Pyrrolobenzodiazepine intermediates and uses thereof

Abstract

Pyrrolobenzodiazepine (PBD) intermediates, including JPEG2025509424000051.jpg37137, are useful in the preparation of advanced intermediates, PBDs, and PBD dimers. Such PBD intermediates offer several advantages, including simplified synthesis of the desired PBD products, higher substrate compatibility, and safer reaction conditions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 269,205, filed March 11, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Discovered in 1963, pyrrolobenzodiazepines (PBDs) are a class of naturally derived compounds with antibiotic and antitumor properties. Originally derived from the genus Streptomyces, PBDs are potent DNA damaging agents, far more potent than current systemic chemotherapy drugs. Although early applications were limited due to concerns about toxicity, PBDs have been shown to be effective as payloads for antibody-drug conjugates (ADCs). Currently, there is one PBD-containing ADC commercially available, and many others are in clinical trials.

[0003] Currently, there are no commercially available intermediates for the synthesis of PBD payloads. Scientists must develop new synthetic methods for each and then develop GMP methods to scale up and produce PBDs. Novel intermediates useful for the synthesis of PBDs are needed. Summary of the Invention

[0004] overview Formula I useful for the synthesis of pyrrolobenzodiazepines [ka] where the dashed line represents a single or double bond.

[0005] R1 is a protecting group selected from tert-butyldimethylsilyl ether (TBS) and acetate (Ac).

[0006] R2 is -H, ═O, -OTf, -OH, C1-C5 alkyl, C3-C6 cycloalkyl, C1-C5 alkenyl, C5-C 10 Aryl, C5-C 10 Optionally, R2 may be substituted with one or more substituents selected from halo, nitro, cyano, ether, carboxy, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene, and a linker.

[0007] R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halo, where R is C1-C4 alkyl, C3-C 10 Heterocyclyl and C5-C 10 aryl. R may be optionally substituted with C1-C4 alkyl or phenyl.

[0008] R4 is selected from -H, -R, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halo, where R and R' are independently C1-C4 alkyl, C3-C 10 Heterocyclyl and C5-C 10 aryl. R and R' may optionally have substituents selected from C1-C4 alkyl and phenyl.

[0009] R5 is a protecting group selected from tert-butyldiphenylsilyl ether (TBDPS) and triisopropylsilyl ether (TIPS).

[0010] R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halo, where R is C1-C4 alkyl, C3-C 10 Heterocyclyl and C5-C 10 aryl, and R may optionally bear substituents selected from C1-C4 alkyl, phenyl.

[0011] In some embodiments, R3 and R6 are each -H.

[0012] In some embodiments, R2 is selected from -H, =O, -OTf, -OH, methyl, cyclopropyl, =CH2, ethylenyl, propenyl, phenyl, 4-fluorophenyl, indole, and a linker. R2 can be optionally substituted with one or more substituents selected from halo, nitro, cyano, ether, carboxy, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene, and a linker.

[0013] In some embodiments, R4 is selected from -H, -OCH3, -OCH2Ph, -N(CH3)2, morpholino, piperidinyl, and -N-Me-piperazinyl.

[0014] In some embodiments, R2 is =O, and in some embodiments, R2 is -OTf.

[0015] In some embodiments, R4 is OCH3.

[0016] Also provided is a process for preparing an advanced PBD intermediate, the process comprising the steps of providing a compound of formula I, wherein R2 is a ketone, performing a CC bond forming reaction with the ketone, and optionally deprotecting the amine.

[0017] In some embodiments, the method comprises converting the ketone to a triflate, then performing a CC bond forming reaction on the triflate, and optionally deprotecting the amine.

[0018] In some embodiments, the CC bond forming reaction is Suzuki coupling.

[0019] Also provided is a process for preparing advanced PBD intermediates from compounds of formula I where R2 is a triflate, comprising carrying out a CC bond forming reaction on the triflate and optionally deprotecting the amine.

[0020] In some embodiments of this method, the CC bond forming reaction is a Suzuki coupling.

[0021] In some embodiments of these methods, when the amine is deprotected, the method includes the further step of coupling a linker to the deprotected amine. [Brief description of the drawings]

[0022] [Figure 1] Figure 1 provides the structures of two PBD intermediates described herein: Figure 1A shows PBD core A, and Figure 1B shows PBD core A triflate.

[0023] [Diagram 2] FIG. 2 provides a scheme for the synthesis of PBD core A from commercially available starting materials.

[0024] [Diagram 3] Figure 3 provides a scheme for the generation of PBD core A via advanced PBD intermediates to PBD fragments. Figure 3A highlights the synthesis of fragment A via a common intermediate disclosed herein, and Figure 3B highlights the synthesis of fragment B via a common intermediate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] detail Novel pyrrolobenzodiazepine (PBD) cores and advanced intermediates are provided. These compounds are particularly suitable for the synthesis of PBD payloads used in antibody drug conjugates. These compounds reduce the number of steps required to synthesize PBD payloads, simplifying the process. These PBD intermediates are highly useful for developers interested in pursuing multiple analogs of PBD payloads due to their synthetic flexibility.

[0026] The PBD intermediates and methods provided herein solve the problem of drug researchers having to develop lengthy new syntheses to obtain individual PBD payloads. The PBD intermediates provided herein can be used to produce many of the PBD-derived payloads that are in clinical trials.

[0027] Representative PBD intermediates described herein include PBD Core A and PBD Core A Triflates: [ka] Includes:

[0028] One feature of the PBD intermediates described herein is the Boc-protected nitrogen versus the commonly utilized nitro group. The Boc group provides versatility in synthetic options for downstream chemistry. It offers the opportunity to prepare novel PBDs that cannot tolerate nitro reduction conditions. Furthermore, early reduction avoids the hazardous zinc dust reduction required for advanced intermediates. Furthermore, the Boc group can be selectively removed or allowed to persist throughout the pathway, providing great flexibility.

[0029] Older methods utilize the -NO2 group, which must be reduced after the ketone functionalization step. Changing the order of chemical steps allows for greater substrate compatibility, reduces the number of post-differentiation steps, and avoids the use of explosive zinc dust. Late introduction of R groups provides a late diversification point that simplifies the reaction sequence. Use of Boc protecting groups allows for selective removal if desired, or can be allowed to persist throughout the reaction sequence and removed by simple acidic deprotection in the final step.

[0030] The ketone and triflate functionalities exemplified by PBD Core A and PBD Core A Triflate provide additional flexibility in the preparation of PBDs or advanced intermediates. The ketone of PBD Core A is suitable for ketone functionalization. The triflate group of PBD Core A shows high regioselectivity. The production of PBD Core A Triflate from PBD Core A is in high yield and provides excellent selectivity for the desired enol triflate stereoisomer.

[0031] In certain embodiments, the PBD intermediate has formula I: [ka] It is a PBD intermediate.

[0032] Here, the dashed line represents a single or double bond. R1 and R5 are each an alcohol protecting group, and are selected so that each protecting group can be removed separately. In other words, R1 and R5 are selected so that they are orthogonal to each other. In some embodiments, R1 is tert-butyldimethylsilyl ether (TBS). In other embodiments, R1 is acetate (Ac). In some embodiments, R5 is tert-butyldiphenylsilyl ether (TBDPS), and in other embodiments, triisopropylsilyl ether (TIPS). In a preferred embodiment, R1 is TBS and R5 is TBDPS.

[0033] R2 may be selected from -H, =O, -OTf (trifluoromethanesulfonate functional group, CF3, SO3-, or triflate), -OH, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, and linker. Optionally, R2 may be substituted with one or more substituents selected from halo, nitro, cyano, ether, carboxy, ester, alkyl, alkenyl, heterocyclyl, bis-oxy-C1-C3 alkylene, and linker.

