INHIBITOR OF PROSTATE SPECIFIC MEMBRANE ANTIGEN AND PHARMACEUTICAL USE THEREOF

Abstract

An inhibitor of a prostate specific membrane antigen and a pharmaceutical use thereof. Specifically, the present solution belongs to the field of radiopharmaceuticals and relates to a compound represented by formula (IV) or a pharmaceutically acceptable salt thereof.

##STR00001##

Claims

1. A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, ##STR00158## wherein: Q is selected from the group consisting of H and a protecting group, preferably from H; R.sub.1 and R.sub.2 are each independently selected from the group consisting of H and C.sub.1-4 alkyl and are preferably both H; the C.sub.1-4 alkyl is optionally substituted with one or more substituents P or is unsubstituted; each occurrence of Q, R.sub.1, and R.sub.2 may be the same or different; Y.sub.1 is S or O, preferably O; T is selected from the group consisting of NR.sub.4(CO), NR.sub.4 (SO.sub.2), and NR.sub.4 (CH.sub.2); R.sub.4 is selected from the group consisting of H, C.sub.1-6 alkyl, 6-10 membered aryl, and 5-12 membered heteroaryl; the C.sub.1-6 alkyl, 6-10 membered aryl, or 5-12 membered heteroaryl is optionally substituted with one or more substituents P or is unsubstituted; ring A is selected from 3-12 membered nitrogen-containing heterocyclyl, wherein the 3-12 membered nitrogen-containing heterocyclyl is optionally substituted with one or more substituents P or is unsubstituted; W is selected from the group consisting of 6-10 membered aryl and 5-12 membered heteroaryl; the 10 membered aryl or 5-12 membered heteroaryl is optionally substituted with one or more substituents P or is unsubstituted; the substituents P are selected from the group consisting of C.sub.1-C.sub.6 alkyl, halogen, deuterium, hydroxy, sulfhydryl, NR.sub.iR.sub.j, oxo, thio, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthioether group, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused cycloaryl, and 5- to 12-membered fused heteroaryl; R.sub.i and R.sub.j are each independently selected from the group consisting of a hydrogen atom, hydroxy, C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6 alkoxy; R.sub.k is independently selected from the group consisting of a hydrogen atom, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 alkoxy, hydroxy, and NR.sub.iR.sub.j, wherein the alkyl, alkoxy, or haloalkyl is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkyl, halogen, hydrogen, sulfhydryl, NR.sub.iR.sub.j, oxo, thio, carboxyl, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthioether group, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; y, z, g, and h are each independently integers of 0-6; R.sub.3 is selected from the group consisting of H and a chelating agent.

2. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein T is NH(CO); ring A is a 5-12 membered nitrogen-containing spiroheterocyclyl group, preferably a 5-12 membered nitrogen-containing monospiroheterocyclyl group, more preferably a 3-membered/4-membered, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, 5-membered/6-membered, or 6-membered/6-membered nitrogen-containing monospiroheterocyclyl group, most preferably ##STR00159## ##STR00160## and particularly preferably ##STR00161##

3. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein W is selected from 6-10 membered aryl, preferably naphthyl.

4. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein Y.sub.1 is O.

5. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 and R.sub.2 are each independently H.

6. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein Q is selected from the group consisting of H and a protecting group, preferably from H.

7. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein y and h are each independently selected from the group consisting of 0, 1, and 2, preferably from 1.

8. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein g is selected from the group consisting of 3 and 4, preferably from 3.

9. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, wherein z is selected from the group consisting of 0 and 1, preferably from 0.

10. The compounds or the pharmaceutically acceptable salts thereof according to claim 1, wherein the chelating agent is selected from the group consisting of: ##STR00162## ##STR00163## ##STR00164## preferably from ##STR00165##

11. The compounds according to claim 1, being selected from the group consisting of ##STR00166## ##STR00167## ##STR00168## ##STR00169## ##STR00170## ##STR00171## wherein R.sub.3 is selected from the group consisting of H and ##STR00172##

12. The compound represented by formula (IV) or the pharmaceutically acceptable salt thereof according to claim 1, being ##STR00173## and most preferably ##STR00174##

13. The compound according to claim 1, wherein the chelating agent comprises a radionuclide.

14. The compound or the pharmaceutically acceptable salt thereof according to claim 13, wherein the radionuclide is selected from at least one of .sup.18F, .sup.11C, .sup.68Ga, .sup.124I, .sup.89Zr, .sup.64Cu, .sup.86Y, .sup.99mTc, .sup.111In, .sup.123I, .sup.90Y, .sup.125I, .sup.131I, .sup.177Lu, .sup.211At, .sup.153Sm, .sup.186Re, .sup.188Re, .sup.67Cu, .sup.212Pb, .sup.225Ac, .sup.213Bi, .sup.212Bi, .sup.212Pb, and .sup.67Ga, preferably from the group consisting of .sup.68Ga and .sup.177Lu.

15. A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, being ##STR00175## wherein the chelating agent comprises a radionuclide, and the radionuclide is .sup.68Ga.

16. A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, being ##STR00176## wherein the chelating agent comprises a radionuclide and the radionuclide is .sup.177Lu.

17. A pharmaceutical composition, comprising the compound or the pharmaceutically acceptable salt thereof according to claim 1, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

18. (canceled)

19. (canceled)

20. A method of treating and/or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 wherein preferably, the cancer is prostate cancer.

21. A preparation method for a compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (IV) is the compound represented by formula v or a pharmaceutically acceptable salt thereof; the preparation method comprises the step of removing tert-butyl groups from the compound represented by formula v-5: ##STR00177##

22. (canceled)

23. A preparation method for the compound according to claim 13, comprising the step of preparing the compound represented by formula (IV) and further comprising the step of complexing the chelating agent in the compound represented by formula (IV) or the pharmaceutically acceptable salt thereof with the radionuclide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0241] FIG. 1 shows a comparison of the enzyme activity experiments of PSMA-617, compound v, and compound x.

[0242] FIG. 2 shows 2-h biodistributions of 68Ga-v and 68Ga-x in LnCaP tumor-bearing mice.

[0243] FIG. 3 shows curves of the metabolisms of .sup.68Ga-PSMA-617 and .sup.68Ga-v (Example 9) in the blood of normal mice.

DETAILED DESCRIPTION

[0244] The present disclosure is explained below in more detail with reference to examples. The examples of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and the essence and scope of the present disclosure are not limited to these examples. Unless otherwise specified, all the starting materials used in the present disclosure are normal, commercially available products.

[0245] The NMR shifts (6) are given in 10.sup.6 (ppm). The NMR analyses were performed on a Bruker AVANCE-400 nuclear magnetic resonance instrument, with dimethyl sulfoxide-D6 (DMSO-d.sup.6), chloroform-D (CDCl.sub.3), and methanol-D4 (CD.sub.3OD) as solvents and tetramethylsilane (TMS) as an internal standard.

[0246] The MS analyses were performed on a Shimadzu 2010 Mass Spectrometer or Agilent 6110A MSD Mass Spectrometer.

[0247] The HPLC analyses were performed on Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or Agilent 1200 LC high-performance liquid chromatograph (Ultimate XB-C18 3.0150 mm chromatography column or Xtimate C18 2.130 mm chromatography column).

[0248] In the chiral HPLC analyses, Chiralpak IC-3 1004.6 mm I.D., 3 m, Chiralpak AD-3 1504.6 mm I.D., 3 m, Chiralpak AD-3 504.6 mm I.D., 3 m, Chiralpak AS-3 1504.6 mm I.D., 3 m, Chiralpak AS-3 1004.6 mm I.D., 3 m, ChiralCel OD-3 1504.6 mm I.D., 3 m, Chiralcel OD-3 1004.6 mm I.D., 3 m, ChiralCel OJ-H 1504.6 mm I.D., 5 m, and Chiralcel OJ-3 1504.6 mm I.D., 3 m chromatography columns were used.

