Multi-arm targeting anti-cancer conjugate

Abstract

A multi-branched drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof. In the formula, R is an organic center, 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. The symbol “*” in L represents a junction point of the multivalent linker L and the targeting molecule T, “#” represents a junction point of the multivalent linker L and the active agent D, and “%” represents a junction point of the multivalent linker L and POLY. 1 is any integer between 2 and 20, and m and n are each an integer between 0 and 10. T is iRGD, cRGD, tLyp-1, Lyp-1, RPARPAR, Angiopep2, or GE11. D is a camptothecin drug. ##STR00001##

Claims

1. A multi-branched drug conjugate having the following structural formula (I) or a pharmaceutically acceptable salt thereof: ##STR00070## 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 L is ##STR00071## a 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, wherein I is any integer between 2 and 20, and m and n are any integer between 0 and 10 respectively; T is an RGD peptide containing a sequence of “arginine-glycine-aspartic acid”, tLyp-1, Lyp-1, RPARPAR, Angiopep2, or GE11; D is a camptothecin-based drug as represented by formula (II): ##STR00072## wherein R.sub.1 to R.sub.5 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 the pharmaceutically acceptable salt thereof according to claim 1, wherein T is cRGD or iRGD.

3. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 1, wherein POLY is polyethylene glycol.

4. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 3, wherein POLY is ##STR00073## wherein “” represents a site for attachment of atoms, an oxygen atom marked with “&” is the atom attached to the organic core “R”, k is an integer in a range of 50 to 200, and r is any integer between 1 and 10.

5. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 4, which is as represented by structural formula (III), (IV), or (V): ##STR00074##

6. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 5, wherein POLY is ##STR00075##

7. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 6, which is ##STR00076##

8. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 7, wherein k has an average value of 113.

9. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 8, wherein L is ##STR00077##

10. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 9, wherein D is irinotecan, SN-38, 10-hydroxycamptothecin, or rubitecan.

11. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 10, which is ##STR00078## ##STR00079##

12. The multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 11, which is ##STR00080## ##STR00081##

13. A pharmaceutical composition, comprising the multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according claim 1, and a pharmaceutically acceptable excipient.

14. A method for treating a cancer selected from the group consisting of colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and a malignant sarcoma, a cancer and a 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 the pharmaceutically acceptable salt thereof according to claim 1.

15. A method for treating a cancer selected from the group consisting of colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and a malignant sarcoma, a cancer and a 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 pharmaceutical composition according to claim 13.

16. A pharmaceutical composition, comprising the multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 5, and a pharmaceutically acceptable excipient.

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

18. A pharmaceutical composition, comprising the multi-branched drug conjugate or the pharmaceutically acceptable salt thereof according to claim 12, and a pharmaceutically acceptable excipient.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the chemical structure of Compound a.

(2) FIG. 2 shows the chemical structure of Compound e.

(3) FIG. 3 shows the chemical structure of Compound f.

DETAILED DESCRIPTION

(4) The present disclosure will be described in detail below. However, the present disclosure may be specifically 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. Among them, 4ARM-PEG20K-SCM is purchased from Beijing Jenkem Technology Co., Ltd.

(5) Noun Explanations

(6) TABLE-US-00002 Abbreviations Noun explanations DMF N,N-dimethylformamide DCM dichloromethane Boc-Gly-OH DMAP 4-dimethylaminopyridine DCC dicyclohexylcarbodiimide IPA isopropanol EA ethyl acetate DEPC diethyl cyanophosphonate Pbf DIC N,N-diisopropylcarbodiimide DPPA diphenyl phosphorazidate NMM N-methylmorpholine MTBE tert-butyl methyl ether TFA trifluoroacetic acid TBME tert-butyl methyl ether Fmoc-OSU 9-fluorenylmethyl-N-succinimidyl carbonate DME ethylene glycol dimethyl ether HOBT 1-hydroxybenzotriazole THF tetrahydrofuran DIEA N,N-diisopropylethylamine DEA triethylamine H-Lys (Boc)-OBzl•HCl TFE trifluoroethanol SPPS solid-phase peptide synthesis TIS triisopropylsilane

Example 1

(7) ##STR00044##

(8) Preparation of Compound 2

(9) To a 250 mL round-bottom flask, 3.50 g of Compound 1 (1.0 eq) 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 hours. 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 hour, filtered, washed twice with n-heptane, and dried to obtain 4.02 g of Compound 2 as a pale yellow solid.

(10) Preparation of Compound 3

(11) To a 100 mL three-necked flask, 4.02 g of Compound 2 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 the 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 4.00 g of Compound 3 as a pale yellow solid.

Example 2

(12) ##STR00045##

(13) Preparation of Compound 5

(14) To a 250 mL three-necked flask, 6.9 g of Compound 4 and 30 mL of EA were added, stirred and dissolved, and then cooled to 0° C. 40 mL of 0.3 M HC/EA was added, and the reaction was carried out for 2 h while the temperature was kept still. The completion of the reaction was monitored by TLC, and the mixture was concentrated to dryness so as to obtain Compound 5, which was directly subjected to the next reaction.

(15) Preparation of Compound 6

(16) Compound 5 (1.0 eq) was dissolved in 50 mL of purified water, and 3.96 g of sodium bicarbonate (2.0 eq) was added. 5.30 g of Fmoc-OSU (1.0 eq) was dissolved in 50 mL of DME, the mixture was added to the reaction flask containing Compound 5, 25 mL of THF was added additionally, and the mixture was stirred at room temperature for 2 hours. After the completion of the reaction was monitored by TLC, the organic solvent was removed by distillation, and EA was used to extract the impurities. The pH of the aqueous phase was adjusted to 3 to 4 with dilute hydrochloric acid, the mixture was extracted twice with EA, and the organic phases were combined and washed once with water. After being washed with saturated saline, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 8.4 g of Compound 6 as a pale yellow oil.

