CA2895779A1 - Legumain activated doxorubicin derivative as well as preparation method and application thereof - Google Patents
Legumain activated doxorubicin derivative as well as preparation method and application thereof Download PDFInfo
- Publication number
- CA2895779A1 CA2895779A1 CA2895779A CA2895779A CA2895779A1 CA 2895779 A1 CA2895779 A1 CA 2895779A1 CA 2895779 A CA2895779 A CA 2895779A CA 2895779 A CA2895779 A CA 2895779A CA 2895779 A1 CA2895779 A1 CA 2895779A1
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- Prior art keywords
- asn
- ala
- leu
- doxorubicin
- compound
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- 230000004071 biological effect Effects 0.000 description 1
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- 231100000366 bone marrow toxicity Toxicity 0.000 description 1
- NKWPZUCBCARRDP-UHFFFAOYSA-L calcium bicarbonate Chemical compound [Ca+2].OC([O-])=O.OC([O-])=O NKWPZUCBCARRDP-UHFFFAOYSA-L 0.000 description 1
- 229910000020 calcium bicarbonate Inorganic materials 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- MQYQOVYIJOLTNX-UHFFFAOYSA-N dichloromethane;n,n-dimethylformamide Chemical compound ClCCl.CN(C)C=O MQYQOVYIJOLTNX-UHFFFAOYSA-N 0.000 description 1
- 229940035857 doxorubicin hydrochloride 10 mg Drugs 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- XLYMOEINVGRTEX-UHFFFAOYSA-N fumaric acid monoethyl ester Natural products CCOC(=O)C=CC(O)=O XLYMOEINVGRTEX-UHFFFAOYSA-N 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
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- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- GNOIPBMMFNIUFM-UHFFFAOYSA-N hexamethylphosphoric triamide Chemical compound CN(C)P(=O)(N(C)C)N(C)C GNOIPBMMFNIUFM-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
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- 230000002427 irreversible effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 description 1
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 description 1
- 229910052808 lithium carbonate Inorganic materials 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
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- QWDJLDTYWNBUKE-UHFFFAOYSA-L magnesium bicarbonate Chemical compound [Mg+2].OC([O-])=O.OC([O-])=O QWDJLDTYWNBUKE-UHFFFAOYSA-L 0.000 description 1
- 229910000022 magnesium bicarbonate Inorganic materials 0.000 description 1
- 239000002370 magnesium bicarbonate Substances 0.000 description 1
- 235000014824 magnesium bicarbonate Nutrition 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000014380 magnesium carbonate Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
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- 230000002107 myocardial effect Effects 0.000 description 1
- WVKNBCACIPKHEW-UHFFFAOYSA-N n,n-diethylethanamine;n,n-dimethylformamide Chemical compound CN(C)C=O.CCN(CC)CC WVKNBCACIPKHEW-UHFFFAOYSA-N 0.000 description 1
- TYRGLVWXHJRKMT-QMMMGPOBSA-N n-benzyloxycarbonyl-l-serine-betalactone Chemical compound OC(=O)[C@H](C)NC(=O)OCC1=CC=CC=C1 TYRGLVWXHJRKMT-QMMMGPOBSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 208000011932 ovarian sarcoma Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000003880 polar aprotic solvent Substances 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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- 238000001953 recrystallisation Methods 0.000 description 1
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- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
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- 238000001356 surgical procedure Methods 0.000 description 1
- HBEJJYHFTZDAHZ-QMMMGPOBSA-N tert-butyl (2s)-2-amino-4-methylpentanoate Chemical compound CC(C)C[C@H](N)C(=O)OC(C)(C)C HBEJJYHFTZDAHZ-QMMMGPOBSA-N 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
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Abstract
The present invention discloses doxorubicin derivatives for targeted activation by legumain, its preparation method and use. The doxorubicin derivatives are obtained by condensation between the amino group of compound A and the carboxyl goup of compound B
and have the following structure:
(see above structures) compounds A and B have the following structures, respectively:
(see above compounds) wherein R3 in compound B is Leu or absent; R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn; R6 is (see formula I), wherein n=1-20; or (see formula II), wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated C1-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon. The doxorubicin derivatives of the present invention are specifically tumor-targeted and have a long in vivo metabolic half-life, as compared with doxorubicin. They exhibit an efficient and safe anti-tumor effect and could be use to prepare an anti-tumor drug.
and have the following structure:
(see above structures) compounds A and B have the following structures, respectively:
(see above compounds) wherein R3 in compound B is Leu or absent; R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn; R6 is (see formula I), wherein n=1-20; or (see formula II), wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated C1-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon. The doxorubicin derivatives of the present invention are specifically tumor-targeted and have a long in vivo metabolic half-life, as compared with doxorubicin. They exhibit an efficient and safe anti-tumor effect and could be use to prepare an anti-tumor drug.
Description
Legumain activated Doxorubicin derivative as well as preparation method and application thereof Technical Field The present invention relates to an anti-tumor compound. Specifically, the present invention relates to a doxorubicin derivative for targeted activation by legumain, with a long metabolic half-life in vivo, and its preparation method and use.
Technical Background Doxorubicin (DOX) hydrochloride and epirubicin are commercially available anti-tumor antibiotics with a broad anti-tumor spectrum, killing many kinds of tumor cells, and the mechanism underlying is mainly the inhibition on the synthesis of nucleic acids upon the doxorubicin molecule inserting into DNA. Doxorubicin hydrochloride and epirubicin can be used to treat hematological tumors and solid tumors, such as breast cancer, ovarian cancer, sarcoma, and many other solid tumors. However, the dose of this kind of anthracycline compounds is restricted in clinical use because they bring serious toxicity or side effect.
Doxorubicin hydrochloride can cause various unwanted effects, including bone marrow toxicity, gastrointestinal diseases, stomatitis, alopecia, exosmosis, acute and cumulative cardiac toxicity. A main limitation of doxorubicin hydrochloride lies in that, after each course of treatment, a great dose of doxorubicin hydrochloride leads to sharp reduction of monocytes and platelets in bone marrow and blood. A major concern is that a cumulative cardiac toxicity may induce a myocardial congestive heart failure, which is irreversible.
Doxorubicin (DOX) and epirubicin were functionally modified to produce effective doxorubicin based anti-tumor drugs having lower side effect.
Inventors of the subject application reported the structures and biological effects of Legubicin (BOC-AANL-DOX) and LEG3 (Succinyl-AANL-DOX) in Cancer Research in 2003 and 2006. However, further investigation on the drugs showed that each of both compounds is only provided with a single-targeting ability. And they had a short metabolic half-life, which leads to insufficient concentration and duration necessary for drug activation at the tumor site, and in turn, low efficacy in an animal model. Thus, they are not ideal anti-tumor drugs.
Therefore, targeting doxorubicin- and/or epirubicin-based drugs with reduced toxicity of doxorubicin hydrochloride and epirubicin and high efficacy as anti-tumor agents are in special need.
Disclosure of the Invention The present invention intends to provide a targeting doxorubicin derivative for targeted activation by legumain, which exhibits a long metabolic half-life in vivo. The derivative only targets to and is only activated at the tumor site. Thus, it has greatly reduced toxicity and greatly increased efficacy.
To achieve the purpose, the present invention provides a doxorubicin derivative for targeted activation by legumain, which exhibits a long metabolic half-life in vivo and has the following structural formula:
OH
OH
.1 0 0 OH 0 õõN-113¨
Asn¨ R4 ¨R5¨R6 (s) 0, The doxorubicin derivative is prepared by condensation between amino of compound A
and carboxyl of compound B, wherein compounds A and B have the following structures, respectively:
40000,0H OH
R
OCH30 OH 0.,,,NH2 3-Asn-R4-Compound A Compound B
wherein R3 in compound B is Leu or absent, if R3 is absent then compound B is a tripeptide, that is, the carboxyl of Asn covalently condensates with the amino of compound A
directly to produce apolypeptide doxorubicin; if R3 is Leu, then compound B is a tetrapeptide, that is Leu-Asn-R4-R5-;
R4 is any one amino acid selected from the group consisting of Ala and Thr;
R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn;
Technical Background Doxorubicin (DOX) hydrochloride and epirubicin are commercially available anti-tumor antibiotics with a broad anti-tumor spectrum, killing many kinds of tumor cells, and the mechanism underlying is mainly the inhibition on the synthesis of nucleic acids upon the doxorubicin molecule inserting into DNA. Doxorubicin hydrochloride and epirubicin can be used to treat hematological tumors and solid tumors, such as breast cancer, ovarian cancer, sarcoma, and many other solid tumors. However, the dose of this kind of anthracycline compounds is restricted in clinical use because they bring serious toxicity or side effect.
Doxorubicin hydrochloride can cause various unwanted effects, including bone marrow toxicity, gastrointestinal diseases, stomatitis, alopecia, exosmosis, acute and cumulative cardiac toxicity. A main limitation of doxorubicin hydrochloride lies in that, after each course of treatment, a great dose of doxorubicin hydrochloride leads to sharp reduction of monocytes and platelets in bone marrow and blood. A major concern is that a cumulative cardiac toxicity may induce a myocardial congestive heart failure, which is irreversible.
Doxorubicin (DOX) and epirubicin were functionally modified to produce effective doxorubicin based anti-tumor drugs having lower side effect.
Inventors of the subject application reported the structures and biological effects of Legubicin (BOC-AANL-DOX) and LEG3 (Succinyl-AANL-DOX) in Cancer Research in 2003 and 2006. However, further investigation on the drugs showed that each of both compounds is only provided with a single-targeting ability. And they had a short metabolic half-life, which leads to insufficient concentration and duration necessary for drug activation at the tumor site, and in turn, low efficacy in an animal model. Thus, they are not ideal anti-tumor drugs.
Therefore, targeting doxorubicin- and/or epirubicin-based drugs with reduced toxicity of doxorubicin hydrochloride and epirubicin and high efficacy as anti-tumor agents are in special need.
Disclosure of the Invention The present invention intends to provide a targeting doxorubicin derivative for targeted activation by legumain, which exhibits a long metabolic half-life in vivo. The derivative only targets to and is only activated at the tumor site. Thus, it has greatly reduced toxicity and greatly increased efficacy.
To achieve the purpose, the present invention provides a doxorubicin derivative for targeted activation by legumain, which exhibits a long metabolic half-life in vivo and has the following structural formula:
OH
OH
.1 0 0 OH 0 õõN-113¨
Asn¨ R4 ¨R5¨R6 (s) 0, The doxorubicin derivative is prepared by condensation between amino of compound A
and carboxyl of compound B, wherein compounds A and B have the following structures, respectively:
40000,0H OH
R
OCH30 OH 0.,,,NH2 3-Asn-R4-Compound A Compound B
wherein R3 in compound B is Leu or absent, if R3 is absent then compound B is a tripeptide, that is, the carboxyl of Asn covalently condensates with the amino of compound A
directly to produce apolypeptide doxorubicin; if R3 is Leu, then compound B is a tetrapeptide, that is Leu-Asn-R4-R5-;
R4 is any one amino acid selected from the group consisting of Ala and Thr;
R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn;
2 R6 is 0 , wherein n=1-20; or 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl -C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
Compound B (R3-Asn-R4-R6) consists of a short peptide R3-Asn-R4-R5- which is specifically hydrolyzed by legumain, and a functional group R6 which improves the metabolic half-life of the drug. legumain can cleave the peptide fragment, by hydrolysis, at the position before Asn to release compound A-Asn or compound A.
In the above doxorubicin derivative for targeted activation by legumain, compound A may be doxorubicin or epirubicin, wherein doxorubicin has the following structure:
0 Oid I
õ
OH
doxorubicin wherein epirubicin is an isomer of doxorubicin and has the following structure:
*OH
0 0 OH 0õ 12 , OH
In the above doxorubicin derivative for targeted activation by Legumain, preferably, R6 is:
N , -or The present invention further provides a method for preparing the above doxorubicin
Compound B (R3-Asn-R4-R6) consists of a short peptide R3-Asn-R4-R5- which is specifically hydrolyzed by legumain, and a functional group R6 which improves the metabolic half-life of the drug. legumain can cleave the peptide fragment, by hydrolysis, at the position before Asn to release compound A-Asn or compound A.