[0249] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates, 0.15 mm-0.2 mm layer thickness, were used in the thin-layer chromatography (TLC) analyses and 0.4 mm-0.5 mm layer thickness in the TLC separations and purifications.

[0250] Yantai Huanghai silica gel of 100-200 mesh, 200-300 mesh, or 300-400 mesh was generally used as a carrier in the column chromatography purifications.

[0251] In the preparative chiral chromatography purifications, a DAICEL CHIRALPAK IC (250 mm30 mm, 10 m) or Phenomenex-Amylose-1 (250 mm30 mm, 5 m) column was used.

[0252] The CombiFlash preparative flash chromatograph used was Combiflash Rf150 (TELEDYNE ISCO).

[0253] The kinase mean inhibition rates and IC.sub.50 values were measured using a NovoStar microplate reader (BMG, Germany).

[0254] Known starting materials in the present disclosure may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals, and other companies.

[0255] In the examples, the reactions can all be performed in an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

[0256] The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen gas.

[0257] The hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen gas.

[0258] The pressurized hydrogenation reactions were performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogenator, or an HC2-SS hydrogenator.

[0259] The hydrogenation reactions generally involved 3 cycles of vacuumization/hydrogen filling.

[0260] The microwave reactions were performed using a CEM Discover-S 908860 microwave reactor.

[0261] In the examples, the solutions refer to aqueous solutions unless otherwise specified.

[0262] In the examples, the reaction temperature is room temperature, i.e., 20 C.-30 C., unless otherwise specified.

[0263] The monitoring of the reaction processes in the examples was conducted using thin-layer chromatography (TLC). The developing solvents for the reactions, the eluent systems of column chromatography for compound purification, and the developing solvent systems of thin-layer chromatography include: A: a dichloromethane/methanol system, B: a n-hexane/ethyl acetate system, C: a petroleum ether/ethyl acetate system, and D: a petroleum ether/ethyl acetate/methanol system. The volume ratio of the solvents was adjusted depending on the polarity of the compound, or adjusted by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

[0264] The abbreviations used in the experiments below have the following meanings:

[0265] EtOAc (EA): ethyl acetate; DCM: dichloromethane; THF: tetrahydrofuran; DIPEA: N,N-diisopropylethylamine; PPTS: pyridinium p-toluenesulfonate; Boc: t-butoxycarbonyl; MeOH: methanol; HATU: 2-(7-azabenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate; DIEA: N,N-diisopropylethylamine.

Example 1

[0266] ##STR00127##

(((S)-5-((S)-2-(4-(aminomethyl)piperidine-1-carboxamido)-3-(2-naphthyl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0267] ##STR00128##

Step 1 (Preparation of Resin Compound S-4)

[0268] ##STR00129##

[0269] The starting materials and resin needed were taken out, placed in a desiccator, and equilibrated to room temperature. 25 g of Wang Resin (sub=0.38 mmol/g, 8.75 mmol) (Wang Resin, purchased from Xi'an Sunresin Tech Ltd.) was weighed out and placed into a 500 mL single-necked flask, and 250 mL of DMF was added. The flask was placed in a shaker and shaken for 30 min. Fmoc-Lys(Alloc)-OH (19.8 g, 43.75 mmol) (N-[(9H-fluoren-9-methoxy)carbonyl]-N-[(2-propenyloxy)carbonyl]-L-lysine, purchased from GL Biochem), DIC (5.5 g, 43.75 mmol), HOBt (13.0 g, 43.75 mmol) (1-hydroxybenzotriazole, purchased from Meryer), and DMAP (0.11 g, 0.875 mmol) (4-dimethylaminopyridine, purchased from Energy) were added, and the mixture was left to react on the shaker at room temperature for 23 h. The resin was transferred into a solid-phase reaction column, and the reaction solution was removed. The resin was washed 3 times with DMF, 300 mL per wash. The resin was blocked with 150 mL of pyridine:acetic anhydride=1:1 (V:V). The resin was blocked for 8 h, and the peptide resin was shrunk and dried with methanol to give the product S-1 (8.75 mmol).

[0270] The product S-1 (7 mmol) was swollen with 150 mL of DMF at room temperature, and then 200 mL of 20% DBLK (a 20% piperidine/DMF solution, purchased from Energy) was added for deprotection (10 min). After the liquid was removed, another 200 mL of 20% DBLK was added for deprotection (10 min). A Kaiser test of the resin yielded blue. The resin was drained and washed with DMF until it was neutral, and the product S-2 was obtained.

[0271] The product S-2 (7 mmol) was added to a glutamyl isocyanate reaction solution at room temperature. The reaction was stirred slowly on a thermostatic shaker for 18 h, and a Kaiser test of the resin yielded no color change. The resin was transferred into a solid-phase reaction column, and the reaction solution was removed. The resin was washed 3 times with DMF, 300 mL per wash, to give the product S-3.

[0272] The product S-3 (7 mmol) was added to a reaction column at room temperature. Phenylsilane (4.6 g, 42 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.81 g, 0.7 mmol) were dissolved in 180 mL of DCM, and the resulting solution was added to the reaction column. Nitrogen was bubbled through the mixture for 0.5 h, and the solvent was removed. The procedure was repeated 2 times, and a Kaiser test of the resin yielded bluish black. After the reaction was complete, the solvent was removed. The resin was washed with DMF three times, drained, shrunk three times with methanol, and then dried in vacuo at 30 C. for 2 h to give the product S-4 (22.2 g, 7 mmol) for later use.

Step 2

[0273] The resin compound S-4 (4.0 g, 1.28 mmol) was swollen with dichloromethane (purchased from Sinopharm Chemical Reagent Co., Ltd.) at room temperature for 0.5 h, drained, washed three times with DMF, and drained for later use.

[0274] Fmoc-2-NAL-OH (1.68 g, 3.84 mmol) (Fmoc-3-(2-naphthyl)-L-alanine, purchased from Meryer), HOBt (0.52 g, 3.84 mmol) (1-hydroxybenzotriazole, purchased from Meryer), HATU (1.46 g, 3.84 mmol) 2-(7-azabenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, purchased from Macklin), and DIEA (1.01 g, 7.68 mmol) (N,N-diisopropylethylamine, purchased from Energy) were weighed out and dissolved in DMF (25 mL) (N,N-dimethylformamide, purchased from Energy), and the resulting solution was then added to a solid-phase reaction column containing compound S-4. After 2 h of reaction, a Kaiser test of the resin yielded no color change. The reaction solution was removed, and the resin was washed three times with DMF to give the title product a-1.

Step 3

[0275] At room temperature, 40 mL of 20% DBLK (a 20% piperidine/DMF solution, purchased from Energy) was added to a solid-phase reaction column containing a-1 for deprotection (10 min). After the liquid was removed, another 40 mL of 20% DBLK was added for deprotection (10 min). A Kaiser test of the resin yielded blue. The resin was drained and washed with DMF until it was neutral, and the product, compound a-2, was obtained.

Step 4

[0276] Compound a-2 (1.0 mmol) and triphosgene (0.2 g, 0.68 mmoL) were added to 6 mL of DCM solution at room temperature. The mixture was cooled to 0 C., and DIEA (0.65 g, 5 mmol) was added dropwise. After 2 h of reaction at that temperature, a Kaiser test of the resin yielded no color change. 4-Boc-aminomethylpiperidine was added, and the mixture was warmed to room temperature and left to react for 3 h. The reaction solution was removed, and the resin was washed three times with DMF (N,N-dimethylformamide), shrunk three times with methanol, and then dried in vacuo at 30 C. for 2 h to give the product, compound a-3, for later use.

Step 5

[0277] A 40 mL cleavage solution (TFA (trifluoroacetic acid):H.sub.2O:Tis (triisopropylsilane, purchased from Macklin)=95:2.5:2.5) was prepared at room temperature, and the peptide resin compound 4 was added with stirring. The mixture was left to react for 2 h. Then the mixture was filtered under reduced pressure. The resin was removed, and the filtrate was concentrated by rotary evaporation. The concentrate was added to 100 mL of isopropyl ether. The mixture was filtered under reduced pressure, and the filter cake was dried under reduced pressure to give a crude peptide (0.40 g, 60.9% yield). The crude product was purified by high-pressure preparative liquid chromatography to give the title product, compound a (105 mg, 26.2% yield).