[0034] In some embodiments, R2 is -H, =O, -OTf, -OH, C1-C5 alkyl, C3-C6 cycloalkyl, C1-C5 alkenyl, C5-C 10 Aryl, C5-C 10 Optionally, R2 may be substituted with one or more substituents selected from halo, nitro, cyano, ether, carboxy, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene, and a linker.

[0035] R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halo, where R is selected from alkyl, heterocyclyl, and aryl. R can be optionally substituted with an alkyl or aryl group.

[0036] In some embodiments, R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halo, where R is C1-C4 alkyl, C3-C 10 Heterocyclyl and C5-C 10 aryl. R may be optionally substituted with C1-C4 alkyl or phenyl.

[0037] R4 is selected from -H, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halo, where R and R' are independently selected from alkyl, heterocyclyl and aryl. R and R' may optionally bear substituents selected from alkyl and aryl.

[0038] In some embodiments, R4 is selected from -H, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halo, where R and R' are independently C1-C4 alkyl, C3-C 10 Heterocyclyl and C5-C 10 aryl. R and R' may optionally have substituents selected from C1-C4 alkyl and phenyl.

[0039] R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halo, where R is selected from alkyl, heterocyclyl and aryl, and R may optionally bear substituents selected from alkyl and aryl.

[0040] In some embodiments, R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halo, where R is C1-C4 alkyl, C3-C 10 Heterocyclyl and C5-C 10 aryl, and R may optionally bear substituents selected from C1-C4 alkyl, phenyl.

[0041] In some embodiments, R3 and R6 are each -H.

[0042] In some embodiments, R2 is selected from -H, =O, -OTf, -OH, methyl, cyclopropyl, =CH2, ethylenyl, propenyl, phenyl, 4-fluorophenyl, indole, and linker. R2 can be optionally substituted with one or more substituents selected from halo, nitro, cyano, ether, carboxy, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene, and linker. In some embodiments, R2 is =O, and in some embodiments, R2 is -OTf.

[0043] In some embodiments, R4 is selected from -H, -OCH3, -OCH2Ph, -N(CH3)2, morpholino, piperidinyl, and -N-Me-piperazinyl. In some embodiments, R4 is OCH3.

[0044] In embodiments that include a linker, suitable linkers include those suitable for use in ADCs. Such linkers can include, for example, cleavable or non-cleavable linkers. The choice of linker depends on factors such as the point of attachment to the PBD core intermediate, the point of attachment to the specific antibody that is ultimately used, and the point of attachment to the desired release point at the target. Suitable linkers can be identified by those skilled in the art. In some embodiments, the linker includes a 4-aminobenzyl (PAB) group attached to a dipeptide such as valine-citrulline.

[0045] In some embodiments, the compound of formula I is [ka] It is one of the following.

[0046] In another embodiment, the compound of formula I is [ka] It is one of the following.

[0047] In yet another embodiment, the compound of formula I is [ka] It could be.

[0048] Also provided are analogs of Formula I in which one or more protecting groups have been removed. Exemplary compounds include: [ka] Includes:

[0049] In certain other embodiments, the compound of formula II: [ka] wherein R7 is selected from -H, -C(O)O-TBS, Alloc, and a linker, such as a protected PABC-peptide linker, and R8 is selected from H and TBS. Exemplary compounds of formula II include: [ka] Includes:

[0050] Also, Formula III: [ka] wherein R9 is selected from Alloc and a linker, such as a protected PABC-peptide linker, and R8 is selected from -H and TBS. Also provided are intermediates of the formula: Exemplary compounds of formula III include: [ka] Includes:

[0051] The intermediates provided herein can be prepared using commercially available starting materials, as described below.

[0052] Also provided is a method for preparing an advanced PBD intermediate, which includes further reaction at R2. In particular, the presence of a ketone or triflate group at R2 is suitable for carbon-carbon cross-coupling reaction. Many carbon-carbon cross-coupling reactions, or CC cross-coupling reactions, are known to those skilled in the art. Exemplary CC cross-coupling reactions include, but are not limited to, Suzuki-Miyaura or Suzuki coupling, Wittig reaction, aldol reaction, α-CH arylation reaction, Heck coupling, Negishi coupling, Stille coupling, and the like. Many different CC cross-coupling reactions are suitable according to the methods provided herein. In some embodiments, Suzuki coupling is the preferred method for CC cross-coupling.

[0053] In certain embodiments, the method includes providing a compound of formula I, where R2 is a ketone, performing a CC bond forming reaction with the ketone, and optionally deprotecting the amine.

[0054] In some embodiments, the method comprises converting the ketone to a triflate, then performing a CC bond forming reaction on the triflate, and optionally deprotecting the amine.

[0055] In some embodiments, the CC bond forming reaction is Suzuki coupling.

[0056] Also provided is a method for preparing advanced PBD intermediates from compounds of formula I where R2 is a triflate, comprising carrying out a CC bond forming reaction on the triflate and optionally deprotecting the amine.

[0057] In some embodiments of this method, the CC bond forming reaction is a Suzuki coupling.

[0058] In some embodiments of these methods, when the amine is deprotected, the method includes the further step of coupling a linker to the deprotected amine.

[0059] The following intermediates and advanced intermediates were prepared as outlined below: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0060] The examples below illustrate the preparation and use of the intermediates described herein. EXAMPLES

[0061] Example 1. Preparation of PBD Core A [ka] tert-Butyl-(2-((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate: A solution of PBD-benzoic acid (5.0 g, 9.58 mmol, 1.0 equiv) in ethyl acetate (10 vol) was cooled to 0° C. To this solution was added 2-hydroxypyridine 1-oxide (HOPO, 1.28 g, 11.5 mmol, 1.2 equiv) followed by DIC (1.78 mL, 4.79 mmol, 1.2 equiv) at a rate to keep the temperature below 5° C. The reaction mixture was stirred for 30 minutes before PBD-pyrrolidine (2.2 g, 9.58 mmol, 1.0 equiv) was charged in two portions. The mixture was then warmed to 20° C. and stirred for 3.5 hours. When complete by HPLC, the mixture was filtered through a pad of Celite. The filtrate was washed once with water (10 vol) and the aqueous layer was extracted with ethyl acetate (5 vol). The organic layers were combined and washed with 1 M HCl (2x5 vol), followed by 1 M sodium bicarbonate (5 vol), and twice with 2 M brine (5 vol). The solvent was replaced by heptane (5 vol) via quantitative distillation and the resulting precipitate was removed by filtration through Celite. The filtrate was concentrated in vacuo to give PBD-INT as an off-white solid, which was used crude in the next step (yield 80-90%).

[0062] tert-Butyl-(S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-oxopyrrolidine-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate: A solution of sulfur trioxide-pyridine complex (7.07 g, 44.4 mmol, 6.0 equiv) in DMSO (10 vol) was prepared and stirred for 20 min. Pyridine (3.59 mL, 44.4 mmol, 6.0 equiv) was added to the sulfur trioxide-pyridine solution and stirred for an additional 30 min. In a separate flask, a solution of crude PBD-INT (5.44 g, 7.40 mmol, 1.0 equiv) in 10 vol DCM was prepared and cooled to 0° C. To the cooled solution was added triethylamine (8.25 mL, 59.2 mmol, 8.0 equiv). The sulfur trioxide-pyridine solution was added to the cooled PBD-INT solution at such a rate as to keep the temperature below 10° C. The reaction mixture was warmed to 20° C. and stirred for 3 h. When the reaction was complete as shown by HPLC, the mixture was cooled to 0° C. and quenched with 1 M sodium bicarbonate (5 vol). The organic layer was collected and the aqueous layer was extracted with 5 vol DCM. The organic layers were combined and washed three times with 1 M brine (3x5 vol). The organic layer was concentrated in vacuo and the product was purified by column chromatography (12-22% ethyl acetate / heptane). The product-containing fractions were combined and isolated to give PBD core A (purity >95%, yield 75%).

