Azido prodrug compound as well as preparation method and application thereof

Abstract

The invention discloses an azide prodrug compound as well as a preparation method and application thereof, and belongs to the technical field of medicines. The invention provides an azide prodrug compound with a structure as shown in a formula (1), and D is derived from active small molecules. The conversion rate of the prodrug after irradiation of the azide prodrug compound is relatively high, and meanwhile, obvious generation of an original drug / intermediate transition state is detected, so that the azide prodrug compound shows better drug release performance; meanwhile, compared with the original medicine, the toxicity is lower. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a class of azide prodrug compounds, a preparation method and application thereof, and belongs to the technical field of medicines. Background Art

[0002] At present, the treatment of cancer with chemotherapy drugs is still considered to be the most effective treatment method to prolong the survival time of patients. However, since cancer cells are mutated from healthy cells of the body, the selectivity of existing chemotherapy drugs makes it difficult to distinguish cancer cells from healthy tissue cells. Therefore, it is difficult to kill cancer cells while ensuring the health and safety of patients when using chemotherapy drugs. In order to achieve precision chemotherapy, the research on prodrug activation strategy has become one of the current hot directions. Through chemical means, the active sites such as carboxyl, amino, and hydroxyl groups in the more toxic drug molecules are bonded to specific protective groups, thereby shielding their activity and reducing toxicity. After administration, the protective groups are removed in situ at the tumor location by chemical reactions triggered by the specific endogenous environment of the biological tumor or exogenous stimulation, restoring their activity, and finally achieving controlled release of the drug. This strategy can not only effectively reduce the toxic side effects in the traditional chemotherapy process and overcome the dose limitation of the parent drug, but also improve the bioavailability of the original drug molecules through the design and optimization of the protective groups, prolong the blood circulation time, and promote its application in clinical practice.

[0003] Endogenously driven prodrug activation mainly relies on the tumor-specific microenvironment, such as overexpressed enzymes, reactive oxygen species, glutathione, and lower pH in the tumor lesion area. However, this prodrug conversion strategy that relies on the tumor microenvironment has defects that cannot be ignored, such as poor adjustability, few types, and premature drug release. In order to circumvent the various obstacles of endogenous stimulation, researchers have turned their attention to smart prodrug activation systems mediated by non-invasive signals (such as light, sound, magnetic fields, etc.). These exogenous response systems use the energy input of external signals as a switch to control drug release, accurately converting low-toxic small molecule prodrugs into highly toxic drugs in the affected area, and ultimately killing tumor cells. Compared with traditional direct drug delivery methods, non-invasive prodrug activation has better biosafety. However, due to many factors such as poor tissue traditionality, difficult material preparation, and poor system efficiency, the actual application of prodrug activation treatment methods such as light and sound has been hindered, making it difficult to truly achieve clinical treatment.

[0004] In contrast, radiation has excellent tissue penetration. At the focal point, the chemical ionization of water by radiation produces a large number of active free radicals, which can accurately kill tumor cells. At present, radiotherapy has become one of the three main treatments for tumors. In addition, the X-ray technology used for medical radiotherapy is developing rapidly at this stage, and can accurately locate tumors at the submillimeter level. Therefore, the X-ray-mediated prodrug activation strategy has the advantages of time and space control, dose control, and good tissue penetration. It can better achieve the controlled release of anticancer drugs at the tumor site, greatly reduce the killing of normal cells, and achieve precise tumor treatment on the basis of combined radiotherapy and chemotherapy. Compared with other activation methods, X-ray-mediated prodrug activation systems have been less studied, and related reports have only appeared in the past five years. According to existing research, the activation of prodrugs by X-rays mainly depends on the upregulated enzymes (such as caspase-3) in the tumor site after X-ray irradiation and the hydroxyl radicals (·OH), hydrogen radicals (·H), and hydrated electrons (e - aq ) etc. Although the above-mentioned prodrug activation system has achieved certain results in in vitro activation and in vivo efficacy experiments, it is still in the early stage of exploration and faces challenges such as unstable activation efficiency and limited application scope of the original drug. Therefore, it is very necessary to further develop a radiotherapy-responsive prodrug activation system with novel structure, good safety and stable activation for a variety of chemotherapeutic drugs with broad-spectrum anti-tumor activity. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a class of azide prodrug compounds having a structure shown in formula (1) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] In the formula, R 1 is mono- to tetra-substituted, specifically selected from any one or more of the following substituents: H, halogen (F, Cl, Br, I), substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted C 1-6 Alkoxy, -C(=O)N(R a )-(CHR b )-COOR c The substituted substituent is selected from halogen atoms, hydroxyl groups, C 1-6 Alkyl and C 1-6 Epoxy group; R a , R b , R c are independently selected from H, C 1-6 alkyl;

[0008] A is a vacant bond, -C(=O)-, or

[0009] D is derived from an active small molecule, and the active small molecule is selected from the following compounds or pharmaceutically acceptable salts thereof: daunorubicin (DAU), doxorubicin (DOX), camptothecin (CPT), mitomycin C (MMC), monomethyl auristatin E (MMAE), bufalin (BUF), 7-ethyl-10-hydroxycamptothecin (SN-38), gemcitabine (GEM), resiquimod (RES), exitecan (EXA), paclitaxel (PTX); R 2 , R 3 are independently selected from: H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted aryl, -CH 2 -C(=O)N(R 4 )CH 2 COOR 5 ; The substitution on the aryl group is one to four substitutions, and the aryl or C 1-6 The substituents on the alkyl group are selected from any one or more of the following: halogen (F, Cl, Br, I), azido (-N 3 );R 4 , R 5 are independently selected from H, C 1-6 alkyl;

[0010] Furthermore, when the active small molecule is daunorubicin or mitomycin C, R 2 , R 3 Not H at the same time.

[0011] In one embodiment of the present invention, the aryl group is a benzene ring or a naphthalene ring.

[0012] In one embodiment of the present invention, when A is a blank bond, the structure of the corresponding general formula is

[0013] In one embodiment of the present invention, preferably: D is R 2 For H, R 3 is a substituted or unsubstituted phenyl group; the substituent is selected from any one or more of the following: halogen (F, Cl, Br, I), azido (-N 3 ).

[0014] In one embodiment of the present invention, preferably: D is R 2 For H, R 3 -CH 2 -C(=O)N(R 4 )CH 2 COOR5 ; R 4 , R 5 are independently selected from H, C 1-6 alkyl.

[0015] In one embodiment of the present invention, preferably: D is R 1 The mono- to tetra-substituted substituents are H, halogen (F, Cl, Br, I); more preferably, mono- to di-halogen (F, Cl, Br, I) substitutions.

[0016] In one embodiment of the present invention, D is specifically selected from the following structures:

[0017]

[0018]

[0019] In one embodiment of the present invention, the pharmaceutically acceptable salt thereof includes inorganic salt or organic salt; wherein the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate, sodium salt, calcium salt, potassium salt, magnesium salt, silver salt, lithium salt. The organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate, meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, terbutylamine salt, N,N-dibenzylethylenediamine salt.

[0020] The present invention also provides a pharmaceutical composition comprising the above-mentioned azide prodrug compound or a pharmaceutically acceptable salt thereof.

[0021] In one embodiment of the present invention, the pharmaceutical composition further comprises pharmaceutical excipients. The pharmaceutical excipients include: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids, and release retardants.

[0022] In one embodiment of the present invention, the pharmaceutical composition further comprises a drug carrier, which is selected from microcapsules, microspheres, nanoparticles and liposomes.

[0023] In one embodiment of the present invention, the dosage forms of the pharmaceutical composition include: injection, lyophilized powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

[0024] The present invention also provides an antibody-small molecule drug conjugate, which is obtained by conjugating the above-mentioned azide prodrug compound or a pharmaceutically acceptable salt thereof with an antibody.

[0025] In one embodiment of the present invention, the structure of the antibody-small molecule drug conjugate is as follows:

[0026]

[0027] or

[0028] Wherein, x is the ratio of small molecule drug to antibody, x is 3-5; Ab is trastuzumab; R 1 , R 2 , R 3 , R 4 , R a Same definition as above.

[0029] Beneficial effects:

[0030] The peak area of ​​the prodrug compound of the present invention after irradiation is significantly reduced, the conversion rate of the prodrug is high, and obvious original drug / intermediate transition state generation is detected, showing better drug release performance.

[0031] In addition, the prodrug compounds of the present invention have lower toxicity than the original drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison chart of the toxicity results of the compound in Example 4.

[0033] Figure 2 This is a comparison chart of the toxicity results of the compound in Example 5.

[0034] Figure 3 This is a comparison chart of the toxicity results of the compound in Example 6.

[0035] Figure 4 This is a comparison chart of the toxicity results of the compound in Example 7.

[0036] Figure 5 This is a comparison chart of the toxicity results of the compound in Example 9.

[0037] Figure 6 This is a comparison chart of the toxicity results of the compound in Example 10.

[0038] Figure 7 This is a comparison chart of the toxicity results of the compound in Example 12.

[0039] Figure 8 This is a comparison chart of the toxicity results of the compound in Example 18.

[0040] Fig. 9 This is the Maldi (time-of-flight mass spectrometry) of ADC and naked antibody in Example 27.

[0041] Fig.10 This is the ESI (high resolution protein spectrum) of XP-106-ADC001 before irradiation in Example 27.

[0042] Fig.11 This is the ESI (high resolution protein spectrum) after irradiation (100 Gy) in Example 27.

[0043] Fig.12 This is a comparison chart of the toxicity results of the compound in Example 27. DETAILED DESCRIPTION

[0044] In the present invention, "C 1-6 "Alkyl" refers to a saturated, linear or branched, monovalent hydrocarbon radical of 1 to 6 carbon atoms. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl and 2-methyl-2-propyl.

[0045] In the present invention, "C 3-6 "Cycloalkyl" refers to cycloalkyl groups of 3 to 6 carbon atoms.

[0046] In the present invention, "substituted" means that one or more hydrogen atoms in the group are replaced by the same or different substituents.

[0047] The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0048] The HPLC test process involved in the present invention comprises:

[0049] The chromatographic conditions involved in HPLC are as follows:

[0050]

[0051] The irradiation parameter conditions involved in the present invention are as follows: the instrument is a small animal X-ray irradiator, model RS2000-PRO-225, the irradiation parameters selected are 225KV, 17.7mA, the position is placed at the fifth layer, 60-100Gy, and the required time is 9-15min. The sample is first dissolved in DMSO, an appropriate amount of surfactant Triton X-100 is added, and then diluted with pure water to a concentration of 10uM-20uM, the prepared liquid is placed in a glass bottle and ultrasonicated for 5-10min, 1mL of the sample is taken, and the remaining liquid is passed through argon for 20min and then sealed, and irradiated at room temperature.

