Discovery of 1,4-Benzodiazepine-2,5-dione (BZD) Derivatives as Dual

Article pubs.acs.org/jmc

Discovery of 1,4-Benzodiazepine-2,5-dione (BZD) Derivatives as Dual Nucleotide Binding Oligomerization Domain Containing 1/2 (NOD1/NOD2) Antagonists Sensitizing Paclitaxel (PTX) To Suppress Lewis Lung Carcinoma (LLC) Growth in Vivo Suhua Wang,†,§ Jingshu Yang,‡,§ Xueyuan Li,‡ Zijie Liu,† Youzhen Wu,‡ Guangxu Si,‡ Yiran Tao,‡ Nan Zhao,† Xiao Hu,† Yao Ma,*,†,‡ and Gang Liu*,‡ †

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, P. R. China ‡ School of Pharmaceutical Sciences, Tsinghua University, Haidian District, Beijing 100084, P. R. China S Supporting Information *

ABSTRACT: Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular sensors of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Previously, we reported nucleotide-binding oligomerization domain-containing protein 1 (NOD1) antagonists (11, 12) and a NOD2 antagonist (9) that sensitized docetaxel (DTX) or paclitaxel (PTX) treatment for breast or lung cancer. In this article, we describe for the first time a 1,4-benzodiazepine-2,5-dione (BZD) derivative (26bh) that acts as a dual NOD1/NOD2 antagonist and inhibits both nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) inflammatory signaling, thereby sensitizing PTX to suppress Lewis lung carcinoma (LLC) growth. After investigation of the compound’s cytotoxicity, a systematic structure−activity relationship (SAR) was completed and revealed several key factors that were necessary to maintain antagonistic ability. This study establishes the possibility for using adjuvant treatment to combat cancer by antagonizing both NOD1 and NOD2 signaling.

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INTRODUCTION Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular sensors of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).1 Nucleotide-binding oligomerization domain-containing proteins 1 and 2 (NOD1 and NOD2, respectively), which are two important NLRs, are cytoplasmic receptors with a characteristic tripartite domain architecture comprising (i) a C-terminal sensor domain consisting of leucine-rich repeats (LRRs), which mediate ligand recognition; (ii) a central nucleotide-binding oligomerization domain (NACHT), which can bind adenosine triphosphate (ATP), self-oligomerize, and be activated; and (iii) an N-terminal effector domain containing one or two caspase-recruitment repeats for signal transduction. Upon recognition of their ligands, both NOD1 and NOD2 self-oligomerize, undergo a conformational change, and recruit receptor-interacting protein 2 (RIP2) to form a multiprotein signaling complex termed the “NODosome”, which leads to the activation of the transcription factor nuclear factor κB (NF-κB) and mitogen-associated protein kinase (MAPK) pathways.2 NOD1 recognizes peptidoglycan motifs from the bacterial cell wall, specifically, L-Ala-γ- D -Glu-meso-diaminopimelic acid (L-Ala-γ-D-Glu-meso-DAP), which is mostly found in © 2017 American Chemical Society

Gram-negative bacteria. By contrast, NOD2 selectively detects N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP, 1), which is the smallest glycopeptide of the bacterial glycans recognized by the human immune system and is present in both Gram-positive and Gram-negative bacteria.3 Studies have indicated that 1 can nonspecifically modulate human macrophages, bone marrow mononuclear cells, neutrophils, and T and B lymphocytes. Thus, as an adjuvant, it can inhibit bacterial infections (e.g., Bacillus pneumonia, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans) and has anticancer activity.4 However, the use of 1 in clinical applications has been limited by its severe side effects and nondruggable profile, including its pyrogenicity, poor penetration of cell membranes, and rapid elimination.5 Structural modifications of 1 and its analogues have been extensively conducted, and its structure−activity relationships (SARs) have been comprehensively summarized.6 Several compounds have successfully moved into clinical trials (Figure 1). For instance, murabutide (2) was investigated in a phase I trial as an anti-HIV drug candidate.7 Another drug candidate, Received: April 21, 2017 Published: May 25, 2017 5162

DOI: 10.1021/acs.jmedchem.7b00608 J. Med. Chem. 2017, 60, 5162−5192

Journal of Medicinal Chemistry

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Figure 1. Structures of MDP and its analogues.

Figure 2. Structures of PTX-MDP conjugates.

romurtide (3), was also evaluated in six lung cancer patients with cytologically positive malignant pleural effusion. Treatment with 3 increased the levels of chemotactic cytokines, including interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and proinflammatory cytokines (tumor necrosis factor α (TNF-α), IL-1β, and IL-6).8a,b It was documented that administration of 3 to 89 patients with leukopenia caused a marked increase of neutrophils and thus reached restorative 3000 WBC/mm3 that benefit patients in reduction of the risk of infectious diseases.8c Mifamurtide (4), a liposome of muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE), was approved for the treatment of osteosarcoma in combination with other chemotherapeutics.9

Free L-MTP-PE was demonstrated to activate monocytes or macrophages and increase the production of proinflammatory cytokines, including TNF-α, IL-1, IL-6, and IL-8. It was speculated that L-MTP-PE activated NOD2 signaling as an MDP prodrug.10 Our lab identified MDP-C (5), which effectively stimulated murine bone-marrow-derived dendritic cells (BMDCs) to produce IL-2 and IL-12, and cytotoxic T lymphocytes (CTLs) to produce interferon-γ.5 Compound 5 remarkably enhanced the immune system’s responsiveness to hepatitis B surface antigen (HBsAg) in hepatitis B virus transgenic mice in terms of both antibody production and specific HBsAg T-cell responses ex vivo. Furthermore, it was discovered that the muramic acid moiety of MDP could be 5163



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Figure 3. MDP focused libraries.

Figure 4. Hit compounds from library 16, which is specified as 26 scaffold in further.

noncleavable MTC-220 (Figure 2, 10) as an antagonist of NOD2 signaling that enhanced the antitumor and antimetastatic efficacy of PTX in LLC-tumor-bearing mice, very likely via blocking DAMPs derived from chemotherapy.15 Conjugates (Figure 2, 11 and 12) of docetaxel (DTX) and MDP derivative were also developed for clinical investigations in a recent study.16 Both 11 and 12 demonstrated antagonism of NOD1 signaling and were superior to DTX in breast cancer treatment. These observations revealed that both NOD1 and NOD2 are required for tumor survival and invasion. Furthermore, effective inhibitors for cancer treatment should antagonize both NOD1 and NOD2 signaling. Herein, we found that small-molecule heterocyclic 1,4-benzodiazepine-2,5-diones (BZDs) (Figure 3, 26) could be used as a new class MDP mimics and can block both NOD1 and NOD2 signaling and significantly sensitize PTX treatment to reduce the tumor size in LLC-tumor-bearing mice. This article also reports the first alternative strategy in which the core structure of 1 is replaced with heterocyclic molecules.

replaced by various aromatic groups, which led to compounds that exhibited improved immunological activity.11 All the above compounds could successfully stimulate target cells (e.g., macrophage and dendritic cells) and thus induce tremendous amounts of inflammatory cytokines. In addition to these compounds, we previously proposed conjugates of paclitaxel (PTX) and MDP analogues as part of our efforts to discover superior compounds that not only inhibit tumor growth but also prevent tumor metastasis. When conjugating PTX (at the 2′-O, 3′-N, or 7-O positions) and an MDP analogue featuring a muramic acid moiety (Figure 2, 2′-O-MTC-01 (6), 3′-N-MTC01 (7), 7-O-MTC-01 (8)), only compound 6 synergistically induced TNF-α and IL-12 production in murine peritoneal macrophages; however, it did not inhibit tumor metastasis in Lewis lung carcinoma (LLC) bearing mice, although 6 displayed an ability inhibiting tumor growth in mice.12 Interestingly, when the muramic acid moiety was replaced by a cinnamic acid derivative, the resulting MTC-220 (9) clearly displayed a dual function as an inhibitor of both tumor growth and metastasis in LLC- and 4T1-tumor-bearing mice.13 Compound 9 is currently under an investigational new drug (IND) application.14 The study indicated that treatment with 9 sufficiently suppressed myeloid-derived suppressor cells (MDSCs) accumulation in the spleen and bone marrow of a tumorbearing host and decreased the mRNA levels of several inflammatory cytokines, including TNF-α in the tumor tissue, compared to PTX treatment alone.13 Recently, we reported

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RESULTS AND DISCUSSION Cell-Based Approach for Identifying Dual NOD1/ NOD2 Antagonists. Screening for dual inhibitors of the NOD1/2 signaling pathways was conducted using the HEKBlue hNOD1-secreted alkaline phosphatase (SEAP) reporter and HEK-Blue hNOD2 SEAP reporter cells activated by lauroyl-γ-D-Glu-mDAP (C12-iE-DAP) and MDP, respectively. 5164



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Table 1. SAR Investigation of D-Glu BZD Analogues with R3 Modifications in C12-iE-DAP (50 ng/mL) Stimulated HEK-Blue hNOD1 Cells and MDP (100 ng/mL) Stimulated HEK-Blue hNOD2 Cellsa

a

ND: not determined.

Hit compounds were then profiled in dose−response assays and counterscreened to eliminate cytotoxic compounds by the sulforhodamine B assay, as previously described.17 Subsequently, direct inhibitors of RIP2 kinase activity could be eliminated by an established luminescent kinase assay measuring RIP2 kinase activity by quantifying the amount of adenosine diphosphate (ADP) produced during a kinase reaction.18 Several focused libraries (approximately 3000 compounds in total) were screened. These libraries, in which the D-Glu pharmacophore has been integrated (Figure 3, 14−17), were previously synthesized in our lab.19

The initial concentration of the compounds identified during the primary screening was set at 10 μM. Compounds from the linear L-Ala-isoGln library (Figure 3, 14) exhibited weakly antagonistic activity against both NOD1 and NOD2 activation by C12-iE-DAP or MDP stimulation, respectively, under the tested conditions (Table S1 in Supporting Information). However, compounds with substitutents from the BZD library 16, but not 15, displayed relatively higher inhibition, e.g., 26e, 26h, and 26j (Figure 4, Table S2) that were further specified as 26 scaffold in Figure 3. We were then interested in compounds of scaffold 26 with 7-position and 8-position subsitituents because this structure not only does allow diversified substitution 5165



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a

ND: not determined.

for further optimization but also was a privileged structure with good druggability.

After considering compounds 26e, 26j, and 26h, 26h was chosen for further studies because of its relatively high inhibition 5166



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Table 3. continued

a

ND: not determined.

32 and 39 in Figure 3 (Tables 5 and 6) were used to investigate the effects of C-7 and C-8 substitutions. The synthetic route toward the BZDs is illustrated in Scheme 1. Nitration of 3-chlorobenzoic acid (18) was first achieved in the presence of KNO3 and conc H2SO4 to provide intermediate 19. After a nucleophilic aromatic substitution reaction with phenols, alcohols, or aliphatic amines, the resulting intermediate (20) was further reacted with the corresponding amino acid hydrochloride to afford amide product 21. Intermediates 23a−23c were acquired in a tandem reduction− cyclization reaction from 21 via intermediate 22. Intermediates 23a and 23b were transformed to 26dl and 26dm by reaction with the corresponding acyl chloride. Intermediate 23c was transformed to 24 using the same method. Next, 24 was hydrolyzed with LiOH to provide 25. Intermediate 25 was amidated with various amines to produce the targeted compounds (26) in good yield after purification by chromatography on silica gel. The syntheses of C-7 (Table 5) and C-8 (Table 6) chlorinated compounds are illustrated in Schemes 2 and 3, respectively. For C-7 chlorinated compounds, intermediate 27 was used as the starting material and reacted with dimethyl D-glutamate hydrochloride to give amide product 28, which was further reduced with Fe powder and subsequently converted into cyclized compound 29 in AcOH at 90 °C. Next, R6-Br was reacted with

and comparatively low cell toxicity (data not shown). Additional diversified compounds based on 26h were prepared by introducing different substituents at R3, R4, and R5 of 26 (Figure 3). These compounds included 26aa−ag, which were prepared by introducing chlorine atoms on the C-7 m-dimethylamine phenol group to give a variety of R3 substituents (Table 1); 26ba−26bp, which were prepared with various R4 groups by the formation of different amides (Table 2); and 26ca−26cr (R5), which were prepared to determine the contributions of electron-withdrawing and electron-rich groups and halogen substituents (Table 3). It should be noted that 26cs was designed to replace oxygen with a carbon atom (Table 3). 26ct and 26cu were designed for nonaromatic substitutions (Table 3). Table 4 summarizes the other synthesized compounds (26da−26dhh) with multiple simultaneous substitutions. To further evaluate the effects of different configurations and species of amino acid, 26da, 26db, 26dd, 26dl, and 26dm were designed (Table 4). On the basis of the collected inhibitory activities of the above compounds, different substituent groups associated with relatively high inhibition were combined to afford 26de, 26df, 26dh, 26di, 26dj, and 26dk. Compounds 26dc and 26dg were designed to explore the influence of the dimethylamino moiety in the R3 group. 26dn−26dz, 26daa−26dhh were designed aiming at further diversifying substituents at R3, R4, and R5. Scaffolds 5168



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Table 4. SAR Investigation of D-Glu BZD Analogues with Various Modifications in C12-iE-DAP (50 ng/mL) Stimulated HEK-Blue hNOD1 Cells and MDP (100 ng/mL) Stimulated HEK-Blue hNOD2 Cellsa

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Table 4. continued

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Table 4. continued

a

ND: not determined.

1-NH of 29 selectively in the presence of Cs2CO3 to give 30. Intermediate 30 was transformed into 31, which was subsequently reacted with various amines to yield the final product (32). Similarly, as shown in Scheme 3, intermediate 33 was amidated with dimethyl D-glutamate hydrochloride to give amide product 34. After methylation with MeI, intermediate 35 was reduced by Fe powder and subsequently converted into cyclized compound 36 in AcOH at 90 °C. Similar procedures were implemented in the next three steps to yield the final product (39). Antagonistic NOD1/2 Activity of Synthesized Compounds and Their SARs. After primary screening (Tables S1 and S2), 93 BZD compounds (Tables 1−6) were successfully prepared and screened. Table 1 presents the results of the hit 26h analogues with a variety of chlorine substituents in the R3 group at the C-7 position of BZD. The 50% inhibitory concentration (IC50) values of compounds 26ab and 26af were measured in this study, but those of other compounds were not because they exhibited relatively low antagonistic ability. Indeed, in general, the IC50 value of any compound displaying an inhibitory percentage less than 50% at the primary tested concentration was not determined. When either the N,N-dimethyl group was removed (26dc) or the C-7 m-dimethylamine phenol

group was replaced with a naphthol group (26dt, 26dy− 26dbb) or 3,4-dimethyl phenol group (26dv−26dx), no improved activity was observed (Table 4). However, when the N,N-dimethyl group of 26h at the R3 position was replaced with a cyclopentylamine (26dg, Table 4), higher NOD1/2 inhibitory activities were achieved, with IC50 of 1.39 μM (NOD1) and 1.50 μM (NOD2), respectively. Interestingly, the presence of a free carboxyl acid group (26bp, Table 2) at R4 of 26h led to a complete loss of the compound’s antagonistic ability for both NOD1 and NOD2 signaling, indicating that the acidity of the D-Glu δ-carboxyl acid group of the BZD derivatives exerted a negative influence.20 Various amidation and esterification reactions of the δ-carboxyl group were investigated (Table 2). With the exception of 26bd−26bf with long aliphatic chain substitutions, 26bl−26bm with 1-methylpiperazine and piperazine amides, and 26bo with an NH2 group, which all showed lower activity, the designed compounds (26ba−26bc, 26bg−26bk, and 26bn) exhibited the anticipated blocking effects on NOD1/2 activation in vitro. R5 was defined as the α-benzyl ether acetyl group at the C-8 NH-position of BZD. Upon removal of the O atom from the R5 chain (26cs, Table 3), the activity disappeared, implying that a hydrogen bond might involve this O atom as the acceptor. 5171



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When the benzene ring in R5 was replaced with an alkenyl group (26ct) or dodecylamine (26cu), lower activity or the complete loss of activity was observed. Thus, little room is available for saturated and unsaturated aliphatic chain substitutions. The ortho-, meta-, and para-fluorine, bromine, and chloride phenyl groups of R5 were introduced into compounds 26ca−26ch and 26cl, 26cm, and 26cp, respectively. All m-Cl substitutions (26ca, 26cd, 26cl, and 26cm) decreased the antagonistic ability of the BZD compounds, whereas all other halogen substitutions of the R5 group (26cb−26cc, 26ce− 26ch, 26cp) increased the activity of the compounds toward blocking C12-iE-DAP and MDP stimulation. For example, 26cp bearing a 4-chlorophenyl substituent showed the greatest potency with IC50 values of 0.61 μM for NOD1 and 1.24 μM for NOD2. The improved NOD1/2 inhibitory activities of compounds with halogen substituents could be ascribed to their better liposolubility and improved hydrophobic interactions with the target. Electrostatic interaction may also play an important role in improving these compounds’ potency. In this study, derivatives of 26h were prepared by introducing phenyl groups with p-trifluoromethyl (26ci), p-methoxyl (26cj), and p-cyano (26ck) substituents and naphthyl (26cr) and p-t-Buphenyl groups (26cq) at R5. Compounds with para-electronwithdrawing groups, especially 26ci and 26ck, exhibited significantly increased inhibitory activities. Further conversion of the phenyl ring at R5 into furan and thiophene rings resulted in 26cn and 26co, which had decreased activities. To explore the role of the configuration of the key amino acid (D-Glu at the C3-position), L-Glu, L-Ala, and Gly were integrated into BZD scaffold 26. The Gly-incorporated compound (26dl) clearly lost its activity; however, replacement with L-Ala (26dm) maintained the function of BZD in blocking NOD1/2 signaling. Surprisingly, changing D-Glu to L-Glu (26da, 26db, and 26dd) did not affect the ability of the compounds to antagonize NOD1/2 activation relative to the corresponding compounds, 26bh (Table 2) and 26cd and 26ci (Table 3), respectively. Thus, the C3-position of BZD must be substituted, and maintaining a chiral isomer is not necessary. Among the compounds 26de, 26df, 26dh, 26di, 26dj, 26dk, 26dn, and 26do shown in Table 4, 4-fluoropiperidyl (26dn) and 4,4-difluoropiperidyl (26do) substitutions at R4 resulted in the highest inhibitory activities; unfortunately, both of these compounds also exhibited cytotoxicity toward HEK-Blue cell lines at tested concentration (Supporting Information Table S3). All other compounds in Table 4 (26dp−26dhh) were designed to mimic 26j (Figure 4) and displayed no interesting results. Thus, the IC50 was not measured for any of these compounds. Tables 5 and 6 show the synthesized 32 and 39 compounds (Figure 3) and their potencies in vitro. Clearly, compounds without substitutions at the 7- or 8-position displayed no activity, whereas 1-NH or 4-NH substitution did not affect either the antagonizing or the agonizing potency. Therefore, substitutions at C-7 and C-8 played a key role in determining the resulting compounds’ abilities to inhibit NOD1/2 signaling. In summary, in this study, a number of BZD compounds were designed and prepared and further assayed as NOD1/2 dual antagonists. Systematic SAR analyses (Figure 5) revealed that the specific configuration of D-Glu at the C-3 position of BZD was not necessary; however, activity was lost when the side chain of D-Glu was completely removed. The hydrogen atoms at 1-NH and 4-NH of BZD also did not contribute to the activity because their substitution did not affect the corresponding compound’s activity. R3 at C-7, R5 at C-8, and

Table 5. SAR Investigation of (R)-3-(7-Chloro-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propionyl Derivatives in C12-iE-DAP (50 ng/mL) Stimulated HEK-Blue hNOD1 Cells and MDP (100 ng/mL) Stimulated HEK-Blue hNOD2 Cells

R4 at the Glu C-terminal of BZD (C-3) played key roles in mediating NOD1/2 dual antagonistic activity. The small SAR of the R3 group led to the conclusion that m-N,N-dimethylamine phenol ether linkage was required. Modification at R3 did not improve the potency of the compounds in vitro. N,N-Dimethyl substitutions of amines have generally been considered metabolis soft spots. Further strategies to overcome this barrier for in vivo investigations should be developed. A free carboxyl acid group (26bp) at Glu of 26h appeared to result in the complete loss of antagonistic activity.20 The negative charge of 26bp could explain this result because it prevented this compound from interacting with NOD1/2. The amide groups at R4 clearly contributed significantly to the antagonistic activity; however, some of the potent compounds appeared to be cytotoxic to the tested cells (26dn−26do). Carefully selecting R4 should facilitate choosing appropriate compound(s) for a proof-of-concept demonstration in an animal model. The type of chain at R5 between the BZD core structure and the substituted benzene ring was also important because a total loss of activity was observed when the oxygen atom was removed. Most importantly, p-halogen substituents on the phenyl group significantly improved the potency of the BZD derivatives in vitro, and the p-trifluoromethyl moiety (26ci) gave the best potency, however, with some toxicity following animal treatment. On the basis of the SARs described above, in terms of cytotoxicity and potency in vitro and tested in animal, compound 26bh was finally selected to support our discovery of adjuvant chemotherapy both in vitro and in vivo. Identification of Dual Inhibitors of NOD1/2 Signaling Pathways. Upon recognition of C12-iE-DAP or MDP, NOD1 and NOD2 signaling led to the activation of transcription factor NF-κB, which in turn induces proinflammatory responses. To identify dual inhibitors of NOD1 and NOD2 signaling, 5172