(17) Preparation of Compound 7

(18) To a 100 mL reaction flask, 4.00 g of Compound 6 (1.0 eq) and 2.92 g of H-Lys (Boc)-OBzl.HCl were added, and 40 mL of DCM was used for dissolution. 2.76 g of DIEA (3.0 eq) and 1.74 g of DEPC (1.5 eq) were added, and the mixture was stirred at room temperature for 2 hours. After the completion of the reaction was monitored by TLC, the mixture was washed with an acetic acid aqueous solution, washed with a sodium bicarbonate solution, washed once with water, and washed once with saturated saline. Then, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 7.0 g of Compound 7 as a pale yellow oil, which was directly subjected to the next reaction without purification. (Compound 16 was prepared by using the same method.)

(19) Preparation of Compound 8

(20) 140 mL of 25% DEA/DCM was used to dissolve 7.0 g of Compound 7, and the mixture was stirred at room temperature for 6 hours. After the completion of the reaction was monitored by TLC, the mixture was concentrated to dryness, and 150 mL of EA were added. pH was adjusted to 3 to 4 by dilute hydrochloric acid, the mixture was subjected to liquid separation, and the aqueous phase was extracted twice with EA and then concentrated to dryness, so as to obtain 3.5 g of Compound 8 as a pale yellow solid. (Compound 17 was prepared by using the same method)

Example 3 Preparation of a Targeting Molecule cRGD with an Attached Protecting Group (Compound 11)

(21) ##STR00046##

(22) Preparation of Compound 9

(23) 2Cl-Trt Resin and Fmoc protection method were used, HOBT/DIC was adopted as a coupling reagent, DMF was a reaction solvent, the reaction was monitored by ninhydrin detection, and the following protecting amino acids were sequentially attached to the resin: Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OBzl)-OH, Fmoc-D-Phe-OH, and Fmoc-Asp(OtBu)OH. Fmoc was removed. The mixture was dried after being washed with DMF, DCM, and methanol. The cleavage reagent (acetic acid/TFE/DCM=1/2/7) was added, and the mixture was reacted for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed and dried to obtain Compound 9 as an off-white solid.

(24) Preparation of Compound 10

(25) To a 2 L three-necked flask, 14.0 g of Compound 9 (1.0 eq) was added, 1 L of DMF was added, the mixture was cooled to 0° C., and 9.2 g of sodium bicarbonate (8.0 eq) was added. After the mixture was dissolved to give a clear solution, 15.1 g of DPPA (4.0 eq) was added, and the temperature of the mixture was kept still overnight. After the completion of the reaction was monitored by TLC, the mixture was poured into 5 L of water, extracted twice with EA, washed with water, and washed with a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 11.5 g of Compound 10 as an off-white solid.

(26) Preparation of Compound 11

(27) To a 1 L hydrogenation reactor, 11.5 g of Compound 10, 1 L of methanol and 2.5 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight. After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 11.0 g of Compound 11 as a gray solid.

Example 4 Preparation of a Targeting Molecule iRGD with an Attached Protecting Group (Compound 20)

(28) ##STR00047##

(29) Fmoc-Sieber 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-Cys(Acm)-OH, Fmoc-Asp(Alloc)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 being washed with DMF, thallium trifluoroacetate (2.0 eq) was added. After being stirred for 18 hours, 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. A solution of 3 eq of Pd (PPh.sub.3).sub.4 in CHCl.sub.3:AcOH:NMM (18:1:0.5) was added, the reaction was carried out for 2 h, and Alloc was removed. Then the mixture was washed with chloroform (6×20 mL), a solution of 20% HOAc in DCM, DCM, and DMF. The mixture was dried after being washed with DMF, DCM and methanol. 1% TFA/DCM was added, and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed and dried to obtain Compound 20 as an off-white solid.

Example 5 Preparation of a Targeting Molecule tLyP-1 with an Attached Protecting Group (Compound 30)

(30) ##STR00048##

(31) 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 Boc-Cys(Trt)-OH. The cleavage reagent (acetic acid/TFE/DCM=1/2/7) was added, and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed and dried to obtain Compound 30 as an off-white solid.

Example 6 Preparation of a Targeting Molecule RPARPAR with an Attached Protecting Group (Compound 40)

(32) ##STR00049##

(33) 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-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, and Boc-Arg(Pbf)-OH. The cleavage reagent (acetic acid/TFE/DCM=1/2/7) was added, and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed and dried to obtain Compound 40 as an off-white solid.

Example 7 Preparation of a Targeting Molecule Angiopep-2 with an Attached Protecting Group (Compound 50)

(34) ##STR00050##

(35) The sequence of Angiopep-2 was TFFYGGSRGKRNNFKTEEY.

(36) 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-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, and Boc-Thr(tBu)-OH. The cleavage reagent (acetic acid/TFE/DCM=1/2/7) was added, and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed and dried to obtain Compound 50 as an off-white solid.

Example 8 Preparation of a Targeting Molecule GE11 with an Attached Protecting Group (Compound 60)

(37) ##STR00051##

(38) The sequence of GE11 was YHWYGYTPQNVI.

(39) 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 to protecting amino acids were sequentially attached to the resin: Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH, and Boc-Tyr(tBu)-OH. The cleavage reagent (acetic acid/TFE/DCM=1/2/7) was added, and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed and dried to obtain an off-white solid 60.

Example 9 Preparation of Compound a and Compound A

(40) ##STR00052## ##STR00053##

(41) Preparation of Compound 12

(42) To a 5 mL reaction flask, 480 mg of Compound 11 (1.0 eq), 380 mg of Compound 8 (1.1 eq), 1 mL of DMF, 203 mg of DIEA (3.0 eq) and 128 mg of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 10 mL of water, extracted twice with EA, and washed with dilute hydrochloric acid, a sodium bicarbonate solution and a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 0.8 g of Compound 12 as a jelly-like solid, which was directly subjected to the next reaction.

(43) Preparation of Compound 13

(44) To a 200 mL hydrogenation reactor, 0.8 g of Compound 10, 30 mL of methanol and 0.28 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight. After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 0.66 g of Compound 13 as a gray solid.

(45) Preparation of Compound 14

(46) To a 100 mL reaction flask, 6.60 g of Compound 13 (1.0 eq), 3.59 g of Compound 3 (1.05 eq), 66 mL of DMF, 1.16 g of DIEA (3.0 eq) and 1.10 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 700 mL of TBME and subjected to suction filtration after pulping. After the solid was dissolved in 150 mL of DCM, the mixture was poured into 1.5 L of TBME, subjected to pulping and suction filtration, and dried to obtain 9.0 g of Compound 14 as gray powder, which was directly subjected to the next reaction.

