SIDEROPHORE-DIHYDROFOLATE REDUCTASE INHIBITOR CONJUGATE AND APPLICATION THEREOF

Abstract

A conjugate provided. The conjugate is a compound represented by Formula (I), or the conjugate is a stereoisomer, tautomer, homologue, solvate, metabolite, pharmaceutically acceptable salt, or prodrug of the compound represented by Formula (I):

##STR00001##

where A is a linker, and B is a dihydrofolate reductase inhibitor; R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently selected from H or C.sub.1-6 alkyl; and n1, n2, and n3 are each an integer selected from 0 to 6.

Claims

1. A conjugate, being a compound represented by Formula (I), or being a stereoisomer, tautomer, homologue, solvate, metabolite, pharmaceutically acceptable salt, or prodrug of the compound represented by Formula (I): ##STR00029## wherein A is a linker, and B is a dihydrofolate reductase inhibitor; R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently selected from H or C.sub.1-6 alkyl; and n1, n2, and n3 are each an integer selected from 0 to 6.

2. The conjugate according to claim 1, wherein the linker comprises at least one of alkane, alkene, alkyne, aromatic hydrocarbon, heteroatom-containing alkane, heteroatom-containing alkene, heteroatom-containing alkyne, heteroatom-containing aromatic hydrocarbon, amino acid, ketone, ester, amide, sulfonamide, urea, or enamine; optionally, the conjugate is substituted with protium or deuterium; optionally, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently C.sub.1-4 alkyl; and optionally, n1, n2, and n3 are each an integer selected from 1 to 3.

3. The conjugate according to claim 1, wherein the dihydrofolate reductase inhibitor comprises at least one of methotrexate, a derivative of methotrexate, trimethoprim, or a derivative of trimethoprim.

4. The conjugate according to claim 3, wherein the derivative of methotrexate has any one of the following structures: ##STR00030##

5. The conjugate according to claim 4, being a compound represented by any one of Formula (II) to Formula (VII), or being a stereoisomer, tautomer, homologue, solvate, metabolite, pharmaceutically acceptable salt, or prodrug of the compound represented by any one of Formula (II) to Formula (VII): ##STR00031## ##STR00032## ##STR00033##

6. The conjugate according to claim 3, wherein the derivative of trimethoprim has any one of the following structures: ##STR00034##

7. The conjugate according to claim 6, being a compound represented by any one of Formula (II) to Formula (VII), or being a stereoisomer, tautomer, homologue, solvate, metabolite, pharmaceutically acceptable salt, or prodrug of the compound represented by any one of Formula (II) to Formula (VII): ##STR00035## ##STR00036## ##STR00037##

8. The conjugate according to claim 2, wherein the linker is further connected to one or more of an aromatic ring compound, an alkane ring compound, or a heterocyclic compound.

9. The conjugate according to claim 8, wherein the aromatic ring compound is optionally substituted benzene, tea polyphenol, aniline, polyaminobenzene, halogenated benzene, hydroxybenzene, polyhydroxybenzene, anthracene, chrysene, perylene, or benzopyrene.

10. The conjugate according to claim 8, wherein the alkane ring compound is optionally substituted cyclopropane, cyclobutane, cyclopentane, cyclohexane, or cycloheptane.

11. The conjugate according to claim 8, wherein the heterocyclic compound is optionally substituted furan, thiophene, pyrrole, thiazole, imidazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, quinoline, pteridine, acridine, ethylene oxide, ethylene sulfide, aziridine, β-propiolactone, β-propiolactam, maleic anhydride, γ-butyrolactone, caprolactam, oxepine, thiepine, or 1H-azepine.

12. The conjugate according to claim 2, wherein the amino acid is a natural and/or unnatural amino acid, and optionally, the linker is a single amino acid, a repeating chain of a single amino acid, or a hybrid chain of different amino acids.

13. The conjugate according to claim 2, wherein the alkane or the heteroatom-containing alkane is C2-C30 alkane or heterocycloalkane; and optionally, the heterocycloalkane comprises tetrahydrofuran, tetrahydrothiophene, pyrrolidine, piperidine, piperazine, morpholine, dioxane, butylene oxide, azetidine, ethylene oxide, and aziridine.