In the above doxorubicin derivative for targeted activation by legumain, compound A may be doxorubicin or epirubicin, wherein doxorubicin has the following structure:
0 Oid I
õ
OH
doxorubicin wherein epirubicin is an isomer of doxorubicin and has the following structure:
*OH
0 0 OH 0õ 12 , OH
In the above doxorubicin derivative for targeted activation by Legumain, preferably, R6 is:
N , -or The present invention further provides a method for preparing the above doxorubicin
3 derivative for targeted activation by Legumain, comprising the following steps:
Step 1, preparing a tripeptide or a tetrapeptide, R3-Asn-R4-R5, by conjugating the amino acid residues together and isolating to obtain the formed tripeptide or tetrapeptide R3-Asn-R4-R5;
Step 2, preparing compound B by reacting R3-Asn-R4-R5 obtained in step 1 with the acyl or carboxyl of R6-C1 or R6-OH to obtain R3-Asn-R4-R5-R6;
Step 3, covalently combining the carboxyl in R3 of the compound R3-Asn-R4-R5-obtained in step 2 with the amino of compound A to form the doxorubicin derivative for targeted activation by Legumain, having a long half-life.
In the method for preparing the above doxorubicin derivative for targeted activation by Legumain, R3 in compound B is Leu or absent; R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn; and R6 is a drug targeting functional group, selected from group N R7, )===õ\:
0 , wherein n=1-20; or group 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
In the method for preparing the above doxorubicin derivative for targeted activation by Legumain, R6 is:
N
) 41 c 11 0`
or The present invention also provides use of the above doxorubicin derivative for targeted activation by Legumain in the preparation of an anti-tumor drug.
To reduce toxicity brought by an anti-tumor drug on normal cells and tissues of human body, great efforts have been made to enhance the biological specificity and selectivity of anti-tumor drugs. Recently, with development in tumor molecular biology and other basic sciences, great improvement in research and development of drugs for targeted treatment of tumors has been made. The molecular targets for the above doxorubicin derivative for targeted activation by Legumain are cathepsin (Cathepsin) and Legumain (Legumain) that generally expressed in malignant tumor cells. Both cathepsin and Legumain are proteolytic enzymes highly expressed at the tumor site. They present in most of the solid tumors and tumor microenvironment, and
Step 1, preparing a tripeptide or a tetrapeptide, R3-Asn-R4-R5, by conjugating the amino acid residues together and isolating to obtain the formed tripeptide or tetrapeptide R3-Asn-R4-R5;
Step 2, preparing compound B by reacting R3-Asn-R4-R5 obtained in step 1 with the acyl or carboxyl of R6-C1 or R6-OH to obtain R3-Asn-R4-R5-R6;
Step 3, covalently combining the carboxyl in R3 of the compound R3-Asn-R4-R5-obtained in step 2 with the amino of compound A to form the doxorubicin derivative for targeted activation by Legumain, having a long half-life.
In the method for preparing the above doxorubicin derivative for targeted activation by Legumain, R3 in compound B is Leu or absent; R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn; and R6 is a drug targeting functional group, selected from group N R7, )===õ\:
0 , wherein n=1-20; or group 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
In the method for preparing the above doxorubicin derivative for targeted activation by Legumain, R6 is:
N
) 41 c 11 0`
or The present invention also provides use of the above doxorubicin derivative for targeted activation by Legumain in the preparation of an anti-tumor drug.
To reduce toxicity brought by an anti-tumor drug on normal cells and tissues of human body, great efforts have been made to enhance the biological specificity and selectivity of anti-tumor drugs. Recently, with development in tumor molecular biology and other basic sciences, great improvement in research and development of drugs for targeted treatment of tumors has been made. The molecular targets for the above doxorubicin derivative for targeted activation by Legumain are cathepsin (Cathepsin) and Legumain (Legumain) that generally expressed in malignant tumor cells. Both cathepsin and Legumain are proteolytic enzymes highly expressed at the tumor site. They present in most of the solid tumors and tumor microenvironment, and
4 also distributed in a great amount in immune infiltrating macrophages and endothelial cells of the new blood vessels. Overexpression of such enzymes is highly associated with invasion of the tumor cells to the normal histiocytes, tumor metastasis and apoptosis of tumor cells.
Because of the increased permeability of blood vessels along with the growth of the tumor, high expression of the Legumain, Legumain, within the tumor microenvironment, and low pH in the local acidic microenvironment on the surface of the tumor, the modified polypeptide doxorubicin hydrochloride which is tumor microenvironment-targeting and activated, as a specific substrate of Legumain, will be effectively hydrolyzed and activated only in the tumor microenvironment, thereby releasing its cytotoxicity. Although there are also a small amount of Legumains expressed in other normal cells of a human body, this enzyme is not active in the microenvironment on the surface of normal cells. Thus, the polypeptide doxorubicin hydrochloride for targeted activation could not be hydrolyzed and activated on the surface of normal cells, and in turn causes no cytotoxicity to normal cells.
Consequently, the targeted activation produces cytotoxicity only to tumor cells.
Additionally, since the drug can be completely released after the peptide chain is hydrolyzed by Legumain, we can conjugate a targeted group that can improve the efficacy of the drug to the other end of the peptide chain. After screening by experiments, the drug release and activation will not be affected and the retention of the drug at the tumor site and its anti-tumor efficiency can be improved when R6 is preferably 6-maleimidocaproic (8-maleimidocaproic, EMC) or trans-butanedioic acid monoester (EFA).
R6 includes 0 , wherein n=1-20; or 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl -C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
Preferably, R6 is 6-maleimidocaproic (6- maleimidocaproic, EMC) or trans-butanedioic acid monoester (EFA).
Therefore, as compared with doxorubicin, the doxorubicin derivative for targeted activation by Legumain synthesized in the present invention exhibits both tumor-specific targeting and tumor-specific activation, and a highly efficient and safe anti-tumor effect.
In summary, the present invention provides a targeted doxorubicin derivative for targeted activation by Legumain, having a long metabolic half¨life in vivo, wherein Legumain can cleave the peptide by hydrolysis at the position before Asn of the drug to release compound A-Leu or compound A. As a result, the doxorubicin of the present invention is tumor-targeted and has a long half-life. As compared with doxorubicin and epirubicin, the derivative of the present invention has a greatly improved efficacy and a greatly reduced toxicity and is thus provided
Because of the increased permeability of blood vessels along with the growth of the tumor, high expression of the Legumain, Legumain, within the tumor microenvironment, and low pH in the local acidic microenvironment on the surface of the tumor, the modified polypeptide doxorubicin hydrochloride which is tumor microenvironment-targeting and activated, as a specific substrate of Legumain, will be effectively hydrolyzed and activated only in the tumor microenvironment, thereby releasing its cytotoxicity. Although there are also a small amount of Legumains expressed in other normal cells of a human body, this enzyme is not active in the microenvironment on the surface of normal cells. Thus, the polypeptide doxorubicin hydrochloride for targeted activation could not be hydrolyzed and activated on the surface of normal cells, and in turn causes no cytotoxicity to normal cells.
Consequently, the targeted activation produces cytotoxicity only to tumor cells.
Additionally, since the drug can be completely released after the peptide chain is hydrolyzed by Legumain, we can conjugate a targeted group that can improve the efficacy of the drug to the other end of the peptide chain. After screening by experiments, the drug release and activation will not be affected and the retention of the drug at the tumor site and its anti-tumor efficiency can be improved when R6 is preferably 6-maleimidocaproic (8-maleimidocaproic, EMC) or trans-butanedioic acid monoester (EFA).
R6 includes 0 , wherein n=1-20; or 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl -C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
Preferably, R6 is 6-maleimidocaproic (6- maleimidocaproic, EMC) or trans-butanedioic acid monoester (EFA).
Therefore, as compared with doxorubicin, the doxorubicin derivative for targeted activation by Legumain synthesized in the present invention exhibits both tumor-specific targeting and tumor-specific activation, and a highly efficient and safe anti-tumor effect.
In summary, the present invention provides a targeted doxorubicin derivative for targeted activation by Legumain, having a long metabolic half¨life in vivo, wherein Legumain can cleave the peptide by hydrolysis at the position before Asn of the drug to release compound A-Leu or compound A. As a result, the doxorubicin of the present invention is tumor-targeted and has a long half-life. As compared with doxorubicin and epirubicin, the derivative of the present invention has a greatly improved efficacy and a greatly reduced toxicity and is thus provided
5 with a promising application prospect.
Brief Description of Drawing Figure 1 is a histogram experimental result of the binding effect of Si , Succinyl-AANL-DOX and Si +E-64 to cathepsin. The result showing that Si can bind cathepsin and this binding can be inhibited by cathepsin inhibitor E-64.
Figure 2 is a curve showing the experimental results obtained from inhibition of enzymatic activity of cathepsin by Si .The result showing that cross linking between Si and cysteine of the active center of the enzyme.
Figure 3 shows accumulative effects of Si, Dox and Succinyl-AANL-DOX at the cancer cell. The result showing that the binding between Si and cathepsin have the effect to accumulate the drug at tumor cell. After cleaving by legumain, Dox and Leu-DOX
become cell penetrating and remain inside cancer cell.
Figure 4 shows distribution of Si, Dox and Succinyl-AANL-DOX in the tumor and heart tissues, indicating accumulation of S1 in the tumor site and reduced accumulation in the heart.
The result showing that the binding and activation cause drug selectly accumulate in tumor tissue. With a low concentration in normal tissue, the Si have no toxicity to heart.
Figure 5 is a curve showing the tumor-inhibiting effects of Si of the present invention, Dox, Succinyl-AANL-DOX, Legubicin and solvent control (Vehicle) on the 4T1 breast cancer model, indicating that EMC-AANL-DOX exhibits a superior inhibitory effect on tumor growth over Dox, BOC-AANL-DOX and Succinyl-AANL-DOX.
Figure 6 is a curve showing the tumor-inhibiting effects of Si of the present invention, used in different doses, on a human non-small cell lung cancer model (A549), indicating that the efficacy of EMC-AANL-DOX satisfies our requirements on further clinical development.
Best Mode for Carrying Out the Invention The technical solution of the present invention is further illustrated by making reference to the Examples.
The present invention provides a method for preparing a polypeptide-doxorubicin for targeted activation by Legumain in tumor microenvironment, comprising the following steps:
firstly, conjugating amino acid residues by a known chemical, biological or recombinant technique, and isolating to obtain the formed polypeptide R3-Asn-R4-R5;
secondly, reacting the N-terminal of formed polypeptide with the carboxyl or acyl of R6 which can bind to albumin, by a known chemical or biological method, to form a covalent conjugate R3-Asn-R4-R5-R6;
Brief Description of Drawing Figure 1 is a histogram experimental result of the binding effect of Si , Succinyl-AANL-DOX and Si +E-64 to cathepsin. The result showing that Si can bind cathepsin and this binding can be inhibited by cathepsin inhibitor E-64.
Figure 2 is a curve showing the experimental results obtained from inhibition of enzymatic activity of cathepsin by Si .The result showing that cross linking between Si and cysteine of the active center of the enzyme.
Figure 3 shows accumulative effects of Si, Dox and Succinyl-AANL-DOX at the cancer cell. The result showing that the binding between Si and cathepsin have the effect to accumulate the drug at tumor cell. After cleaving by legumain, Dox and Leu-DOX
become cell penetrating and remain inside cancer cell.
Figure 4 shows distribution of Si, Dox and Succinyl-AANL-DOX in the tumor and heart tissues, indicating accumulation of S1 in the tumor site and reduced accumulation in the heart.
The result showing that the binding and activation cause drug selectly accumulate in tumor tissue. With a low concentration in normal tissue, the Si have no toxicity to heart.
Figure 5 is a curve showing the tumor-inhibiting effects of Si of the present invention, Dox, Succinyl-AANL-DOX, Legubicin and solvent control (Vehicle) on the 4T1 breast cancer model, indicating that EMC-AANL-DOX exhibits a superior inhibitory effect on tumor growth over Dox, BOC-AANL-DOX and Succinyl-AANL-DOX.