[0278] MS m/z (ESI): 657.3 [M+1].sup.+

[0279] 1H NMR (400 MHz, Deuterium Oxide) 7.82 (t, J=9.1 Hz, 3H), 7.64 (s, 1H), 7.46 (s, 2H), 7.37 (d, J=8.4 Hz, 1H), 4.51 (t, J=8.1 Hz, 1H), 4.14 (s, 1H), 3.85 (dd, J=30.1, 11.1 Hz, 2H), 3.71 (d, J=13.8 Hz, 1H), 3.22 (dd, J=13.6, 7.5 Hz, 1H), 3.14-3.01 (m, 1H), 2.97-2.83 (m, 1H), 2.67 (dt, J=33.6, 12.7 Hz, 2H), 2.40 (dt, J=15.4, 6.9 Hz, 4H), 2.12-1.97 (m, 1H), 1.84 (dd, J=14.3, 7.5 Hz, 1H), 1.68 (s, 1H), 1.49 (d, J=19.0 Hz, 4H), 1.36 (s, 1H), 1.13 (d, J=7.4 Hz, 2H), 0.93 (s, 2H), 0.66 (d, J=12.2 Hz, 1H), 0.54 (s, 1H).

Example 2

[0280] ##STR00130##

(((S)-5-((S)-2-((((1R,4S)-4-(aminomethyl)cyclohexyl)methyl)amino)-3-(2-naphthyl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0281] ##STR00131## ##STR00132##

Step 1

[0282] Trans-4-(Boc-aminomethyl)cyclohexanemethanol compound e-1 (3 g, 12 mmol) was dissolved in 60 mL of DCM at room temperature. The solution was cooled to 60 C., and a DMP/DCM solution (7.95 g, 18 mmol, DCM 60 mL) was added. After the addition, the mixture was naturally warmed to room temperature and stirred for 4 h. TLC monitoring showed the reaction was complete. The reaction solution was washed with 100 mL of an aqueous solution of Na.sub.2CO.sub.3 and Na.sub.2S2O.sub.3 and then with 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=10:1-4:1 to give compound e-2 (1.8 g, 59.7% yield).

[0283] MS m/z (ESI): 242.3 [M+1].sup.+

Step 2

[0284] Compound e-3 (1.56 g, 6.8 mmol) and compound e-2 (1.48 g, 6.1 mmol) were dissolved in 40 mL of a solution of DCM/THF (V1:V2=1:1) at room temperature, and the resulting solution was stirred for 2 h. Sodium cyanoborohydride (0.5 g, 7.9 mmol) and acetic acid (0.3 mL) were slowly added, and the mixture was stirred for 3 h. The reaction solution was washed with water and extracted with DCM (40 mL3). The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=10:1-4:1 to give compound e-4 (1.54 g, 55.6% yield).

[0285] MS m/z (ESI): 455.3 [M+1].sup.+

Step 3

[0286] Compound e-4 (1.5 g, 3.3 mmol) was dissolved in 12 mL of THE and 5 mL of water at room temperature, and lithium hydroxide (0.24 g, 9.9 mmol) was added. After the addition, the mixture was left to react at room temperature overnight. 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL2). The aqueous phases were combined and cooled to 0 C. in an ice bath, and the pH was adjusted to 3-4 with 0.5 N citric acid. A solid precipitated. 100 mL of H.sub.2O and 50 mL of DCM were added, and the mixture was stirred for 0.5 h. A pH test showed the pH did not change. The mixture was filtered, and the filter cake was dried in vacuo (40 C., 4 h) to constant weight to give the product, compound e-5 (1.1 g, 75.9% yield).

[0287] MS m/z (ESI): 441.3 [M+1].sup.+

Step 4

[0288] Compound e-5 (0.13 g, 0.3 mmol), HATU (0.16 g, 0.42 mmol), DIEA (0.18 g, 1.41 mmol), and DCM (1 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. Compound e-6 was added, and the mixture was stirred overnight. The reaction solution was washed with water and extracted with EA (30 mL3). The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=EA (0%-90%) to give the product, compound e-7 (0.13 g, 59.4% yield).

[0289] MS m/z (ESI): 784.4 [M+1].sup.+

Step 5

[0290] Compound e-7 (0.13 g, 0.16 mmol) was dissolved in 2 mL of ethyl acetate at room temperature, and a 2 M HCL/EA solution (2 mL, 4 mmol) was added with stirring. The mixture was stirred for 2 h. TLC monitoring showed the reaction was complete. The reaction solution was concentrated under reduced pressure to constant weight to give the product, compound e-8 (0.11 g, 92% yield).

[0291] MS m/z (ESI): 684.3 [M+1].sup.+

Step 6

[0292] Compound e-8 (0.11 g, 0.16 mmol) was dissolved in 2 mL of THE and 1 mL of water at room temperature, and lithium hydroxide (29 mg, 1.2 mmol) was added. After the addition, the mixture was left to react at room temperature overnight. TFA was added to the reaction solution to adjust the pH to 2-3. The solution was stirred for 0.5 h, and a pH test showed the pH did not change. The reaction solution was purified by high-pressure preparative liquid chromatography to give the title product, compound e (80 mg, 78.4% yield).

[0293] MS m/z (ESI): 640.3 [M1].sup.

[0294] 1H NMR (400 MHz, Deuterium Oxide) 7.94 (t, J=6.7 Hz, 3H), 7.91-7.80 (m, 1H), 7.54 (p, J=7.2 Hz, 1H), 7.46 (t, J=7.8 Hz, 1H), 7.38 (d, J=7.1 Hz, 1H), 4.18 (dt, J=9.1, 5.6 Hz, 2H), 4.05 (d, J=7.6 Hz, 2H), 3.69 (dd, J=13.3, 4.9 Hz, 2H), 3.53 (t, J=12.3 Hz, 2H), 2.94-2.70 (m, 3H), 2.42 (s, 1H), 2.08 (s, 2H), 1.80 (s, 1H), 1.68 (s, 2H), 1.58 (s, 2H), 1.39 (d, J=9.7 Hz, 2H), 1.27 (s, 3H), 1.02 (q, J=10.5, 9.9 Hz, 3H), 0.63 (s, 2H), 0.46 (s, 2H).

Example 3

[0295] ##STR00133##

(((S)-5-((S)-2-(4-(aminomethyl)-1-pyrazolyl)-3-phenylpropanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0296] ##STR00134##

Step 1

[0297] Boc-L-tyrosine methyl ester compound g-1 (0.5 g, 2.78 mmol) was dissolved in 120 mL of DCM at room temperature, and pyridine (1.2 mL, 14.61 mmol) was added. The mixture was cooled to 0 C., and trifluoromethanesulfonic anhydride (2.4 mL, 14.10 mmol) was added. After the addition, the mixture was naturally warmed to room temperature and stirred for 3 h. TLC monitoring showed the reaction was complete. The reaction solution was washed with 100 mL of saturated sodium bicarbonate solution, with 100 mL of 1 N HCL, and then with 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give compound g-2 (0.84 g, 99.0% yield).

Step 2

[0298] 4-(Boc-aminomethyl)pyrazole (0.61 g, 3.01 mmol) was dissolved in 25 mL of DCM at room temperature, and DIEA (0.49 g, 3.72 mmol) was added. The mixture was stirred for 1 h. A solution of compound 2 (0.84 g, 2.69 mmol) in DCM was added dropwise, and the mixture was stirred overnight. The reaction solution was washed with 30 mL of saturated sodium bicarbonate solution and then with 30 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=10:1-3:1 to give compound g-3 (0.51 g, 48.4% yield).