(47) Preparation of Compound 15

(48) To a 250 mL reaction flask, 9.0 g of Compound 14 was added, and 92.5% TFA/2.5% water/2.5% TIS was added as a cleavage reagent. The mixture was stirred at room temperature for 2 h, ice-cold MTBE was used for precipitation, and the mixture was centrifuged and washed. The crude product was purified by reverse-phase HPLC and lyophilized to obtain 5.0 g of Compound 15 as a pale yellow floccule.

(49) Preparation of Compound a

(50) To a reaction flask, 2.3 g of Compound 15 (4.5 eq), 6.0 g of 4ARM-PEG20K-SCM (1.0 eq), 60 mL of DMF and 0.27 g of TEA (9.0 eq) were added, and the mixture was reacted at room temperature. After there was no significant reaction progress monitored by HPLC, the mixture was poured into 1000 mL of TBME, and subjected to pulping and suction filtration to obtain 7.6 g of crude product a as off-white powder. The crude product was desalted after being purified by HPLC. The organic solvent was removed by concentration, and the resultant was lyophilized to obtain 3.4 g of Compound a as off-white powder.

(51) Preparation of Compound A

(52) After obtaining a powdery crude product of Compound a, the crude product was desalted after being purified by HPLC. The organic solvent was removed by concentration, pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 3.4 g of Compound A as yellow-green powder.

(53) The molecular weight detected by MALDI-TOF was 25480.27.

Example 10 Preparation of Compound b and Compound B

(54) ##STR00054## ##STR00055## ##STR00056##

(55) Preparation of Compound 21

(56) To a 100 mL reaction flask, 5.00 g of Compound 20 (1.0 eq), 2.36 g of Compound 17 (1.1 eq), 50 ml of DMF, 1.11 g of DIEA (3.0 eq) and 0.71 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 300 mL of water, extracted twice with EA, and washed with an acetic acid aqueous solution, a sodium bicarbonate solution and a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 7.18 g of pale yellow solid 21, which was directly subjected to the next reaction.

(57) Preparation of Compound 22

(58) To a 200 mL hydrogenation reactor, 7.00 g of Compound 21, 120 mL of methanol and 0.35 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight.

(59) After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 7.05 g of Compound 22 as a gray solid.

(60) Preparation of Compound 23

(61) To a 100 mL reaction flask, 7.00 g of Compound 22 (1.0 eq), 2.22 g of Compound 3 (1.05 eq), 70 mL of DMF, 1.10 g of DIEA (3.0 eq) and 0.70 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 700 mL of TBME and subjected to suction filtration after pulping. After the solid was dissolved in 100 mL of DCM, the mixture was poured into 1.0 L of TBME, subjected to pulping and suction filtration, and dried to obtain 8.60 g of Compound 23 as gray powder, which was directly subjected to the next reaction.

(62) Preparation of Compound 24

(63) 8.60 g of Compound 23 was added to 200 mL of the cleavage reagent (acetic acid/TFE/DCM=1/2/7), and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed, dried, and purified by HPLC to obtain 2.10 g of off-white solid 24.

(64) Preparation of Compound 25

(65) To a 50 mL reaction flask, 1.23 g of Compound 24 (4.5 eq), 2.00 g of 4ARM-PEG20K-SCM (1.0 eq), 20 mL of DMF and 0.09 g of TEA (9.0 eq) were added, and the mixture was reacted at room temperature. After there was no significant reaction progress monitored by HPLC, the mixture was poured into 400 mL of TBME, and subjected to pulping and suction filtration to obtain 3.05 g of Compound 25, which was directly subjected to the next reaction.

(66) Preparation of Compound b

(67) To a 50 mL reaction flask, 3.0 g of Compound 25 and 30 mL of the cleavage reagent (92.5% TFA/2.5% water/2.5% TIS) were added, and the mixture was stirred at room temperature for 2 h. Ice-cold MTBE was used for precipitation, and the mixture was centrifuged and washed. The crude product was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, and the resultant was lyophilized to obtain 1.02 g of Compound b as off-white powder.

(68) Preparation of Compound B

(69) The crude product of Compound b was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 1.02 g of Compound B as yellow-green powder.

(70) The molecular weight detected by MALDI-TOF was 29013.19.

Example 11 Preparation of Compound c and Compound C

(71) ##STR00057## ##STR00058## ##STR00059## ##STR00060##

(72) Preparation of Compound 31

(73) To a 100 mL reaction flask, 5.60 g of Compound 30 (1.0 eq), 2.39 g of Compound 17 (1.1 eq), 60 mL of DMF, 1.03 g of DIEA (3.0 eq) and 0.65 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 300 mL of water, extracted twice with EA, and washed with an acetic acid aqueous solution, a sodium bicarbonate solution and a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 7.08 g of Compound 31 as a pale yellow solid, which was directly subjected to the next reaction.

(74) Preparation of Compound 32

(75) To a 200 mL hydrogenation reactor, 7.05 g of Compound 31, 150 mL of methanol and 0.35 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight.

(76) After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 6.95 g of Compound 32 as a gray solid.

(77) Preparation of Compound 33

(78) To a 100 mL reaction flask, 6.80 g of Compound 32 (1.0 eq), 1.89 g of Compound 3 (1.05 eq), 70 mL of DMF, 0.94 g of DIEA (3.0 eq) and 0.59 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 700 mL of TBME and subjected to suction filtration after pulping. After the solid was dissolved in 100 mL of DCM, the mixture was poured into 1.0 L of TBME, subjected to pulping and suction filtration, and dried to obtain 8.20 g of Compound 33 as gray powder, which was directly subjected to the next reaction.

(79) Preparation of Compound 34

(80) 8.20 g of Compound 33 was added into 160 mL of the cleavage reagent (acetic acid/TFE/DCM=1/2/7), and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed, dried, and purified by HPLC to obtain 5.6 g of Compound 34 as an off-white solid.