14. The conjugate according to claim 1, wherein the linker is connected to ##STR00038## or the dihydrofolate reductase inhibitor through an amide bond, an ester bond, an ether bond, a sulfonamide bond, a carbon-carbon bond or a carbon-sulfur bond.

15. The conjugate according to claim 2, wherein the linker comprises any one of the following structures: ##STR00039## ##STR00040## ##STR00041##

16. A conjugate, being any one of the following compounds, or being a stereoisomer, tautomer, homologue, solvate, metabolite, pharmaceutically acceptable salt, or prodrug of any one of the following compounds: ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##

17. A pharmaceutical composition, comprising the conjugate according to claim 1.

18. The pharmaceutical composition according to claim 17, further comprising a pharmaceutically acceptable excipient.

19. A method for inhibiting pathogenic bacteria, comprising: administrating the conjugate according to claim 1 to a subject in need thereof.

20. The method according to claim 19, wherein the pathogenic bacteria are Streptococcus pneumoniae.

Description

DESCRIPTION OF EMBODIMENTS

[0118] The embodiments of the present disclosure are described in detail below. The embodiments described below with reference to the accompanying drawings are illustrative and are intended to explain the present disclosure, but should not be construed as limiting the present disclosure.

Example 1

[0119] Synthesis of compound 3-4a: as shown in the following scheme, siderophore fragment 2-81 and compound 3-5 are condensed under HATU and DIPEA conditions to obtain compound 3-1a. Compound 3-1a was deprotected by removing Boc with trifluoroacetic acid, and then coupled with methotrexate 3-8 protected with a tert-butyl ester to obtain compound 3-3a. Similarly, the tert-butyl ester protection was removed using trifluoroacetic acid, and then K.sub.2CO.sub.3 was used to remove the remaining Bz and Fmoc protecting groups to obtain the target product 3-4a.

##STR00024## ##STR00025##

[0120] 1) Preparation of Compound 3-1a

[0121] Under the protection of nitrogen, compound 2-81 (300 mg, 0.281 mmol, 1.0 equiv), compound 3-5 (45 mg, 0.281 mmol, 1.0 equiv), and HATU (160 mg, 0.421 mmol, 1.5 equiv) were dissolved in dry DMF (5 mL), placed in a low-temperature reactor at −15° C. and stirred, and DIPEA (93 μL, 0.562 mmol, 2.0 equiv) was slowly added dropwise. After reacting for 2 hours, pH of the reaction solution was adjusted to be neutral using a 2M HCl solution. The solvent was removed through rotary evaporation using an oil pump under reduced pressure, then the remaining solution was dissolved in ethyl acetate, and washed respectively with 0.5M HCl solution, saturated NaHCO.sub.3 solution and saturated brine. Then, subsequent to drying over anhydrous sodium sulfate, filtering and concentration, the obtained crude product was subjected to silica gel column chromatography to obtain compound 3-1a.

[0122] NMR data of compound 3-1a: .sup.1H NMR (600 MHz, CDCl.sub.3) δ 8.07 (d, J=7.7 Hz, 4H), 8.05 (d, J=7.9 Hz, 2H), 7.74 (d, J=7.5 Hz, 2H), 7.65 (t, J=6.4 Hz, 3H), 7.62 (d, J=6.9 Hz, 2H), 7.50 (t, J=7.1 Hz, 6H), 7.37 (t, J=7.4 Hz, 2H), 7.28 (t, J=7.4 Hz, 2H), 7.17 (d, J=7.1 Hz, 1H), 6.89 (brs, 1H), 6.14-5.94 (m, 1H), 5.64 (brs, 1H), 4.53-4.28 (m, 4H), 4.25 (brs, 1H), 4.20 (t, J=7.0 Hz, 1H), 4.07 (brs, 1H), 3.95 (brs, 2H), 3.85-3.61 (m, 3H), 3.41-3.14 (m, 4H), 2.13-1.93 (m, 12H), 1.87-1.65 (m, 9H), 1.46-1.34 (m, 9H). .sup.13C NMR (150 MHz, CDCl.sub.3) δ 173.5, 171.7, 171.6, 164.8, 164.7, 164.5, 157.1, 156.2, 143.7, 143.7, 141.2, 134.7, 130.0, 129.0, 127.7, 127.1, 126.4, 125.1, 119.9, 79.0, 67.2, 55.1, 54.2, 52.8, 47.3, 47.0, 40.2, 40.0, 29.2, 28.4, 27.9, 24.2, 23.9, 23.7, 20.3, 20.3.