Figure 6 is a curve showing the tumor-inhibiting effects of Si of the present invention, used in different doses, on a human non-small cell lung cancer model (A549), indicating that the efficacy of EMC-AANL-DOX satisfies our requirements on further clinical development.
Best Mode for Carrying Out the Invention The technical solution of the present invention is further illustrated by making reference to the Examples.
The present invention provides a method for preparing a polypeptide-doxorubicin for targeted activation by Legumain in tumor microenvironment, comprising the following steps:
firstly, conjugating amino acid residues by a known chemical, biological or recombinant technique, and isolating to obtain the formed polypeptide R3-Asn-R4-R5;
secondly, reacting the N-terminal of formed polypeptide with the carboxyl or acyl of R6 which can bind to albumin, by a known chemical or biological method, to form a covalent conjugate R3-Asn-R4-R5-R6;
6 _ thirdly, covalently binding the carboxyl of R3 in R3-Asn-R4-R5-R6 (or the carboxyl of Asn when R3 is absent) to the amino of doxorubicin or salt thereof or doxorubicin derivative or salt thereof (compound A) to form a doxorubicin analogue, i.e., compound A-R3-Asn-R4-R5-R6, which has a short peptide and a targeting group. The reaction scheme is showed as follows:
Amino acids used in the present invention Conjugating the amino acid residues by a known chemical, biological or recombinant technique, and isolating V
A short peptide, protective group-R3-Asn-R4-R0112), containing a suitable protective group for amino acid Condensation agents, Acid, ester or acyl chloride containing bases, polar aprotic the R6 group solvents V
R6-R5-R4-Asn-R3-protective group De-protected i R6-R5-ft4-Asn-R3 Condensation agents, A: doxorubicin or its hydrochloride, or bases, polar aprotic epirubicin or its hydrochloride solvents V
A-R3-Asn-R4-R5-R6 , The condensating agent includes the known chemical agents used for condensation of a carboxylic acid with an amino group to form an amide, which can be used alone or in combination, such as 1-hydroxylbenzotriazol (HOBT), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 2-(7-azabenzotriazol-1-y1)-N,N,N',Nt-tetramethyluronium tetrafluoroborate (TATU), 0-(benzotriazol-1-y1)-N,N,N,N-tetramethyluronium tetrafluoroborate (TBTU), N-hydroxy-7-azabenzotriazole (HOAT), benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), etc.
The base may include an inorganic base or an aprotic organic base. The inorganic base may include sodium carbonate, potassium carbonate, lithium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, etc. The aprotic organic base may include triethylamine, N,N-
Amino acids used in the present invention Conjugating the amino acid residues by a known chemical, biological or recombinant technique, and isolating V
A short peptide, protective group-R3-Asn-R4-R0112), containing a suitable protective group for amino acid Condensation agents, Acid, ester or acyl chloride containing bases, polar aprotic the R6 group solvents V
R6-R5-R4-Asn-R3-protective group De-protected i R6-R5-ft4-Asn-R3 Condensation agents, A: doxorubicin or its hydrochloride, or bases, polar aprotic epirubicin or its hydrochloride solvents V
A-R3-Asn-R4-R5-R6 , The condensating agent includes the known chemical agents used for condensation of a carboxylic acid with an amino group to form an amide, which can be used alone or in combination, such as 1-hydroxylbenzotriazol (HOBT), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 2-(7-azabenzotriazol-1-y1)-N,N,N',Nt-tetramethyluronium tetrafluoroborate (TATU), 0-(benzotriazol-1-y1)-N,N,N,N-tetramethyluronium tetrafluoroborate (TBTU), N-hydroxy-7-azabenzotriazole (HOAT), benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), etc.
The base may include an inorganic base or an aprotic organic base. The inorganic base may include sodium carbonate, potassium carbonate, lithium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, etc. The aprotic organic base may include triethylamine, N,N-
7 diisopropylethylamine, pyridine, 4-dimethylaminopyridine, and N-methylmorpholine, etc.
Polar aprotic solvent may include N,N-dimethylformamide, dichloromethane, trichloromethane, ethyl acetate, tetrahydrofuran, acetonitrile, dioxane, methyl t-butyl ether, ethylene glycol dimethyl ether, dimethylsulfoxide, and hexamethylphosphoramide, etc.
Example 1: Synthesis of the polypeptide doxorubicins Si and S2 for targeted activation in the tumor microenvironment Si and S2 were synthesized as follows:
L-Ala-OMe LION
Cbz-L-Ala-OH Cbz-L-Ala-L-Ala-OMe THF, H2O - Cbz-L-Ala-L-Ala-OH
HOBt, EDCHCI, DCM, Et3N
II
L-Leu-OtBu Fmoc-L-Asn(Trt)-OH FMoc-L-Asn(Trt)-L-Leu-OtBu piperidine--L-Asn(Trt)-L-Leu-OtBu HOBt, EDCHCI, DCM, Et3N DMF
Ill IV
Cbz-L-Ala-L-Ala-OH________ Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF Pd/C
V VI
___________________ - R6-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu R6-L-Ala-L-Ala-L-Asn-Leu-OH
HBTU, DIPEA, DMF DCM
VII R6 = EMC IX R6 = EMC
VIII R6 = EFA X R6= EFC
OH IXor X OH
0H 0400, _____________________________ iosiolp-bH
HBTU, DMF
OCH30 OH 6 õNH2HCI OCH30 OH õNyL-Leu-L-Asn-L-Ala-L-Ala-Rs "OH C) "OH
0 0 Si R6 = EMC
R6 = Or C) 0 S2 R6 = EFA
1) Synthesis of Cbz-L-Ala-L-Ala-Ome (I) L-Ala-OMe Cbz-L-Ala-OH Cbz-L-Ala-L-Ala-OMe HOBt, EDCHCI, DCM, Et3N
N-benzyloxycarbonyl-L-alanine (100g, 0.45mol) was dissolved in a dried N,N-dimethylformamide (3L), and 1-hydroxylbenzotriazole (72.6g, 0.54mo1) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (103.3g, 0.54mol) were added while
Polar aprotic solvent may include N,N-dimethylformamide, dichloromethane, trichloromethane, ethyl acetate, tetrahydrofuran, acetonitrile, dioxane, methyl t-butyl ether, ethylene glycol dimethyl ether, dimethylsulfoxide, and hexamethylphosphoramide, etc.
Example 1: Synthesis of the polypeptide doxorubicins Si and S2 for targeted activation in the tumor microenvironment Si and S2 were synthesized as follows:
L-Ala-OMe LION
Cbz-L-Ala-OH Cbz-L-Ala-L-Ala-OMe THF, H2O - Cbz-L-Ala-L-Ala-OH
HOBt, EDCHCI, DCM, Et3N
II
L-Leu-OtBu Fmoc-L-Asn(Trt)-OH FMoc-L-Asn(Trt)-L-Leu-OtBu piperidine--L-Asn(Trt)-L-Leu-OtBu HOBt, EDCHCI, DCM, Et3N DMF
Ill IV
Cbz-L-Ala-L-Ala-OH________ Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF Pd/C
V VI
___________________ - R6-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu R6-L-Ala-L-Ala-L-Asn-Leu-OH
HBTU, DIPEA, DMF DCM
VII R6 = EMC IX R6 = EMC
VIII R6 = EFA X R6= EFC
OH IXor X OH
0H 0400, _____________________________ iosiolp-bH
HBTU, DMF
OCH30 OH 6 õNH2HCI OCH30 OH õNyL-Leu-L-Asn-L-Ala-L-Ala-Rs "OH C) "OH
0 0 Si R6 = EMC
R6 = Or C) 0 S2 R6 = EFA
1) Synthesis of Cbz-L-Ala-L-Ala-Ome (I) L-Ala-OMe Cbz-L-Ala-OH Cbz-L-Ala-L-Ala-OMe HOBt, EDCHCI, DCM, Et3N
N-benzyloxycarbonyl-L-alanine (100g, 0.45mol) was dissolved in a dried N,N-dimethylformamide (3L), and 1-hydroxylbenzotriazole (72.6g, 0.54mo1) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (103.3g, 0.54mol) were added while
8 stirring. After stirring for reaction for 1 hour, the mixture was subjected to an ice bath until the temperature reached 0 C. L-alanine methyl ester (46.2g, 0.45mo1) and N,N-diisopropyl ethylamine (173.8g, 1.34mol) dissolved in an N,N-dimethylformamide (1L) solution were dropped into the mixture. After dropping, the mixture was stirred under ambient temperature for 10 hours and the solvents were removed by evaporation under reduced pressure.
The crude product was dissolved in dichloromethane (2L) and washed successively with saturated ammonia chloride solution, water and saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate and the solvents were removed by evaporation under reduced pressure. The crude product was re-crystallized by ethyl acetate/petroleum ether to obtain a pure product, which was a white solid I, i.e., Cbz-L-Ala-L-Ala-OMe (101g; Yield, 73.1%).
2) Synthesis of Cbz-L-Ala-L-Ala-OH (II) Cbz-L-Ala-L-Ala-OMe LIONTHF, H20 ) Cbz-L-Ala-L-Ala-OH
I II
Cbz-L-Ala-L-Ala-OMe (100 g, 0.34 mol) was dissolved in a mixed solution of tetrahydrofuran (2 L) and water (I L) and cooled to 0 C. 1 mol/L lithium hydroxide solution (400 mL) was dropped to the mixture and then stirred and reacted for 10 hours.
Concentrated hydrochloric acid was dropped to the mixture to neutralize its pH to below 6.
Tetrahydrofuran was removed by evaporation under reduced pressure. The residual water phase was extracted by dichloromethane (1 Lx3). The organic phase was dried with anhydrous sodium sulfate and removed by evaporation under reduced pressure to obtain a white solid II, i.e., Cbz-Ala-Ala-OH
(88g; Yield, 92.2%).
3) Synthesis of Fmoc-L-Asn(Trt)-L-Leu-013u (III) L-Leu-OtBu Fmoc-L-Asn(Trt)-OH 1P- FMoc-L-Asn(Trt)-L-Leu-OtBu HOBt, EDCHCI, DCM, Et3N
III
L-leucine t-butyl ester (22.4 g, 0.1 ml), N-Fmoc-N'-tribenzyl asparagine (59.6 g, 0.1 mol) were disclosed in N,N-dimethylformamide (1000 mL) in a three-necked bottle. 1-hydroxylbenzotriazol (14.85 g, 0.11 mol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide
The crude product was dissolved in dichloromethane (2L) and washed successively with saturated ammonia chloride solution, water and saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate and the solvents were removed by evaporation under reduced pressure. The crude product was re-crystallized by ethyl acetate/petroleum ether to obtain a pure product, which was a white solid I, i.e., Cbz-L-Ala-L-Ala-OMe (101g; Yield, 73.1%).
2) Synthesis of Cbz-L-Ala-L-Ala-OH (II) Cbz-L-Ala-L-Ala-OMe LIONTHF, H20 ) Cbz-L-Ala-L-Ala-OH
I II
Cbz-L-Ala-L-Ala-OMe (100 g, 0.34 mol) was dissolved in a mixed solution of tetrahydrofuran (2 L) and water (I L) and cooled to 0 C. 1 mol/L lithium hydroxide solution (400 mL) was dropped to the mixture and then stirred and reacted for 10 hours.
Concentrated hydrochloric acid was dropped to the mixture to neutralize its pH to below 6.
Tetrahydrofuran was removed by evaporation under reduced pressure. The residual water phase was extracted by dichloromethane (1 Lx3). The organic phase was dried with anhydrous sodium sulfate and removed by evaporation under reduced pressure to obtain a white solid II, i.e., Cbz-Ala-Ala-OH
(88g; Yield, 92.2%).
