ES?MS(m-H)
-1379.2，(m+H)
+1381.2.
ES?MS(mH)
-949.2；
1H?NMR(D
2O)δ1.55(s，3H)，1.95(m，2H)，2.05(s，3H)，2.45(s，3H)，2.75(dd，2H)，2.95(dd，2H)，3.05(s，3H)，3.3(dd，2H)，3.35(d，2H)，3.45(t，2H)，4.85(q，2H)，6.5(d，2H)，7.45(d，2H)，8.5(s，1H).
Embodiment 28
1H?NMR(DMSO-d
6)δ1.5(s)，2.25(t)，2.75(m)，3.9(q)，4.6(d)，4.85(t)，6.6(d)，7.6(d)，7.9(d)，8.15(d)，8.25(t)，8.65(s)，8.7(m)，9.3(m)，10.2(t).
Embodiment 29
ES?MS(m-H)
-1413.5，(m+H)
+1415.3.
ES?MS(m+H)
+1530.2；
1H?NMR(DMSO-d
6/D
2O)δ1.2(s，1H)，2.9(t，1H)，3.65(t，1H)，4.15(t，1H)，4.25(t，1H)，4.35(t，1H)，6.7(d，2H)，7.0(s，1H)，8.1(d，2H)，8.25(s，1H)，8.7(s，1H).
Embodiment 31
1H?NMR(DMSO-d
6)δ1.75(s，1H)，1.85(s，1H)，2.1(t，2H)，4.3(t，1H)，4.6(d，1H)，4.9(t，1H)，6.6(d，2H)，8.15(s，2H)，8.6(s，1H).
Embodiment 32
ES?MS(m+H)
+1408.4.
Embodiment 33
ES?MS(m-H)
-1491.1，(m+H)
+1493.1；
1H?NMR(DMSO-d
6/D
2O)δ4.15(q，1H)，4.6(d，1H)，4.9(t，1H)，6.6(d，2H)，7.25(s，1H)，7.4(d，1H)，7.9(d，1H)，7.95(d，2H)，8.15(d，2H)，8.6(s，1H).
Embodiment 34
1H?NMR(DMSO-d
6/D
2O)δ2.1(t，2H)，2.75(q，2H)，4.3(t，1H)，4.65(d，1H)，4.9(t，1H)，6.6(d，2H)，7.9(d，1H)，8.0(d，2H)，8.2(t，2H)，8.6(s，1H).
Embodiment 35
Embodiment 36
ES?MS(m+H)
+1680.4；
1H?NMR(DMSO-d
6/D
2O)δ0.3(s，3H)，0.35(s，3H)，1.05(s，9H)，2.15(t，2H)，4.15(t，1H)，4.85)t，1H)，6.6(d，2H)，7.55(t，4H)，7.9(d，1H)，8.0(s，1H)，8.05(d，1H)，8.15(s，1H)，8.6(s，1H).
Embodiment 37
1H?NMR(DMSO-d
6/D
2O)δ1.1(s，3H)，1.8(s，1H)，4.55(d，1H)，4.8(t，1H)，6.6(d，2H)，7.8(d，1H)，8.1(d，1H)，8.15(s，1H)，8.6(s，1H).
Embodiment 38
Embodiment 39
Embodiment 41
Embodiment 42
With the inhibition of EC112 treatment to the mouse tumor growth
The chemical compound (EC112) of estimating the chemical compound (EC111) of embodiment 9b and embodiment 9c is having anti-tumor activity in the Balb/c mice of subcutaneous M109 tumor when intravenous (i.v.) when giving tumor animal, its Chinese medicine is a daunorubicin.With 1 * 10
6The right subcutaneous tissue of axilla of M109 cell tumor inoculation is after 4 days, with the embodiment 9b of 2-10 μ mol/kg or the chemical compound of embodiment 9c, or gives mice (5/group) intravenous injection 2 times weekly, lasting 4 weeks with unconjugated daunorubicin or PBS.Each treatment group was measured tumor growth every 3 days or 4 days with caliber gauge.With equation V=a * b
2/ 2 calculate gross tumor volume, and wherein " a " is length of tumor, and " b " is width, and unit is a millimeter.The weight of animals is also every 3 days or 4 days weighings.
As depicted in figs. 1 and 2, effectively postpone the M109 growth of tumor with the treatment of the chemical compound of embodiment 9c, and do not have overt toxicity (based on the weight of animals).Unconjugated amycin also provides the antitumor reaction, but with the toxicity based on body weight.
Embodiment 43
With the inhibition of EC105 treatment to the mouse tumor growth
Scheme is as described in the embodiment 42, and what only be to use is the chemical compound (EC105) of embodiment 10a, and its Chinese medicine is two-indyl-open loop-CBI.The chemical compound of embodiment 10a is with the dosage injection of 0.3 μ mol/kg.Equally, test two Subcutaneous tumor models, comprised M109 model (the folacin receptor positive) and 4T1 model (folacin receptor feminine gender), in some animals, 67 times of excessive free folic acid (20 μ mol/kg; FA) with described conjugate (being the chemical compound of embodiment 10a) injection altogether.