[0052] The preparation of intermediate 5 ((4-azido-2,3,5,6-tetrafluorophenyl)methyl-4-nitrophenyl carbonate) of the present invention is as follows:

[0053] The corresponding synthetic route is shown below:

[0054]

[0055] The specific steps include:

[0056] first step

[0057]

[0058] Operation: Dissolve 5.5 g of pentafluorobenzaldehyde in 50 mL of DMF, add 3.3 g (1.05 eq) of trimethylsilane at room temperature, then slowly drop 2.56 mL of tetrabutylammonium fluoride (1 mol / L tetrahydrofuran solution, 10 mol%), and react at room temperature for half an hour.

[0059] Detection: TLC detection (PE:EA=40:1).

[0060] Post-treatment: slowly pour the reaction solution into 500 mL of water, extract the liquid with petroleum ether / ether (10 / 1) (110 mL x 3), combine the organic phases and wash twice with saturated brine (150 mL x 2), dry with anhydrous sodium sulfate, filter, remove the petroleum ether / ether (recyclable) by rotary evaporation at 40 ° C, and then pump dry with an oil pump to obtain 5.5 g of off-white solid. (No purification required for the next step)

[0061] Step 2

[0062]

[0063] Operation: Dissolve 5.5 g of 4-azido-2,3,5,6-tetrafluorobenzaldehyde in 50 mL of methanol. After dissolution, cool to 0°C in an ice bath. Add 260 mg of sodium borohydride gradually in portions and detect immediately after addition.

[0064] Detection: TLC detection (PE:EA=15:1).

[0065] Post-treatment: After the reaction is confirmed to be complete after detection, quench immediately, add 20mL of dilute hydrochloric acid solution (1mol / L) and 30mL of water (a large amount of white solid precipitates) in an ice bath, stir for 10min, spin off methanol (recyclable) at 45℃, then add 20mL of dilute hydrochloric acid solution (1mol / L) and 30mL of water, stir for 10min (pH>1.5 at this time), filter, rinse the filter cake with 30mL of water, dry, and then pump dry with an oil pump to obtain 5g of white solid product. (No purification required, directly used in the next step)

[0066] Step 3

[0067]

[0068] Operation: Dissolve 5 g of 4-azido-2,3,5,6-tetrafluorobenzyl alcohol in 50 mL of DCM, add 5.47 g of p-nitrophenyl chloroformate, cool to 0°C in an ice bath, add 0.5 g of DMAP, and then slowly drop 3.5 mL of triethylamine, and react in an ice bath for 5-30 min (mainly for detection).

[0069] Detection: TLC detection (PE:EA=10:1).

[0070] Post-treatment: After the reaction is basically completed, add water (150 mL) and 50 mL DCM to extract the liquid, wash the organic phase with 100 mL of dilute hydrochloric acid solution (0.4 mol / L) and then with 100 mL of saturated sodium carbonate solution, then wash the organic phase with water (100 mL x 2) and 100 mL of saturated brine, dry over anhydrous sodium sulfate, filter, remove DCM (recoverable) by rotary evaporation at 45 ° C, and then dry with an oil pump to obtain 8.5 g of an off-white solid crude product.

[0071] Purification: Use 50mL PE:EA=100:1 to make pulp for 2h, filter and dry, use 40mL ethanol to completely dissolve at 80℃, remove the heat source, cool naturally for 10h (recrystallization), filter, rinse the filter cake with 5mL ethanol and 20mL petroleum ether, and then pump dry with an oil pump to obtain 54.8g of off-white solid intermediate. (Acid and alkali washing can wash away one impurity, but another impurity is enhanced under TLC ultraviolet. Before recrystallization, TLC spot plate should be used to see whether the product is the main point. You can use a centrifuge tube to try whether it can be recrystallized.)

[0072] 1 H NMR (400 MHz, CDCl 3 )δ8.29(d,J=9.2Hz,2H),7.40(d,J=9.2Hz,2H),5.40(s,2H).

[0073] The preparation of intermediate 13 involved in the present invention:

[0074] Preparation method of N-(3-(4-azido-2,3,5,6-tetrafluorophenyl)-3-(((4-nitrophenoxy)carbonyl)oxy)propanoyl)-N-methylglycine linker:

[0075]

[0076] first step

[0077]

[0078] Operation: 2.0 g of 4-azido-2,3,5,6-tetrafluorobenzaldehyde was dissolved in 40 mL of DCM, and 2.58 g of DIPEA, 0.5 g of glacial acetic acid and 4.05 g of TMSOTf were added at room temperature, and the mixture was reacted for two hours at room temperature.

[0079] Detection: TLC detection (PE:EA=10:1).

[0080] Post-treatment: After the reaction is complete, it is directly used in the next step without purification and post-treatment.

[0081] Step 2

[0082]

[0083] Operation: Add 40 mL of ethanol, slowly add 2.25 mL of TFA, stir at room temperature for 5 minutes, and then spin dry at 48 °C.

[0084] Detection: TLC detection (PE:EA=10:1).

[0085] Post-treatment: Dissolve with DCM and wet sample, column chromatography (PE:EA = 1:0, 10:1, 5:1, 3:1, 1:1), spin dry, pump for 2 hours to obtain 1.03g product

[0086] Step 3

[0087]

[0088] Procedure: Dissolve 1.03g of compund 9 in 20mL of DCM, add 0.94mL of LTEA, 0.8g of compound 10,

[0089] 1.52g DCC and 0.6g HOBt, react at room temperature for 16h.

[0090] Detection: TLC detection (PE:EA=1:1).

[0091] Post-treatment: Filter and spin-dry the filtrate.

[0092] Purification: Dissolve with DCM and then wet load, column chromatography (PE:EA=1:0, 10:1, 5:1, 4:1, 3.5:1), spin dry, and pump the oil pump for 2 hours to obtain 1.2 g of product.

[0093] 1 H NMR (400 MHz, CDCl 3 )δ5.55(dt,J=10.0,2.8Hz,1H),4.33(t,J=3.6Hz,1H),4.12–3.98(m,2H),3.03(d,J=11.6Hz,2H),1.70–1.62(m,3H),1.47(s,9H).

[0094] Step 4

[0095]

[0096] Operation: Dissolve 1.2g of compund 11 in 20mL of DCM, add 0.63g of compound 4, cool to 0°C in an ice bath, add 0.12g of DMAP, slowly drop 0.55mL of triethylamine, and react in an ice bath for 30-60min (mainly for detection).

[0097] Detection: TLC detection (PE:EA=1:1).

[0098] Post-treatment: add water (50 mL) and 30 mL DCM to extract the solution, wash with saturated brine, and spin-dry the organic phase to obtain 1.6 g of crude product.

[0099] Step 5

[0100]

[0101] Procedure: Dissolve 1.6 g of compund 12 (crude product) in 6 mL of DCM, slowly add 6 mL of TFA dropwise, and react at room temperature for 2 h.

[0102] Detection: TLC detection (PE:EA=5:1) and (DCM:MeOH=20:1).

[0103] Post-treatment: spin dry at 40℃.

[0104] Purification: Dissolved with DCM and wet loaded, column chromatography (DCM: MeOH = 1:0, 100:1, 80:1, 40:1), spin dried, pumped on the oil pump for 2 h to obtain 430 mg of the product (intermediate 13).

[0105] 1 H NMR (400 MHz, CDCl 3 )δ8.26(d,J=9.2Hz,2H),7.38(d,J=9.2Hz,2H),6.52(t,J=7.2Hz,1H),4.14(t,J=12.0Hz,2H),3.46(dd,J=16.8,7.6Hz,2H),3.18–3.06(m,3H).

[0106] The preparation of intermediate 22 involved in the present invention:

[0107] Preparation method of linker 4-azido-3,5-difluorobenzyl alcohol:

[0108] Synthesis method:

[0109]

[0110] first step

[0111]

[0112] Procedure: Dissolve 2.0 g of compound 19 in 20 mL of anhydrous DMF and add 1.73 g of TMSN. 3 Then 1.25 mL TBAF was added dropwise at 0 °C and reacted at room temperature for 24 h.

[0113] Detection: TLC detection (PE:EA=10:1).

[0114] Post-treatment and purification: The reaction solution was slowly poured into 500 mL of water, extracted with petroleum ether / ether (10 / 1) (110 mL x 3), the organic phases were combined and washed twice with saturated brine (150 mL x 2), dried over anhydrous sodium sulfate, filtered, and the petroleum ether / ether (recoverable) was removed by rotary evaporation at 40 ° C, and then dried with an oil pump to obtain 2.2 g of an off-white solid.

[0115] Step 2

[0116]

[0117] Operation: Dissolve 250 mg of 4-azido-3,5-tetrafluorobenzaldehyde in 10 mL of methanol. After dissolution, cool to 0°C in an ice bath. Add 11 mg of sodium borohydride gradually in portions and detect immediately after addition.

[0118] Detection: TLC detection (PE:EA=10:1).

[0119] Post-treatment: After the reaction is confirmed to be complete after detection, it is immediately quenched, 4 mL of dilute hydrochloric acid solution (1 mol / L) and 20 mL of water (a large amount of white solid precipitates) are added under an ice bath, stirred for 10 min, methanol (recoverable) is removed at 45 ° C, and then 20 mL of dilute hydrochloric acid solution (1 mol / L) and 30 mL of water are added, stirred for 10 min (pH>1.5 at this time), filtered, the filter cake is rinsed with 30 mL of water, dried, and then pumped dry with an oil pump to obtain 250 mg of a white solid product.

[0120] Step 3

[0121]

[0122] Procedure: Dissolve 134.8 mg of 4-azido-3,5-tetrafluorobenzyl alcohol in 5 mL of DCM, add 190.89 mg of p-nitrophenyl chloroformate, cool to 0°C in an ice bath, add 13.5 mg of DMAP, and then slowly drop 0.15 mL of triethylamine, and react in an ice bath for 30-60 min (mainly for detection).

[0123] Detection: TLC detection (PE:EA=10:1).

[0124] Post-treatment and purification: After treatment, add water to quench, pay attention to controlling the low temperature, extract with ethyl acetate, and purify by column chromatography.

[0125] Example 1

[0126] Preparation of Azide Prodrug XP-101-DAU-000:

[0127]

[0128] Synthesis method:

[0129]

[0130] Procedure: Dissolve 60 mg (4-azido-2,3,5,6-tetrafluorophenyl)methyl-4-nitrophenyl carbonate (intermediate 5) in 4 mL of anhydrous DMF at room temperature, then add molecular sieves and 25 μL of triethylamine, and stir for 10 min. Dissolve 30 mg of daunorubicin hydrochloride in 4 mL of anhydrous DMF, add 25 μL of triethylamine, and add the above solution; react at room temperature for 19-24 h under nitrogen protection.

[0131] Detection: The reaction was monitored by TLC (5% methanol:DCM).

[0132] Workup: dilute with water, extract with EtOAc, saturated NaHCO 3 The solution was washed with water, anhydrous Na 2 SO 4 Dry and concentrate by rotary evaporation.