3) Synthesis of Fmoc-L-Asn(Trt)-L-Leu-013u (III) L-Leu-OtBu Fmoc-L-Asn(Trt)-OH 1P- FMoc-L-Asn(Trt)-L-Leu-OtBu HOBt, EDCHCI, DCM, Et3N
III
L-leucine t-butyl ester (22.4 g, 0.1 ml), N-Fmoc-N'-tribenzyl asparagine (59.6 g, 0.1 mol) were disclosed in N,N-dimethylformamide (1000 mL) in a three-necked bottle. 1-hydroxylbenzotriazol (14.85 g, 0.11 mol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide
9 hydrochloride (23 g, 0.12 mol) were added under stirring and then the mixture was subjected to an ice bath until the temperature reached 0 C. N,N-diisopropyl ethylamine (25.8 g, 0.2 mol) was dropped and then the mixture was stirred for 10 hours. Then the solvents were removed by evaporation under reduced pressure. The crude product was dissolved in chloroform (1000 ml) and washed successively with saturated ammonia chloride solution, saturated sodium chloride solution and water. The organic phase was dried with anhydrous sodium sulfate and filtered.
The solvents were removed by evaporation under reduced pressure to obtain a crude product.
The crude product was purified by recrystallization (dichloromethane:ethyl acetate=1:1, by volume) to obtain a white solid III, i.e., Fmoc-L-Asn(Trt)-L-Leu-013u (42.4 g;
Yield, 55.4%).
4) Synthesis of L-Asn(Trt)-L-Leu-OtBu (IV) FMoc-L-Asn(Trt)-L-Leu-0 tBu piperidine L-Asn(Trt)-L-Leu-OtBu DMF
III IV
Fmoc-L-Asn(Trt)-L-Leu-Ch3u (7.65 g, 0.01 mol) was dissolved in a mixed solution of dichloromethane (100 mL) and N,N-dimethylformamide (100 mL) and then piperidine (40 ml) was added. The mixture was stirring under ambient temperature for 5 hours. The solvents were removed by evaporation under reduced pressure and a small amount of residual piperidine was removed by drying under high vacuum in a high vacuum oven to produce L-Asn(Trt)-L-Leu-O'Bu (IV), which was a pale yellow solid and could be used without further purification.
5) Synthesis of Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (V) L-Asn(Trt)-L-Leu-OtBu Cbz-L-Ala-L-Ala-OHCbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF
IV V
The crude L-Asn(Trt)-L-Leu-OtBu obtained in step 4) was dissolved in N,N-dimethylformamide (200 mL), and Cbz-L-Ala-L-Ala-OH (2.94 g, 0.012 mol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (6.07 g, 0.016 mol) were added. The mixture was subjected to an ice bath until its temperature reached 0 C. N,N-diisopropyl ethylamine (2.6 g, 0.02 mol) was added and then the mixture was stirred overnight under ambient temperature. The solvents were removed by evaporation under reduced pressure.
The residue was dissolved in chloroform (100 ml), washed successively with saturated ammonia chloride solution and saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered. Then the solvents were removed by evaporation.
The resultant crude product was subject to silica column chromatography (dichloromethane:methano1=50:1 -20:1, by volume) to produce compound V, i.e., Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu, which was a white solid (3.1 g; total yield in two steps, 37.8%).
6) Synthesis of L-Ala-LAla-L-Asn(Trt)-Leu-OtBu (VI) Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu Pd/C L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu V VI
Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (10 g, 12.2 mmol) was dissolved in methanol (400mL) and 10% palladium-carbon (1g) was added. After introducing hydrogen gas, the mixture was stirred for reaction under normal pressure and temperature for 4 hours. Then the reaction mixture was filtered to remove palladium-carbon and washed with methanol. The filtrate and the wash solution were pooled and the solvents contained therein were removed by evaporation under reduced pressure to obtain VI, i.e., L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu, which was a white solid (7.6 g; Yield, 91%).
7) Synthesis of EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (VII) OH
L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu 0 r EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF
vi vii 6-Maleimidocaproic acid (1.02 g, 4.82 mmol) was dissolved in N,N-dimethylformamide (60 mL), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (2.49 g, 6.57 mmol) was added. Then the mixture was stirred under ambient temperature for half an hour and subjected to an ice bath until its temperature reached 0 C. L-Ala-L-Ala-L-Asn-L-Leu-O'Bu (3g, 4.38mmol) and N,N-diisopropyl ethylamine (1.13 g, 8.76 mmol) dissolved in N,N-dimethylformamide (60 mL) were dropped into the mixture. After dropping, the mixture was waimed up to ambient temperature and then stirred for 10 hours. The solvents were removed by evaporation under reduced pressure. The residue was dissolved in dichloromethane (200 mL) and washed successively with saturated ammonia chloride solution and saturated sodium chloride solution, dried withanhydrous sodium sulfate, and filtered. Then the solvents were removed by evaporation under reduced pressure. The resultant crude product was subjected to silica column chromatography (dichloromethane:methano1=50:1 - 20:1, by volume) to produce compound VII, i.e., EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu, which was a pale yellow solid (2.52 g; total yield in two steps, 65.46%).
8) Synthesis of EFA-L-Ala-L-Ala-L-Asn-L-Leu- OtBu (VIII) L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu __________________ EFA-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF
VI VIII
Monoethyl fumarate (0.69 g, 4.82 mmol) was dissolved in N,N-dimethylformamide (60 mL), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (2.49 g, 6.57mmol) was added. Then the mixture was stirred under ambient temperature for half an hour and subjected to an ice bath until its temperature was below OE. L-Ala-L-Ala-L-Asn-L-Leu-O'Bu (3 g, 4.38 mmol) and N,N-diisopropyl ethylamine (1.13 g, 8.76 mmol) dissolved in N,N-dimethylformamide (60 mL) were dropped into the mixture. After dropping, the mixture was warmed up to ambient temperature and then stirred for 10 hours. The solvents were removed by evaporation under reduced pressure. The residue was dissolved in dichloromethane (200 mL) and washed successively with saturated ammonia chloride solution and saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered. Then the solvents were removed by evaporation under reduced pressure. The resultant crude product was subjected to silica column chromatography (dichloromethane:methano1=50:1 - 20:1, by volume) to produce compound VIII, i.e., EFA-L-Ala-L-Ala-L-Asn(TrO-L-Leu-OtBu, which was a pale yellow solid (2.10 g; Yield, 59.15%).
9) Synthesis of EMC-L-Ala-L-Ala-L-Asn-L-Leu-OH (IX) TD Fc Am EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu EMC-L-Ala-L-Ala-L-Asn-Leu-OH
VII IX
EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (1g, 1.68mmol) was dissolved in dichloromethane (50mL), and trifluoroacetic acid (10mL) was added. The mixture was stirred under ambient temperature for 10 hours. The reaction solution was washed with water and then separated. The organic phase was dried with anhydrous sodium sulfate and the solvents were removed by evaporation under reduced pressure. The residual trifluoroacetic acid was removed by evaporation under high vacuum to produce a white solid IX, i.e., EMC-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.60g; Yield, 90.9%).
The solvents were removed by evaporation under reduced pressure to obtain a crude product.
The crude product was purified by recrystallization (dichloromethane:ethyl acetate=1:1, by volume) to obtain a white solid III, i.e., Fmoc-L-Asn(Trt)-L-Leu-013u (42.4 g;
Yield, 55.4%).
4) Synthesis of L-Asn(Trt)-L-Leu-OtBu (IV) FMoc-L-Asn(Trt)-L-Leu-0 tBu piperidine L-Asn(Trt)-L-Leu-OtBu DMF
III IV
Fmoc-L-Asn(Trt)-L-Leu-Ch3u (7.65 g, 0.01 mol) was dissolved in a mixed solution of dichloromethane (100 mL) and N,N-dimethylformamide (100 mL) and then piperidine (40 ml) was added. The mixture was stirring under ambient temperature for 5 hours. The solvents were removed by evaporation under reduced pressure and a small amount of residual piperidine was removed by drying under high vacuum in a high vacuum oven to produce L-Asn(Trt)-L-Leu-O'Bu (IV), which was a pale yellow solid and could be used without further purification.
5) Synthesis of Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (V) L-Asn(Trt)-L-Leu-OtBu Cbz-L-Ala-L-Ala-OHCbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF
IV V
The crude L-Asn(Trt)-L-Leu-OtBu obtained in step 4) was dissolved in N,N-dimethylformamide (200 mL), and Cbz-L-Ala-L-Ala-OH (2.94 g, 0.012 mol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (6.07 g, 0.016 mol) were added. The mixture was subjected to an ice bath until its temperature reached 0 C. N,N-diisopropyl ethylamine (2.6 g, 0.02 mol) was added and then the mixture was stirred overnight under ambient temperature. The solvents were removed by evaporation under reduced pressure.
The residue was dissolved in chloroform (100 ml), washed successively with saturated ammonia chloride solution and saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered. Then the solvents were removed by evaporation.
The resultant crude product was subject to silica column chromatography (dichloromethane:methano1=50:1 -20:1, by volume) to produce compound V, i.e., Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu, which was a white solid (3.1 g; total yield in two steps, 37.8%).
6) Synthesis of L-Ala-LAla-L-Asn(Trt)-Leu-OtBu (VI) Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu Pd/C L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu V VI
Cbz-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (10 g, 12.2 mmol) was dissolved in methanol (400mL) and 10% palladium-carbon (1g) was added. After introducing hydrogen gas, the mixture was stirred for reaction under normal pressure and temperature for 4 hours. Then the reaction mixture was filtered to remove palladium-carbon and washed with methanol. The filtrate and the wash solution were pooled and the solvents contained therein were removed by evaporation under reduced pressure to obtain VI, i.e., L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu, which was a white solid (7.6 g; Yield, 91%).
7) Synthesis of EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (VII) OH
L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu 0 r EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF
vi vii 6-Maleimidocaproic acid (1.02 g, 4.82 mmol) was dissolved in N,N-dimethylformamide (60 mL), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (2.49 g, 6.57 mmol) was added. Then the mixture was stirred under ambient temperature for half an hour and subjected to an ice bath until its temperature reached 0 C. L-Ala-L-Ala-L-Asn-L-Leu-O'Bu (3g, 4.38mmol) and N,N-diisopropyl ethylamine (1.13 g, 8.76 mmol) dissolved in N,N-dimethylformamide (60 mL) were dropped into the mixture. After dropping, the mixture was waimed up to ambient temperature and then stirred for 10 hours. The solvents were removed by evaporation under reduced pressure. The residue was dissolved in dichloromethane (200 mL) and washed successively with saturated ammonia chloride solution and saturated sodium chloride solution, dried withanhydrous sodium sulfate, and filtered. Then the solvents were removed by evaporation under reduced pressure. The resultant crude product was subjected to silica column chromatography (dichloromethane:methano1=50:1 - 20:1, by volume) to produce compound VII, i.e., EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu, which was a pale yellow solid (2.52 g; total yield in two steps, 65.46%).
8) Synthesis of EFA-L-Ala-L-Ala-L-Asn-L-Leu- OtBu (VIII) L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu __________________ EFA-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu HBTU, DIPEA, DMF
VI VIII
Monoethyl fumarate (0.69 g, 4.82 mmol) was dissolved in N,N-dimethylformamide (60 mL), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (2.49 g, 6.57mmol) was added. Then the mixture was stirred under ambient temperature for half an hour and subjected to an ice bath until its temperature was below OE. L-Ala-L-Ala-L-Asn-L-Leu-O'Bu (3 g, 4.38 mmol) and N,N-diisopropyl ethylamine (1.13 g, 8.76 mmol) dissolved in N,N-dimethylformamide (60 mL) were dropped into the mixture. After dropping, the mixture was warmed up to ambient temperature and then stirred for 10 hours. The solvents were removed by evaporation under reduced pressure. The residue was dissolved in dichloromethane (200 mL) and washed successively with saturated ammonia chloride solution and saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered. Then the solvents were removed by evaporation under reduced pressure. The resultant crude product was subjected to silica column chromatography (dichloromethane:methano1=50:1 - 20:1, by volume) to produce compound VIII, i.e., EFA-L-Ala-L-Ala-L-Asn(TrO-L-Leu-OtBu, which was a pale yellow solid (2.10 g; Yield, 59.15%).
