Pyrrolobenzodiazepine intermediates and uses thereof

Abstract

Pyrrolobenzodiazepine # imgabs0 # (PBD) intermediates, including those useful in the preparation of higher intermediates PBD and PBD dimers. Such PBD intermediates provide several advantages, including simplified synthesis of the desired PBD product, greater substrate compatibility, and safer reaction conditions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 269,205, filed on March 11, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Pyrrolobenzodiazepines discovered in 1963 (PBD) is a class of naturally occurring compounds with antibiotic and antitumor properties. Originally derived from Streptomyces, PBD is an effective DNA damaging agent that is significantly more effective than current systemic chemotherapy drugs. Although toxicity involves limited initial applications, PBD has been shown to be effective as a payload in antibody-drug conjugates (ADC). There is currently a commercial ADC containing PBD, and many other ADCs are currently in clinical trials.

[0004] Currently, there are no commercially available intermediates for the synthesis of PBD payloads. Scientists must develop each synthesis from scratch and then further scale up and develop GMP processes for manufacturing PBDs. There is a need for new intermediates that can be used to synthesize PBDs. Summary of the invention

[0005] Provided are methods for synthesizing pyrrolobenzodiazepines The compound of formula I

[0006]

[0007] The dashed lines represent single or double bonds.

[0008] R1 is a protecting group selected from tert-butyldimethylsilyl ether (TBS) and acetate (Ac).

[0009] R2 is selected from -H, =O, -OTf, -OH, C1-C5 alkyl, C3-C6 cycloalkyl, C1-C5 alkenyl, C5-C 10 Aryl, C5-C 10 Heteroaryl and linking group. Optionally, R2 may be substituted by one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxyl, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclic group, bis-oxy-C1-C3 alkylene and linking group.

[0010] R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10R may be optionally substituted by a C1-C4 alkyl group or a phenyl group.

[0011] R4 is selected from -H, -R, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halogen, wherein R and R' are independently selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 R and R' may optionally have a substituent selected from the group consisting of a C1-C4 alkyl group and a phenyl group.

[0012] R5 is a protecting group selected from tert-butyldiphenylsilyl ether (TBDPS) and triisopropylsilyl ether (TIPS).

[0013] R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 aryl group, and R may optionally have a substituent selected from C1-C4 alkyl group, phenyl group.

[0014] In some embodiments, R3 and R6 are each -H.

[0015] In some embodiments, R2 is selected from -H, =O, -OTf, -OH, methyl, cyclopropyl, =CH2, vinyl, propenyl, phenyl, 4-fluorophenyl, indole and a linker. R2 may be optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxyl, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene and a linker.

[0016] In some embodiments, R4 is selected from -H, -OCH3, -OCH2Ph, -N(CH3)2, morpholino, piperidinyl, and -N-Me-piperazinyl.

[0017] In some embodiments, R2 is =0, and in some embodiments, R2 is -OTf.

[0018] In some embodiments, R4 is OCH3.

[0019] 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 at the ketone, and optionally, deprotecting the amine.

[0020] In some embodiments, the method comprises the steps of converting the ketone to a triflate, then performing a CC bond forming reaction on the triflate, and optionally, deprotecting the amine.

[0021] In some embodiments, the CC bond forming reaction is a Suzuki coupling.

[0022] Also provided is a process for preparing an advanced PBD intermediate from a compound of formula I wherein R2 is a triflate, the process comprising the steps of performing a CC bond forming reaction on the triflate, and optionally, deprotecting the amine.

[0023] In some embodiments of the method, the CC bond forming reaction is a Suzuki coupling.

[0024] In some embodiments of these methods, when the amine is deprotected, the method comprises the further step of coupling a linker to the deprotected amine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 provides the structures of two PBD intermediates described herein. Figure 1A Shows PBD core A, while Figure 1B PBD core A triflate is shown.

[0026] Figure 2 A protocol for the synthesis of PBD core A from commercially available starting materials is provided.

[0027] Figure 3 A scheme for the processing of PBD core A into PBD fragments via higher-order PBD intermediates is provided. Figure 3A The synthesis of fragment A via the common intermediate disclosed herein is highlighted; Figure 3B The synthesis of fragment B via common intermediates is highlighted. DETAILED DESCRIPTION

[0028] Provided are new pyrrolobenzodiazepines (PBD) cores and advanced intermediates. These compounds are particularly easy to synthesize PBD payloads for antibody-drug conjugates. These compounds reduce the number of steps required to synthesize PBD payloads, simplifying the process. Due to the synthetic flexibility provided, these PBD intermediates provide considerable utility for developers interested in a variety of analogs dedicated to PBD payloads.

[0029] The PBD intermediates and methods provided herein solve the problem that pharmaceutical researchers need to develop lengthy de novo syntheses to obtain a single PBD payload. The PBD intermediates provided herein can be used to prepare many PBD-derived payloads that have entered the clinic.

[0030] Representative PBD intermediates described herein include PBD Core A and PBD Core A triflate:

[0031]

[0032] A feature of the PBD intermediates described herein is the Boc-protected nitrogen relative to the commonly used nitro group. The Boc-group provides versatility in the synthetic options in downstream chemistry. It provides the opportunity to prepare novel PBDs that are not tolerant to nitro-reducing conditions. In addition, early reduction avoids harmful zinc powder reduction, which is otherwise necessary for advanced intermediates. In addition, the Boc-group can be selectively removed or carried through this approach, providing greater flexibility.

[0033] Earlier methods utilized a -NO2 group that had to be reduced after the keto functionalization step. The change in the order of the chemical steps means that the sequence has greater substrate compatibility, reduces the number of post-differentiation steps, and avoids the use of explosive zinc dust. The later introduction of the R group provides a later diversification point, which simplifies the sequence. The use of a Boc-protecting group allows for selective removal if desired, or it can be performed throughout the sequence and removed by a simple acidic deprotection in the final step.