[0063] Example 2. Trifluoration / Suzuki coupling / Boc deprotection [ka] (S)-1-(2-((tert-butoxycarbonyl)amino)-4-((tert-butyldiphenylsilyl)oxy)-5-methoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate: A solution of PBD core A (500.0 mg, 0.682 mmol, 1.0 equiv) in THF (40 mL) was prepared under nitrogen atmosphere. To this solution was added N-(2-pyridyl)bis(trifluoromethanesulfonimide) (489.0 mg, 1.36 mmol, 2.0 equiv) and cooled to -40°C. To this solution was then added lithium tert-butoxide (1 M solution in THF, 1.71 mL, 1.71 mmol, 2.50 equiv) in one portion. Stirred at -40°C until reaction was complete by HPLC (2.5 h). The reaction was quenched with 10% citric acid in ethyl acetate (40 vol) and warmed to 0°C. To the reaction mixture was added 40 vol water and the organic layer was collected, washed with 2 M brine (10 vol), dried over sodium sulfate and concentrated in vacuo. The residue was taken up in heptane (5 vol) and filtered through celite. The filtrate was concentrated in vacuo and purified by column chromatography (0-10% ethyl acetate / heptane). The product containing fractions were combined and concentrated in vacuo to give the vinyl triflate (75-80%).

[0064] (S)-1-(2-((tert-butoxycarbonyl)amino)-4-((tert-butyldiphenylsilyl)oxy)-5-methoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate: A suspension of potassium phosphate tribasic (5.8 g, 27 mmol, 5.0 equiv) and cesium acetate (1.0 g, 5.5 mmol, 1.0 equiv) was prepared in degassed toluene (6.0 vol) under nitrogen. The suspension was heated to 45 °C. Pd(dppf)Cl2·DCM was then added to the suspension. In a separate flask, a solution of PBD vinyl triflate (4.7 g, 5.5 mmol, 1.0 equiv) in degassed toluene (6.0 vol) was prepared under nitrogen. The vinyl triflate solution was added dropwise to the heated suspension. Once the addition of the vinyl triflate was complete, methyl boronate (1.3 g, 22 mmol, 4.0 equiv) was added in portions. The reaction was stirred at 45 °C until the reaction was complete by HPLC (typically 1.5 h). The mixture was cooled to room temperature and poured into water (12 vol). The organic layer was collected, concentrated in vacuo, and purified by column chromatography (0-15% ethyl acetate / heptane). The product-containing fractions were combined and isolated to give the product (30-50% yield).

[0065] (S)-(2-amino-4-((tert-butyldiphenylsilyl)oxy)-5-methoxyphenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone: A solution of the Suzuki product (230.0 mg, 0.315 mmol, 1.0 equiv) in DCM (10 vol) was prepared. To this solution was added 2,6-lutidine (0.729 mL, 6.29 mmol, 20.0 equiv), followed by TBSOTf (0.722 mL, 3.15 mmol, 10.0 equiv). The solution was then heated to 40° C. and stirred for 15 h. Upon completion as shown by HPLC, the mixture was quenched with saturated ammonium chloride (10 vol). The aqueous layer was extracted with 10 vol DCM and the organic layers were combined, washed with 2 M brine (10 vol), dried over magnesium sulfate and then concentrated in vacuo. The residue was taken up in THF (10 vol) and cooled to 0° C. To this solution was added TBAF (1 M solution in THF, 0.157 mL, 0.16 mmol, 0.5 equiv) and stirred at 0° C. for 30 min. The solution was then diluted with 10 vol ethyl acetate and quenched with 10 vol water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 vol). The organic layers were combined, dried over magnesium sulfate and concentrated in vacuo. The product was purified by column chromatography (15% ethyl acetate / heptane). Fractions containing the product were combined and isolated to give the free aniline (40-60% yield).

[0066] Example 3. Preparation of PBD Benzoic Acid: [ka] 2-((tert-butoxycarbonyl)amino)-4-hydroxy-5-methoxybenzoic acid (2): Benzoic acid 1 (1.0 equiv) was dissolved in 15 vol MeOH. To the resulting mixture was added 10% palladium on carbon (0.03 equiv, 10% w / w) and Boc2O (5 equiv). The reaction vessel was evacuated and back-filled with nitrogen three times. The reaction vessel was fitted with a hydrogen-filled balloon, then evacuated and back-filled with hydrogen three times. The reaction was stirred overnight until complete conversion to 2 was observed by TLC. After completion of the reaction, the mixture was solvent substituted with hexanes (10 vol) and stirred at room temperature for 1 h. The residue was filtered through Celite. The filtrate was concentrated in vacuo to give the final product as a light brown to pink solid in 86% yield.1 H NMR (400 MHz, DMSO-d6)δ13.10 (br s, 1H), 10.49 (s, 1H), 10.15 (br s, 1H), 7.88 (s, 1 HRMS: C 13 H 16 Calculated value of NO6-: 282.0983 Measured value: 282.0984.

[0067] 2-((tert-butoxycarbonyl)amino)-4-hydroxy-5-methoxybenzoic acid: To a solution of 2 (1.1 equiv) in THF (20 vol) was added triethylamine (8 equiv). The mixture was stirred for 30 min. Then tert-butyl(chloro)diphenylsilane (3.0 equiv) was slowly added to the solution. The reaction was stirred at room temperature for 5.5 h. The reaction was quenched with 1 M HCl (12 vol) and the mixture was concentrated in vacuo to remove THF. Ethyl acetate (15 vol) was added to the mixture. The aqueous layer was back extracted with ethyl acetate (5 vol), the organic layers were combined and washed with 2 M brine (5 vol). The aqueous layer was back extracted with ethyl acetate (5 vol.), the organic layers were combined and concentrated in vacuo. 15% MTBE / 85% heptane was added to the crude product and the resulting slurry was heated to 50° C. The resulting solution was stirred at 50° C. for 1.5 h, then cooled to room temperature and stirred at room temperature for 2 h. The material was filtered and PBD-benzoic acid was isolated as a light brown to off-white solid (57% yield, 97% purity). The solid was then dissolved in MTBE (10 vol) and CUNO Type 2 activated carbon (10% w / w) was added to the solution. The mixture was stirred for 2 h and then filtered through Celite. The filtrate was concentrated in vacuo to give the product as a white foam (88% recovery, 99.5% purity). 1H NMR (400 MHz, DMSO-d6)δ13.30 (br s, 1H), 10.30 (s, 1H), 7.86 (s, 1H), 7.64 (dd, J=1.47, 7.82 Hz, 4H), 7.36 - 7.47 (m, 6H), 7.32 - 7.36 (s, 1H), 3.39 (s, 3H), 1.39 (s, 9H), 1.05 (s, 9H); HRMS: C 29 H 34 NO6Si - Calculated value: 520.2161 Measured value: 520.2110.