[0299] MS m/z (ESI): 360.2 [M+1].sup.+

Step 3

[0300] Compound g-3 (0.51 g, 1.4 mmol) was dissolved in 1.5 mL of THE and 1 mL of water at room temperature, and lithium hydroxide was added. After the addition, the mixture was left to react at room temperature overnight. 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL2). The aqueous phases were combined and cooled to 0 C. in an ice bath, and the pH was adjusted to 3-4 with 0.5 N citric acid. An ethyl ester (10 mL3) was added for extraction. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the title product, compound g-4 (0.39 g, 80.0% yield).

[0301] MS m/z (ESI): 344.5 [M1].sup.

Step 4

[0302] The resin compound S-4 prepared in Example 1 (1.1 g, 0.38 mmol) was swollen with DCM at room temperature for 0.5 h, drained, and washed three times with DMF for later use.

[0303] Compound g-4 (0.36 g, 1.04 mmol), HATU (0.4 g, 1.04 mmol), HOBt (0.14 g, 1.04 mmol), DIEA (0.27 g, 2.08 mmol), and DMF (10 mL) were added to a reaction flask, and the flask was shaken to form a clear solution. The swollen resin prepared in advance was added to the reaction flask, and the flask was shaken overnight. A Kaiser test of the resin yielded no color change. The reaction solution was removed, and the resin was washed three times with DMF, shrunk with methanol, and then dried for later use. The product, compound g-5, was obtained.

Step 5

[0304] A 10 mL cleavage solution (TFA:H2O:Tis=95:2.5:2.5) was prepared at room temperature, and the peptide resin compound g-5 was added with stirring. The mixture was left to react for 2 h. Then the mixture was filtered under reduced pressure. The resin was removed, and the filtrate was concentrated by rotary evaporation. The concentrate was added to 40 mL of isopropyl ether, and a solid precipitated. The mixture was filtered under reduced pressure, and the resulting solid was dried under reduced pressure to give a crude peptide (0.15 g, 72.1% yield). The crude product was purified by high-pressure preparative liquid chromatography to give the title product, compound g (26 mg, 17.3% yield).

[0305] MS m/z (ESI): 547.8 [M+1].sup.+

[0306] 1H NMR (400 MHz, Deuterium Oxide) 7.77 (s, 1H), 7.54 (s, 1H), 7.26-7.13 (m, 3H), 7.13-7.06 (m, 2H), 5.07 (t, J=8.2 Hz, 1H), 4.17-4.08 (m, 1H), 3.95 (s, 3H), 3.32 (d, J=8.2 Hz, 2H), 3.06 (dt, J=12.8, 6.2 Hz, 1H), 2.92 (dt, J=13.4, 6.5 Hz, 1H), 2.37 (t, J=7.3 Hz, 2H), 2.04 (dq, J=13.1, 7.2 Hz, 1H), 1.83 (dq, J=14.9, 7.3 Hz, 1H), 1.59 (s, 1H), 1.47 (dd, J=9.4, 4.7 Hz, 1H), 1.25-1.17 (m, 2H), 0.98 (d, J=6.7 Hz, 2H).

Example 4

[0307] ##STR00135##

(((S)-5-((S)-2-amino-3-(4-pyridinyl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0308] ##STR00136##

Step 1

[0309] The resin compound S-4 (1.7 g, 0.65 mmol) was swollen with DCM at room temperature for 0.5 h, drained, and washed three times with DMF for later use.

[0310] Boc-3-(4-pyridinyl)-L-alanine (0.54 g, 2.01 mmol), HATU (0.76 g, 2.01 mmol), HOBt (0.27 g, 2.01 mmol), DIEA (0.52 g, 4.02 mmol), and DMF (15 mL) were added to a reaction flask, and the flask was shaken to form a clear solution. The swollen resin prepared in advance was added to the reaction flask, and the flask was shaken for 2 h. A Kaiser test of the resin yielded no color change. The reaction solution was removed, and the resin was washed three times with DMF, shrunk with methanol, and then dried for later use. The title product, compound j-1, was obtained.

Step 2

[0311] A 20 mL cleavage solution (TFA:H.sub.2O:Tis=95:2.5:2.5) was prepared at room temperature, and the peptide resin compound 2 was added with stirring. The mixture was left to react for 2 h. Then the mixture was filtered under reduced pressure. The resin was removed, and the filtrate was concentrated by rotary evaporation. The concentrate was added to 50 mL of isopropyl ether, and a solid precipitated. The mixture was filtered under reduced pressure, and the resulting solid was dried under reduced pressure to give a crude peptide (0.20 g, 66.7% yield). The crude product was purified by high-pressure preparative liquid chromatography to give the title product, compound j (75 mg, 37.5% yield).

[0312] MS m/z (ESI): 468.8 [M+1].sup.+

[0313] 1H NMR (400 MHz, Deuterium Oxide) 8.73-8.66 (m, 2H), 7.90 (d, J=6.3 Hz, 2H), 4.17 (ddd, J=23.5, 9.1, 5.6 Hz, 2H), 4.00 (dd, J=8.9, 5.0 Hz, 1H), 3.50-3.30 (m, 2H), 3.08 (dt, J=13.6, 6.8 Hz, 1H), 2.95 (dt, J=13.5, 6.8 Hz, 1H), 2.40 (t, J=7.3 Hz, 2H), 2.07 (dq, J=13.2, 7.2 Hz, 1H), 1.85 (ddd, J=16.1, 14.1, 7.1 Hz, 1H), 1.59 (dtd, J=55.3, 14.5, 14.1, 7.9 Hz, 2H), 1.25 (s, 2H), 1.08 (d, J=9.0 Hz, 2H).

Example 5

[0314] ##STR00137##

(((S)-5-(2-(4-((S)-2-(2-aminoacetamido)-2-carboxyethyl)phenoxy)acetamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0315] ##STR00138##

Step 1

[0316] Fmoc-L-tyrosine tert-butyl ester (5.0 g, 10.9 mmol) was dissolved in 50 mL of DMF at room temperature, and potassium carbonate (1.81 g, 13.1 mmol) was added. The mixture was stirred at room temperature for 12 h. Boc-glycine (2.29 g, 13.1 mmol) and HATU (4.98 g, 13.1 mmol) were added, and the mixture was cooled to 0 C. in an ice bath. DIEA was added dropwise, and the mixture was stirred at room temperature for 2 h. 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL2). The organic phases were combined, washed with saturated sodium bicarbonate solution (100 mL), with saturated ammonium chloride solution (100 mL2), and with saturated sodium chloride solution (100 mL2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of PE/EA=100:1-50:50 to give the product, compound 0-1 (3.8 g, 70% yield).

[0317] MS m/z (ESI): 395.4 [M+1].sup.+

Step 2

[0318] Compound O-1 (3.8 g, 9.7 mmol) was dissolved in 40 mL of DMF at room temperature, and potassium carbonate (1.81 g, 13.1 mmol) and methyl bromoacetate (2.22 g, 14.5 mmol) were added. The mixture was heated to 80 C. and stirred for 5-10 h. 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL2). The organic phases were combined, washed with saturated sodium chloride solution (100 mL2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the product, compound 0-2 (4.2 g, 110% yield).

[0319] MS m/z (ESI): 467.4 [M+1].sup.+

Step 3

[0320] Compound O-2 (4.2 g, 9.0 mmol) was dissolved in 50 mL of THE and 50 mL of water at room temperature. The solution was cooled to 0 C. in an ice bath, and an aqueous solution of lithium hydroxide was slowly added. After the addition, the mixture was left to react at room temperature for 2 h. The reaction solution was extracted with ethyl acetate (100 mL2). The aqueous phases were combined and cooled to 0 C. in an ice bath, and the pH was adjusted to 3-4 with 0.5 N dilute hydrochloric acid. Ethyl acetate (100 mL2) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of DCM/MeOH=100:1-20:1 to give the product, compound 0-3 (3.5 g, 80% yield).

[0321] MS m/z (ESI): 451.4 [M1].sup.