[0034] The ketone and triflate functionalities exemplified in PBD core A and PBD core A triflate provide further flexibility in preparing PBD or advanced intermediates. The ketone of PBD core A is easy to ketone functionalization. The triflate group of PBD core A triflate is highly regioselective. The processing from PBD core A to PBD core A triflate is high yield and provides excellent selectivity for desired enol triflate stereoisomers.

[0035] In one embodiment, the PBD intermediates are those of Formula I:

[0036]

[0037] Wherein dotted line represents single bond or double bond. R1 and R5 are each alcohol protecting group, and select it so that each protecting group can be removed respectively. In other words, R1 and R5 are selected to be 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, R5 is triisopropylsilyl ether (TIPS). In preferred embodiments, R1 is TBS, and R5 is TBDPS.

[0038] R2 can be selected from -H, =O, -OTf (trifluoromethanesulfonate functional group, CF3, SO3- or trifluoromethanesulfonate), -OH, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl and linking group.Optionally, R2 can be substituted by one or more substituents selected from the following: halogen, nitro, cyano, ether, carboxyl, ester, alkyl, alkenyl, heterocyclic radical, bis-oxy-C1-C3 alkylene and linking group.

[0039] In some embodiments, R2 can be selected from -H, =O, -OTf, -OH, C1-C5 alkyl, C3-C6 cycloalkyl, C1-C5 alkenyl, C5-C 10 Aryl, C5-C 10 Heteroaryl and linking group. Optionally, R2 may be substituted by one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxyl, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclic group, bis-oxy-C1-C3 alkylene and linking group.

[0040] R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from alkyl, heterocyclic and aryl. R may be optionally substituted by alkyl or aryl.

[0041] In some embodiments, R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 R may be optionally substituted by a C1-C4 alkyl group or a phenyl group.

[0042] R4 is selected from -H, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halogen, wherein R and R' are independently selected from alkyl, heterocyclic and aryl. R and R' may optionally have a substituent selected from alkyl and aryl.

[0043] In some embodiments, R4 is selected from -H, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halogen, wherein R and R' are independently selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 R and R' may optionally have a substituent selected from the group consisting of a C1-C4 alkyl group and a phenyl group.

[0044] R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from alkyl, heterocyclic and aryl, and R may optionally have a substituent selected from alkyl and aryl.

[0045] In some embodiments, R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 aryl group, and R may optionally have a substituent selected from C1-C4 alkyl group, phenyl group.

[0046] In some embodiments, R3 and R6 are each -H.

[0047] In some embodiments, R2 is selected from -H, =O, -OTf, -OH, methyl, cyclopropyl, =CH2, vinyl, propenyl, phenyl, 4-fluorophenyl, indole and linking group. R2 can be optionally substituted by one or more substituents selected from the following: halogen, nitro, cyano, ether, carboxyl, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclic radical, bis-oxy-C1-C3 alkylene and linking group. In some embodiments, R2 is =O, and in some embodiments, R2 is -OTf.

[0048] 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.

[0049] In the embodiment including a linking group, suitable linking groups include linking groups suitable for ADC. Such linking groups can include, for example, cleavable linking groups or non-cleavable linking groups. The selection of linking groups will depend on the following factors, such as the connection point with the PBD core intermediate, the connection point with the final specific antibody used, and the connection point with the release point expected at the target. Suitable linking groups can be identified by those skilled in the art. In some embodiments, linking groups include 4-aminobenzyl (PAB) groups connected to a dipeptide (e.g., valine-citrulline).

[0050] In some embodiments, the compound of Formula I is one of the following

[0051]

[0052] In other embodiments, the compound of Formula I is one of the following

[0053]

[0054] In yet other embodiments, the compound of Formula I may be:

[0055]

[0056] Also provided are analogs of Formula I in which one or more protecting groups have been removed. Exemplary compounds include:

[0057]

[0058] In certain other embodiments, compounds of Formula II are provided:

[0059]

[0060] 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:

[0061]

[0062] Also provided are intermediates of formula III:

[0063]

[0064] Wherein R9 is selected from Alloc and a linker, such as a protected PABC-peptide linker, and R8 is selected from -H and TBS. Exemplary compounds of Formula III include:

[0065]

[0066] The intermediates provided herein can be prepared using commercially available starting materials as described below.

[0067] Also provided is a method for preparing a high-level PBD intermediate, the method comprising further reaction at R2. In particular, the presence of a ketone or triflate group at R2 is prone to carbon-carbon cross-coupling reactions. Many carbon-carbon cross-coupling reactions or CC cross-coupling reactions are known to those skilled in the art. Some exemplary CC cross-coupling reactions include, but are not limited to, Suzuki-Miyaura coupling or Suzuki coupling, Wittig reaction, aldol reaction, α-CH arylation, Heck coupling, Negishi coupling, Stille coupling, etc. According to the method provided herein, many different CC cross-coupling reactions are suitable. In some embodiments, Suzuki coupling is a preferred method for CC cross-coupling.

[0068] In one embodiment, the method comprises the steps of providing a compound of formula I wherein R2 is a ketone, performing a CC bond forming reaction at the ketone, and optionally, deprotecting the amine.

[0069] In some embodiments, the method comprises the steps of converting the ketone to a triflate, then performing a CC bond forming reaction on the triflate, and optionally, deprotecting the amine.

[0070] In some embodiments, the CC bond forming reaction is a Suzuki coupling.

[0071] Also provided is a process for preparing an advanced PBD intermediate from a compound of formula I, wherein R2 is a triflate, the process comprising the steps of performing a CC bond forming reaction on the triflate, and optionally, deprotecting the amine.

[0072] In some embodiments of the method, the CC bond forming reaction is a Suzuki coupling.

[0073] In some embodiments of these methods, when the amine is deprotected, the method comprises the further step of coupling a linker to the deprotected amine.

[0074] The following intermediates and higher intermediates have been prepared as described below:

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] The following examples illustrate the preparation and use of the intermediates described herein.