(81) Preparation of Compound 35

(82) To a 50 mL reaction flask, 0.75 g of Compound 34 (4.5 eq), 1.00 g of 4ARM-PEG20K-SCM (1.0 eq), 10 mL of DMF and 0.05 g of TEA (9.0 eq) were added, and the mixture was reacted at room temperature. After there was no significant reaction progress monitored by HPLC, the mixture was poured into 200 mL of TBME, and subjected to pulping and suction filtration to obtain 1.69 g of Compound 35, which was directly subjected to the next reaction.

(83) Preparation of Compound c

(84) To a 50 mL reaction flask, 1.65 g of Compound 25 and 20 mL of the cleavage reagent (92.5% TFA/2.5% water/2.5% TIS) were added, and the mixture was stirred at room temperature for 2 h. Ice-cold MTBE was used for precipitation, and the mixture was centrifuged and washed. The crude product was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, and the resultant was lyophilized to obtain 0.84 g of Compound c as off-white powder.

(85) Preparation of Compound C

(86) The crude product of Compound c was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 0.84 g of Compound C as yellow-green powder.

(87) The molecular weight detected by MALDI-TOF was 28076.21.

Example 12 Preparation of Compound d and Compound D

(88) ##STR00061## ##STR00062## ##STR00063##

(89) Preparation of Compound 41

(90) To a 100 mL reaction flask, 6.20 g of Compound 40 (1.0 eq), 3.30 g of Compound 17 (1.1 eq), 62 mL of DMF, 1.43 g of DIEA (3.0 eq) and 0.90 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 310 mL of water, extracted twice with EA, and washed with an acetic acid aqueous solution, a sodium bicarbonate solution and a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 8.84 g of Compound 41 as a pale yellow solid, which was directly subjected to the next reaction.

(91) Preparation of Compound 42

(92) To a 200 mL hydrogenation reactor, 8.80 g of Compound 41, 150 mL of methanol and 0.44 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight.

(93) After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 8.84 g of Compound 42 as a gray solid.

(94) Preparation of Compound 43

(95) To a 100 mL reaction flask, 8.50 g of Compound 42 (1.0 eq), 2.77 g of Compound 3 (1.05 eq), 85 mL of DMF, 1.38 g of DIEA (3.0 eq) and 0.87 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 850 mL of TBME and subjected to suction filtration after pulping. After the solid was dissolved in 110 mL of DCM, the mixture was poured into 1.1 L of TBME, subjected to pulping and suction filtration, and dried to obtain 9.86 g of Compound 43 as gray powder, which was directly subjected to the next reaction.

(96) Preparation of Compound 44

(97) 9.80 g of Compound 43 was added to 200 mL of the cleavage reagent (acetic acid/TFE/DCM=1/2/7), and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed, dried, and purified by HPLC to obtain 5.92 g of Compound 44 as an off-white solid.

(98) Preparation of Compound 45

(99) To a 50 mL reaction flask, 0.60 g of Compound 34 (4.5 eq), 1.00 g of 4ARM-PEG20K-SCM (1.0 eq), 10 mL of DMF and 0.05 g of TEA (9.0 eq) were added, and the mixture was reacted at room temperature. After there was no significant reaction progress monitored by HPLC, the mixture was poured into 200 mL of TBME, and subjected to pulping and suction filtration to obtain 1.45 g of Compound 45, which was directly subjected to the next reaction.

(100) Preparation of Compound d

(101) To a 50 mL reaction flask, 1.45 g of Compound 45 and 15 mL of the cleavage reagent (92.5% TFA/2.5% water/2.5% TIS) were added, and the mixture was stirred at room temperature for 2 h. Ice-cold MTBE was used for precipitation, and the mixture was centrifuged and washed. The crude product was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, and the resultant was lyophilized to obtain 0.57 g of Compound d as off-white powder.

(102) Preparation of Compound D

(103) The crude product of Compound d was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 0.57 g of Compound D as yellow-green powder.

(104) The molecular weight detected by MALDI-TOF was 27963.54.

Example 13 Preparation of Compound e and Compound E

(105) ##STR00064## ##STR00065## ##STR00066##

(106) Preparation of Compound 51

(107) To a 100 mL reaction flask, 6.20 g of Compound 50 (1.0 eq), 1.39 g of Compound 17 (1.1 eq), 62 mL of DMF, 0.60 g of DIEA (3.0 eq) and 0.38 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 10 mL of water, extracted twice with EA, and washed with an acetic acid aqueous solution, a sodium bicarbonate solution and a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 6.5 g of Compound 51 as a jelly-like solid, which was directly subjected to the next reaction.

(108) Preparation of Compound 52

(109) To a 200 mL hydrogenation reactor, 6.53 g of Compound 51, 150 mL of methanol and 0.33 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight. After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 6.50 g of Compound 52 as a gray solid.

(110) Preparation of Compound 53

(111) To a 100 mL reaction flask, 6.50 g of Compound 52 (1.0 eq), 1.08 g of Compound 3 (1.05 eq), 65 mL of DMF, 0.54 g of DIEA (3.0 eq) and 0.34 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 650 mL of TBME and subjected to suction filtration after pulping. After the solid was dissolved in 100 mL of DCM, the mixture was poured into 1.0 L of TBME, subjected to pulping and suction filtration, and dried to obtain 6.71 g of Compound 53 as gray powder, which was directly subjected to the next reaction.

(112) Preparation of Compound 54

(113) 2.5 g of Compound 53 was added to 50 mL of the cleavage reagent (acetic acid/TFE/DCM=1/2/7), and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed, dried, and purified by HPLC to obtain 0.97 g of Compound 54 as an off-white solid.

(114) Preparation of Compound 55

(115) To a 50 mL reaction flask, 2.91 g of Compound 54 (4.5 eq), 3.00 g of 4ARM-PEG20K-SCM (1.0 eq), 30 mL of DMF and 0.13 g of TEA (9.0 eq) were added, and the mixture was reacted at room temperature. After there was no significant reaction progress monitored by HPLC, the mixture was poured into 300 mL of TBME, and subjected to pulping and suction filtration to obtain 5.83 g of Compound 55, which was directly subjected to the next reaction.

(116) Preparation of Compound e

(117) To a 50 mL reaction flask, 2.04 g of Compound 55 and 30 mL of the cleavage reagent (92.5% TFA/2.5% water/2.5% TIS) were added, and the mixture was stirred at room temperature for 2 h. Ice-cold MTBE was used for precipitation, and the mixture was centrifuged and washed. The crude product was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, and the resultant was lyophilized to obtain 0.42 g of off-white powder e.