Step 4

[0322] Compound O-3 (500 mg, 1.1 mmol) was dissolved in 30 mL of DCM at room temperature, and HATU was added. The mixture was cooled to 0 C. in an ice bath, and DIEA was added dropwise. The mixture was stirred at room temperature for 0.5 h. The mixture was cooled to 0 C. in an ice bath, and compound 0-4 was added. The mixture was stirred at room temperature for 2-5 h. 100 mL of water was added to the reaction solution, and the mixture was extracted with DCM (100 mL2). The organic phases were combined, washed with saturated sodium bicarbonate solution (100 mL) and with saturated sodium chloride solution (100 mL2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of DCM/MeOH=100:1-20:1 to give the product, compound 0-5 (970 mg, 80% yield).

[0323] MS m/z (ESI): 796.5 [M+1].sup.+

Step 5

[0324] Compound O-5 (970 mg, 1.22 mmol) was dissolved in 5 mL of ethyl acetate at room temperature, and a 2 N solution of hydrogen chloride in ethyl acetate (15 mL) was added. The mixture was stirred for 1-2 h. The reaction solution was concentrated under reduced pressure to give the product, compound 0-6 (1.2 g, 110% yield).

[0325] MS m/z (ESI): 696.5 [M+1].sup.+

Step 6

[0326] Compound O-6 (200 mg, 0.29 mmol) was dissolved in 10 mL of THE and 10 mL of water at room temperature. The solution was cooled to 0 C. in an ice bath, and lithium hydroxide (41.76 mg, 1.74 mmol) was slowly added. After the addition, the mixture was left to react at room temperature for 12 h. The reaction solution was extracted with ethyl acetate (20 mL2). The aqueous phases were combined and cooled to 0 C. in an ice bath, and the pH was adjusted to 2-3 with 1 N dilute hydrochloric acid. The mixture was purified by high-pressure preparative liquid chromatography to give the title product, compound O (20 mg, 20% yield).

[0327] MS m/z (ESI): 598.3 [M+1].sup.+

[0328] 1H NMR (400 MHz, D2O) 7.15 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.7 Hz, 2H), 4.52 (s, 2H), 4.44 (dd, J=8.9, 5.1 Hz, 1H), 4.09 (dd, J=8.6, 5.0 Hz, 1H), 3.99 (dd, J=8.2, 4.9 Hz, 1H), 3.73-3.58 (m, 3H), 3.18 (t, J=6.6 Hz, 2H), 3.10-3.04 (m, 1H), 2.84 (dd, J=14.6, 9.1 Hz, 1H), 2.37 (t, J=7.3 Hz, 2H), 2.07-2.00 (m, 1H), 1.88-1.79 (m, 1H), 1.67 (s, 1H), 1.55 (d, J=7.5 Hz, 1H), 1.46-1.39 (m, 2H), 1.20 (d, J=15.5 Hz, 3H).

Example 6

[0329] ##STR00139##

(((S)-5-(6-(4-(aminomethyl)benzamido)picolinamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0330] ##STR00140##

Step 1

[0331] Compound q-1 (3 g, 11.94 mmol) was dissolved in 60 mL of dichloromethane at room temperature, and EDCI (5.49 g, 28.66 mmol) (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, purchased from Macklin) and DMAP (4.37 g, 35.82 mmol) were added. The mixture was stirred in a N.sub.2 atmosphere for 20 min, and compound q-2 (2.17 g, 14.33 mmol) was added. The mixture was stirred at room temperature for 16-18 h. 60 mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane (100 mL3). The organic phases were combined, with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with an eluent system (PE/EA=100%-50%) to give the product, compound q-3 (2.0 g, 43.5% yield).

[0332] MS m/z (ESI): 386.2 [M+1].sup.+

Step 2

[0333] Compound q-3 (1 g, 2.59 mmol) was dissolved in 6 mL of tetrahydrofuran at room temperature, and 4 mL of an aqueous solution of LiOH (187 mg, 7.77 mmol) was added. The mixture was stirred at room temperature for 16-18 h. 20 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL2). The pH of the aqueous phase was adjusted to 3-4 with 0.5 mol/L citric acid, and the aqueous phase was then extracted with ethyl acetate (50 mL4). The organic phases were combined, with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the title product, compound q-4 (0.6 g, 62.5% yield).

[0334] MS m/z (ESI): 372.2 [M+1].sup.+

Step 3

[0335] The resin compound S-4 (2.11 g, 0.68 mmol) was swollen with DMF (20 mL) at room temperature for 30 min. Compound q-4 (760 mg, 2.05 mmol), HATU (779 mg, 2.05 mmol), HOBt (277 mg, 2.05 mmol), and DIEA (529 mg, 4.09 mmol) were dissolved in DMF (15 mL), and the solution was then added to the swollen resin. The mixture was left to react at room temperature for 2.5 h. A small amount of resin was collected, filtered under reduced pressure, and then washed with DMF (2 mL3). A ninhydrin test of the resin yielded no color change. The reaction resin was filtered under reduced pressure and then washed with DMF (50 mL3), DCM (50 mL3), and isopropyl ether (50 mL3) to give a wet resin compound q-5.

Step 4

[0336] The resin compound q-5 obtained in the previous step was added to TFA:Tis:H.sub.2O=95:2.5:2.5 (20 mL) at room temperature, and the mixture was left to react at room temperature for 2 h. The resin was filtered under reduced pressure and with TFA (5 mL3). The filtrate was concentrated under reduced pressure until no significant fraction was produced. The resulting oily liquid was added dropwise to isopropyl ether (20 mL). After filtration, the filter cake was purified by preparative chromatography to give compound q (20 mg).

[0337] MS m/z (ESI): 573.2 [M+1]+.

[0338] 1H NMR (400 MHz, D2O): 8.04 (d, 1H), 7.86 (dd, 3H), 7.65 (d, 1H), 7.49 (d, 2H), 4.18 (s, 2H), 4.18 (s, 2H), 4.12-4.06 (m, 2H), 3.29 (s, 1H), 3.36-3.20 (m, 2H), 2.32 (t, 2H), 2.03-1.94 (m, 1H), 1.87-1.72 (m, 2H), 1.70-1.47 (m, 3H), 1.37 (d, 2H).

Example 7

[0339] ##STR00141##

(((S)-5-((S)-2-(3-(aminomethyl)bicyclo[1.1.1]pentane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid

[0340] ##STR00142##

Step 1

[0341] r-1 (106 mg, 0.44 mmol), HATU (166 mg, 0.44 mmol), DIEA (113 mg, 0.88 mmol), and DCM (10 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. r-2 (200 mg, 0.29 mmol) was added, and the mixture was stirred overnight. TLC monitoring showed the reaction was complete. The reaction solution was washed with water and extracted with DCM (30 mL3). The organic phases were combined, washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol=methanol (0%-7%) to give r-3 (200 mg, 75.4% yield).

[0342] MS m/z (ESI): 908.9 [M+1].sup.+

Step 2

[0343] r-3 (200 mg, 0.42 mmoL) was dissolved in TFA (5 mL) at room temperature. After the addition, the solution was left to react at 33 C. overnight. The reaction solution was concentrated to dryness and purified by high-pressure preparative liquid chromatography to give the title product r (71.5 mg, 50.7% yield).

[0344] MS m/z (ESI): 640.4 [M1].sup.

Example 8

[0345] ##STR00143##

(((1S)-1-carboxy-5-((2S)-3-(naphthalen-2-yl)-2-(6-azaspiro[2.5]octane-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid

[0346] ##STR00144##

Step 1

[0347] N-E-Benzyloxycarbonyl-L-lysine tert-butyl ester hydrochloride (10 g, 0.03 mol) and DIEA (3.84 g, 0.03 mol) were dissolved in 120 mL of DCM. The solution was cooled to 10 C.-0 C. and stirred for 0.5 h, and triphosgene (4.4 g, 0.015 mol) was added. After the addition, DIEA (19.2 g, 0.149 mol) was added dropwise at 10 C.-0 C. After the dropwise addition, the mixture was left to react at that temperature for 3 h. L-Glutamic acid di-tert-butyl ester hydrochloride (10 g, 0.039 mol) was added, and the mixture was naturally warmed to room temperature and stirred overnight. TLC monitoring showed the reaction was complete. The reaction solution was washed with 100 mL of saturated NaHCO.sub.3 solution and then with 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=10:1-1:1. The solvent was evaporated, and t-3 was obtained as an oil (9.8 g, 53.2% yield).