[0081] Example 1. Preparation of PBD Core A:

[0082]

[0083] 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 volumes) was cooled to 0°C. To the 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 and then 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 hours. When the display is completed by HPLC, the mixture is filtered through a pad of diatomaceous earth. The filtrate is then washed once with water (10 volumes), and the aqueous layer is extracted with ethyl acetate (5 volumes). The organic layers are combined and washed twice (2×5 volumes) with 1M HCl, then washed with 1M sodium bicarbonate (5 volumes) and 2M saline (5 volumes). Solvent exchange is carried out to heptane (5 volumes) via isochoric distillation, and the resulting precipitation is removed by filtering through diatomaceous earth. The filtrate is concentrated in vacuo to provide PBD-INT, which is an off-white solid, which is used in the next step as a crude product (80-90% yield).

[0084] (S)-tert-butyl(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 volumes) was prepared and stirred for 20 minutes. Pyridine (3.59 mL, 44.4 mmol, 6.0 equiv) was added to the sulfur trioxide-pyridine solution and stirred for an additional 30 minutes. In a separate flask, a solution of crude PBD-INT (5.44 g, 7.40 mmol, 1.0 equiv) in 10 volumes of DCM was prepared and cooled to 0°C. To the cooled solution was added triethylamine (8.25 mL, 59.2 mmol, 8.0 equiv). Sulfur trioxide-pyridine solution is added to the solution of cooling PBD-INT at a rate keeping the temperature below 10 ℃. The reaction mixture is warmed to 20 ℃, and stirred for 3 hours. When the reaction is completed by HPLC, the mixture is cooled to 0 ℃, and quenched with 1M sodium bicarbonate (5 volumes). Collect organic phase, and extract aqueous phase with 5 volumes of DCM. Merge organic phase, and wash three times (3×5 volumes) with 1M salt water. Organic layer is concentrated in vacuo, and purified by column chromatography (12-22% ethyl acetate / heptane). Merge fractions containing product, and separate, to obtain PBD core A (>95% purity, 75% yield).

[0085] Example 2. Triflation / Suzuki coupling / Boc-deprotection

[0086]

[0087] (S)-1-(2-((tert-butyloxycarbonyl)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 a nitrogen atmosphere. N-(2-pyridyl)bis(trifluoromethanesulfonimide) (489.0 mg, 1.36 mmol, 2.0 equiv) was added to the solution and cooled to -40°C. Lithium tert-butoxide (1 M solution in THF, 1.71 mL, 1.71 mmol, 2.50 equiv) was then added to the solution in one portion. The reaction was stirred at -40°C until complete by HPLC (2.5 hours). The reaction was quenched with 10% citric acid in ethyl acetate (40 volumes) and then warmed to 0°C. 40 volumes of water were added to the reaction mixture, and the organic layer was collected and washed with 2M saline (10 volumes), dried over sodium sulfate, and concentrated in vacuo. The residue was dissolved in heptane (5 volumes) and filtered through celite. The filtrate was concentrated in vacuo and purified via column chromatography (0-10% ethyl acetate / heptane). The fractions containing the product were combined and concentrated in vacuo to obtain vinyl triflate (75-80%).

[0088] (S)-1-(2-((tert-Butyloxycarbonyl)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 (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 vols) under nitrogen. The suspension was heated to 45 °C and Pd(dppf)Cl2.DCM was added to the suspension. In a separate flask, a solution of PBD vinyl triflate (4.7 g, 5.5 mmol, 1.0 equiv) was prepared in degassed toluene (6.0 vols) under nitrogen. The vinyl triflate solution was added dropwise to the heated suspension. Once the addition of vinyl triflate is complete, methylboronic acid (1.3 g, 22 mmol, 4.0 equiv) is added in portions. The reaction is stirred at 45 °C until completion as indicated by HPLC (usually 1.5 hours). The mixture is then cooled to room temperature and poured into water (12 volumes). The organic phase is collected, concentrated in vacuo, and purified via column chromatography (0-15% ethyl acetate / heptane). The fractions containing the product are combined and separated to obtain the product (30-50% yield).

[0089] (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 volumes) was prepared. To the 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 hours. When complete by HPLC, the mixture was quenched with saturated ammonium chloride (10 volumes). The aqueous layer was extracted with 10 volumes of DCM and the organic layers were combined, washed with 2M brine (10 volumes), dried over magnesium sulfate, and concentrated in vacuo. The residue was dissolved in THF (10 volumes) and cooled to 0°C. TBAF (1M solution in THF, 0.157 mL, 0.16 mmol, 0.5 equivalent) was added to the solution and stirred for 30 minutes at 0°C. The solution was then diluted with 10 volumes of ethyl acetate and quenched with 10 volumes of water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 volumes). The organic layers were combined, dried over magnesium sulfate, and concentrated in vacuo. The product was purified by column chromatography (15% ethyl acetate / heptane). The fractions containing the product were combined and separated to obtain free aniline (aniline) (40-60% yield).

[0090] Example 3 Preparation of PBD benzoic acid:

[0091]

[0092] 2-((tert-Butyloxycarbonyl)amino)-4-hydroxy-5-methoxybenzoic acid (2): Benzoic acid 1 (1.0 eq) was dissolved in 15 volumes of MeOH. To the resulting mixture was added 10% palladium on carbon (0.03 eq, 10% w / w) and Boc2O (5 eq). The reaction vessel was evacuated and backfilled 3 times with nitrogen. The reaction vessel was equipped with a hydrogen-filled balloon, which was then evacuated and backfilled 3 times with hydrogen. The reaction was stirred overnight until complete conversion to 2 was observed by TLC. After completion of the reaction, the mixture was solvent exchanged with hexanes (10 volumes) and stirred at room temperature for 1 hour. The residue was then filtered through diatomaceous earth. The filtrate was concentrated in vacuo to give the final product as a light brown to pink solid in 86% yield. 1H NMR (400 MHz, DMSO-d6) δ 13.10 (br s, 1H), 10.49 (s, 1H), 10.15 (br s, 1H), 7.88 (s, 1H), 7.39 (s, 1H), 3.74 (s, 3H) 1.47 (s, 9H); HRMS: calculated value C 13 H 16 NO6-: 282.0983, measured value: 282.0984.