[0068] Example 4. Preparation of PBD core: [ka] tert-Butyl-(2-((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate: A solution of PBD-benzoic acid (5.0 g, 9.58 mmol, 1.0 equiv) in ethyl acetate (10 vol) was cooled to 0° C. To this solution was added 2-hydroxypyridine 1-oxide (HOPO, 1.28 g, 11.5 mmol, 1.2 equiv), followed by DIC (1.78 mL, 4.79 mmol, 1.2 equiv) at a rate to keep the temperature below 5° C. The reaction mixture was stirred for 30 min, after which PBD-pyrrolidine (2.2 g, 9.58 mmol, 1.0 equiv) was added in two portions. The mixture was then warmed to 20° C. and stirred for 3.5 h. When HPLC showed completion, the mixture was filtered through a pad of Celite. The filtrate was then washed once with water (10 vol) and the aqueous layer was extracted with ethyl acetate (5 vol). The organic layers were combined and washed with 1 M HCl (2x5 vol), followed by 1 M sodium bicarbonate (5 vol) and two times with 2 M brine (5 vol). The solvent was replaced by heptane (5 vol) via quantitative distillation and the resulting precipitate was removed by filtration through Celite. The filtrate was concentrated in vacuo to give PBD-INT as an off-white solid, which was used crude in the next step (yield 80-90%). 1 H NMR (400 MHz, DMSO-d6.)δ8.85 (s, 1H), 7.65 (ddd, J = 7.86, 3.64, 1.47 Hz, 4H), 7.36 - 7.49 (m, 7H), 6.70 (s, 1H), 4.94 - 4.82 (m, 1H), 4.28 (m, 1H), 4.12 - 4.22 (m, 1H), 3.85 - 3.67 (m, 2H), 3.52 - 3.43 (m, 1H), 3.38 (s, 3H), 3.21 (br d, J = 10.76 Hz, 1H), 2.07 - 1.85 (m, 2H), 1.37 (s, 9H), 1.08 - 1.05 (m, 9H), 0.90 - 0.81 (m, 9H), 0.06 - -0.02 (m, 6H); HRMS: C 40 H 59 N2O7Si2 + Calculated value: 735.3855 Measured value: 735.3864.

[0069] tert-Butyl-(S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-oxopyrrolidine-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate: A solution of sulfur trioxide-pyridine complex (7.07 g, 44.4 mmol, 6.0 equiv) in DMSO (10 vol) was prepared and stirred for 20 min. Pyridine (3.59 mL, 44.4 mmol, 6.0 equiv) was added to the sulfur trioxide-pyridine solution and stirred for an additional 30 min. In a separate flask, a solution of crude PBD-INT (5.44 g, 7.40 mmol, 1.0 equiv) in 10 vol DCM was prepared and cooled to 0° C. Triethylamine (8.25 mL, 59.2 mmol, 8.0 equiv) was added to the cooled solution. The sulfur trioxide-pyridine solution was added to the cooled PBD-INT solution at a rate that kept the temperature below 10° C. The reaction mixture was warmed to 20° C. and stirred for 3 h. When the reaction was complete as shown by HPLC, the mixture was cooled to 0° C. and quenched with 1 M sodium bicarbonate (5 vol). The organic layer was collected and the aqueous layer was extracted with 5 vol DCM. The organic layers were combined and washed three times with 1 M brine (3x5 vol). The organic layer was concentrated in vacuo and the product was purified by column chromatography (12-22% ethyl acetate / heptane). The product-containing fractions were combined and isolated to give PBD core A (purity >95%, yield 75%). 1 H NMR (400 MHz, DMSO-d6)δ8.41 (s, 1H), 7.68 - 7.62 (m, 4H), 7.45 - 7.34 (m, 7H), 7.15 (s, 1H), 6.74 (s, 1H), 4.51 - 4.73 (s, 1H), 3.91 - 3.77 (m, 1H), 3.69 (s, 1H), 3.67 - 3.58 (m, 1H), 3.44 (m, 3H), 2.87 - 2.83 (dd, J=17.52, 9.16 Hz, 1H), 2.52 - 2.46 (d, J=17.72, 1H), 1.35 (s, 9H), 1.08 (s, 9H), 0.82 (s, 9H), 0.00 (s, 6H); 13C NMR (400 MHz, DMSO-d6)δ210.1, 168.6, 153.2, 146.6, 146.1, 135.4, 133.2, 130.27, 128.03, 115.5, 111.9, 79.6, 56.0, 31.7, 28.5, 27.0, 26.0, 22.5, 19.8 18.2, 14.2, -5.4; IR (Neat) 3344, 3073, 2954, 2930, 2896, 2857, 1765, 1726, 1627, 1589, 1516, 1471, 1427, 1391, 1366, 1251, 1238, 1155, 1106, 1013, 942, 874, 834, 776, 700; HRMS:C 40 H 57 N2O7Si2 + Calculated value: 733.3699 Measured value: 733.3700.

[0070] Examples 5-8 Downstream Chemistry Example 5. Preparation of PBD vinyl triflate [ka] (S)-1-(2-((tert-butoxycarbonyl)amino)-4-((tert-butyldiphenylsilyl)oxy)-5-methoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate: A solution of PBD core (1.0 equiv) in CPME or THF (40 vol) was prepared under nitrogen atmosphere. To this solution was added N-(2-pyridyl)bis(trifluoromethanesulfonimide) (1.2 equiv) and cooled to -40°C. To this solution was then added lithium tert-butoxide (1 M solution in THF, 1.71 mL, 1.71 mmol, 2.50 equiv) in one portion. Stirred at -40°C until reaction was complete by HPLC (2.5 h). Reaction was quenched with 10% citric acid in ethyl acetate (40 vol) and then warmed to 0°C. 40 vol water was added to the reaction mixture and the organic layer was collected, washed with 2 M brine (10 vol), dried over sodium sulfate and concentrated in vacuo. The residue was taken up in heptane (5 vol) and filtered through celite. The filtrate was concentrated in vacuo and purified by column chromatography (0-10% ethyl acetate / heptane). The product containing fractions were combined and concentrated in vacuo to give the vinyl triflate (75-80%). HRMS: C 41 H 56 F3N2O9SSi2 + Calculated value: 865.3192 Measured value: 865.3157.

[0071] Example 6. Cross-coupling procedure [ka]

[0072] Example 6A. Suzuki Coupling A suspension of potassium phosphate tribasic (5.0 equiv.) and cesium acetate (1.0 equiv.) was prepared in degassed toluene (6.0 vol.) under nitrogen. The suspension was heated to 45°C. Pd(dppf)Cl2·DCM was then added to the suspension. In a separate flask, a solution of PBD-vinyl triflate (1.0 equiv.) in degassed toluene (6.0 vol.) was prepared under nitrogen. The vinyl triflate solution was added dropwise to the heated suspension. Once the addition of vinyl triflate was complete, methylboronic acid (4.0 equiv.) was added in portions. The mixture was stirred at 45°C until the reaction was complete as indicated by HPLC (typically 1.5 h). The mixture was then cooled to room temperature and poured into water (12 vol.). The organic layer was collected, concentrated in vacuo, and purified by column chromatography (0–15% ethyl acetate / heptane). The product-containing fractions were combined and isolated to give the product (30–50% yield).

[0073] Example 6B. Copper Coupling A suspension of copper(I) cyanide di(lithium chloride) complex in THF or CPME (10 vol) (5.0 eq) was prepared and cooled to -40°C. To this suspension was added methylmagnesium bromide (3.0 M in diethyl ether, 10.0 eq) dropwise. The mixture was warmed to 0°C, stirred for 10 min and then cooled back to -40°C. A solution of PBD vinyl triflate (1.0 eq) in THF or CPME (10 vol) was added dropwise over 10 min. The reaction was stirred at -40°C until complete by HPLC (usually 1 h). After completion of the reaction, it was warmed to 0°C and slowly quenched with saturated ammonium chloride (20 vol). The organic layer was collected and washed with saturated ammonium chloride (20 vol), saturated sodium bicarbonate (20 vol) and saturated brine (20 vol). The organic layer was collected and solvent exchanged into toluene (20 vol). SiliaMetS DOTA was then added to this solution and stirred at room temperature for 1 hour. The mixture was filtered and the filtrate was concentrated in vacuo to give the crude product as a clear yellow oil. The product was then purified by column chromatography (0-7% ethyl acetate / heptane). The fractions containing the product were combined and concentrated in vacuo to give the product (40% yield).