[0133] Purification: Finally, purification by flash chromatography (5% MeOH:EtOAc) gave 35 mg of a brick-red product, which was the azide prodrug XP-101-DAU-000.

[0134] 1 H NMR (400 MHz, CDCl 3 )δ13.99(s,1H),13.29(s,1H),8.17–7.94(m,1H),7.79(t,J=8.0Hz,1H),7.39(d,J=8.4Hz,1H),5. 49(d,J=3.6Hz,1H),5.26(s,1H),5.11(s,2H),4.42(s,1H),4.21(d,J=6.0Hz,1H),4.08(s,3H),3. 87(s,1H),3.65(s,1H),3.24(d,J=18.8Hz,1H),3.01–2.83(m,3H),2.41(s,2H),2.30(d,J=14.8Hz ,1H),2.11(dd,J=14.8,3.9Hz,1H),1.89(d,J=13.6Hz,2H),1.80–1.70(m,1H),1.33–1.23(m,3H).

[0135] HRMS(ESI):m / z Calcd for C 35 H 30 F 4 N 4 O 12 Na,[M+Na] + :797.16886,found:797.16913.

[0136] HPLC detection: The HPLC peak time of the original drug molecule DAU is 17.284min. The peak area (HPLC 254nm) of the prodrug before and after irradiation is reduced by 32.4% (100Gy, 20uM), and the new peaks generated are more complex.

[0137]

[0138] Conversion rate involved = (peak area of ​​prodrug before irradiation - peak area of ​​prodrug after irradiation) / peak area of ​​prodrug before irradiation.

[0139] Example 2

[0140] Preparation of prodrug XP-104-CPT-000:

[0141]

[0142] Synthesis method:

[0143]

[0144] Procedure: Dissolve 105 mg of camptothecin in 1 mL of anhydrous DCM, add 150 mg of p-nitrophenyl chloroformate, cool to 0°C in an ice bath, and stir for 10 min.

[0145] 37 mg of DMAP was dissolved in 1 mL of anhydrous DCM and added dropwise to the reaction system. Stirring was continued for 10 min. The reaction system was taken out of the ice bath and stirred at room temperature for 2 h.

[0146] Detection: Use TLC to detect whether camptothecin has reacted completely.

[0147] Work-up: dilute with EtOAc, wash with 0.1M HCl, wash with water, wash with Na 2 SO 4 Dry and concentrate by rotary evaporation.

[0148] Purification: Finally, the desired product was purified by flash chromatography (EtOAc).

[0149]

[0150] Procedure: Dissolve 65 mg of camptothecin-4-nitrophenyl carbonate and 100 mg of 4-azido-2,3,5,6-tetrafluorobenzyl alcohol in 3 mL of anhydrous DCM at room temperature and stir for 10 min. Dissolve 24 mg of DMAP and 30 μL of triethylamine in 1 mL of anhydrous DCM and slowly add dropwise to the above solution; react at room temperature for 24 h under nitrogen protection.

[0151] Detection: Monitor the reaction by TLC.

[0152] Work-up: dilute with water, extract with EtOAc, saturated NaHCO 3 Solution, 0.1M hydrochloric acid and saturated brine, anhydrous Na 2 SO 4 Dry and concentrate by rotary evaporation.

[0153] Purification: Finally, the product was purified by flash chromatography (EtOAc).

[0154] 1 H NMR (400 MHz, CDCl 3)δ8.42(s,1H),7.96(d,J=8.1Hz,1H),7.86(t,J=7.7Hz,1H),7.70(dt,J=1 0.6,5.3Hz,2H),7.53(dd,J=5.6,3.3Hz,1H),5.72(d,J=17.3Hz,1H),5.42( d,J=17.3Hz,1H),5.34–5.27(m,2H),4.30(t,J=6.7Hz,2H),2.37(td,J=14 .8,7.3Hz,1H),2.23(ddd,J=25.4,16.4,9.0Hz,1H),1.07(t,J=7.5Hz,3H).

[0155] HRMS(ESI):m / z Calcd for C 28 H 17 O 6 N 5 F 4 Na,[M+Na] + :618.10072,found:618.10070.

[0156] HPLC detection: The HPLC peak time of the original drug molecule CPT is 18.442min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 95.5% (100Gy, 10uM), producing the original drug CPT peak and the linker peak compound 3-a (HPLC254nm peak time is 17.0min).

[0157]

[0158] Among them, the structure of compound 3-a is

[0159] Intracellular toxicity assay

[0160] The corresponding cells were seeded in a 96-well cell culture plate at a density of 4000 per well and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; then the cells were treated with different concentrations of XP-104-CPT-000 and original drug CPT, and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio with the culture medium was 1:10) was added to each well, and after constant temperature incubation for 2 hours, the ultraviolet absorption at 450nm in each well was detected with an enzyme marker, and the cell activity value was obtained by comparing the absorbance of the treated cells with that of the untreated cells, and the IC50 value of the compound was calculated.

[0161] The specific results are shown in Table 1. For different cells, the IC50 of the prodrug XP-104-CPT-000 is significantly lower than that of the original drug CPT.

[0162] Example 3

[0163] Preparation of prodrug XP-106-MMAE-000:

[0164]

[0165] Synthesis method:

[0166]

[0167] Operation: Dissolve 150 mg MMAE in 0.5 mL anhydrous DMF, add 140 mg (4-azido-2,3,5,6-tetrafluorophenyl)methyl-nitrophenyl carbonate, then add 60 mg HOBt and dropwise 150 uL triethylamine, and react at room temperature for 24 h.

[0168] Detection: TLC detection (EA).

[0169] Post-treatment and purification: At 40°C, N,N-dimethylformamide was spin-dried with an oil pump, redissolved with dichloromethane and purified with a silica gel plate, the eluent ratio (DCM:MeOH=20:1). The product (205 mg, 84.3% yield) was obtained as a white solid.

[0170] 1 H NMR (400 MHz, CDCl 3 )δ7.33(dd,J=19.2,12.6Hz,5H),6.51(d,J=12.8Hz,2H),5.43–5.11(m,3H),4 .96(s,1H),4.70(s,2H),4.40–3.99(m,6H),3.86(dd,J=31.2,17.4Hz,2H),3. 65–3.26(m,9H),3.12–2.74(m,6H),2.40(dt,J=24.0,15.2Hz,3H),2.29–2.14 (m,2H),2.01(d,J=24.0Hz,4H),1.85(s,2H),1.26(s,3H),1.06–0.77(m,20H).

[0171] HRMS(ESI):m / z Calcd for C 47 H 68 O 9 N 8 F 4 Na,[M+Na] +:987.49376,found:987.49377.

[0172] HPLC detection: The HPLC peak time of the original drug molecule MMAE is about 17.018min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 83.2% (100Gy, 10uM), and new peaks are generated, including the intermediate state XP-106-MMAE-000-cp1, the original drug MME (220nm) and the linker peak compound 3_a.

[0173]

[0174] in,

[0175] Example 4

[0176] Preparation of prodrug XP-107-BUF-000:

[0177]

[0178] Synthesis method:

[0179]

[0180] Procedure: Dissolve 100 mg of bufalin in 1 mL of anhydrous DCM, add 120 mg of p-nitrophenyl chloroformate, add 38 mg of DMAP and 60 uL of TEA at 0°C, and react at room temperature for 24 h.

[0181] Detection: TLC detection (PE:EA=1:1)

[0182] Post-treatment and purification: spin-dry at 37°C, dissolve again in dichloromethane and separate by wet column chromatography (PE:EA=1:0, 5:1, 3:1, 1:1). Finally, the product compound 7 (110 mg, 77.1% yield) was obtained as a white solid.

[0183]

[0184] Procedure: 60 mg of compound 7 was dissolved in 2 mL of anhydrous DCM, 28 mg of (4-azido-2,3,5,6-tetrafluorophenyl)methanol, 26 mg of DMAP and 40 uL of TEA were added, and the mixture was reacted at room temperature for 48 h.

[0185] Detection: TLC detection (PE:EA=1:1)

[0186] Post-treatment and purification: spin-dry at 37°C, dissolve again in dichloromethane and separate on a silica gel plate (PE:EA=1:1). Finally, the product XP-107-BUF-000 (50 mg, 72.5% yield) was obtained as a white solid.

[0187] 1 H NMR (400MHz, CDCl3) δ7.83 (dd, J=9.6, 2.5Hz, 1H), 7.23 (d, J=2.0Hz, 1H), 6.26 (d, J=9.6Hz,1H),5.23(s,2H),5.00(s,1H),2.46(dd,J=9.2,6.4Hz,1H),2.26–2.13(m ,1H),2.12–1.98(m,2H),1.99–1.81(m,3H),1.80–1.63(m,5H),1.57(d,J=3.6Hz,3 H),1.49(d,J=3.6Hz,1H),1.45–1.14(m,8H),0.92(d,J=18.0Hz,3H),0.70(s,3H).

[0188] HRMS(ESI):m / z Calcd for C 32 H 36 O 6 N 3 F 4 ,[M+H] + :634.25348,found:634.25378.

[0189] HPLC detection: The HPLC peak time of the original drug molecule BUF is 21.813min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 72.3% (100Gy, 10uM), and a small amount of original drug BUF and linker compound 3_a are produced.

[0190]

[0191] Intracellular toxicity assay

[0192] The corresponding cells were seeded in a 96-well cell culture plate at a density of 4000 per well and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (with a ratio of 1:10 to the culture medium) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450nm in each well was detected with an ELISA instrument, and the cell activity value was obtained by comparing the absorbance of the treated cells with that of the untreated cells, and the IC50 value of the compound was calculated.

[0193] The specific results are shown in Table 4 and Figure 1 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0194] Example 5

[0195] Prodrug XP-107-BUF-004

[0196]

[0197] Synthesis method:

[0198]

[0199] Procedure: Dissolve 120 mg of compound 5 in 5 mL of anhydrous DCM, add 40 mg of Piperazine and 56 u of LTEA, and react at room temperature for 2 h.

[0200] Detection: TLC detection (PE:EA=1:1)

[0201] Post-treatment and purification: spin-dry at 37°C, re-separate with dichloromethane-assisted wet column chromatography (PE:EA=1:0,0:1), (EA:MeOH=20:1,10:1,5:1) and add 1 / 1000 of ammonia water. Finally, product 6 (92.2 mg, 92% yield) was obtained as a white solid.

[0202]

[0203] Procedure: Dissolve 40 mg of compound 7 in 2 mL of anhydrous DCM, add 29 mg of compound 6 and 40 u of LTEA, and react at room temperature for 24 h.

[0204] Detection: TLC detection (PE:EA=1:1)

[0205] Post-treatment and purification: spin-dry at 37°C, dissolve in dichloromethane again, and separate on a silica gel plate (DCM:MeOH=30:1). The obtained white solid is then ultrasonically washed with (PE:EA=1:1, 1 mL) and filtered. Finally, the product XP-107-BUF-004 (30 mg, 50.1% yield) is obtained as a white solid.