9) Synthesis of EMC-L-Ala-L-Ala-L-Asn-L-Leu-OH (IX) TD Fc Am EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu EMC-L-Ala-L-Ala-L-Asn-Leu-OH
VII IX
EMC-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (1g, 1.68mmol) was dissolved in dichloromethane (50mL), and trifluoroacetic acid (10mL) was added. The mixture was stirred under ambient temperature for 10 hours. The reaction solution was washed with water and then separated. The organic phase was dried with anhydrous sodium sulfate and the solvents were removed by evaporation under reduced pressure. The residual trifluoroacetic acid was removed by evaporation under high vacuum to produce a white solid IX, i.e., EMC-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.60g; Yield, 90.9%).
10) Synthesis of EFA-L-Ala-L-Ala-L-Asn-L-Leu-OH (X) EFA-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu TFA
EFA-L-Ala-L-Ala-L-Asn-Leu-OH
DCM
VIII X
EFA-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (1g, 1.23mmol) was dissolved in dichloromethane (50mL), and trifluoroacetic acid (10mL) was added. The mixture was stirred under ambient temperature for 10 hours. The reaction solution was washed with water and then separated. The organic phase was dried with anhydrous sodium sulfate and the solvents were removed by evaporation under reduced pressure. The residual trifluoroacetic acid was removed by evaporation under high vacuum to produce a white solid X, i.e., EFA-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.51g; Yield, 80.9%).
EFA-L-Ala-L-Ala-L-Asn-Leu-OH
DCM
VIII X
EFA-L-Ala-L-Ala-L-Asn(Trt)-L-Leu-OtBu (1g, 1.23mmol) was dissolved in dichloromethane (50mL), and trifluoroacetic acid (10mL) was added. The mixture was stirred under ambient temperature for 10 hours. The reaction solution was washed with water and then separated. The organic phase was dried with anhydrous sodium sulfate and the solvents were removed by evaporation under reduced pressure. The residual trifluoroacetic acid was removed by evaporation under high vacuum to produce a white solid X, i.e., EFA-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.51g; Yield, 80.9%).
11) Synthesis of EMC-AANL-Doxorubicin (51) HBTU, DMF
OH OH
4040400 IX , loose -0H
OCH30 OH ,,NH2HCI OCH30 OH 0 ,õNyL-Leu-L-Asn-L-Ala-L-Ala-EMC
`=:-7.'*OH
Si EMC-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.5g, 0.85mmol) and N-methyl morpholine (0.18g, 1.78mmol) were dissolved in anhydrous N,N-dimethylformamide (20mL) and cooled to On or below. Benzotriazole-N,N,N',N-tetramethyluronium hexatluophosphate (HBTU) (0.49g, 1.28mmol) was added. After stirring for half an hour, doxorubicin hydrochloride (0.45g, 0.78mmol) was added. Away from light, the reaction temperature was slowly warmed up to ambient temperature and then stirred for 5 hours. The reaction solution was added to 200mL, 0.1% acetate aqueous solution. Dichloromethane was added for extraction. The organic phases were pooled, washed with water, and dried with anhydrous sodium sulfate. The solvents were removed by evaporation under reduced pressure to obtain a crude product, which was orange red. The crude product was purified by silica column chromatography (dichloromethane/methanol) to produce the title product Si, i.e., EMC-AANL-Doxorubicin, which was a red solid (0.45g; Yield, 52.24%). The molecular weight was 1105.45. After analysis of S1 by HPLC-MASS, its purity at the 6.99 eluting peak was 97%, and the corresponded MASS result was 1105.45. Thus the target product was confirmed as Si.
OH OH
4040400 IX , loose -0H
OCH30 OH ,,NH2HCI OCH30 OH 0 ,õNyL-Leu-L-Asn-L-Ala-L-Ala-EMC
`=:-7.'*OH
Si EMC-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.5g, 0.85mmol) and N-methyl morpholine (0.18g, 1.78mmol) were dissolved in anhydrous N,N-dimethylformamide (20mL) and cooled to On or below. Benzotriazole-N,N,N',N-tetramethyluronium hexatluophosphate (HBTU) (0.49g, 1.28mmol) was added. After stirring for half an hour, doxorubicin hydrochloride (0.45g, 0.78mmol) was added. Away from light, the reaction temperature was slowly warmed up to ambient temperature and then stirred for 5 hours. The reaction solution was added to 200mL, 0.1% acetate aqueous solution. Dichloromethane was added for extraction. The organic phases were pooled, washed with water, and dried with anhydrous sodium sulfate. The solvents were removed by evaporation under reduced pressure to obtain a crude product, which was orange red. The crude product was purified by silica column chromatography (dichloromethane/methanol) to produce the title product Si, i.e., EMC-AANL-Doxorubicin, which was a red solid (0.45g; Yield, 52.24%). The molecular weight was 1105.45. After analysis of S1 by HPLC-MASS, its purity at the 6.99 eluting peak was 97%, and the corresponded MASS result was 1105.45. Thus the target product was confirmed as Si.
12) Synthesis of EFA-AANL-Doxorubicin (S2) OH
400400,0H OH HBTU, DMF
____________________________________________________ *op. OH
ocHp OH (3 õNH2HCI OCH30 OH 0,r- õN yL-Leu-L-Asn-L-Ala-L-Ala-EFA
, 0 "OH H. 0 EFA-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.44g, 0.85mmol) and N-methyl morpholine (0.18g, 1.78mmol) were dissolved in anhydrous N,N-dimethylformamide (20mL) and cooled to OH or below. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (0.49g, 1.28mmol) was added. After stirring for half an hour, doxorubicin hydrochloride (0.45g, 0.78mmol) was added. Away from light, the reaction temperature was slowly warmed up to ambient temperature and then stirred for 5 hours. The reaction solution was added to 200mL, 0.1% acetate aqueous solution. Dichloromethane was added for extraction. The organic phases were pooled, washed by water, and dried with anhydrous sodium sulfate. The solvents were removed by evaporation under reduced pressure to obtain a crude product, which was orange red. The crude product was purified by silica column chromatography (dichloromethane/methanol) to produce the title product S2, i.e., EFA-AANL-DOX, which was a red solid (0.40g; Yield, 49.43%). After analysis by HPLC-MASS, the purity at the 6.99 eluting peak was 96%, and the corresponding MASS result was 11138.41. Thus the target product was confirmed as S2.
E-Maleimidocaproic acid was synthesized according to Synthesis, 2008(8), 1316-and its reaction scheme is showed below:
AcOH , Phhyle / 0 + H2N W-00 OH i.
EMC
Example 2: Preparation of an injection The synthesized Si and S2 were dried under vacuum to produce red powders, sterilized by gas sterilization, and separately packaged in a sterile room. Before animal experiment, the powder was dissolved in water for injection containing 50% alcohol in the sterile room and then diluted with water for injection to the desired concentration.
Example 3: Method for determining the contents of Si and S2 and their content ranges Si and S2 samples were analyzed by analytical type HPLC (Agilent 1100 series, equipped with C8 column of 5um and 4.6 mm IDx250 mm, and the mobile phase being 0 -95% acetonitrile (CAN). Results showed that the purity was in the range of 95%
to 99%.
The beneficial effects of the present invention were demonstrated by the following drug tolerance assays and efficacy assays.
Test Example 1: Measurement of maximum tolerated dose (MTD) by intravenous administration of the test drug Test purpose: to investigate the acute toxicity of the subject new drug formulation via MTD assay by intravenous administration to mice.
Test drug: Si and S2 injections, diluted to the corresponding concentrations with physiological saline when tested.
Animal: the first class BALB/C mice, weighing 19-21 g and all mice being female.
Method and results: 36 female BALB/C mice weighing 19-21 g were randomly divided into 6 groups according to their body weights, with 6 mice in each group. As shown in Table 1, the mice were intraperitoneally injected with Si or S2 for just one time in a dose of 0 mg/kg, 50mg/kg, 100 mg/kg, 150mg/kg, 200 mg/kg or 300mg/kg. Control tests were performed by injecting 0.2m1 physiological saline or doxorubicin hydrochloride. Animals were observed for 17 continuous days for presence or absence of the following behaviors on each day: pilo-erection, hair tousle and lackluster, lethargy, stoop and irritable reaction, and body weight and death were recorded. Blood samples were taken on the 3, 5 and 14 days for counting the whole blood cells. Anmals were anatomized on day 14 to take the heart, liver, kidney, lung, spleen, and pancreas for HE staining.
Table 1: Comparison of mortality rates of test mice receiving different doses of Si and S2 injections, physiological saline or doxorubicin hydrochloride injection Group Dose (mg/kg) Number Number of Mortality of animal dead rate (%) animal 1 physiological saline 0 mg/kg 6 0 0 2 Si 50 mg/kg 6 0 0 3 51 100 mg/kg 6 0 0 4 Si 150 mg/kg 6 0 0 5 Si 200 mg/kg 6 2 33.3%
6 Si 300 mg/kg 6 5 100%
7 S2 50 mg/kg 6 0 0 8 S2 100 mg/kg 6 0 0 9 S2 150 mg/kg 6 0 0 S2 200 mg/kg 6 2 33.3%
11 S2 300 mg/kg 6 3 50%
17 doxorubicin hydrochloride 10 mg/kg 6 6 100%
Results and discussion: no pilo-erection, hair tousle and lackluster, lethargy, stoop, irritable reaction and death were observed in mice receiving 150 mg/kg Si and S2 injection. As 10 shown in Table 1, the MTD of the Si and S2 injections were far beyond 100 mol/kg, and were significantly higher than the MTD of doxorubicin hydrochloride (4 - 8 limol/kg). The MTD for intravenous administration of a test drug is an important reference index for drug toxicity. The results indicate that the toxicity of the doxorubicin hydrochloride derivative, which binds to serum albumin, is significantly reduced as compared with doxorubicin hydrochloride.
Test Example 2: Study on efficacy of S1 and S2 in nude mice Test purpose: to investigate the anti-tumor efficacy of S1 and S2 via mouse tumor treatment model.
Test drug: S1 and S2 injections, and doxorubicin hydrochloride injection, diluted to corresponding concentrations by physiological saline when testing.
Method and results:
1. Animal: nude mice of 6-8 weeks old, all female.
2. Production of tumor model 1) MDA-MB231 cells were purchased from American type culture collection (ATCC) and identified according the specification provided by ATCC. Cells were cultivated in dulbecco's minimum essential medium (DMEM culture medium) containing 10% fetal bovine serum at 37 C and 5% CO2. The cells were passaged for every three days and cells within the 15th Passage were used.
2) Production of tumor. 106MDA-MB231 cells were subcutaneously injected to the back of the nude mice. Mice were randomly grouped after the diameter of the tumor reached about 0.3 cm to 0.4 cm. Then treatment began.
3) Course of treatment According to the clinical application of Si and S2, drugs were intraperitoneally injected.
A dose of 30 mg/kg (<1/2 MTD) was used for the Si treatment group, 30 mg/kg (<1/2 MTD) for the S2 treatment group, and 4mg/kg (>1/2 MTD) for doxorubicin hydrochloride. The control group was administered with physiological saline. The drugs were administered twice weekly for three weeks.
4) Grouping and test results are shown in Table 2.
Table 2: Treatment effects of Sl, S2, doxorubicin hydrochloride and control on tumors of nude mice Group Number of Size of tumor (mm3) Inhibitory rate of animal tumor 20 days 38 days 20 days 38 days S1 group 10 287.42+30.52 678.49+37.7 25.8% 68.5%
S2 group 10 254.59+34.19 618.49+51.27 34.3% 71.3%
Control Group 10 358.7+39.7 881.2+86.5 7.4% 29.1%
(doxorubicin hydrochloride) Model Control 10 387.5+35.6 2155.44+325.
5) Results and discussion: as shown in Table 2, inhibitory effect on tumor in the nude mice after administering with Si and S2 by intraperitoneal injection was greatly improved as compared with the doxorubicin hydrochloride control group. And S2 could diminish and eliminate the tumor. Results show that this kind of drugs exhibit excellent inhibiting efficacy on tumor growth.
