Multi-Arm Polymeric Targeting Anti-Cancer Conjugate

Abstract

Disclosed is a multi-arm targeting drug conjugate modified by a water-soluble polymer; the drug conjugate has the structural formula of (III). In formula (III), R is an organic core, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is a camptothecin-based drug, and q is any integer between 3 and 8. The drug conjugate may improve the poor water solubility, high toxicity and low bioavailability of camptothecin-based drugs.

Claims

1. A multi-branched drug conjugate having the following structural formula (III) or a pharmaceutically acceptable salt thereof: ##STR00134## wherein R is an organic core, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, and q is any integer between 3 and 8, wherein D is a camptothecin-based drug represented by the following formula: ##STR00135## wherein Ri to R5 are selected from the following groups independently from each other: hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxyl, cyano, nitro, azido, amido, hydrazine, amine group, substituted amine group, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, and alkylsulfonyloxy; R.sub.6 is H or OR.sub.8; R.sub.8 is alkyl, alkenyl, cycloalkyl, halogenated alkyl, or hydroxyalkyl; and R.sub.7 is hydroxyl.

2. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein D is irinotecan, SN-38, 10-hydroxycamptothecin, or rubitecan.

3. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein POLY is polyethylene glycol, polypropylene glycol, poly(vinyl pyrrolidone), poly(hydroxyalkyl methyl acrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(a-hydroxy acid), poly(acrylic acid), poly(vinyl acetate), polyphosphazine, polyoxazoline, or poly(N-acryloyl morpholine).

4. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 , wherein POLY is: ##STR00136## n is 5 to 500.

5. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the multivalent linker L is: ##STR00137## symbol * represents an attachment point of the multivalent linker L and the targeting molecule T, # represents an attachment point of the multivalent linker L and the active agent D, % represents an attachment point of the multivalent linker L and POLY, wherein m and m are any integer between 1 and 20 respectively, and l and k are any integer between 1 and 10 respectively.

6. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the multivalent linker L is: ##STR00138##

7. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein T is an arginine-glycine-aspartic acid sequence-containing RGD peptide, tLyp-1, Lyp-1, RPARPAR, Angiopep2, GE11, or folic acid.

8. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein T is iRGD or cRGD.

9. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein said multi-branched drug conjugate has a structure represented by structural formula (IV), (V), or (VI): ##STR00139## wherein n is 5 to 500.

10. A multi-branched drug conjugate having the following structure or a pharmaceutically acceptable salt thereof: ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150##

11. A drug composition containing the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

12. A method for treating colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and malignant sarcoma, cancer and lymphoma of breast, ovary, colon, kidney, bile duct, lung and brain, comprising administering to a subject in need thereof a therapeutic effective amount of the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1.

13. A preparation method of a multi-branched drug conjugate or a pharmaceutically acceptable salt thereof, comprising: ##STR00151## wherein R is an organic core, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, q is any integer between 3 and 8, and D is a camptothecin-based drug represented by the following formula: ##STR00152## wherein R.sub.1 to R5 are selected from the following groups independently from each other: hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxyl, cyano, nitro, azido, amido, hydrazine, amine group, substituted amine group, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, and alkylsulfonyloxy; R.sub.6 is H or OR.sub.8; R.sub.8 is alkyl, alkenyl, cycloalkyl, halogenated alkyl, or hydroxyalkyl; and R.sub.7 is hydroxyl; (1) the active agent D is attached to the multivalent linker L to obtain a D-L portion; (2) the D-L portion is attached to a multi-arm polymer RPOLY).sub.q to obtain RPOLY-L-D.sub.q; and (3) RPOLY-L-D).sub.q obtained in the previous step is attached to the targeting molecule T.

14. A preparation method of the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein D is irinotecan, SN-38, 10-hydroxycamptothecin, or rubitecan; the multi-arm polymer RPOLY).sub.q is 3armPEG20K-SCM, 4armPEG20K-SCM, or 8armPEG20K-SCM; L is ##STR00153## symbol * represents an attachment point of the multivalent linker L and the targeting molecule T, # represents an attachment point of the multivalent linker L and the active agent D, and % represents an attachment point of the multivalent linker L and POLY; and T is iRGD, cRGD, tLyp-1, Lyp-1, RPARPAR, Angiopep2, GE11, or folic acid.

15. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein POLY is: ##STR00154## n is 50 to 200.

16. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein POLY is: ##STR00155## n is 113.

17. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein said multi-branched drug conjugate has a structure represented by structural formula (IV), (V), or (VI): ##STR00156## wherein n is 50 to 200.

18. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein said multi-branched drug conjugate has a structure represented by structural formula (IV), (V), or (VI): ##STR00157## wherein n is 113.

19. A drug composition containing the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 10, and a pharmaceutically acceptable excipient.

20. A method for treating colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and malignant sarcoma, cancer and lymphoma of breast, ovary, colon, kidney, bile duct, lung and brain, comprising administering to a subject in need thereof a therapeutic effective amount of the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 10.

Description

DETAILED DESCRIPTION

[0137] The present disclosure will be described in detail below. However, the present disclosure may be embodied in many different forms, and it should not be limited to the examples described herein. The purpose of providing these examples is to make the disclosed content more complete and more comprehensive. The reagents and raw materials used are all commercially available except for those with preparation method provided.

[0138] Unless otherwise defined, all technical terms in the present description have the same meaning as those commonly understood by those skilled in the art of the claimed subjected matters.

[0139] Unless otherwise specified, the terms used herein have the following meanings:

[0140] DMF: N, N-dimethylformamide

[0141] DCM: dichloromethane

[0142] Boc-Gly-OH:

##STR00066##

[0143] DMAP: 4-dimethylaminopyridine

[0144] DCC: dicyclohexylcarbodiimide

[0145] IPA: isopropanol

[0146] TFA: trifluoroacetic acid

[0147] TBME: tert-butyl methyl ether

[0148] EA: ethyl acetate

[0149] DME: ethylene glycol dimethyl ether

[0150] HOSU: N-succinimidyl carbonate

[0151] THF: tetrahydrofuran

[0152] Boc-Lys-OH:

##STR00067##

[0153] DIEA: N,N-diisopropylethylamine

[0154] DEPC: diethyl cyanophosphonate

[0155] Phf:

##STR00068##

[0156] HOBT: 1-hydroxybenzotriazole

[0157] DIC: N,N-diisopropylcarbodiimide

[0158] MTBE: tert-butyl methyl ether

[0159] EDT: dithioglycol

[0160] PBS: phosphate buffer

[0161] EDC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

EXAMPLE 1

[0162] Preparation of iRGD

##STR00069##

[0163] Fmoc was removed from 10.0 g of Fmoc-Rink MBHA Amide Resin. HOBT/DIC was adopted as a coupling reagent, DMF was a reaction solvent, the reaction was monitored by ninhydrin detection method, and the following protecting amino acids were sequentially attached to the resin: Fmoc-Cys(Acm)-OH, Fmoc-Asp(OtBu)OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Cys(Acm)-OH. After washing the mixture with DMF, thallium trifluoroacetate (2.0 eq) was added. After being stirred for 18 h, the mixture was washed with DMF, and Fmoc was removed. Fmoc-Cys(Trt)-OH was condensed, the mixture was washed with DMF, and Fmoc was removed. Acetic anhydride and pyridine were added to react for 20 min, and the mixture was washed with DMF, DCM and methanol, followed by drying. 82.5% TFA/5% phenol/5% water/2.5% EDT/5% thioanisole was added as a cleavage reagent, ice-cold MTBE was added to precipitate, and the mixture was washed. The crude product was purified by reverse phase HPLC and lyophilized to obtain a white floc iRGD (1.56 g).

Preparation of the L Portion

[0164] ##STR00070##

1. Preparation of BP103a01

[0165] Under nitrogen protection, to a 1000 mL three-necked flask, 200 mL of pyridine (120 g 1 (1.0 eq)) was added, stirred and cooled to 0 C. 151.8 g (1.0 eq) of TsCl was added in batch and stirred for 1 h. Then, the temperature was raised slowly to room temperature, and stirring was continued for 3 h to 4 h. After the reaction was complete, the reaction solution was poured into an ice-cold dilute hydrochloric acid solution, and EA was added for extraction. The EA layer was washed once with dilute hydrochloric acid, washed with saturated sodium bicarbonate and saturated saline, and dried over anhydrous Na.sub.2SO.sub.4. The solvent was removed by distillation under reduced pressure, and 55 g of pure BP103a01 was obtained by silica gel column chromatography.

2. Preparation of BP103a02

[0166] To a 1000 mL three-necked flask, 55 g (1.0 eq) of BP103a01 and 160 mL of DMSO were added and stirred until homogeneous, and 23.52 g (2.0 eq) of NaN3 was then added. The mixture was heated to 50 C. to react for 3 h and cooled to room temperature. The reaction solution was poured into water and EA was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain 29.2 g of BP103a02 as a colorless liquid.

3. Preparation of BP103a03

[0167] To a 1 L hydrogenation reaction kettle, 29 g of Compound 3, 360 mL of methanol, and 5.0 g of palladium on carbon were added, stirred and purged with nitrogen. Hydrogen was fed to react for 3 h to 4 h. After the completion of the reaction was monitored by TLC, the reaction solution was filtered, and the filtrate was concentrated to obtain 23.5 g of BP103a03 as an oil-like substance.

4. Preparation of BP103a04

[0168] To a 1 L three-necked flask, 23.5 g (1.0 eq) of Compound BP103a03, 68.6 g (2.0 eq) of (Boc)20, and 500 mL of a mixed solution of methanol:triethylamine (9:1) were added, stirred, the temperature was raised to reflux, and reacted for 1 h. After the completion of the reaction was monitored by TLC, methanol and triethylamine were removed by distillation, water was added for dissolution, and the mixture was extracted 3 times with dichloromethane. The organic layers were combined, washed once with water, and dried over anhydrous sodium sulfate. The solvent was removed by distillation, and the mixture was dried to obtain 34.8 g of BP103a04 as a solid.

[0169] s5. Preparation of BP103a05

[0170] To a 1000 mL three-necked flask, 34.8 g (1.0 eq) of Compound BP103a04, toluene and THF (each 150 mL), and 58.2 g (3 eq) of bromoacetic acid were added, stirred and heated to 45 C. to 50 C. 33.5 g (6 eq) of sodium hydroxide was then added, and reacted overnight. After the completion of the reaction was monitored by TLC, the reaction solution was removed by distillation, water and EA were added for extraction, and the pH of the aqueous phase was adjusted to 3. The aqueous phase was extracted with dichloromethane, and the dichloromethane layers were combined. After being dried over anhydrous sodium sulfate, the mixture was concentrated to obtain 18 g of BP103a05 as an oily compound.

6. Preparation of BP103a

[0171] To a 250 mL three-necked flask, 18 g of Compound BP103a05 and 100 mLof EA were added, stirred and dissolved, then cooled to 0 C. 150 mL (3.5M) of EA/HCl was added, and the temperature was kept at 0 C. The completion of the reaction was monitored by TLC, the mixture was filtered, and the filter cake was washed with TBME to obtain 10.4 g of BP103a as a white solid.

##STR00071##

[0172] Preparation of M2

[0173] To a 100 mL flask, 3.0 g (1.0 eq) of BP103a, 4.0 g (1.0 eq) of Compound M1, 40 mL of DCM, and 4.0 mL (2.0 eq) of DIEA were added and stirred under room temperature. The completion of the reaction was monitored by TLC, the organic solvent was removed by distillation, and 5.2 g of oil-like substance M2 was obtained by column chromatography.

[0174] Preparation of M3

[0175] To a 200 mL three-necked flask, 9.00 g (1.0 eq) of Compound M2, 3.96 g (1.53 eq) of HOSU, 90 mL of DCM, and 6.60 g (1.53 eq) of EDC.HCl were added and reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then washed twice with a 50 mmol/L aqueous solution of potassium dihydrogen phosphate (pH=6.0), washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain 5.9 g of Compound M3 as a colorless oil-like substance.

[0176] Preparation of M4

[0177] To a 200 mL flask, 2.93 g (1.0 eq) of Compound Boc-Lys-OH, 60 mL of water and 2.00 g (2.0 eq) of NaHCO3 were added and stirred. A solution of 5.9 g (1.0 eq) of Compound M3 in 60 mL of DME (ethylene glycol dimethyl ether) was added dropwise, 60 mL of THF was further added, and the mixture was stirred overnight. The completion of the reaction was monitored by TLC, the organic solvent was removed by distillation, and the pH was adjusted to 4 with acetic acid. The mixture was extracted with EA, dried over anhydrous sodium sulfate, and concentrated to obtain 4.50 g of Compound M4 as a colorless oil-like substance.

[0178] Preparation of M5

[0179] To a 100 mL flask, 3.81 g (1.0 eq) of Compound M4, 40 mL of DCM, 1.07 g of HOSU and 1.78 g of EDC.HCl were added, stirred, and reacted at room temperature for 4 h. The completion of the reaction was monitored by TLC. After being diluted with DCM, the mixture was washed twice with a 50 mM aqueous solution of potassium dihydrogen phosphate, washed once with purified water, washed once with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain 4.1 g of Compound M5 as a colorless oil-like substance.

Preparation of the Conjugate

[0180] ##STR00072##

[0181] Preparation of M7

[0182] To a 250 mL round bottom flask, 3.50 g (1.0 eq) of Compound M6 and 52.5 mL of DMF were added and heated to 60 C. for dissolution. After 5 to 10 min, DMF was removed by distillation under reduced pressure. 300 mL of n-heptane was added and distilled under reduced pressure, and the procedure was repeated three times. After the mixture was dried by rotary evaporation, 105 mL of DCM, 1.08 g of Boc-Gly-OH (1.2 eq) and 63 mg of DMAP (0.1 eq) were added, a solution of 1.59 g of DCC (1.5 eq) in 10 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 120 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 150 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 4.02 g of Compound M7 as a pale yellow solid.

[0183] Preparation of M8

[0184] To a 100 mL three-necked flask, 4.02 g of Compound M7 and 50 mL of DCM were added. After the mixture was stirred and dissolved, 11.6 mL of TFA was added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M8 (4.00 g).

[0185] Preparation of M9

[0186] To a 200 mL three-necked flask, 2.95 g of Compound M8, 80 mL of DCM, 2.57 g (1.05 eq) of Compound 4, 2.16 mL of DIEA (3.0 eq) and 0.96 mL of DEPC (1.5 eq) were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then the mixture was washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M9 (1.48 g).

##STR00073##

[0187] Preparation of M10

[0188] To a 50 mL round bottom flask, 260 mg of Compound M9 and 10 mL of 20% TFA/DCM were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, and dried to obtain a pale yellow solid M10 (210 mg).

[0189] Preparation of M11

[0190] To a 10 mL round bottom flask, 51 mg (4.0 eq) of Compound M10, 2 mL of DCM, 11 L (8.0 eq) of TEA and 201 mg (1.0 eq) of 4armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid M11 (240 mg).

##STR00074##

[0191] Preparation of Compound 1 and H1

[0192] To a 10 mL round bottom flask, 50 mg (1.0 eq) of Compound M11 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 10.6 mg of iRGD in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 1. The crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H1 (Hydrochloride of compound 1)(50 mg).