Observe the surprising antitumor reaction of chemical compound of embodiment 10a, do not have overt toxicity (referring to Fig. 3 and Fig. 4) simultaneously based on the weight of animals.The antitumor reaction of embodiment 10a chemical compound is proved the specificity (referring to Fig. 3) of this reaction by excessive free folic acid blocking-up.As shown in Figure 5, in 4T1 model (folacin receptor feminine gender), do not observe anti-tumor activity, prove the specificity of this reaction once more.
Embodiment 44
With the inhibition of EC145 treatment to the mouse tumor growth
The chemical compound (EC145) of estimating embodiment 16b is having anti-tumor activity in the Balb/c mice of subcutaneous M109 tumor when intravenous (i.v.) when giving tumor animal, its Chinese medicine is for taking off acetyl vinblastine one hydrazides.With 1 * 10
6(mean tumour volume is at t after about 11 days for the right subcutaneous tissue of axilla of M109 cell tumor inoculation
o=60mm
3), with the EC145 of 1500nmol/kg, or give mice (5/organize) intravenous injection 2 times (BIW) weekly, lasting 3 weeks with the PBS (contrast) of equal dose volume.Each treatment group was measured tumor growth every 2 days or 3 days with caliber gauge.With equation V=a * b
2/ 2 calculate gross tumor volume, and wherein " a " is length of tumor, and " b " is width, and unit is a millimeter.
As shown in Figure 6, with respect to the M109 growth of tumor in the animal of brine treatment, treat effective delay M109 growth of tumor with EC145.
Embodiment 45
With the inhibition of EC140 treatment to the mouse tumor growth
The chemical compound (EC140) of estimating embodiment 17a is having anti-tumor activity in the Balb/c mice of subcutaneous M109 tumor when intravenous (i.V.) when giving tumor animal, its Chinese medicine is for taking off acetyl vinblastine one hydrazides.With 1 * 10
6(mean tumour volume is at t after about 11 days for the right subcutaneous tissue of axilla of M109 cell tumor inoculation
o=60mm
3), with the EC140 of 1500nmol/kg, or give mice (5/organize) intravenous injection 3 times (TIW) weekly, lasting 3 weeks with the PBS (contrast) of equal dose volume.Each treatment group was measured tumor growth every 2 days or 3 days with caliber gauge.With equation V=a * b
2/ 2 calculate gross tumor volume, and wherein " a " is length of tumor, and " b " is width, and unit is a millimeter.
As shown in Figure 7, with respect to the M109 growth of tumor in the animal of brine treatment, treat effective delay M109 growth of tumor with EC140.
Embodiment 46
With the inhibition of EC136 treatment to the mouse tumor growth
The chemical compound (EC136) of estimating embodiment 10b is having anti-tumor activity in the DBA mice of subcutaneous L1210A tumor when intravenous (i.V.) when giving tumor animal, its Chinese medicine is CBI.With 0.25 * 10
5(mean tumour volume is at t after about 5 days for the right subcutaneous tissue of axilla of L1210A cell tumor inoculation
o～50mm
35 mice/groups), use the EC136 of 400nmol/kg, or give animal (5/group) intravenous injection 3 times (TIW) weekly, continued for 3 weeks with the independent PBS (contrast) of equal dose volume.Each treatment group was measured tumor growth every 2 days or 3 days with caliber gauge.With equation V=a * b
2/ 2 calculate gross tumor volume, and wherein " a " is length of tumor, and " b " is width, and unit is a millimeter.
As shown in Figure 8, with respect to the L1210A growth of tumor in the animal of brine treatment, treat effective delay L1210A growth of tumor with EC136.
Embodiment 47
Various folic acid-synthetic the inhibition of drug conjugate pair cell DNA
Shown in Fig. 9-16, the dose dependent cytotoxicity is measurable, in most of the cases, and IC
50Value (reduces
3The H-thymidine mixes the required drug conjugate concentration of new synthetic DNA 50%) in low nanomole scope.In addition, the cytotoxicity of these conjugates is lowered in the presence of excessive free folic acid, illustrates that observed cell killing is by being attached on the folacin receptor and mediate.
With EC158 and the cell line that comprises IGROV (known cell line), A549-Clone-4 (the A549 cell of personnel selection folacin receptor cDNA transfection), New Line-01 (in the body folacin receptor being expressed Line-01 cell mutation strain selectively), M109,4T1 Clone-2 (with the 4T1 cell of Mus folacin receptor cDNA transfection) and HeLa cell, in the test of this type, all obtain similar result.
Claims (64)
1. vitamin receptor binding drug delivery conjugates, described conjugate comprises:
(a) vitamin receptor bound fraction;
(b) bivalence joint; With
(c) medicine or its analog or derivant;
Wherein said vitamin receptor bound fraction is covalently bound to described bivalence joint;
Described medicine or its analog or derivant are covalently bound to described bivalence joint;
Described bivalence joint comprises one or more and is selected from following component: but spacer nipple releasing-joint and hetero atom joint and combination thereof;
But being described bivalence joint, condition comprises that at least one is not the releasing-joint of disulphide.