[0206] 1 H NMR(400MHz, CDCl3)δ7.85(dd,J=9.6,2.5Hz,1H),7.25(s,1H),6.29(d,J=9.6Hz ,1H),5.25(s,2H),5.06(s,1H),3.75(d,J=7.2Hz,1H),3.49(s,8H),2.56–2.41(m ,1H),2.22(d,J=12.4Hz,2H),2.07(t,J=10.8Hz,2H),1.92(dd,J=17.2,7.2Hz,2 H),1.76(dd,J=25.2,11.9Hz,4H),1.57–1.14(m,12H),0.97(s,3H),0.72(s,3H).

[0207] HRMS(ESI):m / z Calcd for C 37 H 44 O 7 N 5 F 4 ,[M+H] + :746.31714,found:746.31757.

[0208] HPLC detection: The HPLC peak time of the original drug molecule is 17.077min. The peak area (HPLC 254nm) of prodrug before and after irradiation decreased by 48.6% (100Gy, 10uM). There was no new peak after the first 100Gy irradiation. After two consecutive 100Gy irradiations, a small amount of original drug 3-2-3 and linker compound 3_a were produced at 220nm.

[0209]

[0210]

[0211] Intracellular toxicity assay

[0212] The corresponding cells were seeded in a 96-well cell culture plate at a density of 4000 per well and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value, and the IC50 value of the compound was calculated.

[0213] The specific results are shown in Table 4 and Figure 2 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0214] Example 6

[0215] Prodrug XP-102-DOX-001

[0216]

[0217] Synthesis method:

[0218]

[0219] Procedure: Dissolve doxorubicin (50.0 mg, 0.09 mmol, 1.0 eq) and compound 13 (60 mg) in anhydrous N,N-dimethylformamide (1 mL). Add triethylamine (0.04 mL) to the reaction solution under ice bath. React the reaction solution at 0°C for 24 h.

[0220] Detection: TLC (DCM:MeOH=10:1) showed that the doxorubicin in raw material 1 had been completely reacted, and two new spots with very close polarities were generated.

[0221] Post-treatment and purification: Filter with 0.2um filter membrane, separate by preparative HPLC, and obtain peak 1 (21.882min). Peak 2 (22.279min) was not clean (there were two peaks, isomers), and peak 1 (21.882min) was used for experiments.

[0222] HRMS(ESI):m / z Calcd for C 40 H 37 O 16 N 5 F 4 Na,[M+Na] + :942.20636,found:942.20648.

[0223] HPLC detection: The HPLC peak time of the original drug molecule is about 16.265min. The peak area (HPLC 254nm) of prodrug before and after irradiation decreased by 46.2% (100Gy, 10uM), but there was no obvious new peak.

[0224]

[0225] Intracellular toxicity assay

[0226] The corresponding cells were seeded at a density of 10,000 per well in a 96-well cell culture plate and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 24 hours; after 24 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value, and the IC50 value of the compound was calculated.

[0227] Specific results such as Figure 3 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0228] Example 7

[0229] Preparation of prodrug XP-102-DOX-002

[0230]

[0231] Synthesis method:

[0232]

[0233] Procedure: Dissolve 27 mg of compound 14 in 5 mL of anhydrous DCM, add 22 mg of p-nitrophenyl chloroformate, add 3 mg of DMAP and 15 u of LTEA at 0°C, and react at room temperature for 2 h.

[0234] Detection: TLC detection (PE:EA=5:1)

[0235] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (PE:EA=3:1).

[0236] Finally, the product compound 15 (14 mg) was obtained in the form of a white solid.

[0237]

[0238] Procedure: Dissolve 20 mg DOX in 0.5 mL anhydrous DMF, add 18 mg compound 15, add 3 mg HOBt and 15 uL TEA at 0°C, and react at room temperature for 24 h.

[0239] Detection: TLC detection (DCM: MeOH = 10: 1)

[0240] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (DCM:MeOH=10:1). Finally, the product XP-102-DOX-002 (5 mg) was obtained as a brick-red solid.

[0241] HPLC showed two peaks, indicating isomerism.

[0242] HRMS(ESI):m / z Calcd for C 41 H 34 O 13 N 4 F 4 Na,[M+Na] + :889.19507,found:889.19537.

[0243] HPLC detection: The HPLC peak time of the original drug molecule is about 16.265min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 62.1% (100Gy, 10uM), and two new peaks XP-102-DOX-002-cp1 (22.884; 23.132min) are generated.

[0244]

[0245] in,

[0246] Intracellular toxicity assay

[0247] The corresponding cells were seeded at a density of 10,000 per well in a 96-well cell culture plate and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 24 hours; after 24 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value, and the IC50 value of the compound was calculated.

[0248] Specific results such as Figure 4As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0249] Example 8

[0250] Preparation of prodrug XP-102-DOX-004

[0251]

[0252] Synthesis method:

[0253]

[0254] Procedure: Dissolve 70 mg of compound 16 in 0.5 mL of anhydrous DCM, add 45 mg of compound 4, add 7 mg of DMAP and 24 uL of TEA at 0°C, and react at room temperature for 1-2 h.

[0255] Detection: TLC detection (PE:EA=5:1)

[0256] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (DCM:MeOH=20:1). Finally, the product compound 17 was obtained as a brick red solid.

[0257]

[0258] Procedure: Dissolve 20 mg DOX in 0.5 mL anhydrous DMF, add 27 mg compound 17, add 3 mg HOBt and 18 uL TEA at 0°C, and react at room temperature for 24 h.

[0259] Detection: TLC detection (EA)

[0260] Post-treatment and purification: spin-dry at 37°C, dissolve again in dichloromethane and separate on a silica gel plate (DCM:MeOH=20:1). Finally, the product XP-102-DOX-004 is obtained as a brick-red solid.

[0261] HRMS(ESI):m / z Calcd for C 41 H 29 O 13 N 7 F 8 Na,[M+Na] + :1002.15878,found:1002.15906.

[0262] HPLC detection: The HPLC peak time of the original drug molecule is about 16.265min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 20.4% (100Gy, 10uM), and multiple small new peaks are generated with very weak absorption.

[0263]

[0264] Example 9

[0265] Prodrug XP-106-MMAE-001

[0266]

[0267] Synthesis method:

[0268]

[0269] Procedure: Dissolve 73 mg MMAE in 2 mL anhydrous DMF, add 90 mg compound 12, then add 30 mg HOBt and dropwise 70 uL triethylamine, and react at room temperature in the dark for 24 h.

[0270] Detection: TLC detection (EA).

[0271] Post-treatment and purification: At 30°C, N,N-dimethylformamide was spin-dried with an oil pump, and the sample was re-dissolved in dichloromethane and wet-loaded, and then column chromatography (n-hexane: ethyl acetate = 1:0, 1:1, 0:1) was changed to (ethyl acetate: methanol = 20:1). The product (11 mg, 85% yield) was obtained as a white solid.

[0272] HRMS(ESI):m / z Calcd for C 56 H 83 O 12 N 9 F 4 Na,[M+Na] + found:1172.59924.

[0273] 1 H NMR (400 MHz, CDCl 3)δ8.06(d,J=15.2Hz,1H),7.61–7.32(m,5H),6.80–6.68(m,1H),6.37(s,2H),4.99(s,1H),4.71(s,2H),4.36–3.90(m,7H) ,3.76(d,J=4.8Hz,2H),3.65–3.22(m,9H),3.23–2.72(m,12H),2.55–1.98(m,9H),1.59–1.14(m,17H),1.16–0.68(m,17H).

[0274]

[0275] Procedure: 100 mg of compound 18 was dissolved in 5 mL of anhydrous DCM, and 0.3 mL of TMSOTf dissolved in 1 mL of DCM was added dropwise at 0°C. The mixture was reacted in an ice bath in the dark for 1 h. If unreacted, 0.2 mL of TMSOTf was added and the reaction was continued for another half an hour.

[0276] Detection: TLC detection (EA).

[0277] Workup and purification: NaHCO 3 The solution was quenched (pH < 5), 10 mL of DCM was added, and the organic phase was extracted. The organic phase was washed with brine and dried over anhydrous sodium sulfate. The sample was re-dissolved in dichloromethane and wet loaded, column chromatography (ethyl acetate: methanol = 1:0, 100:1, 20:1, 10:1, 1:1), spin-dried, and the DCM solution was filtered with a filter membrane (except silica gel). The mixture was slurried with n-hexane: ethyl acetate (20:1) for 2 h, and the solid (60 mg, 63.1% yield) was obtained by centrifugation. The solid was white solid.

[0278] HRMS(ESI):m / z Calcd for C 52 H 75 O 12 N 9 F 4 Na,[M+Na] + ,found:1116.53931.

[0279] HPLC detection: The HPLC peak time of the original drug molecule is about 17.018min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 72.7% (100Gy, 10uM), producing the intermediate XP-106-MMAE-001cp1, the original drug MMAE and the linker peak compound 13-a.

[0280]

[0281]

[0282] Among them, the structures of XP-106-MMAE-001cp1 and the linker peak compound 13-a are as follows:

[0283]

[0284] Intracellular toxicity assay

[0285] The corresponding cells were seeded at a density of 10,000 per well in a 96-well cell culture plate and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 24 hours; after 24 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value, and the IC50 value of the compound was calculated.

[0286] Specific results such as Figure 5 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0287] Example 10

[0288] Prodrug XP-106-MMAE-002

[0289]

[0290] Synthesis method:

[0291]

[0292] Procedure: Dissolve 20 mg MMAE in 0.5 mL anhydrous DMF, add 14 mg compound 15, then add 3 mg HOBt and dropwise 10 uL triethylamine, and react at room temperature for 24 h.

[0293] Detection: TLC detection (EA).

[0294] Post-treatment and purification: At 40°C, N,N-dimethylformamide was spin-dried with an oil pump, redissolved with dichloromethane and purified with a silica gel plate, eluent ratio (EA). Finally, the product XP-106-MMAE-002 (20 mg) was obtained as a white solid.

[0295] HRMS(ESI):m / z Calcd for C 53 H 72 O9 N 8 F 4 Na,[M+Na] + ,found:1041.5437.

[0296] HPLC detection: The HPLC peak time of the original drug molecule is about 17.018min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 13.1% (100Gy, 20uM), producing the intermediate XP-106-MMAE-002cp1 and the original drug MMAE.

[0297]

[0298] Among them, the structure of the intermediate state XP-106-MMAE-002cp1 is as follows:

[0299]

[0300] Intracellular toxicity assay

[0301] The corresponding cells were seeded at a density of 10,000 per well in a 96-well cell culture plate and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 24 hours; after 24 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value, and the IC50 value of the compound was calculated.