[0193] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.328(s,2H), 5.480(s,2H),7.0-8.5(m NH)

EXAMPLE 2

[0194] ##STR00075##

[0195] 10.0 g of 2Cl-Trt Resin was used, HOBT/DIC was adopted as a coupling reagent, DMF was a reaction solvent, the reaction was monitored by ninhydrin detection method, and the following protecting amino acids were sequentially attached to the resin: Fmoc-Arg(pbf)-OH , Fmoc-Thr(tBu)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Cys(Trt)-OH. 82.5% TFA/5% phenol/5% water/2.5% EDT/5% thioanisole was added as a cleavage reagent, ice-cold MTBE was added to precipitate, and the mixture was washed. The crude product was purified by reverse phase HPLC and lyophilized to obtain a white floc (5.5 g).

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0196] ##STR00076##

[0197] Preparation of Compound 2 and H2

[0198] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M11 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 139 mg of tLyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 2, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H2 (Hydrochloride of compound 2) (1.0 g).

[0199] .sup.1HNMR(DMSO+D.sub.2O): 0.903(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.332(s,2H), 5.484(s,2H), 7.0-8.5 (m NH)

EXAMPLE 3

Preparation of Lyp-1

[0200] ##STR00077##

[0201] Fmoc was removed from 10.0 g of Fmoc-Rink MBHA Amide Resin. HOBT/DIC was adopted as a coupling reagent, DMF was a reaction solvent, the reaction was monitored by ninhydrin detection method, and the following protecting amino acids were sequentially attached to the resin: Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH , Fmoc-Arg(Pbf)-OH , Fmoc-Thr(tBu)-OH, Fmoc-Arg(pbf-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Cys(Acm)-OH. After washing the mixture with DMF, thallium trifluoroacetate (2.0 eq) was added. After being stirred for 18 h, the mixture was washed with DMF, and Fmoc was removed. Fmoc-Cys(Trt)-OH was condensed, the mixture was washed with DMF, Fmoc was removed, and then the mixture was dried. 82.5% TFA/5% phenol/5% water/2.5% EDT/5% thioanisole was added as a cleavage reagent, ice-cold MTBE was added to precipitate, and the mixture was washed. The crude product was purified by reverse phase HPLC and lyophilized to obtain a white floc LyP-1 (765 mg).

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0202] ##STR00078##

[0203] Preparation of Compound 3and H3

[0204] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M11 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 182 mg of LyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixutre was dialyzed and concentrated to obtain compound 3. The crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H3 (Hydrochloride of compound 3) (1.05 g).

[0205] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.328(s,2H), 5.481(s,2H), 7.0-8.5 (m NH)

EXAMPLE 4

Preparation of CPARPAR

[0206] 10.0 g of 2C1-Trt Resin was used, HOBT/DIC was adopted as a coupling reagent, DMF was a reaction solvent, the reaction was monitored by ninhydrin detection method, and the following protecting amino acids were sequentially attached to the resin: Fmoc-Arg(pbf)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Cys(Trt)-OH. 82.5% TFA/5% phenol/5% water/2.5% EDT/5% thioanisole was added as a cleavage reagent, ice-cold MTBE was added to precipitate, and the mixture was washed. The crude product was purified by reverse phase HPLC and lyophilized to obtain a white floc CRPARPAR (6.1 g).

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0207] ##STR00079##

[0208] Preparation of Compound 4 and H4

[0209] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M11 and 20 ml of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 154 mg of CRPARPAR in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 4, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H4 (Hydrochloride of compound 4) (1.09 g).

[0210] .sup.1HNMR(DMSO+D.sub.2O): 0.905(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.335(s,2H), 5.486(s,2H), 7.0-8.5 (m NH)

EXAMPLE 5

[0211] Preparation of cRGD

##STR00080##

[0212] cRGD used in the present disclosure was obtained from outsourcing.

Preparation of the L Portion was the Same as Example 1

[0213] ##STR00081##

[0214] Preparation of Compound 5 and H5

[0215] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M11 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 96.4 g of cRGD in 4 mL of PBS (pH=7, 0.01M) and 6 mL of methanol was added.

[0216] After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 5, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H5 (Hydrochloride of compound 5) (1.06 g).

[0217] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.326(s,2H), 5.480(s,2H), 7.0-8.5 (m NH)

EXAMPLE 6

[0218] Preparation of iRGD was the Same as Example 1

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0219] ##STR00082## ##STR00083##

1. Preparation of M21

[0220] To a 250 mL round bottom flask, 5.00 g (1.0 eq) of Compound M20, 100 mL of DCM and 3.89 g (3.0 eq) of TEA were added, and a solution of 3.50 g (1.0 eq) of TBDPS-Cl in 20 mL of DCM was added dropwise. After the completion of the reaction was monitored by TLC, the mixture was washed with water, washed with saturated saline, dried over anhydrous sodium sulfate, and then subjected to column chromatography to obtain 3.62 g of Compound M21 as a pale yellow solid.

2. Preparation of M22

[0221] To a 250 mL round bottom flask, 4.80 g (1.0 eq) of Compound M21, 145 mL of DCM, 1.64 g (1.2 eq) of Boc-Gly-OH and 95 mg (0.1 eq) of DMAP were added, a solution of 2.41 g (1.5 eq) of DCC in 10 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 130 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 160 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 4.65 g of Compound M22 as a pale yellow solid.

3. Preparation of M23

[0222] To a 100 mL three-necked flask, 4.65 g of Compound M22 and 50 mL of DCM were added. After the mixture was stirred and dissolved, 11.6 mL of TFA was added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30 min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M23 (2.48 g).

4. Preparation of M24

[0223] To a 200 mL three-necked flask, 2.3 g of Compound M23, 45 mL of DCM, 3.196 g (1.05 eq) of Compound M5 and 1.75 mL (3.0 eq) of TEA were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M24 (2.20 g).

5. Preparation of M25

[0224] To a 200 mL round bottom flask, 2.0 g of Compound M24 and 60 mL of 20% TFA/DCM were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, and dried to obtain a pale yellow solid M25 (1.75 g).

6. Preparation of M26

[0225] To a 500 mL round bottom flask, 1.51 g (4.0 eq) of Compound M25, 140 mL of DCM, 390 L (8.0 eq) of TEA and 7.0 g (1.0 eq) of 4armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid M26 (7.9 g).

##STR00084##

Preparation of Compound 6 and H6

[0226] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M26 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 188 mg of iRGD in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 6, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H6 (Hydrochloride of compound 6) (980 mg).

[0227] .sup.1HNMR(DMSO+D.sub.2O): 0.905(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.333(s,2H), 5.487(s,2H), 7.0-8.5 (m NH)

EXAMPLE 7

[0228] Preparation of tLyp-1 was the Same as Example 2

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0229] ##STR00085##

[0230] Preparation of Compound 7 and H7

[0231] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M26 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 144 mg of tLyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 7, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H7 (Hydrochloride of compound 7) (1.01 g).

[0232] .sup.1HNMR(DMSO+D.sub.2O): 0.904(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.335(s,2H), 5.488(s,2H), 7.0-8.5 (m NH)

EXAMPLE 8

Preparation of Lyp-1 was the Same as Example 3

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0233] ##STR00086##

[0234] Preparation of Compound 8 and H8

[0235] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M26 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 188 mg of LyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 8, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H8 (Hydrochloride of compound 8) (1.04 g).

[0236] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.328(s,2H), 5.481(s,2H), 7.0-8.5 (m NH)

EXAMPLE 9

Preparation of CRPARPAR was the Same as Example 4

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0237] ##STR00087##

[0238] Preparation of Compound 9 and H9

[0239] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M26 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 159 mg of CRPARPAR in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 9, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H9 (Hydrochloride of compound 9) (1.04 g).

[0240] .sup.1HNMR(DMSO+D.sub.2O): 0.910(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.328(s,2H), 5.480(s,2H), 7.0-8.5 (m NH)

EXAMPLE 10

[0241] Preparation of cRGD was the Same as Example 5

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0242] ##STR00088##

[0243] Preparation of Compound 10 and H10

[0244] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M26 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 99.5 mg of cRGD in 4 mL of PBS (pH=7, 0.01M) and 6 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 10, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H10 (Hydrochloride of compound 10) (1.03 g).

[0245] .sup.1HNMR(DMSO+D.sub.2O): 0.915(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.337(s,2H), 5.484(s,2H), 7.0-8.5 (m NH)

EXAMPLE 11

[0246] Preparation of iRGD was the Same as Example 1

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0247] ##STR00089##

[0248] Preparation of M31

[0249] To a 250 mL round bottom flask, 6.00 g (1.0 eq) of Compound M30, 120 mL of DCM and 5.00 g (3.0 eq) of TEA were added, and a solution of 4.53 g (1.0 eq) of TBDPS-Cl in 20 mL of DCM was added dropwise. After the completion of the reaction was monitored by TLC, the mixture was washed with water and saturated saline, dried over anhydrous sodium sulfate, and then subjected to column chromatography to obtain 4.32 g of Compound M31 as a pale yellow solid.

[0250] Preparation of M32

[0251] To a 250 mL round bottom flask, 5.0 g (1.0 eq) of Compound M31, 150 mL of DCM, 1.73 g (1.2 eq) of Boc-Gly-OH and 101 mg (0.1 eq) of DMAP were added, a solution of 2.55 g (1.5 eq) of DCC in 10 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 130 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 160 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 4.52 g of Compound M32 as a pale yellow solid.

[0252] Preparation of M33

[0253] To a 100 mL three-necked flask, 4.40 g of Compound M32 and 50 mL of DCM were added. After the mixture was stirred and dissolved, 11.6 mL of TFA was added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30 min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M33 (2.32 g).

##STR00090##

[0254] Preparation of M34

[0255] To a 200 mL three-necked flask, 2.3 g of Compound M33, 45 mL of DCM, 2.77 g (1.05 eq) of Compound M5 and 1.1 g (3.0 eq) of TEA were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M34 (2.02 g).

[0256] Preparation of M35

[0257] To a 200 mL round bottom flask, 2.0 g of Compound M34 and 60 mL of 20% TFA/DCM were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, and dried to obtain a pale yellow solid M35 (1.69 g).

[0258] Preparation of M36

[0259] To a 500 mL round bottom flask, 1.50 g (4.0 eq) of Compound M35, 140 mL of DCM, 390 L (8.0 eq) of TEA and 7.0 g (1.0 eq) of 4armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid M36 (7.64 g).

##STR00091##

[0260] Preparation of Compound 11 and H11

[0261] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M36 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 189 mg of iRGD in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 11, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H11 (Hydrochloride of compound 11) (1.08 g).

[0262] .sup.1HNMR(DMSO+D.sub.2O): 0.909(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.333(s,2H), 5.481(s,2H), 7.0-8.5 (m NH)

EXAMPLE 12

[0263] Preparation of tLyp-1 was the Same as Example 2

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0264] ##STR00092##

[0265] Preparation of Compound 12 and H12

[0266] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M36 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 144 mg of tLyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 12, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H12 (Hydrochloride of compound 12) (0.99 g).

[0267] .sup.1HNMR(DMSO+D.sub.2O): 0.915(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.340 (s,2H), 5.487(s,2H), 7.0-8.5 (m NH)

EXAMPLE 13

Preparation of Lyp-1 was the Same as Example 3

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0268] ##STR00093##

[0269] Preparation of Compound 13 and H13

[0270] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M36 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 189 mg of LyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 13, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H13 (Hydrochloride of compound 13) (1.06 g).

[0271] .sup.1HNMR(DMSO+D.sub.2O): 0.908(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.335 (s,2H), 5.484(s,2H), 7.0-8.5 (m NH)

EXAMPLE 14

Preparation of CRPARPAR was the Same as Example 4

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0272] ##STR00094##

[0273] Preparation of Compound 14 and H14

[0274] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M36 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 160 mg of CRPARPAR in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 14, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H14 (Hydrochloride of compound 14) (1.01 g).

[0275] .sup.1HNMR(DMSO+D.sub.2O): 0.910(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.336 (s,2H), 5.485(s,2H), 7.0-8.5 (m NH)

EXAMPLE 15

[0276] Preparation of cRGD was the Same as Example 5

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0277] ##STR00095##

[0278] Preparation of Compound 15 and H15

[0279] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M36 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 100 mg of cRGD in 4 mL of PBS (pH=7, 0.01M) and 6 mL of methanol was added.

[0280] After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 15, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H15 (Hydrochloride of compound 15) (1.05 g).

[0281] .sup.1HNMR(DMSO+D.sub.2O): 0.914(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.340 (s,2H), 5.489(s,2H), 7.0-8.5 (m NH)

EXAMPLE 16

[0282] Preparation of iRGD was the Same as Example 1

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0283] ##STR00096##

[0284] 1. Preparation of M41

[0285] To a 250 mL round bottom flask, 5.0 g (1.0 eq) of Compound M40, 150 mL of DCM, 2.67 g (1.2 eq) of Boc-Gly-OH and 155 mg (0.1 eq) of DMAP were added, a solution of 3.93 g (1.5 eq) of DCC in 15 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 130 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 160 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 6.85 g of Compound M41 as a pale yellow solid.

[0286] 2. Preparation of M42

[0287] To a 100 mL three-necked flask, 4.00 g of Compound M41 and 50 mL of DCM were added. After the mixture was stirred and dissolved, 11.6 mL of TFA was added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30 min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M42 (3.54 g).

##STR00097##

[0288] 1. Preparation of M43

[0289] To a 200 mL three-necked flask, 3.00 g of Compound M42, 45 mL of DCM, 3.46 g (1.05 eq) of Compound M5 and 1.38 g (3.0 eq) of TEA were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then the mixture was washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M43 (2.68 g).

[0290] 2. Preparation of M44

[0291] To a 200 mL round bottom flask, 2.0 g of Compound M43 and 60 mL of 20% TFA/DCM were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, dried to obtain a pale yellow solid M44 (1.61g).

[0292] 3. Preparation of M45

[0293] To a 500 mL round bottom flask, 1.50 g (4.0 eq) of Compound M44, 140 mL of DCM, 391 L (8.0 eq) of TEA and 7.0 g (1.0 eq) of 4armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid M45 (7.61 g).

##STR00098##

[0294] Preparation of Compound 16 and H16

[0295] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M45 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 188 mg of iRGD in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 16, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H16 (Hydrochloride of compound 16) (1.09 g).

[0296] .sup.1HNMR(DMSO+D.sub.2O): 0.909(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.335(s,2H), 5.482(s,2H), 7.0-8.5 (m NH)

EXAMPLE 17

[0297] Preparation of tLyp-1 was the Same as Example 2

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0298] ##STR00099##

Preparation of Compound 17 and H17

[0299] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M45 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 144 mg of tLyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 17, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H17 (Hydrochloride of compound 17) (1.02 g).

[0300] .sup.1HNMR(DMSO+D.sub.2O): 0.912(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.336(s,2H), 5.485(s,2H), 7.0-8.5 (m NH)

EXAMPLE 18

Preparation of Lyp-1 was the Same as Example 3

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0301] ##STR00100##

Preparation of Compound 18 and H18

[0302] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M45 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 188 mg of LyP-1 in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 18, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H18 (Hydrochloride of compound 18) (1.06 g).

[0303] .sup.1HNMR(DMSO+D.sub.2O): 0.917(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.340(s,2H), 5.491(s,2H), 7.0-8.5 (m NH)

EXAMPLE 19

Preparation of CRPARPAR was the Same as Example 4

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0304] ##STR00101##

Preparation of Compound 19 and H19

[0305] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M45 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 159 mg of CRPARPAR in 10 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 19, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H19 (Hydrochloride of compound 19) (1.07 g).