[0348] MS m/z (ESI): 622.3 [M+1].sup.+

Step 2

[0349] t-3 (9.8 g, 15.8 mmol) was dissolved in 100 mL of methanol solution at room temperature, and the mixture was stirred to form a clear solution. Pd/C (4.9 g, 58% water content) was added. The reaction flask was purged with nitrogen 3 times and hydrogen 3 times. The mixture was stirred at room temperature for 5 h, and TLC monitoring showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to give t-4 (6.2 g, 80.7% yield).

[0350] MS m/z (ESI): 488.3 [M+1].sup.+

Step 3

[0351] Fmoc-3-(2-naphthyl)-L-alanine (2.3 g, 5.3 mmol), HATU (2.0 g, 5.3 mmol), DIEA (2.1 g, 16.4 mmol), and DCM (20 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. t-4 (2 g, 4.1 mmol) was added, and the mixture was stirred overnight. TLC monitoring showed the reaction was complete. The reaction solution was washed with water and extracted with EA (30 mL3). The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol=methanol (0%-10%) to give t-5 (2.8 g, 76% yield).

[0352] MS m/z (ESI): 907.5 [M+1].sup.+

Step 4

[0353] t-5 (2.8 g, 3 mmol) and DCM (20 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. Diethylamine was added, and the mixture was stirred overnight. The reaction solution was washed with water and extracted with EA (30 mL3). The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=EA (0%-100%) to give the title product t-6 (1.4 g, 66.7% yield).

[0354] MS m/z (ESI): 685.4 [M+1].sup.+

Step 5

[0355] 6-Boc-6-azaspiro[2.5]octane-1-carboxylic acid (145 mg, 0.57 mmol), HATU (216 mg, 0.57 mmoL), DIEA (226 mg, 1.75 mmoL), and DCM (4 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. t-6 (300 mg, 0.44 mmol) was added, and the mixture was stirred overnight. TLC monitoring showed the reaction was complete. The reaction solution was washed with water and extracted with EA (30 mL3). The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol=methanol (0%-10%) to give the title product t-8 (300 mg, 74.2% yield).

[0356] MS m/z (ESI): 922.8 [M+1].sup.+

Step 6

[0357] t-8 (300 mg, 325 mmol) was dissolved in 2 mL of DCM at room temperature, and TFA (3 mL) was added. After the addition, the mixture was left to react at 30 C. overnight. The reaction solution was concentrated to dryness and purified by high-pressure preparative liquid chromatography to give the title product t (70 mg, 32.8% yield).

[0358] MS m/z (ESI): 654.3 [M1].sup.

Example 9

[0359] ##STR00145##

(((S)-1-carboxy-5-((S)-3-(2-naphthyl)-2-((6S,9r)-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecanyl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid

[0360] ##STR00146##

Step 1

[0361] DOTA (1636 mg, 2.86 mmol), HATU (1087 mg, 2.86 mmol), DIEA (N,N-diisopropylethylamine, 851 mg, 6.6 mmol), and DCM (10 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. v-1 (500 mg, 2.20 mmol) was added, and the mixture was stirred overnight. TLC monitoring showed the reaction was complete. The reaction solution was washed with water and extracted with EA (30 mL3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol to give the title product v-2 (1290 mg, 75.1% yield).

[0362] MS m/z (ESI): 782.5 [M+1].sup.+

Step 2

[0363] v-2 (800 mg, 1.01 mmol) was dissolved in 12 mL of THF and 10 mL of water at room temperature, and lithium hydroxide (73 mg, 3.2 mmol) was added. After the addition, the mixture was left to react at room temperature overnight. Dilute hydrochloric acid was added to the reaction solution to adjust the pH to 2-3. The reaction solution was stirred for 0.5 h, and a pH test showed the pH did not change. The reaction solution was extracted with an ethyl ester. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give the title product v-3 (675 mg, 87.5% yield).

[0364] MS m/z (ESI): 754.4 [M+1].sup.+

Step 3

[0365] v-3 (600 mg, 0.80 mmol), HATU (303 mg, 0.80 mmol), DIEA (316 mg, 2.45 mmol), and DCM (8 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. v-4 (420 mg, 0.61 mmol, see t-6 of Example 8 for the preparation method) was added, and the mixture was stirred. TLC monitoring showed the reaction was complete. The reaction solution was washed with water and extracted with EA (30 mL3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol to give v-5 (638 mg, 73.3% yield).

[0366] MS m/z (ESI): 1421 [M+1].sup.+

Step 4

[0367] v-5 (500 mg, 0.35 mmol) was dissolved in 4 mL of DCM at room temperature, and TFA (5 mL) was added. After the addition, the mixture was left to react at 30 C. overnight. The reaction solution was concentrated to dryness and purified by high-pressure preparative liquid chromatography to give the title product v (27 mg, 7.1% yield).

[0368] MS m/z (ESI): 1084.5 [M1].sup.

[0369] 1H NMR (400 MHz, Deuterium Oxide) 7.84 (t, 3H), 7.65 (s, 1H), 7.47-7.49 (m, 2H), 7.38 (d, 1H), 4.55 (t, 1H), 4.18-4.19 (m, 1H), 3.10-3.87 (m, 33H), 2.42 (t, 2H), 1.91 (dt, 1H), 1.89 (m, 1H), 1.87 (m, 3H), 0.84-1.42 (m, 14H).

Example 10

[0370] ##STR00147##

(((S)-1-carboxy-5-((S)-3-(2-naphthyl)-2-(4-((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecanyl)-1-acetyl)methyl)piperidine-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid

[0371] ##STR00148##

Step 1

[0372] w-2 (3 g, 11.3 mmol) and DIEA (1.45 g, 11.3 mmol) were dissolved in 120 mL of DCM at room temperature. The solution was cooled to 10 C.-0 C. and stirred for 0.5 h, and triphosgene (1.7 g, 5.7 mmol) was added. After the addition, DIEA (7.3 g, 56.6 mmol) was added dropwise at 10 C.-0 C. After the dropwise addition, the mixture was left to react at that temperature for 3 h. w-1 (3.2 g, 14.7 mmol) was added, and the mixture was naturally warmed to room temperature and stirred overnight. TLC monitoring showed the reaction was complete. The reaction solution was washed with 100 mL of saturated NaHCO.sub.3 solution and then with 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of n-heptane/ethyl ester=10:1-1:1. The solvent was evaporated, and w-3 was obtained as a white solid (4.0 g, 75.5% yield).

[0373] MS m/z (ESI): 470.6 [M+1].sup.+

Step 2

[0374] w-3 (4.0 g, 8.5 mmol) was dissolved in 20 mL of THE and 10 mL of water at room temperature, and lithium hydroxide (0.62 g, 25.6 mmol) was added. After the addition, the mixture was left to react at room temperature overnight. 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL2). The aqueous phases were combined and cooled to 0 C. in an ice bath, and the pH was adjusted to 3-4 with 0.5 N citric acid. A solid precipitated. The mixture was stirred for 0.5 h, and a pH test showed the pH did not change. The mixture was filtered, and the filter cake was dried in vacuo (40 C., 4 h) to constant weight to give the title product w-4 (3.4 g, 87.6% yield).

[0375] MS m/z (ESI): 456.6 [M+1].sup.+

Step 3

[0376] w-4 (1.0 g, 2.2 mmol), HATU (1.02 g, 2.7 mmol), DIEA (1.39 g, 10.8 mmol), and DCM (20 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. w-5 (0.66 g, 1.8 mmol) was added, and the mixture was stirred overnight. The reaction solution was washed with water and extracted with DCM (40 mL3). The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol=methanol (0%-10%) to give the title product w-6 (0.86 g, 59.4% yield).