[0302] Specific results such as Figure 6 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0303] Embodiment 11

[0304] Prodrug XP-106-MMAE-004

[0305]

[0306] Synthesis method:

[0307]

[0308] Procedure: 30 mg MMAE was dissolved in 0.5 mL anhydrous DMF, 27 mg compound 17 was added, and then 3 mg HOBt and 18 uL triethylamine were added dropwise, and the mixture was reacted at room temperature for 24 h.

[0309] Detection: TLC detection (EA).

[0310] Post-treatment and purification: At 40°C, spin-dry N,N-dimethylformamide with an oil pump, dissolve it again with dichloromethane and purify it with a silica gel plate, eluent ratio (EA). Finally, the product XP-106-MMAE-004 is obtained as a white solid. HRMS (ESI): m / z Calcd for C 53 H 67 O 9 N 11 F 8 Na,[M+Na] + :1176.48877,found:1176.48889.

[0311] HPLC detection: The HPLC peak time of the original drug molecule is about 17.018min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 99.0% (100Gy, 20uM), producing the intermediate XP-106-MMAE-004cp1 and the original drug MMAE.

[0312]

[0313] Among them, the structure of the intermediate state XP-106-MMAE-004cp1 is as follows:

[0314]

[0315] Example 12

[0316] Prodrug XP-106-MMAE-005

[0317]

[0318] Synthesis method:

[0319]

[0320] Procedure: Dissolve 34.46 mg MMAE in 0.5 mL anhydrous DMF, add 20 mg compound 22, then add 10 mg HOBt and dropwise 8 uL triethylamine, and react at room temperature in the dark for 24 h.

[0321] Detection: TLC detection (EA).

[0322] Post-treatment and purification: At 40°C, N,N-dimethylformamide was spin-dried with an oil pump, redissolved with dichloromethane and purified with a silica gel plate, and the eluent was pure EA. Finally, the product XP-106-MMAE-005 (40 mg, 89.7% yield) was obtained as a white solid.

[0323] 1 H NMR (400MHz, CDCl3) δ7.79 (dd, J=57.2, 8.0Hz, 1H), 7.55-7.30 (m, 3H), 6.91 (d, J=8.8Hz, 1H), 6.50 (d, J=9.2Hz,1H),5.32(d,J=19.2Hz,1H),5.16-4.91(m,2H),4.69(d,J=7.6Hz,2H),4.37–4.23(m,1H),4.1 6(s,1H),4.08(s,1H),3.84(d,J=8.0Hz,1H),3.58-3.17(m,5H),3.06-2.62(m,4H),2.49-2.30(m,2H) ,2.03(dd,J=31.6,21.7Hz,3H),1.85(s,1H),1.37-1.17(m,7H),1.09–0.94(m,3H),0.91-0.67(m,7H).

[0324] HRMS(ESI):m / z Calcd for C 47 H 70 O 9 N 8 F 2 Na,[M+Na] + :951.51260,found:951.51257.

[0325] HPLC detection: The HPLC peak time of the original drug molecule is about 17.018min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 99.0% (100Gy, 10uM), producing the intermediate XP-106-MMAE-005cp1 and the original drug MMAE.

[0326]

[0327] Among them, the structure of the intermediate state XP-106-MMAE-005cp1 is as follows:

[0328]

[0329] Intracellular toxicity assay

[0330] The corresponding cells were seeded in a 96-well cell culture plate at a density of 10,000 per well and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the cell activity value was obtained by comparing the absorbance of the treated cells with that of the untreated cells, and the IC50 value of the compound was calculated.

[0331] Specific results such as Figure 7 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0332] Embodiment 13

[0333] Prodrug XP-107-BUF-001

[0334]

[0335] Synthesis method:

[0336]

[0337] Procedure: Dissolve 20 mg of compound 3-2-3 in 0.5 mL of anhydrous DCM, add 20 mg of compound 13, and then add 20 uL of triethylamine, and react at room temperature for 24 h.

[0338] Detection: TLC detection (DCM:MeOH=10:1).

[0339] Post-treatment and purification: DCM was dried by spin drying at 37°C and purified by HPLC. The product (5 mg, 84.3% yield) was obtained as a white solid.

[0340] HRMS(ESI):m / z Calcd for C 42 H 51 O 10 N 6 F 4 ,[M+Na] + :875.35973,found:875.36023.

[0341] HPLC detection: The HPLC peak time of the original drug molecule is about 17.077min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 69.1% (100Gy, 20uM), producing linker compound 13-a, intermediate XP-107-BUF-001cp1 and original drug 3-2-3.

[0342]

[0343]

[0344] Embodiment 14

[0345] Prodrug XP-104-CPT-002

[0346]

[0347] Synthesis method:

[0348]

[0349] Procedure: 106 mg of compound 6 was dissolved in 2 mL of anhydrous DCM and 1 mL of DMF, 48.5 mg of compound 21 was added, and then 8 mg of DMAP and 35 uL of triethylamine were added dropwise, and the mixture was reacted at room temperature for 16 h.

[0350] Detection: TLC detection (DCM:MeOH=20:1).

[0351] Post-treatment and purification: At 37°C, first spin-dry DCM and then spin-dry N,N-dimethylformamide using an oil pump, re-dissolve it in dichloromethane and purify it by column chromatography to obtain the product XP-104-CPT-002 (54 mg) as an off-white solid.

[0352] 1 H NMR (400MHz, CDCl3) δ8.41(s,1H),8.23(d,J=8.4Hz,1H),7.95(d,J=8.0Hz,1 H),7.89–7.83(m,1H),7.69(t,J=7.2Hz,1H),7.25(s,1H),6.97–6.88(m,2H) ,5.71(d,J=17.2Hz,1H),5.38(d,J=17.2Hz,1H),5.30(s,2H),5.08–4.99(m, 2H), 2.27(dt,J=15.2,7.6Hz,1H),2.20–2.12(m,1H),1.02(t,J=7.6Hz,3H).

[0353] HRMS(ESI):m / z Calcd for C 28 H 20 O 6 N 5 F 2 ,[M+Na] + :560.13762,found:560.13812.

[0354] HPLC detection: The HPLC peak time of the original drug molecule is about 18.442 min. The peak area (HPLC 254 nm) of prodrug before and after irradiation is reduced by 63.1%, producing the original drug CPT and the intermediate XP-104-CPT-002cp1.

[0355]

[0356]

[0357]

[0358] Embodiment 15

[0359] Prodrug XP-104-CPT-004

[0360]

[0361] Synthesis method:

[0362]

[0363] Dissolve 2-amino-5-fluorobenzyl alcohol (50.0 mg, 1.0 eq) in 5M hydrochloric acid (2.0 mL) and stir in an ice bath. Slowly drop sodium nitrite (27.0 mg, 1.1 eq) aqueous solution into the reaction solution. After 10 minutes, slowly add sodium azide (92.0 mg, 4.0 eq) aqueous solution into the reaction solution and stir in an ice bath for 1 hour. After the reaction is completed, adjust the pH to 8 with sodium bicarbonate, extract with ethyl acetate, and spin dry the organic phase (temperature <35°C). Purify with prep-TLC (EA:PE=1:1) to obtain the product 2-azido-5-fluorobenzyl alcohol (50.0 mg, 84% Yield) as a brown solid.

[0364]

[0365] Procedure: Intermediate 6 (100 mg, 1.0 eq.) was dissolved in dry DCM (3 mL) and DMF (0.5 mL), TEA (82 μL, 3.0 eq), DMAP (10 mg, 0.1 eq) and 2-azido-5-fluorobenzyl alcohol (40 mg, 1.1 eq) were added under ice bath and stirred at room temperature for 24 hours.

[0366] Detection: TLC detection (DCM:MeOH=20:1).

[0367] Post-treatment and purification: At 37°C, the reaction solution was spin-dried with DCM and purified by prep-TLC (EA) to give the product (6 mg, 5% Yield) as a white solid.

[0368] 1 H NMR (400MHz, CDCl3) δ8.43(s,1H),8.26(d,J=8.4Hz,1H),7.98(d,J=8.4Hz,1H),7.88(t,J=7 .2Hz,1H),7.71(t,J=7.6Hz,1H),7.34(d,J=5.6Hz,1H),7.15(dd,J=8.8,2.8Hz,1H),7.09(d d,J=8.8,4.5Hz,1H),7.04–6.97(m,1H),5.73(d,J=17.2Hz,1H),5.42(d,J=17.2Hz,1H),5.3 3(s,2H),5.12(d,J=2.0Hz,2H),2.35–2.30(m,1H),2.24–2.16(m,1H),1.04(t,J=7.6Hz,3H).

[0369] LCMS(ESI):m / z found:542.3.

[0370] HPLC detection: The HPLC peak time of the original drug molecule is about 18.544min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 58.1% (100Gy, 10uM), producing original drug CPT.

[0371]

[0372] Example 16

[0373] Prodrug XP-104-CPT-005

[0374]

[0375] Synthesis method:

[0376]

[0377] Procedure: 80 mg of compound 6 was dissolved in 5 mL of anhydrous DCM, 27.9 mg of compound 27 was added, and then 5.7 mg of DMAP and 26 uL of triethylamine were added dropwise, and the mixture was reacted at room temperature for 16 h.

[0378] Detection: TLC detection (DCM:MeOH=20:1).

[0379] Post-treatment and purification: At 37°C, N,N-dimethylformamide was spin-dried with an oil pump, redissolved with dichloromethane and purified using a large silica gel plate with an eluent ratio of (DCM:MeOH=10:1) to obtain the product XP-104-CPT-005 (44 mg) as an off-white solid.

[0380] 1 H NMR (400MHz, CDCl3) δ8.41(s,1H),8.23(d,J=8.6Hz,1H),7.96(d,J=8.0Hz,1H),7.93–7.83(m,1H),7.76–7.66(m,1H),7.33(d,J=8.4Hz,2H),7 .26(s,2H),6.92(d,J=8.4Hz,1H),5.70(d,J=17.2Hz,1H),5.49–5.28(m ,3H),5.09(d,J=14.4Hz,2H),2.32–2.10(m,2H),1.01(t,J=7.6Hz,3H).

[0381] HRMS(ESI):m / z Calcd for C 28 H 22 O 6 N 5 ,[M+Na] + :524.15756,found:524.15680.

[0382] HPLC detection: The HPLC peak time of the original drug molecule is about 18.490min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 68.1% (100Gy, 10uM), producing original drug CPT.

[0383]

[0384] Embodiment 17

[0385] Prodrug XP-106-MMAE-008

[0386]

[0387] Synthesis method:

[0388]

[0389] Dissolve p-azidobenzyl alcohol (35 mg, 0.23 mmol, 1.0 eq) in anhydrous DCM, dissolve compound 4 (55.6 mg, 0.27 mmol, 1.2 eq) in DCM, add to the reaction bottle under ice bath conditions, stir in ice bath for five minutes, then slowly drop DMAP (3.5 mg, 10%, 0.5 ml) in dichloromethane solution, TEA (47.8 uL, 0.34 mmol, 1.5 eq) in 0.5 mL dichloromethane solution, react for 10 min, monitor the reaction with PE:EA=1:1, the product Rf is about 0.8. Directly scrape the plate for purification to obtain a white solid product (53 mg, yield: 73.4%).