[0306] .sup.1HNMR(DMSO+D.sub.2O): 0.911(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.332(s,2H), 5.480(s,2H), 7.0-8.5 (m NH)

EXAMPLE 20

[0307] Preparation of cRGD was the Same as Example 5

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0308] ##STR00102##

[0309] Preparation of Compound 20 and H20

[0310] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound M45 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 99.5 mg of cRGD in 4 mL of PBS (pH=7, 0.01M) and 6 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated to obtain compound 20, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid H20 (Hydrochloride of compound 20) (1.03 g).

[0311] .sup.1HNMR(DMSO+D.sub.2O): 0.909(t,CH.sub.2CH.sub.3), 3.5(br m PEG),5.331(s,2H), 5.478(s,2H), 7.0-8.5 (m NH)

EXAMPLE 21

[0312] Preparation of iRGD was the Same as Example 1

Preparation of the L Portion was the Same as Example 1

[0313] ##STR00103##

[0314] Preparation of 50

[0315] To a 10 mL round bottom flask, 52 mg (3.0 eq) of Compound M10, 2 mL of DCM, 11 L (6.0 eq) of TEA and 273 mg (1.0 eq) of 3armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid 50 (310 mg).

##STR00104##

[0316] Preparation of Compound 21

[0317] To a 10 mL round bottom flask, 50 mg (1.0 eq) of Compound 50 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 7.2 mg of iRGD in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated , the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 21 (49 mg).

[0318] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.328(s,2H), 5.480(s,2H), 7.0-8.5(m NH)

EXAMPLE 22

Preparation of the L Portion was the Same as Example 1

[0319] ##STR00105##

[0320] Preparation of Compound 22

[0321] To a 10 mL round bottom flask, 55 mg (1.0 eq) of Compound 50 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 4.2 mg of cRGD in 1 mL of PBS (pH=7, 0.01M) and 1.5 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 22 (50 mg).

[0322] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.333(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 23

[0323] Preparation of iRGD was the Same as Example 1

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0324] ##STR00106##

[0325] Preparation of 60

[0326] To a 100 mL round bottom flask, 476 mg (8.0 eq) of Compound M10, 15 mL of DCM, 105 L (16.0 eq) of TEA, and 1.0 g (1.0 eq) of 8armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid 60 (1.37 g).

##STR00107##

[0327] Preparation of Compound 23

[0328] To a 10 mL round bottom flask, 50 mg (1.0 eq) of Compound 60 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 14.5 mg of iRGD in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 23 (54 mg).

[0329] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.328(s,2H), 5.480(s,2H), 7.0-8.5(m NH)

EXAMPLE 24

Preparation of Lyp-1 was the Same as Example 3

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0330] ##STR00108##

[0331] Preparation of Compound 24

[0332] To a 10 mL round bottom flask, 60 mg (1.0 eq) of Compound 60 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 17.4 mg of LyP-1 in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 24 (68 mg).

[0333] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.329(s,2H), 5.481(s,2H), 7.0-8.5(m NH)

EXAMPLE 25

[0334] ##STR00109##

[0335] Preparation of Compound 25

[0336] To a 10 mL round bottom flask, 45 mg (1.0 eq) of Compound 60 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 10.0 mg of tLyP-1 in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 25(45 mg).

[0337] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.330(s,2H), 5.482(s,2H), 7.0-8.5(m NH)

EXAMPLE 26

[0338] ##STR00110##

[0339] Preparation of Compound 26

[0340] To a 10 mL round bottom flask, 55 mg (1.0 eq) of Compound 60 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 13.5 mg of CRPARPAR in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 26 (62 mg).

[0341] .sup.1HNMR(DMSO+D.sub.2O): 60. 901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.331(s,2H), 5.480(s,2H), 7.0-8.5(m NH)

EXAMPLE 27

[0342] ##STR00111##

[0343] Preparation of Compound 27

[0344] To a 10 mL round bottom flask, 50 mg (1.0 eq) of Compound 60 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 7.7 mg of cRGD in 1 mL of PBS (pH=7, 0.01M) and 1.5 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 27 (50 mg).

[0345] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.328(s,2H), 5.481(s,2H), 7.0-8.5(m NH)

EXAMPLE 28

[0346] Preparation of iRGD was the Same as Example 1

Preparation of the L Portion was the Same as Example 1

Preparation of the Conjugate

[0347] ##STR00112## ##STR00113##

[0348] Preparation of M21

[0349] To a 250 mL round bottom flask, 5.00 g (1.0 eq) of Compound M20, 100 mL of DCM, and 3.87 g (3.0 eq) of TEA were added, and a solution of 3.49 g (1.0 eq) of TBDPS-Cl in 20 mL of DCM was added dropwise. After the completion of the reaction was monitored by TLC, the mixture was washed with water and saturated saline, dried over anhydrous sodium sulfate, and then subjected to column chromatography to obtain 3.52g of Compound M21 as a pale yellow solid.

[0350] Preparation of M22

[0351] To a 250 mL round bottom flask, 4.50 g (1.0 eq) of Compound M21, 135 mL of DCM, 1.5 g (1.2 eq) of Boc-Gly-OH, and 87 mg (0.1eq) of DMAP were added, a solution of 2.21 g (1.5 eq) of DCC in 10 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 120 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 150 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 4.95 g of Compound M22 as a pale yellow solid.

[0352] Preparation of M23

[0353] To a 100 mL three-necked flask, 4.95 g of Compound M22 and 55 mL of DCM were added. After being stirred and dissolved, 13.7 mL of TFA were added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30 min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M23 (2.5 g).

[0354] Preparation of M24

[0355] To a 200 mL three-necked flask, 2.3 g of Compound M23, 45 mL of DCM, 3.9 g (1.05 eq) of Compound 5 and 2.14 mL (3.0 eq) of TEA were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M24 (2.45 g).

[0356] Preparation of M25

[0357] To a 200 mL round bottom flask, 2.2 g of Compound M24 and 65 mL of 20% TFA/DCM were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, and dried to obtain a pale yellow solid M25 (1.7 g).

[0358] Preparation of 70

[0359] To a 100 mL round bottom flask, 1.21 g (8.0 eq) of Compound M25, 60 mL of DCM, 314 L (16.0 eq) of TEA and 3.0 g (1.0 eq) of 4armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid 70 (3.9 g).

##STR00114##

[0360] Preparation of Compound 28

[0361] To a 10 mL round bottom flask, 50 mg (1.0 eq) of Compound 70 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 15.3 mg of iRGD in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 28 (55 mg).

[0362] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.335(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 29

[0363] ##STR00115##

[0364] Preparation of Compound 29

[0365] To a 10 mL round bottom flask, 60 mg (1.0 eq) of Compound 70 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 18.3 mg of LyP-1 in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 29 (68 mg).

[0366] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.333(s,2H), 5.486(s,2H), 7.0-8.5(m NH)

EXAMPLE 30

[0367] ##STR00116##

[0368] Preparation of Compound 30

[0369] To a 10 mL round bottom flask, 55 mg (1.0 eq) of Compound 70 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 12.8 mg of tLyP-1 in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 30(55 mg).

[0370] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.335(s,2H), 5.488(s,2H), 7.0-8.5(m NH)

EXAMPLE 31

[0371] ##STR00117##

[0372] Preparation of Compound 31

[0373] To a 10 mL round bottom flask, 55 mg (1.0 eq) of Compound 70 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 14.2 mg of CRPARPAR in 1 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 31(60 mg).

[0374] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.332(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 32

[0375] ##STR00118##

[0376] Preparation of Compound 32

[0377] To a 10 mL round bottom flask, 60 mg (1.0 eq) of Compound 70 and 1.5 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 9.7 mg of cRGD in 1 mL of PBS (pH=7, 0.01M) and 1.5 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 32 (62 mg).

[0378] .sup.1HNMR(DMSO+D.sub.2O): 0.900(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.333(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 33

[0379] ##STR00119##

[0380] Preparation of M31

[0381] To a 250 mL round bottom flask, 6.00 g (1.0 eq) of Compound M30, 120 mL of DCM and 5.00 g (3.0 eq) of TEA were added, and a solution of 4.53 g (1.0 eq) of TBDPS-Cl in 20 mL of DCM was added. After the completion of the reaction was monitored by TLC, the mixture was washed with water and saturated saline, dried over anhydrous sodium sulfate, and then subjected to column chromatography to obtain 4.32 g of Compound M31 as a pale yellow solid.

[0382] Preparation of M32

[0383] To a 250 mL round bottom flask, 5.0 g (1.0 eq) of Compound M31, 150 mL of DCM, 1.73 g (1.2 eq) of Boc-Gly-OH, and 101 mg (0.1 eq) of DMAP were added, a solution of 2.55 g (1.5 eq) of DCC in 10 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 130 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 160 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 4.52 g of Compound M32 as a pale yellow solid.

[0384] Preparation of M33

[0385] To a 100 mL three-necked flask, 4.40 g of Compound M32 and 50 mL of DCM were added. After the mixture was stirred and dissolved, 11.6 mL of TFA was added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30 min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M33 (2.32 g).

##STR00120##

[0386] Preparation of M34

[0387] To a 200 mL three-necked flask, 2.3 g of Compound M33, 45 mL of DCM, 2.77 g (1.05 eq) of Compound M5, and 1.1 g (3.0 eq) of TEA were added and reacted at room temperature for 4h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M34 (2.02 g).

[0388] Preparation of M35

[0389] To a 200 mL round bottom flask, 2.0 g of Compound M34 and 60 mL of 20% TFA/DCM were added and reacted at room temperature for 4h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, and dried to obtain a pale yellow solid M35 (1.69 g).

[0390] Preparation of 80

[0391] To a 500 mL round bottom flask, 2.75 g (8.0 eq) of Compound M35, 140 mL of DCM, 730 L (16.0 eq) of TEA and 7.0 g (1.0 eq) of 8armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid 80 (8.64 g).

##STR00121##

[0392] Preparation of Compound 33

[0393] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound 80 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 307 mg of iRGD in 15 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 33 (1.18 g).

[0394] .sup.1HNMR(DMSO+D.sub.2O): 0.903(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.334(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 34

Preparation of Compound 34

[0395] ##STR00122##

EXAMPLE 35

[0396] ##STR00123##

[0397] Preparation of Compound 35

[0398] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound 80 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 235 mg of tLyP-1 in 15 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 35 (1.05 g).

[0399] .sup.1HNMR(DMSO+D.sub.2O): 0.904(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.334(s,2H), 5.489(s,2H), 7.0-8.5(m NH)

EXAMPLE 36

Preparation of Compound 36

[0400] ##STR00124##

EXAMPLE 37

[0401] ##STR00125##

[0402] Preparation of Compound 37

[0403] To a 100 mL round bottom flask, 1.0 g (1.0 eq) of Compound 80 and 20 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 163 mg of cRGD in 6 mL of PBS (pH=7, 0.01M) and 10 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 37 (1.00 g).

[0404] .sup.1HNMR(DMSO+D.sub.2O): 0.903(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.334(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 38

[0405] ##STR00126##

[0406] Preparation of M41

[0407] To a 250 mL round bottom flask, 5.0 g (1.0 eq) of Compound M40, 150 mL of DCM, 2.67 g (1.2eq) of Boc-Gly-OH and 155 mg (0.1 eq) of DMAP were added, a solution of 3.93 g (1.5 eq) of DCC in 15 mL of DCM was added dropwise, and the mixture was reacted at 20 C. for 4 h. After the completion of the reaction was monitored by TLC, the mixture was filtered, and 130 mL of IPA was added when the mixture was concentrated to 25% of its total volume. 75% of the solvent was removed by distillation, and 160 mL of n-heptane was added. The mixture was stirred at room temperature for 1 h, filtered, washed twice with n-heptane, and dried to obtain 6.85 g of Compound M41 as a pale yellow solid.

[0408] Preparation of M42

[0409] To a 100 mL three-necked flask, 4.00 g of Compound M41 and 50 mL of DCM were added. After the mixture was stirred and dissolved, 11.6 mL of TFA was added dropwise, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, 150 mL of acetonitrile was added. After 120 mL of solvent was distilled under reduced pressure, the mixture was poured into 320 mL of TBME solution, stirred for 30 min, and filtered. The filter cake was washed with TBME to obtain a pale yellow solid M42 (3.54 g).

##STR00127##

[0410] Preparation of M43

[0411] To a 200 mL three-necked flask, 3.00 g of Compound M42, 45 mL of DCM, 3.46 g (1.05 eq) of Compound M5, and 1.38 g (3.0 eq) of TEA were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was diluted with DCM, then washed twice with water, washed once with saturated saline, dried, concentrated, purified by HPLC, and then lyophilized to obtain a pale yellow solid M43 (2.68 g).

[0412] Preparation of M44

[0413] To a 200 mL round bottom flask, 2.0 g of Compound M43 and 60 mL of 20% TFA/DCM were added and reacted at room temperature for 4 h. After the completion of the reaction was monitored by TLC, the mixture was poured into TBME, centrifuged, and dried to obtain a pale yellow solid M44 (1.61 g).

[0414] Preparation of 90

[0415] To a 250 mL round bottom flask, 1.61 g (8.0 eq) of Compound M44, 80 mL of DCM, 418 L (16.0 eq) of TEA, and 4.0 g (1.0 eq) of 8armPEG20K-SCM were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, and dried to obtain an off-white solid 90 (5.15 g).

##STR00128##

[0416] Preparation of Compound 38

[0417] To a 50 mL round bottom flask, 0.5 g (1.0 eq) of Compound 90 and 10 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 152 mg of iRGD in 5 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 38 (0.53 g).

[0418] .sup.1HNMR(DMSO+D.sub.2O): 0.902(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.333(s,2H), 5.488(s,2H), 7.0-8.5(m NH)

EXAMPLE 39

[0419] ##STR00129##

[0420] Preparation of Compound 39

[0421] To a 50 mL round bottom flask, 0.5 g (1.0 eq) of Compound 90 and 10 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 153 mg of LyP-1 in 5 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 39 (0.57 g).

[0422] .sup.1HNMR(DMSO+D.sub.2O): 0.900(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.332(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 40

Preparation of Compound 40

[0423] ##STR00130##

EXAMPLE 41

[0424] ##STR00131##

[0425] Preparation of Compound 41

[0426] To a 50 mL round bottom flask, 0.5 g (1.0 eq) of Compound 90 and 10 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a solution of 129 mg of CRPARPAR in 5 mL of PBS (pH=7, 0.01M) was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 41 (0.55 g).

[0427] .sup.1HNMR(DMSO+D.sub.2O): 0.901(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.331(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

EXAMPLE 42

[0428] ##STR00132##

[0429] Preparation of Compound42

[0430] To a 50 mL round bottom flask, 0.5 g (1.0 eq) of Compound 90 and 10 mL of PBS (pH=7, 0.01M) were added. After the mixture was dissolved to give a clear solution, a mixed solution of 80.8 mg of cRGD in 4 mL of PBS (pH=7, 0.O1M) and 6 mL of methanol was added. After reacting at room temperature for 4 h, the mixture was dialyzed and concentrated, the crude product was dissolved in methanol, and a solution of HCl/EA was added. The final mixture was concentrated, added to TBME, centrifuged, and dried to obtain a yellow solid 42 (0.50 g).

[0431] .sup.1HNMR(DMSO+D.sub.2O): 0.903(t,CH.sub.2CH.sub.3), 3.5(br m PEG), 5.330(s,2H), 5.487(s,2H), 7.0-8.5(m NH)

[0432] In the preparation of Compounds 23 to 42, R was

##STR00133##

EXAMPLE 43

Inhibitory Effect on In Vivo Tumor Growth in a Human Colon Cancer HT-29 Transplated Tumor Model in Nude Mice

[0433] 1. Experimental Purpose

[0434] The inhibitory effects of Compounds 1 to 42 on in vivo tumor growth in the human colon cancer HT-29 transplanted tumor model in nude mice were tested.