[0123] 2) Preparation of Compound 3-3a

[0124] Compound 3-1a (200 mg, 0.165 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL), placed and stirred in a low temperature reactor at 0° C., and then trifluoroacetic acid (0.8 mL) was slowly added. After reacting for 3 hours, toluene (5 mL) was added and then trifluoroacetic acid was removed through spin drying, and this operation was repeated three times. The obtained crude product was directly used in the next reaction without further purification.

[0125] Under the protection of nitrogen, the crude product obtained in the previous step, compound 3-8 (151 mg, 0.165 mmol, 1.0 equiv), and HATU (94 mg, 0.247 mmol, 1.5 equiv) were dissolved in dry DMF (4 mL), and the mixture was placed and stirred in a low-temperature reactor at 15° C. Then, DIPEA (55 μL, 0.330 mmol, 2.0 equiv) was slowly added dropwise. After reacting for 2 hours, the reaction solution was adjusted to a neutral pH using a 2M HCl solution. The solvent was removed through rotary evaporation with an oil pump under reduced pressure, then the remaining solution was dissolved in ethyl acetate and washed respectively with a 0.5M HCl solution, a saturated NaHCO.sub.3 solution and saturated brine. Then, subsequent to drying over anhydrous sodium sulfate, filtering and concentration, the obtained crude product was subjected to silica gel column chromatography to obtain compound 3-3a.

[0126] 3) Preparation of Compound 3-4a

[0127] Compound 3-3a (100 mg, 0.059 mmol, 1.0 equiv) was dissolved in dichloromethane (3 mL) and placed and stirred in a low temperature reactor at 0° C., then trifluoroacetic acid (1.5 mL) and water (5 μL, 0.295 mmol, 5.0 equiv) were slowly added. After reacting for 3 hours, toluene (5 mL) was added, trifluoroacetic acid was removed through spin drying, and then this operation was repeated three times. The obtained crude product was directly used in the next step reaction without further purification.

[0128] The crude product prepared in the previous step was dissolved in methanol (3 mL), and 81 μL of potassium carbonate aqueous solution (200 mg/mL) was added. Then, 81 μL of the potassium carbonate solution (200 mg/mL) was added every 1 hour until the reaction was completed. The reaction solution was subjected to reverse phase column chromatography to obtain compound 3-4a.

[0129] NMR data of compound 3-4a: .sup.1H NMR (600 MHz, D.sub.2O) δ 8.34 (s, 1H), 7.50 (s, 2H), 6.51 (s, 2H), 4.49 (brs, 2H), 4.41-4.29 (m, 2H), 4.24-4.12 (m, 1H), 4.04 (brs, 1H), 3.66-3.42 (m, 6H), 3.32-3.11 (m, 4H), 3.00 (s, 3H), 2.29 (brs, 2H), 2.23-2.13 (m, 1H), 2.13-1.97 (m, 10H), 1.89-1.78 (m, 2H), 1.77-1.45 (m, 10H). .sup.13C NMR (150 MHz, D.sub.2O) δ 178.2, 175.6, 173.7, 173.6, 173.5, 173.3, 172.8, 169.5, 168.4, 162.5, 160.2, 151.1, 151.0, 148.8, 148.7, 128.6, 121.8, 120.1, 111.2, 54.9, 54.6, 53.7, 53.3, 52.5, 50.8, 50.6, 47.1, 47.0, 46.9, 38.8, 38.5, 32.5, 28.1, 28.0, 27.9, 22.4, 22.2, 21.4, 19.5, 19.2.