[0093]

[0136] 2-((tert-Butyloxycarbonyl)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. Tert-butyl(chloro)diphenylsilane (3.0 equiv.) was then 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 then added to the mixture. The aqueous layer was back extracted with ethyl acetate (5 vol.) and the organic phases were combined and washed with 2 M brine (5 vol.). The aqueous layer was back extracted with ethyl acetate (5 vol.) and the organic phases were combined and concentrated in vacuo. 15% MTBE / 85% heptane was then 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 to separate PBD-benzoic acid as a light brown to off-white solid (57% yield, 97% purity). The solid was then dissolved in MTBE (10 volumes) and CUNO 2 type activated carbon (10% w / w) was added to the solution. The mixture was stirred for 2 hours and then filtered through diatomaceous earth. The filtrate was concentrated in vacuo to provide the product as a white foam (88% recovery, 99.5% purity). 1 H NMR (400 MHz, DMSO-d6) δ 13.30 (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: calculated value C 29 H 34 NO6Si - :520.2161, measured value:520.2110.

[0094] Example 4. Preparation of PBD core:

[0095]

[0096] 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 volumes) was cooled to 0°C. To the 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 and then 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 hours. When the display is completed by HPLC, the mixture is filtered through a pad of diatomaceous earth. The filtrate is then washed once with water (10 volumes), and the aqueous layer is extracted with ethyl acetate (5 volumes). The organic layers are combined and washed twice (2×5 volumes) with 1M HCl, then washed with 1M sodium bicarbonate (5 volumes) and 2M saline (5 volumes). Solvent exchange is carried out to heptane (5 volumes) via isochoric distillation, and the resulting precipitation is removed by filtering through diatomaceous earth. The filtrate is concentrated in vacuo to provide PBD-INT, which is an off-white solid, which is used in the next step as a crude product (80-90% yield). 1 H NMR (400MHz, DMSO-d6) δ8.85 (s, 1H), 7.65 (ddd, J=7.86, 3.64, 1.47Hz, 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: calculated value C 40 H 59 N2O7Si2 + : 735.3855, measured value: 735.3864.

[0097] (S)-tert-butyl(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 volumes) was prepared and stirred for 20 minutes. Pyridine (3.59 mL, 44.4 mmol, 6.0 equiv) was added to the sulfur trioxide-pyridine solution and stirred for an additional 30 minutes. In a separate flask, a solution of crude PBD-INT (5.44 g, 7.40 mmol, 1.0 equiv) in 10 volumes of DCM was prepared and cooled to 0°C. To the cooled solution was added triethylamine (8.25 mL, 59.2 mmol, 8.0 equiv). Sulfur trioxide-pyridine solution is added to the solution of cooling PBD-INT at a rate keeping the temperature below 10 ℃. The reaction mixture is warmed to 20 ℃, and stirred for 3 hours. When the reaction is completed by HPLC, the mixture is cooled to 0 ℃, and quenched with 1M sodium bicarbonate (5 volumes). Collect organic phase, and extract aqueous phase with 5 volumes of DCM. Merge organic phase, and wash three times (3×5 volumes) with 1M salt water. Organic layer is concentrated in vacuo, and purified by column chromatography (12-22% ethyl acetate / heptane). Merge fractions containing product, and separate, to obtain PBD core A (>95% purity, 75% yield). 1 HNMR (400MHz, 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, 9.16Hz, 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 (400MHz, DMSO-d6) δ210.1, 168.6, 153.2, 146.6, 146.1, 135.4, 133.2, 1 30.27, 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 (net) 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: calc. C 40 H 57 N2O7Si2 + : 733.3699, measured value: 733.3700.

[0098] Example 5-8 Downstream Chemistry

[0099] Example 5. Preparation of PBD vinyl trifluoromethanesulfonate

[0100]

[0101] (S)-1-(2-((tert-Butyloxycarbonyl)amino)-4-((tert-butyldiphenylsilyl)oxy)-5-methoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate: A solution of the PBD core (1.0 equiv) in CPME or THF (40 vol) was prepared under a nitrogen atmosphere. N-(2-pyridyl)bis(trifluoromethanesulfonimide) (1.2 equiv) was added to the solution and cooled to -40 °C. Lithium tert-butoxide (1 M solution in THF, 1.71 mL, 1.71 mmol, 2.50 equiv) was then added to the solution in one portion. The reaction was stirred at -40 °C until complete by HPLC (2.5 h). The reaction was quenched with 10% citric acid in ethyl acetate (40 vol) and then warmed to 0 °C. 40 volumes of water were added to the reaction mixture, and the organic layer was collected and washed with 2M brine (10 volumes), dried over sodium sulfate, and concentrated in vacuo. The residue was dissolved in heptane (5 volumes) and filtered through celite. The filtrate was concentrated in vacuo and purified via column chromatography (0-10% ethyl acetate / heptane). The fractions containing the product were combined and concentrated in vacuo to give vinyl triflate (75-80%). HRMS: calculated value C 41 H 56 F3N2O9SSi2 +:865.3192, measured value:865.3157.

[0102] Example 6. Cross-coupling procedure

[0103]

[0104] Example 6A. Suzuki coupling

[0105] Under nitrogen, a suspension of potassium phosphate (5.0 equivalents) and cesium acetate (1.0 equivalents) was prepared in degassed toluene (6.0 volumes). The suspension was heated to 45°C and Pd(dppf)Cl2·DCM was added to the suspension. In a separate flask, a solution of PBD vinyl triflate (1.0 equivalents) in degassed toluene (6.0 volumes) was prepared under nitrogen. The vinyl triflate solution was added dropwise to the heated suspension. Once the addition of vinyl triflate was complete, methylboric acid (4.0 equivalents) was added in batches. The reaction was stirred at 45°C until completion (usually 1.5 hours) was shown by HPLC. The mixture was then cooled to room temperature and poured into water (12 volumes). The organic phase was collected, concentrated in vacuo, and purified via column chromatography (0-15% ethyl acetate / heptane). The fractions containing the product were combined and separated to obtain the product (30-50% yield).