2. the drug delivery conjugates of claim 1, wherein said vitamin receptor bound fraction are selected from vitamin and vitamin receptor thereof in conjunction with analog and derivant.
3. the drug delivery conjugates of claim 1, wherein said hetero atom joint is nitrogen-atoms, oxygen atom or sulphur atom, perhaps is selected from-NHR
1NHR
2-,-SO-,-S (O)
2-and-NR
3O-, wherein R
1, R
2And R
3Independently be selected from hydrogen, alkyl, aryl, aryl alkyl, substituted aryl, substituted aryl alkyl, heteroaryl, substituted heteroaryl and alkoxyalkyl separately.
4. the drug delivery conjugates of claim 1; wherein said spacer nipple is selected from carbonyl; thiocarbonyl; alkylidene; cycloalkylidene; the alkylidene cycloalkyl; the alkylidene carbonyl; the cycloalkylidene carbonyl; the carbonylic alkyl carbonyl; 1-alkylidene butanimide-3-base; 1-(carbonylic alkyl) butanimide-3-base; the alkylidene sulfonic group; the sulfonyl alkyl; alkylidene sulfonic group alkyl; alkylidene sulfonyl alkyl; carbonyl tetrahydrochysene-2H-pyranose; the carbonyl tetrahydrofuran base; 1-(carbonyl tetrahydrochysene-2H-pyranose) butanimide-3-base and 1-(carbonyl tetrahydrofuran base) butanimide-3-base, wherein each described spacer nipple is optional by one or more substituent X
1Replace;
Each substituent X wherein
1Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately.
5. the drug delivery conjugates of claim 4, wherein said hetero atom joint is a nitrogen, and wherein said substituent X
1Be combined together to form heterocycle with described hetero atom joint with the spacer nipple that is connected them.
6. the drug delivery conjugates of claim 5, wherein said heterocycle is selected from pyrrolidine, piperidines, oxazolidine, isoxazole alkyl, Thiazolidine, isothiazolidine, ketopyrrolidine, piperidones, oxazolidone, isoxazole alkyl ketone, thiazolidone, isothiazolidine ketone and butanimide.
7. the drug delivery conjugates of claim 1; but wherein said releasing-joint is selected from methylene; 1-alkoxyl alkylidene; 1-alkoxyl cycloalkylidene; 1-alkoxyl alkylidene carbonyl; 1-alkoxyl cycloalkylidene carbonyl; the carbonyl aryl carbonyl; carbonyl (carboxyl aryl) carbonyl; carbonyl (two carboxyl aryl) carbonyl; halo alkylidene carbonyl; alkylidene (dialkyl group silicyl); alkylidene (alkylaryl silicyl); alkylidene (diaryl silicyl); (dialkyl group silicyl) aryl; (alkylaryl silicyl) aryl; (diaryl silicyl) aryl; oxygen base ketonic oxygen base; oxygen base ketonic oxygen base alkyl; the sulfonyl alkyl; the imino group alkylidene; the carbonyl alkylen group imino group; the imino group cycloalkylidene; carbonyl cycloalkylidene imino group; the alkylidene sulfonyl; the alkylidene sulfenyl; alkylidene aryl sulfenyl and carbonylic alkyl sulfenyl, but wherein each described releasing-joint is optional by one or more substituent X
2Replace;
Each substituent X wherein
2Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately.
8. the drug delivery conjugates of claim 7, wherein said hetero atom joint is a nitrogen, and wherein said substituent X
2But be combined together to form heterocycle with the releasing-joint that is connected them with described hetero atom joint.
9. the drug delivery conjugates of claim 8, wherein said heterocycle is selected from pyrrolidine, piperidines, oxazolidine, isoxazole alkyl, Thiazolidine, isothiazolidine, ketopyrrolidine, piperidones, oxazolidone, isoxazole alkyl ketone, thiazolidone, isothiazolidine ketone and butanimide.
10. the drug delivery conjugates of claim 1; wherein said hetero atom joint is a nitrogen; and but wherein said releasing-joint and described hetero atom joint are combined together to form bilvalent radical; described bilvalent radical comprises alkylidene aziridine-1-base, alkylidene carbonyl aziridine-1-base, carbonylic alkyl aziridine-1-base, alkylidene sulfonic group aziridine-1-base, sulfonic group alkyl aziridine-1-base, sulfonyl alkyl aziridine-1-base or alkylidene sulfonyl aziridine-1-base, but wherein each described releasing-joint is optional by one or more substituent X
2Replace;
Each substituent X wherein
2Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately.