Test Example 3: Study on efficacy of Si and S2 in a tumor metastasis model from BALB/C mice Test purpose: to investigate the anti-tumor efficacy of S1 and S2 in a tumor metastasis treatment model from BALB/C mice.
Test drug: S1 and S2 injections, and doxorubicin hydrochloride injection, diluted to corresponding concentrations with physiological saline when testing.
1. Animal: BALB/C mice of 6-8 weeks old, all female.
2. Production of tumor model 1) 4T1 cells were purchased from American type culture collection (ATCC) and identified according the specification provided by ATCC. Cells were cultivated in DMEM
culture medium containing 10% fetal bovine serum at 37 C and 5% CO2. The cells were passaged for every three days and cells within the 15th passage were used.
2) Production of tumor metastasis. 106T1 cells were subcutaneously injected to the back of the BALB/C mice. Mice were randomly grouped after the tumor grew to about 1.5 cm. The subcutaneous tumor was removed by surgery and drug treatment began. Mice were killed after anesthesia on day 27. The whole lung was taken out and put into Bouin's solution for staining.
The number of the tumor metastasized to lung was counted with anatomical microscope.
3) Course of treatment. According to the clinical application of S1 and S2, drugs were intraperitoneally injected. A dose of 10 pmol/kg (<1/10 MTD) was used for the Si treatment group, 10umol/kg (<1/10 MTD) for the S2 treatment group, and 3 iumol/kg (>1/2 MTD) for doxorubicin hydrochloride. The control group was administered with physiological saline. The drugs were administered daily for 8 days.
4) Grouping and test results are shown in Table 3.
Table 3: Effects of SI, S2, doxorubicin hydrochloride and control on inhibition of tumor metastasis in nude mice Group Animal Number of metastasized Inhibitory rate on tumor metastasis S I group 10 12+4 91.6%
S2 group 10 10+7 93%
doxorubicin hydrochloride 10 98 18 31.4%
control group Model control 10 143.0 29 Inhibitory rate on metastasis11-(number of metastasized tumors in the treatment group)/(number of metastasized tumors in the control group)]*100%
5) Results and discussion. As shown in Table 3, the inhibitory effect on tumor metastasis of BALB/C mice was greatly improved after intraperitoneal injection of Si and S2, as compared with the doxorubicin hydrochloride control group, indicating that this kind of drugs exhibits an excellent efficacy on anti-tumor metastasis.
In some Examples of the present invention (Examples 4-15, synthesized by the same method as in Examples 1-3), toxicity, inhibitory rate on tumor and inhibitory rate on metastasis of some doxorubicin derivatives, which have different substituents and amino acids were tested respectively by the same methods as in the above Test Examples 1-3 and the results are shown in Table 4.
Table 4: Activation activity, tumor-inhibitory rate and metastasis-inhibitory rate of Examples 4-15 Item RI R2 MDA-MB231 Tumor-inhibitory Metastasis-rate (day 38) inhibitory rate Example 4 Ala Ala 150 55.6% 84.7%
Example 5 Ala Thr 120 46.2 % 74.5%
Example 6 Ala Asn 130 49.5% 81.6%
Example 7 Thr Ala 120 51. 3% 77.4%
Example 8 Ala ALa 120 45. 8% 79.3%
Example 9 Ala Thr 120 68.3% 66.8%
Example 10 Ala Asn 110 58.3% 84.8%
Example 11 Thr Ala 120 63.8% 82.1%
Example 12 Ala Ala 140 55.2% 68.3%
Example 13 Ala Ala 120 47.8% 71.4%
Example 14 Ala Ala 140 46.4% 68.9%
Example 15 Ala Ala 140 54.6% 63.5%
doxorubicin 7 mg/kg 29.1% 31.4%
hydrochloride control group Model control 0 0 According to Table 4, the doxorubicin derivatives prepared from condensation of the amino group of the following compound A and the carboxyl of the following compound B
could greatly reduce the toxicity of doxorubicin and greatly improve the anti-tumor effect:
Compound A is doxorubicin or its derivative epirubicin;
In compound B, R3 is Leu or absent, R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, R7, Thr and Asn; and R6 is 0 , wherein n=1-20; or 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
The polypeptide doxorubicin of the present invention was functionally modified for tumor targeting by R6, which is preferably EMC or EFA and will be exemplified by EMC
below. This modification allows the drug to have the following advantages.
1. Having the ability of targeting tumor The R6 group allows the drug to be able to target cathepsin (Cathepsin B) in the tumor microenvironment and inhibit the activity of cathepsin necessary for tumor growth, development and metastasis. On the contrary, Succinyl-AANL-DOX does not exhibit this targeted function. The R6 group also allows the drug to have an additional function of targeting tumor, thus the drug has two targeted effects. In the in vitro binding assay, EMC-AANL-DOX
can efficiently bind to Cathepsin B.
The test method was described as follows. Cathepsin was absorbed on a 96-well plate and blocked with BSA. Succinyl-AANL-DOX, Si and a mixture of Si and E-64 (an inhibitor for specific binding of cathepsin) were added to allow them to bind to the cathepsin on the 96-well plate. The unbound drugs were removed and the fluorescence of the bound drug was detected.
The results showed that Succinyl-AANL-DOX did not bind to absorbed cathepsin while S1 bound to the cathepsin. The competitive inhibitory assay indicated that the Si 's binding site on the cathepsin was close to the E-64's binding site on the cathepsin, and binding of E-64 could competitively inhibit the binding of SI, as shown in Figure 1.
Furthermore, Z-Phe-Arg-NMec was used as substrate to analyze the activity of cathepsin under pH 6Ø Results showed that the substrate could be activated by the cathepsin (Cathepsin B) over the time, and the fluorescent intensity was improved. Addition of Si could effectively inhibit the enzymatic activity of cathepsin, as shown in Figure 2. Cathepsin B
was highly expressed in the lysosome of the tumor cell. It could degrade type I collagen, activate interstitial procollagenase and type IV procollagenase, and degrade type I, II, III and IV
collagen fibers in the stroma, thus anticipating infiltration and metastasis of tumor. Inhibiting the enzymatic activity of cathepsin could improve inhibition of infiltration and metastasis of tumor, and inhibiting cathepsin could also inhibit bone metastasis of tumor (such as breast cancer).
2. Improving retention of drug at the tumor site After intravenous injection of EMC-AANL-DOX, EMC-AANL-DOX could accumulate at the tumor site due to the targeting property of the EMC group, as compared with Succinyl-AANL-DOX and more EMC-AANL-DOX were distributed in the tumor tissue. Since the drug itself could produce fluorescence, we detected distribution of Succinyl-AANL-DOX and EMC-AANL-DOX in the tumor tissue sections by fluorescence microscope 12 hours after intravenous injection of 10 mol/kg Succinyl-AANL-DOX and EMC-AANL-DOX. The nuclear was stained by 4',6-diamidino-2-phenylindole (DAPI). As shown in Figure 3, significantly more EMC-AANL-DOX were distributed in the tumor tissue, indicating that EMC-AANL-DOX
improved retention of drug in the tumor site. Figure 4 shows the distribution of D1, Dox and Succinyl-AANL-DOX in the tumor and heart tissues. As shown in Figure 4, EMC-AANL-DOX
accumulated in a higher concentration in the tumor tissue as compared with doxorubicin and Succinyl-AANL-DOX, with no accumulation in the heart tissue. These results demonstrated that EMC-AANL-DOX could improve the targeting to the tumor and avoid heart toxicity caused by accumulation of Dox in the heart.
3. Improving the efficacy The efficacy of Succinyl-AANL-DOX and EMC-AANL-DOX in BALB/C mice were studied and compared.
Test purpose: to investigate the anti-tumor efficacy of Succinyl-AANL-DOX and EMC-AANL-DOX in a 4T1 breast cancer treatment model from BALB/C mice.
Treatment drug: Succinyl-AANL-DOX and EMC-AANL-DOX injections, and doxorubicin hydrochloride injection, diluted to corresponding concentrations by physiological saline when testing.
1. Animal: BALB/C mice of 6-8 weeks old, all female.
2. Production of tumor model 1) 4T1 cells were purchased from American type culture collection (ATCC) and identified according the specification provided by ATCC. Cells were cultivated in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum at 37 C and 5% CO2. The cells were passaged for every three days and cells within the 15th passage were used.
2) Production of tumor. 106 4T1 cells were subcutaneously injected to the back of the BALB/C mice. Mice were randomly grouped after the diameter of the tumor reached about 0.3 cm to 0.4 cm. Then treatment began.
3) Course of treatment Succinyl-AANL-DOX, Legubicin and EMC-AANL-DOX were intraperitoneally injected.
The dose of Succinyl-AANL-DOX, Legubicin and EMC-AANL-DOX were all 28.161,imol/kg.
The dose of doxorubicin hydrochloride was 3.448 vimol/kg. The control group was administered with physiological saline. The drugs were administered twice weekly for three _ weeks.
4) Results and discussion. Figure 5 showed the curve indicating inhibitory effect on tumor growth in the 4T1 breast cancer model by Si, Dox, Succinyl-AANL-DOX, Legubicin and solvent control (Vehicle). As shown in Figure 5, EMC-AANL-DOX could significantly improve the inhibitory effect on tumor growth in the BALB/C mice after intraperitoneal inject, as compared with Succinyl-AANL-DOX, Dox, and BOC-AANL-DOX.
In some examples and many other tumor treatment modes of the present invention, efficacies of Succinyl-AANL-DOX and EMC-AANL-DOX were also studied and compared according to the same method as described above. It was found that the inhibitory effect of EMC-AANL-DOX on tumor growth was greatly improved in the 4T1 breast cancer model, etc., which was about 4 - 5 folds of Succinyl-AANL-DOX. And, in efficacy assays using different doses, it was found that only EMC-AANL-DOX could completely kill the tumor, with no recurrence of tumor in the later stage of the treatment, as shown by the human non-small cell lung cancer A549 model in Figure 6. Thus, EMC-AANL-DOX could satisfy our requirements on clinical development.
4. Changing in drug metabolism and greatly improving metabolic half-life of the drug The R6 group changed the metabolic state of the drug, such as Sl. After intravenous injection, Succinyl-AANL-DOX did not bind to serum albumin in the blood plasma and was metabolized in the blood as free small molecule. However, after entering into blood, S1 could bind to the serum albumin. Si and Succinyl-AANL-DOX were intravenously injected at the tail. Bloods were taken after 10 minutes and centrifuged to obtain serum. The proteins in the serum were precipitated by 70% ethanol. The results showed that after entering into the blood, most of Si could bind to serum albumin and thus precipitated. On the contrary, Succinyl-AANL-DOX was still present in the supernate. These results demonstrated that doxorubicin drug, such as Si, modified by R6, was a drug having a completely different metabolism.
In some examples of the present invention, it was also found that, after entering into the blood, most of Si bound to the plasma protein and the metabolic half-life of Si in the mice blood was increased to above 74 hours, as compared with 42 minutes for BOC-AANL-DOX
and 37 hours for Succinyl-AANL-DOX.
In summary, the present invention synthesizes derivatives of doxorubicin hydrochloride, which could be activated by Legumain and target to cathep sin and were demonstrated by toxicity and efficacy assays to provide lower toxicity, more significant anti-tumor activity and inhibition of tumor metastasis than doxorubicin hydrochloride and Succinyl-AANL-DOX.
Although the contents of the present invention were detailedly illustrated via the above preferred embodiments, it should be understood that the above descriptions shall not be construed as limitation on the present invention. Many modifications and replacements are apparent to the skilled artisan after reading the above contents. Therefore, the protection scope of the present invention should be defined by the attached claims.