[0377] MS m/z (ESI): 799.4 [M+1].sup.+

Step 4

[0378] w-6 (0.86 g, 1.1 mmol) was dissolved in 2 mL of ethyl acetate at room temperature, and a 4 M HCl/EA solution (8 mL, 32 mmol) was added with stirring. The mixture was stirred for 2 h. TLC monitoring showed the reaction was complete. The reaction solution was concentrated under reduced pressure to constant weight to give the title product w-7 (0.82 g, 95.3% yield).

[0379] MS m/z (ESI): 699.3 [M+1].sup.+

Step 5

[0380] DOTA-tris(t-Bu ester) (209 mg, 0.36 mmol), HATU (137 mg, 0.36 mmol), DIEA (464 mg, 3.6 mmol), and DCM (3 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. w-7 (170 mg, 0.24 mmol) was added, and the mixture was stirred overnight. There was starting material left. More DOTA-tris(t-Bu ester) (139 mg, 0.24 mmol) and HATU (91 mg, 0.24 mmol) were added. After another 2 h, the reaction was complete. The reaction solution was washed with water and extracted with DCM (20 mL3). The organic phases were combined, washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with an eluent system of dichloromethane/methanol=methanol (0%-20%) to give w-8 (110 mg, 36.6% yield).

[0381] MS m/z (ESI): 1253.4 [M+1].sup.+

Step 6

[0382] w-8 (110 mg) was dissolved in 2 mL of THE and 1 mL of water at room temperature, and lithium hydroxide was added. After the addition, the mixture was left to react at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure to give w-9.

Step 7

[0383] w-9 and TFA (2 mL) were added to a reaction flask at room temperature and stirred to form a clear solution. After the addition, the solution was left to react at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure and purified by high-pressure preparative liquid chromatography to give the title product w (13 mg).

[0384] MS m/z (ESI): 1043.6 [M+1].sup.+

Example 11

[0385] ##STR00149##

(((S)-1-carboxy-5-((S)-3-(2-naphthyl)-2-((6R,9s)-4-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecanyl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid

[0386] Compound x was obtained using the preparation method of Example 9.

Example 12. Preparation of Compound .SUP.177.Lu-v

[0387] The total volume of the reaction was 400 L, including 15 nmol of compound v and 15 mCi of .sup.177Lu. To a 1.5 mL centrifuge tube was added 321 L of acetic acid-sodium acetate buffer (0.1 M, pH 4.5), followed by 15 L of a solution of compound v. 7 L of the nuclide .sup.177LuCl3 (activity: 15.23 mCi) was taken. The reaction was shaken on a thermostatic mixer at 95 C., and the reaction time was 15 min. Activity: 15.15 mCi. The HPLC result reached >99%.

Example 13. Preparation of Compound .SUP.68.Ga-v

[0388] 13.5 mg of compound v was weighed out and dissolved in ultrapure water to form a 25 mL solution. 136 mg of sodium acetate trihydrate was weighed out and dissolved in 1 mL of ultrapure water. 20 L of the solution obtained in step 1 was transferred to a reaction vial using a pipette, and 4.5 mL of a hydrochloric acid eluate of .sup.68GaCl.sub.3 and 0.5 mL of the buffer of step 2 were added sequentially. The vial was gently shaken to mix the contents, left to stand at 95 C. for 10 min, and naturally cooled to room temperature. The reaction mixture was sent for analysis and used.

Test Example 1. Tests for Inhibitory Activity Against PSMA

I. Experimental Materials and Instruments

[0389] 1. Multifunctional microplate reader (SPARK, TECAN) [0390] 2. rhPSMA (R&D, 4234-ZN) [0391] 3. N-Acetyl-Asp-Glu (Sigma, A5930) [0392] 4. OPA (Sigma, P0657)

II. Experimental Procedure

[0393] PSMA inhibitors can bind to the enzyme PSMA to prevent the enzyme PSMA from decomposing the substrate N-Acetyl-Asp-Glu. In this experiment, the extent to which the substrate was decomposed and the resulting ultraviolet absorption changes were measured to evaluate the capacities of the PSMA inhibitors to bind to the enzyme PSMA, and the activity of the compounds was evaluated with IC.sub.50 values.

[0394] Buffer 1 (50 mM HEPES, 0.1 M NaCl, pH 7.5) was used to prepare a 0.4 g/mL rhPSMA solution and a 40 M solution of the substrate N-Acetyl-Asp-Glu. rhPSMA was mixed with the small molecules to be tested in a 96-well plate, with a constant rhPSMA content of 50 ng/well maintained. Meanwhile, the small molecules were step-wise diluted to final concentrations of 1 M, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.137 nM, 0.045 nM, and 0 nM. In addition, a positive control was set up using PSMA-617. The rhPSMA-small molecules were taken at 40 L/well and well mixed with the 40 M solution of the substrate N-Acetyl-Asp-Glu (40 L/well). The mixtures were incubated at 37 C. in the dark for 1 h, were heated at 70 C. for 5 min to quench the reactions, and were cooled to room temperature. Buffer 2 (0.2 M NaOH, 0.1% beta-Mercaptoethanol) was used to prepare a 15 mM OPA solution. The OPA solution was added to the reaction systems at 80 L/well and well mixed, and then the plate was incubated at room temperature for 10 min. The mixtures were taken at 100 L/well and added to a 96-well Flat Black. With the excitation wavelength set to 330 nm and the emission wavelength to 465 nm, the intensity of signals was measured. IC.sub.50 values were obtained from dose-response curves.

III. Experimental Data

[0395] The capacities of the compounds of the present disclosure to bind to the enzyme GCPII can be measured through the assay described above. The ICso measurements are shown in Table 1.

TABLE-US-00002 TABLE 1 The IC.sub.50 values of compounds Compound structure IC.sub.50 (nM) [00150] 6.232 [00151] 142.6 [00152] 177.5 [00153] 2.422 [00154] 2.833 [00155] 2.396 [00156] 1.518 [00157] 4.135 PSMA-617 2.358

Test Example 2. Affinity Assays Using Enzyme Activity Method

[0396] Buffer 1 (50 mM HEPES, 0.1 M NaCl, pH 7.5) was used to prepare a 0.4 g/mL rhPSMA solution and a 40 M solution of the substrate N-Acetyl-Asp-Glu. rhPSMA was mixed with the small molecules to be tested in a 96-well plate, with a constant rhPSMA content of 50 ng/well maintained. Meanwhile, the small molecules were step-wise diluted to final concentrations of 1 M, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.137 nM, 0.045 nM, and 0 nM. In addition, a positive control was set up using PSMA-617. The rhPSMA-small molecules were taken at 40 L/well and well mixed with the 40 M solution of the substrate N-Acetyl-Asp-Glu (40 L/well). The mixtures were incubated at 37 C. in the dark for 1 h, were heated at 70 C. for 5 min to quench the reactions, and were cooled to room temperature. Buffer 2 (0.2 M NaOH, 0.1% beta-Mercaptoethanol) was used to prepare a 15 mM OPA solution. The OPA solution was added to the reaction systems at 80 L/well and well mixed, and then the plate was incubated at room temperature for 10 min. The mixtures were taken at 100 L/well and added to a 96-well Flat Black. With the excitation wavelength set to 330 nm and the emission wavelength to 465 nm, the intensity of signals was measured. IC.sub.50 values were obtained from dose-response curves.

TABLE-US-00003 TABLE 2 Compound IC.sub.50 (nM) Ratio to PSMA-617 PSMA-617 1.654 1.00 Example 9 1.231 0.74 Example 11 13.01 7.85

[0397] The specific structure is shown in FIG. 1. Through the enzyme activity experiment, it can be determined that compound v of Example 9 has a better affinity than compound x of Example 11.