[0106] Example 6B. Copper coupling

[0107] Prepare a suspension of copper (I) cyanide di(lithium chloride) complex (5.0 equiv) in THF or CPME (10 volumes) and cool to -40°C. Methylmagnesium bromide (3.0 M in diethyl ether, 10.0 equiv) is added dropwise to the suspension. The mixture is warmed to 0°C and stirred for 10 minutes, then cooled back to -40°C. A solution of PBD vinyl triflate (1.0 equiv) in THF or CPME (10 volumes) is added dropwise over 10 minutes. The reaction is stirred at -40°C until completion as shown by HPLC (usually 1 hour). Upon completion, the reaction is warmed to 0°C and slowly quenched with saturated ammonium chloride (20 volumes). The organic layer is collected and washed with saturated ammonium chloride (20 volumes), saturated sodium bicarbonate (20 volumes), and saturated brine (20 volumes). The organic layer is collected and solvent exchanged to toluene (20 volumes). SiliaMetSDOTA is then added to the 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 via column chromatography (0-7% ethyl acetate / heptane). The fractions containing the product were combined and concentrated in vacuo to give the product (40% yield).

[0108] (S)-tert-Butyl (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(400MHz, 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) δ 165.3, 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: calcd. 41 H 59 N2O6Si2 + :731.3906, measured value:731.3960.

[0109] Example 7. Formal Synthesis of Tesirine

[0110]

[0111]

[0136] (S)-tert-Butyl(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 the 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 as indicated by HPLC. The mixture was then 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) and then with saturated brine (5 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give free phenol as a clear, light brown oil. The intermediate was dissolved in THF (2 mL) and cooled to 0 ° C. Triethylamine (81.6 μL, 0.585 mmol, 2.0 equiv) was added to the solution, followed by TIPSCl (68.9 μL, 0.322 mmol, 2.0 equiv). The reaction mixture was allowed to reach room temperature and stirred for 15 hours. When the reaction was complete as shown by HPLC, the mixture was filtered to remove the solids and the solids were washed with THF (2 mL). The organic phases were combined and washed with saturated sodium bicarbonate (2 mL) and brine (2 mL). The organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (0-15% ethyl acetate / heptane), and the fractions containing the product were combined and separated to provide 9 (96.9 mg, 0.149 mmol, two-step yield 51%). HRMS: calculated value C 34 H 61 N2O6Si2 + : 649.4063, measured value: 649.4049.

[0112]

[0113] (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 volumes) was prepared. To the 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 hours. When complete by HPLC, 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 in vacuo. The residue was dissolved in THF (2 mL) and cooled to 0°C. TBAF (14.9 μL, 0.2 eq) was added to the solution and stirred at 0°C for 30 minutes. The solution was then diluted with 10 volumes of ethyl acetate and quenched with 10 volumes of water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 volumes). The organic layers were combined, dried over magnesium sulfate, and concentrated in vacuo. The product matched the reported NMR data. 1b,2 , and used immediately in the next step. HRMS: calculated value C 29 H 53 N2O4Si2 + : 549.3538, measured value: 549.3558.

[0114]

[0115]

[0136] (S)-Allyl(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate: A solution of the free amine 10 (41 mg, 0.075 mmol, 1.0 equiv) in DCM (12 vols) 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 hour). The reaction mixture was quenched with 10% citric acid solution (8 vols). The organic layer was collected and washed with saturated aqueous sodium bicarbonate solution (8 vols) and brine (8 vols). The organic phase was collected, dried over sodium sulfate, and filtered. The solution was concentrated in vacuo to give the crude product as an orange to brown oil. The product was purified via column chromatography (0-15% ethyl acetate / heptane), and the fractions containing the product were combined and separated to provide 11 (28 mg, 0.075 mmol, 59% yield over two steps). The isolated product matched the reported NMR data. 1b,2 HRMS: Calculated value C 33 H 57 N2O6Si2 + : 633.3750, measured value: 633.3773.

[0116] Example 8 Preparation of advanced intermediates from PBD core.

[0117] Example 8A. General Procedure for Boc Deprotection

[0118]

[0119] A solution of 8 (1.0 eq) in DCM (10 vols) was prepared. 2,6-lutidine (20 eq) and TBSOTf (10.0 eq) were added to the solution. The solution was then heated to 40 °C and stirred for 15 hours. When completed by HPLC, the mixture was quenched with saturated ammonium chloride (5 vols). The aqueous layer was back extracted with DCM (5 vols), and the organic layers were combined, washed with saturated brine (5 vols), and dried over magnesium sulfate. The solution was concentrated in vacuo. Carbamate 12 (HRMS: calculated value C 43 H 65 N2O6Si3 +: 789.4145, found: 789.4115) was dissolved in THF (10 volumes) and cooled to 0°C. TBAF (0.2 eq) was added to the solution and stirred at 0°C for 30 minutes. The solution was then diluted with 10 volumes of ethyl acetate and quenched with 10 volumes of water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 volumes). The organic layers were combined, dried over magnesium sulfate, and concentrated in vacuo to provide 13. The material was used immediately to avoid decomposition.

[0120] (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: calculated value C 29 H 53 N2O4Si2 + : 549.3538, measured value: 549.3558.

[0121] Example 8B

[0122]

[0123]

[0136] (S)-Allyl(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 vols) was prepared and cooled to 0 °C. To the solution was added pyridine (301 μL, 3.72 mmol, 2.2 equiv) 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 hour). The reaction mixture was quenched with 10% citric acid solution (8 vols). The organic layer was collected and washed with saturated aqueous sodium bicarbonate solution (8 vols) and brine (8 vols). The organic phase was collected, dried over sodium sulfate, and filtered. The solution was concentrated in vacuo to give the crude product as an orange to brown oil. The product was purified via column chromatography (0-15% ethyl acetate / heptane), and the fractions containing the product were combined and separated to provide 14. HRMS: Calculated C 40 H 55 N2O6Si2 + :715.3593, measured value:715.3606.