Examples 2 to 14

[0130] The synthesis of Example 3 to 12 was the same as that of Example 1, and accordingly, compounds 3-4b, 3-4c, 3-4f, 3-4j, 3-4k, 3-41, 3-4n, 3-4p, 3-4r, 3-4u, 3-4v, 3-4w, and 3-4x were synthesized, respectively.

[0131] NMR data of compound 3-4v: .sup.1H NMR (400 MHz, D2O)δ8.43 (br, 1H), 7.51 (br, 2H), 6.68 (br, 2H), 4.34-3.90 (m, 4H), 3.55-3.05 (m, 16H), 2.45-0.82 (m, 25H).

[0132] NMR data of compound 3-4w: .sup.1H NMR (400 MHz, D2O)δ, 8.38 (br, 1H), 7.43 (br, 2H), 6.56 (br, 2H), 4.30-4.18 (m, 5H), 3.77-2.74 (m, 20H), 2.55-1.49 (m, 18H).

[0133] NMR data of compound 3-4x: .sup.1H NMR (400 MHz, D2O)δ, 8.31 (br, 1H), 7.35 (br, 2H), 6.42 (br, 2H), 4.47 (s, 2H), 4.29-3.86 (m, 4H), 3.61-2.80 (m, 17H), 2.33-1.66 (m, 13H), 1.31-1.20 (m, 15H).

Example 15

[0134] Synthesis of compound 3-4e: as shown in the following scheme, siderophore fragment 2-81 and compound 3-5 were condensed under HATU and DIPEA conditions to obtain compound 3-1e. Compound 3-1e was deprotected by removing Boc groups with trifluoroacetic acid, and then coupled with serine 3-7 protected by Boc to obtain compound 3-2e. Similarly, the Boc protection was removed by using trifluoroacetic acid, and then the deprotected product and tert-butyl ester protected methotrexate 3-8 were condensed to obtain compound 3-3e. Finally, the tert-butyl ester protection was removed by using trifluoroacetic acid, and then the remaining Bz and Fmoc protecting groups were removed by using K.sub.2CO.sub.3 to obtain the target product 3-4e.

##STR00026## ##STR00027##

[0135] 1) Preparation of Compound 3-1a

[0136] Under the protection of nitrogen, compound 2-81 (300 mg, 0.281 mmol, 1.0 equiv), compound 3-5 (45 mg, 0.281 mmol, 1.0 equiv), and HATU (160 mg, 0.421 mmol, 1.5 equiv) were dissolved in dry DMF (5 mL), and the mixture was placed and stirred in a low-temperature reactor at −15° C. Then, DIPEA (93 μL, 0.562 mmol, 2.0 equiv) was slowly added dropwise. After reacting for 2 hours, the reaction solution was adjusted to a neutral pH using a 2 M HCl solution. The solvent was removed through rotary evaporation with an oil pump under reduced pressure, then dissolved in ethyl acetate, and washed respectively with a 0.5M HCl solution, a saturated NaHCO.sub.3 solution and saturated brine. Then, subsequent to drying over anhydrous sodium sulfate, filtering, and concentration, the obtained crude product was subjected to silica gel column chromatography to obtain compound 3-1a.

[0137] 2) Preparation of Compound 3-2e

[0138] Compound 3-1a (200 mg, 0.165 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL) and then placed and stirred in a low temperature reactor at 0° C., and then trifluoroacetic acid (0.8 mL) was slowly added. After reacting for 3 hours, toluene (5 mL) was added, trifluoroacetic acid was removed through spin drying, and this operation was repeated three times. The obtained crude product was directly used in the next reaction without further purification.

[0139] Under the protection of nitrogen, the crude product obtained in the previous step, compound 3-7 (34 mg, 0.165 mmol, 1.0 equiv) and HATU (94 mg, 0.247 mmol, 1.5 equiv) were dissolved in dry DMF (4 mL), and the mixture was placed and stirred in a low-temperature reactor at 15° C. DIPEA (55 μL, 0.330 mmol, 2.0 equiv) was slowly added dropwise. After reacting for 2 hours, the reaction solution was adjusted to a neutral pH using 2M HCl solution. The solvent was removed through rotary evaporation with an oil pump under reduced pressure, and then the remaining solution was dissolved in ethyl acetate, and washed respectively with a 0.5M HCl solution, a saturated NaHCO.sub.3 solution and saturated brine. Then, subsequent to drying over anhydrous sodium sulfate, filtering and concentration, the obtained crude product was subjected to silica gel column chromatography to obtain compound 3-2e.