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(RT-qPCR). As shown in Figure 8, 26bh dose-dependently inhibited the transcription of IL-6 and TNF-α stimulated by C12-iE-DAP and MDP, respectively. When the C12-iE-DAP concentration was 500 ng/mL, 26bh (10 μM) remarkably decreased IL-6 and TNF-α expression at the mRNA level with inhibition rates of 54.62% and 35.16%, respectively, comparable to the positive control NOD1 inhibitor (NOD1 Inh, Supporting Information Figure S3).22 Compound 26bh (10 μM) also resulted in an increased level of inhibition of the MDP (5 μg/mL) induced expression of IL-6 and TNF-α in PBMCderived macrophages relative to the NOD2-selective inhibitor (NOD2 Inh, Supporting Information Figure S3).18 These data suggested that 26bh inhibited both NOD1 and NOD2 signaling pathways in primary human immune cells. Meanwhile, 26bh exhibited dose-dependent inhibition of IL-8 secretion in C12-iE-DAP and MDP-induced 293-hNOD1 and 293-hNOD2 cells, respectively (Supporting Information Figure S4). Inhibition of NOD1/2-Induced NF-κB and MAPK Pathways. The activation of NOD1 and NOD2 signaling leading to the release of proinflammatory chemokines is mediated by the phosphorylation of a cascade of effector proteins, including RIP2, extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), and IκBα. The phosphorylation of IκBα, followed by degradation, leads to the transcription of the components of NF-κB in the nucleus and eventually activates NF-κB signaling.23 Meanwhile, the phosphorylation of ERK, p38, and JNK induces the activation of the MAPK pathway, which is NF-κB-independent.24 In THP1 cells, 500 ng/mL C12-iE-DAP induced NOD1 activation and resulted in increases in the levels of phospho-RIP2, phospho-p38, and phospho-JNK, a decrease in the level of IκBα, and a barely discernible increase in phosphor-ERK (Figure 9). Pretreatment of cells with 26bh at 1 and 10 μM markedly prevented the increases in p-RIP2, p-p38, and p-JNK and the decrease in IκBα. 26bh treatment significantly inhibited the MDP-induced phosphorylation of RIP2, ERK, p38, and JNK and the reduction of IκBα in a similar, dose-dependent manner. Furthermore, the inhibitory activity of 26bh on C12-iE-DAP- or MDP-induced NF-κB and MAPK signaling was also evident in human PBMC-derived macrophages (Supporting Information Figure S5). Although 26bh was identified as a dual inhibitor of NOD1 and NOD2, the target protein and the definitive mechanism of this compound remained unknown. The RIP2 kinase assay demonstrated that 26bh did not inhibit the activity of RIP2 kinase. Because the NOD1 and NOD2 signal pathways converge at RIP2, 26bh may target RIP2 recruitment or act upstream of RIP2, potentially affecting NOD self-oligomerization, ligand binding, or ATP binding.2 However, the last mechanism (affecting the ATP-binding site) was considered unlikely because both NOD1 and NOD2 have strong binding preference for triphosphate-containing adenine nucleotides25 and because the chemical structure of 26bh was not similar to that of the nucleotides. In addition, mutational analysis revealed that human NOD1 and NOD2 NACHT domains may employ a different model of activation,26 which suggested that targeting NOD self-oligomerization was unlikely to be the mechanism of this dual NOD1/2 inhibitory compound. Further elaboration of the mechanism of 26bh would be of great interest. 26bh Enhanced the Antitumor Efficacy of PTX in LLCTumor-Bearing Mice. NOD1 and NOD2 are members of the family of PRRs, which play an important role in the innate immune system and are closely related to various autoimmune

Table 6. SAR Investigation of (R)-3-(8-Chloro-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propionyl Derivatives in C12-iE-DAP (50 ng/mL) Stimulated HEK-Blue hNOD1 Cells and MDP (100 ng/mL) Stimulated HEK-Blue hNOD2 Cells

approximately 3000 compounds were initially screened for inhibition of C12-iE-DAP or MDP-stimulated SEAP production in HEK-Blue hNOD1 or HEK-Blue hNOD2 cells. Following confirmation by repeat screening in triplicate and the sulforhodamine B (SRB) assay to exclude cytotoxicity, lead compound 26bh was finally identified as a dual inhibitor of the NOD1 and NOD2 signaling pathways (Figure 6, IC50 = 2.36 μM/4.16 μM for NOD1/2 inhibition, respectively) with minimal or no effect on TLR2, TLR4, and TNF-α signaling pathways as well as cell growth (Supporting Information Figure S1 and Figure S2). Additional information to support 26bh as a true antagonist was provided by its promising inability to inhibit RIP2 kinase phosphorylation (Figure 7) and SEAP hydrolysis of its substrate directly (data not shown) at 10 μM. Thus, it was chosen as the lead compound for further proof-of-concept studies. Inhibition of NOD1/2 Stimulated Cytokines in Human Peripheral Blood Mononuclear Cell (PBMC) Derived Macrophages. To test whether compound 26bh blocked NOD1 and NOD2-mediated signaling in primary human immune cells, we measured the expression of IL-6 and TNF-α at the mRNA level in human PBMC-derived macrophages by reverse-transcription quantitative polymerase chain reaction 5173



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Scheme 1. Preparation of D-Glu BZD Analoguesa

Reagents and conditions: (a) i, KNO3, conc H2SO4, 80 °C, 0.5 h; ii, 110 °C, 2 h; iii, 130 °C, 2 h; (b) phenol derivatives, NaHCO3, H2O, 110 °C, 2 h; or alcohol derivatives, KOH, MeOH, room temperature (rt), 12 h; or aliphatic amines, Et3N, tetrahydrofuran (THF), rt; (c) HCl·H-DGlu(OMe)-OMe or HCl·H-L-Glu(OMe)-OMe or L-alanine methyl ester hydrochloride or glycine methyl ester hydrochloride, N,N′diisopropylcarbodiimide (DIC), THF, rt, 4 h. (d, e) Method 1: i, Pd/C, HCOONH4, THF−EtOH (1:1), rt, 2 h; ii, AcOH, 90 °C, 4 h. Method 2: Fe, AcOH, 90 °C, 5 h. (f) Acyl chlorides, Et3N, dichloromethane (DCM) or THF, 0 °C, 0.5 h; (g) LiOH, H2O, THF, 20 min; (h) i, HOSu, DIC, THF or dimethylformamide (DMF), rt, overnight; ii, amines, THF or DMF, rt, 12 h. a

Scheme 2. Preparation of (R)-3-(7-Chloro-2,5-dioxo-2,3,4,5-tetrahydro-1H -benzo[e][1,4]diazepin-3-yl)propionyl Derivativesa

Reagents and conditions: (a) dimethyl D-glutamate hydrochloride, DIC, THF, rt, 12 h; (b) Fe powder, AcOH, 90 °C, 4 h; (c) R6-Br, Cs2CO3, DMF, rt, 12 h; (d) LiOH, H2O, THF, 20 min; (e) i, HOSu, DIC, THF or DMF, rt, overnight; ii, amines, THF or DMF, rt, 12 h.

a

antitumor agents: compounds 11 and 12.16 Compounds 11 and 12 are conjugates of DTX and MDP derivatives that were superior to DTX in its ability to prevent 4T1 tumor growth and metastasis. Compound 12 decreased MDSCs accumulation and blood neutrophil counts in lung tissue and attenuated matrix metallopeptidase 9 (MMP9), tissue inhibitor metallopeptidase 1 (TIMP-1), and S100 calcium-binding protein A9 (S100A9) levels in lung tissue (will be published elsewhere). The strong beneficial effects of 11 and 12 were associated with their inhibitory effects on the NOD1 signaling pathway. Thus, pharmacologically targeting both the NOD1 and NOD2

and chronic inflammatory diseases, such as Crohn’s disease (CD), inflammatory bowel disease (IBD), and Blau syndrome (BS).27 In recent years, NOD1/2 have also been reported to be involved in tumor progression, including in stomach, lung, breast, colon, rectum, and pancreatic cancers; gastric mucosa carcinoma; and melanoma.28 Our previous work demonstrated that the inhibition of NOD2 inflammatory signaling by 9 has a sensitizing effect on chemotherapy of PTX, probably by preventing the formation of an inflammatory tumor microenvironment (TME) by chemotherapy.15 A similar action of sensitizing chemotherapy of DTX was also observed in other 5174



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Scheme 3. Preparation of (R)-3-(8-Chloro-2,5-dioxo-2,3,4,5-tetrahydro-1H- benzo[e][1,4]diazepin-3-yl)propionyl Derivativesa

a Reagents and conditions: (a) dimethyl D-glutamate hydrochloride, DIC, THF, rt, 12 h; (b) MeI, Cs2CO3, DMF, rt, 2 h; (c) Fe powder, AcOH, 90 °C, 4 h; (d) R8-Br, Cs2CO3, DMF, rt, 12 h; (e) LiOH, H2O, THF, 20 min; (f) i, HOSu, DIC, THF or DMF, rt, overnight; ii, amines, THF or DMF, rt, 12 h.

Figure 5. Schematic representation of the generic rules for the D-Glu BZD compounds acting as NOD1/2 dual antagonists.

bacteria: Enterococcus hirae and Barnesiella intestinihominis in an NOD2-dependent manner.29 NOD2 limited the CTX-induced cancer immunosurveillance and bioactivity of these microbes. Their work demonstrated that NOD2 receptor agonists (e.g., MDP) could suppress the antitumor effects of CTX in wildtype (WT) mice. CTX-treated sarcoma growing in Nod2−/− mice had a higher intratumoral CD8/Treg ratio and higher proportions of innate IFN-γ-producing γδT cells than sarcoma growing in WT mice. These cells were useful for suppressing tumor growth in mice. Therefore, accumulating evidence indicates that both NOD1 and NOD2 signaling could exhibit their biological functions that contribute to cancer survival via multiple approaches (e.g., PAMPs, DAMPs, or intestinal bacteria). Targeting NOD1/2 signaling with an adjuvanting therapeutic regimen to control cancer could be superior to antagonizing only one of them. However, to fully address this question, future detailed mechanistic investigations are required.

signaling pathways was expected to have broad therapeutic utility for cancer treatment. To determine whether 26bh effected a similar sensitization of PTX in vivo, we tested the efficacy of PTX in combination with 26bh in a previously described LLC model that mimics human non-small-cell lung cancer (NSCLC) that responds poorly to PTX.15 As expected, the combination therapy significantly reduced tumor growth compared to PTX treatment alone (p < 0.05), and the tumor weight inhibitory percentage increased from 34.54% to 67.35% (Figure 10, Figure S6). Compared to the control group, the combination of 26bh with PTX showed marked improvement in terms of therapeutic efficacy in LLCbearing mice, whereas 26bh or PTX alone showed no statistically significant beneficial effects. The mechanism by which 26bh remarkably enhanced the antitumor efficacy of PTX remains to be determined. On the basis of our previous report, the combination treatment could lead to TME remodeling.15 Further studies on the stromal compartment and immune cell infiltration in TME would be of great interest. In addition, Zitvogel’s group recently reported that the efficacy of cyclophosphamide (CTX) relies on two intestinal

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DISCUSSION Cancer is a major threat to public health worldwide. Currently, chemotherapy is still one of the most important regimens in 5175



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Abolghasem Ajami and his colleagues reported that the chronic activation of NOD1 and NOD2 could play a role in the development of gastric cancer.39 They found that the relative expression levels of NOD1 in patients with gastric cancer were higher than those in patients with nonulcer dyspepsia (NUD) and peptic ulcer disease (PUD) (p < 0.001 and p < 0.001, respectively). Similarly, the PUD group showed a significantly higher level of NOD1 expression than the NUD group (p < 0.01). Moreover, among Helicobacter pylori positive patients, the expression levels of NOD2 were also higher in the gastric cancer group than in the NUD and PUD groups (p < 0.05 and p < 0.01, respectively).39 Couturier-Maillard et al. found that NOD2 could induce a proinflammatory microenvironment that enhanced epithelial dysplasia following chemical injury.40 To reach this conclusion, the authors employed a dextran sodium sulfate (DSS)−azoxymethane (AOM) combination-induced inflammation-driven colon carcinogenesis animal model. DSS is known to mimic epithelial tissue disruption, potentially leading to microbial translocation. AOM is an alkylating agent that promotes DNA mutation during replication.41 An animal intestinal inflammation model was used for NOD2-driven dysbiosis. However, Philpott et al. have questioned whether a phenotype of increased susceptibility to cancer is truly genotype driven or whether additional studies are needed. Two recent independent studies by us and others have revealed that NOD1 and NOD2 antagonists and NOD2 knockout synergized with chemotherapy.15,29 We have speculated that the activation of NOD1 or NOD2 by DAMPs induces chemotherapeutic resistance, whereas Zitvogel and co-workers have reported that NOD2 limits CTX-induced cancer immunosurveillance by limiting the relocation of microbes. Here, we showed that combination with a dual antagonist of NOD1 and NOD2 (26bh) significantly improved the therapeutic efficacy of PTX. Although many of the roles played by NOD1 and NOD2 in cancer remain unclear, the above findings could allow us to conclude that appropriate inhibition of NOD1 and NOD2 signaling can suppress tumor growth, at least in mice. Thereafter, NOD1 and NOD2 dual antagonists should be moved into clinical investigation as soon as possible to support the concept of novel adjuvant cancer therapy by antagonizing, not agonizing, NOD1/2 signaling.

Figure 6. 26bh inhibits C12-iE-DAP-induced and MDP-induced NF-κB activation. HEK-Blue hNOD1 and HEK-Blue hNOD2 cells were preincubated with different concentrations of 26bh for 3 h and then stimulated with C12-iE-DAP (50 ng/mL) and MDP (100 ng/mL) for an additional 20 h. SEAP were quantified as described in the Experimental Section. Data are presented as mean ± standard deviation (SD) (n = 3).

Figure 7. 26bh has no effect on RIP2 kinase activity. The direct effects of 26bh on RIP2 were determined by ADP-Glu assay. RIP2 (50 ng/mL) or myelin basic protein (MBP) (1 μg/mL) was preincubated with 26bh in reaction buffer and then with 50 μM ATP for 60 min. Next, the addition of 5 μL of ADP-Glo reagent stopped the reaction for 40 min. The amount of ADP generated was measured as described in Materials and Methods. Kinase activities in the absence of compound were set to 100%, and remaining activities with compound are expressed relative to this value. SB203580 was used as a positive control.21

cancer treatment; however, drug resistance greatly limits its efficacy and leads to the recurrence of cancer in the short term.30 A recent study revealed that most potential therapeutic targets identified in primary tumors do not exist in recurrent tumors (50%, compounds were retested, and the SRB assay was conducted to exclude cytotoxicity. Next, the compounds were profiled to the selectivity assays to identify the inhibitory activity of Pam3CSK4, LPS-EK, and rhTNF-α mediated TLR2, TLR4, and TNF-α signaling pathways. The IC50 values were determined using the GraphPad Prism 5 software.15 The direct effects of RIP2 were excluded by the ADP-Glo kinase assay. ADP-Glo in Vitro Kinase Assays. As previously described,15 the direct effects of compounds on RIP2 kinase activity were measured using ADP-Glo assays (Promega, WI, USA). For the ADP-Glo assays, 1 μg of myelin basic protein (MBP) or 50 ng of RIP2 (Promega) was diluted in reaction buffer (40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 0.5 mM dithiothreitol [DTT], and 0.01% bovine serum albumin [BSA]) supplemented with 50 μM ATP for 60 min at rt. Reactions were stopped by the addition of 5 μL of ADP-Glo reagent for 40 min. The amount of ADP generated was measured using the ADP-Glo universal ADP detection assay for kinases according to the manufacturer’s protocol. Kinase activities in the absence of compound were set to 100%, and remaining activities with compound are expressed relative to this value. SB203580 (Selleckchem, USA) was used as a positive control. Human PBMC Isolation and Culture. As we previously described,15 the human PBMCs used in these studies were isolated from the buffy coats of anticoagulated blood from disease-free volunteers (Beijing Red Cross Blood Center, China) by densitygradient centrifugation using Lymphoprep (Axis-Shield, Dundee, Scotland), and the monocytes were further purified by positive magnetic separation of CD14+ cells (Miltenyi Biotec, Cologne, Germany). Human monocytes were cultured in DMEM (Gibco) supplemented with 10% FBS and 20 ng/mL macrophage colonystimulating factor (M-CSF) (Peprotech, RH, USA) for 3 days. Then, 20 mL of fresh medium was added for another 4 days to obtain the

PBMC-derived macrophages. These cells were guaranteed to be mycoplasma-free. RNA Isolation and qRT-PCR. Human PBMC-derived macrophages were seeded at 1.0 × 106 cells/well in a 12-well plate and pretreated with compounds and the positive control for 1 h. Then, they were stimulated with C12-iE-DAP (500 ng/mL) or MDP (5 μg/mL) for 90 min. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol, and 1 μg of total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Invitrogen). Real-time PCR was performed in 96-well format using TaqMan Gene Expression Master Mix (Invitrogen) with an ABI 7500 PCR system (Applied Biosystems, Foster City, CA). The primer and probes from Applied Biosystems were as follows: human IL-6 (Hs00985639_m1), human TNF-α (Hs01113624_g1), and human GAPDH (Hs02758991_g1). The gene expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control. Activator of response of C12-iE-DAP or MDP was set to 100%. Compounds were dissolved in DMSO and further diluted in cell medium. The final concentration of DMSO is 0.1%. NT was the control cells treated with 0.1% DMSO. Western Blotting. The human monocytic leukemia THP1 cell line was cultured in RPMI-1640 containing 10% FBS in a humidified atmosphere containing 5% CO2 at 37 °C. THP1 cells were seeded at 2 × 106 cells per well in a 6-well plate and treated with PMA (20 ng/mL) for 48 h to induce differentiation into macrophages. Then, they were washed three times with phosphate buffered saline (PBS) and cultured in PMA-free RPMI-1640 complete medium for 24 h. Next, the cells were serum-starved with 0.1% FBS overnight. The next day, the cells were pretreated with 26bh or NOD1 Inh as a positive control for 1 h, followed by C12-iE-DAP (50 ng/mL) for 45 min. The cells were washed with cold PBS, lysed in 1× cell lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with protease inhibitor (Sigma, St. Louis, MO, USA) and phosphatase inhibitor sets II and III (Sigma-Aldrich), and stored at −80 °C. 293-hNOD2 cells were seeded in 6-well plates at 2 × 106 cells/well, serum-starved overnight, preincubated for 1 h with the compound or positive control, and then stimulated for 1 h with 5 μg/mL MDP, as 5178