[0435] 2. Experimental Materials

[0436] 2.1 Test Articles

[0437] Irinotecan (bulk drug) and nktr-102 (bulk drug) were obtained by purchasing, and Compounds 1 to 42 were all provided by Brightgene Bio-Medical Technology Co., Ltd.

[0438] 2.2 Reagents

[0439] McCoy's 5A liquid culture medium, fetal bovine serum (FBS), trypsin, penicillin-streptomycin bispecific antibody, water for injection, lactic acid, and sorbitol.

[0440] 2.3 Experimental Animals

[0441] Female BALB/c nude mice (number of animals: 300, weeks of age: 5 to 7 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housed in an SPF animal room. The temperature was 20 C. to 25 C., the relative humidity was 40% to 70%, and the light and dark were respectively 12 h. The animals had free access to water and food. After about one week of normal feeding, upon a veterinary inspection, mice with good physical signsand conditions could be selected for the experiment. A marking pen was used to mark the base of the tails of the animals before the grouping, and each animal was marked by ear clipping after the grouping.

[0442] 2.4 Transplantable Tumor Cell Line

[0443] Human colon cancer cell HT-29, derived from Cell Bank of Committee on Type Culture Collection of Chinese Academy of Science (CAS, stored frozen in liquid nitrogen in the laboratory).

[0444] 3 Experimental Methods

[0445] 3.1 Culture of HT-29 Cells

[0446] Under a culture condition of 5% CO2 and 37 C., HT-29 cells were subjected to conventional cell cultivation in McCoy's 5A liquid culture medium containing 10% fetal bovine serum, digested with 0.25% trypsin, and passaged. According to the condition of cell growth, the cells were passaged 2 to 3 times every week, and were passaged in a ratio of 1:3 to 1:8.

[0447] 3.2 Preparation of the Animal Model

[0448] HT-29 cells in logarithmic phase of growth were collected. The cells were resuspended in an McCoy's 5A culture medium free of serum after being counted, and the concentration of the cells was adjusted to 610.sup.7 cells/mL. The cells were charged into a 50 mL centrifuge tube after being pipetted with a pipettor to make them evenly dispersed, and the centrifuge tube was placed in an ice box. Cell suspension was aspirated with a 1 mL syringe, and injected subcutaneously to the anterior right armpit of nude mice. Each animal was inoculated with 100 L (610.sup.6 cells/animal), and the HT-29 transplated tumor model in nude mice was established. Animal status and the condition of tumor growth were observed regularly after inoculation, an electronic vernier caliper was used to measure the tumor diameters, the data was directly input to an Excel spreadsheet, and the tumor volumes were calculated. When the tumor volumes reached 100 mm.sup.3 to 300 mm.sup.3, 225 animals with good health condition and similar tumor volume were selected and divided into 45 groups using randomized block method (n=5). The tumor diameters were measured twice a week after the initiation of the experiment, the tumor volumes were calculated, and the body weights of the animals were weighed and recorded at the same time.

[0449] The calculation formula of the tumor volume (TV) was as follows:

TV(mm.sup.3)=lw.sup.2/.sub.2

[0450] wherein l represented the long diameter of a tumor (mm), and w represented the short diameter of a tumor (mm).

[0451] 3.3 Preparation of the Solvent

[0452] 0.5 g of sorbitol was weighed and charged into a 50 mL centrifuge tube, 50 mL of water for injection was added to the centrifuge tube, and the solid substance was dissolved completely by vortexing, so that an aqueous sorbitol solution (w/v) with a concentration of 1% was formulated. The solution was stored in a refrigerator at 4 C. until use.

[0453] 3.4 Preparation of the Dosing Formulations

[0454] 3.4.1 Preparation of the Dosing Formulation of Irinotecan

[0455] 12.0 mg of irinotecan was weighed, 0.15 mL of 1% lactic acid was added, the drug was dissolved completely by vortexing, and 2.85 mL of 1% aqueous sorbitol solution was then added respectively. The mixture was mixed evenly by vortexing, and the ratio of 1% lactic acid to 1% aqueous sorbitol solution was approximately 5:95 (v/v) in the solution. The concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0456] 3.4.2 Preparation of the Dosing Formulation of nktr-102

[0457] Before each administration, 88.55 mg of nktr-102 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0458] 3.4.3 Preparation of the Dosing Formulations of Compounds 1 to 5

[0459] Before each administration, 113.85 mg of Compound 1 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0460] Before each administration, 109.80 mg of Compound 2 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0461] Before each administration, 113.88 mg of Compound 3 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0462] Before each administration, 111.24 mg of Compound 4 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0463] Before each administration, 105.78 mg of Compound 5 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0464] 3.4.4 Preparation of the Dosing Formulations of Compounds 6 to 10

[0465] Before each administration, 113.09 mg of Compound 6 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0466] Before each administration, 109.05 mg of Compound 7 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0467] Before each administration, 113.12 mg of Compound 8 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0468] Before each administration, 110.48 mg of Compound 9 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0469] Before each administration, 105.02 mg of Compound 10 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0470] 3.4.5 Preparation of the Dosing Formulations of Compounds 11 to 15

[0471] Before each administration, 112.98 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0472] Before each administration, 108.94 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0473] Before each administration, 113.01 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0474] Before each administration, 110.37 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0475] Before each administration, 104.91 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0476] 3.4.6 Preparation of the Dosing Formulations of Compounds 16 to 20

[0477] Before each administration, 113.09 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0478] Before each administration, 109.05 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0479] Before each administration, 113.12 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0480] Before each administration, 110.48 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0481] Before each administration, 105.02 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0482] 3.4.7 Preparation of the Dosing Formulations of Compounds 21 to 42

[0483] The preparation methods were as described above, so that the concentration of the free active agent in the solution was 4.0 mg.Math.mL.sup.1.

[0484] 3.5 Grouping of Animals and Administration

[0485] The grouping of animals and the dosing regimens were as shown in Table 1.

[0486] Groups with irinotecan as the active agent (Groups 1 to 8). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Group 1 was a solvent control group, and was given blank solvent by intravenous injection via tail once every 4 days for a total of 3 times (Q4D3). Groups 2 to 8 were given irinotecan, nktr-102, and Compounds 1 to 5 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of irinotecan), Q4D3.

[0487] Groups with SN-38 as the active agent (Groups 9 to 13). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 9 to 13 were given Compounds 6 to 10 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of SN-38), Q4D3.

[0488] Groups with 10-hydroxycamptothecin as the active agent (Groups 14 to 18). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 14 to 18 were given Compounds 11 to 15 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of 10-hydroxycamptothecin), Q4D3.

[0489] Groups with rubitecan as the active agent (Groups 19 to 23). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 19 to 23 were given Compounds 16 to 20 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of rubitecan), Q4D3.

[0490] The modes of administration of Groups 24 to 25 were as described above.

TABLE-US-00001 TABLE 1 Dosing regimens of the pharmacodynamic experiments in transplanted tomor models in nude mice Volume of Number of Dosage administration Route of Period of Group Sample animals (mg .Math. kg.sup.1) (mL .Math. kg.sup.1) administration administration 1 blank solvent 5 10 IV Q4D 3 2 irinotecan 5 40 10 IV Q4D 3 3 nktr-102 5 40 10 IV Q4D 3 4 Compound 1 5 40 10 IV Q4D 3 5 Compound 2 5 40 10 IV Q4D 3 6 Compound 3 5 40 10 IV Q4D 3 7 Compound 4 5 40 10 IV Q4D 3 8 Compound 5 5 40 10 IV Q4D 3 9 Compound 6 5 40 10 IV Q4D 3 10 Compound 7 5 40 10 IV Q4D 3 11 Compound 8 5 40 10 IV Q4D 3 12 Compound 9 5 40 10 IV Q4D 3 13 Compound 10 5 40 10 IV Q4D 3 14 Compound 11 5 40 10 IV Q4D 3 15 Compound 12 5 40 10 IV Q4D 3 16 Compound 13 5 40 10 IV Q4D 3 17 Compound 14 5 40 10 IV Q4D 3 18 Compound 15 5 40 10 IV Q4D 3 19 Compound 16 5 40 10 IV Q4D 3 20 Compound 17 5 40 10 IV Q4D 3 21 Compound 18 5 40 10 IV Q4D 3 22 Compound 19 5 40 10 IV Q4D 3 23 Compound 20 5 40 10 IV Q4D 3 24 Compound 21 5 40 10 IV Q4D 3 25 Compound 22 5 40 10 IV Q4D 3 26 Compound 23 5 40 10 IV Q4D 3 27 Compound 24 5 40 10 IV Q4D 3 28 Compound 25 5 40 10 IV Q4D 3 29 Compound 26 5 40 10 IV Q4D 3 30 Compound 27 5 40 10 IV Q4D 3 31 Compound 28 5 40 10 IV Q4D 3 32 Compound 29 5 40 10 IV Q4D 3 33 Compound 30 5 40 10 IV Q4D 3 34 Compound 31 5 40 10 IV Q4D 3 35 Compound 32 5 40 10 IV Q4D 3 36 Compound 33 5 40 10 IV Q4D 3 37 Compound 34 5 40 10 IV Q4D 3 38 Compound 35 5 40 10 IV Q4D 3 39 Compound 36 5 40 10 IV Q4D 3 40 Compound 37 5 40 10 IV Q4D 3 41 Compound 38 5 40 10 IV Q4D 3 42 Compound 39 5 40 10 IV Q4D 3 43 Compound 40 5 40 10 IV Q4D 3 44 Compound 41 5 40 10 IV Q4D 3 45 Compound 42 5 40 10 IV Q4D 3

[0491] 3.6 End of the Experiment

[0492] On the last day of the experiment, the animals were euthanized (CO.sub.2) after the body weights were weighed and the tumor diameters were measured. The tumor tissues were excised, weighed and photographed (respectively photographed according to the following combinations), and the tumor weight inhibition rates were calculated. The animals were subjected to gross anatomy, and whether there was abnormality in internal organs were observed by naked eyes.

[0493] 4 Data Recording and Calculation Formulae

[0494] The calculation formula of the relative tumor volume (RTV) was:

RTV=TV.sub.t/TV.sub.initial

[0495] wherein TV.sub.initial was the tumor volume measured at the time of grouping and first administration, and TV.sub.t was the tumor volume at each measurement during administration.

[0496] The calculation formula of the relative tumor proliferation rate (% T/C) was:

% T/C=100%(RTV.sub.T/RTV.sub.C)

[0497] wherein RTV.sub.T represented the RTV of a treatment group, and RTV.sub.C represented the RTV of the solvent control group.

[0498] The calculation formula of the tumor growth inhibition rate (TGI (%)) was:

TGI=100%[1(TV.sub.t(T)TV.sub.initial(T))/(TV.sub.t(C)TV.sub.initial(C))]

[0499] wherein TV.sub.t(T) represented the tumor volume of a treatment group at each measurement, TV.sub.initial(T) represented the tumor volume of a treatment group measured at the time of grouping and first administration, TV.sub.t(C) represented the tumor volume of the solvent control group at each measurement, and TV.sub.initial(C) represented the tumor volume of the solvent control group measured at the time of grouping and first administration.

[0500] The calculation formula of the decline rate of animal body weight was:

decline rate of animal body weight=100%(BW.sub.initialBW.sub.t)/BW.sub.initial

[0501] wherein BW.sub.t represented the animal body weight at each measurement during administration, and BW.sub.initial represented the animal body weight at the time of grouping and first administration.

[0502] The calculation formula of the tumor weight inhibition rate (IR (%)) was:

IR(%)=100%(W.sub.CW.sub.T)/W.sub.C

[0503] wherein W.sub.C represented the tumor weight of the control group, and W.sub.T represented the tumor weight of a treatment group.

[0504] 5 Method for Statistical Analysis

[0505] The experimental data was subjected to calculation and related statistical treatments using Microsoft Office Excel 2007 Software. Unless otherwise specified, the data was represented by meanstandard error (MeanSE), and t test was adopted for the comparison between two groups.

[0506] 6 Experimental Observation

[0507] During the experiment, experimenter(s) and veterinarian(s) needed to continuously observe the physical signs and health status of the experimental animals. Any abnormal performance of the animals, such as pain, depression, and decreased activity, should be recorded in the original experiment record. If abnormal performances of the experimental animals exceeded the provisions of the IACUC-related animal welfare documents, the veterinarian(s) could judge whether to suspend the experiment and notify the person in charge of the experimental project.

[0508] 7 Results

[0509] As for the human cancer xenograft models, the relative tumor proliferation rate T/C (%) was recommended to be adopted as the evaluation index of the experiment. Lower proliferation rate indicated better tumor inhibitory effect, as shown in Table 2.

TABLE-US-00002 TABLE 2 Relative tumor proliferation rate T/C (%) Relative Relative tumor Number of tumor volume proliferation Group Sample animals (RTV) rate (% T/C) 1 blank solvent 5 13.78 2 irinotecan 5 9.52 69% 3 nktr-102 5 6.37 46% 4 Compound 1 5 2.07* 15%.sup.# 5 Compound 2 5 2.48* 18%.sup.# 6 Compound 3 5 2.70* 19.5%.sup.# 7 Compound 4 5 2.65* 19.2%.sup.# 8 Compound 5 5 2.46* 17.9%.sup.# 9 Compound 6 5 2.27* 16.5%.sup.# 10 Compound 7 5 2.50* 18.1%.sup.# 11 Compound 8 5 3.18* 23.1%.sup.# 12 Compound 9 5 2.90* 21.1%.sup.# 13 Compound 10 5 2.16* 15.7%.sup.# 14 Compound 11 5 2.61* 18.9%.sup.# 15 Compound 12 5 2.53* 18.3%.sup.# 16 Compound 13 5 2.35* 17%.sup.# 17 Compound 14 5 2.47* 17.9%.sup.# 18 Compound 15 5 2.20* 16.8%.sup.# 19 Compound 16 5 2.48* 18%.sup.# 20 Compound 17 5 2.65* 19.2%.sup.# 21 Compound 18 5 2.78* 20.2%.sup.# 22 Compound 19 5 2.68* 19.4%.sup.# 23 Compound 20 5 2.33* 16.9%.sup.# 24 Compound 21 5 2.14* 15.5%.sup.# 25 Compound 22 5 2.31* 16.7%.sup.# 26 Compound 23 5 2.25* 16.3%.sup.# 27 Compound 24 5 2.47* 17.9%.sup.# 28 Compound 25 5 2.76* 20.0%.sup.# 29 Compound 26 5 2.69* 19.5%.sup.# 30 Compound 27 5 2.37* 17.2%.sup.# 31 Compound 28 5 2.93* 21.2%.sup.# 32 Compound 29 5 2.72* 19.7%.sup.# 33 Compound 30 5 2.54* 18.4%.sup.# 34 Compound 31 5 2.31* 16.7%.sup.# 35 Compound 32 5 2.57* 18.6%.sup.# 36 Compound 33 5 3.01* 21.8%.sup.# 37 Compound 34 5 2.67* 19.3%.sup.# 38 Compound 35 5 2.29* 16.6%.sup.# 39 Compound 36 5 2.46* 17.8%.sup.# 40 Compound 37 5 2.39* 17.3%.sup.# 41 Compound 38 5 2.84* 20.6%.sup.# 42 Compound 39 5 2.57* 18.6%.sup.# 43 Compound 40 5 2.48* 18.0%.sup.# 44 Compound 41 5 2.76* 20.0%.sup.# 45 Compound 42 5 2.64* 19.1%.sup.# *P < 0.05, as compared with the RTV of the blank solvent group, irinotecan group and nktr-102 group. .sup.#P < 0.05, as compared with % T/C of the blank solvent group, irinotecan group and nktr-102 group.

