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Abstract
Discovery Of The Clinical Candidate Sonrotoclax (BGB-11417), A Highly Potent And Selective Inhibitor For Both WT And G101V Mutant Bcl‑2
Shuaishuai Chen, Taichang Zhang, Yue Wu, Dan Su, Yutong Zhu, Aiying Xu, Haipeng Xu, Shasha Yang, Zhijun Zheng, Junhua Liu, Xuefei Yang, Xi Yuan, Yuan Hong, Xuebing Sun, Yin Guo, Changyou Zhou,
Drug Annotationpubs.Acs.Org/Jmc
130.6, 129.1, 127.4 (2C), 126.5, 125.5, 125.1, 124.7, 120.7 (2C),
113.7, 109.1, 108.0, 101.3, 100.5, 70.5, 62.5, 52.8, 50.5, 48.5, 44.4,
Data Availability Statement
Accession Codes
Corresponding Authors
Authors
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Regimen For Previously Untreated Chronic Lymphocytic Leukemia
2019210828 A1, 2019.
Abstract
The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax’s limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.
Discovery of the Clinical Candidate Sonrotoclax (BGB-11417), a Highly Potent and Selective Inhibitor for Both WT and G101V Mutant Bcl‑2
Yunhang Guo,*,# Hai Xue,# Nan Hu, Ye Liu, Hanzi Sun, Desheng Yu, Ling Qin, Gongyin Shi, Fan Wang, Lei Xin, Weihua Sun, Fan Zhang, Xiaomin Song, Shuran Li, Qiang Wei, Ying Guo, Yong Li, Xiaoxin Liu,
Shuaishuai Chen, Taichang Zhang, Yue Wu, Dan Su, Yutong Zhu, Aiying Xu, Haipeng Xu, Shasha Yang, Zhijun Zheng, Junhua Liu, Xuefei Yang, Xi Yuan, Yuan Hong, Xuebing Sun, Yin Guo, Changyou Zhou,
Xuesong Liu, Lai Wang, and Zhiwei Wang* Cite This: https://doi.org/10.1021/acs.jmedchem.4c00027 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax’s limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment. ■ INTRODUCTION Apoptosis, a form of programmed cell death, is crucial for tissue homeostasis and plays an important role in causing and preventing some important biological processes.1−5 Evading apoptosis is critical in tumor development and maintenance and is ultimately a universal hallmark of cancer.2,3 Apoptosis can be triggered via two main pathways: the extrinsic or death receptor mediated pathway and the intrinsic or mitochondrial pathway.1 Cell death mediated through the intrinsic pathway is regulated by proteins in the B-cell lymphoma-2 (Bcl-2) family, which contains three subfamilies: the proapoptotic BAX/BAKlike proteins are the essential effectors of apoptosis; the antiapoptotic proteins (such as Bcl-2, Bcl-xL, Bcl-W, and Mcl1) promote cell survival by inhibiting their proapoptotic counterparts; the BH3-only proteins, including BIM, PUMA, BID, NOXA, BMF, BIK, and HRK, initiate and stimulate apoptosis either by directly interacting with proapoptotic proteins or by releasing them by binding to the corresponding antiapoptotic proteins.6,7 Defects in apoptotic signaling are a common requirement for oncogenesis and are often driven by the overexpression of antiapoptotic factors. One such factor is Bcl-2, which was found to be different from other identified oncogenes in that it promotes cell survival instead of cell proliferation.8,9 Bcl-2 plays a dominant role in the survival of lymphoid malignancies as well as in some solid tumors2,3 and is frequently highly expressed in those cancers, exerting its functions by sequestering proapoptotic proteins through binding to their BH3 domains. 10 Inhibiting these intracellular protein−protein interactions is therefore an attractive strategy to target the aberrant survival of cancer cells caused by Bcl-2 dysregulation.11 Tremendous efforts have been made in the past two decades to develop BH3-mimetic small molecule inhibitors (Figure 1) to abolish the biological activity of the Bcl-2 protein. Encouraged by the discovery of the Bcl-2/Bcl-xL dual inhibitors ABT-737 (1a)11−13 and its orally bioavailable analogue ABT-263 (navitoclax, 1b)14,15 via a high-throughput NMR-based screening method at Abbott Laboratories (now Received: January 4, 2024 Revised: March 22, 2024 Accepted: April 23, 2024
Drug Annotationpubs.acs.org/jmc