(118) Preparation of Compound E

(119) The crude product of Compound e was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 0.42 g of yellow-green powder E.

(120) The molecular weight detected by MALDI-TOF was 33812.65.

Example 14 Preparation of Compound f and Compound F

(121) ##STR00067## ##STR00068## ##STR00069##

(122) Preparation of Compound 61

(123) To a 100 mL reaction flask, 5.00 g of Compound 60 (1.0 eq), 1.66 g of Compound 17 (1.1 eq), 50 mL of DMF, 0.72 g of DIEA (3.0 eq) and 0.45 g of DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 10 mL of water, extracted twice with EA, and washed with an acetic acid aqueous solution, a sodium bicarbonate solution and a saturated sodium chloride solution. Afterwards, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain 6.14 g of a jelly-like solid 61, which was directly subjected to the next reaction.

(124) Preparation of Compound 62

(125) To a 200 mL hydrogenation reactor, 6.10 g of Compound 61, 150 mL of methanol and 0.31 g of Pd/C were added, and the mixture was subjected to hydrogenation overnight. After the completion of the reaction was monitored by TLC, the mixture was filtered and concentrated to obtain 5.98 g of Compound 62 as a gray solid.

(126) Preparation of Compound 63

(127) To a 100 mL reaction flask, 5.95 g of Compound 62 (1.0 eq), 1.36 g of Compound 3 (1.05 eq), 60 mL of DMF, 0.68 g of DIEA (3.0 eq) and 0.43 g DEPC (1.5 eq) were added, and the mixture was reacted at room temperature for 2 h. After the completion of the reaction was monitored by TLC, the mixture was poured into 600 mL of TBME and subjected to suction filtration after pulping. After the solid was dissolved in 100 mL of DCM, the mixture was poured into 1.0 L of TBME, subjected to pulping and suction filtration, and dried to obtain 6.53 g of Compound 63 as gray powder, which was directly subjected to the next reaction.

(128) Preparation of Compound 64

(129) 6.50 g of Compound 63 was added to 130 mL of the cleavage reagent (acetic acid/TFE/DCM=1/2/7), and the reaction was carried out for 2 hours. Ice-cold MTBE was used for precipitation, and the mixture was washed, dried, and purified by HPLC to obtain 3.63 g of Compound 64 as an off-white solid.

(130) Preparation of Compound 65

(131) To a 50 mL reaction flask, 2.06 g of Compound 64 (4.5 eq), 3.00 g of 4ARM-PEG20K-SCM (1.0 eq), 30 mL of DMF and 0.13 g of TEA (9.0 eq) were added, and the mixture was reacted at room temperature. After there was no significant reaction progress monitored by HPLC, the mixture was poured into 300 mL of TBME, and subjected to pulping and suction filtration to obtain 4.67 g of Compound 65, which was directly subjected to the next reaction.

(132) Synthesis of Compound f

(133) To a 200 mL reaction flask, 4.60 g of Compound 65 and 100 mL of the cleavage reagent (92.5% TFA/2.5% water/2.5% TIS) were added, and the mixture was stirred at room temperature for 2 h. Ice-cold MTBE was used for precipitation, and the mixture was centrifuged and washed. The crude product was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, and the resultant was lyophilized to obtain 1.34 g of Compound f as off-white powder.

(134) Synthesis of Compound F

(135) The crude product of Compound f was desalted after being purified by reverse-phase HPLC. The organic solvent was removed by concentration, pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 1.34 g of Compound F as yellow-green powder.

(136) The molecular weight detected by MALDI-TOF was 30907.82.

(137) The sources of the test articles, reagents, instruments, and the like which were used in Examples 15 to 19 of the present disclosure were as follows.

(138) Irinotecan (bulk drug) was obtained by purchasing.

(139) As for the preparation method of nktr-102, the method disclosed in CN102711837A was referred and the method was as follows.

(140) Compound 3 (829 mg, 4.5 eq) in Example 1 was added to a 250 mL reaction flask, and DCM (50 mL) and triethylamine (221 mg, 9.0 eq) were added. After the mixture was dissolved, 4ARM-PEG20K-SCM (5.00 g, 1.0 eq) was added to this reaction flask. After there was no significant reaction progress monitored by HPLC, about 20 mL of DCM was distilled off under reduced pressure. The solution was poured into 300 mL of TBME, stirred, and subjected to precipitation and filtration, so as to obtain 5.4 g of crude product. The crude product was purified by HPLC and desalted. pH was adjusted to 5 to 6 by dilute hydrochloric acid, and the resultant was lyophilized to obtain 2.71 g of nktr-102 as pale green powder.

(141) Physiological saline was purchased from Shanghai Huayuan Changfu Pharmaceutical (Group) Co., Ltd. 1 mL sterile syringes were purchased from Shanghai Kindly Enterprise Development Group Co., Ltd (Shanghai, China). 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. Matrigel (BD Matrigel) was purchased from Becton, Dickinson and Company (BD, USA).

(142) Biosafe cabinet (model: AC2-6E1) was purchased from ESCO; water-jacketed CO.sub.2 cell incubator (model: 3111) was purchased from Thermo Scientific Forma; the inverted microscope (model: CKX41SF) was purchased from Olympus; electric suction apparatus (model: YX930D) was purchased from Shanghai Medical Instruments (Group) Co., Ltd.; balance (METTLER-TOLEDO AB135-S) was purchased from METTLER-TOLEDO; the low speed centrifuge (model: LD5-2A) was purchased from Beijing Lab Centrifuge Co., Ltd.; and digimatic caliper (model: SF2000) was purchased from Guilin Guanglu Measuring Instrument Co., Ltd.

(143) In Examples 15 to 19 of the present disclosure, 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. According to “Technical guidelines for non-clinical research on cytotoxic antitumor drugs” (November, 2006) by Chinese SFDA, the treatment was considered to be effective when % T/C≤40% and P<0.05 according to statistical analysis.