[0510] The experimental results showed that the compounds of the present disclosure had good inhibitory effects on in vivo tumor growth in the human colon cancer HT-29 transplanted tumor model in nude mice, and were superior to irinotecan and nktr-102. Example 44 Inhibitory effect in a human breast cancer MDA-MB-231 xenograft model in nude mice

[0511] 1. Experimental Purpose

[0512] The human breast cancer MDA-MB-231 transplanted tumor model in nude mice was used in this study to evaluate the in vivo antitumor activities of Compounds 1 to 42.

[0513] 2. Experimental Animals

[0514] 2.1 Animal species

[0515] Mice.

[0516] 2.2 Strain

[0517] BALB/c nude mice.

[0518] 2.3 Sex

[0519] Female.

[0520] 2.4 Number

[0521] 320 animals were inoculated, and 225 animals were used for the experiment.

[0522] 2.5 Age

[0523] 6 to 8 week old.

[0524] 2.6 Body weight

[0525] 20 to 22 g20% of the average body weight.

[0526] 2.7 Animal source (supplier)

[0527] Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. (BK), license number: SCXK (Shanghai) 2008-0016.

[0528] 2.8 Management of Experimental Animals

[0529] All experimental animals were housed in an SPF-level laboratory. Experimenters were responsible for daily care and experimental research.

[0530] 2.8.1 Identification Method of Animal Identity

[0531] Each mice cage had an identification card with information such as the experimental number, experimental group, name(s) of the experimenter(s), and the strain and sex of mice. The mice were marked with earrings.

[0532] 2.8.2 Random Grouping

[0533] After the tumor volumes reached 150 mm.sup.3 to 200mm.sup.3, the animals were grouped using randomized block method with 5 mice in each group, ensuring the tumor volumes and the body weights of the mice were uniform between each group. The difference between the mean value of the tumor volumes in each group and the mean value of the tumor volumes of all experimental animals was no more than 10%.

[0534] 2.8.3 Operating and Managing Practice

[0535] The operation and management of all experimental animals strictly adhered to the guideline of the use and management of animals.

[0536] 2.8.4 Feeding Conditions

[0537] Living conditions: IVC system, 5 animals per cage

[0538] Temperature: 20 C. to 26 C.

[0539] Humidity: 40% to 70%

[0540] Illumination: 12 h light/dark cycle

[0541] 2.8.5 Feed

[0542] Irradiated feed for rat and mice, purchased from Beijing Keao Xieli Feed Co., Ltd. The animals had free access to food.

[0543] 2.8.6 Drinking Water

[0544] City tap water, used for drinking after being filtered and autoclaved.

[0545] 2.8.7 Beddings

[0546] Corn cobs (Shanghai Maoshengyan Biologic Science & Technology Co., Ltd.) were used after being autoclaved. Beddings were changed twice a week.

[0547] 2.8.8 Acclimation Period

[0548] The mice were given at least one week of acclimation period for the environment before the experiment.

[0549] 3. Experimental Materials

[0550] 3.1 Test Drugs

[0551] Irinotecan (bulk drug) and nktr-102 (bulk drug) were obtained by purchasing, and Compounds 1 to 42 were all provided by Brightgene Bio-Medical Technology Co., Ltd.

[0552] 3.2 Other Chemical Reagents and Materials

[0553] 3.2.1 Physiological Saline

[0554] Physiological saline was purchased from Shanghai Huayuan Changfu Pharmaceutical (Group) Co., Ltd.

[0555] 3.2.2 Sterile Syringe

[0556] 1 mL sterile syringes were purchased from Shanghai Kindly Enterprise Development Group Co., Ltd (Shanghai, China).

[0557] 3.2.3 Cell Strain

[0558] Human breast cancer MDA-MB-231 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences.

[0559] MDA-MB-231 cells were cultured in DMEM culture medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA), and cultured in a 37 C. incubator containing 5% CO.sub.2.

[0560] 3.2.4 Matrigel (BD Matrigel)

[0561] Matrigel was purchased from Becton, Dickinson and Company (BD), the USA.

[0562] 3.3 Instruments

[0563] Biosafe cabinet (model: AC2-6E1), purchased from ESCO; water jacketed CO.sub.2 cell incubator (model: 3111), purchased from Thermo Scientific Forma;

[0564] inverted microscope (model: CKX41SF), purchased from Olympus;

[0565] electric suction apparatus (model: YX930D), purchased from Shanghai Medical Instruments (Group) Co., Ltd.;

[0566] balance (METTLER-TOLEDO AB135-S), purchased from METTLER-TOLEDO;

[0567] low speed centrifuge (model: LD5-2A), purchased from Beijing Lab Centrifuge Co., Ltd.; digimatic caliper (model: SF2000), purchased from Guilin Guanglu Measuring Instrument Co., Ltd.

[0568] 4. Experimental Design

[0569] A human breast cancer MDA-MB-231 subcutaneous transplanted tumor model in nude mice was established, and each animal was inoculated with 1 10.sup.6 cells.

[0570] The following dosages of administration and dosing regimens (Table 3) were designed for the experiment.

TABLE-US-00003 TABLE 3 Antitumor effect in the human breast cancer MDA-MB-231 transplanted tumor model in nude mice Volume of Number of Route of Dosage administration Period of Group animals Sample administration (mg .Math. kg.sup.1) (mL .Math. kg.sup.1) administration 1 5 physiological saline IV N/A 10 Q4D 3 2 5 irinotecan IV 40 10 Q4D 3 3 5 nktr-102 IV 40 10 Q4D 3 4 5 Compound 1 IV 40 10 Q4D 3 5 5 Compound 2 IV 40 10 Q4D 3 6 5 Compound 3 IV 40 10 Q4D 3 7 5 Compound 4 IV 40 10 Q4D 3 8 5 Compound 5 IV 40 10 Q4D 3 9 5 Compound 6 IV 40 10 Q4D 3 10 5 Compound 7 IV 40 10 Q4D 3 11 5 Compound 8 IV 40 10 Q4D 3 12 5 Compound 9 IV 40 10 Q4D 3 13 5 Compound 10 IV 40 10 Q4D 3 14 5 Compound 11 IV 40 10 Q4D 3 15 5 Compound 12 IV 40 10 Q4D 3 16 5 Compound 13 IV 40 10 Q4D 3 17 5 Compound 14 IV 40 10 Q4D 3 18 5 Compound 15 IV 40 10 Q4D 3 19 5 Compound 16 IV 40 10 Q4D 3 20 5 Compound 17 IV 40 10 Q4D 3 21 5 Compound 18 IV 40 10 Q4D 3 22 5 Compound 19 IV 40 10 Q4D 3 23 5 Compound 20 IV 40 10 Q4D 3 24 5 Compound 21 IV N/A 10 Q4D 3 25 5 Compound 22 IV 40 10 Q4D 3 26 5 Compound 23 IV 40 10 Q4D 3 27 5 Compound 24 IV 40 10 Q4D 3 28 5 Compound 25 IV 40 10 Q4D 3 29 5 Compound 26 IV 40 10 Q4D 3 30 5 Compound 27 IV N/A 10 Q4D 3 31 5 Compound 28 IV 40 10 Q4D 3 32 5 Compound 29 IV 40 10 Q4D 3 33 5 Compound 30 IV 40 10 Q4D 3 34 5 Compound 31 IV 40 10 Q4D 3 35 5 Compound 32 IV 40 10 Q4D 3 36 5 Compound 33 IV N/A 10 Q4D 3 37 5 Compound 34 IV 40 10 Q4D 3 38 5 Compound 35 IV 40 10 Q4D 3 39 5 Compound 36 IV 40 10 Q4D 3 40 5 Compound 37 IV 40 10 Q4D 3 41 5 Compound 38 IV 40 10 Q4D 3 42 5 Compound 39 IV N/A 10 Q4D 3 43 5 Compound 40 IV 40 10 Q4D 3 44 5 Compound 41 IV 40 10 Q4D 3 45 5 Compound 42 IV 40 10 Q4D 3

[0571] 5. Preparation of the Dosing Formulations of the Compounds

[0572] The preparation methods were provided by Brightgene Bio-Medical Technology Co., Ltd. Volume required for a single dose:

[0573] 20 g (body weight)10 (animals)'10 mL/kg/10001.5=3 mL

[0574] 5.1 Preparation of the Dosing Formulation of Irinotecan

[0575] 12.0 mg of irinotecan was weighed, 0.15 mL of 1% lactic acid was added, the drug was dissolved completely by vortexing, and 2.85 mL of 1% aqueous sorbitol solution was then added respectively. The mixture was mixed evenly by vortexing, and the ratio of 1% lactic acid to 1% aqueous sorbitol solution was approximately 5:95 (v/v) in the solution. The concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0576] 5.2 Preparation of the Dosing Formulation of nktr-102

[0577] Before each administration, 88.55 mg of nktr-102 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0578] 5.3 Preparation of the Dosing Formulations of Compounds 1 to 5

[0579] Before each administration, 113.85 mg of Compound 1 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0580] Before each administration, 109.80 mg of Compound 2 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0581] Before each administration, 113.88 mg of Compound 3 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0582] Before each administration, 111.24 mg of Compound 4 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg. mL.sup.1.

[0583] Before each administration, 105.78 mg of Compound 5 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0584] 5.4 Preparation of the Dosing Formulations of Compounds 6 to 10

[0585] Before each administration, 113.09 mg of Compound 6 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0586] Before each administration, 109.05 mg of Compound 7 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0587] Before each administration, 113.12 mg of Compound 8 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0588] Before each administration, 110.48 mg of Compound 9 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0589] Before each administration, 105.02 mg of Compound 10 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0590] 5.4 Preparation of the Dosing Formulations of Compounds 11 to 15

[0591] Before each administration, 112.98 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0592] Before each administration, 108.94 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0593] Before each administration, 113.01 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0594] Before each administration, 110.37 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0595] Before each administration, 104.91 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0596] 5.5 Preparation of the Dosing Formulations of Compounds 16 to 20

[0597] Before each administration, 113.09 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0598] Before each administration, 109.05 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0599] Before each administration, 113.12 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0600] Before each administration, 110.48 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0601] Before each administration, 105.02 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0602] 5.6 Preparation of the Dosing Formulations of Compounds 21 to 42

[0603] The preparation methods were as described above, so that the concentration of the free active agent in the solution was 4.0 mg.Math.mL.sup.1.

[0604] 6. Experimental Methods

[0605] MDA-MB-231 cells were cultured in DMEM, which contained 10% fetal bovine serum FBS (GIBCO, USA). The cells were placed in a 37 C. incubator containing 5% CO.sub.2 to cultivate.

[0606] A subcutaneous tumor xenograft model in nude mice was established by cell inoculation method: tumor cells in logarithmic phase of growth were collected, counted and then resuspended in 1xPBS, and the concentration of the cell suspension was adjusted to 110.sup.7/ mL. 1 mL syringes (4 gauge needle) were used to inoculate the tumor cells to the right back of the nude mice subcutaneously, 110.sup.6/0.1 mL/mice.

[0607] When the tumor volumes reached 100 mm.sup.3 to 200 mm.sup.3, the animals were grouped randomly according to randomized block method, so that the difference of the tumor volumes between each group was less than 10% of the mean value. There were 5 animals in each group, Day 0 was the day when the animals were grouped, and the animals were administered on the same day when being grouped.

[0608] The experimental period lasted for 3 weeks, and the body weights and the tumor sizes of the animals were measured twice a week during the experiment. The clinical symptoms were observed and recorded daily. On the last day of the experiment, the animals were sacrificed, the body weights were weighed, and the tumors were excised, weighed and recorded by photographing.

[0609] The operation of all animal experiments strictly adhered to the principles of the use and management of animals. For the calculation of the tumor-related parameters, Technical guidelines for non-clinical research on cytotoxic antitumor drugs by Chinese CFDA was referred.

[0610] The calculation formula of the tumor volume (TV) was: TV=ab.sup.2/2, wherein a and b represented the measured length and width of the tumor, respectively. The calculation formula of the relative tumor volume (RTV) was: RTV=V.sub.t/V.sub.0, wherein V.sub.0 was the tumor volume measured at the time of grouping and first administration, and V.sub.t was the tumor volume at the time of a measurement. The evaluation indexes of the antitumor activity were the relative tumor proliferation rate (T/C (%)) and the tumor inhibition rate (%), and the calculation formulae were T/C(%)=(T.sub.RTV/C.sub.RTV)100%,wherein TRTV was the RTV of a treatment group, and CRTv was the RTV of the negative control group; and tumor inhibition rate (%)=(the average tumor weight of the negative control group - the average tumor weight of a treatment group)/the average tumor weight of the negative control group100%, respectively.

[0611] Body weight change (%) of a tumor-bearing animal was calculated as follows: (the body weight at the time of a measurementthe body weight at the time of grouping)/the body weight at the time of grouping100.

[0612] 7. Data Analysis

[0613] The experimental data was subjected to calculation and related statistical treatments using Microsoft Office Excel 2007 Software. Unless otherwise specified, the data was represented by meanstandard error (Mean SE), and t test was adopted for the comparison between two groups.

[0614] 8. Results and Reports

[0615] According to Technical guidelines for non-clinical research on cytotoxic antitumor drugs (November 2006) by Chinese CFDA, the treatment was considered to be effective when T/C (%) 40% and p<0.05 according to statistical analysis, as shown in Table 4.

TABLE-US-00004 TABLE 4 Relative tumor proliferation rate T/C (%) Relative Relative tumor Number of tumor volume proliferation Group Sample animals (RTV) rate (% T/C) 1 blank solvent 5 12.56 2 irinotecan 5 6.19 44.8%.sup. 3 nktr-102 5 4.46 32.2%.sup. 4 Compound 1 5 1.28* 9.3%.sup.# 5 Compound 2 5 1.66* 12.1%.sup.# 6 Compound 3 5 1.89* 13.65%.sup.# 7 Compound 4 5 1.59* 11.5%.sup.# 8 Compound 5 5 1.97* 14.3%.sup.# 9 Compound 6 5 1.34* 9.7%.sup.# 10 Compound 7 5 1.80* 13.1%.sup.# 11 Compound 8 5 2.03* 14.8%.sup.# 12 Compound 9 5 1.97* 14.3%.sup.# 13 Compound 10 5 1.40* 10.2%.sup.# 14 Compound 11 5 1.88* 13.6%.sup.# 15 Compound 12 5 1.65* 11.9.sup.# 16 Compound 13 5 1.60* 11.6%.sup.# 17 Compound 14 5 1.53* 11.10%.sup.# 18 Compound 15 5 1.40* 10.8%.sup.# 19 Compound 16 5 1.79* 13.0%.sup.# 20 Compound 17 5 1.59* 11.5%.sup.# 21 Compound 18 5 1.75* 12.7%.sup.# 22 Compound 19 5 1.77* 12.8%.sup.# 23 Compound 20 5 1.51* 11.0%.sup.# 24 Compound 21 5 1.38* 10.0%.sup.# 25 Compound 22 5 1.52* 11.0%.sup.# 26 Compound 23 5 1.29* 9.3%.sup.# 27 Compound 24 5 1.49* 10.8%.sup.# 28 Compound 25 5 1.73* 12.6%.sup.# 29 Compound 26 5 1.64* 10.3%.sup.# 30 Compound 27 5 1.67* 12.1%.sup.# 31 Compound 28 5 1.57* 11.4%.sup.# 32 Compound 29 5 1.72* 12.5%.sup.# 33 Compound 30 5 1.49* 10.8%.sup.# 34 Compound 31 5 1.52* 11.0%.sup.# 35 Compound 32 5 1.87* 13.6%.sup.# 36 Compound 33 5 1.63* 11.8%.sup.# 37 Compound 34 5 1.50* 9.7%.sup.# 38 Compound 35 5 1.46* 10.6%.sup.# 39 Compound 36 5 1.73* 12.6%.sup.# 40 Compound 37 5 1.41* 10.2%.sup.# 41 Compound 38 5 1.54* 11.2%.sup.# 42 Compound 39 5 1.73* 12.6%.sup.# 43 Compound 40 5 1.87* 13.6%.sup.# 44 Compound 41 5 1.69* 12.3%.sup.# 45 Compound 42 5 1.76* 12.8%.sup.# *P < 0.05, as compared with the RTV of the blank solvent group, irinotecan group and nktr-102 group. .sup.#P < 0.05, as compared with % T/C of the blank solvent group, irinotecan group and nktr-102 group.