© XXXX The Authors. Published by American Chemical Society A https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX This article is licensed under CC-BY-NC-ND 4.0 D ow nl oa de d vi a 10 3. 41 .1 72 .9 o n M ay 7 , 2 02 4 at 0 3: 24 :0 3 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s. AbbVie), the first selective Bcl-2 (Bcl-xL-sparing) inhibitor ABT-199 (venetoclax, 1c) was rationally developed to treat Bcl-2-dependent malignancies.16 This was accomplished by exploiting the subtle differences between the binding interfaces of Bcl-2 and Bcl-xL to mitigate Bcl-xL-related platelet toxicity (thrombocytopenia). On the basis of its remarkable clinical efficacy and tolerable safety, venetoclax has been approved by the U.S. Food and Drug Administration (FDA) for chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) treatment.17,18 Servier, in collaboration with Vernalis and Novartis, disclosed another Bcl-2-specific inhibitor, S65487 (2), which has a partially overlapping but distinct binding mode to the hydrophobic groove of Bcl-2 compared to ABT-199 due to its different structure.19 S65487 was reported to be active against both wild-type (WT) Bcl-2 and Bcl-2 mutants resistant to venetoclax, and intravenously administered S65487 is currently in phase I clinical evaluations.20 Several other selective Bcl-2 inhibitors, such as lisaftoclax (APG-2575, 3), lacutoclax (LP-108, 4), LOXO-338, and ZNd5,21 have also been reported and entered the clinical stage in the last 10 years, as well as other Bcl-2/Bcl-xL dual inhibitors, such as AZD4320 (5) and APG-1252 (6).22 AZD4320 is the active substance of the dendrimer conjugate AZD046623 that is in phase II clinical trials, and APG-1252 is a prodrug under phase II evaluations. Although venetoclax has shown significant efficacy, partic- ularly in CLL patients, and encouraging activity against multiple myeloma24 (MM) and estrogen receptor positive breast cancer,25 the low overall response rate (ORR = 18%) and the short estimated median progression-free survival (mPFS = 1 month) for diffuse large B-cell lymphoma (DLBCL) patients treated with venetoclax even at the highest dose (1200 mg) indicated that venetoclax may not be potent enough to treat DLBCL.26 Furthermore, continuous treatment with venetoclax resulted in nearly half of the patients becoming refractory after 2−3 years, as has been reported in trials with heavily pretreated CLL patients.27,28 Several Bcl-2 mutations, suggested to be potential resistance mechanisms, were identified in relapsed patients, among which the Gly101Val (G101V) mutation was the most frequent.29,30 Therefore, we embarked on a drug development effort to obtain a novel Bcl-2 inhibitor with improved potency for both WT and mutant Bcl2, especially G101V. Generation of a Chemical Starting Point. To rationally design a Bcl-2 inhibitor that can more potently inhibit the WT Bcl-2 protein and cover mutations such as G101V, we carefully investigated the binding modes of reported Bcl-2 inhibitors. As https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX B illustrated in the X-ray cocrystal structures of Bcl-2 with inhibitors (Figure 2), these compounds bind to the P2 and P4 pockets (ABT-263, Figure 2a) or only the P2 pocket (S55746, Figure 2b) of Bcl-2 to achieve good binding affinity, and the P2 pocket is relatively flexible and compatible with various structures. In addition, the Bcl-2 G101V:venetoclax cocrystal structure (PDB: 6O0L) reveals the molecular basis of the acquired resistance of the G101V mutant after treatment with venetoclax.31 Because the chlorobenzene moiety of venetoclax inserts deeply into the P2 pocket, when G101 is mutated into the bulkier valine, one key residue, Glu152, is pushed into the base of the P2 pocket and generates a steric clash with the chlorobenzene; this leads to an ∼180-fold decrease in binding affinity of venetoclax to Bcl-2 G101V.29,30 Thus, we hypothesized that modifying or fine-tuning this moiety to have a shallow and broad interaction with the P2 pocket may enhance the binding affinity of