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≥95% pure as detected by analytical high-performance LC (HPLC) and 1H NMR. General Procedures for the Synthesis of Compounds 26a− 26k, 26aa−26ag, 26ba−26bp, 26ca−26cu, 26da−26dhh (Scheme 1). To Prepare Intermediate 19. Intermediate 18 (20 g) was dissolved in 240 mL of sulfuric acid at rt; then, KNO3 (33 g, 2.6 equiv) was added in portions. After the solids had dissolved, they were allowed to react for 30 min at 80 °C, then 2 h at 110 °C, and finally 2 h at 130 °C. After reaction completion, the reaction mixture was cooled to rt and added to 660 g of ice slowly. After the ice dissolved completely, the mixture was filtered to yield a pale-yellow solid. Recrystallization with EtOH and H2O (volume ratio, 1:5) precipitated a solid to afford intermediate 19 as a yellow powder in 44% yield. 1H NMR (400 MHz, DMSO-d6) δ 14.61 (s, 1H), 8.86 (s, 1H), 8.30 (s, 1H). To Prepare Intermediate 20. Phenol Substitution. To a solution of 19 (4.9 g, 19.8 mmol) in 36 mL of H2O were added NaHCO3 (3.5 g, 42.33 mmol, 2.1 equiv) and a phenol derivative (1.1 equiv); the resulting mixture was heated at 110 °C to reflux for 2 h. Then, the reaction mixture was treated with a solution of 6 N HCl in an ice−water bath. Finally, the crude intermediate (20) with phenol substitution was filtered, washed, and dried to give the product in 50−70% yield. Aliphatic Amine Substitution. To a solution of 19 (2.5 g) in 30 mL of THF were added Et3N (3.5 mL, 2.5 equiv) and an aliphatic amine (1.1 equiv), which were allowed to react at rt for 3 h. Upon completion, as monitored by LC−MS, the reaction mixture was adjusted to pH = 6−7 using aqueous HCl and concentrated under vacuum. The mixture was extracted with DCM, washed with brine, dried over anhydrous MgSO4, and filtered. The filtrate was concentrated under vacuum to give intermediate 20 with aliphatic amine substitution. Alcohol Substitution. To a solution of 19 (2.5 g) in 30 mL of MeOH were added KOH (1.4 g, 2.5 equiv) and an alcohol derivative (1.1 equiv). The reaction mixture was allowed to react at 65 °C for 5 h. Upon completion, as monitored by LC−MS, the reaction mixture was cooled, adjusted to pH = 6−7 using aqueous HCl, and concentrated under vacuum. The residue was diluted in DCM and washed with brine. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under vacuum to give intermediate 20 with alcohol substitution. To Prepare Intermediate 21. To a solution of 20 (4.6 g, 13.1 mmol) in 120 mL of THF were added the corresponding amino acid hydrochloride (13.8 mmol, 1.1 equiv) and DIC (3.1 mL, 19.7 mmol, 1.5 equiv). The resulting solution was allowed to react at rt for 4 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was dissolved in DCM and washed with brine. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under vacuum. The crude product was chromatographed on silica gel to give intermediate 21 in 70−85% yield. To Prepare Intermediates 22 and 23a−23c. Method 1. To a solution of 21 (4.8 g, 9.6 mmol) in 50 mL of MeOH and 50 mL of THF were added Pd/C (5%, 4.8 g, wet) and ammonium formate (9.6 g, 0.2 mol). The resulting solution was allowed to react at rt for 2 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was dissolved in DCM and washed with brine. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under vacuum to give intermediate 22. Intermediate 22 was dissolved in 100 mL of acetic acid and heated at 90 °C for 2.5 h. Upon completion, as detected by LC−MS, the reaction mixture was filtered and concentrated under vacuum. Then, the resulting residue was chromatographed on silica gel to give intermediate 23a−23c in a 40−50% yield. Method 2. To a solution of 21 (250 mg, 0.5 mmol) in 10 mL of acetic acid was added iron powder (520 mg, 9.4 mmol, 20 equiv). The resulting solution was allowed to react at 90 °C for 1.5−2.5 h. Upon completion, as detected by LC−MS, the reaction mixture was cooled and diluted with 20 mL of DCM. The mixture was filtered through

previously described. Whole-cell lysates were harvested as described above. The cell lysates were separated by 8% sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) and then electrophoretically transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% (v/v) Tween 20 containing 5% nonfat milk for 2 h at rt. The membrane was then probed with the primary antibody at 4 °C overnight, followed by incubation with the appropriate horseradish peroxidase (HRP) conjugated secondary antibody for 1 h at rt. The blots were developed by ChemiDoc XRS+ (Bio-Rad). The levels of phosphorylated and total p38, JNK, ERK1/2, and RIP2 and total IκBα were determined in each sample using the following antibodies: phosphor-p38 (no. 4631), p38 (no. 9212S), phosphor-JNK (no. CST9251S), JNK (no. 9252S), phosphor-RIP2 (no. 14397S), RIP2 (no. 4142), and IκBα (no. 9242S) antibodies, which were purchased from Cell Signaling Technology, Danvers, USA; phosphor-ERK1/2 (sc-7383) and ERK1/2 (sc-94) antibodies, which were purchased from Santa Cruz Biotechnology; goat polyclonal secondary antibody to rabbit IgG-H&L-HRP (Abcam, ab6721); and rabbit polyclonal secondary antibody to mouse IgG-H&L-HRP (Abcam, ab6728). LLC Animal Model. The LLC tumor was implanted subcutaneously into the right hind calf of the mice, as reported previously.15 One day after the implantation of the LLC tumor tissue, the mice were divided randomly into four groups and received intravenous injections of the control vehicle or PTX (12 mg/kg) every 4 days or 26bh (20 mg/kg) or PTX combined with 26bh daily. PTX and 26bh were formulated in DMSO/Cremophor EL/saline at 5:5:90 (v:v:v). Beginning on day 7 after implantation, primary tumor growth was measured using vernier calipers to determine the two orthogonal axes. The tumor volume was calculated by the formula (1/2)a2b, where a is the shorter axis and b is the longer axis. Eleven days after implantation, all mice were sacrificed by cervical dislocation. The primary tumors were removed, weighed, and photographed. Tumor weight inhibition was calculated by the following formula: [(C − T)/C] × 100 (C, tumor weight of control group; T, tumor weight of treated group). Statistical Analysis. Statistical analyses were performed by twotailed Student’s t test. P < 0.05 was considered statistically significant. Chemistry. Materials and Methods. General Information on Synthesis. DCM, THF, DMF, MeOH, and other commercial reagents were purchased from domestic corporations and used without further purification. Analytical thin-layer chromatography (TLC) plates, preparative TLC plates, and silica gel for column chromatography were purchased from Qingdao Haiyang Chemical and Special Silica Gel Co, Ltd. The automatic liquid chromatography−mass spectrometry (LC−MS) analysis was performed on a Waters SQ Advantage mass spectrometer equipped with an ultraperformance LC (UPLC) system and an eluent splitter (5% eluent was split into the MS system). High-resolution LC−MS was conducted using Agilent LC/MSD TOF with an Agilent ZORBAX SB-C18 (rapid resolution, 3.5 μm, 2.1 mm × 30 mm) column at a flow rate of 0.40 mL/min. The solvent was MeOH/water (75:25 (v/v)) containing 5 mmol/L ammonium formate. The ion source was electrospray ionization (ESI). Infrared (IR) spectra were recorded on a Thermo Nicolet Avatar 330 FT-IR spectrometer. Proton nuclear magnetic resonance was performed on a Bruker Advance 300 NMR, 400 NMR, or 600 NMR spectrometer depending on its necessary. The chemical shifts of the 1H NMR spectra are reported in units of parts per million (ppm) downfield from SiMe4 (δ 0.0) and relative to the signals of chloroform-d (δ = 7.26 ppm, singlet), (CD3)2CO (δ = 2.05 ppm), and dimethyl sulfoxide (DMSO-d6) (δ = 2.50 ppm, quintet). Multiplicities are described as follows: s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and m (multiplets). The number of protons (n) for a given resonance is indicated by nH. The chemical shifts of the 13 C NMR spectra are reported in units of ppm downfield from SiMe4 (δ 0.0) and relative to the signals of chloroform-d (δ = 77.16 ppm, triplet), (CD3)2CO (δ = 29.84 ppm), and DMSO-d6 (δ = 39.52 ppm, septet). All compounds were purified by a semipreparative reversed phase C18 column chromatography with H2O and MeOH until 5179



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kieselguhr and concentrated under vacuum. Then, the resulting residue was chromatographed on silica gel to give intermediate 23a−23c in 30−40% yield. To Prepare Intermediate 24, Compounds 26dl and 26dm. To a solution of 23a (32.6 mg, 0.1 mmol) and triethylamine (69.6 μL, 0.5 mmol) in 4 mL of THF was added 56 (Supporting Information Scheme S5) (0.2 mmol, 2 equiv) with 1 mL of THF. The resulting solution was allowed to react at rt for 30 min. Upon completion, as detected by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 26dl in 73% yield. To a solution of 23b (34 mg, 0.1 mmol) and triethylamine (69.6 μL, 0.5 mmol) in 4 mL of THF was added 56 (0.2 mmol, 2 equiv) with 1 mL of THF. The resulting solution was allowed to react at rt for 30 min. Upon completion, as detected by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 26dm in 88% yield. To a solution of 23c (0.1 mmol) and triethylamine (69.6 μL, 0.5 mmol) in 4 mL of THF was added an acyl chloride (0.2 mmol, 2 equiv) with 1 mL of THF. The resulting solution was allowed to react at rt for 30 min. Upon completion, as detected by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 24 in 50−70% yield. To Prepare Intermediate 25. To a solution of 24 (71.8 mg, 1 equiv) in 6 mL of THF and 3 mL of H2O was added LiOH (8.2 mg, 3 equiv). The resulting solution was allowed to react at rt for 30 min. Upon completion, as monitored by LC−MS, the reaction mixture was adjusted to pH = 6−7 using aqueous HCl (2.0 N) and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 25 in 70−82% yield. To Prepare Compound 26. To a solution of 25 (170 mg) and DIC (128.4 μL, 3 equiv) in 30 mL of THF was added HOSu (95.5 mg, 3 equiv). The resulting solution was allowed to react at rt overnight. Upon the disappearance of the starting material, as monitored by LC−MS, to the reaction mixture was added an amine derivative (5 equiv) at rt for 3 h. Upon completion, the mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give final compound 26 in 12−83% yield. General Procedure for the Synthesis of Compounds 32a− 32f (Scheme 2). To a solution of 27 (13.1 mmol) in 120 mL of THF were added dimethyl D-glutamate hydrochloride (13.8 mmol, 1.1 equiv) and DIC (3.1 mL, 19.7 mmol, 1.5 equiv). The resulting solution was allowed to react at rt for 12 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was dissolved in DCM and washed with brine. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under vacuum. The crude product was chromatographed on silica gel to give intermediate 28. To a solution of 28 (0.5 mmol) in 10 mL of acetic acid was added iron powder (520 mg, 9.4 mmol, 20 equiv), and the mixture was allowed to react at 90 °C for 4 h. Upon completion, as monitored by LC−MS, the reaction mixture was cooled, and 20 mL of DCM was added. The mixture was filtered through kieselguhr, concentrated under vacuum, and then chromatographed on silica gel to give intermediate 29 in 30−40% yield. To a solution of 29 (0.1 mmol) and Cs2CO3 (0.5 mmol) in 4 mL of DMF was added R6-Br (0.2 mmol, 2 equiv) at rt for 12 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 30. To a solution of 30 (1 equiv) in 6 mL of THF and 3 mL of H2O was added LiOH (8.2 mg, 3 equiv) at rt for 20 min. Upon completion, as monitored by LC−MS, the reaction mixture was adjusted to pH = 6−7 using aqueous HCl (2 N) and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 31. To a solution of 31 (1 equiv) and DIC (128.4 μL, 3 equiv) in 30 mL of THF was added HOSu (95.5 mg, 3 equiv). The resulting

solution was allowed to react at rt overnight. Upon the disappearance of the starting material, as monitored by LC−MS, to the reaction mixture was added an amine derivative (5 equiv) at rt for 12 h. After completion, the mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give compound 32. General Procedure for the Synthesis of Compounds 39a− 39i (Scheme 3). To a solution of 33 (13.1 mmol) in 120 mL of THF were added dimethyl D-glutamate hydrochloride (13.8 mmol, 1.1 equiv) and DIC (3.1 mL, 19.7 mmol, 1.5 equiv), and the resulting mixture was allowed to react at rt for 12 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was dissolved in DCM, and the combined extracts were washed with brine, dried over anhydrous MgSO4, and filtered. The filtrate was concentrated under vacuum. The crude product was chromatographed on silica gel to give intermediate 34. To a solution of 34 (0.1 mmol) and Cs2CO3 (0.5 mmol) in 4 mL of DMF was added MeI (0.2 mmol, 2 equiv), and the mixture was then allowed to react at rt for 2 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 35. To a solution of 35 (0.5 mmol) in 10 mL of acetic acid was added iron powder (520 mg, 9.4 mmol, 20 equiv). The resulting solution was allowed to react at 90 °C for 4 h. Upon completion, as monitored by LC−MS, the reaction mixture was cooled, and 20 mL of DCM was added. The mixture was filtered through kieselguhr and concentrated under vacuum. Then, the resulting residue was chromatographed on silica gel to give intermediate 36. To a solution of 36 (0.1 mmol) and Cs2CO3 (0.5 mmol) in 4 mL of DMF was added R8-Br (0.2 mmol, 2 equiv). The resulting solution was allowed to react at rt for 12 h. Upon completion, as monitored by LC−MS, the reaction mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 37. To a solution of 37 (1 equiv) in 6 mL of THF and 3 mL of H2O was added LiOH (8.2 mg, 3 equiv). The resulting solution was allowed to react at rt for 20 min. Upon completion, as monitored by LC−MS, the reaction mixture was adjusted to pH = 6−7 using aqueous HCl (2 N) and concentrated under vacuum. The residue was chromatographed on silica gel to give intermediate 38. To a solution of 38 (1 equiv) and DIC (128.4 μL, 3 equiv) in 30 mL of THF was added HOSu (95.5 mg, 3 equiv). The resulting solution was allowed to react at rt overnight. Upon the disappearance of the starting material, as monitored by LC−MS, to the reaction mixture was added an amine derivative (5 equiv) at rt for 12 h. After completion, the mixture was filtered and concentrated under vacuum. The residue was chromatographed on silica gel to give final compound 39. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-7-(2-chloro-3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26aa). The product was obtained as white powder with the yield of 34%; mp 191−198 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.46 (s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.20 (s, 1H), 7.34 (dd, J = 14.6, 7.0 Hz, 3H), 7.25−7.19 (m, 3H), 7.09 (d, J = 8.1 Hz, 1H), 6.96 (s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 4.63 (s, 2H), 4.27−4.19 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.78 (s, 6H), 2.413−2.40 (m, 2H), 2.03 (td, J = 14.2, 6.7 Hz, 1H), 1.81 (td, J = 14.8, 7.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.83, 170.92, 168.19, 166.70, 152.30, 151.58, 142.44, 137.20, 132.73, 131.13, 128.17, 127.70, 127.50, 121.40, 119.01, 116.78, 116.03, 114.97, 112.38, 72.55, 69.25, 51.28, 50.91, 43.25, 29.65, 23.13. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4Cl, 595.1954; found, 595.1941. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-7-(4-chloro-3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ab). The product was obtained as white powder with the yield of 61%; mp 173−177 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 9.37 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 8.14 (s, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.31−7.26(m, 5180



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5H), 7.20 (s, 1H), 6.87 (d, J = 2.6 Hz, 1H), 6.61 (dd, J = 8.7, 2.6 Hz, 1H), 4.57 (s, 2H), 4.22−4.14 (m, 2H), 3.76−3.71 (m, 1H), 3.56 (s, 3H), 2.71 (s, 6H), 2.42 (dd, J = 11.5, 6.9 Hz, 2H), 2.04 (td, J = 14.3, 6.9 Hz, 1H), 1.83 (td, J = 14.8, 7.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.80, 170.95, 168.19, 166.69, 155.20, 151.57, 142.17, 137.22, 133.11, 132.06, 131.43, 128.24, 127.73, 127.57, 121.78, 121.66, 118.61, 112.83, 112.42, 110.97, 72.52, 69.24, 51.28, 50.95, 42.92, 29.66, 23.13. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4Cl, 595.1954; found, 595.1939. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-7-(2-chloro-5(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ac). The product was obtained as gray powder with the yield of 34%; mp 221−228 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.50 (s, 1H), 8.45 (d, J = 5.3 Hz, 1H), 8.19 (s, 1H), 7.40−7.37 (m, 3H), 7.27−7.22 (m,3H), 6.91 (s, 1H), 6.68 (dd, J = 9.0, 2.9 Hz, 1H), 6.62 (d, J = 2.8 Hz, 1H), 4.66 (s, 2H), 4.30−4.21 (m, 2H), 3.72−3.67 (m, 1H), 3.56 (s, 3H), 2.91 (s, 6H), 2.44−2.40 (m, 2H), 2.03 (dt, J = 14.6, 6.5 Hz, 1H), 1.81 (td, J = 14.6, 7.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.83, 170.91, 168.19, 166.78, 150.74, 150.31, 142.94, 137.23, 132.26, 130.63, 130.56, 128.21, 127.76, 127.57, 121.29, 114.99, 112.29, 111.17, 110.56, 106.00, 72.59, 69.28, 51.27, 50.90, 29.63, 23.12. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4Cl, 595.1954; found, 595.1941. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-7-(3-chloro-5(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ad). The product was obtained as white powder with the yield of 68%; mp 98−101 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.35 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 8.14 (s, 1H), 7.30−7.28 (m, 5H), 7.20 (s, 1H), 6.55 (s, 1H), 6.36 (s, 1H), 6.28 (s, 1H), 4.58 (s, 2H), 4.23−4.14 (m, 2H), 3.77−3.72 (m, 1H), 3.56 (s, 3H), 2.89 (s, 6H), 2.45−2.41 (m, 2H), 2.04 (td, J = 14.5, 7.2 Hz, 1H), 1.83 (td, J = 14.8, 7.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.80, 170.96, 168.17, 166.72, 157.33, 152.30, 142.04, 137.20, 134.55, 133.11, 132.04, 128.23, 127.72, 127.56, 121.76, 118.75, 112.73, 107.65, 105.27, 100.97, 72.52, 69.24, 51.26, 50.93, 29.66, 23.13. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4Cl, 595.1954; found, 595.1938. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-7-(2,3-dichloro-5(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ae). The product was obtained as light-yellow powder with the yield of 24%; mp 240− 242 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.46 (s, 1H), 8.45 (d, J = 4.7 Hz, 1H), 8.17 (s, 1H), 7.36−7.34 (m, 2H), 7.25− 7.24(m, 3H), 6.95 (s, 1H), 6.85 (s, 1H), 6.58 (s, 1H), 4.64 (s, 2H), 4.28−4.19 (m, 2H), 3.72−3.67 (m, 1H), 3.56 (s, 3H), 2.91 (s, 6H), 2.45−2.37 (m, 2H), 2.06−2.00 (m, 1H), 1.86−1.79 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.81, 170.91, 168.21, 166.68, 151.73, 149.97, 142.47, 137.23, 133.11, 132.65, 130.84, 128.17, 127.74, 127.52, 121.40, 115.48, 112.54, 110.08, 109.56, 104.52, 72.55, 69.25, 51.26, 50.92, 30.63, 29.63, 23.11. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4Cl2, 629.1564; found, 629.1554. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-2,5-dioxo-7(2,4,5-trichloro-3-(dimethylamino)phenoxy)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26af). The product was obtained as white powder with the yield of 38%; mp 186−188 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.42 (s, 1H), 8.48 (d, J = 4.8 Hz, 1H), 8.13 (s, 1H), 7.33−7.25 (m, 6H), 7.12 (s, 1H), 4.61 (s, 2H), 4.25−4.16 (m, 2H), 3.77−3.72 (m, 1H), 3.56 (s, 3H), 2.87 (s, 6H), 2.42 (s, 2H), 2.07−2.01 (m, 1H), 1.85− 1.80(m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.81, 170.97, 168.30, 166.58, 150.72, 148.78, 141.78, 137.25, 133.55, 131.63, 131.13, 128.66, 128.15, 127.69, 127.47, 125.15, 121.89, 117.96, 117.64, 113.17, 72.49, 69.24, 51.29, 50.91, 41.82, 29.67, 23.12. HRMS (ESI): m/z (M + H)+ calcd for C30H30O7N4Cl3, 663.1175; found, 663.1160. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-2,5-dioxo-7(2,3,4-trichloro-5-(dimethylamino)phenoxy)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26ag). The product was obtained as white powder with the yield of 36%; mp 112−114 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 9.46 (s, 1H), 8.46 (d, J = 5.5 Hz, 1H), 8.14 (s, 1H), 7.33−7.31 (m, 2H), 7.25−7.23 (m, 3H), 7.02 (d, J = 11.0 Hz, 2H), 4.62 (s, 2H),