[0616] The experimental results showed that the compounds of the present disclosure had good inhibitory effects on human breast cancer MDA-MB-231 transplanted tumor in nude mice, and were superior to irinotecan and nktr-102.

Example 45

Inhibitory Effect in a Human Pancreatic Cancer MIA Paca-2 Xenograft model in Nude Mice 1. Experimental Purpose

[0617] The human pancreatic cancer MIA Paca-2 transplanted tumor model in nude mice was used in the study to evaluate the in vivo antitumor activities of Compounds 1 to 42.

[0618] 2. Experimental Animals

[0619] 2.1 Animal Species

[0620] Mice.

[0621] 2.2 Strain

[0622] BALB/c-nu/nu nude mice.

[0623] 2.3 Sex

[0624] Female.

[0625] 2.4 Number

[0626] 300.

[0627] 2.5. Age

[0628] 6 to 8 week old.

[0629] 2.6. Body weight

[0630] 20 to 22 g 20% of the average body weight.

[0631] 2.7. Animal Source (Supplier)

[0632] Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. (BK), license number: SCXK (Shanghai) 2008-0016.

[0633] 2.8. Management of the Experimental Animals

[0634] All experimental animals were housed in an SPF-level laboratory.

[0635] 2.8.1 Identification Method of Animal Identity

[0636] Each mice cage had an identification card with information such as the experimental number, experimental group, name(s) of experimenter(s), and the strain and sex of mice. The mice were marked with earrings.

[0637] 2.8.2 Random grouping After the tumor volumes reached 150 mm.sup.3 to 200 mm.sup.3, the animals were grouped using randomized block method with 5 mice in each group, ensuring the tumor volumes and the body weights of the mice were uniform between each group. The difference between the mean value of the tumor volumes in each group and the mean value of the tumor volumes of all experimental animals was no more than 10%. 2.8.3 Operating and managing practice

[0638] The operation and management of all experimental animals strictly adhered to the guideline of the use and management of experimental animals.

[0639] 2.8.4 Feeding Conditions

[0640] Living conditions: IVC system, 5 animals per cage

[0641] Temperature: 25 C.1 C.

[0642] Humidity: 65%10%

[0643] Illumination: 12 h light/dark cycle

[0644] 2.8.5 Feed

[0645] Irradiated feed for rat and mice, purchased from Beijing Keao Xieli Feed Co., Ltd. The animals had free access to food.

[0646] 2.8.6 Drinking Water

[0647] City tap water, used for drinking after being filtered and autoclaved.

[0648] 2.8.7 Bedding

[0649] Corn cobs (Shanghai Maoshengyan Biologic Science & Technology Co., Ltd.) were used after being autoclaved. Beddings were changed twice a week.

[0650] 2.8.8 Acclimation Period

[0651] The mice were given at least one week of acclimation period for the environment before the experiment.

[0652] 3. Experimental Materials

[0653] 3.1 Test Drugs

[0654] Irinotecan (bulk drug) and nktr-102 (bulk drug) were obtained by purchasing, and Compounds 1 to 42 were all provided by Brightgene Bio-Medical Technology Co., Ltd.

[0655] 3.2 Other Chemical Reagents and Materials

[0656] 3.2.1 Physiological Saline

[0657] Physiological saline was purchased from Shanghai Huayuan Changfu Pharmaceutical (Group) Co., Ltd. (Shanghai, China).

[0658] 3.2.2 Sterile Syringe

[0659] 1 mL sterile syringes were purchased from Shanghai Kindly Enterprise Development Group Co., Ltd (Shanghai, China).

[0660] 3.2.3 Cell Strain

[0661] Human pancreatic cancer MIA Paca-2 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences.

[0662] MIA Paca-2 cells were cultured in DMEM culture medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and 2.5% HS, and cultured in a 37 C. incubator containing 5% CO.sub.2.

[0663] 3.2.4 Matrigel (BD Matrigel)

[0664] Matrigel was purchased from Becton, Dickinson and Company (BD), the USA.

[0665] 3.3 Instruments

[0666] Biosafe cabinet (model: AC2-6E1), purchased from ESCO;

[0667] water jacketed CO2 cell incubator (model: 3111), purchased from Thermo Scientific Forma;

[0668] inverted microscope (model: CKX41SF), purchased from Olympus;

[0669] wlectric suction apparatus (model: YX930D), purchased from Shanghai Medical Instruments (Group) Co., Ltd.;

[0670] balance (METTLER-TOLEDOAB135-S), purchased from METTLER-TOLEDO;

[0671] low speed centrifuge (model: LD5-2A), purchased from Beijing Lab Centrifuge Co., Ltd.;

[0672] digimatic caliper (model: SF2000), purchased from Guilin Guanglu Measuring Instrument Co., Ltd.

[0673] 4. Experimental Design

[0674] A human pancreatic cancer MIA Paca-2 subcutaneous transplanted tumor model in nude mice was established, and each animal was inoculated with 310.sup.6 cells.

[0675] The following dosages of administration and dosing regimen were designed for the experiment (Table 5).

TABLE-US-00005 TABLE 5 Antitumor effect in the human pancreatic cancer MIA Paca-2 transplanted tumor model in nude mice Tumor cell strain: MIA Paca-2; a total of 225 animals were inoculated. Volume of Number of Route of Dosage administration Period of Group animals Sample administration (mg .Math. kg.sup.1) (mL .Math. kg.sup.1) administration 1 5 physiological saline IV N/A 10 Q4D 3 2 5 irinotecan IV 40 10 Q4D 3 3 5 nktr-102 IV 40 10 Q4D 3 4 5 Compound 1 IV 40 10 Q4D 3 5 5 Compound 2 IV 40 10 Q4D 3 6 5 Compound 3 IV 40 10 Q4D 3 7 5 Compound 4 IV 40 10 Q4D 3 8 5 Compound 5 IV 40 10 Q4D 3 9 5 Compound 6 IV 40 10 Q4D 3 10 5 Compound 7 IV 40 10 Q4D 3 11 5 Compound 8 IV 40 10 Q4D 3 12 5 Compound 9 IV 40 10 Q4D 3 13 5 Compound 10 IV 40 10 Q4D 3 14 5 Compound 11 IV 40 10 Q4D 3 15 5 Compound 12 IV 40 10 Q4D 3 16 5 Compound 13 IV 40 10 Q4D 3 17 5 Compound 14 IV 40 10 Q4D 3 18 5 Compound 15 IV 40 10 Q4D 3 19 5 Compound 16 IV 40 10 Q4D 3 20 5 Compound 17 IV 40 10 Q4D 3 21 5 Compound 18 IV 40 10 Q4D 3 22 5 Compound 19 IV 40 10 Q4D 3 23 5 Compound 20 IV 40 10 Q4D 3 24 5 Compound 21 IV N/A 10 Q4D 3 25 5 Compound 22 IV 40 10 Q4D 3 26 5 Compound 23 IV 40 10 Q4D 3 27 5 Compound 24 IV 40 10 Q4D 3 28 5 Compound 25 IV 40 10 Q4D 3 29 5 Compound 26 IV 40 10 Q4D 3 30 5 Compound 27 IV N/A 10 Q4D 3 31 5 Compound 28 IV 40 10 Q4D 3 32 5 Compound 29 IV 40 10 Q4D 3 33 5 Compound 30 IV 40 10 Q4D 3 34 5 Compound 31 IV 40 10 Q4D 3 35 5 Compound 32 IV 40 10 Q4D 3 36 5 Compound 33 IV N/A 10 Q4D 3 37 5 Compound 34 IV 40 10 Q4D 3 38 5 Compound 35 IV 40 10 Q4D 3 39 5 Compound 36 IV 40 10 Q4D 3 40 5 Compound 37 IV 40 10 Q4D 3 41 5 Compound 38 IV 40 10 Q4D 3 42 5 Compound 39 IV N/A 10 Q4D 3 43 5 Compound 40 IV 40 10 Q4D 3 44 5 Compound 41 IV 40 10 Q4D 3 45 5 Compound 42 IV 40 10 Q4D 3

[0676] 5. Preparation of the Dosing Formulations of the Compounds

[0677] The preparation methods were provided by Brightgene Bio-Medical Technology Co., Ltd.

[0678] Volume required for a single dose:

[0679] 20 g (body weight)10 (animals)10 mL/kg/10001.5=3 mL

[0680] 5.1 Preparation of the Dosing Formulation of Irinotecan

[0681] 12.0 mg of irinotecan was weighed, 0.15 mL of 1% lactic acid was added, the drug was dissolved completely by vortexing, and 2.85 mL of 1% aqueous sorbitol solution was then added respectively. The mixture was mixed evenly by vortexing, and the ratio of 1% lactic acid to 1% aqueous sorbitol solution was approximately 5:95 (v/v) in the solution. The concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0682] 5.2 Preparation of the Dosing Formulation of nktr-102

[0683] Before each administration, 88.55 mg of nktr-102 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0684] 5.3 Preparation of the Dosing Formulations of Compounds 1 to 5

[0685] Before each administration, 113.85 mg of Compound 1 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0686] Before each administration, 109.80 mg of Compound 2 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0687] Before each administration, 113.88 mg of Compound 3 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0688] Before each administration, 111.24 mg of Compound 4 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0689] Before each administration, 105.78 mg of Compound 5 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0690] 5.4 Preparation of the Dosing Formulations of Compounds 6 to 10

[0691] Before each administration, 113.09 mg of Compound 6 was weighed accurately 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0692] Before each administration, 109.05 mg of Compound 7 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0693] Before each administration, 113.12 mg of Compound 8 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0694] Before each administration, 110.48 mg of Compound 9 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0695] Before each administration, 105.02 mg of Compound 10 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0696] 5.5 Preparation of the Dosing Formulations of Compounds 11 to 15

[0697] Before each administration, 112.98 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0698] Before each administration, 108.94 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0699] Before each administration, 113.01 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0700] Before each administration, 110.37 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0701] Before each administration, 104.91 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0702] 5.6 Preparation of the Dosing Formulations of Compounds 16 to 20

[0703] Before each administration, 113.09 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0704] Before each administration, 109.05 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0705] Before each administration, 113.12 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0706] Before each administration, 110.48 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0707] Before each administration, 105.02 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0708] 5.7 Preparation of the Dosing Formulations of Compounds 21 to 42

[0709] The preparation methods were as described above, so that the concentration of the free active agent in the solution was 4.0 mg. mL.sup.1.

[0710] 6. Experimental Methods

[0711] MIA Paca-2 cells were cultured in DMEM, which contained 10% fetal bovine serum FBS (GIBCO, USA) and 2.5% HS. The cells were placed in a 37 C. incubator containing 5% CO.sub.2 to cultivate.

[0712] A subcutaneous tumor xenograft model in nude mice was established by cell inoculation method: tumor cells in logarithmic phase of growth were collected, counted and then resuspended in 1PBS, and the concentration of the cell suspension was adjusted to 310.sup.7/mL. 1 mL syringes (4 gauge needle) were used to inoculate the tumor cells to the right back of the nude mice subcutaneously, 310.sup.6/0.1 mL/mice.

[0713] When the tumor volumes reached 100 mm.sup.3 to 200 mm.sup.3, the animals were grouped randomly according to randomized block method, so that the difference of the tumor volumes between each group was less than 10% of the mean value. There were 5 animals in each group, Day 0 was the day when the animals were grouped, and the animals were administered on the same day when being grouped.

[0714] The experimental period lasted for 3 weeks, and the body weights and the tumor sizes of the animals were measured twice a week during the experiment. The clinical symptoms were observed and recorded daily. On the last day of the experiment, the animals were sacrificed, the body weights were weighed, and the tumors were excised, weighed and recorded by photographing.

[0715] The operation of all animal experiments strictly adhered to the principles of the use and management of animals. For the calculation of the tumor-related parameters, Technical guidelines for non-clinical research on cytotoxic antitumor drugs by Chinese SFDA was referred.

[0716] The calculation formula of the tumor volume (TV) was: TV=ab.sup.2/2, wherein a and b represented the measured length and width of the tumor, respectively. The calculation formula of the relative tumor volume (RTV) was: RTV=V.sub.i/V.sub.0, wherein V.sub.0 was the tumor volume measured at the time of grouping and first administration, and V.sub.i was the tumor volume at the time of a measurement. The evaluation indexes of the antitumor activity were the relative tumor proliferation rate (T/C (%)) and the tumor inhibition rate (%), the calculation formulae were T/C (%)=(T.sub.RTV/C.sub.RTV)100%, wherein TRTV was the RTV of a treatment group, and C.sub.RTV was the RTV of the negative control group; and tumor inhibition rate (%)=(the average tumor weight of the negative control groupthe average tumor weight of a treatment group)/the average tumor weight of the negative control group100%, respectively.

[0717] Body weight change (%) of a tumor-bearing animal was calculated as follows: (the body weight at the time of a measurementthe body weight at the time of grouping)/the body weight at the time of grouping100.

[0718] According to Technical guidelines for non-clinical research on cytotoxic antitumor drugs (November 2016) by Chinese SFDA, the treatment was considered to be effective when T/C (%) 40% and P<0.05 according to statistical analysis.

[0719] 7. Data Analysis

[0720] The experimental data was subjected to calculation and related statistical treatments using Microsoft Office Excel 2007 Software. Unless otherwise specified, the data was represented by mean standard error (MeanSE), t test was adopted for the comparison between groups, and P<0.05 was considered as a significant difference.

[0721] 8. Results and Reports

[0722] According to Technical guidelines for non-clinical research on cytotoxic antitumor drugs (Nov 2006) by Chinese CFDA, the treatment was considered to be effective when T/C (%) 40% and P<0.05 according to statistical analysis, as shown in Table 6.