11. the drug delivery conjugates of claim 10; wherein said hetero atom joint is a nitrogen; and but described releasing-joint and described hetero atom joint be combined together to form bilvalent radical, and described bilvalent radical comprises alkylidene aziridine-1-base, carbonylic alkyl aziridine-1-base, sulfonic group alkyl aziridine-1-base or sulfonyl alkyl aziridine-1-base.
12. the drug delivery conjugates of claim 11, wherein said spacer nipple is selected from carbonyl, thiocarbonyl, alkylidene carbonyl, cycloalkylidene carbonyl, carbonylic alkyl carbonyl and 1-(carbonylic alkyl) butanimide-3-base, and wherein each described spacer nipple is optional by one or more substituent X
1Replace;
Each substituent X wherein
1Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately; And
But wherein said spacer nipple is connected to and forms the aziridine amide on the releasing-joint.
13. the drug delivery conjugates of claim 1, wherein said medicine is mitomycin, E09 or mitomycin analogs, but described releasing-joint is selected from carbonylic alkyl sulfenyl, carbonyl tetrahydrochysene-2H-pyranose, carbonyl tetrahydrofuran base, 1-(carbonyl tetrahydrochysene-2H-pyranose) butanimide-3-base and 1-(carbonyl tetrahydrofuran base) butanimide-3-base, but wherein each described releasing-joint is optional by one or more substituent X
2Replace,
Each substituent X wherein
2Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately;
And but the aziridine of wherein said mitomycin is connected to formation acyl group aziridine on the described releasing-joint.
14. the drug delivery conjugates of claim 1, wherein said medicine comprises two key nitrogen-atoms, but wherein said releasing-joint is selected from alkylidene carbonylamino and 1-(alkylidene carbonylamino) butanimide-3-base, but and wherein said releasing-joint be connected on the described medicine nitrogen and form hydrazone.
15. the drug delivery conjugates of claim 1, wherein said medicine comprises sulphur atom, but described releasing-joint is selected from alkylidene sulfenyl and carbonylic alkyl sulfenyl, but and wherein said releasing-joint be connected on the described medicine sulfur and form disulphide.
16. the drug delivery conjugates of claim 4, wherein said vitamin receptor bound fraction is the folic acid that comprises nitrogen, and described spacer nipple is selected from alkylidene carbonyl, cycloalkylidene carbonyl, carbonylic alkyl carbonyl and 1-(carbonylic alkyl) butanimide-3-base, and wherein said spacer nipple is connected to and forms imidodicarbonic diamide or alkylamide on the folic acid nitrogen.
17. the drug delivery conjugates of claim 16, wherein each substituent X
1Independently be selected from alkyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, carboxyl, carboxyalkyl, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately.
18. the drug delivery conjugates of claim 4, wherein said hetero atom joint is a nitrogen, and described spacer nipple is selected from alkylidene carbonyl, cycloalkylidene carbonyl, carbonylic alkyl carbonyl and 1-(carbonylic alkyl) butanimide-3-base, and wherein each described spacer nipple is optional by one or more substituent X
1Replace, and described spacer nipple is connected on the described nitrogen and forms amide.
19. the drug delivery conjugates of claim 4, wherein said hetero atom joint is a sulfur, and described spacer nipple is selected from alkylidene and cycloalkylidene, and wherein each described spacer nipple is optional by carboxyl substituted, and described spacer nipple is connected on the described sulfur and forms mercaptan.
20. the drug delivery conjugates of claim 4, wherein said hetero atom joint is a sulfur, and described spacer nipple is selected from 1-alkylidene butanimide-3-base and 1-(carbonylic alkyl) butanimide-3-base, and described spacer nipple is connected to and forms the basic mercaptan of butanimide-3-on the described sulfur.
21. the drug delivery conjugates of claim 7, wherein said hetero atom joint is an oxygen, and but described releasing-joint is selected from methylene, 1-alkoxyl alkylidene, 1-alkoxyl cycloalkylidene, 1-alkoxyl alkylidene carbonyl and 1-alkoxyl cycloalkylidene carbonyl, but wherein each described releasing-joint is optional by one or more substituent X
2Replace, but and described releasing-joint be connected to and form acetal or ketal on the described oxygen.
22. the drug delivery conjugates of claim 7, wherein said hetero atom joint is an oxygen, but and described releasing-joint be methylene, aryl that wherein said methylene is optionally substituted replaces, but and described releasing-joint be connected to and form acetal or ketal on the described oxygen.
23. the drug delivery conjugates of claim 7, wherein said medicine comprises nitrogen-atoms, described hetero atom joint is a nitrogen, and but described releasing-joint is selected from carbonyl aryl carbonyl, carbonyl (carboxyl aryl) carbonyl, carbonyl (two carboxyl aryl) carbonyl, and but described releasing-joint is connected on the described hetero atom joint nitrogen and forms amide, and is connected on the described medicine nitrogen and forms amide.