4.26−4.18 (m, 2H), 3.73−3.69 (m, 1H), 3.56 (s, 3H), 2.74 (s, 6H), 2.41 (dd, J = 11.0, 7.3 Hz, 2H), 2.03 (dt, J = 14.2, 6.7 Hz, 1H), 1.82 (td, J = 14.6, 7.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.79, 170.95, 168.28, 166.60, 151.30, 150.22, 141.94, 137.24, 133.16, 132.55, 131.21, 128.15, 127.69, 127.46, 122.54, 121.71, 117.50, 116.55, 113.07, 111.55, 72.52, 69.25, 51.27, 42.96, 29.63, 23.09. HRMS (ESI): m/z (M + H)+ calcd for C30H30O7N4Cl3, 663.1175; found, 663.1165. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-butylpropanamide (26ba). The product was obtained as white powder with the yield of 47%; mp 183−185 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.39 (s, 1H), 8.44 (d, J = 4.7 Hz, 1H), 8.18 (s, 1H), 7.77 (t, J = 4.8 Hz, 1H), 7.32−7.20 (m, 6H), 7.14 (s, 1H), 6.59 (d, J = 7.9 Hz, 1H), 6.44 (s, 1H), 6.29 (d, J = 7.4 Hz, 1H), 4.60 (s, 2H), 4.25−4.16 (m, 2H), 3.67−3.63 (m, 1H), 2.98 (m, 2H), 2.90 (s, 6H), 2.18 (t, J = 6.2 Hz, 2H), 2.00 (dd, J = 13.1, 6.7 Hz, 1H), 1.79 (dd, J = 13.8, 6.9 Hz, 1H), 1.31−1.24(m, 2H), 1.20−1.15 (m, 2H), 0.79 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.22, 171.00, 168.15, 166.79, 156.42, 152.06, 142.79, 137.15, 132.47, 131.66, 130.24, 128.28, 127.73, 127.63, 121.58, 117.65, 112.21, 108.56, 105.96, 102.94, 72.58, 69.23, 37.93, 31.23, 31.10, 23.73, 19.47, 13.50. HRMS (ESI): m/z (M + H)+ calcd for C33H40O6N5, 602.2973; found, 602.2956. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-diethylpropanamide (26bb). The product was obtained as light-yellow powder with the yield of 12%; mp 102−104 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.39 (s, 1H), 8.44 (d, J = 5.3 Hz, 1H), 8.17 (s, 1H), 7.33−7.20 (m, 6H), 7.14 (s, 1H), 6.59 (dd, J = 8.4, 2.1 Hz, 1H), 6.45−6.44 (m, 1H), 6.29 (dd, J = 7.9, 1.8 Hz, 1H), 4.60 (s, 2H), 4.25−4.16 (m, 2H), 3.75−3.70 (m, 1H), 3.26−3.17 (m, 4H), 2.90 (s, 6H), 2.41−2.37 (m, 2H), 2.05− 1.97 (m, 1H), 1.80 (td, J = 14.9, 7.7 Hz, 1H), 1.06 (t, J = 7.0 Hz, 3H), 0.96 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.22, 170.44, 168.16, 166.83, 156.47, 152.07, 142.83, 137.20, 132.53, 131.68, 130.25, 128.28, 127.76, 127.64, 121.62, 117.73, 112.28, 108.55, 105.98, 102.97, 72.58, 69.28, 41.16, 28.40, 23.66, 14.12, 13.05. HRMS (ESI): m/z (M + H)+ calcd for C33H40O6N5, 602.2973; found, 602.2958. (R)-2-(Benzyloxy)-N-(7-(3-(dimethylamino)phenoxy)-2,5dioxo-3-(3-oxo-3-(piperidin-1-yl)propyl)-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-8-yl)acetamide (26bc). The product was obtained as yellow powder with the yield of 46%; mp 114− 116 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.40 (s, 1H), 8.43 (d, J = 5.0 Hz, 1H), 8.17 (s, 1H), 7.34−7.20 (m, 6H), 7.13 (s, 1H), 6.59 (d, J = 8.2 Hz, 1H), 6.45 (s, 1H), 6.30 (d, J = 7.8 Hz, 1H), 4.60 (s, 2H), 4.20 (d, J = 2.2 Hz, 2H), 3.72−3.69 (m, 1H), 3.36 (s, 4H), 2.90 (s, 6H), 2.39 (t, J = 7.1 Hz, 2H), 2.01−1.98 (m, 1H), 1.82−1.77 (m, 1H), 1.53 (s, 2H), 1.44 (s, 2H), 1.37 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 171.19, 169.65, 168.16, 166.84, 156.45, 152.07, 142.86, 137.20, 132.51, 131.66, 130.25, 128.28, 127.76, 127.64, 121.64, 117.67, 112.30, 108.56, 106.01, 103.01, 72.58, 69.28, 45.71, 41.88, 28.63, 26.01, 25.29, 24.01, 23.60. HRMS (ESI): m/z (M + H)+ calcd for C34H40O6N5, 614.2973; found, 614.2956. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-octadecylpropanamide (26bd). The product was obtained as white powder with the yield of 56%; mp 185− 189 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.38 (s, 1H), 8.43 (d, J = 5.3 Hz, 1H), 8.19 (s, 1H), 7.75 (t, J = 5.5 Hz, 1H), 7.32 (dd, J = 7.0, 2.3 Hz, 2H), 7.28−7.20 (m, 4H), 7.15 (s, 1H), 6.59 (dd, J = 8.4, 2.1 Hz, 1H), 6.44−6.43 (m, 1H), 6.29 (dd, J = 7.9, 1.8 Hz, 1H), 4.60 (s, 2H), 4.24−4.15 (m, 2H), 3.67−3.62 (m, 1H), 2.98− 2.93(m, 2H), 2.90 (s, 6H), 2.20−2.16 (m, 2H), 2.00 (td, J = 13.6, 7.0 Hz, 1H), 1.79 (td, J = 13.9, 6.9 Hz, 1H), 1.28−1.17 (m, 32H), 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.17, 171.01, 168.06, 166.78, 156.47, 152.03, 142.67, 137.16, 132.51, 131.71, 130.20, 128.24, 127.71, 127.56, 121.55, 117.71, 112.15, 108.50, 105.86, 102.84, 72.56, 69.27, 38.27, 31.25, 31.22, 28.98, 28.96, 28.91, 28.66, 28.62, 26.25, 23.72, 22.05, 13.90. HRMS (ESI): m/z (M + H)+ calcd for C47H68O6N5, 798.5164; found, 798.5141. 5181



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(R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-dodecylpropanamide (26be). The product was obtained as white powder with the yield of 83%; mp 185−188 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.37 (s, 1H), 8.42 (d, J = 5.3 Hz, 1H), 8.18 (s, 1H), 7.75 (t, J = 5.5 Hz, 1H), 7.31 (dd, J = 7.0, 2.2 Hz, 2H), 7.26−7.19 (m, 4H), 7.14 (s, 1H), 6.58 (dd, J = 8.3, 2.1 Hz, 1H), 6.43−6.42 (m, 1H), 6.28 (dd, J = 7.9, 1.8 Hz, 1H), 4.59 (s, 2H), 4.24−4.14 (m, 2H), 3.66−3.61 (m, 1H), 2.98−2.93 (m, 2H), 2.89 (s, 6H), 2.19−2.16 (m, 2H), 1.99 (td, J = 14.3, 7.5 Hz, 1H), 1.78 (td, J = 14.5, 7.4 Hz, 1H), 1.31−1.16 (m, 20H), 0.84 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.17, 171.01, 168.07, 166.78, 156.47, 152.04, 142.68, 137.17, 132.51, 131.71, 130.20, 128.25, 127.72, 127.57, 121.55, 117.71, 112.16, 108.51, 105.87, 102.85, 72.56, 69.27, 38.27, 31.25, 31.22, 29.01, 28.99, 28.97, 28.96, 28.90, 28.66, 28.61, 26.24, 23.72, 22.04, 13.90. HRMS (ESI): m/z (M + H)+ calcd for C41H56O6N5, 714.4225; found, 714.4213. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-hexadecylpropanamide (26bf). The product was obtained as white powder with the yield of 61%; mp 180− 188 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.37 (s, 1H), 8.42 (d, J = 5.3 Hz, 1H), 8.18 (s, 1H), 7.75 (t, J = 5.5 Hz, 1H), 7.31 (dd, J = 6.9, 2.2 Hz, 2H), 7.28−7.17 (m, 4H), 7.14 (s, 1H), 6.58 (dd, J = 8.3, 2.0 Hz, 1H), 6.43 (m, 1H), 6.28 (dd, J = 7.9, 1.7 Hz, 1H), 4.59 (s, 2H), 4.26−4.12 (m, 2H), 3.64 (m, 1H), 2.95 (m, 2H), 2.89 (s, 6H), 2.17 (m, 2H), 2.06−1.93 (m, 1H), 1.78 (td, J = 14.2, 7.0 Hz, 1H), 1.23 (m, 28H), 0.85 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.17, 171.01, 168.06, 166.77, 156.47, 152.03, 142.68, 137.16, 132.51, 131.71, 130.20, 128.24, 127.71, 127.56, 121.55, 117.71, 112.16, 108.50, 105.86, 102.84, 72.56, 69.27, 38.27, 31.25, 31.22, 28.99, 28.97, 28.95, 28.90, 28.65, 28.61, 26.24, 23.72, 22.05, 13.90. HRMS (ESI): m/z (M + H)+ calcd for C45H64O6N5, 770.4851; found, 770.483. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-dimethylpropanamide (26bg). The product was obtained as khaki powder with the yield of 64%; mp 110−116 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.38 (s, 1H), 8.42 (d, J = 5.2 Hz, 1H), 8.16 (s, 1H), 7.32 (dd, J = 6.9, 2.4 Hz, 2H), 7.27− 7.20 (m, 4H), 7.13 (s, 1H), 6.58 (dd, J = 8.4, 2.0 Hz, 1H), 6.45−6.44 (m, 1H), 6.29 (dd, J = 7.9, 1.8 Hz, 1H), 4.59 (s, 2H), 4.24−4.15 (m, 2H), 3.75−3.70 (m, 1H), 2.91 (s, 3H), 2.89 (s, 6H), 2.77 (s, 3H), 2.40−2.37 (m, 2H), 2.04−1.95 (m, 1H), 1.79 (td, J = 14.6, 7.3 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 171.41, 171.18, 168.15, 166.81, 156.45, 152.06, 142.85, 137.19, 132.51, 131.66, 130.24, 128.27, 127.74, 127.63, 121.60, 117.70, 112.30, 108.55, 106.02, 102.99, 72.57, 69.27, 36.52, 34.77, 28.71, 23.39. HRMS (ESI): m/z (M + H)+ calcd for C31H36O6N5, 574.2660; found, 574.2640. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-propylpropanamide (26bh). The product was obtained as khaki powder with the yield of 70%; mp 110−112 °C, [α]20D −164.1 (c 0.13, DMSO). IR (νmax): 3362, 3292, 2929, 1715, 1693, 1673, 1490, 1472, 1281 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.38 (s, 1H), 8.43 (d, J = 5.1 Hz, 1H), 8.17 (s, 1H), 7.78−7.76 (m, 1H), 7.32−7.19 (m, 6H), 7.13 (s, 1H), 6.59−6.57 (m, 1H), 6.44 (s, 1H), 6.29−6.27 (m, 1H), 4.59 (s, 2H), 4.24−4.15 (m, 2H), 3.68−3.63 (m, 1H), 2.96−2.93 (m, 2H), 2.89 (s, 6H), 2.20−2.17 (m, 2H), 2.04−1.97 (m, 1H), 1.78 (dt, J = 21.2, 7.3 Hz, 1H), 1.31 (dt, J = 14.0, 7.0 Hz, 2H), 0.75 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.29, 171.07, 168.15, 166.81, 156.48, 152.06, 142.78, 137.20, 132.51, 131.70, 130.23, 128.27, 127.75, 127.63, 121.60, 117.71, 112.25, 108.54, 105.93, 102.92, 72.58, 69.28, 51.20, 31.21, 23.71, 22.30, 11.27. HRMS (ESI): m/z (M + H)+ calcd for C32H38O6N5, 588.2817; found, 588.2800. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-dibutylpropanamide (26bi). The product was obtained as light-yellow powder with the yield of 53%; mp 82−84 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.39 (s, 1H),

8.44 (d, J = 5.3 Hz, 1H), 8.17 (s, 1H), 7.33−7.25 (m, 5H), 7.24−7.19 (m, 1H), 7.12 (s, 1H), 6.58 (dd, J = 8.4, 2.1 Hz, 1H), 6.44−6.43 (m, 1H), 6.28 (dd, J = 7.9, 1.9 Hz, 1H), 4.59 (s, 2H), 4.24−4.15 (m, 2H), 3.70−3.66 (m, 1H), 3.23−3.10 (m, 4H), 2.89 (s, 6H), 2.46−2.31 (m, 2H), 2.05−1.96 (m, 1H), 1.79 (td, J = 13.9, 7.3 Hz, 1H), 1.46−1.39 (m, 2H), 1.38−1.30 (m, 2H), 1.25−1.22 (m, 2H), 1.18−1.13 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H), 0.81 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.20, 170.79, 168.18, 166.87, 156.43, 152.08, 142.88, 137.21, 132.49, 131.67, 130.26, 128.30, 127.78, 127.66, 121.62, 117.60, 112.25, 108.59, 106.04, 103.01, 72.58, 69.28, 46.73, 44.64, 30.70, 29.47, 28.29, 23.75, 19.60, 19.48, 13.74, 13.71. HRMS (ESI): m/z (M + H)+ calcd for C37H48O6N5, 658.3599; found, 658.3584. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-(tert-butyl)propanamide (26bj). The product was obtained as khaki powder with the yield of 20%; mp 115−117 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.37 (s, 1H), 8.38 (d, J = 5.2 Hz, 1H), 8.17 (s, 1H), 7.36 (s, 1H), 7.32−7.25 (m, 5H), 7.20 (t, J = 8.2 Hz, 1H), 7.13 (s, 1H), 6.59−6.57 (m, 1H), 6.43 (s, 1H), 6.26 (dd, J = 7.8, 1.3 Hz, 1H), 4.59 (s, 2H), 4.23−4.15 (m, 2H), 3.68−3.63 (m, 1H), 2.89 (s, 6H), 2.15−2.11 (m, 2H), 1.96 (dt, J = 13.1, 8.7 Hz, 1H), 1.75 (dt, J = 20.2, 6.5 Hz, 1H), 1.17 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 171.13, 171.10, 168.13, 166.78, 156.54, 152.05, 142.71, 137.19, 132.55, 131.72, 130.19, 128.25, 127.73, 127.61, 121.61, 117.79, 112.23, 108.50, 105.81, 102.85, 72.56, 69.27, 49.80, 31.96, 28.43, 23.82. HRMS (ESI): m/z (M + H)+ calcd for C33H40O6N5, 602.2973; found, 602.2957. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-cyclopropylpropanamide (26bk). The product was obtained as white powder with the yield of 64%; mp 117−118 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.37 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 8.16 (s, 1H), 7.85 (d, J = 3.6 Hz, 1H), 7.32− 7.25(m, 5H), 7.23−7.19(m, 1H), 7.13 (s, 1H), 6.58 (dd, J = 8.6, 1.5 Hz, 1H), 6.44 (s, 1H), 6.30−6.28 (m, 1H), 4.59 (s, 2H), 4.23−4.15 (m, 2H), 3.68−3.63 (m, 1H), 2.89 (s, 6H), 2.57−2.53 (m, 1H), 2.15− 2.12 (m, 2H), 1.98 (dt, J = 13.9, 7.0 Hz, 1H), 1.76 (td, J = 14.6, 7.6 Hz, 1H), 0.57−0.51 (m, 2H), 0.33−0.26 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.64, 171.08, 168.17, 166.80, 156.50, 152.08, 142.79, 137.20, 132.52, 131.74, 130.22, 128.28, 127.76, 127.64, 121.58, 117.75, 112.26, 108.54, 105.93, 102.92, 72.60, 69.30, 51.27, 31.02, 23.60, 22.13, 5.59, 5.52. HRMS (ESI): m/z (M + H)+ calcd for C32H36O6N5, 586.2660; found, 586.2646. (R)-2-(Benzyloxy)-N-(7-(3-(dimethylamino)phenoxy)-3-(3-(4methylpiperazin-1-yl)-3-oxopropyl)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)acetamide (26bl). The product was obtained as white powder with the yield of 42%; mp 214− 215 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.39 (s, 1H), 8.43−8.42 (m, 1H), 8.16 (s, 1H), 7.33−7.20 (m, 6H), 7.12 (s, 1H), 6.58 (d, J = 9.2 Hz, 1H), 6.44 (s, 1H), 6.29 (d, J = 7.9 Hz, 1H), 4.59 (s, 2H), 4.24−4.15 (m, 2H), 3.73−3.68 (m, 1H), 3.38 (d, J = 0.8 Hz, 4H), 2.89 (s, 6H), 2.41−2.38 (m, 2H), 2.23−2.14 (m, 7H), 2.01−1.96 (m, 1H), 1.82−1.75 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 171.18, 170.02, 168.20, 166.86, 156.46, 152.09, 142.91, 137.22, 132.51, 131.67, 130.29, 128.30, 127.78, 127.67, 121.65, 117.67, 112.33, 108.59, 106.07, 103.03, 72.59, 69.29, 54.77, 54.33, 45.62, 44.66, 40.92, 28.62, 23.53. HRMS (ESI): m/z (M + H)+ calcd for C34H41O6N6, 629.3082; found, 629.3068. (R)-2-(Benzyloxy)-N-(7-(3-(dimethylamino)phenoxy)-2,5dioxo-3-(3-oxo-3-(piperazin-1-yl)propyl)-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-8-yl)acetamide (26bm). The product was obtained as white powder with the yield of 35%; mp 120−121 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.39 (s, 1H), 8.42 (d, J = 4.2 Hz, 1H), 8.16 (s, 1H), 7.31−7.13 (m, 6H), 7.13 (s, 1H), 6.58 (dd, J = 7.6, 1.5 Hz, 1H), 6.44 (s, 1H), 6.30−6.28 (m, 1H), 4.59 (s, 2H), 4.24−4.15 (m, 2H), 3.72−3.70 (m, 1H), 3.43−3.38 (m, 4H), 2.89 (s, 6H), 2.62−2.58 (m, 4H), 2.40−2.36 (m, 2H), 2.02−1.97 (m, 2H), 1.82−1.76 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 171.16, 169.93, 168.15, 166.82, 156.46, 152.06, 142.84, 137.19, 132.51, 131.66, 130.24, 128.27, 127.74, 127.63, 121.63, 117.71, 112.31, 108.55, 106.00, 5182



Article

160.42, 156.53, 152.06, 142.77, 133.50 (d, J = 3.0 Hz), 132.52, 131.77, 130.24, 129.83 (d, J = 8.3 Hz), 121.58, 117.90, 115.03 (d, J = 21.3 Hz), 112.35, 108.50, 105.85, 102.86, 71.80, 69.25, 51.29, 50.94, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4F, 579.2249; found, 579.2233. (R)-Methyl 3-(8-(2-((3-Chlorobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cc). The product was obtained as light-yellow powder with the yield of 56%; mp 96−97 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.40 (s, 1H), 8.44 (d, J = 5.3 Hz, 1H), 8.15 (s, 1H), 7.42 (s, 1H), 7.36−7.32 (m, 1H), 7.30−7.29 (m, 2H), 7.23−7.18 (m, 1H), 7.14 (s, 1H), 6.57 (dd, J = 8.3, 2.1 Hz, 1H), 6.43−6.42 (m, 1H), 6.29 (dd, J = 7.9, 1.9 Hz, 1H), 4.61 (s, 2H), 4.27−4.18 (m, 2H), 3.75−3.70 (m, 1H), 3.57 (s, 3H), 2.89 (s, 6H), 2.45−2.40 (m, 2H), 2.07−2.00 (m, 1H), 1.82 (td, J = 14.7, 7.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 170.97, 168.06, 166.84, 156.45, 152.03, 143.04, 139.89, 133.04, 132.43, 131.70, 130.21, 130.15, 127.64, 127.26, 126.02, 121.61, 117.75, 112.57, 108.53, 106.06, 102.98, 71.60, 69.43, 51.30, 50.94, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4Cl, 595.1954; found, 595.1941. (R)-Methyl 3-(8-(2-((2-Chloro-4-fluorobenzyl)oxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26cd). The product was obtained as white powder with the yield of 50%; mp 230−237 °C, [α]20D −166.1 (c 0.12, DMSO). 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.36 (s, 1H), 8.43 (d, J = 5.4 Hz, 1H), 8.15 (s, 1H), 7.54−7.50 (m, 1H), 7.42−7.39 (m, 1H), 7.21−7.17 (m, 1H), 7.13 (s, 1H), 7.10−7.05 (m, 1H), 6.56 (dd, J = 8.4, 2.1 Hz, 1H), 6.40−6.39 (m, 1H), 6.25 (dd, J = 7.9, 1.8 Hz, 1H), 4.65 (s, 2H), 4.31−4.22 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.44−2.39 (m, 2H), 2.08−1.97 (m, 1H), 1.81 (td, J = 14.7, 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 170.97, 167.98, 166.82, 162.66, 160.20, 156.49, 152.00, 142.82, 133.19 (d, J = 10.8 Hz), 132.48, 131.74, 131.21 (dd, J = 6.1, 4.6 Hz), 130.19, 121.61, 117.85, 116.60 (d, J = 25.1 Hz), 114.19 (d, J = 20.9 Hz), 112.45, 108.46, 105.83, 102.83, 69.56, 69.29, 51.30, 50.94, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4ClF, 613.1859; found, 613.1844. (R)-Methyl 3-(8-(2-((3,4-Difluorobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ce). The product was obtained as light-yellow powder with the yield of 78%; mp 102− 103 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.37 (s, 1H), 8.44 (d, J = 5.2 Hz, 1H), 8.14 (s, 1H), 7.42−7.37 (m, 1H), 7.33−7.26 (m, 1H), 7.21−7.17 (m, 2H), 7.13 (s, 1H), 6.56 (dd, J = 8.3, 1.8 Hz, 1H), 6.40 (s, 1H), 6.26 (dd, J = 7.9, 1.5 Hz, 1H), 4.57 (s, 2H), 4.25−4.17 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.44−2.37 (m, 2H), 2.03 (dt, J = 14.3, 6.9 Hz, 1H), 1.81 (td, J = 14.4, 7.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 170.97, 168.06, 166.83, 156.49, 152.03, 150.27 (dd, J = 37.8, 12.5 Hz), 147.83 (dd, J = 37.1, 12.6 Hz), 142.95, 135.18 (dd, J = 5.7, 3.7 Hz), 132.47, 131.75, 130.19, 124.33 (dd, J = 6.5, 3.5 Hz), 121.63, 117.87, 117.26 (d, J = 17.1 Hz), 116.59 (d, J = 17.3 Hz), 112.55, 108.49, 108.43, 105.88, 102.84, 71.18 (d, J = 1.0 Hz), 69.39, 51.30, 50.93, 39.89, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4F2, 597.2155; found, 597.2138. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-8-(2-((3fluorobenzyl)oxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cf). The product was obtained as khaki powder with the yield of 64%; mp 96−97 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.40 (s, 1H), 8.45 (d, J = 5.3 Hz, 1H), 8.16 (s, 1H), 7.34−7.28 (m, 1H), 7.23−7.09 (m, 5H), 6.57 (dd, J = 8.4, 2.0 Hz, 1H), 6.43 (s, 1H), 6.28 (dd, J = 7.9, 1.7 Hz, 1H), 4.62 (s, 2H), 4.27−4.18 (m, 2H), 3.75−3.70 (m, 1H), 3.57 (s, 3H), 2.89 (s, 6H), 2.45−2.38 (m, 2H), 2.04 (dt, J = 14.5, 6.8 Hz, 1H), 1.82 (td, J = 14.7, 7.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.81, 171.06, 168.03, 166.90, 163.34, 160.92, 156.47, 152.05, 142.99, 140.26 (d, J = 7.4 Hz), 132.46, 131.72, 130.26 (d, J = 8.4 Hz), 123.37 (d, J = 2.7 Hz), 121.62, 117.78, 114.48 (d, J = 20.9 Hz), 114.12 (d, J = 21.6 Hz), 112.53, 108.53, 105.99, 102.92, 71.68, 69.42, 51.23, 50.87,