[0140] NMR data of compound 3-2e: .sup.1H NMR (600 MHz, CDCl.sub.3) δ 8.11-8.03 (m, 5H), 7.73 (d, J=7.4 Hz, 2H), 7.69-7.56 (m, 5H), 7.50 (t, J=7.4 Hz, 6H), 7.37 (t, J=7.3 Hz, 3H), 7.27 (t, J=7.3 Hz, 2H), 7.20 (brs, 1H), 6.97 (brs, 1H), 6.45-6.11 (m, 1H), 5.88 (brs, 1H), 4.46-4.25 (m, 4H), 4.25-4.13 (m, 3H), 4.08-3.89 (m, 3H), 3.88-3.57 (m, 5H), 3.47-3.27 (m, 3H), 2.12-1.89 (m, 12H), 1.88-1.62 (m, 9H), 1.40 (s, 9H). .sup.13C NMR (150 MHz, CDCl.sup.3) δ 172.3, 171.5, 164.5, 157.1, 155.7, 143.7, 141.2, 134.7, 130.0, 129.0, 127.7, 127.1, 126.3, 125.1, 125.1, 119.9, 79.7, 67.3, 63.4, 56.0, 55.3, 54.5, 53.7, 47.4, 47.2, 47.0, 39.4, 38.7, 29.0, 28.3, 27.8, 24.2, 23.8, 20.3.

[0141] 3) Preparation of Compound 3-3e

[0142] Compound 3-2e (180 mg, 0.139 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL) and then placed and stirred in a low temperature reactor at 0° C. Then, trifluoroacetic acid (0.8 mL) was slowly added. After reacting for 3 hours, toluene (5 mL) was added, trifluoroacetic acid was removed through spin drying, and this operation was repeated three times. The obtained crude product was directly used in the next reaction without further purification.

[0143] Under the protection of nitrogen, the crude product obtained in the previous step, compound 3-8 (71 mg, 0.139 mmol, 1.0 equiv), and HATU (79 mg, 0.208 mmol, 1.5 equiv) were dissolved in dry DMF (4 mL), and the mixture was placed and stirred in a low temperature reactor at −15° C. Then, DIPEA (46 μL, 0.278 mmol, 2.0 equiv) was slowly added dropwise. After reacting for 2 hours, the reaction solution was adjusted to a neutral pH using a 2M HCl solution. The solvent was removed through rotary evaporation with an oil pump under reduced pressure, and the remaining solution was mixed with silica gel and subjected to silica gel column chromatography to remove most of the by-products. Then, the obtained crude product was further purified with a silica gel plate to obtain compound 3-3e.

[0144] 4) Preparation of Compound 3-4e

[0145] Compound 3-3e (100 mg, 0.059 mmol, 1.0 equiv) was dissolved in dichloromethane (3 mL), and then placed and stirred in a low temperature reactor at 0° C. Trifluoroacetic acid (1.5 mL) and water (5 μL, 0.295 mmol, 5.0 equiv) were slowly added. After reacting for 3 hours, toluene (5 mL) was added and trifluoroacetic acid was removed through spin drying, and this operation was repeated three times. The obtained crude product was directly used in the next reaction without further purification.

[0146] The crude product obtained in the previous step was dissolved in methanol (3 mL), 81 μL of potassium carbonate aqueous solution (200 mg/mL) was added. Then, 81 μL of the potassium carbonate solution (200 mg/mL) was added every 1 hour until the reaction was completed. The reaction solution was subjected to reverse phase column chromatography to obtain compound 3-4e.