[0390]

[0391] Procedure: Dissolve MMAE (43.8 mg, 0.06 mmol, 1.0 eq) in anhydrous DMF, the solution becomes colorless and transparent, add intermediate 28 (23 mg, 0.073, 1.2 eq), the solution turns yellow. Add HOBt DMF solution (10 mg, 30%, 0.5 ml), the reaction solution turns light yellow, add TEA (10 uL, 1.2 eq), the reaction solution turns darker and light yellow. React at room temperature overnight in the dark.

[0392] Detection: The reaction was judged to be complete after monitoring the reaction with pure EA by TLC.

[0393] Post-treatment and purification: At 38°C, the DMF was removed by oil pump and redissolved on a large plate with a small amount of DCM. Pure EA was run on a large plate for purification to obtain 12.8 mg of a white solid product (yield: 23.5%).

[0394] 1H NMR (400MHz, CDCl3) δ7.50–7.31(m,5H),7.00(d,J=7.6Hz,2H),6.53(s,2H),6.32–6.18(m ,1H),5.39–5.29(m,1H),5.11(d,J=26.4Hz,3H),4.95(s,1H),4.68(s,3H),4.39–3.98(m, 6H),3.86(s,2H),3.65–3.27(m,9H),3.18–2.84(m,7H),2.83–2.70(m,2H),2.39(d,J=21. 2Hz,4H),2.19(d,J=20.4Hz,4H),1.98(d,J=44.8Hz,6H),1.84(s,4H),1.13–0.93(m,9H).

[0395] HRMS(ESI):m / z Calcd for C 47 H 72 O 9 N 8 Na,[M+Na] + :915.53145,found:915.53156.

[0396] HPLC detection: The HPLC peak time of the original drug molecule is about 17.043min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 69.1% (100Gy, 10uM), producing the original drug MMAE.

[0397]

[0398] Embodiment 18

[0399] Prodrug XP-106-MMAE-009

[0400]

[0401] Synthesis method:

[0402]

[0403] Procedure: Dissolve 50 mg of compound 29 in 5 mL of ethanol: water (7:3), add 40 mg of cuprous iodide, NaN 3 27.3 mg and 7.4 mg of L-Proline, 2.5 mg of NaOH (dissolved in 10 mL of ethanol: water (7:3)) was added dropwise under argon and reacted at 70°C for 2 h.

[0404] Detection: LCMS monitoring.

[0405] Post-treatment and purification: The cooled mixture was extracted with ethyl acetate and water (if the substrate has a carboxylic acid group, the reaction mixture should be acidified to pH = 1 before extraction). The organic layer was separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine and washed with Na 2 SO 4 Dry and concentrate in vacuo. The crude product is then subjected to the next step.

[0406]

[0407] Procedure: 70 mg of compound 30 was dissolved in 2 mL of anhydrous DCM, 79 mg of compound 31a was added, and then 89.61 mg of DCC, 58.7 mg of HOBt and 203 uL of triethylamine were added, and the mixture was reacted at room temperature for 16 h.

[0408] Detection: TLC detection (PE:EA=1:1).

[0409] Post-treatment and purification: spin-drying, re-dissolving in dichloromethane and purifying on a large silica gel plate with an eluent ratio of (PE:EA=1:1) to obtain 18.8 mg of a yellow oily product.

[0410]

[0411] Procedure: 18.8 mg of compound 31 was dissolved in 1 mL of anhydrous DCM, 23.5 mg of compound 31a was added, and then 1 mg of DMAP and 17 uL of triethylamine were added dropwise at 0°C, and the mixture was reacted at room temperature for 16 h.

[0412] Detection: TLC detection (PE:EA=1:1).

[0413] Post-treatment and purification: Purification was performed using a large silica gel plate with an eluent ratio of (PE:EA=1:1) to obtain 6 mg of the product as a yellow solid.

[0414]

[0415] Procedure: Dissolve 5 mg MMAE in 5 mL anhydrous DMF, add 6 mg compound 32, then add 1.89 mg HOBt and dropwise add 2 uL triethylamine, and react at room temperature for 16 h.

[0416] Detection: TLC detection (EA 100%).

[0417] Post-treatment and purification: At 37°C, N,N-dimethylformamide was spin-dried with an oil pump, redissolved with dichloromethane and purified using a large silica gel plate with an eluent ratio of (EA 100%) to obtain the product XP-106-MMAE-009 (4.5 mg) as an off-white solid.

[0418] LCMS(ESI):m / z found:1065.0.

[0419] HPLC detection: The HPLC peak time of the original drug molecule is about 17.025min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 73.34% (60Gy, 10uM), producing the original drug MMAE.

[0420]

[0421] Intracellular toxicity assay

[0422] The corresponding cells were seeded in a 96-well cell culture plate at a density of 10,000 per well and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; the cells were then treated with drugs at different concentrations and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (the ratio to the culture medium was 1:10) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450 nm in each well was detected with an enzyme marker, and the cell activity value was obtained by comparing the absorbance of the treated cells with that of the untreated cells, and the IC50 value of the compound was calculated.

[0423] Specific results such as Figure 8 As shown in Figure 3, for different cells, the IC50 of the prodrug is significantly lower than that of the original drug.

[0424] Embodiment 19

[0425] Prodrug XP-108-GEM-000

[0426]

[0427] Synthesis method:

[0428]

[0429] Procedure: Gemcitabine (100 mg) was dissolved in DMF (2 mL), and imidazole (28 mg), triethylamine (58 μL) and dimethyl-tert-butylsilyl chloride (143 mg) were added at room temperature, and the mixture was stirred at room temperature overnight.

[0430] Detection: TLC detection (EA).

[0431] Post-treatment: The DMF in the reaction solution was dried and washed with NaHCO 3 The reaction was quenched with the solution and extracted with EA (10 mL x 3). The organic phases were combined, washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and EA was removed by rotary evaporation at 40 °C. The product was purified by pure EA to obtain compound 33 as a white solid (57 mg, 30% yield).

[0432]

[0433] Procedure: Compound 33 (57 mg) was dissolved in DCM (1 mL), triethylamine (16 μL) and 3 (50 mg) were added at room temperature, and the mixture was stirred at room temperature overnight.

[0434] Detection: TLC detection (EA:PE=1:3).

[0435] Post-treatment: The reaction solution was directly purified by plate washing to obtain the product compound 34 (47 mg, 54% yield) as a colorless oily liquid.

[0436]

[0437] Procedure: Dissolve compound 34 (47 mg) in THF (1 mL), add tetrabutylammonium fluoride (0.2 μL) at room temperature and stir for 20 min.

[0438] Detection: TLC detection (MeOH:DCM=1:10).

[0439] Post-treatment and purification: The reaction solution was directly purified by running on a plate to obtain the product XP-108-GEM-000 (20 mg, 61% yield) as a white solid.

[0440] 1 H NMR (400MHz, DMSO) δ11.02(s,1H),8.24(d,J=7.6Hz,1H),7.05(d,J=7.6Hz,1H),6.31(d,J=6.4Hz,1H),6.16(t,J=7.6Hz,1 H),5.37–5.18(m,2H),4.27–4.10(m,1H),3.89(d,J=8.4Hz,1H),3.85–3.75(m,1H),3.71–3.55(m,1H),1.38–1.18(m,2H).

[0441] LCMS(ESI):m / z Calcd for C 17 H 13 O 6 N6 F 6 ,[M+H] + :511.1,found:511.2.

[0442] HPLC detection: The HPLC peak time of the original drug molecule is about 4.559min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 36.58% (100Gy, 10uM), producing the original drug GEM. The time of this HPLC method is 15min.

[0443]

[0444]

[0445] Embodiment 20

[0446] Prodrug XP-111-Res-000

[0447]

[0448] Synthesis method:

[0449]

[0450] Operation: Dissolve 44 mg of 4-azido-2,3,5,6-tetrafluorobenzyl (4-nitrophenyl) carbonate in 5 mL of DMF, add 30 mg of 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol dissolved in 5 mL of DMF, stir for 5 min, add 13 mg of HOBt, dilute 38 μL of triethylamine with 5 mL of DMF, and react at room temperature for 16 h.

[0451] Detection: TLC detection (EA).

[0452] Post-treatment and purification: Use EA extraction, at 40°C, first use a water pump to spin dry the EA, then use an oil pump to spin dry the N,N-dimethylformamide, re-dissolve it with dichloromethane and purify it with a large silica gel plate, the eluent ratio is (EA:PE=50:1). Finally, the product is obtained as a white solid.

[0453] LCMS(ESI):m / z Calcd for C 25 H 23 F 4 N 7 O 4 ,[M+H] + :562.4,found:562.4.

[0454] 1 H NMR (400MHz, DMSO) δ8.54(d,J=8.0Hz,1H),7.92(d,J=8.4Hz,1H),7.63(t,J=7.6Hz,1H),7.55(t,J=7.6Hz,1H) ,5.29(s,2H),4.91(s,2H),3.52(dd,J=13.6,6.8Hz,2H),1.15(dd,J=10.8,3.6Hz,6H),1.12(d,J=6.0Hz,3H).

[0455] HPLC detection: The HPLC peak time of the original drug molecule is about 15.1min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 97.6% (100Gy, 10uM), producing the original drug RES and linker 3-a.

[0456]

[0457] Embodiment 21

[0458] Prodrug XP-111-Res-001

[0459]

[0460] Synthesis method:

[0461]

[0462] Procedure: 35.8 mg of compound 28 was dissolved in 5 mL of DMF, 30 mg of 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol dissolved in 5 mL of DMF was added, and stirred for 5 min. After adding 13 mg of HOBt, 76 μL of triethylamine was diluted with 5 mL of DMF and reacted at room temperature for 16 h.

[0463] Detection: TLC detection (EA).

[0464] Post-treatment and purification: Use EA extraction, at 40°C, first use a water pump to spin dry the EA, then use an oil pump to spin dry the N,N-dimethylformamide, re-dissolve it with dichloromethane and purify it with a large silica gel plate, the eluent ratio is (EA:PE=50:1). Finally, the product is obtained as a white solid.

[0465] LCMS(ESI):m / z Calcd for C 25 H 27 N 7 O 4 ,[M+H] +:490.4,found:490.4.

[0466] 1 H NMR (400MHz, DMSO) δ8.53(d,J=8.4Hz,1H),7.93(d,J=8.0Hz,1H),7.67–7.60(m,1H),7.54(d,J=12.6Hz,1H),7.51(d,J= 8.0Hz,2H),7.14(d,J=8.0Hz,2H),5.18(s,2H),3.53(dd,J=14.0,7.0Hz,2H),1.19(s,3H),1.13(dd,J=7.6,6.4Hz,6H).