TABLE-US-00006 TABLE 6 Relative tumor proliferation rate T/C (%) Relative Relative tumor Number of tumor volume proliferation Group Sample animals (RTV) rate (% T/C) 1 blank solvent 5 11.95 2 irinotecan 5 7.79 59.4% 3 nktr-102 5 5.42 37.5% 4 Compound 1 5 2.21* 12.1%.sup.# 5 Compound 2 5 2.65* 14.5%.sup.# 6 Compound 3 5 2.57* 13.3%.sup.# 7 Compound 4 5 2.06* 10.6%.sup.# 8 Compound 5 5 2.68* 13.8%.sup.# 9 Compound 6 5 3.21* 16.6%.sup.# 10 Compound 7 5 2.25* 11.6%.sup.# 11 Compound 8 5 2.48* 12.8%.sup.# 12 Compound 9 5 2.98* 15.3%.sup.# 13 Compound 10 5 2.68* 13.8%.sup.# 14 Compound 11 5 2.36* 12.1%.sup.# 15 Compound 12 5 2.48* 12.71.sup.# 16 Compound 13 5 2.73* 14.0%.sup.# 17 Compound 14 5 2.24* 11.5%.sup.# 18 Compound 15 5 2.69* 13.8%.sup.# 19 Compound 16 5 3.23* 16.6%.sup.# 20 Compound 17 5 2.75* 14.1%.sup.# 21 Compound 18 5 2.48* 12.7%.sup.# 22 Compound 19 5 2.78* 14.2%.sup.# 23 Compound 20 5 2.67* 13.6%.sup.# 24 Compound 21 5 2.49* 12.1%.sup.# 25 Compound 22 5 2.73* 14.9%.sup.# 26 Compound 23 5 2.39* 13.1%.sup.# 27 Compound 24 5 2.84* 15.5%.sup.# 28 Compound 25 5 3.07* 16.8%.sup.# 29 Compound 26 5 2.92* 16.0%.sup.# 30 Compound 27 5 2.59* 14.2%.sup.# 31 Compound 28 5 2.67* 14.6%.sup.# 32 Compound 29 5 2.74* 15.0%.sup.# 33 Compound 30 5 2.68* 14.7%.sup.# 34 Compound 31 5 2.43* 13.3%.sup.# 35 Compound 32 5 2.79* 15.3%.sup.# 36 Compound 33 5 3.13* 17.1%.sup.# 37 Compound 34 5 2.74* 15.0%.sup.# 38 Compound 35 5 2.57* 14.1%.sup.# 39 Compound 36 5 2.65* 14.5%.sup.# 40 Compound 37 5 2.76* 15.1%.sup.# 41 Compound 38 5 2.91* 15.9%.sup.# 42 Compound 39 5 2.73* 14.9%.sup.# 43 Compound 40 5 2.93* 16.0%.sup.# 44 Compound 41 5 2.84* 15.5%.sup.# 45 Compound 42 5 2.91* 15.9%.sup.# *P < 0.05, as compared with the RTV of the blank solvent group, irinotecan group and nktr-102 group. .sup.#P < 0.05, as compared with % T/C of the blank solvent group, irinotecan group and nktr-102 group.

[0723] The experimental results showed that the compounds of the present disclosure had good inhibitory effects on the human pancreatic cancer MIA Paca-2 transplated tumor in nude mice, and were superior to irinotecan and nktr-102.

Example 46

Inhibitory Effect on In Vivo Tumor Growth in a Human Gastric Cancer NCI-N87 Cell Strain Transplanted Model in Nude Mice

[0724] 1. Experimental Purpose

[0725] The inhibitory effects of the test compounds 1 to 42 on in vivo tumor growth in the human gastric cancer NCI-N87 cell strain transplanted tumor model in nude mice were evaluated.

[0726] 2. Experimental Materials

[0727] 2.1 Test Articles

[0728] Irinotecan (bulk drug), SN-38, 10-hydroxycamptothecin, rubitecan, and nktr-102 were obtained by purchasing, and Compounds 1 to 42 were all provided by Brightgene Bio-Medical Technology Co., Ltd.

[0729] 2.2 Reagents

[0730] RPMI-1640 liquid culture medium, fetal bovine serum (FBS), trypsin, penicillin-streptomycin, and physiological saline.

[0731] 2.3 Experimental Animals

[0732] Female BALB/c nude mice (number of animals: 150, weeks of age: 6 to 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housed in an SPF animal room in Suzhou Shengsu New Drug Development Co., Ltd., the temperature was 20 C. to 25 C., the relative humidity was 40% to 70%, and the light and dark were respectively 12 h. The animals had free access to water and food. After about one week of normal feeding, upon a veterinary inspection, mice with good physical signs and conditions could be selected for the experiment. A marking pen was used to mark the base of the tails of the animals before the grouping, and each animal was marked by ear clipping after the grouping.

[0733] 2.4 Transplantable Tumor Cell Strain

[0734] Human gastric cancer cell NCI-N87, derived from Cell Bank of Committee on Type Culture Collection of Chinese Academy of Science (CAS, stored frozen in liquid nitrogen in the laboratory).

[0735] 3 Experimental Methods

[0736] 3.1 Culture of NCI-N87 Cells

[0737] Under a culture condition of 5% CO.sub.2 and 37 C., NCI-N87 cells were subjected to conventional cell cultivation in RPMI-1640 liquid culture medium containing 10% fetal bovine serum, digested with 0.25% trypsin, and passaged. According to the condition of cell growth, the cells were passaged once or twice every week, and were passaged in a ratio of 1:2 to 1:6.

3.2 Preparation of the Animal Model

[0738] NCI-N87 cells in logarithmic phase of growth were collected. The cells were resuspended in RPMI-1640 culture medium free of serum after being counted, and the concentration of the cells was adjusted to 510.sup.7 cells/mL. The cells were charged into a 50 mL centrifuge tube after being pipetted with a pipettor to make them evenly dispersed, and the centrifuge tube was placed in an ice box. Cell suspension was aspirated with a 1 mL syringe, and injected subcutaneously to the anterior right armpit of the nude mice. Each animal was inoculated with 100 L (510.sup.6 cells/animal), and the NCI-N87 transplanted tumor model in nude mice was established. Animal status and the condition of tumor growth were observed regularly after inoculation, an electronic vernier caliper was used to measure the tumor diameters, the data was directly input to an Excel spreadsheet, and the tumor volumes were calculated. When the tumor volumes reached 100 mm.sup.3 to 300 mm.sup.3, 225 animals with good health condition and similar tumor volume were selected and divided into 45 groups using randomized block method (n=5). The tumor diameters were measured twice a week after the initiation of the experiment, the tumor volumes were calculated, and the body weights of the animals were weighed and recorded at the same time.

[0739] The calculation formula of the tumor volume (TV) was as follows:

TV(mm.sup.3)=lw.sup.2/2

wherein l represented the long diameter of a tumor (mm), and w represented the short diameter of a tumor (mm).

[0740] 3.3 Preparation of the Solvent

[0741] 0.5 g of sorbitol was weighed and charged into a 50 mL centrifuge tube, 50 mL of water for injection was added to the centrifuge tube, and the solid substance was dissolved completely by vortexing, so that an aqueous sorbitol solution (w/v) with a concentration of 1% was formulated. The solution was stored in a refrigerator at 4 C. until use.

[0742] 3.4 Preparation of the Dosing Formulations

[0743] 3.4.1 Preparation of the Dosing Formulation of Irinotecan

[0744] 12.0 mg of irinotecan was weighed, 0.15 mL of 1% lactic acid was added, the drug was dissolved completely by vortexing, and 2.85 mL of 1% aqueous sorbitol solution was then added respectively. The mixture was mixed evenly by vortexing, and the ratio of 1% lactic acid to 1% aqueous sorbitol solution was approximately 5:95 (v/v) in the solution. The concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0745] 3.4.2 Preparation of the Dosing Formulation of nktr-102

[0746] Before each administration, 88.55 mg of nktr-102 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0747] 3.4.3 Preparation of the Dosing Formulations of Compounds 1 to 5

[0748] Before each administration, 113.85 mg of Compound 1 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0749] Before each administration, 109.80 mg of Compound 2 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0750] Before each administration, 113.88 mg of Compound 3 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0751] Before each administration, 111.24 mg of Compound 4 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0752] Before each administration, 105.78 mg of Compound 5 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0753] 3.4.4 Preparation of the Dosing Formulations of Compounds 6 to 10

[0754] Before each administration, 113.09 mg of Compound 6 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0755] Before each administration, 109.05 mg of Compound 7 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0756] Before each administration, 113.12 mg of Compound 8 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0757] Before each administration, 110.48 mg of Compound 9 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0758] Before each administration, 105.02 mg of Compound 10 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0759] 3.4.5 Preparation of the Dosing Formulations of Compounds 11 to 15

[0760] Before each administration, 112.98 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0761] Before each administration, 108.94 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0762] Before each administration, 113.01 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0763] Before each administration, 110.37 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0764] Before each administration, 104.91 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0765] 3.4.6 Preparation of the Dosing Formulations of Compounds 16 to 20

[0766] Before each administration, 113.09 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0767] Before each administration, 109.05 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0768] Before each administration, 113.12 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0769] Before each administration, 110.48 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0770] Before each administration, 105.02 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0771] 3.4.7 Preparation of the Dosing Formulations of Compounds 21 to 42

[0772] The preparation methods were as described above, so that the concentration of the free active agent in the solution was 4.0 mg.Math.mL.sup.1.

[0773] 3.5 Grouping of Animals and Administration

[0774] The grouping of animals and the dosing regimens were as shown in Table 7.

[0775] Groups with irinotecan as the active agent (Groups 1 to 8). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Group 1 was a solvent control group, and was given blank solvent by intravenous injection via tail once every 4 days for a total of 3 times (Q4D3). Groups 2 to 8 were given irinotecan, nktr-102 and Compounds 1 to 5 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of irinotecan), Q4D3.

[0776] Groups with SN-38 as the active agent (Groups 9 to 13). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 9 to 13 were given Compounds 6 to 10 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of SN-38), Q4D3.

[0777] Groups with 10-hydroxycamptothecin as the active agent (Groups 14 to 18). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 14 to 18 were given Compounds 11 to 15 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of 10-hydroxycamptothecin), Q4D3.

[0778] Groups with rubitecan as the active agent (Groups 19 to 23). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 19 to 23 were given Compounds 16 to 20 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of rubitecan), Q4D3.

[0779] The modes of administration of Groups 24 to 25 were as described above.

TABLE-US-00007 TABLE 7 Dosing regimens of the pharmacodynamic experiments in transplanted tumor model in nude mice Volume of Number Dosage administration Route of Period of Group Sample of animals (mg .Math. kg.sup.1) (mL .Math. kg.sup.1) administration administration 1 blank solvent 5 10 IV Q4D 3 2 irinotecan 5 40 10 IV Q4D 3 3 nktr-102 5 40 10 IV Q4D 3 4 Compound 1 5 40 10 IV Q4D 3 5 Compound 2 5 40 10 IV Q4D 3 6 Compound 3 5 40 10 IV Q4D 3 7 Compound 4 5 40 10 IV Q4D 3 8 Compound 5 5 40 10 IV Q4D 3 9 Compound 6 5 40 10 IV Q4D 3 10 Compound 7 5 40 10 IV Q4D 3 11 Compound 8 5 40 10 IV Q4D 3 12 Compound 9 5 40 10 IV Q4D 3 13 Compound 10 5 40 10 IV Q4D 3 14 Compound 11 5 40 10 IV Q4D 3 15 Compound 12 5 40 10 IV Q4D 3 16 Compound 13 5 40 10 IV Q4D 3 17 Compound 14 5 40 10 IV Q4D 3 18 Compound 15 5 40 10 IV Q4D 3 19 Compound 16 5 40 10 IV Q4D 3 20 Compound 17 5 40 10 IV Q4D 3 21 Compound 18 5 40 10 IV Q4D 3 22 Compound 19 5 40 10 IV Q4D 3 23 Compound 20 5 40 10 IV Q4D 3 24 Compound 21 5 40 10 IV Q4D 3 25 Compound 22 5 40 10 IV Q4D 3 26 Compound 23 5 40 10 IV Q4D 3 27 Compound 24 5 40 10 IV Q4D 3 28 Compound 25 5 40 10 IV Q4D 3 29 Compound 26 5 40 10 IV Q4D 3 30 Compound 27 5 40 10 IV Q4D 3 31 Compound 28 5 40 10 IV Q4D 3 32 Compound 29 5 40 10 IV Q4D 3 33 Compound 30 5 40 10 IV Q4D 3 34 Compound 31 5 40 10 IV Q4D 3 35 Compound 32 5 40 10 IV Q4D 3 36 Compound 33 5 40 10 IV Q4D 3 37 Compound 34 5 40 10 IV Q4D 3 38 Compound 35 5 40 10 IV Q4D 3 39 Compound 36 5 40 10 IV Q4D 3 40 Compound 37 5 40 10 IV Q4D 3 41 Compound 38 5 40 10 IV Q4D 3 42 Compound 39 5 40 10 IV Q4D 3 43 Compound 40 5 40 10 IV Q4D 3 44 Compound 41 5 40 10 IV Q4D 3 45 Compound 42 5 40 10 IV Q4D 3

[0780] 3.6 End of the Experiment

[0781] After the experiment was ended, the animals were euthanized (CO.sub.2) after the body weights were weighed and the tumor diameters were measured. The tumor tissues were excised and weighed, and the tumor weight inhibition rates were calculated. After being weighed, the tumor tissues were transferred to a refrigerator below 70 C. for subsequent analysis.

[0782] 4. Data Recording and Calculation Formulas

[0783] The calculation formula of the relative tumor volume (RTV) was:

RTV=TV.sub.t/TV.sub.initial

[0784] wherein TV.sub.initial was the tumor volume measured at the time of grouping and first administration, and TV.sub.t was the tumor volume at each measurement during administration.

[0785] The calculation formula of the relative tumor proliferation rate (% T/C) was:

% T/C=100%(RTV.sub.T/RTV.sub.C)

[0786] wherein RTV.sub.T represented the RTV of a treatment group, and RTV.sub.C represented the RTV of the solvent control group.

[0787] The calculation formula of the tumor growth inhibition rate (TGI (%)) was:

TGI=100%[1(TV.sub.t(T)TV.sub.initial(T))/(TV.sub.t(C)TV.sub.initial(C))]

[0788] wherein TV.sub.t(T) represented the tumor volume of a treatment group at each measurement, TV.sub.initial(T) represented the tumor volume of a treatment group measured at the time of grouping and first administration, TV.sub.t(C) represented the tumor volume of the solvent control group at each measurement, and TV.sub.initial(C) represented the tumor volume of the solvent control group measured at the time of grouping and first administration.

[0789] The calculation formula of the decline rate of animal body weight was:

decline rate of animal body weight=100%(BW.sub.initialBW.sub.t)/BW.sub.initial

[0790] wherein BW.sub.t represented the animal body weight at each measurement during administration, and BW.sub.initial represented the animal body weight at the time of grouping and first administration.

[0791] The calculation formula of the tumor weight inhibition rate (IR (%)) was:

IR(%)=100%(W.sub.CW.sub.T)/W.sub.C

[0792] wherein W.sub.C represented the tumor weight of the control group, and W.sub.T represented the tumor weight of a treatment group.

[0793] 5. Method for Statistical Analysis

[0794] The experimental data was subjected to calculation and related statistical treatments using Microsoft Office Excel 2007 Software. Unless otherwise specified, the data was represented by mean standard error (MeanSE), and t test was adopted for the comparison between two groups.

[0795] 6. Experimental Observation

[0796] During the experiment, experimenter(s) and veterinarian(s) needed to continuously observe the physical signs and health status of the experimental animals. Any abnormal performance of the animals, such as pain, depression, and decreased activity, should be recorded in the original record of the experiment. If the abnormal performance of the experimental animals exceeded the provisions of the IACUC-related animal welfare documents, the veterinarian(s) could judge whether to suspend the experiment, and notify the person in charge of the experimental project.