(144) The calculation formula of the tumor volume (TV) was as follows: TV (mm.sup.3)=l×w.sup.2/2

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

(146) The calculation formula of the relative tumor volume (RTV) was: RTV=TV.sub.t/TV.sub.initial

(147) wherein TV.sub.initial was the tumor volume measured at the time of grouping and the first administration, and TV.sub.t was the tumor volume at each measurement during administration.

(148) The calculation formula of the relative tumor proliferation rate (% T/C) was: % T/C=100%×(RTV.sub.T/RTV.sub.C)

(149) wherein RTV.sub.T represented the RTV of a treatment group, and RTV.sub.C represented the RTV of the solvent control group.

(150) 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))]

(151) 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 the 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 the first administration.

(152) The calculation formula of the decline rate of animal body weight was: decline rate of animal body weight=100%×(BW.sub.initial−BW.sub.t)/BW.sub.initial

(153) 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 the first administration.

(154) The calculation formula of the tumor weight inhibition rate (IR (%)) was: IR (%)=100%×(W.sub.C−W.sub.T)/W.sub.C

(155) wherein W.sub.C represented the tumor weight of the control group, and W.sub.T represented the tumor weight of a treatment group.

(156) 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 (Mean±SE), t-test was adopted for the comparison between groups, and P<0.05 was considered as having significant difference.

Example 15 In Vivo Efficacy Evaluation of a Series of Compounds in a Human Colon Cancer HT-29 Cell Strain Transplanted Tumor Model in Nude Mice

(157) Test articles: irinotecan, nktr-102, and 12 compounds of the present disclosure.

(158) Reagents: McCoy's 5A liquid culture medium, fetal bovine serum (FBS), trypsin, penicillin-streptomycin, water for injection, physiological saline, lactic acid, and sorbitol.

(159) Experimental animals: Female BALB/c nude mice (number of animals: 150; weeks of age: 6 to 7 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and 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 hours. 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 this 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.

(160) Transplantable tumor cell line: 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).

(161) Culture of HT-29 cells: Under a culture condition of 5% CO.sub.2 and 37° C., HT-29 cells were subjected to conventional cell cultivation in an 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 1:4 to 1:6.

(162) Preparation of the animal model: 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 4×10.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 μL (4×10.sup.6 cells/animal), and an HT-29 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, 90 animals with good health condition and similar tumor volumes were selected and divided into 15 groups (n=6) using randomized block method. 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.

(163) Preparation of the solvent: 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 with a concentration of 1% (w/v) was formulated and stored in a refrigerator at 4° C. until use.

(164) Preparation of the dosing formulation of irinotecan: 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 effective concentration of irinotecan in the solution was 4.0 mg.Math.mL.sup.−1.

(165) Preparation of the dosing formulation of nktr-102: Before each administration, 101.5 mg of nktr-102 was weighed accurately, 2.5 mL of physiological saline was added, the drug was dissolved completely by vortexing, and the effective concentration of nktr-102 in the solution was 4.0 mg.Math.mL.sup.−1.

(166) Preparation of the dosing formulations of the compounds of the present disclosure: Before each administration, 120.3 mg of Compound a and Compound A, 137.0 mg of Compound b and Compound B, 132.6 mg of Compound c and Compound C, 132.0 mg of Compound d and Compound D, 159.6 mg of Compound e and Compound E, and 145.9 mg of Compound f and Compound F were respectively weighed accurately. 2.5 mL of physiological saline was added, and the drug was dissolved completely by vortexing and sonication (if needed). The effective concentration of the compounds of the present disclosure in the solution was 4.0 mg.Math.mL.sup.−1.

(167) Grouping of animals and dosage regimens: The first administration was initiated on the day of grouping, the experiment was ended after about 21 days, and the volumes of administration were all 10 mL.Math.kg.sup.−1. The equivalent effective doses of irinotecan were all 40 mg.Math.kg.sup.−1. Group 1 was a solvent control group, and physiological saline was administered by intravenous injection via tail once every 4 days for a total of 3 times (Q4D×3). Groups 2 to 15 were respectively given the following test samples by intravenous injection via tail once every 4 days, Q4D×3: irinotecan, nktr-102, Compound a, Compound A, Compound b, Compound B, Compound c, Compound C, Compound d, Compound D, Compound e, Compound E, Compound f, and Compound F.

(168) 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 and weighed. 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 relative tumor proliferation rate indicated better tumor inhibitory effect, and the results were as shown in Table 1.

(169) TABLE-US-00003 TABLE 1 Relative tumor proliferation rate (% T/C) Relative tumor Number of Relative tumor proliferation Group Sample animals volume (RTV) rate (% T/C) 1 blank solvent 6 15.04 — 2 irinotecan 6 9.52  .sup. 69% 3 nktr-102 6 5.23 34.70%.sup.  4 Compound a 6 2.57* 17.08%.sup.# 5 Compound A 6 2.55* 16.95%.sup.# 6 Compound b 6 3.00* 20.01%.sup.# 7 Compound B 6 2.91* 19.41%.sup.# 8 Compound c 6 3.18* 21.29%.sup.# 9 Compound C 6 3.18* 21.29%.sup.# 10 Compound d 6 2.69* 17.98%.sup.# 11 Compound D 6 2.76* 18.45%.sup.# 12 Compound e 6 3.40* 22.70%.sup.# 13 Compound E 6 3.43* 22.90%.sup.# 14 Compound f 6 3.71* 24.73%.sup.# 15 Compound F 6 3.56* 23.73%.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.

(170) 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 16 Inhibitory Effect in a Human Breast Cancer MDA-MB-231 Xenograft Model in Nude Mice

(171) Test articles: irinotecan, nktr-102, and 12 compounds of the present disclosure.

(172) Experimental animals: Female BALB/c nude mice. 150 animals were inoculated, and 90 animals were used for the experiment. The animals were 6 to 8 week old and had a body weight of 20 g to 22 g f 20% of the average body weight. The animal source was Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. (BK), license number: SCXK (Shanghai) 2008-0016. All experimental animals were housed in an SPF-level laboratory. Experimenters were responsible for daily care and experimental research. 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.

(173) Random grouping: After the tumor volumes reached 150 mm.sup.3 to 200 mm.sup.3, the animals were divided into 15 groups with 6 mice in each group using randomized block method, so as to ensure that 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%.