[0467] HPLC detection: The HPLC peak time of the original drug molecule is about 15.1min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 64.3% (100Gy, 10uM), producing the original drug RES.

[0468]

[0469] Embodiment 22

[0470] Prodrug XP-105-MMC-001

[0471]

[0472] Synthesis method:

[0473]

[0474] Procedure: 100 mg of compound 13 was dissolved in 2 mL of anhydrous DCM, 59 mg of MMC was added, and then 30 mg of HOBt and 19.9 uL of triethylamine were added dropwise, and the mixture was reacted at room temperature for 16 h.

[0475] Detection: TLC detection (DCM:MeOH=20:1).

[0476] Post-treatment and purification: At 37°C, N,N-dimethylformamide was spin-dried with an oil pump, redissolved with dichloromethane and purified using a large silica gel plate with an eluent ratio of (DCM:MeOH=10:1) to obtain the product XP-105-MMC-001 (57.2 mg) as a purple solid.

[0477] 1H NMR (400MHz, DMSO) δ7.08 (s, 2H), 6.58 (d, J = 58.4Hz, 2H), 6.08 (ddd, J = 29.2, 14. 0,7.2Hz,1H),4.91(dd,J=10.8,4.8Hz,1H),4.77(td,J=10.8,4.4Hz,1H),4.32–4 .20(m,1H),3.99–3.78(m,2H),3.78–3.63(m,2H),3.55–3.48(m,4H),3.15–3.08 (m,3H),2.92(d,J=12.4Hz,1H),2.71(s,1H),1.65(d,J=5.6Hz,3H),1.23(s,1H).

[0478] LCMS(ESI):m / z Calcd for C 28 H 27 O 10 N 8 F 4 ,[M+H] + :711.2,found:711.4.

[0479] HPLC showed two peaks, indicating the presence of diastereomers.

[0480] HPLC detection: The HPLC peak time of the original drug molecule is about 14.105min. The peak area (HPLC 254nm) of prodrug before and after irradiation decreased by 81.0%; 85.5% (60Gy, 10uM), but the corresponding original drug MMC and linker 3-a were not produced.

[0481]

[0482] Embodiment 23

[0483] Prodrug XP-103-SN38-003

[0484]

[0485] Synthesis method:

[0486]

[0487] Dissolve difluorobenzyl azido alcohol (500 mg, 1.0 eq) in dry DCM (10 mL), add pyridine (110 μL, 0.5 eq), and stir in a 45°C oil bath. Slowly add phosphorus tribromide (50 μL x 3) and reflux for 3 hours. TLC detects that the raw material has reacted completely. Cool the reaction solution to room temperature and add isopropanol. Stir for ten minutes. Add saturated NaHCO3 The mixture was quenched and extracted with EA (10 mL x 3). The organic phases were combined and spin-dried to give the product (620 mg, crude) as a brown oily liquid.

[0488]

[0489] Procedure: SN-38 (50 mg, 1.0 eq) was dissolved in dry DCM (2 mL) and DMF (2 mL), 40 mg of compound 35 was added, and K 2 CO 3 (18 mg), stirred at room temperature for 24 hours.

[0490] Detection: TLC detection (DCM:MeOH=10:1).

[0491] Post-treatment and purification: DMSO was added to the reaction solution to completely dissolve it, and the filtrate was filtered to obtain the product (4 mg, 5% Yield) as a yellow solid.

[0492] 1 H NMR (400MHz, DMSO) δ8.11(d,J=9.6Hz,1H),7.60(d,J=7.6Hz,2H),7.44(d,J=9.2Hz,2H),7.28(s,1H),6.58(d,J=61. 2Hz,2H),5.59–5.17(m,5H),3.29–3.08(m,2H),2.03–1.83(m,2H),1.34–1.27(m,3H),0.87(dd,J=15.6,8.0Hz,3H).

[0493] LCMS(ESI):m / z Calcd for C 29 H 24 O 5 N 5 F 2 ,[M+Na] + :560.2,found:560.3.

[0494] HPLC detection: The HPLC peak time of the original drug molecule is about 17.852min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 30.2% (60Gy, 10uM), producing the original drug SN-38 and the intermediate XP-103-SN38-003cp1.

[0495]

[0496]

[0497] Embodiment 24

[0498] Prodrug XP-104-CPT-008

[0499]

[0500] Synthesis method:

[0501]

[0502] Procedure: Dissolve camptothecin (40 mg, 1.0 eq) in dry DCM (2 mL) and DMF (2 mL), add 35 mg of compound 35, and add K 2 CO 3 (18 mg), stirred at 60°C for 3 hours.

[0503] Detection: TLC detection (DCM:MeOH=10:1).

[0504] Post-treatment and purification: DMSO was added to the reaction solution to completely dissolve it, and the filtrate was filtered to obtain the product (3.5 mg, 5% Yield) as a white solid.

[0505] LCMS(ESI):m / z Calcd for C 29 H 24 O 5 N 5 F 2 ,[M+Na] + :560.2,found:560.3.

[0506] HPLC detection: The HPLC peak time of the original drug molecule is about 18.480min. The peak area (HPLC 254nm) of the prodrug before and after irradiation is reduced by 61.3% (60Gy, 10uM), producing the intermediate state XP-104-CPT-008cp1.

[0507]

[0508]

[0509] Embodiment 25

[0510] Prodrug XP-109-EXA-000

[0511]

[0512] Synthesis method:

[0513]

[0514] Procedure: Dissolve exotecan (30 mg, 1.0 eq) in dry DMF (1 mL), add 31.9 mg of compound 5, add 19.1 u of LTEA to the stirred reaction solution, and stir at room temperature for 16 hours.

[0515] Detection: TLC detection (PE:EA=1:1).

[0516] Post-treatment and purification: DMSO was added to the reaction solution to completely dissolve it, and the filtrate was filtered to obtain the product (23 mg, 48.9% Yield) as a white solid.

[0517] 1 H NMR (400MHz, DMSO) δ8.17(d,J=8.8Hz,1H),7.76(d,J=10.8Hz,1H),7.30(s,1H),6.50(s,1H),5.43(s,2H),5. 30–5.06(m,4H),2.37(s,2H),2.18(s,3H),1.86(dd,J=16.8,7.2Hz,2H),1.23(s,2H),0.88(t,J=7.2Hz,3H).

[0518] HRMS(ESI):m / z Calcd for C 32 H 22 O 6 N 6 F 5 ,[M+Na] + :681.15265,found:681.15359.

[0519] HPLC detection: The HPLC peak time of the original drug molecule is about 15.155min. The peak area (HPLC 254nm) of prodrug before and after irradiation decreased by 82.8% (100Gy, 10uM), but no new peak was generated.

[0520]

[0521] Embodiment 26

[0522] Preparation of prodrug XP-113-PTX-000

[0523]

[0524] Synthesis method:

[0525]

[0526] Procedure: Dissolve paclitaxel (15 mg, 0.017 mmol) in 1 ml of dichloromethane, add DMAP (1 mg, 0.05 mmol), triethylamine (3 uL, 0.021 mmol), and finally add compound 5 (8.1 mg, 0.021 mmol), and stir at room temperature overnight.

[0527] Detection: TLC monitoring (PE:EA=2:1), paclitaxel is almost completely reacted, and a small amount of excess raw material II remains, which indicates that the reaction is complete.

[0528] Post-treatment and purification: spin dry at 37°C, spin off the solvent DCM, use DMF to prepare the sample, and obtain 9 mg of product.

[0529] LCMS(ESI):m / z Calcd for C 55 H 52 O 16 N 4 F 4 ,[M+H] + :1101.3,found:1101.4.

[0530] 1 HNMR (400MHz, CDCl 3 )δ8.17–8.11(m,2H),7.74–7.69(m,2H),7.61(t,J=7.6Hz,1H),7.50(dd,J=14.4,7.6Hz,3H),7.44–7.34(m,7H),6.8 7(d,J=9.6Hz,1H),6.33–6.24(m,2H),5.99(dd,J=9.2,2.4Hz,1H),5.69(d,J=7.2Hz,1H),5.45(d,J=2.8Hz,1H),5.28 (d,J=14.8Hz,2H),4.98(d,J=8.0Hz,1H),4.44(dd,J=10.8,6.8Hz,1H),4.32(d,J=8.4Hz,1H),4.21(d,J=8.4Hz,1H) ,3.82(d,J=7.0Hz,1H),2.62–2.36(m,7H),2.27–2.16(m,4H),2.08–1.84(m,5H),1.69(s,3H),1.25(d,J=4.0Hz,6H).

[0531] HPLC detection: The HPLC peak time of the original drug molecule is about 23.94min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 33.2% (60Gy, 10uM), producing the original drug PTX and the linker peak compound 3_a.

[0532]

[0533]

[0534] Embodiment 27

[0535] An antibody-small molecule drug conjugate, the structure of which is shown in Formula XP-106-ADC001:

[0536]

[0537] Wherein, x is the ratio of small molecule drug to antibody, x is 3-5, and Ab is trastuzumab.

[0538] The specific preparation process includes:

[0539] (1) XP-106-MMAE-001 was prepared according to Example 9, and then the following esterification process was performed:

[0540]

[0541] Procedure: Dissolve 100 mg XP-106-MMAE-001 (Compound A) in 2 mL of anhydrous DMF, add DMAP (2.0 eq), TEA (2.0 eq), and DSC (2.0 eq), and react at room temperature in the dark for 5 min to obtain Compound B through esterification.

[0542] Detection: TLC detection (EA).

[0543] Post-treatment and purification: After filtering with a 0.22 um filter membrane, directly pass through preparative HPLC and spin dry to obtain a solid (30 mg) as a white solid.

[0544] HRMS(ESI):m / z Calcd for C 56 H 78 O 14 N 10 F 4 Na,[M+Na] + 1213.55276, found:1213.55286.

[0545] (2) ADC synthesis:

[0546]

[0547] Procedure: Dissolve 5 mg of trastuzumab in 5 mL of ddHO 2O, add 500uL PBS, and then slowly drop the solution of compound B dissolved in DMA (1.2mg compound B dissolved in 400uL DMA), and react at room temperature in the dark for 16h.

[0548] Post-treatment and purification: Filter with a filter membrane and then directly ultrafilter (30K, ultrafiltration tube with an inner tube of 4 mL, rotation speed 2500-3000g, 3-5min, number of times 150 times).

[0549] The antibody-drug conjugate XP-106-ADC001 synthesized in this example was compared with the naked antibody in mass spectra to obtain x=(152615.48-148304.325) / 1076.5=4.0. Fig. 9 shown.