[0074] tert-Butyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate (8) 1 H NMR(400 MHz, CDCl3)δ8.10 (br s, 1H), 7.75 - 7.62 (m, 5H), 7.42 - 7.27 (m, 7H), 6.53 (s, 1H), 6.04 (s, 1H), 4.60 (m, 1H), 3.97 - 3.67 (m, 2H), 3.18 (s, 3H), 2.76 - 2.61 (m, 1H), 2.56 - 2.45 (m, 1H), 1.70 - 1.58 (m, 3H) 1.49 - 1.38 (m, 9H) 1.14 - 1.05 (m, 9H) 0.93 - 0.77 (m, 12H) 0.09 - -0.05 (m, 6H); 13 C NMR (101 MHz, CDCl3)δ165.3, 152.7, 147.3, 144.9, 135.2, 129.4, 127.4, 125.4, 125.3, 123.6, 121.9, 113.7 , 112.2, 79.86, 62.5, 59.1, 55.3, 36.3, 29.7, 28.3, 26.7, 25.7, 19.9, 18.1, 14.1, 13.7, .01, -5.5; HRMS: C 41 H 59 N2O6Si2 + Calculated value: 731.3906 Measured value: 731.3960.

[0075] Example 7. Formal synthesis of tesirine [ka] tert-Butyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (9): A solution of 8 (214 mg, 0.293 mmol, 1.0 equiv) in wet DMF (2 mL) was prepared. To this solution was added lithium acetate (19.3 mg, 0.293 mmol, 1.0 equiv) and heated to 40° C. The mixture was stirred until complete by HPLC. The mixture was cooled to room temperature and diluted with ethyl acetate (5 mL). The mixture was washed with water (5 mL). The aqueous layer was collected and back extracted with ethyl acetate (5 mL). The organic layers were combined and washed with 5% citric acid (5 mL) followed by saturated brine (5 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to give the free phenol as a clear light brown oil. The intermediate was dissolved in THF (2 mL) and cooled to 0° C. To this solution was added triethylamine (81.6 μL, 0.585 mmol, 2.0 equiv), followed by TIPSCl (68.9 μL, 0.322 mmol, 2.0 equiv). The reaction mixture was allowed to warm to room temperature and stirred for 15 h. When HPLC showed the reaction to be complete, the mixture was filtered to remove solids and the solids were washed with THF (2 mL). The organic layers were combined and washed with saturated sodium bicarbonate (2 mL) and brine (2 mL). The organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (0-15% ethyl acetate / heptane) and the product-containing fractions were combined and isolated to give 9 (96.9 mg, 0.149 mmol, 51% yield over two steps). HRMS: C 34 H 61 N2O6Si2 + Calculated value: 649.4063 Measured value: 649.4049.

[0076] [ka] (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)-methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone (10): A solution of 10 (96.9 mg, 0.149 mmol, 1.0 equiv) in DCM (10 vol) was prepared. To this solution was added 2,6-lutidine (346 μL, 2.99 mmol, 20 equiv) and TBSOTf (343 μL, 1.49 mmol, 10.0 equiv). The solution was then heated to 40 °C and stirred for 15 h. When HPLC showed completion, the mixture was quenched with saturated ammonium chloride (2 mL). The aqueous layer was back-extracted with DCM (2 mL) and the organic layers were combined, washed with saturated brine (2 mL) and dried over magnesium sulfate. The solution was concentrated under reduced pressure. The residue was taken up in THF (2 mL) and cooled to 0 °C. To the solution was added TBAF (14.9 μL 0.2 equiv) and stirred at 0 °C for 30 min. The solution was then diluted with 10 vol ethyl acetate and quenched with 10 vol water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 vol). The organic layers were combined, dried over magnesium sulfate and concentrated under reduced pressure. The product was determined according to the reported NMR data. 1b、2 It was used immediately in the next step. HRMS: C 29 H 53 N2O4Si2 + Calculated value: 549.3538 Measured value: 549.3558.

[0077] Allyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate: [ka] A solution of the free amine 10 (41 mg, 0.075 mmol, 1.0 equiv) in DCM (12 vol) was prepared and cooled to 0° C. To the solution was added pyridine (13 μL, 0.16 mmol, 2.2 equiv) followed by allyl chloroformate (8.8 μL, .082 mmol, 1.1 equiv). The reaction was stirred at 0° C. until complete by HPLC (typically 1 h). The reaction mixture was quenched with 10% citric acid solution (8 vol). The organic layer was collected and washed with saturated aqueous sodium bicarbonate (8 vol) and brine (8 vol). The organic layer was collected, dried over sodium sulfate, and filtered. The solution was filtered under reduced pressure to give the crude product as an orange to brown oil. The product was purified by column chromatography (0–15% ethyl acetate / heptane) and the product-containing fractions were combined and isolated to give 11 (28 mg, 0.075 mmol, 59% yield over two steps). The isolated product was consistent with the reported NMR data. 1b、2 HRMS: C 33 H 57 N2O6Si2 + Calculated value: 633.3750 Measured value: 633.3773.

[0078] Example 8 Preparation of advanced intermediates from PBD cores.

[0079] Example 8A. General procedure for Boc deprotection [ka] A solution of 8 (1.0 equiv) in DCM (10 vol) was prepared. To the solution was added 2,6-lutidine (20 equiv) and TBSOTf (10.0 equiv). The solution was then heated to 40° C. and stirred for 15 h. When HPLC showed completion, the mixture was quenched with saturated ammonium chloride (5 vol). The aqueous layer was back extracted with DCM (5 vol) and the organic layers were combined, washed with saturated brine (5 vol) and dried over magnesium sulfate. The solution was concentrated under reduced pressure. Carbamate 12 (HRMS: C 43 H 65 N2O6Si3 +Calculated for 789.4145 Found: 789.4115) was taken up in THF (10 vol) and cooled to 0° C. To the solution was added TBAF (0.2 equiv) and stirred at 0° C. for 30 min. The solution was then diluted with 10 vol ethyl acetate and quenched with 10 vol water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 vol). The organic layers were combined, dried over magnesium sulfate and concentrated under reduced pressure to give 13. The material was used immediately to avoid decomposition.

[0080] (S)-(2-Amino-4-((tert-butyldiphenylsilyl)oxy)-5-methoxyphenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone (13): HRMS: C 29 H 53 N2O4Si2 + Calculated value: 549.3538 Measured value: 549.3558.

[0081] Example 8B [ka] Allyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate (14): A solution of the free amine 13 (1.04 g, 1.69 mmol, 1.0 equiv) in DCM (12 vol) was prepared and cooled to 0°C. Pyridine (301 μL, 3.72 mmol, 2.2 equiv) was added to the solution followed by allyl chloroformate (198 μL, 1.86 mmol, 1.1 equiv). The reaction was stirred at 0°C until complete by HPLC (typically 1 h). The reaction mixture was quenched with 10% citric acid solution (8 vol). The organic layer was collected and washed with saturated aqueous sodium bicarbonate (8 vol) and brine (8 vol). The organic layer was collected, dried over sodium sulfate and filtered. The solution was concentrated under reduced pressure to give the crude product as an orange to brown oil. The product was purified by column chromatography (0-15% ethyl acetate / heptane) and the product-containing fractions were combined and isolated to give 14. HRMS: C 40 H 55 N2O6Si2 + Calculated value: 715.3593 Measured value: 715.3606.