Test Example 3. Biodistribution of Compounds in Tumor-Bearing Mice

[0398] After single tail-vein injections into mice, the in-vivo distributions of .sup.68Ga-labeled compound v (Example 9) and compound x (Example 11) in the positive LnCaP tumor-bearing mice were observed.

[0399] At 2 h after the injection, a total of 3 animals were sacrificed by cervical dislocation, and tissue samples were collected, including samples of the blood, the heart, the lungs, the liver, the spleen, the kidneys, the stomach, the intestine, the bones, the flesh, the brain, the salivary gland, the large intestine, the pancreas, and the tumor. The net weight of the tissues was measured first, and the radioactivity counts of the collected tissues were then measured using a -counter. The distributions of the labeled compounds in the different tissues and organs of the mice were measured. Meanwhile, a test sample was accurately diluted 100-fold, and 0.1 mL of the dilution was added to a counting tube and used as standard 1% ID (i.e., one percent of the injected dose). The radioactivity counts of the 1% ID standard and the biological samples were measured simultaneously on a -counter. The biodistribution data were expressed as the percentage of the radioactivity counts per gram of tissue or organ to the total injected dose (radioactivity counts) (% ID/g).

[0400] The specific results are shown in FIG. 2, and the results show that the uptake value of .sup.68Ga-v (Example 9) in the LnCap tumor is the greatest and is 10 Id %/g, followed by those in the kidneys, the liver, the lung, and the spleen, and the uptake values in the other tissues are all very low. The comparison shows that .sup.68Ga-v (Example 9) has a good targeting effect on the LnCap tumor. .sup.68Ga-v (Example 9)'s tumor uptake is better than .sup.68Ga-x (Example 11)'s.

Test Example 4. Pharmacokinetics and Toxicity

4.1. Half-Life of .SUP.68.Ga-v (Example 9) in Blood

[0401] Through single tail-vein injections into mice, the pharmacokinetics of .sup.68Ga-v (Example 9) and PSMA-617 in blood was studied.

[0402] Each mouse was dosed at 50 Ci/100 L, and blood samples were taken from the orbit 0.083, 0.25, 0.5, 1, 2, and 4 h post-dose (4 animals per time point). The blood samples were collected in pre-weighed sample tubes. The tubes were weighed and the weight of the blood samples was recorded. Then radioactivity counting was performed using a -counter. Meanwhile, a test sample was accurately diluted 100-fold, and 0.1 mL of the dilution was added to a counting tube and used as standard 1% ID (i.e., one percent of the injected dose). The radioactivity counts of the 1% ID standard and the biological samples were measured simultaneously on a -counter. The data on blood were expressed as the percentage of the radioactivity counts per gram of blood to the total injected dose (radioactivity counts) (% ID/g). Pharmacokinetic parameters were calculated from blood drug concentration data.

[0403] The uptake results in the blood of normal mice are shown in Table 3 below (n=4).

TABLE-US-00004 TABLE 3 Blood % ID/g .sup.68Ga-v (Example 9) .sup.68Ga-PSMA-617 Time/h 1 2 3 4 1 2 3 4 0.083 5.89 4.98 5.26 4.11 4.22 4.29 3.66 4.22 0.25 1.33 1.11 1.52 1.03 2.12 2.53 2.71 1.73 0.5 1.15 0.52 0.79 0.99 0.55 0.84 0.86 0.55 1 0.33 0.19 0.19 0.16 0.14 0.16 0.10 0.13 2 0.07 0.13 0.10 0.11 0.04 0.07 0.10 0.13 4 0.06 0.02 0.10 0.08 0.29 0.10 / 0.09

[0404] The calculated pharmacokinetic parameters are shown in Table 4 below (0-4 h).

TABLE-US-00005 TABLE 4 Pharmacokinetic .sup.68Ga-V .sup.68Ga- parameters Unit (Example 9) PSMA-617 t.sub.1/2 h 0.13 0.04 0.22 0.02 C.sub.max % ID/g 5.06 0.74 4.10 0.29 AUC.sub.last h* % ID/g 1.67 0.26 1.66 0.14 AUC.sub.INF_obs h* % ID/g 1.66 0.24 1.65 0.13 Vz_obs Ci/% ID/g 4.97 1.09 4.06 1.39 Cl_obs Ci/h* % ID/g 12.17 1.77 12.10 1.0 MRT.sub.last h 0.39 0.02 0.33 0.05

[0405] The above experimental results show that after .sup.68Ga-v (Example 9) and .sup.68Ga-PSMA-617 entered the blood of normal mice, the radioactive substance was rapidly distributed; 0.25 h after the injections, the contents of the radioactive substance in the blood were only 1.250.22% ID/g and 2.270.44% ID/g; the radioactive substance was rapidly cleared from the blood, and the half-lives in the blood were only 0.13 h (7.8 min) and 0.22 h (13.2 min).

[0406] .sup.68Ga-v (Example 9) has a half-life of 0.13 h (7.8 min) in the blood of mice and an elimination phase half-life of 0.758 h. Generally, the time it takes to complete the metabolism is estimated to be 5 times the half-life; therefore, .sup.68Ga-v should be substantially metabolized 3.9 h after dosing. In addition, according to imaging data, few signals were detected from the normal organs after 4 h. According to calculations, the effective half-life is Te=0.45 h; it is 2.27 h, as estimated using 5 effective half-lives.

4.2. Absorbed Doses of Radiation

[0407] The AUC of drug metabolisms was calculated from the biodistribution (Bio-D) data and imported into the OLINDA software to generate doses of radiation absorbed by all the organs.

TABLE-US-00006 TABLE 5 A summary of radiation dose estimates for human organs mSv/MBq .sup.68Ga-V .sup.68Ga- .sup.177Lu- .sup.177Lu-V Target Organ (Example 9) PSMA-617 PSMA-617 (Example 9) Adrenals 8.50E03 7.49E02 2.43E02 1.74E02 Brain 3.99E02 7.80E02 1.38E01 6.03E02 Esophagus 3.77E03 1.79E02 3.39E03 4.33E03 Eyes 1.84E03 1.25E02 2.74E03 2.21E03 Gallbladder Wall 5.52E03 3.22E02 3.58E03 4.67E03 Left colon 6.87E03 1.63E02 4.60E03 4.81E03 Small Intestine 1.31E01 1.00E01 4.48E02 1.73E01 Stomach Wall 8.54E02 1.91E02 8.22E03 2.73E02 Right colon 3.43E03 1.34E02 2.74E03 3.03E03 Rectum 1.99E01 1.83E01 1.49E03 1.91E03 Heart Wall 1.62E02 3.93E02 2.41E02 2.72E02 Kidneys 3.54E02 5.47E01 1.47E+00 8.83E01 Liver 2.50E02 1.75E01 3.15E02 7.43E02 Lungs 1.05E02 1.21E01 1.12E01 1.95E01 Pancreas 9.51E03 1.86E02 4.36E03 5.14E03 Prostate 3.77E03 7.73E03 1.23E03 1.29E03 Salivary Glands 1.81E03 9.39E03 2.18E03 1.67E03 Red Marrow 2.12E03 2.11E02 3.80E03 4.16E03 Osteogenic Cells 4.65E03 3.14E01 5.58E01 6.52E01 Spleen 5.13E02 1.79E01 9.86E01 6.84E01 Testes 3.51E04 3.69E03 5.04E04 5.84E04 Thymus 1.99E03 1.16E02 2.04E03 2.83E03 Thyroid 9.11E04 1.08E02 1.83E03 2.41E03 Urinary Bladder 2.27E03 Wall 5.54E03 7.53E04 9.14E04 Total Body 0.496E02 3.61E02 2.91E02 3.07E02 Effective Dose 2.10E02 4.68E02 4.74E02 5.46E02

[0408] As can be seen from the table above, the absorbed dose of radiation resulting from .sup.68Ga-v (Example 9) is half of that resulting from .sup.68Ga-PSMA-617, and thus the former is safer; the absorbed dose of radiation resulting from .sup.177Lu-v (Example 9) is smaller than that resulting from .sup.177Lu-PSMA-617.