400400,0H OH HBTU, DMF
____________________________________________________ *op. OH
ocHp OH (3 õNH2HCI OCH30 OH 0,r- õN yL-Leu-L-Asn-L-Ala-L-Ala-EFA
, 0 "OH H. 0 EFA-L-Ala-L-Ala-L-Asn-L-Leu-OH (0.44g, 0.85mmol) and N-methyl morpholine (0.18g, 1.78mmol) were dissolved in anhydrous N,N-dimethylformamide (20mL) and cooled to OH or below. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate (HBTU) (0.49g, 1.28mmol) was added. After stirring for half an hour, doxorubicin hydrochloride (0.45g, 0.78mmol) was added. Away from light, the reaction temperature was slowly warmed up to ambient temperature and then stirred for 5 hours. The reaction solution was added to 200mL, 0.1% acetate aqueous solution. Dichloromethane was added for extraction. The organic phases were pooled, washed by water, and dried with anhydrous sodium sulfate. The solvents were removed by evaporation under reduced pressure to obtain a crude product, which was orange red. The crude product was purified by silica column chromatography (dichloromethane/methanol) to produce the title product S2, i.e., EFA-AANL-DOX, which was a red solid (0.40g; Yield, 49.43%). After analysis by HPLC-MASS, the purity at the 6.99 eluting peak was 96%, and the corresponding MASS result was 11138.41. Thus the target product was confirmed as S2.
E-Maleimidocaproic acid was synthesized according to Synthesis, 2008(8), 1316-and its reaction scheme is showed below:
AcOH , Phhyle / 0 + H2N W-00 OH i.
EMC
Example 2: Preparation of an injection The synthesized Si and S2 were dried under vacuum to produce red powders, sterilized by gas sterilization, and separately packaged in a sterile room. Before animal experiment, the powder was dissolved in water for injection containing 50% alcohol in the sterile room and then diluted with water for injection to the desired concentration.
Example 3: Method for determining the contents of Si and S2 and their content ranges Si and S2 samples were analyzed by analytical type HPLC (Agilent 1100 series, equipped with C8 column of 5um and 4.6 mm IDx250 mm, and the mobile phase being 0 -95% acetonitrile (CAN). Results showed that the purity was in the range of 95%
to 99%.
The beneficial effects of the present invention were demonstrated by the following drug tolerance assays and efficacy assays.
Test Example 1: Measurement of maximum tolerated dose (MTD) by intravenous administration of the test drug Test purpose: to investigate the acute toxicity of the subject new drug formulation via MTD assay by intravenous administration to mice.
Test drug: Si and S2 injections, diluted to the corresponding concentrations with physiological saline when tested.
Animal: the first class BALB/C mice, weighing 19-21 g and all mice being female.
Method and results: 36 female BALB/C mice weighing 19-21 g were randomly divided into 6 groups according to their body weights, with 6 mice in each group. As shown in Table 1, the mice were intraperitoneally injected with Si or S2 for just one time in a dose of 0 mg/kg, 50mg/kg, 100 mg/kg, 150mg/kg, 200 mg/kg or 300mg/kg. Control tests were performed by injecting 0.2m1 physiological saline or doxorubicin hydrochloride. Animals were observed for 17 continuous days for presence or absence of the following behaviors on each day: pilo-erection, hair tousle and lackluster, lethargy, stoop and irritable reaction, and body weight and death were recorded. Blood samples were taken on the 3, 5 and 14 days for counting the whole blood cells. Anmals were anatomized on day 14 to take the heart, liver, kidney, lung, spleen, and pancreas for HE staining.
Table 1: Comparison of mortality rates of test mice receiving different doses of Si and S2 injections, physiological saline or doxorubicin hydrochloride injection Group Dose (mg/kg) Number Number of Mortality of animal dead rate (%) animal 1 physiological saline 0 mg/kg 6 0 0 2 Si 50 mg/kg 6 0 0 3 51 100 mg/kg 6 0 0 4 Si 150 mg/kg 6 0 0 5 Si 200 mg/kg 6 2 33.3%
6 Si 300 mg/kg 6 5 100%
7 S2 50 mg/kg 6 0 0 8 S2 100 mg/kg 6 0 0 9 S2 150 mg/kg 6 0 0 S2 200 mg/kg 6 2 33.3%
11 S2 300 mg/kg 6 3 50%
17 doxorubicin hydrochloride 10 mg/kg 6 6 100%
Results and discussion: no pilo-erection, hair tousle and lackluster, lethargy, stoop, irritable reaction and death were observed in mice receiving 150 mg/kg Si and S2 injection. As 10 shown in Table 1, the MTD of the Si and S2 injections were far beyond 100 mol/kg, and were significantly higher than the MTD of doxorubicin hydrochloride (4 - 8 limol/kg). The MTD for intravenous administration of a test drug is an important reference index for drug toxicity. The results indicate that the toxicity of the doxorubicin hydrochloride derivative, which binds to serum albumin, is significantly reduced as compared with doxorubicin hydrochloride.
Test Example 2: Study on efficacy of S1 and S2 in nude mice Test purpose: to investigate the anti-tumor efficacy of S1 and S2 via mouse tumor treatment model.
Test drug: S1 and S2 injections, and doxorubicin hydrochloride injection, diluted to corresponding concentrations by physiological saline when testing.
Method and results:
1. Animal: nude mice of 6-8 weeks old, all female.
2. Production of tumor model 1) MDA-MB231 cells were purchased from American type culture collection (ATCC) and identified according the specification provided by ATCC. Cells were cultivated in dulbecco's minimum essential medium (DMEM culture medium) containing 10% fetal bovine serum at 37 C and 5% CO2. The cells were passaged for every three days and cells within the 15th Passage were used.
2) Production of tumor. 106MDA-MB231 cells were subcutaneously injected to the back of the nude mice. Mice were randomly grouped after the diameter of the tumor reached about 0.3 cm to 0.4 cm. Then treatment began.
3) Course of treatment According to the clinical application of Si and S2, drugs were intraperitoneally injected.
A dose of 30 mg/kg (<1/2 MTD) was used for the Si treatment group, 30 mg/kg (<1/2 MTD) for the S2 treatment group, and 4mg/kg (>1/2 MTD) for doxorubicin hydrochloride. The control group was administered with physiological saline. The drugs were administered twice weekly for three weeks.
4) Grouping and test results are shown in Table 2.
Table 2: Treatment effects of Sl, S2, doxorubicin hydrochloride and control on tumors of nude mice Group Number of Size of tumor (mm3) Inhibitory rate of animal tumor 20 days 38 days 20 days 38 days S1 group 10 287.42+30.52 678.49+37.7 25.8% 68.5%
S2 group 10 254.59+34.19 618.49+51.27 34.3% 71.3%
Control Group 10 358.7+39.7 881.2+86.5 7.4% 29.1%
(doxorubicin hydrochloride) Model Control 10 387.5+35.6 2155.44+325.
5) Results and discussion: as shown in Table 2, inhibitory effect on tumor in the nude mice after administering with Si and S2 by intraperitoneal injection was greatly improved as compared with the doxorubicin hydrochloride control group. And S2 could diminish and eliminate the tumor. Results show that this kind of drugs exhibit excellent inhibiting efficacy on tumor growth.
Test Example 3: Study on efficacy of Si and S2 in a tumor metastasis model from BALB/C mice Test purpose: to investigate the anti-tumor efficacy of S1 and S2 in a tumor metastasis treatment model from BALB/C mice.
Test drug: S1 and S2 injections, and doxorubicin hydrochloride injection, diluted to corresponding concentrations with physiological saline when testing.
1. Animal: BALB/C mice of 6-8 weeks old, all female.
2. Production of tumor model 1) 4T1 cells were purchased from American type culture collection (ATCC) and identified according the specification provided by ATCC. Cells were cultivated in DMEM
culture medium containing 10% fetal bovine serum at 37 C and 5% CO2. The cells were passaged for every three days and cells within the 15th passage were used.
2) Production of tumor metastasis. 106T1 cells were subcutaneously injected to the back of the BALB/C mice. Mice were randomly grouped after the tumor grew to about 1.5 cm. The subcutaneous tumor was removed by surgery and drug treatment began. Mice were killed after anesthesia on day 27. The whole lung was taken out and put into Bouin's solution for staining.
The number of the tumor metastasized to lung was counted with anatomical microscope.
3) Course of treatment. According to the clinical application of S1 and S2, drugs were intraperitoneally injected. A dose of 10 pmol/kg (<1/10 MTD) was used for the Si treatment group, 10umol/kg (<1/10 MTD) for the S2 treatment group, and 3 iumol/kg (>1/2 MTD) for doxorubicin hydrochloride. The control group was administered with physiological saline. The drugs were administered daily for 8 days.
4) Grouping and test results are shown in Table 3.
Table 3: Effects of SI, S2, doxorubicin hydrochloride and control on inhibition of tumor metastasis in nude mice Group Animal Number of metastasized Inhibitory rate on tumor metastasis S I group 10 12+4 91.6%
S2 group 10 10+7 93%
doxorubicin hydrochloride 10 98 18 31.4%
control group Model control 10 143.0 29 Inhibitory rate on metastasis11-(number of metastasized tumors in the treatment group)/(number of metastasized tumors in the control group)]*100%
5) Results and discussion. As shown in Table 3, the inhibitory effect on tumor metastasis of BALB/C mice was greatly improved after intraperitoneal injection of Si and S2, as compared with the doxorubicin hydrochloride control group, indicating that this kind of drugs exhibits an excellent efficacy on anti-tumor metastasis.
In some Examples of the present invention (Examples 4-15, synthesized by the same method as in Examples 1-3), toxicity, inhibitory rate on tumor and inhibitory rate on metastasis of some doxorubicin derivatives, which have different substituents and amino acids were tested respectively by the same methods as in the above Test Examples 1-3 and the results are shown in Table 4.
Table 4: Activation activity, tumor-inhibitory rate and metastasis-inhibitory rate of Examples 4-15 Item RI R2 MDA-MB231 Tumor-inhibitory Metastasis-rate (day 38) inhibitory rate Example 4 Ala Ala 150 55.6% 84.7%
Example 5 Ala Thr 120 46.2 % 74.5%
Example 6 Ala Asn 130 49.5% 81.6%
Example 7 Thr Ala 120 51. 3% 77.4%
Example 8 Ala ALa 120 45. 8% 79.3%
Example 9 Ala Thr 120 68.3% 66.8%
Example 10 Ala Asn 110 58.3% 84.8%
Example 11 Thr Ala 120 63.8% 82.1%
Example 12 Ala Ala 140 55.2% 68.3%
Example 13 Ala Ala 120 47.8% 71.4%
Example 14 Ala Ala 140 46.4% 68.9%
Example 15 Ala Ala 140 54.6% 63.5%
doxorubicin 7 mg/kg 29.1% 31.4%
hydrochloride control group Model control 0 0 According to Table 4, the doxorubicin derivatives prepared from condensation of the amino group of the following compound A and the carboxyl of the following compound B
could greatly reduce the toxicity of doxorubicin and greatly improve the anti-tumor effect:
Compound A is doxorubicin or its derivative epirubicin;
In compound B, R3 is Leu or absent, R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, R7, Thr and Asn; and R6 is 0 , wherein n=1-20; or 0 , wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated Cl-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
The polypeptide doxorubicin of the present invention was functionally modified for tumor targeting by R6, which is preferably EMC or EFA and will be exemplified by EMC
below. This modification allows the drug to have the following advantages.
1. Having the ability of targeting tumor The R6 group allows the drug to be able to target cathepsin (Cathepsin B) in the tumor microenvironment and inhibit the activity of cathepsin necessary for tumor growth, development and metastasis. On the contrary, Succinyl-AANL-DOX does not exhibit this targeted function. The R6 group also allows the drug to have an additional function of targeting tumor, thus the drug has two targeted effects. In the in vitro binding assay, EMC-AANL-DOX
can efficiently bind to Cathepsin B.
The test method was described as follows. Cathepsin was absorbed on a 96-well plate and blocked with BSA. Succinyl-AANL-DOX, Si and a mixture of Si and E-64 (an inhibitor for specific binding of cathepsin) were added to allow them to bind to the cathepsin on the 96-well plate. The unbound drugs were removed and the fluorescence of the bound drug was detected.