[0124] Example 8C.

[0125]

[0126]

[0136] (S)-Allyl (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 vols) and water (0.3 vol) was prepared. p-Toluenesulfonic acid hydrate (39.3 mg, 0.21 mmol, 0.6 equiv) was added to the solution and stirred at room temperature until the reaction was complete as shown by HPLC. The reaction mixture was diluted with ethyl acetate (10 vols) and washed with water (4 vols) / then brine (4 vols). The organic phase was collected, dried over sodium sulfate, and filtered. The solution was concentrated in vacuo to give the crude product as an orange to brown oil. The product was purified via column chromatography (0-15% ethyl acetate / heptane) and the fractions containing the product were combined and separated to provide 15. HRMS: Calculated value C 34 H 41 N2O6Si2 + :601.2728, measured value:601.2735.

[0127] Example 8D.

[0128]

[0129] (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)-allyl formate (16): A solution of oxalyl chloride (9.3 μL, 0.11 mmol, 1.03 equiv) in DCM (9 volumes) was prepared and cooled to -65°C. Dimethyl sulfoxide (18.3 μL, 0.26 mmol, 2.5 equiv) was added to the cooled solution. The reaction mixture was stirred for 30 minutes, then a solution of 15 (62.1 mg, 0.10 mmol, 1.0 equiv) in DCM (8 volumes) was added dropwise. After 30 minutes, triethylamine (72.0 μL, 0.52 mmol, 5.0 equiv) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred until complete as indicated by HPLC. The reaction was quenched with 5% citric acid (10 volumes) and washed with saturated aqueous sodium bicarbonate solution (10 volumes) and brine (10 volumes). The organic phase was collected, dried over sodium sulfate, and filtered. The solution was concentrated in vacuo to give the crude product 17, which was used in the next step without purification (87% yield). HRMS: Calculated C 34 H 39 N2O6Si +: 599.2572, measured value: 599.2549.

[0130] Example 8E.

[0131]

[0132] (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)-allylformate (17): To a solution of 16 (54.1 mg, 0.09 mmol, 1.0 equiv) in DCM (10 volumes) was added 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 trifluoromethanesulfonate (119 μL, 0.52 mmol, 5.0 equiv). The reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for 3 hours. Once the reaction was complete as shown by HPLC, it was quenched with saturated aqueous sodium bicarbonate (10 volumes) and brine (10 volumes). The organic phase was collected, dried over sodium sulfate, and filtered. The solution was concentrated in vacuo to give the crude product 17, which was used in the next step without purification. HRMS: calculated value C 40 H 53 N2O6Si2 + : 713.3437, measured value: 713.3429.

[0133] Example 8F.

[0134]

[0135] (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)-Allyl formate (18): To a solution of 17 (64.2 mg, 0.09 mmol, 1.0 equiv) in wet DMF (15 vols) 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 vols) and washed with water (10 vols) and saturated brine (10 vols). The organic phase was collected, dried over sodium sulfate, and filtered. The solution was concentrated in vacuo to give the crude product 18, which was consistent with the literature. 2HRMS: Calculated value C 24 H 35 N2O6Si + : 475.2259, measured value: 475.2284.

[0136] Example 8G.

[0137]

[0138]

[00136] 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 the HPLC showed completion, the mixture was quenched with saturated ammonium chloride (5 volumes). The aqueous layer was back-extracted with DCM (5 volumes), and the organic layers were combined, washed with saturated brine (5 volumes), and dried over magnesium sulfate. The solution was concentrated in vacuo. The carbamate was dissolved in THF (10 volumes) and cooled to 0 ° C. TBAF (0.16 mL, 1 M, 0.16 mmol, 0.4 equivalent) was added to the solution and stirred for 30 minutes at 0 ° C. The solution was then diluted with 10 volumes of ethyl acetate and quenched with 10 volumes of water. The layers were separated and the aqueous layer was extracted with ethyl acetate (10 volumes). The organic layers were combined, dried over magnesium sulfate, and concentrated in vacuo to provide 14. The material was used immediately to avoid decomposition. 10 volumes of DCM were added to 13 and cooled to -20 ° C. Triphosgene (46.9 mg, 0.16 mmol, 0.40 equivalent) was slowly added to the reaction mixture. Triethylamine (121 μL, 0.87 mmol, 2.2 equiv) was then added to the solution and stirred at -20°C for 30 minutes. At the same time, a mixture of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (156.6 mg, 0.41 mmol, 1.05 equiv) in a solution of triethylamine (83 μL, 0.60 mmol, 1.5 equiv) in DCM (10 volumes) was prepared. The solution was quickly added to the reaction at -20°C. The reaction was stirred for 15 hours. When the reaction was complete as shown by HPLC, the reaction mixture was washed with water (4 volumes). The organic phase was collected, dried over sodium sulfate, and concentrated in vacuo. The resulting mixture was purified via flash chromatography to provide the product as a white solid (321.2 mg, 79% yield in three steps). HRMS: Calculated value C 56 H 76 N5O 10 Si2 + :1034.5125, measured value:1034.5094.

[0139] Example 8H

[0140]

[0141]

[00136] 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. p-Toluenesulfonic acid hydrate (35.4 mg, 0.18 mmol, 0.6 equiv) was added to the solution for 2 h. When the reaction was complete as shown by HPLC, the solution was diluted with ethyl acetate (10 volumes) and washed with water (5 volumes), saturated sodium bicarbonate (5 volumes) and saturated brine (5 volumes). The organic phase was collected, dried over sodium sulfate, and concentrated in vacuo. The product was purified via column chromatography (heptane / ethyl acetate 40 / 60 to 0 / 100) to give the product as a light yellow solid (83% yield). HRMS: calculated value C 50 H 62 N5O 10 Si + :920.4260, measured value:920.4264.