(174) Feeding conditions: living condition: IVC system, 6 animals per cage; temperature: 20° C. to 26° C.; humidity: 40% f 70%; illumination: 12 hours light/dark cycle. Irradiated feed for rat and mice was purchased from Beijing Keao Xieli Feed Co., Ltd. The animals had free access to food. The drinking water was the city tap water, which was used for drinking after being filtered and autoclaved. The beddings were corn cobs (Shanghai Maoshengyan Biologic Science & Technology Co., Ltd.) which were used after being autoclaved. The beddings were changed twice a week. The mice were given at least one week of acclimation period for the environment before the experiment.

(175) Other chemical reagents and materials: Human breast cancer MDA-MB-231 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences.

(176) 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. The volumes of administration were all 10 mL.Math.kg.sup.−1. The equivalent effective doses of irinotecan were all 40 mg.Math.kg.sup.−1. Group 1 was a solvent control group, and physiological saline was administered by intravenous injection via tail once every 4 days for a total of 3 times (Q4D×3). Groups 2 to 15 were respectively given the following test samples by intravenous injection via tail once every 4 days, Q4D×3: irinotecan, nktr-102, Compound a, Compound A, Compound b, Compound B, Compound c, Compound C, Compound d, Compound D, Compound e, Compound E, Compound f, and Compound F.

(177) The formulation of the test articles was as shown in Example 15. The volume required for a single administration was 3 mL.

(178) Experimental methods: MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA). The cells were placed in a 37° C. incubator containing 5% CO.sub.2 for cultivation. 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 1×PBS, and the concentration of the cell suspension was adjusted to 1×10.sup.7 cells/mL. 1 mL syringes (4 gauge needle) were used to inoculate the tumor cells to the right back of the nude mice subcutaneously, 1×10.sup.6 cells/0.1 mL/mice. When the tumor volumes reached 100 mm.sup.3 to 200 mm.sup.3, the animals were subjected to random grouping according to randomized block method and divided into 15 groups, so that the difference of the tumor volumes between each group was less than 10% of the mean value. There were 6 animals in each group. Day 1 was the day when the animals were grouped, and the animals were administered on the same day when being grouped. 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. The results were as shown in Table 2.

(179) TABLE-US-00004 TABLE 2 Relative tumor proliferation rate (% T/C) Relative tumor Number of Relative tumor proliferation Group Sample animals volume (RTV) rate (% T/C) 1 blank solvent 6 12.78 — 2 irinotecan 6 6.25 45.23% 3 nktr-102 6 4.63 33.42% 4 Compound a 6 1.25* 9.32%.sup.# 5 Compound A 6 1.26* 9.39%.sup.# 6 Compound b 6 1.72* 12.82%.sup.# 7 Compound B 6 1.70* 12.67%.sup.# 8 Compound c 6 1.56* 11.63%.sup.# 9 Compound C 6 1.64* 12.22%.sup.# 10 Compound d 6 1.87* 13.94%.sup.# 11 Compound D 6 1.90* 14.16%.sup.# 12 Compound e 6 1.78* 13.27%.sup.# 13 Compound E 6 1.69* 12.60%.sup.# 14 Compound f 6 1.88* 14.02%.sup.# 15 Compound F 6 1.93* 14.39%.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.

(180) 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 17 Inhibitory Effect in a Human Pancreatic Cancer MIA Paca-2 Xenograft Model in Nude Mice

(181) Test articles: irinotecan, nktr-102, and 12 compounds of the present disclosure.

(182) Experimental animals: Female BALB/c nude mice. 150 animals were inoculated, and 90 animals were used for the experiment. The animals were 6 to 8 week old and had a body weight of 20 g to 22 g±20% of the average body weight. The animal source was Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. (BK), license number: SCXK (Shanghai) 2008-0016. All experimental animals were housed in an SPF-level laboratory. Experimenters were responsible for daily care and experimental research. 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. After the tumor volumes reached 150 mm.sup.3 to 200 mm.sup.3, the animals were divided into 15 groups with 6 mice in each group using randomized block method, so as to ensure that 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%.

(183) Feeding conditions: the living condition was an IVC system, 6 animals per cage; temperature: 20° C. to 26° C.; humidity: 40%±70%; illumination: 12 hours light/dark cycle. Irradiated feed for rat and mice was purchased from Beijing Keao Xieli Feed Co., Ltd. The animals had free access to food. The drinking water was the city tap water which was used for drinking after being filtered and autoclaved. The beddings were corn cobs (Shanghai Maoshengyan Biologic Science & Technology Co., Ltd.) which were used after being autoclaved. The beddings were changed twice a week. The mice were given at least one week of acclimation period for the environment before the experiment.

(184) Other chemical reagents and materials: Human pancreatic cancer MIA Paca-2 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences.

(185) A human pancreatic cancer MIA Paca-2 subcutaneous transplanted tumor model in nude mice was established, and each animal was inoculated with 3×10.sup.6 cells. The volumes of administration were all 10 mL.Math.kg.sup.−1. The equivalent effective doses of irinotecan were all 40 mg.Math.kg.sup.−1. Group 1 was a solvent control group, and physiological saline was administered by intravenous injection via tail once every 4 days for a total of 3 times (Q4D×3). Groups 2 to 15 were respectively given the following test samples by intravenous injection via tail once every 4 days, Q4D×3: irinotecan, nktr-102, Compound a, Compound A, Compound b, Compound B, Compound c, Compound C, Compound d, Compound D, Compound e, Compound E, Compound f, and Compound F.

(186) The formulation of the test articles was as shown in Example 15. The volume required for a single administration was 3 mL.

(187) Experimental methods: MIA Paca-2 cells were cultured in DMEM containing 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 for cultivation.

(188) 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 1×PBS, and the concentration of the cell suspension was adjusted to 3×10.sup.7 cells/mL. 1 mL syringes (4 gauge needle) were used to inoculate the tumor cells to the right back of the nude mice subcutaneously, 3×10.sup.6 cells/0.1 mL/mice.

(189) When the tumor volumes reached 100 mm.sup.3 to 200 mm.sup.3, the animals were subjected to random grouping according to randomized block method and divided into 15 groups, so that the difference of the tumor volumes between each group was less than 10% of the mean value. There were 6 animals in each group, Day 1 was the day when the animals were grouped, and the animals were administered on the same day when being grouped.