24. the drug delivery conjugates of claim 7, wherein said medicine comprises oxygen atom, described hetero atom joint is a nitrogen, and but described releasing-joint is selected from carbonyl aryl carbonyl, carbonyl (carboxyl aryl) carbonyl, carbonyl (two carboxyl aryl) carbonyl, and but described releasing-joint is connected on the described hetero atom joint nitrogen and forms amide, and is connected on the described medicine oxygen and forms ester.
25. the drug delivery conjugates of claim 7, wherein said hetero atom joint is a nitrogen, and but described releasing-joint is selected from imino group alkylidene, carbonyl alkylen group imino group, imino group cycloalkylidene and carbonyl cycloalkylidene imino group, but wherein each described releasing-joint is optional by one or more substituent X
2Replace, but and described releasing-joint be connected on the described nitrogen and form hydrazone.
26. the drug delivery conjugates of claim 7, wherein said hetero atom joint is an oxygen, and but described releasing-joint is selected from alkylidene (dialkyl group silicyl), alkylidene (alkylaryl silicyl), alkylidene (diaryl silicyl), (dialkyl group silicyl) aryl, (alkylaryl silicyl) aryl and (diaryl silicyl) aryl, but wherein each described releasing-joint is optional by one or more substituent X
2Replace, but and described releasing-joint be connected on the described oxygen and form silanol.
27. the drug delivery conjugates of claim 1, wherein said medicine comprises nitrogen-atoms, but and described releasing-joint for optional by one or more substituent X
2The halo alkylidene carbonyl that replaces;
Each substituent X wherein
2Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately;
But described releasing-joint is connected on the described medicine nitrogen and forms amide.
28. the drug delivery conjugates of claim 1, wherein said medicine comprises oxygen atom, but and described releasing-joint for optional by one or more substituent X
2The alkylene oxide group carbonyl or the halo alkylidene carbonyl that replace;
Each substituent X wherein
2Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately;
But described releasing-joint is connected to and forms carbonic ester or ester on the described medicine oxygen.
29. the drug delivery conjugates of claim 1, wherein said hetero atom joint is an oxygen, and described spacer nipple is for choosing wantonly by one or more substituent X
1The 1-alkylidene butanimide-3-base that replaces, and but described releasing-joint is selected from methylene, 1-alkoxyl alkylidene, 1-alkoxyl cycloalkylidene, 1-alkoxyl alkylidene carbonyl, 1-alkoxyl cycloalkylidene carbonyl, but wherein each described releasing-joint is optional by one or more substituent X
2Replace;
Each substituent X wherein
2Independently be selected from alkyl, alkoxyl, alkoxyalkyl, hydroxyl, hydroxy alkyl, amino, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl, halogen, haloalkyl, mercaptoalkyl, alkyl sulfenyl alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylic acid ester group, alkyl alkanoic acid ester group, guanidine alkylation, R
4-carbonyl, R
5-carbonylic alkyl, R
6-acylamino-and R
7-amidoalkyl, wherein R
4And R
5Independently be selected from aminoacid, amino acid derivativges and peptide separately, and R wherein
6And R
7Independently be selected from aminoacid, amino acid derivativges and peptide separately;
But wherein said spacer nipple and described releasing-joint are connected to separately and form butanimide-1-base alkyl acetal or ketal on the described hetero atom joint.
30. the drug delivery conjugates of claim 1, but wherein said bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkoxy methyl oxygen base, and wherein said methyl is optional to be replaced by alkyl or substituted aryl.
31. the drug delivery conjugates of claim 1; but wherein said bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkyl-carbonyl, and wherein said carbonyl and described medicine or its analog or derivant form the acyl group aziridine.
32. the drug delivery conjugates of claim 1, but wherein said bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form the inferior cycloalkyloxy of 1-alkoxyl.
33. comprising, the drug delivery conjugates of claim 1, wherein said bivalence joint be combined together to form alkylidene amino carbonyl (dicarboxyl arlydene) but spacer nipple, hetero atom joint and the releasing-joint of carboxylate.
34. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form disulfide group alkyl-carbonyl hydrazides, and wherein said hydrazides and described medicine or its analog or derivant form hydrazone.
35. the drug delivery conjugates of claim 1, but wherein said bivalence joint comprises hetero atom joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkyl-carbonyl hydrazides, and wherein said hydrazides and described medicine or its analog or derivant form hydrazone.
36. the drug delivery conjugates of claim 1; wherein said bivalence joint comprises and is combined together to form 3-sulfenyl alkyl sulphonyl alkyl (two replace silicyl) but hetero atom joint, spacer nipple, hetero atom joint, spacer nipple and the releasing-joint of oxygen base that wherein said two replace silicyls is replaced by alkyl or the optional aryl that replaces.
37. the drug delivery conjugates of claim 1, wherein said bivalence joint comprises many spacer nipples that are selected from naturally occurring aminoacid and stereoisomer thereof.
38. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkoxy carbonyl, and wherein said carbonyl and described medicine or its analog or derivant form carbonic ester.
39. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group aryl-alkoxy carbonyl, wherein said carbonyl and described medicine or its analog or derivant form carbonic ester, and described aryl is optional the replacement.
40. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises hetero atom joint, spacer nipple releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-sulfenyl butanimide-1-base alkoxyl alkoxyl alkylidene, wherein said alkylidene and described medicine or its analog or derivant form hydrazone, each alkyl is independently selected, and described oxygen base alkoxyl is optional by alkyl or the optional aryl replacement that replaces.
41. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkoxy carbonyl hydrazides.
42. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkyl amino, and wherein said amino and described medicine or its analog or derivant form the bivinyl amide.
43. the drug delivery conjugates of claim 42, wherein said alkyl are ethyl.
44. the drug delivery conjugates of claim 1, but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group alkyl amino-carbonyl, and wherein said carbonyl and described medicine or its analog or derivant form carbamate.
45. the drug delivery conjugates of claim 44, wherein said alkyl are ethyl.
46. the drug delivery conjugates of claim 1; but but wherein said bivalence joint comprises releasing-joint, spacer nipple and the releasing-joint that is combined together to form 3-disulfide group aryl-alkoxy carbonyl, and wherein said carbonyl and described medicine or its analog or derivant form carbamate or carbamoyl aziridine.
47. a vitamin receptor binding drug delivery conjugates, described conjugate comprises:
(a) be selected from vitamin and vitamin receptor thereof vitamin receptor bound fraction in conjunction with analog and derivant;
(b) but comprise first, second and the 3rd hetero atom joint, spacer nipple and at least one is not the bivalence joint of the releasing-joint of disulphide; With
(c) medicine or its analog or derivant;
Wherein said vitamin receptor bound fraction is covalently bound to described bivalence joint by the first hetero atom joint;
Described medicine or its analog or derivant are covalently bound to described bivalence joint by the second hetero atom joint;
But described spacer nipple and described releasing-joint are covalently bound mutually by the 3rd hetero atom joint.
48. a vitamin receptor binding drug delivery conjugates, described conjugate comprises:
(a) be selected from vitamin and vitamin receptor thereof vitamin receptor bound fraction in conjunction with analog and derivant;
(b) but comprise hetero atom joint, spacer nipple and at least one is not the bivalence joint of the releasing-joint of disulphide; With
(c) medicine or its analog or derivant;
Wherein said vitamin receptor bound fraction is covalently bound to described bivalence joint;
Described medicine or its analog or derivant are covalently bound to described bivalence joint;
But described spacer nipple and described releasing-joint are covalently bound mutually by the hetero atom joint.
49. a pharmaceutical composition, described pharmaceutical composition comprise among the claim 1-48 each drug delivery conjugates and medicine acceptable carrier, diluent or excipient.
50. an elimination has the method for the interior pathogenic cell colony of host animal body of pathogenic cell colony, the member of wherein said pathogenic cell colony have can and vitamin or the binding site of its analog or derivant, and wherein said binding site is by the unique expression of described pathogenic cell, overexpression or preferential the expression, and described method comprises and gives among described host's claim 1-48 each the drug delivery conjugates or the step of its pharmaceutical composition.
51. a vitamin receptor binding drug delivery conjugates intermediate, described intermediate comprises:
(a) vitamin receptor bound fraction;
(b) has the bivalence joint of first terminal and second end; With
(c) coupling group;
Wherein said coupling group is nucleopilic reagent, electrophilic reagent or its precursor that can form covalent bond with medicine or its analog or derivant;
Described vitamin receptor bound fraction is covalently bound to described bivalence joint at first end of described bivalence joint, and described coupling group is covalently bound to described bivalence joint at second end of described bivalence joint;
Described bivalence joint comprises that one or more are selected from following component: but spacer nipple releasing-joint and hetero atom joint and combination thereof; But being described bivalence joint, condition comprises that at least one is not the releasing-joint of disulphide.
52. vitamin receptor binding drug delivery conjugates with following formula:
V-L-D
Wherein L is selected from (l
s)
a, (l
H)
b(l
r)
cAnd combination; (l wherein
rBut) be releasing-joint, (l
s) be spacer nipple, (l
H) be the hetero atom joint;
V is the vitamin receptor bound fraction, and D is medicine or its analog or derivant; And
A, b and c are 0,1,2,3 or 4 independently of one another;
Condition is that L comprises that at least one is not disulphide (l
r).
53. vitamin receptor binding drug delivery conjugates with following formula:
V-L-D
Wherein L is selected from (l
s)
a(l
H)
bAnd combination; (l wherein
s) be spacer nipple, (l
H) be the hetero atom joint; A and b are 0,1,2,3 or 4 independently of one another; And
V is the vitamin receptor bound fraction, and D is medicine or its analog or derivant.
54. the drug delivery conjugates of claim 53, wherein said medicine are hapten.
55. the drug delivery conjugates of claim 54, wherein said hapten is selected from fluorescein and dinitrophenyl.
56. a pharmaceutical composition, described pharmaceutical composition comprise among the claim 53-55 each drug delivery conjugates and medicine acceptable carrier, diluent or excipient.