[0142] Example 8I.

[0143]

[0144] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutyramido)propionamido)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)-formate (21). A solution of oxalyl chloride (2.03M, 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 minutes, at which time a solution of 20 (236.2 mg, 0.26 mmol, 1.0 equiv) in DCM (5 volumes) was added dropwise. The reaction was stirred at -65 °C for 15 minutes, and then triethylamine (179 μL, 1.28 mmol, 5.0 equiv) was added dropwise. The reaction was stirred at -65 °C for 1 hour. Once the reaction was complete as shown by HPLC, the reaction mixture was warmed to 0 °C and washed with 0.2N HCl (10 volumes), saturated sodium bicarbonate (10 volumes) and brine (10 volumes). The organic phase was collected and concentrated in vacuo. DCM (10 vol) was then added to the residue and the resulting solution was concentrated again in vacuo. This procedure was repeated twice to afford product 21 in 86% yield. The crude product was used immediately in the next step. HRMS: Calculated C 50 H 60 N5O 10 Si + :918.4104, measured value:918.4091.

[0145] Example 8J.

[0146]

[0147] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutyramido)propionamido)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)-formate (22). A solution of 21 (203 mg, 0.22 mmol, 1.0 equiv) in DCM (20 volumes) 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 trifluoromethanesulfonate (295 μL, 1.28 mmol, 5.0 equiv) was added dropwise. The reaction was stirred for 30 minutes under a solution of oxalyl chloride (2.03 M, 152 μL, 0.31 mmol, 1.2 equiv) in DCM, 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 minutes and then at -20 °C for 20 hours. When the reaction was complete as shown by HPLC, it was quenched with 10% citric acid (10 volumes) and washed with saturated sodium bicarbonate (10 volumes) and brine (10 volumes). The organic phase was collected and concentrated in vacuo. DCM (10 volumes) was then added to the residue and the resulting solution was concentrated in vacuo again. This procedure was repeated twice to give the product 22. The crude product was used immediately in the next step. HRMS: calculated value C 56 H 74 N5O 10 Si2 + : 1032.4969, measured value: 1032.4922.

[0148] Embodiment 8K

[0149]

[0150]

[0151] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutyramido)propionamido)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)-formate (23). A solution of crude 22 (265 mg, 0.26 mmol, 1.0 eq) in DMF (10 volumes) and water (0.3 eq) was prepared. Lithium acetate (16.9 mg, 0.26 mmol, 1.0 eq) was added to the solution. The mixture was heated to 40 °C and stirred for 8 hours. Once the reaction was complete as shown by HPLC, the mixture was cooled to room temperature and diluted with Me-THF (10 volumes). The mixture was washed with 10% citric acid (10 volumes), 5% sodium bicarbonate (10 volumes), saturated brine (10 volumes) and water (10 volumes). The organic phase was collected and concentrated in vacuo. 2-MeTHF (10 volumes) was then added to the residue and the resulting solution was concentrated in vacuo again. The procedure was repeated twice to obtain the crude product. The crude product was purified via column chromatography (heptane / ethyl acetate 40 / 60 to 0 / 100) to give the product 23 (80.2 mg, 40% yield over two steps). The product matched the reported NMR data 1b,2 HRMS: Calculated value C 40 H 55 N5O 10 Si + :794.3791, measured value:794.3778.

[0152] Example 8L

[0153]

[0154] (S)-tert-Butyl (2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((tert-butyldiphenylsilyl)oxy)-4-methoxyphenyl)carbamate (24). Preparation of methyltriphenylphosphine bromide (2.92g, 8.17mmol, 2.0 equivalents) in THF (38.5 volumes) and cooled to 0°C. Potassium tert-butoxide (1.0M, 8.18mL, 8.18mmol, 2.0 equivalents) was added dropwise to the solution. The reaction mixture was stirred at 0°C for 15 minutes and then at room temperature for 45 minutes. The reaction mixture was then cooled to 0°C and PBD core A (3.0g, 4.09mmol, 1.0 equivalents) in THF (1.5 volumes) was added dropwise. After the addition of PBD core A was completed, the reaction mixture was warmed to room temperature and stirred until the reaction was complete as shown by HPLC. The reaction was quenched with water (15 volumes). The water layer was collected and washed twice with MTBE (15 volumes). The organic phase was combined and washed with water (15 volumes) and brine (15 volumes), dried over sodium sulfate, and then concentrated in vacuo. The product was purified by column chromatography (heptane / ethyl acetate 85 / 15). The fractions containing the product were isolated to give the product 24 (2.00 g, 2.7 mmol) in 64% yield. HRMS: Calculated value C 41 H 59 N2O6Si2 + :731.3906, measured value:731.3786.

[0155] Embodiment 8M

[0156]

[0157] (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 vols) was prepared. To the solution was added 2,6-lutidine (0.19 mL, 1.6 mmol, 20.0 equiv) and cooled to 0 °C. To the reaction mixture was then added tert-butyldimethylsilyl trifluoromethanesulfonate (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 vols). The organic layer was collected and washed with saturated brine (5 vols). The crude product was isolated (90% yield) and stored at -20°C. HRMS: Calculated C 36 H 51 N2O4Si2 + : 631.3382, measured value: 631.3354.

[0158] Example 8N

[0159]

[0160] (S)-tert-Butyl(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 slowly warm to room temperature and stirred for 2 hours. The reaction was shown to be complete by HPLC. Half of the material was set aside for stability studies. The remaining half was isolated and subjected to Swern conditions (see preparation of 27). HRMS: Calcd. C 35 H 45 N2O6Si + : 617.3041, measured value: 617.3065.