102.98, 72.58, 69.28, 51.30, 46.12, 45.86, 45.41, 42.20, 28.69, 23.54. HRMS (ESI): m/z (M + H)+ calcd for C33H39O6N6, 615.2926; found, 615.2913. (R)-2-(Benzyloxy)-N-(7-(3-(dimethylamino)phenoxy)-2,5dioxo-3-(3-oxo-3-(pyrrolidin-1-yl)propyl)-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-8-yl)acetamide (26bn). The product was obtained as khaki powder with the yield of 95%; mp 112−113 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.38 (s, 1H), 8.43 (d, J = 5.0 Hz, 1H), 8.16 (s, 1H), 7.33−7.26 (m, 5H), 7.24−7.19 (m, 1H), 7.13 (s, 1H), 6.58 (d, J = 8.3 Hz, 1H), 6.44 (s, 1H), 6.29 (d, J = 7.9 Hz, 1H), 4.60 (s, 2H), 4.24−4.15 (m, 2H), 3.76−3.72 (m, 1H), 3.34 (s, 2H), 3.23 (t, J = 6.7 Hz, 2H), 2.89 (s, 6H), 2.36−2.33 (m, 2H), 2.01 (td, J = 13.6, 6.8 Hz, 1H), 1.85−1.78 (m, 3H), 1.76−1.69 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 171.17, 169.82, 168.14, 166.81, 156.45, 152.06, 142.84, 137.18, 132.51, 131.67, 130.23, 128.26, 127.73, 127.61, 121.57, 117.69, 112.29, 108.54, 106.00, 102.98, 72.58, 69.28, 51.27, 45.78, 45.20, 29.91, 25.52, 23.87, 23.06. HRMS (ESI): m/z (M + H)+ calcd for C33H38O6N5, 600.2817; found, 600.2802. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanamide (26bo). mp 130−132 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.38 (s, 1H), 8.43 (d, J = 5.5 Hz, 1H), 8.17 (s, 1H), 7.34−7.20 (m, 7H), 7.13 (s, 1H), 6.73 (s, 1H), 6.60−6.57 (m, 1H), 6.45 (t, J = 2.3 Hz, 1H), 6.31−6.29 (m, 1H), 4.60 (s, 2H), 4.20 (d, J = 2.8 Hz, 2H), 3.65 (s, 1H), 2.89 (s, 6H), 2.17 (d, J = 6.8 Hz, 2H), 2.00−1.95 (m, 1H), 1.81−1.75 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.70, 171.10, 168.19, 166.83, 156.47, 152.08, 142.85, 137.22, 132.52, 131.72, 130.28, 128.30, 127.77, 127.66, 121.56, 117.67, 112.25, 108.57, 106.03, 103.00, 72.60, 69.30, 51.30, 39.98, 30.87, 23.45. HRMS (ESI): m/z (M + H)+ calcd for C29H32O6N5, 546.2347; found, 546.2349. (R)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoic Acid (26bp). 1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 10.41 (s, 1H), 9.38 (s, 1H), 8.42 (d, J = 5.1 Hz, 1H), 8.17 (s, 1H), 7.33−7.31 (m, 2H), 7.27−7.20 (m, 4H), 7.14 (s, 1H), 6.58 (d, J = 8.4 Hz, 1H), 6.44 (s, 1H), 6.29 (d, J = 7.6 Hz, 1H), 4.60 (s, 2H), 4.24- 4.15 (m, 2H), 3.73−3.68 (m, 1H), 2.89 (s, 6H), 2.33 (t, J = 7.0 Hz, 2H), 1.99 (td, J = 14.8, 7.4 Hz, 1H), 1.83−1.75 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 174.02, 171.07, 168.24, 166.91, 156.46, 152.10, 142.92, 137.25, 132.52, 131.73, 130.32, 128.34, 127.82, 127.71, 121.57, 117.68, 112.30, 108.61, 106.09, 103.06, 72.61, 69.30, 51.04, 40.01, 29.96, 23.19. HRMS (ESI): m/z (M + H)+ calcd for C29H31N4O7, 547.2114; found, 547.2144. (R)-Methyl 3-(8-(2-((2-Chlorobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ca). The product was obtained as khaki powder with the yield of 86%; mp 217−221 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.39 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.16 (s, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.34−7.30 (m, 1H), 7.23−7.18 (m, 2H), 7.13 (s, 1H), 6.56 (dd, J = 8.2, 1.3 Hz, 1H), 6.41 (s, 1H), 6.27 (d, J = 7.9 Hz, 1H), 4.69 (s, 2H), 4.32−4.24 (m, 2H), 3.74−3.70 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.44−2.33 (m, 2H), 2.02 (dt, J = 14.8, 7.4 Hz, 1H), 1.81 (td, J = 14.8, 7.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.83, 170.96, 167.99, 166.83, 156.43, 152.01, 142.89, 134.74, 132.44, 132.11, 131.68, 130.20, 129.51, 129.44, 129.18, 127.12, 121.58, 117.70, 112.42, 108.51, 105.98, 102.92, 69.79, 69.59, 51.29, 50.94, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30 H32 O7 N4 Cl, 595.1954; found, 595.1938. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-8-(2-((4fluorobenzyl)oxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cb). The product was obtained as khaki powder with the yield of 68%; mp 182−186 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.36 (s, 1H), 8.43 (d, J = 5.4 Hz, 1H), 8.16 (s, 1H), 7.38−7.34 (m, 2H), 7.23−7.19 (m, 1H), 7.14 (s, 1H), 7.08−7.03 (m, 2H), 6.58 (dd, J = 8.3, 2.1 Hz, 1H), 6.43− 6.42 (m, 1H), 6.27 (dd, J = 7.9, 1.9 Hz, 1H), 4.57 (s, 2H), 4.23−4.15 (m, 2H), 3.74−3.70 (m, 1H), 3.56 (s, 3H), 2.89 (s, 6H), 2.44−2.39 (m, 2H), 2.06−2.00 (m, 1H), 1.82 (td, J = 14.6, 7.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 170.97, 168.13, 166.83, 162.84, 5183



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29.67, 23.08. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4F, 579.2249; found, 579.2231. (R)-Methyl 3-(8-(2-((3-Bromobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cg). The product was obtained as khaki powder with the yield of 69%; mp 98−99 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.40 (s, 1H), 8.44 (d, J = 5.3 Hz, 1H), 8.15 (s, 1H), 7.56 (s, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.22 (m, 2H), 7.14 (s, 1H), 6.57 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 6.29 (d, J = 9.7 Hz, 1H), 4.60 (s, 2H), 4.27−4.18 (m, 2H), 3.72−3.69 (m, 1H), 3.57 (s, 3H), 2.89 (s, 6H), 2.45- 2.36 (m, 2H), 2.04 (dt, J = 14.6, 6.9 Hz, 1H), 1.82 (dt, J = 12.2, 6.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.98, 168.07, 166.85, 156.47, 152.04, 143.05, 140.15, 132.43, 131.71, 130.55, 130.45, 130.22, 130.18, 126.44, 121.62, 117.77, 112.60, 108.53, 106.07, 102.99, 71.56, 69.43, 51.31, 50.94, 29.67, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C30H32Br81N4O7, 641.1527; found, 641.1411. (R)-Methyl 3-(8-(2-((4-Bromobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ch). The product was obtained as khaki powder with the yield of 64%; mp 205−207 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.38 (s, 1H), 8.45 (d, J = 5.3 Hz, 1H), 8.17 (s, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 7.22 (t, J = 8.2 Hz, 1H), 7.16 (s, 1H), 6.59 (dd, J = 8.4, 2.0 Hz, 1H), 6.43 (t, J = 2.0 Hz, 1H), 6.27 (dd, J = 7.9, 1.8 Hz, 1H), 4.57 (s, 2H), 4.26−4.17 (m, 2H), 3.72−3.69 (m, 1H), 3.57 (s, 3H), 2.90 (s, 6H), 2.45−2.378(m, 2H), 2.09−2.00 (m, 1H), 1.82 (td, J = 14.7, 7.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.97, 168.14, 166.88, 156.54, 151.98, 142.73, 136.76, 132.52, 131.82, 131.17, 130.24, 129.64, 121.56, 120.69, 118.05, 112.33, 108.39, 105.67, 102.83, 71.68, 69.33, 51.31, 50.86, 29.67, 23.20. HRMS (ESI): m/z (M + H)+ calcd for C30H32Br81N4O7, 641.1527; found, 641.1410. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-2,5-dioxo-8(2-((4-(trifluoromethyl)benzyl)oxy)acetamido)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26ci). The product was obtained as khaki powder with the yield of 66%; mp 187−189 °C, [α]20D −148.7 (c 0.23, DMSO). 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.41 (s, 1H), 8.44 (d, J = 5.0 Hz, 1H), 8.16 (s, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.22− 7.18 (m, 1H), 7.16 (s, 1H), 6.58−6.56 (m, 1H), 6.43 (s, 1H), 6.27− 6.25 (m, 1H), 4.70 (s, 2H), 4.30−4.21 (m, 2H), 3.75−3.70 (m, 1H), 3.56 (s, 3H),2.87 (s, 6H), 2.44−2.40 (m, 2H), 2.03 (td, J = 14.5, 7.3 Hz, 1H), 1.82 (td, J = 14.7, 7.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.98, 167.97, 166.83, 156.61, 152.04, 142.73, 142.21, 132.58, 131.86, 130.23, 128.14 (d, J = 31.6 Hz), 127.78, 125.08 (q, J = 3.8 Hz), 121.63, 118.03, 112.37, 108.40, 105.69, 102.80, 71.56, 69.57, 51.30, 50.95, 39.86, 29.67, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C31H32O7N4F3, 629.2217; found, 629.2200. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-8-(2-((4methoxybenzyl)oxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-3-yl)propanoate (26cj). The product was obtained as khaki powder with the yield of 59%; mp 197−202 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.35 (s, 1H), 8.43 (d, J = 5.4 Hz, 1H), 8.17 (s, 1H), 7.25−7.20 (m, 3H), 7.14 (s, 1H), 6.77 (d, J = 8.6 Hz, 2H), 6.59 (dd, J = 8.4, 2.1 Hz, 1H), 6.45−6.44 (m, 1H), 6.29 (dd, J = 7.9, 1.9 Hz, 1H), 4.51 (s, 2H), 4.19−4.11 (m, 2H), 3.74−3.71 (m, 1H), 3.70 (s, 3H), 3.56 (s, 3H), 2.89 (s, 6H), 2.44− 2.37 (m, 2H), 2.03 (dt, J = 14.2, 6.4 Hz, 1H), 1.81 (td, J = 14.6, 7.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 170.97, 168.27, 166.84, 158.89, 156.47, 152.07, 142.75, 132.49, 131.71, 130.26, 129.43, 129.07, 121.51, 117.74, 113.63, 112.17, 108.52, 105.97, 102.99, 72.35, 68.99, 54.96, 51.30, 50.95, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C31H35O8N4, 591.2449; found, 591.2435. (R)-Methyl 3-(8-(2-((4-Cyanobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ck). The product was obtained as khaki powder with the yield of 59%; mp 205−207 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.40 (s, 1H), 8.44 (d, J = 5.4 Hz, 1H), 8.14 (s, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.22−7.18 (m, 1H), 7.14 (s, 1H), 6.57 (dd, J = 8.4, 2.1 Hz, 1H), 6.40−6.39 (m, 1H), 6.25 (dd, J = 7.9, 1.9 Hz, 1H), 4.69

(s, 2H), 4.30−4.21 (m, 2H), 3.74−3.70 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.44−2.39 (m, 2H), 2.03 (dt, J = 15.2, 7.0 Hz, 1H), 1.82 (td, J = 14.6, 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.98, 167.97, 166.83, 156.60, 152.01, 143.17, 142.78, 132.56, 132.18, 131.86, 130.23, 127.87, 121.66, 118.69, 118.07, 112.49, 110.29, 108.42, 105.69, 102.78, 71.53, 69.63, 51.31, 50.94, 29.68, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C31H32O7N5, 586.2296; found, 586.2280. (R)-Methyl 3-(8-(2-((2,4-Dichlorobenzyl)oxy)acetamido)-7(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cl). The product was obtained as white powder with the yield of 53%; mp 204−205 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.38 (s, 1H), 8.44 (d, J = 5.6 Hz, 1H), 8.15 (s, 1H), 7.58 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 7.4 Hz, 1H), 7.21−7.17 (m, 1H), 7.13 (s, 1H), 6.56 (dd, J = 8.0, 0.8 Hz, 1H), 6.40 (d, J = 0.7 Hz, 1H), 6.24 (dd, J = 7.2, 0.8 Hz, 1H), 4.66 (s, 2H), 4.28 (s, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.43−2.40 (m, 2H), 2.05−1.99 (m, 1H), 1.84−1.7 (m, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 172.84, 170.97, 167.92, 166.82, 156.52, 151.99, 142.77, 134.02, 133.08, 132.99, 132.52, 131.78, 130.58, 130.19, 128.67, 127.28, 121.63, 117.94, 112.44, 108.42, 105.72, 102.78, 69.65, 69.19, 51.30, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4Cl2, 629.1564; found, 629.1547. (R)-Methyl 3-(8-(2-((3,4-Dichlorobenzyl)oxy)acetamido)-7(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cm). The product was obtained as white powder with the yield of 95%; mp 176−177 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.39 (s, 1H), 8.44 (d, J = 4.4 Hz, 1H), 8.14 (s, 1H), 7.60 (s, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.31 (dd, J = 8.3, 1.1 Hz, 1H), 7.22−7.17 (m, 1H), 7.13 (s, 1H), 6.56 (dd, J = 8.3, 1.8 Hz, 1H), 6.41−6.40 (m, 1H), 6.26 (dd, J = 8.1, 1.7 Hz, 1H), 4.60 (s, 2H), 4.27−4.19 (m, 2H), 3.76−3.69 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.44−2.40 (m, 2H), 2.06−1.99 (m, 1H), 1.84−1.79 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 170.96, 168.00, 166.82, 156.50, 152.01, 142.97, 138.62, 132.46, 131.75, 130.99, 130.44, 130.18, 129.34, 127.59, 121.64, 117.88, 112.58, 108.47, 105.90, 102.90, 70.90, 69.50, 51.30, 50.93, 29.67, 23.14. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4Cl2, 629.1564; found, 629.1549. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-2,5-dioxo-8(2-(thiophen-3-ylmethoxy)acetamido)-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cn). The product was obtained as white powder with the yield of 63%; mp 88−89 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.33 (s, 1H), 8.44 (d, J = 5.3 Hz, 1H), 8.16 (s, 1H), 7.46 (dd, J = 4.9, 2.9 Hz, 1H), 7.40− 7.39 (m, 1H), 7.23−7.19 (m, 1H), 7.14 (s, 1H), 7.06 (dd, J = 4.9, 1.0 Hz, 1H), 6.58 (dd, J = 8.4, 2.2 Hz, 1H), 6.46−6.45 (m, 1H), 6.28 (dd, J = 7.9, 2.0 Hz, 1H), 4.59 (s, 2H), 4.21−4.13 (m, 2H), 3.75−3.70 (m, 1H), 3.56 (s, 3H), 2.91 (s, 6H), 2.44−2.40 (m, 2H), 2.03 (dt, J = 15.0, 6.7 Hz, 1H), 1.82 (td, J = 14.5, 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.94, 171.05, 168.27, 166.91, 156.52, 152.11, 142.85, 138.29, 132.55, 131.78, 130.34, 127.41, 126.69, 123.78, 121.61, 117.88, 112.38, 108.59, 105.94, 102.97, 69.08, 67.95, 51.39, 50.93, 40.03, 29.71, 23.19. HRMS (ESI): m/z (M + H)+ calcd for C28H31O7N4S, 567.1908; found, 567.1892. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-8-(2-(furan-3ylmethoxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26co). The product was obtained as white powder with the yield of 63%; mp 170−171 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.28 (s, 1H), 8.43 (d, J = 5.3 Hz, 1H), 8.15 (s, 1H), 7.64−7.59 (m, 2H), 7.23−7.19 (m, 1H), 7.12 (s, 1H), 6.58 (dd, J = 8.4, 2.1 Hz, 1H), 6.45−6.44 (m, 2H), 6.28 (dd, J = 7.9, 1.9 Hz, 1H), 4.46 (s, 2H), 4.18−4.10 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.90 (s, 6H), 2.44−2.39 (m, 2H), 2.03 (td, J = 14.6, 6.7 Hz, 1H), 1.81 (td, J = 15.0, 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.93, 171.04, 168.30, 166.91, 156.43, 152.12, 143.80, 142.95, 141.36, 132.47, 131.67, 130.33, 121.58, 121.32, 117.70, 112.41, 110.47, 108.63, 106.05, 103.06, 68.85, 64.10, 51.39, 50.95, 40.00, 29.70, 23.18. HRMS (ESI): m/z (M + H)+ calcd for C28H31O8N4, 551.2136; found, 551.2118. (R)-Methyl 3-(8-(2-((4-Chlorobenzyl)oxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cp). The product was 5184



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obtained as khaki powder with the yield of 53%; mp 182−186 °C. 1H NMR (400 MHz, DMSO- d6) δ 10.44 (s, 1H), 9.38 (s, 1H), 8.45 (d, J = 5.3 Hz, 1H), 8.17 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 7.22 (t, J = 8.2 Hz, 1H), 7.15 (s, 1H), 6.59 (dd, J = 8.4, 1.9 Hz, 1H), 6.43 (s, 1H), 6.27 (dd, J = 7.9, 1.6 Hz, 1H), 4.59 (s, 2H), 4.26−4.17 (m, 2H), 3.72−3.69 (m, 1H), 3.53 (s, 3H), 2.90 (s, 6H), 2.45−2.40 (m, 2H), 2.04 (dt, J = 20.8, 7.1 Hz, 1H), 1.82 (td, J = 14.6, 7.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.79, 170.91, 168.04, 166.78, 156.59, 152.05, 142.77, 136.30, 132.52, 132.30, 131.69, 130.14, 129.35, 128.17, 121.57, 117.92, 112.34, 108.46, 105.75, 102.84, 71.65, 69.36, 51.35, 50.88, 29.62, 23.20. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N4Cl, 595.1954; found, 595.1939. (R)-Methyl 3-(8-(2-((4-(tert-Butyl)benzyl)oxy)acetamido)-7(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cq). The product was obtained as yellow powder with the yield of 49%; mp 100−101 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.41 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 8.18 (s, 1H), 7.25−7.18 (m, 5H), 7.13 (s, 1H), 6.60 (dd, J = 8.4, 1.7 Hz, 1H), 6.48 (s, 1H), 6.31 (dd, J = 8.0, 1.1 Hz, 1H), 4.55 (s, 2H), 4.23−4.15 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.90 (s, 6H), 2.44−2.39 (m, 2H), 2.05−1.98 (m, 1H), 1.82 (dt, J = 14.4, 7.2 Hz, 1H), 1.20 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.97, 168.20, 166.85, 156.42, 152.06, 150.17, 142.83, 134.17, 132.47, 131.66, 130.26, 127.47, 124.99, 121.48, 117.54, 112.05, 108.50, 106.14, 103.12, 72.47, 69.27, 51.31, 50.94, 34.15, 31.01, 29.68, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C34H41O7N4, 617.2969; found, 617.2953. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-8-(2-(naphthalen-2-ylmethoxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cr). The product was obtained as white powder with the yield of 47%; mp 140−141 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.45 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.18 (s, 1H), 7.87−7.79 (m, 3H), 7.69−7.67 (m, 1H), 7.51−7.45 (m, 3H), 7.23−7.19 (m, 1H), 7.13 (s, 1H), 6.59 (d, J = 8.5 Hz, 1H), 6.44 (s, 1H), 6.31 (d, J = 8.0 Hz, 1H), 4.78 (s, 2H), 4.31−4.23 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.85 (s, 6H), 2.44−2.39 (m, 2H), 2.03 (dt, J = 14.8, 6.8 Hz, 1H), 1.81 (dt, J = 15.1, 7.3 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.33, 171.46, 168.63, 167.33, 156.91, 152.57, 143.49, 135.33, 133.17, 132.94, 132.91, 132.13, 130.76, 128.41, 128.18, 127.96, 126.63, 126.53, 126.50, 126.09, 122.03, 118.06, 112.79, 109.08, 106.63, 103.63, 73.07, 69.86, 51.78, 51.42, 40.38, 30.16, 23.63. HRMS (ESI): m/z (M + H)+ calcd for C34H35O7N4, 611.2500; found, 611.2486. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-2,5-dioxo-8(4-phenylbutanamido)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26cs). The product was obtained as white powder with the yield of 83%; mp 91−92 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 9.64 (s, 1H), 8.40 (d, J = 5.0 Hz, 1H), 7.99 (s, 1H), 7.29−7.25 (m, 2H), 7.21−7.17 (m, 4H), 7.09 (s, 1H), 6.54 (d, J = 8.2 Hz, 1H), 6.42 (s, 1H), 6.27 (d, J = 7.7 Hz, 1H), 3.73−3.68 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.57 (t, J = 7.6 Hz, 2H), 2.46−2.43 (m, 4H), 2.05−2.00 (m, 1H), 1.87−1.80 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 171.86, 170.95, 166.92, 156.84, 151.95, 143.97, 141.63, 132.83, 132.08, 130.04, 128.28, 128.25, 125.74, 121.45, 117.91, 114.34, 108.23, 106.42, 103.33, 51.29, 50.95, 35.55, 34.52, 29.68, 26.89, 23.17. HRMS (ESI): m/z (M + H)+ calcd for C31H35O6N4, 559.2551; found, 559.2535. (R)-Methyl 3-(8-(2-(Allyloxy)acetamido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26ct). The product was obtained as white powder with the yield of 78%; mp 169−170 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.28 (s, 1H), 8.43 (d, J = 5.0 Hz, 1H), 8.16 (s, 1H), 7.22−7.18 (m, 1H), 7.13 (s, 1H), 6.57 (d, J = 8.3 Hz, 1H), 6.44 (s, 1H), 6.27 (d, J = 7.7 Hz, 1H), 5.86 (ddd, J = 22.0, 10.4, 5.2 Hz, 1H), 5.26 (d, J = 17.2 Hz, 1H), 5.14 (d, J = 10.4 Hz, 1H), 4.13 (d, J = 1.6 Hz, 2H), 4.05 (d, J = 4.9 Hz, 2H), 3.74− 3.70 (m, 1H), 3.56 (s, 3H), 2.90 (s, 6H), 2.44−2.37 (m, 2H), 2.03 (dt, J = 14.7, 7.6 Hz, 1H), 1.81 (td, J = 14.7, 7.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.98, 168.19, 166.83, 156.50, 152.07, 142.72, 134.01, 132.53, 131.70, 130.24, 121.57, 117.91, 117.17, 112.37, 108.52, 105.79, 102.85, 71.49, 69.13, 51.30, 50.93, 29.67,