57. an elimination has the method for the interior pathogenic cell colony of host animal body of pathogenic cell colony, the member of wherein said pathogenic cell colony have can and vitamin or the binding site of its analog or derivant, and wherein said binding site is by the unique expression of described pathogenic cell, overexpression or preferential the expression, and described method comprises and gives among described host's claim 53-55 each the drug delivery conjugates or the step of its pharmaceutical composition.
58. a vitamin receptor binding drug delivery conjugates intermediate, described intermediate comprises:
(a) has the bivalence joint of first terminal and second end;
(b) medicine or its analog or derivant; With
(c) coupling group;
Wherein said bivalence joint comprises that one or more are selected from following component: but spacer nipple releasing-joint and hetero atom joint and combination thereof; But being described bivalence joint, condition comprises that at least one is not the releasing-joint of disulphide;
Described coupling group is first terminal covalently bound to described bivalence joint described bivalence joint, and second terminal covalently bound to described bivalence joint at described bivalence joint of described medicine or its analog or derivant;
Described coupling group is nucleopilic reagent, electrophilic reagent or its precursor that can form covalent bond with the vitamin receptor bound fraction.
59. the intermediate of claim 58, wherein said vitamin receptor bound fraction are that vitamin or its vitamin receptor are in conjunction with analog or derivant; Described coupling group is a michael acceptor; But and described bivalence joint comprise have the releasing-joint that is selected from following formula :-C (O) NHN=,-NHC (O) NHN=and-CH
2C (O) NHN=.
60. the intermediate of claim 58, but wherein said bivalence joint comprises first releasing-joint and first spacer nipple; And but described first releasing-joint, described first spacer nipple and described coupling group are combined together to form the chemical compound with following formula:
Wherein D is medicine or its analog or derivant, and n is selected from 1,2,3 and 4 integer.
61. the intermediate of claim 60, wherein said vitamin receptor bound fraction comprises the alkyl hydrosulfide nucleopilic reagent.
62. one kind prepares the method with following formula: compound:
Wherein V is the vitamin receptor bound fraction; L is selected from (l
r)
c, (l
s)
a(l
H)
bAnd combination, wherein (l
rBut) be releasing-joint, (l
s) be spacer nipple, (l
H) be the hetero atom joint, a, b and c are selected from 0,1,2,3 and 4 integer; D is medicine or its analog or derivant; Described method comprises:
(a) make chemical compound with following formula:
React with chemical compound with following formula:
(b) hydazone derivative of the described medicine of formation or its analog or derivant.
63. one kind prepares the method with following formula: compound:
Wherein V is the vitamin receptor bound fraction; L is selected from (l
r)
c, (l
s)
a(l
H)
bAnd combination, wherein (l
rBut) be releasing-joint, (l
s) be spacer nipple, (l
H) be the hetero atom joint, a, b and c are selected from 0,1,2,3 and 4 integer; D is medicine or its analog or derivant; Described method comprises:
Make chemical compound with following formula:
React with chemical compound with following formula:
64. the method for claim 63, wherein said medicine or its analog or derivant are selected from daunomycin, daunomycin ketone (daunomycinones), daunorubicin, aklavine, paclitaxel, chromomycin and aurilic acids.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US44284503P | 2003-01-27 | 2003-01-27 | |
| US60/442,845 | 2003-01-27 | ||
| US60/492,119 | 2003-08-01 | ||
| US60/516,188 | 2003-10-31 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100815632A Division CN101239190B (en) | 2003-01-27 | 2004-01-27 | Vitamin receptor binding drug delivery conjugates |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1761488A true CN1761488A (en) | 2006-04-19 |
| CN100381177C CN100381177C (en) | 2008-04-16 |
Family
ID=36707301
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004800076791A Expired - Fee Related CN100381177C (en) | 2003-01-27 | 2004-01-27 | Vitamin receptor binding drug delivery conjugates |
| CN2008100815632A Expired - Fee Related CN101239190B (en) | 2003-01-27 | 2004-01-27 | Vitamin receptor binding drug delivery conjugates |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100815632A Expired - Fee Related CN101239190B (en) | 2003-01-27 | 2004-01-27 | Vitamin receptor binding drug delivery conjugates |