[0082] Example 8C.

[0083] [ka] Allyl (S)-(5-((tert-butyldiphenylsilyl)oxy)-2-(2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxyphenyl)carbamate (15): A solution of 14 (246.4 mg, 0.34 mmol, 1.0 equiv) in THF (6 vol) and water (0.3 vol) was prepared. To this solution was added p-toluenesulfonic acid hydrate (39.3 mg, 0.21 mmol, 0.6 equiv) and stirred at room temperature until the reaction was complete as indicated by HPLC. The reaction mixture was diluted with ethyl acetate (10 vol) and washed with water (4 vol) followed by brine (4 vol). The organic layer was collected, dried over sodium sulfate and filtered. The solution was filtered under reduced pressure to give the crude product as an orange to brown oil. The product was purified by column chromatography (0-15% ethyl acetate / heptane) and the product-containing fractions were combined and isolated to give 15. HRMS: C 34 H 41 N2O6Si2 + Calculated value: 601.2728 Measured value: 601.2735.

[0084] Example 8D. [ka]

[0085] Allyl (11S,11aS)-8-((tert-butyldiphenylsilyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (16): A solution of oxalyl chloride (9.3 μL, 0.11 mmol, 1.03 equiv) in DCM (9 vol) was prepared and cooled to -65°C. To the cooled solution was added dimethylsulfoxide (18.3 μL, 0.26 mmol, 2.5 equiv). The reaction mixture was stirred for 30 min, after which a solution of 15 (62.1 mg, 0.10 mmol, 1.0 equiv) in DCM (8 V) was added dropwise. After 30 min, triethylamine (72.0 μL, 0.52 mmol, 5.0 equiv) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred until complete by HPLC. The reaction was quenched with 5% citric acid (10 vol) and washed with saturated aqueous sodium bicarbonate (10 vol) and brine (10 vol). The organic layer was collected, dried over sodium sulfate, and filtered. The solution was concentrated under reduced pressure to give the crude product 17, which was used in the next step without purification (yield 87%). HRMS: C 34 H 39 N2O6Si + Calculated value: 599.2572 Measured value: 599.2549.

[0086] Example 8E. [ka] Allyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-((tert-butyldiphenylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (17): A solution of 16 (54.1 mg, 0.09 mmol, 1.0 equiv) in DCM (10 vol) was added with 2,6-lutidine (71.8 μL, 0.62 mmol, 6.0 equiv) and cooled to 0° C. To the cooled solution was added tert-butyldimethylsilyl triflate (119 μL, 0.52 mmol, 5.0 equiv). The reaction mixture was stirred at 0° C. for 30 min, followed by stirring at room temperature for 3 h. When the reaction was complete as shown by HPLC, it was quenched with saturated aqueous sodium bicarbonate (10 vol) and brine (10 vol). The organic layer was collected, dried over sodium sulfate, and filtered. The solution was concentrated under reduced pressure to give crude product 17, which was used in the next step without purification. HRMS: C 40 H 53 N2O6Si2 + Calculated value: 713.3437 Measured value: 713.3429.

[0087] Example 8F. [ka] Allyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (18): To a solution of 17 (64.2 mg, 0.09 mmol, 1.0 equiv) in wet DMF (15 vol) was added lithium acetate (5.94 mg, 0.09 mmol, 1.0 equiv) and heated to 40° C. The reaction mixture was stirred at 40° C. until complete by HPLC. The reaction mixture was diluted with ethyl acetate (20 vol) and washed with water (10 vol) and saturated brine (10 vol). The organic layer was collected, dried over sodium sulfate and filtered. The solution was concentrated under reduced pressure to give crude product 18, which is consistent with the literature. 2 HRMS: C 24 H 35 N2O6Si + Calculated value: 475.2259 Measured value: 475.2284.

[0088] Example 8G. [ka] Allyl ((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (19): A solution of 8 (288.9 mg, 0.40 mmol, 1.0 equiv) in DCM (10 vol) was prepared. To the solution was added 2,6-lutidine (0.91 mL, 7.9 mmol, 20 equiv) and TBSOTf (10.0 equiv). The solution was then heated to 40° C. and stirred for 15 h. When HPLC showed completion, the mixture was quenched with saturated ammonium chloride (5 vol). The aqueous layer was back-extracted with DCM (5 vol) and the organic layers were combined, washed with saturated brine (5 vol) and dried over magnesium sulfate. The solution was concentrated under reduced pressure. The carbamate was taken up in THF (10 vol) and cooled to 0° C. To the solution was added TBAF (0.16 mL, 1 M, 0.16 mmol, 0.4 equiv) and stirred at 0° C. for 30 min. The solution was then diluted with 10 vol of ethyl acetate and quenched with 10 vol of water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 vol). The organic layers were combined, dried over magnesium sulfate and concentrated under reduced pressure to give 14. The material was used immediately to avoid decomposition. 13 was added with 10 vol of DCM and cooled to -20 °C. Triphosgene (46.9 mg, 0.16 mmol, 0.40 equiv) was added slowly to the reaction mixture. The solution was then charged with triethylamine (121 μL, 0.87 mmol, 2.2 equiv) and stirred at -20 °C for 30 min. Meanwhile, a mixture of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (156.6 mg, 0.41 mmol, 1.05 equiv) in triethylamine (83 μL, 0.60 mmol, 1.5 equiv) in DCM (10 vol) was prepared. This solution was added rapidly to the reaction at -20°C. The reaction was stirred for 15 hours. When HPLC showed the reaction was complete, the reaction mixture was washed with water (4 vol). The organic layer was collected, dried over sodium sulfate and concentrated under reduced pressure. The resulting mixture was purified by flash chromatography to give the product as a white solid (321.2 mg, 79% yield for three steps). HRMS: C 56 H 76 N5O 10 Si2 +Calculated value: 1034.5125 Measured value: 1034.5094.

[0089] Example 8H [ka] Allyl ((S)-1-(((S)-1-((4-((((5-((tert-butyldiphenylsilyl)oxy)-2-((S)-2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (20): A solution of 19 (321.2 mg, 0.31 mmol, 1.0 equiv) in THF (6 vol) and water (0.3 vol) was prepared. To this solution was added para-toluenesulfonic acid hydrate (35.4 mg, 0.18 mmol, 0.6 equiv) over 2 h. When HPLC showed the reaction was complete, the solution was diluted with ethyl acetate (10 vol) and washed with water (5 vol), saturated sodium bicarbonate (5 vol), and saturated brine (5 vol). The organic layer was collected, dried over sodium sulfate, and concentrated under reduced pressure. The product was purified by column chromatography (heptane / ethyl acetate 40 / 60 to 0 / 100) to give the product as a slightly yellow solid (83% yield). HRMS: C 50 H 62 N5O 10 S + Calculated value: 920.4260 Measured value: 920.4264.