23.15. HRMS (ESI): m/z (M + H) +calcd for C26H31O7N4, 511.2187; found, 511.2175. (R)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-8-(2(dodecylamino)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26cu). mp 120−122 °C. 1 H NMR (300 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.46 (d, J = 5.0 Hz, 1H), 8.28 (s, 1H), 7.24 (t, J = 8.2, 1H), 7.17 (s, 1H), 6.60 (dd, J = 8.5, 2.0 Hz, 1H), 6.47 (s, 1H), 6.30 (d, J = 7.9 Hz, 1H), 3.76 (dd, J = 12.4, 6.6 Hz, 1H), 3.61 (s, 3H), 2.95 (s, 6H), 2.49−2.44 (m, 4H), 2.15−2.01 (m, 1H), 1.96−1.74 (m, 1H), 1.37−1.18 1.28 (m, 24H), 0.90 (t, J = 6.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 173.28, 172.90, 171.01, 166.93, 156.66, 152.65, 152.02, 132.63, 132.24, 130.21, 121.06, 117.88, 111.45, 108.42, 105.79, 102.84, 51.36, 31.33, 29.70, 29.08, 29.05, 28.92, 28.75, 26.62, 23.16, 22.13, 14.00HRMS (ESI): m/z (M + H)+ calcd for C35H52O6N5, 638.3912; found, 638.3908. (S)-3-(8-(2-(Benzyloxy)acetamido)-7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-propylpropanamide (26da). The product was obtained as white powder with the yield of 75%; mp 209−210 °C, [α]20D= 175.3(c 0.19, DMSO). 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.38 (s, 1H), 8.43 (d, J = 4.9 Hz, 1H), 8.17 (s, 1H), 7.77 (t, J = 4.9 Hz, 1H), 7.33−7.26 (m, 5H), 7.23−7.19 (m, 1H), 7.12 (s, 1H), 6.58 (d, J = 8.2 Hz, 1H), 6.44 (s, 1H), 6.28 (d, J = 7.9 Hz, 1H), 4.59 (s, 2H), 4.24−4.15 (m, 2H), 3.68−3.63 (m, 1H), 2.96−2.91 (m, 2H), 2.89 (s, 6H), 2.20−2.17 (m, 2H), 2.02−1.97 (m, 1H), 1.78 (td, J = 14.4, 7.5 Hz, 1H), 1.34−1.29 (m, 2H), 0.75 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.28, 171.08, 168.16, 166.81, 156.48, 152.07, 142.80, 137.21, 132.51, 131.70, 130.24, 128.28, 127.75, 127.64, 121.60, 117.69, 112.25, 108.55, 105.95, 102.94, 72.58, 69.28, 31.21, 23.71, 22.31, 11.28. HRMS (ESI): m/z (M + H)+ calcd for C32H38O6N5, 588.2816; found, 588.2803. (S)-Methyl 3-(8-(2-((2-Chloro-4-fluorobenzyl)oxy)acetamido)7-(3-(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-3-yl)propanoate (26db). The product was obtained as white powder with the yield of 31%; mp 218−219 °C, [α]20D 174.6 (c 0.06, DMSO). 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.37 (s, 1H), 8.44 (d, J = 4.2 Hz, 1H), 8.15 (s, 1H), 7.54−7.50 (m, 1H), 7.42−7.40 (m, 1H), 7.21−7.17 (m, 1H), 7.13 (s, 1H), 7.10−7.06 (m, 1H), 6.56 (d, J = 7.9 Hz, 1H), 6.39 (s, 1H), 6.25 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 4.31−4.22 (m, 2H), 3.74−3.69 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.46−2.37 (m, 2H), 2.03 (td, J = 13.2, 6.5 Hz, 1H), 1.86−1.77 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.91, 171.02, 168.06, 166.88, 162.70, 160.24, 156.49, 152.03, 142.89, 133.22 (d, J = 10.8 Hz), 132.50, 131.75, 131.30−131.19 (m), 130.26, 121.64, 117.82, 116.66 (d, J = 25.1 Hz), 114.24 (d, J = 21.0 Hz), 112.47, 108.51, 105.91, 102.90, 69.58, 69.31, 51.37, 50.91, 29.69, 23.17. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4Cl F, 613.1859; found, 613.1844. (R)-Methyl 3-(2,5-Dioxo-7-phenoxy-8-(2-((4-(trifluoromethyl)benzyl)oxy)acetamido)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dc). The product was obtained as white powder with the yield of 46%; mp 205−206 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 9.44 (s, 1H), 8.46 (d, J = 4.9 Hz, 1H), 8.16 (s, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 7.9 Hz, 2H), 7.44−7.40 (m, 2H), 7.23−7.20 (m, 1H), 7.14 (s, 1H), 7.08−7.06 (m, 2H), 4.68 (s, 2H), 4.29−4.20 (m, 2H), 3.76−3.71 (m, 1H), 3.56 (s, 3H), 2.44−2.40 (m, 2H), 2.05−1.97 (m, 1H), 1.82 (td, J = 14.2, 7.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.98, 168.05, 166.75, 155.69, 142.49, 142.23, 132.93, 132.09, 130.19, 129.63, 127.91, 125.21−125.05 (m), 124.33, 121.72, 118.78, 118.38, 112.72, 71.56, 69.51, 51.32, 50.92, 29.69, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C29H27O7N3F3, 586.1795; found, 586.1782. (S)-Methyl 3-(7-(3-(Dimethylamino)phenoxy)-2,5-dioxo-8(2-((4-(trifluoromethyl)benzyl)oxy)acetamido)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dd). The product was obtained as white powder with the yield of 65%; mp 187−188 °C, [α]20D 171.4 (c 0.07, DMSO). 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.42 (s, 1H), 8.45 (d, J = 4.7 Hz, 1H), 8.16 (s, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 7.9 Hz, 2H), 7.22− 7.18 (m, 1H), 7.15 (s, 1H), 6.57 (dd, J = 8.1, 1.1 Hz, 1H), 6.44 (s, 1H), 5185



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(R )-3-(8-(2 -((4-Bromobenzyl)oxy)aceta mido)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)-N-propylpropanamide (26di). The product was obtained as light-yellow powder with the yield of 54%; mp 213−228 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.36 (s, 1H), 8.43 (d, J = 5.3 Hz, 1H), 8.16 (s, 1H), 7.77 (t, J = 5.5 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 7.23−7.18 (m, 1H), 7.13 (s, 1H), 6.58 (dd, J = 8.4, 2.0 Hz, 1H), 6.42 (s, 1H), 6.25 (dd, J = 7.9, 1.7 Hz, 1H), 4.57 (s, 2H), 4.25−4.16 (m, 2H), 3.68−3.63 (m, 1H), 2.96−2.92 (m, 2H), 2.90 (s, 6H), 2.20−2.17 (m,2H), 1.99 (td, J = 13.8, 7.2 Hz, 1H), 1.78 (td, J = 14.0, 6.6 Hz, 1H), 1.36−1.27 (m, 2H), 0.75 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.26, 171.06, 168.03, 166.78, 156.57, 152.03, 142.65, 136.73, 132.57, 131.79, 131.15, 130.21, 129.62, 121.63, 120.76, 117.92, 112.27, 108.43, 105.66, 102.80, 71.67, 69.37, 31.19, 23.70, 22.29, 11.26. HRMS (ESI): m/z (M + H)+ calcd for C32H37O6N5Br, 666.1922; found, 666.1889. (R)-3-(7-(3-(Dimethylamino)phenoxy)-8-(2-((4-fluorobenzyl)oxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-propylpropanamide (26dj). The product was obtained as white powder with the yield of 49%; mp 202−216 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.36 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.15 (s, 1H), 7.77 (t, J = 5.5 Hz, 1H), 7.37−7.34 (m, 2H), 7.23−7.19 (m, 1H), 7.12 (s, 1H), 7.08−7.03 (m, 2H), 6.58 (dd, J = 8.4, 2.1 Hz, 1H), 6.43−6.42 (m, 1H), 6.26 (dd, J = 7.9, 2.0 Hz, 1H), 4.57 (s, 2H), 4.23−4.15 (m, 2H), 3.68−3.63 (m, 1H), 2.96−2.91 (m, 2H), 2.89 (s, 6H), 2.20−2.16 (m, 2H), 1.99 (td, J = 13.7, 6.6 Hz, 1H), 1.78 (td, J = 14.9, 7.6 Hz, 1H), 1.36−1.27 (m, 2H), 0.75 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.27, 171.06, 168.12, 166.79, 156.53, 152.05, 142.74 (d, J = 2.6 Hz), 133.50 (d, J = 3.2 Hz), 132.54, 131.75, 130.22, 129.82 (d, J = 8.2 Hz), 121.61, 117.83, 115.13, 114.92, 112.28, 108.49, 105.80, 102.85, 71.79, 69.25, 31.19, 23.69, 22.30, 11.27. HRMS (ESI): m/z (M + H)+ calcd for C32H37O6N5F, 606.2722; found, 606.2707. (R)-2-((4-Chlorobenzyl)oxy)-N-(7-(3-(dimethylamino)phenoxy)-2,5-dioxo-3-(3-oxo-3-(piperidin-1-yl)propyl)-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)acetamide (26dk). The product was obtained as yellow powder with the yield of 93%; mp 94−120 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.36 (s, 1H), 8.41 (d, J = 5.1 Hz, 1H), 8.15 (s, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.23−7.18 (m, 1H), 7.14 (s, 1H), 6.58 (d, J = 8.3 Hz, 1H), 6.42 (s, 1H), 6.26 (d, J = 7.9 Hz, 1H), 4.58 (s, 2H), 4.25−4.16 (m, 2H), 3.74−3.69 (m, 1H), 3.36−3.34 (m, 4H), 2.89 (s, 6H), 2.41−2.37 (m, 2H), 2.04−1.95 (m, 1H), 1.79 (td, J = 14.5, 7.4 Hz, 1H), 1.54−1.53 (m, 2H), 1.43−1.36 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 171.16, 169.63, 168.03, 166.80, 156.55, 152.04, 142.72, 136.30, 132.57, 132.23, 131.75, 130.20, 129.32, 128.21, 121.67, 117.90, 112.33, 108.45, 105.74, 102.85, 71.64, 69.36, 45.69, 41.87, 28.60, 25.99, 25.26, 23.98, 23.58. HRMS (ESI): m/z (M + H)+ calcd for C34H39O6N5Cl, 648.2583; found, 648.2568. N-(7-(3-(Dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)-2-((4-(trifluoromethyl)benzyl)oxy)acetamide (26dl). The product was obtained as red powder with the yield of 34%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.41 (s, 1H), 8.49 (s, 1H), 8.18 (s, 1H), 7.54 (dd, J = 22.4, 7.3 Hz, 4H), 7.21−7.19 (m, 2H), 6.57−6.55 (m, 1H), 6.43 (s, 1H), 6.26−6.24 (m, 1H), 4.70 (s, 2H), 4.26 (s, 2H), 3.59 (d, J = 4.3 Hz, 2H), 2.87 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 170.75, 167.95, 167.13, 156.68, 152.02, 142.51, 142.22, 133.19, 131.91, 130.21, 129.62, 128.29, 127.77, 125.14−125.02 (m), 120.95, 118.50, 112.31, 108.34, 105.56, 102.70, 71.54, 69.57, 44.49, 39.87. HRMS (ESI): m/z (M + H)+ calcd for C27H26O5N4F3, 543.1849; found, 543.1835. (S)-N-(7-(3-(Dimethylamino)phenoxy)-3-methyl-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)-2-((4(trifluoromethyl)benzyl)oxy)acetamide (26dm). The product was obtained as white powder with the yield of 55%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.41 (s, 1H), 8.38 (d, J = 4.9 Hz, 1H), 8.16 (s, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.2 Hz, 1H), 7.17 (s, 1H), 6.56 (dd, J = 8.4, 2.1 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.24 (dd, J = 7.9, 1.8 Hz, 1H), 4.69 (s, 2H), 4.30−4.21 (m, 2H), 3.86−3.80 (m, 1H), 2.87 (s, 6H), 1.21 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.85, 167.93,

6.27−6.26 (m, 1H), 4.70 (s, 2H), 4.30−4.22 (m, 2H), 3.75−3.70 (m, 1H), 3.56 (s, 3H), 2.88 (s, 6H), 2.44−2.40 (m, 2H), 2.03 (td, J = 14.7, 7.6 Hz, 1H), 1.86−1.79 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.87, 170.99, 167.99, 166.84, 156.61, 152.04, 142.75, 142.23, 132.59, 131.86, 130.25, 128.15 (d, J = 31.8 Hz), 127.80, 125.10 (dd, J = 7.5, 3.7 Hz), 121.64, 118.02, 112.38, 108.42, 105.72, 102.83, 71.55, 69.57, 51.32, 50.97, 39.88, 29.68, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C31H32O7N4F3, 629.2217; found, 629.2199. (R)-Methyl 3-(7-(2-Chloro-3-(dimethylamino)phenoxy)-8-(2((4-chlorobenzyl)oxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26de). The product was obtained as gray powder with the yield of 75%; mp 230−235 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.44 (s, 1H), 8.43 (d, J = 5.1 Hz, 1H), 8.18 (s, 1H), 7.36−7.34 (m, 3H), 7.24 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.1 Hz, 1H), 6.97 (s, 1H), 6.87 (d, J = 8.0 Hz, 1H), 4.62 (s, 2H), 4.28−4.20 (m, 2H), 3.73−3.69 (m, 1H), 3.56 (s, 3H), 2.77 (s, 6H), 2.41−2.39 (m, 2H), 2.02 (dt, J = 14.2, 7.1 Hz, 1H), 1.81 (td, J = 14.7, 7.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.82, 170.91, 168.10, 166.69, 152.29, 151.62, 142.39, 136.34, 132.76, 132.21, 131.18, 129.22, 128.15, 128.12, 121.44, 118.92, 116.71, 116.17, 114.81, 112.41, 71.64, 69.36, 51.27, 50.91, 43.23, 30.64, 29.65, 23.13. HRMS (ESI): m/z (M + H)+ calcd for C30H31O7N4Cl2, 629.1564; found, 629.1551. (R)-Methyl 3-(7-(4-Chloro-3-(dimethylamino)phenoxy)-8-(2((4-chlorobenzyl)oxy)acetamido)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26df). The product was obtained as light-yellow powder with the yield of 73%; mp 187−195 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.36 (s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.13 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.34−7.29 (m, 4H), 7.20 (s, 1H), 6.86 (s, 1H), 6.59 (dd, J = 8.2, 1.2 Hz, 1H), 4.57 (s, 2H), 4.23−4.15 (m, 2H), 3.76−3.71 (m, 1H), 3.56 (s, 3H), 2.72 (s, 6H), 2.44−2.42(m, 2H), 2.08−2.00 (m, 1H), 1.87−1.78 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.80, 170.95, 168.11, 166.68, 155.24, 151.56, 142.19, 136.34, 133.11, 132.26, 132.09, 131.42, 129.31, 128.20, 121.82, 121.64, 118.69, 112.90, 112.35, 110.92, 71.61, 69.32, 51.28, 50.95, 42.90, 41.80, 29.66, 23.13. HRMS (ESI): m/z (M + H) +calcd for C30H31O7N4Cl2, 629.1564; found, 629.1551. (R)-Methyl 3-(8-(2-((4-Chlorobenzyl)oxy)acetamido)-2,5dioxo-7-(3-(pyrrolidin-1-yl)phenoxy)-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26dg). The product was obtained as khaki powder with the yield of 65%; mp 132−134 °C. 1H NMR (400 MHz, DMSO- d6) δ 10.44 (s, 1H), 9.37 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 8.17 (s, 1H), 7.31 (q, J = 8.6 Hz, 4H), 7.21−7.17 (m, 2H), 6.41 (dd, J = 8.3, 1.6 Hz, 1H), 6.23−6.20 (m, 2H), 4.59 (s, 2H), 4.26−4.18 (m, 2H), 3.72−3.69 (m, 1H), 3.57 (s, 3H), 3.19 (t, J = 6.3 Hz, 4H), 2.45−2.40 (m, 2H), 2.09−2.00 (m, 1H), 1.95 (t, J = 6.4 Hz, 4H), 1.82 (dt, J = 14.4, 7.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 170.97, 168.05, 166.83, 156.63, 149.27, 142.61, 136.33, 132.59, 132.22, 131.86, 130.29, 129.29, 128.24, 121.59, 118.14, 112.28, 108.02, 104.75, 102.03, 71.65, 69.40, 51.30, 50.93, 47.32, 29.67, 24.92, 23.15. HRMS (ESI): m/z (M + H)+ calcd for C32H34O7N4Cl, 621.2111; found, 621.2097. (R)-3-(8-(2-((4-Chlorobenz yl)oxy)acetamido)- 7- (3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)-N-propylpropanamide (26dh). The product was obtained as white powder with the yield of 44%; mp 224−230 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.36 (s, 1H), 8.43 (d, J = 5.3 Hz, 1H), 8.15 (s, 1H), 7.77 (t, J = 5.6 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.23−7.19 (m, 1H), 7.13 (s, 1H), 6.58 (dd, J = 8.4, 2.0 Hz, 1H), 6.43−6.42 (m, 1H), 6.25 (dd, J = 7.9, 1.7 Hz, 1H), 4.58 (s, 2H), 4.25−4.16 (m, 2H), 3.68− 3.63 (m, 1H), 2.96−2.91 (m, 2H), 2.90 (m, 6H), 2.20−2.17 (m, 2H), 2.00 (dt, J = 13.4, 6.8 Hz, 1H), 1.78 (td, J = 14.3, 7.0 Hz, 1H), 1.36− 1.27 (m, 2H), 0.75 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSOd6) δ 171.27, 171.06, 168.04, 166.78, 156.57, 152.04, 142.67, 136.31, 132.57, 132.23, 131.79, 130.21, 129.33, 128.23, 121.63, 117.91, 112.28, 108.44, 105.69, 102.81, 71.64, 69.36, 31.20, 23.69, 22.29, 11.26. HRMS (ESI): m/z (M + H)+ calcd for C32H37O6N5Cl, 622.2427; found, 622.2414. 5186