[0147] NMR data of compound 3-4e: .sup.1H NMR (600 MHz, D.sub.2O) δ 8.38 (s, 1H), 7.51 (s, 2H), 6.55 (s, 2H), 4.52 (s, 2H), 4.35 (s, 3H), 4.19 (s, 1H), 4.05 (s, 1H), 3.78 (s, 2H), 3.68-3.45 (m, 5H), 3.37-3.13 (m, 4H), 3.02 (s, 3H), 2.46 (brs, 2H), 2.20 (brs, 1H), 2.08 (s, 9H), 1.90-1.49 (m, 11H). .sup.13C NMR (150 MHz, D.sub.2O) δ 178.3, 175.7, 173.8, 173.6, 173.5, 173.4, 172.8, 171.9, 169.6, 168.5, 162.6, 160.5, 151.2, 148.8, 128.6, 121.9, 120.1, 111.3, 61.2, 55.7, 55.0, 54.7, 53.6, 53.3, 52.5, 47.1, 47.1, 46.9, 38.8, 38.5, 32.2, 28.1, 27.9, 27.7, 22.4, 22.2, 21.4, 19.2.

Examples 16 to 22

[0148] The synthesis schemes of Examples 16 to 22 were the same as the synthetic scheme of Example 15, and accordingly, compounds 3-4h, 3-4g, 3-4i, 3-4m, 3-4s, 3-4q, and 3-4t were synthesized, respectively.

Example 23

[0149] The synthesis of compound 3-4d was as shown in the following scheme.

[0150] Another molecule of tert-butyl glutamate was connected to the carboxyl group at the γ position of methotrexate, and the obtained compound 3-11 was coupled with the siderophore fragment 3-10 connected with ethylenediamine to obtain compound 3-3d. Then, the protecting groups were removed under the same conditions to obtain the target product 3-4d.

##STR00028##

[0151] NMR data of compound 3-4d: .sup.1H NMR (600 MHz, D.sub.2O) δ 8.46 (s, 1H), 7.60 (s, 2H), 6.70 (s, 2H), 4.60 (s, 2H), 4.34 (s, 2H), 4.19 (s, 1H), 4.11 (s, 1H), 3.84 (s, 1H), 3.67-3.46 (m, 6H), 3.34-3.14 (m, 4H), 3.09 (s, 3H), 2.42 (s, 2H), 2.36-2.15 (m, 4H), 2.07 (s, 9H), 1.91-1.47 (m, 13H). .sup.13C NMR (150 MHz, D.sub.2O) δ 178.5, 178.1, 175.4, 174.9, 173.5, 173.5, 173.4, 173.0, 172.0, 169.0, 162.7, 161.8, 153.2, 151.5, 149.1, 148.1, 128.7, 122.1, 120.3, 111.6, 68.0, 54.9, 54.8, 54.4, 53.7, 53.2, 52.9, 50.9, 47.1, 38.8, 38.5, 32.3, 32.2, 29.1, 28.0, 28.

Example 24

[0152] Compound 3-4o was synthesized according to the synthesis strategy of Example 23. Example 25 Detection of inhibitory activity of compounds against Streptococcus pneumoniae

[0153] The minimum inhibitory concentration (MIC) determination was performed in accordance with the standards of the American Association for Clinical and Laboratory Standards (CLSIM07). The MIC determination on Streptococcus pneumoniae was carried out by the microdilution broth method as follows. First, the compound to be tested was dissolved in water to prepare a high-concentration original solution, and then the compound original solution was sequentially diluted by 2 folds, a total of 10 times, to prepare 11 samples of 10× working solution. Then the gradient dilutions of the compound were transferred to a 96-well round bottom plate, the first to eleventh wells contained 10 μL of the 11 samples of 10× working solution prepared above, respectively, and 10 μL of water was added to the twelfth well as a solvent control. Horse blood was added to a MH broth II (adjusted with cations) medium at a ratio of 1:20, 80 μL of 2,2-bipyridine (1 mg/mL) per 5 mL medium was added, fresh monoclonal strains were picked out and prepared into a bacterial suspension in sterilized bacterial physiological saline, the concentration thereof was adjusted to 0.5 McIntosh turbidity. The bacterial suspension was diluted with the above-mentioned MH(II) liquid medium at a ratio of 1:200, and then 90 μL of the bacterial suspension was successively added into wells of the prepared 96-well plate containing the compound. The inoculated 96-well plate was placed in a carbon dioxide incubator (37° C., 5% CO.sub.2) and incubated for 16 hours to 18 hours, and the MIC value of the compound against the bacteria was read by visual observation.