[0550] ESI (high resolution protein spectrum) of XP-106-ADC001 before irradiation Fig.10 XP-106-ADC001 was dissolved in water (the concentration was 0.624 mg / mL as measured by Nanodrop, and diluted to 0.2 mg / mL with water), oxygen was removed by argon gas, and then sealed. After irradiation (100 Gy) by a small animal irradiator (RS2000Pro-225), the ESI (high resolution protein spectrum) was obtained. Fig.11 Compared with the ESI Full ms of the ADC high-resolution mass spectrum before irradiation, it was found that MMAE original drug (m / z=718.5127) was produced after X-ray irradiation (100Gy).

[0551] Intracellular toxicity assay

[0552] The corresponding cells were seeded in a 96-well cell culture plate at a density of 4000 per well and incubated at a constant temperature of 37°C and 4% carbon dioxide for 18 hours to ensure cell adhesion; then the cells were treated with different concentrations of trastuzumab, ADC, and irradiated ADC, and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (with a ratio of 1:10 to the culture medium) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value, such as Fig.12 shown.

[0553] Comparative Example 1

[0554] Preparation of prodrug XP-102-DOX-000:

[0555]

[0556] Synthesis method:

[0557]

[0558] Procedure: Dissolve 60 mg (4-azido-2,3,5,6-tetrafluorophenyl)methyl-4-nitrophenyl carbonate in 4 mL of anhydrous DMF at room temperature, then add molecular sieves and 25 uL of triethylamine, and stir for 10 min. Dissolve 60 mg of doxorubicin hydrochloride in 4 mL of anhydrous DMF, add 25 μL of triethylamine, and add the above solution; react at room temperature under nitrogen protection for 19-24 h.

[0559] Detection: The reaction was monitored by TLC (5% methanol:DCM).

[0560] Workup: dilute with water, extract with EtOAc, saturated NaHCO 3 The solution was washed with water, anhydrous Na 2 SO 4 Dry and concentrate by rotary evaporation.

[0561] Purification: Finally, purification by flash chromatography (5% MeOH: EtOAc) gave 50 mg of solid product, which was the prodrug XP-102-DOX-000.

[0562] 1 H NMR (400 MHz, CDCl 3 )δ14.02(s,1H),13.31(s,1H),8.17–7.95(m,1H),7.80(dd,J=24.8,16.8Hz,1H),7.43(d,J=8.0Hz, 1H),5.52(s,1H),5.33(s,1H),5.15(s,1H),4.78(s,1H),4.52(s,1H),4.14(d,J=14.8Hz,2H),3.89 (s,1H),3.68(s,1H),3.33(d,J=17.6Hz,1H),3.11–2.86(m,3H),2.36(d,J=13.6Hz,1H),2.21(d,J= 10.8Hz,1H),2.05(d,J=15.2Hz,1H),1.93–1.70(m,2H),1.35–1.24(m,3H),0.88(d,J=20.4Hz,3H).

[0563] m / z[M+Na] + :813.17.

[0564] HPLC detection: The HPLC peak time of the original drug molecule DOX is 16.265min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 52.4% (100Gy, 10uM), and the new peak is not the original drug peak.

[0565]

[0566] Comparative Example 2

[0567] Preparation of prodrug XP-105-MMC-000:

[0568]

[0569] Synthesis method:

[0570]

[0571] Operation: Dissolve 4.8 g (4-azido-2,3,5,6-tetrafluorophenyl) methyl-nitrophenyl carbonate in 35 mL DMF, add 3.46 g mitomycin C, stir for 10 min, dissolve 5.2 mL triethylamine in 30 mL DMF, slowly add to the reaction system, and react at room temperature for 48 h.

[0572] Detection: TLC detection (PE:EA=10:1) and (DCM:MeOH=20:1).

[0573] Post-treatment: the reaction solution was poured into 500 mL of water, stirred for 10 min and then filtered. The filter cake was rinsed with 100 mL of water, dried, and then pumped dry with an oil pump to obtain 5.38 g of a purple solid crude product.

[0574] Purification: The crude product was slurried with 100 mL of ethyl acetate overnight, filtered, the filter cake was rinsed with 10 mL of ethyl acetate, and then pumped dry with an oil pump to obtain 4.4 g of a purple solid (HNMR and HPLC showed clean). The slurried mother liquor was poured into 300 mL of n-hexane (solid precipitated), filtered, and the filter cake was dried and continued to be slurried with 40 mL of ethyl acetate overnight. After slurrying, it was filtered, the filter cake was rinsed with 10 mL of ethyl acetate, and then pumped dry with an oil pump to obtain 0.24 g of a purple solid (HNMR and HPLC showed clean), and the remaining mother liquor was spin-dried to obtain 0.5 g of a residue (TLC showed a product content of 70% and about 30% of the raw material mitomycin C).

[0575] 1 H NMR (400 MHz, CDCl 3)δ5.32–5.10(m,3H),4.86(dd,J=10.8,4.7Hz,1H),4.61(s,2H),4.45(d,J=13.2Hz,1H),4.14(dt,J=14.6,9.2H z,1H),3.67(dd,J=11.2,4.8Hz,1H),3.57–3.41(m,2H),3.31(dd,J=4.4,1.6Hz,1H),3.19(s,3H),1.76(s,3H).

[0576] HRMS(ESI):m / z Calcd for C 23 H20F 4 N 7 O 7 ,[M+H] + :582.13547,found:582.13579.

[0577] HPLC detection: The HPLC peak time of the original drug molecule MMC is 14.105min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 67.5% (100Gy, 10uM), and a linker peak compound 3-a is generated (HPLC 254nm peak time is 17.0min).

[0578]

[0579] Among them, the structure of compound 3-a is

[0580] Comparative Example 3

[0581] Prodrug XP-102-DOX-010

[0582]

[0583] Synthesis method:

[0584]

[0585] Procedure: Dissolve doxorubicin (20 mg, 1.00 eq) and compound 5 (13 mg) in anhydrous N,N-dimethylformamide (1 mL). Add triethylamine (0.03 mL) to the reaction solution at room temperature. The reaction solution is reacted at room temperature for 24 hours.

[0586] Detection: TLC (DCM:MeOH=10:1, Rf=0.40) showed a new spot was generated.

[0587] Post-treatment and purification: At 35°C, N,N-dimethylformamide was spin-dried, redissolved in dichloromethane and purified using a silica gel plate with an eluent ratio of (DCM:MeOH=10:1). Finally, the product XP-102-DOX-010 (5.5 mg, 20% yield) was obtained as a red solid.

[0588] 1 H NMR (400 MHz, CDCl 3 )δ13.99(s,1H),13.28(s,1H),8.07(d,J=7.6Hz,1H),7.82(t,J=8.0Hz,1H),7.42(d,J=8.8Hz,1H),5.56(d,J=3. 2Hz,1H),5.43–5.31(m,4H),5.23(d,J=18.4Hz,1H),4.80(s,1H),4.11(s,3H),3.91(d,J=4.8Hz,1H),3.74(s,1H) ,3.52(d,J=3.6Hz,1H),3.31(d,J=19.0Hz,1H),3.07(d,J=19.0Hz,1H),2.83(d,J=11.2Hz,1H),2.47(d,J=14.8H z,2H),2.15(dd,J=14.6,3.8Hz,1H),2.05(d,J=12.8Hz,2H),1.81(dd,J=17.2,8.4Hz,2H),1.41(d,J=7.6Hz,3H).

[0589] HRMS(ESI):m / z Calcd for C 42 H 32 O 13 N 4 F 9 ,[M+H + ]:917.18167,found:917.18190.

[0590] XP-102-DOX-010 had decomposed before irradiation, generating new peaks and was unstable.

[0591] The irradiation release results of each prodrug are shown in Table 1:

[0592] Table 1

[0593]

[0594]

[0595] It can be seen that after irradiation, the prodrug peak area of ​​the example compound of the present invention is significantly reduced, and obvious prodrug / intermediate transition state generation is detected. Compared with the prodrug molecules of comparative examples 1-2, they show better drug release performance.

[0596] The cytotoxicity results of each prodrug are shown in Tables 2-5. Among them, the higher the IC50 ratio of the prodrug / original drug, the lower the cytotoxicity of the prodrug.

[0597] Table 2

[0598]

[0599] Table 3

[0600]

[0601]

[0602] Table 4

[0603]

[0604] Table 5

[0605]

[0606] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A class of azide prodrug compounds having a structure represented by formula (1) or a pharmaceutically acceptable salt thereof: In the formula, R1 is mono- to tetra-substituted, specifically selected from any one or more of the following substituents: H, halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6 Cycloalkyl, substituted or unsubstituted C 1-6 Alkoxy, -C(=O)N(R a )-(CR b )-COOR c The substituted substituent is selected from halogen atoms, hydroxyl groups, C 1-6 Alkyl and C 1-6 Epoxy group; R a , R b , R c are independently selected from H, C 1-6 alkyl; A is a vacant bond, -C(=O)-, or D is derived from an active small molecule, and the active small molecule is selected from the following compounds or pharmaceutically acceptable salts thereof: daunorubicin, doxorubicin, camptothecin, mitomycin C, monomethyl auristatin E, bufalin, 7-ethyl-10-hydroxycamptothecin, gemcitabine, resiquimod, exitecan, paclitaxel; R2 and R3 are independently selected from: H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted aryl, -CH2-C(=O)N(R4)CH2COOR5; the substitution on the aryl is one to four substitutions, aryl or C 1-6 The substituents on the alkyl group are selected from any one or more of the following: halogen, azido; R4 and R5 are independently selected from H, C 1-6 alkyl; Furthermore, when the active small molecule is daunorubicin or mitomycin C, R2 and R3 are not H at the same time.

2. The azide prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The aryl group is a benzene ring or a naphthalene ring.

3. The azide prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: When A is an empty bond, the corresponding general structure is 4. The azide prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: D is specifically selected from the following structures:

5. The azide prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: D is When R2 is H, R3 is substituted or is substituted phenyl; the substituent is selected from any one or more of the following: halogen, azide.

6. The azide prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: D is When R2 is H, R3 is -CH2-C(=O)N(R4)CH2COOR5; R4 and R5 are independently selected from H, C 1-6 alkyl.

7. The azide prodrug compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, characterized in that: The pharmaceutically acceptable salts include inorganic salts or organic salts; wherein the inorganic salts include hydrochlorides, hydrobromides, hydroiodides, perchlorates, sulfates, bisulfates, nitrates, phosphates, acid phosphates, sodium salts, calcium salts, potassium salts, magnesium salts, silver salts, and lithium salts. The organic salts are selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate, meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, terbutylamine salt, and N,N-dibenzylethylenediamine salt.

8. A pharmaceutical composition, characterized in that The invention comprises the azide prodrug compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7.

9. The pharmaceutical composition according to claim 8, characterized in that It also contains pharmaceutical excipients; the pharmaceutical excipients include: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids and release retardants.

10. An antibody-small molecule drug conjugate, characterized in that: The method comprises coupling the azide prodrug compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 to an antibody.

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