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166.75, 156.74, 152.02, 142.52, 142.21, 132.78, 131.88, 130.20, 128.14 (d, J = 31.6 Hz), 127.76, 125.08 (q, J = 3.7 Hz), 124.16 (d, J = 272.0 Hz), 121.68, 118.21, 112.31, 108.32, 105.53, 102.69, 71.55, 69.58, 47.29, 39.87, 13.76. HRMS (ESI): m/z (M + H)+ calcd for C28H28O5N4F3, 557.2006; found, 557.1987. (R)-N-(7-(3-(Dimethylamino)phenoxy)-3-(3-(4-fluoropiperidin-1-yl)-3-oxopropyl)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)-2-((4-(trifluoromethyl)benzyl)oxy)acetamide (26dn). The product was obtained as white powder with the yield of 36% ; mp 111−112 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.40 (s, 1H), 8.40 (s, 1H), 8.15 (s, 1H), 7.57 (d, J = 7.7 Hz, 2H), 7.52 (d, J = 7.2 Hz, 2H), 7.18 (dd, J = 16.9, 8.7 Hz, 2H), 6.57 (d, J = 8.3 Hz, 1H), 6.43 (s, 1H), 6.26 (d, J = 7.6 Hz, 1H), 4.70 (s, 2H), 4.29−4.21 (m, 2H), 3.73−3.72 (m, 1H), 3.55−3.51 (m, 2H), 3.43−3.37 (m, 3H), 2.87 (s, 6H), 2.43 (t, J = 6.4 Hz, 2H), 2.03−1.99 (m, 3H), 1.81−1.79 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.18, 170.02, 167.98, 166.83, 156.60, 156.34, 152.05, 142.78, 142.22, 132.61, 131.82, 130.24, 129.64, 127.79, 125.10 (d, J = 3.8 Hz), 121.72, 117.96, 112.40, 108.43, 105.74, 102.86, 89.11 (d, J = 1.4 Hz), 87.42, 71.56, 69.58, 39.88, 31.27, 28.82, 26.54, 23.57, 22.08. HRMS (ESI): m/z (M + H)+ calcd for C35H38O6N5F4, 700.2753; found, 700.2741. (R)-N-(3-(3-(4,4-Difluoropiperidin-1-yl)-3-oxopropyl)-7-(3(dimethylamino)phenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-8-yl)-2-((4-(trifluoromethyl)benzyl)oxy)acetamide (26do). The product was obtained as white powder with the yield of 20% ; mp 108−110 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.40 (s, 1H), 8.39 (d, J = 3.3 Hz, 1H), 8.15 (s, 1H), 7.57 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.1 Hz, 2H), 7.18 (dd, J = 16.6, 8.2 Hz, 2H), 6.57 (d, J = 8.0 Hz, 1H), 6.43 (s, 1H), 6.26 (d, J = 7.7 Hz, 1H), 4.70 (s, 2H), 4.30−4.21 (m, 2H), 3.75−3.71 (m, 1H), 3.52−3.51 (m, 4H), 2.87 (s, 6H), 2.47 (s, 2H), 2.04−1.97 (m, 3H), 1.88−1.78 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.15, 170.25, 167.98, 166.83, 156.59, 152.05, 142.79, 142.22 (d, J = 1.4 Hz), 132.60, 131.82, 130.24, 129.63, 128.31, 127.99, 127.79, 125.20−125.00 (m), 121.71, 117.96, 112.42 (d, J = 3.8 Hz), 108.44 (d, J = 3.0 Hz), 105.74, 102.86, 71.56, 69.57, 39.87, 33.78, 33.16, 28.56, 26.54, 23.50, 22.07. HRMS (ESI): m/z (M + H)+ calcd for C35H37O6N5F5, 718.2658; found, 718.2651. (R)-Methyl 3-(7-Methoxy-2,5-dioxo-8-(4-(trifluoromethyl)benzamido)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3yl)propanoate (26dp). mp 205−207 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 10.02 (s, 1H), 8.56 (d, J = 5.4 Hz, 1H), 8.32 (dd, J = 14.0, 6.3 Hz, 2H), 8.06 (d, J = 8.1 Hz, 1H), 7.86 (t, J = 8.1 Hz, 1H), 7.78 (s, 1H), 7.42 (s, 1H), 3.96 (s, 3H), 3.80 (dd, J = 13.4, 6.3 Hz, 1H), 3.64 (s, 3H), 2.52 (dd, J = 14.8, 6.4 Hz, 2H), 2.18− 2.07 (m, 1H), 1.92 (td, J = 14.5, 7.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.88, 171.04, 167.33, 165.99, 164.07, 147.55, 135.08, 133.21, 131.96, 131.81, 130.29, 130.10 (d, J = 2.6 Hz), 129.83, 124.38 (q, J = 3.6 Hz), 122.97, 116.17, 111.66, 56.08, 51.33, 50.97, 29.68, 23.18. HRMS (ESI): m/z (M + H)+ calcd for C22H21O6N3F, 480.1377; found, 480.1380. (R,Z)-Methyl 3-(8-(3-(2-Chloro-4-fluorophenyl)acrylamido)7-(naphthalen-1-yloxy)-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (26dq). mp 245−247 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 10.20 (s, 1H), 8.38 (d, J = 5.3 Hz, 1H), 8.32 (s, 1H), 8.05 (t, J = 6.9 Hz, 2H), 7.89−7.80 (m, 3H), 7.65−7.55 (m, 4H), 7.37−7.31 (m, 2H), 7.21 (d, J = 7.5 Hz, 1H), 6.91 (s, 1H), 3.74−3.69 (m, 1H), 3.55 (s, 3H), 2.44−2.37 (m, 2H), 2.07−1.98 (m, 1H), 1.84−1.74 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 175.97, 174.07, 169.83, 166.98, 166.40, 164.73, 154.19, 147.62, 138.16, 137.75, 137.6 (d, J = 5.4 Hz), 135.49, 135.40, 132.52 (d, J = 9.3 Hz), 132.38 (d, J = 3.5 Hz), 131.13, 130.07, 129.69, 129.40, 129.33, 128.25, 127.82, 124.73(d, J = 9.75 Hz), 122.65, 120.41(d, J = 24.9 Hz), 119.65, 118.83, 118.53(d, J = 21.6 Hz), 117.01, 54.41, 53.99, 32.76, 26.26. HRMS (ESI): m/z (M + H)+ calcd for C32H26O6N3ClF, 602.1489; found, 602.1494. (R)-Methyl 3-(8-(2-Naphthamido)-7-methoxy-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dr). mp 218−220 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.76 (s, 1H), 8.47 (d, J = 5.4 Hz, 1H), 8.21 (d, J = 9.1 Hz, 1H), 8.10−7.98 (m, 3H), 7.77 (d, J = 7.0 Hz, 1H), 7.59 (t, J = 3.8 Hz, 3H),

7.32 (s, 1H), 3.84 (s, 3H), 3.77−3.69 (m, 1H), 3.56 (s, 3H), 2.44 (s, 2H), 2.13−1.96 (m, 1H), 1.91−1.75 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 174.40, 173.76, 173.46, 171.98, 168.77, 157.46, 155.11, 144.66, 136.23, 132.59, 131.00, 130.40, 124.49, 123.66, 121.94, 119.78, 119.10, 118.40, 52.85, 52.56, 49.26, 39.22, 32.21, 32.12, 29.40, 28.25, 23.46, 21.44, 19.50, 13.81, 12.97. HRMS (ESI): m/z (M + H)+ calcd for C25H24O6N3, 462.1660; found, 462.1658. (R)-Methyl 3-(8-(3,5-Bis(trifluoromethyl)benzamido)-7-methoxy-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin3-yl)propanoate (26ds). mp 213-215 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.34 (s, 1H), 10.29 (s, 1H), 8.55 (s, 2H), 8.49 (d, J = 5.4 Hz, 1H), 8.37 (s, 1H), 7.62 (s, 1H), 7.35 (s, 1H), 3.87 (s, 3H), 3.72 (d, J = 7.2 Hz, 1H), 3.56 (s, 3H), 2.44−2.40 (m, 2H), 2.10−1.98 (m, 1H), 1.92−1.74 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.88, 171.06, 167.30, 162.97, 147.86, 136.53, 130.94, 130.61, 130.28, 130.05, 129.93, 128.71 (d, J = 3.1 Hz), 125.55−125.22 (m), 124.44, 123.41, 121.72, 116.79, 111.82, 56.05, 51.33, 50.96, 29.67, 23.18. HRMS (ESI): m/z (M + H)+ calcd for C23H20O6N3F6, 548.1251; found, 548.1252. (R)-Methyl 3-(8-(Cyclopropanecarboxamido)-7-(naphthalen1-yloxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dt). mp 166−168 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 10.16 (s, 1H), 8.36 (d, J = 5.3 Hz, 1H), 8.08 (d, J = 5.2 Hz, 2H), 8.03 (d, J = 7.4 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.62−7.53 (m, 3H), 7.14 (d, J = 7.5 Hz, 1H), 6.91 (s, 1H), 3.71−3.66 (m, 1H), 3.55 (s, 3H), 2.41−2.37 (m, 2H), 2.22− 2.17 (m, 1H), 2.05−1.96 (m, 1H), 1.82−1.73 (m, 1H), 0.84−0.80 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 172.87, 172.84, 170.94, 166.79, 151.33, 144.30, 134.62, 132.67, 132.29, 127.97, 126.92, 126.45, 126.27, 126.19, 124.42, 121.72, 121.33, 116.92, 115.18, 114.34, 51.31, 50.90, 29.67, 23.16, 14.26, 7.98, 7.87. HRMS (ESI): m/z (M + H)+ calcd for C27H26O6N3, 488.1816; found, 488.1824. (R)-Methyl 3-(8-(3-(4-Fluorophenyl)ureido)-2,5-dioxo-7-phenoxy-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26du). mp 93−95 °C. 1H NMR (300 MHz, DMSOd6) δ 10.38 (s, 1H), 9.47 (s, 1H), 8.78 (s, 1H), 8.34 (s, 1H), 8.17 (s, 1H), 7.46 (d, J = 6.8 Hz, 4H), 7.27−7.04 (m, 6H), 3.78−3.64 (m, 1H), 3.56 (s, 3H), 2.46−2.37 (m, 2H), 2.11−1.95 (m, 1H), 1.88−1.75 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.93, 170.97, 167.00, 156.35, 156.03, 151.99, 142.03, 135.58 (d, J = 2.3 Hz), 134.45, 133.00, 130.31, 124.39, 119.84 (d, J = 7.8 Hz), 119.33, 117.86, 115.63, 115.41, 110.64, 51.37, 50.97, 29.72, 23.21. HRMS (ESI): m/z (M + H)+ calcd for 269H24O6N4F, 507.1674; found, 507.1678. (R)-Methyl 3-(8-(Cyclopropanecarboxamido)-7-(3,4-dimethylphenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dv). mp 134−136 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.97 (s, 1H), 8.39 (d, J = 5.4 Hz, 1H), 8.03 (s, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.00 (s, 1H), 6.94 (d, J = 2.3 Hz, 1H), 6.85 (dd, J = 8.0, 2.4 Hz, 1H), 3.70 (dd, J = 12.3, 6.8 Hz, 1H), 3.57 (s, 3H), 2.09−1.94 (m, 1H), 0.81 (d, J = 7.4 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 172.91, 172.71, 170.95, 166.94, 153.56, 144.16, 138.33, 132.70, 132.40, 131.98, 130.78, 121.15, 120.97, 117.12, 114.01, 51.36, 50.88, 29.69, 23.18, 19.54, 18.73, 14.22, 7.95, 7.85. HRMS (ESI): m/z (M + H)+ calcd for C25H28O6N3, 466.1973; found, 466.1973. (R)-Methyl 3-(8-(2-(Benzyloxy)acetamido)-7-(3,4-dimethylphenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dw). mp 195−197 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.39 (s, 1H), 8.43 (d, J = 5.4 Hz, 1H), 8.17 (s, 1H), 7.35−7.25 (m, 5H), 7.19 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 6.94−6.87 (m, 1H), 6.81 (dd, J = 8.9, 3.1 Hz, 1H), 4.59 (s, 2H), 4.20 (s, 2H), 3.75−3.68 (m, 1H), 3.56 (s, 3H), 2.41 (d, J = 12.2 Hz, 2H), 2.22 (s, 6H), 2.07−1.99 (m, 1H), 1.87−1.74 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.91, 171.01, 168.23, 166.87, 153.31, 143.07, 138.51, 137.26, 132.61, 132.56, 131.71, 130.87, 128.32, 127.82, 127.66, 121.53, 120.30, 117.49, 116.43, 112.35, 72.58, 69.29, 51.37, 50.89, 29.70, 23.17, 19.53, 18.72. HRMS (ESI): m/z (M + H)+ calcd for C30H32O7N3, 546.2235; found, 546.2239. (R)-3-(8-(Cyclopropanecarboxamido)-7-(3,4-dimethylphenoxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin3-yl)propanoic Acid (26dx). mp 120−122 °C. 1H NMR (400 MHz, 5187



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DMSO-d6) δ 12.085 (s, 1H), 10.28 (s, 1H), 9.96 (s, 1H), 8.43 (d, J = 4.8 Hz, 1H), 8.02 (s, 1H), 7.20 (d, J = 8.2 Hz, 1H), 6.99 (s, 1H), 6.93 (d, J = 2.3 Hz, 1H), 6.84 (dd, J = 8.2, 2.5 Hz, 1H), 3.69−3.64 (m, 1H), 2.30 (t, J = 7.4 Hz, 2H), 2.23 (d, J = 3.4 Hz, 6H), 2.18 (dd, J = 12.4, 6.7 Hz, 1H), 1.99−1.94 (m, 1H), 1.79−1.74 (m, 1H), 0.82−0.79 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 174.03, 172.63, 170.97, 166.88, 153.53, 144.09, 138.25, 132.65, 132.31, 131.95, 130.72, 121.12, 120.89, 117.08, 117.04, 113.96, 30.09, 23.31, 19.48, 18.66, 14.17, 7.86, 7.77. HRMS (ESI): m/z (M + H)+ calcd for C24H26O6N3, 452.1816; found, 452.1814. (R,Z)-3-(8-(3-(2-Chloro-4-fluorophenyl)acrylamido)-7-(naphthalen-1-yloxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoic Acid (26dy). mp 176−178 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 10.46 (s, 1H), 10.21 (s, 1H), 8.39 (d, J = 5.3 Hz, 1H), 8.33 (s, 1H), 8.06 (t, J = 7.9 Hz, 2H), 7.91− 7.80 (m, 3H), 7.65−7.56 (m, 4H), 7.38−7.32 (m, 2H), 7.22 (d, J = 7.4 Hz, 1H), 6.92 (s, 1H), 3.73−3.69 (m, 1H), 2.32 (t, J = 7.8 Hz, 2H), 2.04−1.96 (m, 1H), 1.82−1.74 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.96, 171.01, 166.73, 163.87, 161.21, 151.11, 144.50, 135.06, 134.64, 134.51, 134.40, 132.41, 132.32, 128.02, 126.96, 126.59, 126.30, 126.23, 125.16, 124.70, 121.64 (d, J = 5.7 Hz), 117.43, 117.18, 116.57, 115.69, 115.52, 115.31, 113.90, 50.96, 29.91, 23.17. HRMS (ESI): m/z (M + H)+ calcd for C31H24O6N3ClF, 588.1332; found, 588.1341. (R)-N-(3-(3-Amino-3-oxopropyl)-7-(naphthalen-1-yloxy)-2,5dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)cyclopropanecarboxamide (26dz). mp 234−236 °C 1H NMR (300 MHz, DMSO-d6) δ 10.33 (s, 1H), 10.16 (s, 1H), 8.36 (d, J = 4.8 Hz, 1H), 8.13−7.98 (m, 3H), 7.83 (d, J = 8.3 Hz, 1H), 7.67−7.49 (m, 3H), 7.23 (s, 1H), 7.15 (d, J = 7.2 Hz, 1H), 6.91 (s, 1H), 6.72 (s, 1H), 3.66−3.60 (m, 1H), 2.20−2.13 (m, 3H), 2.03−1.84 (m, 1H), 1.79−1.69 (m, 1H), 0.83 (s, 4H). HRMS (ESI): m/z (M + H)+ calcd for C26H25O5N4, 473.1819; found, 473.1823. (R,Z)-N-(3-(3-Amino-3-oxopropyl)-7-(naphthalen-1-yloxy)2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)-3(2-chloro-4-fluorophenyl)acrylamide (26daa). mp 194−196 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 10.19 (s, 1H), 8.38 (d, J = 5.2 Hz, 1H), 8.31 (s, 1H), 8.05 (t, J = 9.2 Hz, 2H), 7.89−7.80 (m, 3H), 7.65−7.53 (m, 4H), 7.37−7.30 (m, 2H), 7.22 (d, J = 7.7 Hz, 2H), 6.91 (s, 1H), 6.72 (s, 1H), 3.68−3.63 (m, 1H), 2.16 (t, J = 7.2 Hz, 2H), 1.99−1.94 (m, 1H), 1.77−1.72 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 173.69, 171.05, 166.68, 163.85, 163.28, 161.61, 151.10, 144.47, 135.02, 134.63, 134.45(d, J = 10.9 Hz), 132.39, 132.32, 129.40 (d, J = 9.3 Hz), 129.37, 129.26 (d, J = 3.1 Hz), 128.01, 126.95, 126.57, 126.29, 126.22, 125.15, 124.67, 121.63(d, J = 6.3 Hz), 117.29(d, J = 25 Hz), 116.51, 115.68, 115.41(d, J = 21.75 Hz), 113.85, 51.33, 30.83, 23.41. HRMS (ESI): m/z (M + H)+ calcd for C31H25O5N4ClF, 587.1492; found, 587.1495. (R,Z)-3-(2-Chloro-4-fluorophenyl)-N-(3-(3-(diethylamino)-3oxopropyl)-7-(naphthalen-1-yloxy)-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)acrylamide (26dbb). mp 172−174 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 10.20 (s, 1H), 8.38 (d, J = 5.4 Hz, 1H), 8.31 (s, 1H), 8.08−8.03 (m, 2H), 7.89−7.70 (m, 3H), 7.65−7.55 (m, 4H), 7.36−7.32 (m, 2H), 7.21 (d, J = 7.5 Hz, 1H), 6.90 (s, 1H), 3.75−3.68 (m, 1H), 3.24−3.18 (m, 4H), 2.36 (t, J = 7.3 Hz, 2H), 2.03−1.95 (m, 1H), 1.81−1.72 (m, 1H), 1.03 (t, J = 7.1 Hz, 3H), 0.94 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 171.21, 170.45, 166.72, 163.86, 163.29, 161.62, 151.11, 144.52, 135.04, 134.64, 134.49, 134.41, 132.33, 129.45, 129.39, 129.29, 129.26, 128.03, 126.96, 126.60, 126.31, 126.22, 125.15, 124.72, 121.72, 121.59, 117.39, 117.22, 116.50, 115.77, 115.49, 115.35, 113.88, 55.94, 28.38, 23.66, 14.09, 13.11. HRMS (ESI): m/z (M + H)+ calcd for C35H33O5N4ClF, 643.2118; found, 643.2116. (R)-Methyl 3-(8-Acetamido-7-(dibutylamino)-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dcc). mp 66−68 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.05 (s, 1H), 8.38 (d, J = 5.3 Hz, 1H), 8.00 (s, 1H), 7.55 (s, 1H), 3.73−3.68 (m, 1H), 3.56 (s, 3H), 2.86−2.82 (m, 4H), 2.47−2.39 (m, 2H), 2.15 (s, 3H), 2.09−1.96 (m, 1H), 1.88−1.76 (m, 1H), 1.35−1.23 (m, 8H), 0.84 (t, J = 6.9 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 172.87, 170.95, 168.40, 167.36, 138.72, 136.17, 133.86, 124.46, 120.89,