The results showed that Succinyl-AANL-DOX did not bind to absorbed cathepsin while S1 bound to the cathepsin. The competitive inhibitory assay indicated that the Si 's binding site on the cathepsin was close to the E-64's binding site on the cathepsin, and binding of E-64 could competitively inhibit the binding of SI, as shown in Figure 1.
Furthermore, Z-Phe-Arg-NMec was used as substrate to analyze the activity of cathepsin under pH 6Ø Results showed that the substrate could be activated by the cathepsin (Cathepsin B) over the time, and the fluorescent intensity was improved. Addition of Si could effectively inhibit the enzymatic activity of cathepsin, as shown in Figure 2. Cathepsin B
was highly expressed in the lysosome of the tumor cell. It could degrade type I collagen, activate interstitial procollagenase and type IV procollagenase, and degrade type I, II, III and IV
collagen fibers in the stroma, thus anticipating infiltration and metastasis of tumor. Inhibiting the enzymatic activity of cathepsin could improve inhibition of infiltration and metastasis of tumor, and inhibiting cathepsin could also inhibit bone metastasis of tumor (such as breast cancer).
2. Improving retention of drug at the tumor site After intravenous injection of EMC-AANL-DOX, EMC-AANL-DOX could accumulate at the tumor site due to the targeting property of the EMC group, as compared with Succinyl-AANL-DOX and more EMC-AANL-DOX were distributed in the tumor tissue. Since the drug itself could produce fluorescence, we detected distribution of Succinyl-AANL-DOX and EMC-AANL-DOX in the tumor tissue sections by fluorescence microscope 12 hours after intravenous injection of 10 mol/kg Succinyl-AANL-DOX and EMC-AANL-DOX. The nuclear was stained by 4',6-diamidino-2-phenylindole (DAPI). As shown in Figure 3, significantly more EMC-AANL-DOX were distributed in the tumor tissue, indicating that EMC-AANL-DOX
improved retention of drug in the tumor site. Figure 4 shows the distribution of D1, Dox and Succinyl-AANL-DOX in the tumor and heart tissues. As shown in Figure 4, EMC-AANL-DOX
accumulated in a higher concentration in the tumor tissue as compared with doxorubicin and Succinyl-AANL-DOX, with no accumulation in the heart tissue. These results demonstrated that EMC-AANL-DOX could improve the targeting to the tumor and avoid heart toxicity caused by accumulation of Dox in the heart.
3. Improving the efficacy The efficacy of Succinyl-AANL-DOX and EMC-AANL-DOX in BALB/C mice were studied and compared.
Test purpose: to investigate the anti-tumor efficacy of Succinyl-AANL-DOX and EMC-AANL-DOX in a 4T1 breast cancer treatment model from BALB/C mice.
Treatment drug: Succinyl-AANL-DOX and EMC-AANL-DOX injections, and doxorubicin hydrochloride injection, diluted to corresponding concentrations by physiological saline when testing.
1. Animal: BALB/C mice of 6-8 weeks old, all female.
2. Production of tumor model 1) 4T1 cells were purchased from American type culture collection (ATCC) and identified according the specification provided by ATCC. Cells were cultivated in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum at 37 C and 5% CO2. The cells were passaged for every three days and cells within the 15th passage were used.
2) Production of tumor. 106 4T1 cells were subcutaneously injected to the back of the BALB/C mice. Mice were randomly grouped after the diameter of the tumor reached about 0.3 cm to 0.4 cm. Then treatment began.
3) Course of treatment Succinyl-AANL-DOX, Legubicin and EMC-AANL-DOX were intraperitoneally injected.
The dose of Succinyl-AANL-DOX, Legubicin and EMC-AANL-DOX were all 28.161,imol/kg.
The dose of doxorubicin hydrochloride was 3.448 vimol/kg. The control group was administered with physiological saline. The drugs were administered twice weekly for three _ weeks.
4) Results and discussion. Figure 5 showed the curve indicating inhibitory effect on tumor growth in the 4T1 breast cancer model by Si, Dox, Succinyl-AANL-DOX, Legubicin and solvent control (Vehicle). As shown in Figure 5, EMC-AANL-DOX could significantly improve the inhibitory effect on tumor growth in the BALB/C mice after intraperitoneal inject, as compared with Succinyl-AANL-DOX, Dox, and BOC-AANL-DOX.
In some examples and many other tumor treatment modes of the present invention, efficacies of Succinyl-AANL-DOX and EMC-AANL-DOX were also studied and compared according to the same method as described above. It was found that the inhibitory effect of EMC-AANL-DOX on tumor growth was greatly improved in the 4T1 breast cancer model, etc., which was about 4 - 5 folds of Succinyl-AANL-DOX. And, in efficacy assays using different doses, it was found that only EMC-AANL-DOX could completely kill the tumor, with no recurrence of tumor in the later stage of the treatment, as shown by the human non-small cell lung cancer A549 model in Figure 6. Thus, EMC-AANL-DOX could satisfy our requirements on clinical development.
4. Changing in drug metabolism and greatly improving metabolic half-life of the drug The R6 group changed the metabolic state of the drug, such as Sl. After intravenous injection, Succinyl-AANL-DOX did not bind to serum albumin in the blood plasma and was metabolized in the blood as free small molecule. However, after entering into blood, S1 could bind to the serum albumin. Si and Succinyl-AANL-DOX were intravenously injected at the tail. Bloods were taken after 10 minutes and centrifuged to obtain serum. The proteins in the serum were precipitated by 70% ethanol. The results showed that after entering into the blood, most of Si could bind to serum albumin and thus precipitated. On the contrary, Succinyl-AANL-DOX was still present in the supernate. These results demonstrated that doxorubicin drug, such as Si, modified by R6, was a drug having a completely different metabolism.
In some examples of the present invention, it was also found that, after entering into the blood, most of Si bound to the plasma protein and the metabolic half-life of Si in the mice blood was increased to above 74 hours, as compared with 42 minutes for BOC-AANL-DOX
and 37 hours for Succinyl-AANL-DOX.
In summary, the present invention synthesizes derivatives of doxorubicin hydrochloride, which could be activated by Legumain and target to cathep sin and were demonstrated by toxicity and efficacy assays to provide lower toxicity, more significant anti-tumor activity and inhibition of tumor metastasis than doxorubicin hydrochloride and Succinyl-AANL-DOX.
Although the contents of the present invention were detailedly illustrated via the above preferred embodiments, it should be understood that the above descriptions shall not be construed as limitation on the present invention. Many modifications and replacements are apparent to the skilled artisan after reading the above contents. Therefore, the protection scope of the present invention should be defined by the attached claims.
Claims (14)
1. A doxorubicin derivative for targeted activation by Legumain, having the following structural formula:
wherein the doxorubicin derivative is prepared by condensation between the amino group of compound A and the carboxyl group of compound B, and compounds A and B have the following structures, respectively:
wherein compound A is doxorubicin or its derivative;
wherein R3 in compound B is Leu or absent; if R3 is absent then compound B is a tripeptide, that is, the carboxyl of Asn covalently condensates with the amino of compound A
directly to produce a polypeptide doxorubicin; if R3 is Leu, then compound B
is a tetrapeptide, that is Leu-Asn-R4-R5-;
R4 is any one amino acid selected from the group consisting of Ala and Thr;
R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn;
R6 is wherein n=1 -20; or wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated C1-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
wherein the doxorubicin derivative is prepared by condensation between the amino group of compound A and the carboxyl group of compound B, and compounds A and B have the following structures, respectively:
wherein compound A is doxorubicin or its derivative;
wherein R3 in compound B is Leu or absent; if R3 is absent then compound B is a tripeptide, that is, the carboxyl of Asn covalently condensates with the amino of compound A
directly to produce a polypeptide doxorubicin; if R3 is Leu, then compound B
is a tetrapeptide, that is Leu-Asn-R4-R5-;
R4 is any one amino acid selected from the group consisting of Ala and Thr;
R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn;
R6 is wherein n=1 -20; or wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated C1-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
2. The doxorubicin derivative for targeted activation by Legumain of claim 1, wherein compound A is doxorubicin or epirubicin, and epirubicin is an isomer of doxorubicin and has the following structure:
3. The doxorubicin derivative for targeted activation by Legumain of claim 1 or 2, wherein R6 is
4. The doxorubicin derivative for targeted activation by Legumain of claim 3, wherein the doxorubicin derivative is S1 having the following structure:
5. The doxorubicin derivative for targeted activation by Legumain of claim 3, wherein the doxorubicin derivative is S2 having the following structure:
6. A method for preparing the doxorubicin derivative for targeted activation by Legumain of claim 1, comprising the following steps:
Step 1, preparing a tripeptide or a tetrapeptide, R3-Asn-R4-R5, by conjugating the amino acid residues together and isolating to obtain the formed tripeptide or tetrapeptide R3-Asn-R4-R5;
Step 2, preparing compound B by reacting R3-Asn-R4-R5 obtained in step 1 with the acyl or carboxyl of R6-Cl or R6-OH to obtain R3-Asn-R4-R5-R6;
Step 3, covalently condensating the carboxyl group in R3 of the compound R3-Asn-R4-R5-R6 obtained in step 2 with the amino group of compound A to form the doxorubicin derivative for targeted activation by Legumain.
Step 1, preparing a tripeptide or a tetrapeptide, R3-Asn-R4-R5, by conjugating the amino acid residues together and isolating to obtain the formed tripeptide or tetrapeptide R3-Asn-R4-R5;
Step 2, preparing compound B by reacting R3-Asn-R4-R5 obtained in step 1 with the acyl or carboxyl of R6-Cl or R6-OH to obtain R3-Asn-R4-R5-R6;
Step 3, covalently condensating the carboxyl group in R3 of the compound R3-Asn-R4-R5-R6 obtained in step 2 with the amino group of compound A to form the doxorubicin derivative for targeted activation by Legumain.
7. The method for preparing the doxorubicin derivative for targeted activation by Legumain according to claim 6, wherein R3 in compound B is Leu or absent; R4 is any one amino acid selected from the group consisting of Ala and Thr; R5 is any one amino acid selected from the group consisting of Ala, Thr and Asn; and R6 is wherein n=1-20; or wherein R7 is substituted or unsubstituted, linear or branched, saturated or unsaturated C1-C20 fatty hydrocarbon, or substituted or unsubstituted C6-C20 aromatic hydrocarbon.
8. The method for preparing the doxorubicin derivative for targeted activation by Legumain according to claim 6, wherein R6 is:
9. Use of the doxorubicin derivative for targeted activation by Legumain according to claim 1 in the preparation of an anti-tumor drug.
10. The composition of claim 1, wherein the cleavable linker is legumain cleavable and comprises a peptide sequence selected from Leu-Asn-Ala-Ala,Leu-Asn-Ala-Thr,Leu-Asn-Ala-Asn, Leu-Asn-Thr-Ala, Leu-Asn-Thr-Thr, Leu-Asn-Thr-Asn.
11. The composition of claim 1, where in the cleavable linker is legumain cleavable and cleaving at peptide bond between Leu and Asn and then releasing Leu-doxorubicin and Leu-epirubicin
12. A medical comprising at least one compound of Claim 5 for the treatment or prophylaxis of cancer or related neoplastic disease.
13. method for the treatment or prophylaxis of a cancer in a mammal comprising administering to a mammal in need thereof therapeutically effective amounts of at least one compound of Claims 1-6.
14. The method of Claim 13, wherein the cancer is selected from non-small cell lung cancer (NSCLC), liver, colon, kidney, thyroid, pancreatic, head and neck, prostate, ovarian, breast and sarcoma.
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CA (1) | CA2895779A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4108675A4 (en) * | 2020-02-20 | 2024-05-29 | Yafei Shanghai Biolog Medicine Science & Tech Co Ltd | Preparation and use of immunostimulatory coupling complex which is delivered and activated in targeted manner |
-
2015
- 2015-06-26 CA CA2895779A patent/CA2895779A1/en not_active Abandoned
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4108675A4 (en) * | 2020-02-20 | 2024-05-29 | Yafei Shanghai Biolog Medicine Science & Tech Co Ltd | Preparation and use of immunostimulatory coupling complex which is delivered and activated in targeted manner |
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