[0797] 7. Results

[0798] As for the transplanted tumor models of human cancer, the relative tumor proliferation rate T/C (%) was recommended to be adopted as the evaluation index of the experiment. Lower proliferation rate indicated better tumor inhibitory effect, as shown in Table 8.

TABLE-US-00008 TABLE 8 Relative tumor proliferation rate T/C (%) Number Relative Average relative of tumor volume tumor proliferation Group Sample animals (RTV, mm.sup.3) rate (% T/C) 1 Blank solvent 5 2.98 2 irinotecan 5 7.78 63.2% 3 nktr-102 5 6.03 50.0% 4 Compound 1 5 3.62 30.1% 5 Compound 2 5 3.08* 25.6%.sup.# 6 Compound 3 5 3.39* 28.2%.sup.# 7 Compound 4 5 2.92* 24.3%.sup.# 8 Compound 5 5 3.80* 31.6%.sup.# 9 Compound 6 5 3.61* 30.0%.sup.# 10 Compound 7 5 3.18* 26.4%.sup.# 11 Compound 8 5 3.63* 30.1%.sup.# 12 Compound 9 5 3.20* 27.1%.sup.# 13 Compound 10 5 2.98* 25.2%.sup.# 14 Compound 11 5 3.28* 27.7%.sup.# 15 Compound 12 5 3.94* 33.2.sup.# 16 Compound 13 5 2.76* 23.2%.sup.# 17 Compound 14 5 3.31* 27.8%.sup.# 18 Compound 15 5 3.48* 29.2%.sup.# 19 Compound 16 5 3.06* 25.7%.sup.# 20 Compound 17 5 3.37* 28.3%.sup.# 21 Compound 18 5 3.23* 27.2%.sup.# 22 Compound 19 5 3.72* 31.3%.sup.# 23 Compound 20 5 3.46* 29.1%.sup.# 24 Compound 21 5 3.14 26.1% 25 Compound 22 5 3.39 28.2% 26 Compound 23 5 3.29 27.4% 27 Compound 24 5 2.94 24.4% 28 Compound 25 5 3.07 25.5% 29 Compound 26 5 3.36 27.9% 30 Compound 27 5 3.63 30.1% 31 Compound 28 5 2.97 24.7% 32 Compound 29 5 3.15 26.2% 33 Compound 30 5 3.38 28.1% 34 Compound 31 5 3.27 27.2% 35 Compound 32 5 3.21 26.7% 36 Compound 33 5 3.08 25.6% 37 Compound 34 5 3.57 29.7% 38 Compound 35 5 3.18 26.4% 39 Compound 36 5 3.21 26.7% 40 Compound 37 5 3.03 25.2% 41 Compound 38 5 3.12 25.9% 42 Compound 39 5 3.42 28.4% 43 Compound 40 5 3.74 31.1% 44 Compound 41 5 3.56 29.6% 45 Compound 42 5 3.33 27.7% *P < 0.05, as compared with the RTV of the blank solvent group, irinotecan group and nktr-102 group. .sup.#P < 0.05, as compared with % T/C of the blank solvent group, irinotecan group and nktr-102 group.

[0799] The experimental results showed that the compounds of the present disclosure had good inhibitory effects on tumor growth in the human gastric cancer NCI-N87 cell strain transplanted tumor model in nude mice, and were superior to irinotecan and nktr-102.

EXAMPLE 47

Effect on the Survival Rate of an Orthotopic U87MG Brain Glioma Model in Nude Mice

[0800] 1. Experimental Purpose

[0801] The effects of the test compounds 1 to 42 on the survival rate of the orthotopic U87MG brain glioma model in nude mice were evaluated.

[0802] 2. Experimental Materials

[0803] 2.1 Test Articles

[0804] Irinotecan (bulk drug) and nktr-102 (bulk drug) were obtained by purchasing, and Compounds 1 to 42 were all provided by Brightgene Bio-Medical Technology Co., Ltd.

[0805] 2.2 Reagents

[0806] RPMI-1640 liquid culture medium, trypsin, penicillin-streptomycin, and physiological saline.

[0807] 2.3 Experimental Animals

[0808] Female BALB/c nude mice (number of animals: 300, weeks of age: 6 to 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housed in an SPF animal room, the temperature was 20 C. to 25 C., the relative humidity was 40% to 70%, and the light and dark were respectively 12 h. The animals had free access to water and food. After about one week of normal feeding, upon a veterinary inspection, mice with good physical signs and conditions could be selected for the experiment. A marking pen was used to mark the base of the tails of the animals before the grouping, and each animal was marked by ear clipping after the grouping.

[0809] 2.4 Transplantable Tumor Cell Line

[0810] Brain glioma cell U87MG, derived from Cell Bank of Committee on Type Culture Collection of Chinese Academy of Science (CAS, stored frozen in liquid nitrogen in the laboratory).

[0811] 3. Experimental Methods

[0812] Culture of U87MG Cells

[0813] Under a culture condition of 5% CO.sub.2 and 37 C., U87MG cells were subjected to conventional cell cultivation in RPMI-1640 liquid culture medium, digested with 0.25% trypsin, and passaged. According to the condition of cell growth, the cells were passaged once or twice every week, and were passaged in a ratio of 1:2 to 1:6.

[0814] 3.1 Preparation of the Animal Model

[0815] U87MG cells in logarithmic phase of growth were collected. The cells were resuspended in RPMI-1640 culture medium free of serum after being counted, and the concentration of the cells was adjusted to 1 10.sup.8 cells/mL. The cells were charged into a 50 mL centrifuge tube after being pipetted with a pipettor to make them evenly dispersed, and the centrifuge tube was placed in an ice box. Cell suspension was aspirated with a 1 mL syringe, 1 L of human brain glioma cell (U87MG cells) cultured in vitro was inoculated (110.sup.5 cells/animal) using microinjection method via the guidance of a stereotactic apparatus for animals, the orthotopic U87MG brain glioma model was established, and animal status was observed periodically after inoculation. On the 12th day after inoculation, 225 animals were selected and divided into 45 groups using randomized block method (n=5).

[0816] 3.2 Preparation of the Dosing Formulations

[0817] 3.2.1 Preparation of the Dosing Formulation of Irinotecan

[0818] 12.0 mg of irinotecan was weighed, 0.15 mL of 1% lactic acid was added, the drug was dissolved completely by vortexing, and 2.85 mL of 1% aqueous sorbitol solution was then added respectively. The mixture was mixed evenly by vortexing, and the ratio of 1% lactic acid to 1% aqueous sorbitol solution was approximately 5:95 (v/v) in the solution. The concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0819] 3.2.2 Preparation of the Dosing Formulation of nktr-102

[0820] Before each administration, 88.55 mg of nktr-102 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0821] 3.2.3 Preparation of the Dosing Formulations of Compounds 1 to 5

[0822] Before each administration, 113.85 mg of Compound 1 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0823] Before each administration, 109.80 mg of Compound 2 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0824] Before each administration, 113.88 mg of Compound 3 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0825] Before each administration, 111.24 mg of Compound 4 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg. mL.sup.1.

[0826] Before each administration, 105.78 mg of Compound 5 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free irinotecan in the solution was 4.0 mg.Math.mL.sup.1.

[0827] 3.2.4 Preparation of the Dosing Formulations of Compounds 6 to 10

[0828] Before each administration, 113.09 mg of Compound 6 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0829] Before each administration, 109.05 mg of Compound 7 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0830] Before each administration, 113.12 mg of Compound 8 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0831] Before each administration, 110.48 mg of Compound 9 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0832] Before each administration, 105.02 mg of Compound 10 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free SN-38 in the solution was 4.0 mg.Math.mL.sup.1.

[0833] 3.2.5 Preparation of the Dosing Formulations of Compounds 11 to 15

[0834] Before each administration, 112.98 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0835] Before each administration, 108.94 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0836] Before each administration, 113.01 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0837] Before each administration, 110.37 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0838] Before each administration, 104.91 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free 10-hydroxycamptothecin in the solution was 4.0 mg.Math.mL.sup.1.

[0839] 3.2.6 Preparation of the Dosing Formulations of Compounds 16 to 20

[0840] Before each administration, 113.09 mg of Compound 11 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0841] Before each administration, 109.05 mg of Compound 12 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0842] Before each administration, 113.12 mg of Compound 13 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0843] Before each administration, 110.48 mg of Compound 14 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0844] Before each administration, 105.02 mg of Compound 15 was weighed accurately, 2.3 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the concentration of free rubitecan in the solution was 4.0 mg.Math.mL.sup.1.

[0845] 3.2.7 Preparation of the Dosing Formulations of Compounds 21 to 42

[0846] The preparation methods were as described above, so that the concentration of the free active agent in the solution was 4.0 mg.Math.mL.sup.1.

[0847] 3.3 Grouping of Animals and Administration

[0848] The grouping of animals and the dosing regimens were as shown in Table 9.

[0849] Groups with irinotecan as the active agent (Group 1-8). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Group 1 was a solvent control group, and was given blank solvent by intravenous injection via tail once every 4 days for a total of 3 times (Q4D3). Groups 2 to 8 were given irinotecan, nktr-102 and Compounds 1 to 5 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of irinotecan), Q4D3.

[0850] Groups with SN-38 as the active agent (Groups 9 to 13). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 9 to 13 were given Compounds 6 to 10 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1(calculated in terms of the content of SN-38), Q4D3.

[0851] Groups with 10-hydroxycamptothecin as the active agent (Groups 14 to 18). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 14 to 18 were given Compounds 11 to 15 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of 10-hy droxy camptothecin), Q4D3.

[0852] Groups with rubitecan as the active agent (Groups 19 to 23). The first administration was initiated on the day of grouping, the experiment was ended after 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.1. Groups 19 to 23 were given Compounds 16 to 20 as test samples by intravenous injection via tail, respectively, and the dosages of administration were all 40 mg.Math.kg.sup.1 (calculated in terms of the content of rubitecan), Q4D3.

[0853] The modes of administration of Groups 24 to 25 were as described above.

TABLE-US-00009 TABLE 9 Dosing regimens of the pharmacodynamic experiments in transplanted tumor model in nude mice Volume of Number of Dosage administration Route of Period of Group Sample animals (mg .Math. kg.sup.1) (mL .Math. kg.sup.1) administration administration 1 blank solvent 5 10 IV Q4D 3 2 irinotecan 5 40 10 IV Q4D 3 3 nktr-102 5 40 10 IV Q4D 3 4 Compound 1 5 40 10 IV Q4D 3 5 Compound 2 5 40 10 IV Q4D 3 6 Compound 3 5 40 10 IV Q4D 3 7 Compound 4 5 40 10 IV Q4D 3 8 Compound 5 5 40 10 IV Q4D 3 9 Compound 6 5 40 10 IV Q4D 3 10 Compound 7 5 40 10 IV Q4D 3 11 Compound 8 5 40 10 IV Q4D 3 12 Compound 9 5 40 10 IV Q4D 3 13 Compound 10 5 40 10 IV Q4D 3 14 Compound 11 5 40 10 IV Q4D 3 15 Compound 12 5 40 10 IV Q4D 3 16 Compound 13 5 40 10 IV Q4D 3 17 Compound 14 5 40 10 IV Q4D 3 18 Compound 15 5 40 10 IV Q4D 3 19 Compound 16 5 40 10 IV Q4D 3 20 Compound 17 5 40 10 IV Q4D 3 21 Compound 18 5 40 10 IV Q4D 3 22 Compound 19 5 40 10 IV Q4D 3 23 Compound 20 5 40 10 IV Q4D 3 24 Compound 21 5 40 10 IV Q4D 3 25 Compound 22 5 40 10 IV Q4D 3 26 Compound 23 5 40 10 IV Q4D 3 27 Compound 24 5 40 10 IV Q4D 3 28 Compound 25 5 40 10 IV Q4D 3 29 Compound 26 5 40 10 IV Q4D 3 30 Compound 27 5 40 10 IV Q4D 3 31 Compound 28 5 40 10 IV Q4D 3 32 Compound 29 5 40 10 IV Q4D 3 33 Compound 30 5 40 10 IV Q4D 3 34 Compound 31 5 40 10 IV Q4D 3 35 Compound 32 5 40 10 IV Q4D 3 36 Compound 33 5 40 10 IV Q4D 3 37 Compound 34 5 40 10 IV Q4D 3 38 Compound 35 5 40 10 IV Q4D 3 39 Compound 36 5 40 10 IV Q4D 3 40 Compound 37 5 40 10 IV Q4D 3 41 Compound 38 5 40 10 IV Q4D 3 42 Compound 39 5 40 10 IV Q4D 3 43 Compound 40 5 40 10 IV Q4D 3 44 Compound 41 5 40 10 IV Q4D 3 45 Compound 42 5 40 10 IV Q4D 3

[0854] 4. Data recording and Calculation Formulae

[0855] The survival time of the animals were recorded.

[0856] 5. Method for Statistical Analysis

[0857] The experimental data was subjected to calculation and related statistical treatments using Microsoft Office Excel 2007 Software. t test was adopted for the comparison between two groups.

[0858] 6. Results

[0859] See Table 10

TABLE-US-00010 TABLE 10 Survival time of the animals (day) Number of Median Group Sample animals Survival time survival time 1 blank solvent 5 16 to 22 20 2 irinotecan 5 22 to 32 27 3 nktr-102 5 25 to 37 31 4 Compound 1 5 34 to 46 40* 5 Compound 2 5 34 to 43 38* 6 Compound 3 5 33 to 44 36* 7 Compound 4 5 34 to 45 38* 8 Compound 5 5 30 to 39 35* 9 Compound 6 5 34 to 43 41* 10 Compound 7 5 33 to 44 39* 11 Compound 8 5 32 to 45 40* 12 Compound 9 5 33 to 42 38* 13 Compound 10 5 29 to 39 35* 14 Compound 11 5 28 to 42 37* 15 Compound 12 5 35 to 45 42* 16 Compound 13 5 34 to 41 38* 17 Compound 14 5 34 to 44 40* 18 Compound 15 5 35 to 44 41* 19 Compound 16 5 30 to 42 37* 20 Compound 17 5 32 to 43 38* 21 Compound 18 5 31 to 40 36* 22 Compound 19 5 35 to 46 40* 23 Compound 20 5 33 to 43 39* 24 Compound 21 5 34 to 43 41* 25 Compound 22 5 34 to 46 40* 26 Compound 23 5 34 to 41 38* 27 Compound 24 5 34 to 46 40* 28 Compound 25 5 34 to 44 40* 29 Compound 26 5 30 to 42 37* 30 Compound 27 5 30 to 39 35* 31 Compound 28 5 29 to 39 35* 32 Compound 29 5 33 to 44 36* 33 Compound 30 5 35 to 46 40* 34 Compound 31 5 35 to 44 41* 35 Compound 32 5 33 to 44 39* 36 Compound 33 5 33 to 44 39* 37 Compound 34 5 34 to 46 40* 38 Compound 35 5 34 to 43 38* 39 Compound 36 5 30 to 42 37* 40 Compound 37 5 33 to 45 40* 41 Compound 38 5 29 to 39 35* 42 Compound 39 5 34 to 46 40* 43 Compound 40 5 33 to 40 36* 44 Compound 41 5 33 to 44 39* 45 Compound 42 5 30 to 42 38* *P < 0.05, as compared with the median survival time of blank solvent group, irinotecan group and nktr-102 group.

[0860] The experimental results showed that the compounds of the present disclosure had good inhibitory effects on brain glioma, and were superior to irinotecan and nktr-102.