[0154] The activities of the compounds according to the present disclosure against clinical Streptococcus pneumoniae were compared with the activities of ciprofloxacin, vancomycin and methotrexate against Streptococcus pneumoniae. The specific results are shown in Table 1.

TABLE-US-00001 TABLE 1 Comparison of activity of compound against clinical Streptococcus pneumoniae Streptococcus Streptococcus pneumoniae pneumoniae Compound 180918016-S27 180918017-S28 Ciprofloxacin 1 0.0625 Vancomycin 0.25 0.125 Methotrexate 0.125 0.03125 3-4a <0.0078 <0.0078 3-4b 0.0625 <0.0078 3-4c <0.0078 <0.0078 3-4d <0.0078 <0.0078 3-4e <0.0078 <0.0078 3-4f <0.0078 <0.0078 3-4g <0.0078 <0.0078 3-4v <0.0078 <0.0078 3-4w <0.0078 <0.0078 3-4x <0.0078 <0.0078

[0155] The above table indicates that the compounds have each a good inhibitory activity against clinical Streptococcus pneumoniae.

Example 26 Toxicity Test of Compounds

[0156] Cytotoxicity Test:

[0157] The cytotoxicity test was carried out with the MTT colorimetric method. 5×10.sup.3 cells in a logarithmic growth phase were inoculated in each well of a 96-well plate, and incubated overnight at 37° C. in an incubator (5% CO.sub.2). Then, different concentrations of compounds were added and the incubation further continued for 72 hours. Finally, 10 μL of MTT solution was added to each well and he incubation further continued for 4 hours. Then, the culture medium in each well was carefully aspirated. 100 μL of dimethyl sulfoxide was added to each well, and shaken at low speed for 10 minutes on a shaker to fully dissolve crystals. The absorbance of each well was measured at OD490 nm of the enzyme-linked immunometric meter. Three parallel measurements were designed for each group, in order to calculate the standard deviation. Cell survival rate=(compound-treated OD value−blank control OD value)/(normal control OD value−blank control OD value)*100%

[0158] The toxicity of each compound was tested by measuring the half inhibitory concentrations (IC.sub.50) with respect to human normal hepatocytes (L02), human umbilical vein endothelial cells (HUVEC) and human renal epithelial cells (293T), respectively. The specific results are shown in Table 2.

TABLE-US-00002 TABLE 2 Cytotoxicity data of compounds IC.sub.50 (μM) Compound L02 HUVEC 293T 3-4a 10.18 ± 2.20  6.84 ± 0.10 3.07 ± 0.00 3-4b 34.58 ± 14.48 30.98 ± 3.18  8.82 ± 0.08 3-4c 5.46 ± 2.18 4.52 ± 0.21 2.66 ± 0.01 3-4d 7.91 ± 0.67 5.02 ± 0.14 1.95 ± 0.44 3-4f 14.68 ± 1.70  19.16 ± 0.54  3.93 ± 0.03 3-4g 27.00 ± 2.88  18.37 ± 1.82  5.38 ± 0.65 3-4h 34.51 ± 4.01  16.56 ± 2.00  10.59 ± 1.34  MTX 0.01 ± 0.00 <0.078125 0.01 ± 0.00

[0159] In view of the data in the above table, the siderophore-methotrexate conjugates have significantly reduced toxicity to human normal hepatocytes, human umbilical vein endothelial cells and human renal epithelial cells compared with methotrexate. Compounds 3-4b, 3-4g and 3-4h have the least toxicity.

[0160] In the specification, description with reference to the term “one embodiment”, “some embodiments”, “examples”, “specific examples”, or “some examples” etc. means that specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described herein can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and incorporate different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.

[0161] Although the embodiments of the present disclosure have been illustrated and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present disclosure. Those of ordinary skill in the art can make changes, modifications, replacements, and variations to the above-mentioned embodiments within the scope of the present disclosure.