[0090] Example 8I. [ka] 4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-((tert-butyldiphenylsilyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (21): A solution of oxalyl chloride (2.03 M, 152 μL, 0.31 mmol, 1.2 equiv) in DCM was prepared and cooled to -65°C. DMSO (46 μL, 0.64 mmol, 2.5 equiv) was added dropwise. The resulting solution was stirred for 15 min at which point a solution of 20 (236.2 mg, 0.26 mmol, 1.0 equiv) in DCM (5 vol) was added dropwise. The reaction was stirred at -65°C for 15 min after which triethylamine (179 μL, 1.28 mmol, 5.0 equiv) was added dropwise. The reaction was stirred at -65°C for 1 h. When HPLC showed the reaction to be complete, the reaction mixture was warmed to 0°C and washed with 0.2 N HCl (10 vol), saturated sodium bicarbonate (10 vol), and brine (10 vol). The organic layer was collected and concentrated under reduced pressure. The residue was then added with DCM (10 vol) and the resulting solution was concentrated under reduced pressure again. This procedure was repeated twice to give the product 21 in 86% yield. The crude product was used immediately in the next step. HRMS: C 50 H 60 N5O 10 S + Calculated value: 918.4104 Measured value: 918.4091.

[0091] Example 8J. [ka] 4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (11S)-11-((tert-butyldimethylsilyl)oxy)-8-((tert-butyldiphenylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (22): A solution of 21 (203 mg, 0.22 mmol, 1.0 equiv) in DCM (20 vol) was cooled to -15 °C and 2,6-lutidine (178 μL, 1.54 mmol, 6.0 equiv) was added. The reaction mixture was cooled to -40 °C and tert-butyldimethylsilyl triflate (295 μL, 1.28 mmol, 5.0 equiv) was added dropwise. The reaction was stirred for 30 min and then oxalyl chloride in DCM (2.03 M, 152 μL, 0.31 mmol, 1.2 equiv) was prepared and cooled to -65 °C. DMSO (46 μL, 0.64 mmol, 2.5 equiv) was added dropwise. The resulting solution was stirred at -40 °C for 15 min followed by stirring at -20 °C for 20 h. When the reaction was shown to be complete by HPLC, it was quenched with 10% citric acid (10 vol) and washed with saturated sodium bicarbonate (10 vol) and brine (10 vol). The organic layer was collected and concentrated under reduced pressure. DCM (10 vol) was then added to the residue and the resulting solution was concentrated under reduced pressure again. This procedure was repeated twice to give the product 22. The crude product was used immediately in the next step. HRMS: C 56 H 74 N5O 10 Si2 + Calculated value: 1032.4969 Measured value: 1032.4922.

[0092] Example 8K [ka] 4-((S)-2-((S)-2-((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (11S)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (23) A solution of crude 22 (265 mg, 0.26 mmol, 1.0 equiv) in DMF (10 vol) and water (0.3 equiv) was prepared. Lithium acetate (16.9 mg, 0.26 mmol, 1.0 equiv) was added to the solution. The mixture was heated to 40° C. and stirred for 8 h. When HPLC showed the reaction was complete, the mixture was cooled to room temperature and diluted with Me-THF (10 vol). The mixture was washed with 10% citric acid (10 vol), 5% sodium bicarbonate (10 vol), saturated brine (10 vol), and water (10 vol). The organic layer was collected and concentrated under reduced pressure. 2-MeTHF (10 vol) was then added to the residue, and the resulting solution was again concentrated under reduced pressure. This procedure was repeated twice to give the crude product. The crude product was purified by column chromatography (heptane / ethyl acetate 40 / 60 to 0 / 100) to give product 23 (80.2 mg, 40% yield over two steps). 1b、2 HRMS: C 40 H 55 N5O 10 S + Calculated value: 794.3791 Measured value: 794.3778.

[0093] Example 8L [ka] tert-Butyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate (24) A solution of methyltriphenylphosphonium bromide (2.92 g, 8.17 mmol, 2.0 equiv) in THF (38.5 vol) was prepared and cooled to 0°C. Potassium tert-butoxide (1.0 M, 8.18 mL, 8.18 mmol, 2.0 equiv) was added dropwise to the solution. The reaction mixture was stirred at 0°C for 15 min and then at room temperature for 45 min. The reaction mixture was then cooled to 0°C and PBD core A (3.0 g, 4.09 mmol, 1.0 equiv) in THF (1.5 vol) was added dropwise. After complete addition of PBD core A, the reaction mixture was allowed to warm to room temperature and stirred until the reaction was complete as indicated by HPLC. The reaction was quenched with water (15 vol). The aqueous layer was collected and washed twice with MTBE (15 vol). The organic layers were combined, washed with water (15 vol) and brine (15 vol), dried over sodium sulfate, and then concentrated under reduced pressure. The product was purified by column chromatography (heptane / ethyl acetate 85 / 15). The product-containing fractions were isolated to give the product 24 (2.00 g, 2.7 mmol) in 64% yield. HRMS: C 41 H 59 N2O6Si2 + Calculated value: 731.3906 Measured value: 731.3786.

[0094] Example 8M [ka] (S)-(2-amino-4-((tert-butyldiphenylsilyl)oxy)-5-methoxyphenyl)(2-(((tert-butyldimethylsilyl)oxy)-methyl)-4-methylenepyrrolidin-1-yl)methanone (25): A solution of 24 (300.0 mg, 0.40 mmol, 1.0 equiv) in DCM (5 vol) was prepared. 2,6-lutidine (0.19 mL, 1.6 mmol, 20.0 equiv) was added to the solution and cooled to 0 °C. The reaction mixture was then added tert-butyldimethylsilyl triflate (0.18 mL, 0.8 mmol, 2.0 equiv). The solution was stirred at 0 °C for 30 min and then at room temperature for 24 h. The reaction was cooled to 0 °C and 10% aqueous citric acid was added. The resulting mixture was stirred at room temperature for 96 h. The reaction mixture was diluted with DCM (3 vol). The organic layer was collected and washed with saturated brine (5 vol). The crude product was isolated (90% yield) and stored at -20 °C. HRMS: C 36 H 51 N2O4Si2 + Calculated value: 631.3382 Measured value: 631.3354.

[0095] Example 8N [ka] tert-Butyl (S)-(5-((tert-butyldiphenylsilyl)oxy)-2-(2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (26): A solution of 24 (1.90 g, 2.6 mmol, 1.0 equiv) in ethyl acetate (15 vol) was cooled to 0° C. To the solution was added hydrochloric acid (37% reagent, 0.03 mL, 0.8 mmol, 0.3 equiv). The reaction was allowed to warm slowly to room temperature and stirred for 2 h. HPLC showed the reaction to be complete. Half of the material was set aside for stability testing. The other half was isolated and subjected to Swern conditions (see preparation of 27). HRMS: C 35 H 45 N2O6Si + Calculated value: 617.3041 Measured value: 617.3065.

[0096] Example 8O [ka] tert-Butyl (11S,11aS)-8-((tert-butyldiphenylsilyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (27): A solution of 26 (0.65 g, 1.1 mmol, 1.0 equiv) in THF (10 vol) was added with DMSO (0.19 mL, 2.6 mmol, 2.5 equiv) and cooled to -78 °C. Trifluoroacetic anhydride (0.16 mL, 1.2 mmol, 1.1 equiv) was added dropwise to the reaction mixture and then stirred at -78 °C for 30 min. Triethylamine (0.73 mL, 5.3 mmol, 5.0 equiv) was then added slowly to the reaction mixture. The reaction was then quenched with 5% aqueous citric acid and stirred at 5 °C for 15 min. The organic layer was collected, washed with saturated sodium bicarbonate and water, and then dried over magnesium sulfate. The intermediate was isolated and DCM (10 vol) was added. The solution was cooled to -78°C and 2,6-lutidine (0.31 mL, 2.6 mmol, 2.5 equiv) was added followed by tert-butyldimethylsilyl trifluoromethanesulfonate (0.36 mL, 1.6 mmol, 1.5 equiv). The reaction was allowed to warm slowly to room temperature over 30 min. The reaction was stirred at room temperature for 15 h. When HPLC showed the reaction was complete, the reaction mixture was diluted with DCM (45 vol) and quenched with saturated sodium bicarbonate. The organic layer was collected, washed with brine and dried over sodium sulfate. The crude product was isolated under reduced pressure and purified by column chromatography (heptane / ethyl acetate 75 / 25). Fractions containing product were combined to give 27 in 54% yield over three steps. HRMS: C 41 H 57 N2O6Si2 + Calculated value: 729.3750 Measured value: 729.3790.