110.91, 54.39, 51.26, 50.92, 29.68, 28.87, 24.35, 23.18, 19.92, 13.81. HRMS (ESI): m/z (M + H)+ calcd for C23H35O5N4, 447.2602; found, 447.2599. (R)-Methyl 3-(8-(Cyclopropanecarboxamido)-7-morpholino2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26ddd). mp 158−160 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.32 (s, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.46 (s, 1H), 3.87−3.79 (m, 4H), 3.72−3.67 (m, 1H), 3.56 (s, 3H), 2.89−2.84 (m, 2H), 2.78−2.72 (m, 2H), 2.45−2.39 (m, 2H), 2.14−2.07 (m, 1H), 2.05−1.98 (t, J = 14.8 Hz, 1H), 1.86−1.77 (m, 1H), 0.83 (d, J = 6.0 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 172.95, 172.13, 171.01, 167.42, 138.42, 136.23, 133.53, 121.49, 121.21, 112.92, 66.17, 51.94, 51.36, 50.89, 29.68, 23.22, 14.72, 7.91, 7.77. HRMS (ESI): m/z (M + H)+ calcd for C21H27O6N4, 431.1925; found, 431.1933. (R)-Methyl 3-(7-Morpholino-2,5-dioxo-8-(2-phenylacetamido)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (26dee). mp 133−135 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.94 (s, 1H), 8.39 (d, J = 5.4 Hz, 1H), 7.98 (s, 1H), 7.44 (s, 1H), 7.40 (d, J = 4.3 Hz, 4H), 7.36−7.31 (m, 1H), 3.83 (q, J = 15.1 Hz, 2H), 3.71- 3.66 (m, 1H), 3.56 (s, 3H), 3.55−3.46 (m, 4H), 2.68−2.62 (m, 2H), 2.61−2.55 (m, 2H), 2.44− 2.36 (m, 2H), 2.05−1.99 (m, 1H), 1.85−1.78 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 172.87, 170.97, 169.35, 167.28, 138.00, 136.06, 135.13, 133.78, 129.47, 128.83, 127.11, 121.80, 121.31, 111.97, 66.02, 51.84, 51.30, 50.88, 43.83, 29.64, 23.17. HRMS (ESI): m/z (M + H)+ calcd for C25H29O6N3, 481.2082; found, 481.2081. (R)-N-(3-(3-(Dodecylamino)-3-oxopropyl)-7-morpholino-2,5dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)cyclopropanecarboxamide (26dff). mp 166−168 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.29 (s, 1H), 8.35 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.72 (t, J = 5.6 Hz, 1H), 7.44 (s, 1H), 3.83− 3.79 (m, 4H), 3.63−3.58 (m, 1H), 2.96−2.91 (m, 2H), 2.86−2.83 (m, 2H), 2.76−2.71 (m, 2H), 2.16 (t, J = 7.1 Hz, 2H), 2.12−2.05 (m, 1H), 2.01−1.91 (m, 1H), 1.80−1.73 (m, 1H), 1.31−1.16 (m, 20H), 0.85− 0.81 (m, 8H). 13C NMR (150 MHz, DMSO-d6) δ 174.81, 174.05, 173.82, 170.13, 141.02, 139.04, 136.36, 124.21, 124.02, 115.49, 68.81, 54.74, 53.97, 41.08, 34.07, 33.99, 31.81, 31.79, 31.78, 31.73, 31.48, 31.42, 29.06, 26.57, 24.87, 17.51, 16.73, 10.63, 10.50. HRMS (ESI): m/z (M + H)+ calcd for C32H50O5N5, 584.3806; found,584.3806. N-((3R)-7-Morpholino-2,5-dioxo-3-(3-oxo-3-((1,2,3,4tetrahydronaphthalen-1-yl)amino)propyl)-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-8-yl)cyclopropanecarboxamide (26dgg). mp 105−108 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.33 (d, J = 2.3 Hz, 1H), 8.42−8.33 (m, 1H), 8.20 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 4.2 Hz, 1H), 7.47 (d, J = 7.4 Hz, 1H), 7.23− 6.84 (m, 4H), 4.90 (s, 1H), 3.93−3.73 (m, 4H), 3.75−3.56 (m, 1H), 2.96−2.82 (m, 2H), 2.80−2.72 (m, 2H), 2.26 (t, J = 7.1 Hz, 2H), 2.22−2.01 (m, 3H), 1.99−1.72 (m, 4H), 1.73−1.49 (m, 2H), 0.85 (d, J = 5.7 Hz, 4H). HRMS (ESI): m/z (M + H)+ calcd for C30H36O5N5, 546.2711; found, 546.2708. (R)-N-(3-(3-((2-(Cyclohex-1-en-1-yl)ethyl)amino)-3-oxopropyl)-7-morpholino-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)cyclopropanecarboxamide (26dhh). mp 150−152 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 9.30 (s, 1H), 8.37 (d, J = 5.3 Hz, 1H), 7.90 (s, 1H), 7.70 (s, 1H), 7.45 (s, 1H), 5.28 (s, 1H), 3.87−3.78 (m, 4H), 3.65−3.60 (m, 1H), 3.06− 3.01 (m, 2H), 2.89−2.82 (m, 2H), 2.79−2.71 (m, 2H), 2.19−2.15 (m, 2H), 2.13−2.07 (m, 1H), 2.02−1.97 (m, 1H), 1.92−1.87 (m, 4H), 1.85−1.81 (m, 2H), 1.76−1.74 (m, 1H), 1.52−1.42 (m, 4H), 0.83 (d, J = 6.1 Hz, 4H). 13C NMR (150 MHz, DMSO-d6) δ 173.37, 172.55, 172.37, 168.68, 139.57, 137.57, 136.19, 134.89, 123.11, 122.73, 122.49, 114.04, 67.48, 53.27, 38.91, 38.28, 32.56, 28.90, 25.96, 25.15, 23.71, 23.25, 16.05, 9.17, 9.05. HRMS (ESI): m/z (M + H)+calcd for C28H38O5N5, 524.2867; found, 524.2869. (R)-Methyl 3-(7-Chloro-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)propanoate (32a). mp 153−155 °C. IR (νmax): 3287, 3198, 3074, 1726, 1689, 1664 cm−1. 1H NMR (300 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.61 (d, J = 5.3 Hz, 1H), 7.69 (d, J = 2.3 Hz, 1H), 7.59 (dd, J = 8.6, 2.4 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 3.75 (dd, J = 13.4, 6.3 Hz, 1H), 3.57 (s, 3H), 5.50−2.40 5188



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(m,2H),2.10−2.00 (m, 1H), 1.90−1.80 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.82, 171.12, 166.51, 135.62, 132.04, 129.65, 127.95, 127.75, 123.00, 51.34, 50.86, 29.60, 23.07. HRMS (ESI): m/z (M + H)+calcd for C13H14ClO4N2, 297.0637; found, 297.0635. (R)-3-(7-Chloro-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-heptylpropanamide (32b). mp 196− 198 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.61 (d, J = 5.5 Hz, 1H), 7.77 (s, 1H), 7.67 (s, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 3.65 (s, 1H), 3.00−2.92 (m, 2H), 2.19 (t, J = 7.1 Hz, 2H), 2.04−1.94 (m, 1H), 1.84−1.75 (m, 1H), 1.33−1.13 (m, 11H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.19, 166.47, 135.65, 132.07, 129.55, 127.95, 127.75, 122.96, 51.09, 38.31, 31.19, 31.09, 29.07, 28.33, 26.25, 23.61, 22.04, 13.94. HRMS (ESI): m/z (M + H)+ calcd for C19H27O3N3Cl, 380.1735; found, 380.1730. (R)-3-(7-Chloro-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-diethylpropanamide (32c). mp 186− 188 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.61 (d, J = 5.4 Hz, 1H), 7.69 (d, J = 2.5 Hz, 1H), 7.58 (dd, J = 8.6, 2.6 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 3.75 (dd, J = 13.6, 6.1 Hz, 1H), 3.30− 3.14 (m, 4H), 2.40 (t, J = 7.4 Hz, 2H), 2.00 (dd, J = 14.1, 6.7 Hz, 1H), 1.92−1.74 (m, 1H), 1.07 (t, J = 7.0 Hz, 3H), 0.96 (t, J = 7.0 Hz, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 171.38, 170.43, 166.52, 135.68, 132.03, 129.63, 127.92, 127.81, 122.98, 41.16, 28.34, 23.58, 14.11, 13.05. HRMS (ESI): m/z (M + H)+ calcd for C16H21 O3N3Cl, 338.2163; found, 338.2164. Methyl 3-(7-Chloro-1-methyl-2,5-dioxo-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-3-yl)propanoate (32d). mp 82−85 °C. 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 2.4 Hz, 1H), 7.96 (d, J = 5.2 Hz, 1H), 7.68 (dd, J = 8.7, 2.5 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 4.03−3.98 (m, 1H), 3.83 (s, 3H), 3.55 (s, 3H), 2.80−2.67 (m, 2H), 2.55−2.48 (m, 1H), 2.36−2.30 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.88, 170.21, 166.39, 139.55, 131.86, 130.26, 129.31, 128.77, 124.30, 51.38, 50.89, 35.05, 29.54, 23.47. HRMS (ESI): m/z (M + H)+ calcd for C14H15O4N2Cl, 311.0793; found, 311.0793. Methyl 3-(7-Chloro-1-(cyclopropylmethyl)-2,5-dioxo-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (32e). mp 48−50 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 5.8 Hz, 1H), 7.65−7.63 (m, 2H), 7.57 (d, J = 9.5 Hz, 1H), 4.08−4.03 (m, 1H), 3.78−3.73 (m, 1H), 3.60−3.54 (m, 4H), 2.34−2.39 (m, 2H), 2.06−1.98 (m, 1H), 1.92−1.83 (m, 1H), 0.79 (s, 1H), 0.35−0.26 (m, 2H), 0.07−0.06 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 169.57, 166.31, 138.28, 131.78, 131.75, 129.77, 128.78, 125.60, 51.37, 51.04, 50.59, 29.53, 23.39, 9.81, 3.96, 2.85. HRMS (ESI): m/z (M + H)+ calcd for C17H20O4N2Cl, 351.1106; found, 351.1107. Methyl 3-(7-Chloro-1-decyl-2,5-dioxo-2,3,4,5-tetrahydro1H-benzo[e][1,4]diazepin-3-yl)propanoate (32f). 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 2.5 Hz, 1H), 7.52−7.49 (m, 1H), 7.23 (d, J = 8.8 Hz, 1H), 6.47 (d, J = 5.8 Hz, 1H), 4.28−4.18 (m, 1H), 3.82−3.77 (m, 1H), 3.67 (s, 3H), 3.58−3.51 (m, 1H), 2.63−2.56 (m, 1H), 2.49−2.39 (m, 1H), 2.32−2.24 (m, 1H), 2.17−2.09 (m, 1H), 1.63−1.42 (m, 3H), 1.29−1.20 (m, 13H), 0.86 (t, J = 6.9 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 173.42, 169.11, 167.01, 138.20, 132.61, 131.83, 130.60, 130.18, 124.10, 52.02, 52.02, 48.24, 31.80, 29.86, 29.37, 29.16, 29.09, 27.83, 26.60, 23.83, 22.64, 14.10. HRMS (ESI): m/z (M + H)+ calcd for C22H32O4N2Cl, 423.2045; found, 423.2046. (R)-Methyl 3-(8-Chloro-4-methyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (39a). mp 165− 167 °C. 1H NMR (300 MHz, acetone) δ 9.41 (s, 1H), 7.70 (s, 1H), 7.18−7.05 (m, 2H), 4.09 (s, 1H), 3.45 (s, 3H), 2.6 (s, 2H), 2.27 (s, 2H), 1.92 (d, J = 2.0 Hz, 2H). HRMS (ESI): m/z (M + H)+ calcd for C14H16O4N2Cl, 311.0793; found, 311.0793. (R)-3-(8-Chloro-4-methyl-2,5-dioxo-2,3,4,5-tetrahydro-1Hbenzo[e][1,4]diazepin-3-yl)-N,N-diethylpropanamide (39b). mp 66−68 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 7.69 (t, J = 7.8 Hz, 2H), 7.30 (t, J = 7.9 Hz, 1H), 5.48 (d, J = 7.4 Hz, 1H), 4.07 (t, J = 7.2 Hz, 1H), 3.72−3.51 (m, 1H), 3.22−3.16 (m, 5H), 2.95 (s, 3H), 2.27 (d, J = 7.2 Hz, 2H), 2.23−2.10 (m, 1H), 2.05 (d, J = 6.8 Hz, 1H), 1.07−0.87 (m, 13H). 13C NMR (150 MHz, DMSO-d6) δ 169.94,

168.91, 166.88, 133.14, 132.40, 130.48, 129.33, 125.78, 125.24, 54.05, 41.24, 40.65, 28.31, 23.28, 22.02, 14.21, 12.99. HRMS (ESI): m/z (M + H)+ calcd for C17H23O3N3Cl, 352.1422; found, 352.1415. (R)-Methyl 3-(8-Chloro-4-methyl-1-nonyl-2,5-dioxo-2,3,4,5tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanoate (39c). 1 H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.7 Hz, 1H), 7.61−7.57 (m, 1H), 7.34 (t, J = 7.9 Hz, 1H), 4.36−4.30 (m, 1H), 4.02−3.97 (m, 1H), 3.61 (s, 3H), 3.46−3.42 (m, 1H), 3.07 (s, 3H), 2.44- 2.35 (m, 2H), 2.30−2.22 (m, 1H), 2.17−2.12 (m, 1H), 1.27−1.11 (m, 14H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 172.57, 168.14, 167.56, 136.64, 134.25, 133.58, 129.63, 128.31, 128.14, 55.35, 51.76, 47.36, 31.75, 30.34, 29.37, 29.13, 29.07, 28.32, 27.30, 26.53, 22.61, 22.02, 14.06. HRMS (ESI): m/z (M + H)+ calcd for C23H34O4N2Cl, 437.2202; found, 437.2203. (R)-3-(8-Chloro-4-methyl-1-nonyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanamide (39d). 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 5.42 (d, J = 69.8 Hz, 2H), 4.32 (dd, J = 13.7, 8.8 Hz, 1H), 4.03 (dd, J = 9.6, 4.9 Hz, 1H), 3.48−3.40 (m, 1H), 3.07 (s, 3H), 2.46−2.37 (m, 1H), 2.35−2.26 (m, 1H), 2.18 (ddd, J = 17.1, 11.0, 7.0 Hz, 2H), 1.18 (s, 14H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 173.23, 168.49, 167.45, 136.58, 134.29, 133.60, 129.58, 128.42, 128.25, 55.40, 47.43, 31.78, 31.63, 29.40, 29.16, 29.10, 28.40, 27.32, 26.57, 22.64, 22.32, 14.09. HRMS (ESI): m/z (M + H)+ calcd for C22H33O3N3Cl, 422.2205; found, 422.2204. (R)-8-Chloro-4-methyl-3-(3-morpholino-3-oxopropyl)-1nonyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione (39e). 1 H NMR (300 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 4.39−4.28 (m, 1H), 4.03−3.98 (m, 1H), 3.67−3.42 (m, 9H), 3.08 (s, 3H), 2.48−2.38 (m, 2H), 2.25− 2.11 (m, 2H), 1.16−1.274 (m, 14H), 0.85 (t, J = 6.7 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 169.84, 168.55, 167.41, 136.56, 134.31, 133.60, 129.56, 128.49, 128.23, 66.83, 66.62, 55.67, 47.49, 45.92, 42.02, 31.78, 29.40, 29.25, 29.16, 29.10, 28.47, 27.31, 26.57, 23.24, 22.64, 22.42, 14.09. HRMS (ESI): m/z (M + H)+ calcd for C26H39O4N3Cl, 492.2624; found, 492.2622. (R)-3-(8-Chloro-4-methyl-1-nonyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N-(pyridin-3-ylmethyl)propanamide (39f). 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 2H), 7.73−7.71 (m, 1H), 7.59−7.57 (m, 2H), 7.35 (t, J = 7.9 Hz, 1H), 7.27−7.23 (m, 1H), 6.23 (s, 1H), 4.37 (d, J = 5.8 Hz, 2H), 4.35−4.27 (m, 1H), 4.03−3.99 (m, 1H), 3.47−3.40 (m, 1H), 3.06 (s, 3H), 2.45− 2.37 (m, 1H), 2.30−2.12 (m, 3H), 1.35−1.11 (m, 15H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 171.30, 168.45, 167.41, 148.82, 148.56, 136.51, 135.98, 134.25, 133.60, 129.52, 128.38, 128.28, 123.78, 55.51, 47.43, 41.03, 32.45, 31.77, 29.39, 29.15, 29.09, 28.41, 27.30, 26.55, 22.63, 22.59, 14.09. HRMS (ESI): m/z (M + H)+ calcd for C28H38O3N4Cl, 513.2627; found, 513.2626. N-((S)-1-Amino-1-oxopropan-2-yl)-3-((R)-8-chloro-4-methyl1-nonyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)propanamide (39g). mp 47−49 °C. 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 6.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 6.51 (s, 1H), 6.24 (s, 1H), 5.65 (s, 1H), 4.42 (t, J = 7.0 Hz, 1H), 4.34−4.28 (m, 1H), 4.02−3.95 (m, 1H), 3.50−3.39 (m, 1H), 3.06 (s, 3H), 2.45−2.25 (m, 2H), 2.20−2.11 (m, 2H), 1.36−1.09 (m, 17H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 174.59, 171.61, 168.74, 167.78, 136.93, 134.62, 133.89, 129.94, 128.76, 128.56, 55.80, 48.61, 47.75, 32.67, 32.11, 29.73, 29.49, 29.43, 28.75, 27.65, 26.89, 22.97, 22.83, 18.45, 14.43. HRMS (ESI): m/z (M + H)+ calcd for C25H38O4N4Cl, 493.2576; found, 493.2574. (R)-3-(8-Chloro-1-isopentyl-4-methyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-diethylpropanamide (39h). 1H NMR (400 MHz, CDCl3) δ7.74−7.72 (m, 1H), 7.60−7.55 (m, 1H), 7.34 (t, J = 7.9 Hz, 1H), 4.41−4.34 (m, 1H), 4.04−4.00 (m, 1H), 3.51−3.44 (m, 1H), 3.31−3.22 (m, 4H), 3.08 (s, 3H), 2.43−2.36 (m, 2H), 2.27−2.18 (m, 2H), 1.43−1.35 (m, 1H), 1.29−1.22 (m, 1H), 1.15−1.13 (m, 3H), 1.09−1.02 (m, 1H), 0.98 (t, J = 7.1 Hz, 3H), 0.81−0.79 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 170.30, 168.69, 167.59, 136.69, 134.54, 133.62, 129.62, 128.55, 128.31, 5189



Article

55.65, 46.05, 42.36, 40.46, 36.03, 29.02, 28.53, 25.99, 23.61, 14.57, 13.09. HRMS (ESI): m/z (M + H)+ calcd for C22H33O3N3Cl, 422.2205; found, 422.2197. (R)-3-(8-Chloro-1-(cyclopropylmethyl)-4-methyl-2,5-dioxo2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-3-yl)-N,N-diethylpropanamide (39i). 1H NMR (300 MHz, DMSO-d6) δ 7.75 (d, J = 7.9 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 4.30− 4.22 (m, 1H), 4.14 (t, J = 7.1 Hz, 1H), 3.22−3.09 (m, 5H), 2.93 (s, 3H), 2.27−2.21 (m, 2H), 2.15−1.98 (m, 2H), 1.02 (t, J = 6.9 Hz, 3H), 0.88 (t, J = 6.9 Hz, 3H), 0.69−0.53 (m, 1H), 0.27−0.12 (m, 2H), 0.05−0.03 (m, 1H), −0.21 − −0.26 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 169.85, 168.55, 166.86, 136.37, 134.37, 133.21, 128.74, 128.56, 128.13, 54.73, 50.90, 41.35, 28.09, 27.73, 22.29, 14.26, 12.97, 9.33, 3.29, 2.38. HRMS (ESI): m/z (M + H) + calcd for C21H29O3N3Cl, 406.1892; found, 406.1889.

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Gly, glycine; HOAc, acetic acid; HOSu, N-hydroxysuccinimide; HPLC, high performance liquid chromatography; HR-MS, high resolution mass spectrometry; Hz, hertz; IC50, 50% inhibitory concentration; IBD, inflammatory bowel disease; IKK, inhibitor of nuclear factor κB kinase; LC−MS, liquid chromatography− mass spectrometry; IL-6, interleukin-1; IND, investigational new drug; LLC, Lewis lung carcinoma; LRR, leucine-rich repeat; m, multiplet; MCP-1, monocyte chemotactic protein 1; mp, melting point; MAPK, mitogen-associated protein kinase; MDP, N-acetylmuramyl dipeptide; MDSC, myeloid-derived suppressor cell; MeOH, methanol; MMP9, matrix metalloproteinase; NACHT, nucleotide-binding domain; NF-κB, transcription factor nuclear factor κB; NMR, nuclear magnetic resonance; NOD, nucleotide-binding oligomerization domain; PAMP, pathogen-associated molecular pattern; PBMC, peripheral blood mononuclear cell; PTX, paclitaxel; q, quartet; RIP2, receptor-interacting sering/threonine-protein kinase 2; SAR, structure−activity relationship; SEAP, secreted embryonic alkaline phosphatase; SRB, sulforhodamine B; t, triplet; TAB, transforming growth factor binding protein; TAK1, transforming growth factor β activated kinase 1; TIMP, tissue inhibitor of metalloproteinase; t-Bu, tertiary butyl; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; TME, tumor microevironment; TOF, time-of-flight; TNF-α, tumor necrosis factor α

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00608. General synthetic route of linear L-Ala-isoGln library compounds, chemical structures, and inhibitory percentage of NOD1/2 for 14a−14h and 26a−26k, 1H and 13C NMR data and spectra, NOD1/2 selectivity assay, SRB of 26bh, inhibiton of IL-8 release by ELISA, blocking of NOD1 and NOD2 mediated NF-κB and MAPKs pathways in PBMCs-derived macrophages by Western blotting, and photograph of tumors (PDF) Molecular formula strings and some data (CSV)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Y.M.: phone, 86-10-62787371; e-mail, [email protected]. *G.L.: phone, 86-10-62797740; e-mail: gangliu27@tsinghua. edu.cn. ORCID

Gang Liu: 0000-0001-5549-5686 Author Contributions §

S.W. and J.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the funding support of grants from the National Natural Science Foundation of China (Grants 81573289, 81273364, and 91213303).

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ABBREVIATIONS USED Ac, acetyl group; ACN, acetonitrile; AcOEt, acetic ether; Ala, alanine; Bn, benzyl group; Boc, tert-butoxycarbonyl; BMDM, bone marrow derived macrophage; Bz, benzoyl; BZD, 1,4-benzodiazepine-2,5(H)-dione; BS, Blau syndrome; C12-ieDAP, lauroyl-γ-D-glutamyl-meso-diaminopimelic acid; CARD, caspase activation and recruitment domain; calcd, calculated; Cbz (Z), benzyloxycarbonyl; CD, Crohn’s disease; CTX, cyclophosphamide; d, doublet; DAMP, damage/danger-associated molecular pattern; DCM, dichloromethane; DIC, N,N′diisopropylcarbodiimide; DIPEA, N-ethyldiisopropylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N′-dimethylformamide; DMSO, dimethyl sulfoxide; DTX, docetaxel; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; EtOAc, ethyl acetate; EtOH, ethanol; Fmoc, fluorenylmethyloxycarbonyl; GI50, 50% growth inhibition; Glu, glutamic acid; 5190



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Discovery of 1,4-Benzodiazepine-2,5-dione (BZD) Derivatives as Dual

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