[0161] Embodiment 80

[0162]

[0163] (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)-tert-Butyl formate (27): To a solution of 26 (0.65 g, 1.1 mmol, 1.0 equiv) in THF (10 volumes) was added 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 minutes. Triethylamine (0.73 mL, 5.3 mmol, 5.0 equiv) was then slowly added to the reaction mixture. The reaction was then quenched with 5% aqueous citric acid and stirred at 5°C for 15 minutes. The organic phase was collected and washed with saturated sodium bicarbonate and water and then dried over magnesium sulfate. The intermediate was isolated and DCM (10 volumes) 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 slowly warmed to room temperature over 30 minutes. The reaction was stirred at room temperature for 15 hours. When the reaction was complete as shown by HPLC, the reaction mixture was diluted with DCM (45 volumes) and quenched with saturated sodium bicarbonate. The organic layer was collected, washed with brine, and dried over sodium sulfate. The crude product was separated in vacuo and purified by column chromatography (heptane / ethyl acetate 75 / 25). The fractions containing the product were combined to give 27 with a three-step yield of 54%. HRMS: calculated value C 41 H 57 N2O6Si2 + :729.3750, measured value:729.3790.

[0164] Example 8P

[0165]

[0166] (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)-tert-butyl formate (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 hours. Once the reaction was complete as indicated by HPLC, 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 phase was collected, dried over sodium sulfate, and concentrated in vacuo. The crude product was purified via column chromatography (heptane / ethyl acetate 30 / 70) to give product 28 (80.2 mg, 40% yield over two steps). The product matched the reported NMR data. 1b,2 HRMS: Calculated value C 25 H 39 N2O6Si + : 491.2572, measured value: 491.2520.

[0167] The examples herein are for illustrative purposes only and are not intended to limit the scope of the invention which is defined by the claims.

[0168] References:

[0169] (a) Gregson, SJ; Howard, PW; Gullick, DR; Hamaguchi, A.; Corcoran, KE; Brooks, NA; Hartley, JA; Jenkins, TC; Bulow, C.; Barry, C.; Ge, H.; Noti, C.; Leiris, FC; McCormick, M.; Howard, PW; Parker, JSOrg.ProcessRes.Dev.2018, 22(9), 1241-1256.

[0170] (a)Tiberghien,AC;Levy,J.-N.;Masterson,LA;Patel,NV;Adams,LR;Corbett,S.;Williams,DG;Hartley,JA;Howard,PWACS Med.Chem.Lett.2016,7(11),983-987;(b)Tiberghien,AC;Howard,PW;Goundry,WRF;McCormick,M.;Parkers,JSJOrg.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.;Ouyan g,L.;Gou,L.;Chen,H.;Wang,Y.;Yang,JJMed.Chem.2022,65(17),11679-11702.

Claims

1. Compounds of Formula I in Dashed lines represent single or double bonds. R1 is a protecting group selected from tert-butyldimethylsilyl ether (TBS) and acetate (Ac), R2 is selected from -H, =O, -OTf, C1-C5 alkyl, C3-C6 cycloalkyl, C1-C5 alkenyl, C5-C 10 Aryl, C5-C 10 heteroaryl and a linker, wherein R2 may be optionally substituted by one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxyl, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene and a linker, R3 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 aryl, and R may optionally have a substituent selected from C1-C4 alkyl and phenyl, R4 is selected from -H, -R, -OH, -OR, -SH, -SR, -NH2, -NHR, -NHRR', nitro and halogen, wherein R and R' are independently selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 aryl, and R and R' may optionally have a substituent selected from C1-C4 alkyl and phenyl, R5 is a protecting group selected from tert-butyldiphenylsilyl ether (TBDPS) and triisopropylsilyl ether (TIPS), and R6 is selected from -H, -OH, -OR, -SH, -NH2, -NO2, halogen, wherein R is selected from C1-C4 alkyl, C3-C 10 Heterocyclic and C5-C 10 aryl group, and R may optionally have a substituent selected from C1-C4 alkyl group, phenyl group.

2. The compound according to claim 1, wherein R3 and R6 are each -H.

3. The compound according to claim 1 or 2, wherein R2 is selected from -H, =O, -OTf, -OH, methyl, cyclopropyl, =CH2, vinyl, propenyl, phenyl, 4-fluorophenyl, indole and a linker, and R2 may be optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxyl, ester, C1-C7 alkyl, C1-C7 alkenyl, C3-C7 heterocyclyl, bis-oxy-C1-C3 alkylene and a linker, and R4 is selected from -H, -OCH3, -OCH2Ph, -N(CH3)2, morpholino, piperidinyl and -N-Me-piperazinyl.

4. The compound according to any one of claims 1 to 3, wherein R2 is =0.

5. The compound according to any one of claims 1 to 3, wherein R2 is -OTf.

6. The compound according to any one of claims 1 to 5, wherein R4 is OCH3.

7. The compound according to claim 1 or 4, selected from 8. The compound according to claim 1 or 5, selected from 9. The compound according to claim 1, which is selected from 10. A compound selected from 11. Compounds of formula II: wherein R7 is selected from -H, -C(O)O-TBS, Alloc and a linker, and R8 is selected from -H and TBS.

12. The compound according to claim 11, selected from 13. Compounds of formula III: wherein R9 is selected from Alloc and a linker, and R8 is selected from -H and TBS.

14. The compound according to claim 13, which is selected from:

15. Used in the preparation of advanced pyrrolobenzodiazepines (PBD) intermediate method, which comprises the following steps Provided is a compound according to any one of claims 4, 6 or 7, performing a C—C bond forming reaction at the ketone, and Optionally, the amine is deprotected.

16. A method for preparing an advanced PBD intermediate, comprising the following steps: Provided is a compound according to any one of claims 4, 6 or 7, converting the ketone into a triflate, performing a CC bond forming reaction on the triflate, and Optionally, the amine is deprotected.

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

18. A method for preparing an advanced PBD intermediate, comprising the following steps: Provided is a compound according to any one of claims 5, 6 or 8, performing a C—C bond forming reaction on the triflate, and Optionally, the amine is deprotected.

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

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

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