[0097] Example 8P [ka] tert-Butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (28) A solution of crude 27 (205 mg, 0.28 mmol, 1.0 equiv) in DMF (10 vol) and water (0.3 equiv) was prepared. Lithium acetate (18 mg, 0.28 mmol, 1.0 equiv) was added to the solution. The mixture was stirred at room temperature for 15 h. When HPLC showed the reaction was complete, the reaction mixture was diluted with ethyl acetate (25 vol) and washed twice with water (20 vol) and twice with brine (20 vol). The organic layer was collected, dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (heptane / ethyl acetate 30 / 70) to give product 28 (80.2 mg, 40% yield for two steps). The product was determined to be 100% according to the reported NMR data. 1b、2 HRMS: C 25 H 39 N2O6Si + Calculated value: 491.2572 Measured value: 491.2520.

[0098] The examples herein are for illustrative purposes only and are not meant to limit the scope of the invention, which is defined by the claims.

[0099] References: (a) Gregson, S.J.; Howard, P.W.; Gullick, D.R.; Hamaguchi, A.; Corcoran, K.E.; Brooks, N.A.; Hartley, J.A.; Jenkins, T.C.; Patel, S.; Guille, M.J.; Thurston, DEJ Med. Chem. 2004, 57 (5), 1161-1174; (b) Tiberghien, A.C.; von Bulow, C.; Barry, C.; Ge, H.; Noti, C.; Leiris, F.C.; McCormick, M.; Howard, P.W.; Parker, J. S. Org. Process Res. Dev. 2018, 22 (9), 1241-1256. (a) Tiberghien, AC; Levy, J.-N.; Masterson, LA; Patel, N.V.; Adams, L.R.; Corbett, S.; Williams, D.G.; Hartley, J.A.; Howard, PW ACS Med. Chem. Lett. 2016, 7 (11), 983-987; (b) Tiberghien, A.C.; Howard, P.W.; Goundry, W.R.F.; McCormick, M.; Parkers, JSJ Org. Chem. 2019, 84 (8), 4830-4836; (c) Lai, W.; Zhao, S.; Lai, Q.; Zhou, W.; Wu, M.; Jiang, X.; Wang, X.; Peng, Y.; Wei, X.; Ouyang, L.; Gou, L.; Chen, H.; Wang, Y.; Yang, JJ Med. Chem. 2022, 65 (17), 11679-11702.

Claims

1. Formula I 【Chemistry 1】 [During the ceremony, The dashed lines are single or double bonds. R 1 is a protecting group selected from tert-butyldimethylsilyl ether (TBS) and acetate (Ac); R 2 -H, =O, -OTf, C 1 -C 5 Alkyl, C 3 -C 6 Cycloalkyl, C 1 -C 5 Alkenyl, C 5 -C 10 Aryl, C 5 -C 10 heteroaryl, and a linker, wherein R 2 is optionally halo, nitro, cyano, ether, carboxy, ester, C 1 -C 7 Alkyl, C 1 -C 7 Alkenyl, C 3 -C 7 Heterocyclyl, bis-oxy-C 1 -C 3 may be substituted with one or more substituents selected from the group consisting of alkylene and a linker; R 3 -H, -OH, -OR, -SH, -NH 2 , -NO 2 , halo, where R is C 1 -C 4 Alkyl, C 3 -C 10 Heterocyclyl and C 5 -C 10 aryl, and R is optionally selected from the group consisting of C 1 -C 4 may have substituents selected from the group consisting of alkyl and phenyl; R 4 -H, -R, -OH, -OR, -SH, -SR, -NH 2 , -NHR, -NHRR', nitro and halo, where R and R' are independently selected from the group consisting of C 1 -C 4 Alkyl, C 3 -C 10 Heterocyclyl and C 5 -C 10 aryl, and R and R' are optionally selected from the group consisting of C 1 -C 4 may have substituents selected from the group consisting of alkyl and phenyl; R 5 is a protecting group selected from tert-butyldiphenylsilyl ether (TBDPS) and triisopropylsilyl ether (TIPS), and R 6 -H, -OH, -OR, -SH, -NH 2 , -NO 2 , halo, where R is C 1 -C 4 Alkyl, C 3 -C 10 Heterocyclyl and C 5 -C 10 aryl, and R is optionally selected from the group consisting of C 1 -C 4 The alkyl group may have a substituent selected from the group consisting of alkyl, phenyl, and the like. Compound.

2. R 3 and R 6 The compound of claim 1, wherein each is -H.

3. During the ceremony, R 2 -H, =O, -OTf, -OH, methyl, cyclopropyl, =CH 2 , ethylenyl, propenyl, phenyl, 4-fluorophenyl, indole and a linker, and R 2 optionally halo, nitro, cyano, ether, carboxy, ester, C 1 -C 7 Alkyl, C 1 -C 7 Alkenyl, C 3 -C 7 Heterocyclyl, bis-oxy-C 1 -C 3 may be substituted with one or more substituents selected from the group consisting of alkylene and a linker; R 4 -H, -OCH 3 , -OCH 2 Ph, -N(CH 3 ) 2 , morpholino, piperidinyl, and -N-Me-piperazinyl; 3. A compound according to claim 1 or 2.

4. R 2 The compound according to any one of claims 1 to 3, wherein is =O.

5. R 2 The compound according to any one of claims 1 to 3, wherein is -OTf.

6. R 4 But OCH 3 The compound according to any one of claims 1 to 5, 【Request 7】 【Chemical 2】 5. The compound according to claim 1 or 4, selected from: 【Request 8】 【Chemical 3】 6. The compound according to claim 1 or 5, selected from:

9. 【Chemical 4】 2. The compound of claim 1, selected from:

10. 【Chemical 5】 A compound selected from:

11. Formula II: 【Chemistry 6】 [In the formula, R 7 is selected from the group consisting of -H, -C(O)O-TBS, Alloc, and a linker, and R 8 is selected from -H and TBS. Compound.

12. 【Chemical 7】 12. The compound of claim 11, selected from:

13. Formula III: 【Chemistry 8】 [In the formula, R 9 is selected from Alloc and a linker, and R 8 is selected from -H and TBS. Compound.

14. 【Chemical 9】 14. The compound of claim 13, selected from:

15. Providing a compound according to any one of claims 4, 6 or 7, performing a C-C bond forming reaction with a ketone; and Optionally, deprotecting the amine.

2. A process for producing an advanced pyrrolobenzodiazepine (PBD) intermediate comprising:

16. Providing a compound according to any one of claims 4, 6 or 7, converting the ketone to a triflate; performing a C-C bond forming reaction on the triflate; and Optionally, deprotecting the amine. A method for producing an advanced PBD intermediate comprising:

17. The method according to claim 15 or 16, wherein the CC bond forming reaction is Suzuki coupling.

18. Providing a compound according to any one of claims 5, 6 or 8, performing a C-C bond forming reaction on the triflate; and Optionally, deprotecting the amine. A method for producing an advanced PBD intermediate comprising:

19. The method of claim 18, wherein the CC bond forming reaction is Suzuki coupling.

20. 20. The method of any one of claims 15 to 19, further comprising the step of deprotecting the amine and coupling a linker to the deprotected amine.

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