Country Status (3)
| Country | Link |
|---|---|
| CN (2) | CN100381177C (en) |
| SI (1) | SI1592457T1 (en) |
| ZA (1) | ZA200506672B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103403552A (en) * | 2010-11-12 | 2013-11-20 | 恩多塞特公司 | Methods of treating cancer |
| CN103861116A (en) * | 2014-03-06 | 2014-06-18 | 沈阳药大制剂新技术有限公司 | Prodrug of antitumor medicament as well as preparation method and application thereof |
| WO2014101134A1 (en) * | 2012-12-28 | 2014-07-03 | Yan Wenguang | Folic acid derivative, and preparing method and application thereof |
| CN101784565B (en) * | 2007-06-25 | 2014-12-10 | 恩多塞特公司 | Conjugates containing hydrophilic spacer linkers |
| CN104784699A (en) * | 2014-01-20 | 2015-07-22 | 博瑞生物医药技术(苏州)有限公司 | Folate receptor binding ligand-drug conjugate |
| CN105801604A (en) * | 2016-03-07 | 2016-07-27 | 孙关 | Eriocalyxin B derivatives with tumor targeting as well as preparation method and application |
| CN105849568A (en) * | 2013-11-14 | 2016-08-10 | 恩多塞特公司 | Compounds for positron emission tomography |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701061B (en) * | 2009-10-30 | 2011-09-28 | 北京化工大学 | Drug delivering material taking Vitamin C as hydrophilic fragments and preparation method thereof |
| RU2012154914A (en) * | 2010-05-19 | 2014-06-27 | Эндосайт, Инк. | IMPROVED METHOD FOR OBTAINING FOLATED TARGETS |
| KR102238317B1 (en) * | 2012-05-17 | 2021-04-12 | 익스텐드 바이오사이언시즈, 인크. | Carriers for improved drug delivery |
| US9616109B2 (en) | 2014-10-22 | 2017-04-11 | Extend Biosciences, Inc. | Insulin vitamin D conjugates |
| US9585934B2 (en) | 2014-10-22 | 2017-03-07 | Extend Biosciences, Inc. | Therapeutic vitamin D conjugates |
| CN109912683B (en) * | 2017-12-13 | 2023-01-06 | 杭州多禧生物科技有限公司 | Cytotoxin molecule, conjugate, preparation method and application thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5688488A (en) * | 1989-04-03 | 1997-11-18 | Purdue Research Foundation | Composition and method for tumor imaging |
| US5837533A (en) * | 1994-09-28 | 1998-11-17 | American Home Products Corporation | Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent |
| GB9600272D0 (en) * | 1996-01-06 | 1996-03-06 | Univ Nottingham | Polymers |
| AUPQ071299A0 (en) * | 1999-06-02 | 1999-06-24 | Access Pharmaceuticals Australia Pty Limited | Vitamin directed dual targeting therapy |
| JP5448284B2 (en) * | 2000-06-02 | 2014-03-19 | ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム | Ethylenedicysteine (EC) -drug conjugate |
| KR20040015234A (en) * | 2001-05-02 | 2004-02-18 | 펄듀 리서치 파운데이션 | Treatment and diagnosis of macrophage mediated disease |
| AU2003243226A1 (en) * | 2002-05-15 | 2003-12-02 | Endocyte, Inc. | Vitamin-mitomycin conjugates |
-
2004
- 2004-01-27 CN CNB2004800076791A patent/CN100381177C/en not_active Expired - Fee Related
- 2004-01-27 SI SI200431940T patent/SI1592457T1/en unknown
- 2004-01-27 CN CN2008100815632A patent/CN101239190B/en not_active Expired - Fee Related
-
2005
- 2005-08-19 ZA ZA200506672A patent/ZA200506672B/en unknown
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101784565B (en) * | 2007-06-25 | 2014-12-10 | 恩多塞特公司 | Conjugates containing hydrophilic spacer linkers |
| CN104383553A (en) * | 2007-06-25 | 2015-03-04 | 恩多塞特公司 | Conjugates containing hydrophilic spacer linkers |
| CN103403552A (en) * | 2010-11-12 | 2013-11-20 | 恩多塞特公司 | Methods of treating cancer |
| CN103403552B (en) * | 2010-11-12 | 2016-01-06 | 恩多塞特公司 | The method of Therapeutic cancer |
| WO2014101134A1 (en) * | 2012-12-28 | 2014-07-03 | Yan Wenguang | Folic acid derivative, and preparing method and application thereof |
| CN105849568A (en) * | 2013-11-14 | 2016-08-10 | 恩多塞特公司 | Compounds for positron emission tomography |
| CN105849568B (en) * | 2013-11-14 | 2018-05-04 | 恩多塞特公司 | Compound for positron emission fault art |
| CN104784699A (en) * | 2014-01-20 | 2015-07-22 | 博瑞生物医药技术(苏州)有限公司 | Folate receptor binding ligand-drug conjugate |
| CN104784699B (en) * | 2014-01-20 | 2019-05-03 | 博瑞生物医药（苏州）股份有限公司 | Folate receptor binding ligand-drug conjugates |
| CN103861116A (en) * | 2014-03-06 | 2014-06-18 | 沈阳药大制剂新技术有限公司 | Prodrug of antitumor medicament as well as preparation method and application thereof |
| CN105801604A (en) * | 2016-03-07 | 2016-07-27 | 孙关 | Eriocalyxin B derivatives with tumor targeting as well as preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| SI1592457T1 (en) | 2012-12-31 |
| ZA200506672B (en) | 2010-08-25 |
| CN100381177C (en) | 2008-04-16 |
| CN101239190A (en) | 2008-08-13 |
| CN101239190B (en) | 2013-09-25 |
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