(190) 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. The results were as shown in Table 3.

(191) TABLE-US-00005 TABLE 3 Relative tumor proliferation rate (% T/C) Relative tumor Number of Relative tumor proliferation Group Sample animals volume (RTV) rate (% T/C) 1 blank solvent 6 11.82 — 2 irinotecan 6 8.01 61.08% 3 nktr-102 6 5.37 37.15% 4 Compound a 6 2.38* 13.02%.sup.# 5 Compound A 6 2.45* 13.41%.sup.# 6 Compound b 6 2.90* 15.86%.sup.# 7 Compound B 6 2.87* 15.70%.sup.# 8 Compound c 6 2.65* 13.71%.sup.# 9 Compound C 6 2.66* 13.76%.sup.# 10 Compound d 6 3.12* 16.06%.sup.# 11 Compound D 6 3.07* 15.80%.sup.# 12 Compound e 6 2.78* 14.31%.sup.# 13 Compound E 6 2.84* 14.62%.sup.# 14 Compound f 6 2.69* 14.70%.sup.# 15 Compound F 6 2.71* 14.81%.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.

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

Example 18 Inhibitory Effect on In Vivo Tumor Growth in a Human Gastric Cancer NCI-N87 Cell Strain Transplanted Tumor Model in Nude Mice

(193) Test articles: irinotecan, nktr-102, and 12 compounds of the present disclosure.

(194) Reagents: RPMI-1640 liquid culture medium, fetal bovine serum (FBS), trypsin, penicillin-streptomycin, and physiological saline.

(195) Experimental animals: 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., and 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 hours. 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 this 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.

(196) Transplantable tumor cell strain: 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).

(197) Experimental Methods

(198) Culture of NCI-N87 cells: Under a culture condition of 5% CO.sub.2 and 37° C., NCI-N87 cells were subjected to conventional cell cultivation in an 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.

(199) Preparation of the animal model: NCI-N87 cells in logarithmic phase of growth were collected. The cells were resuspended in an RPMI-1640 culture medium free of serum after being counted, and the concentration of the cells was adjusted to 5×10.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 μL (5×10.sup.6 cells/animal), and an 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, 90 animals with good health condition and similar tumor volumes were selected and divided into 15 groups (n=6) using randomized block method. 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.

(200) The formulation of the test articles were as shown in Example 15. The volume required for a single administration was 3 mL.

(201) Grouping of animals and administration: 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 (Q4D×3). Groups 2 to 15 were respectively given the following test samples by intravenous injection via tail: irinotecan, nktr-102, Compound a, Compound A, Compound b, Compound B, Compound c, Compound C, Compound d, Compound D, Compound e, Compound E, Compound f, and Compound F; and the dosages of administration were all 40 mg.Math.kg.sup.−1 (calculated in terms of the content of irinotecan), Q4D×3.

(202) 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. The results were as shown in Table 4.

(203) TABLE-US-00006 TABLE 4 Relative tumor proliferation rate (% T/C) Average Relative tumor relative tumor Number of volume proliferation Group Sample animals (RTV, mm.sup.3) rate (% T/C) 1 blank solvent 6 3.01 — 2 irinotecan 6 8.02 65.15% 3 nktr-102 6 6.14 50.91% 4 Compound a 6 2.80* 23.27%.sup.# 5 Compound A 6 2.87* 23.86%.sup.# 6 Compound b 6 3.30* 27.43%.sup.# 7 Compound B 6 3.25* 27.01%.sup.# 8 Compound c 6 3.44* 28.61%.sup.# 9 Compound C 6 3.49* 29.03%.sup.# 10 Compound d 6 3.41* 28.37%.sup.# 11 Compound D 6 3.27* 27.21%.sup.# 12 Compound e 6 3.79* 31.51%.sup.# 13 Compound E 6 3.80* 31.60%.sup.# 14 Compound f 6 3.59* 29.87%.sup.# 15 Compound F 6 3.54* 29.45%.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.

(204) 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 19 Effect on the Survival Rate of an Orthotopic U87MG Brain Glioma Model in Nude Mice

(205) Test articles: irinotecan, nktr-102, and 12 compounds of the present disclosure.

(206) Reagents: RPMI-1640 liquid culture medium, trypsin, penicillin-streptomycin, and physiological saline.

(207) Experimental animals: 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., and 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 hours. 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 this 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.

(208) Transplantable tumor cell strain: 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).

(209) Experimental Methods:

(210) Culture of U87MG cells: Under a culture condition of 5% CO.sub.2 and 37° C., U87MG cells were subjected to conventional cell cultivation in an 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.

(211) Preparation of the animal model: U87MG cells in logarithmic phase of growth were collected. The cells were resuspended in an 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 μL of human brain glioma cell (U87MG cells) cultured in vitro was inoculated (1×10.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 the animal status was observed periodically after inoculation. On the 12th day after inoculation, 90 animals were selected and divided into 15 groups (n=6) using randomized block method.

(212) Preparation of the dosing formulations: The formulation of the test articles was as shown in Example 15. The volume required for a single administration was 3 mL.

(213) Grouping of animals and administration: 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 (Q4D×3). Groups 2 to 15 were given irinotecan, nktr-102 and test compounds 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), Q4D×3.

(214) Data recording and calculation formula: The survival time of the animals were recorded. 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. The results were as shown in Table 5.

(215) TABLE-US-00007 TABLE 5 Survival time of the animals (day) Median Number of Survival survival Group Sample animals time time 1 blank solvent 6 16 to 22 20  2 irinotecan 6 22 to 32 27  3 nktr-102 6 25 to 37 31  4 Compound a 6 34 to 46 40* 5 Compound A 6 34 to 46 40* 6 Compound b 6 34 to 41 38* 7 Compound B 6 34 to 41 38* 8 Compound c 6 34 to 45 38* 9 Compound C 6 34 to 45 38* 10 Compound d 6 30 to 39 35* 11 Compound D 6 30 to 39 35* 12 Compound e 6 33 to 44 39* 13 Compound E 6 33 to 44 39* 14 Compound f 6 30 to 42 38* 15 Compound F 6 30 to 42 38* *P < 0.05, as compared with the median survival time of the blank solvent group, irinotecan group and nktr-102 group.

(216) 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.