the compound for Bcl-2 WT and restore the inhibitory effect on mutants, especially G101V. To compare compounds’ potencies, biochemical compet- itive binding assays, testing the disruption of the Bcl-2:BAK, Bcl-2 G101V mutant:BAK, or Bcl-xL:BAK complexes using TR-FRET methodology, were set up to evaluate their binding affinities to target proteins. Cell viability assays were used to test their cellular potencies: RS4;11 is a Bcl-2 dependent cell line and chosen for on-target activity evaluation and MOLT-4 is a Bcl-xL dependent cell line and chosen for major off-target activity evaluation. We initially varied the piperidine linker in venetoclax to explore the volume and flexibility of the P2 pocket and focus on Bcl-2 WT for preliminary SAR. As expected, many linkers were well tolerated,32 and even the long and twisted linker bicyclic tetrahydropyrazolo[1,5-a]pyrimidine in compound 7 (Figure 3) gave promising potency in the Bcl-2 WT biochemical assay (IC50 = 45 nM), which indicated the large volume and flexibility of the P2 pocket. The flexibility was further indicated by removing the dimethyl cyclohexene motif to dramatically shorten the linker in compound 8, and only a 1- fold drop in potency was found (IC50 = 105 nM). Considering that the bicyclic linker might be too rigid and/or short to occupy the P2 pocket in an appropriate direction and/or with a reasonable pose, compound 9a was synthesized, having two longer and more flexible separated rings: a 1,4-disubstituted phenyl ring connected to a pyrrolidine ring and a pchlorophenyl group substitution at the 2-position of pyrrolidine. Compound 9a presented a nearly 4-fold increase in biological activity, indicating that this modification was heading in the right direction, and deserved to be further optimized as a promising chemical starting point. Exploration of the Binding Motif of 9a in the P2 Pocket. Considering that the p-chlorobenzene moiety in 9a may not favor the desired shallow and broad interaction and/ or clash with the protein due to its linear configuration, we first walked the Cl atom around the phenyl ring (Table 1, 9a−9c) and surprisingly found that the o-Cl analogue 9c was 4-fold more potent, while the m-Cl analogue 9b was 1-fold less potent than the original p-Cl compound 9a. Further introduction of a F atom at another ortho position (9d) or removal of the Cl atom (9e) resulted in a slight drop in potency. Moreover, the introduction of an extra methyl group to the 2-position of pyrrolidine in 9e gave 9f with nearly 2-fold higher biochemical and cellular potencies, but 9f was not chosen for further optimization due to its synthetic challenge. Further fine-tuning the direction of the phenyl ring in 9e by replacing the fivemembered pyrrolidine with four-membered azetidine (9g) and six-membered piperidine (9h) or shifting the phenyl ring to the meta position of the pyrrolidine (9i) was proven to be insufficient according to the decrease in biochemical and/or cellular potency, indicating that the ortho position of pyrrolidine provided the most favorable direction. We then switched the two connected groups on pyrrolidine (9j), introduced an extra N atom on the benzene ring (9k), and fully saturated the benzene ring (9l); unfortunately, all these modifications decreased the biochemical and/or cellular potency. Next, we tested the compatibility of the ortho position on the phenyl ring. A smaller F atom (9m) or a phenyl ring (9n) led to a drop in potency, while a hydrophobic methyl group (9o) was tolerable. Interestingly, as the size of the alkyl group increased, the potency first improved but then worsened (9o−9v). These data demonstrated that a relatively small alkyl group formed a good hydrophobic interaction with the P2 pocket of the Bcl-2 protein, among which the compound with a cyclopropyl group (9s) was the most potent analogue. Notably, the two enantiomers of 9s were synthesized from commercially available starting materials with known absolute configurations, and the R enantiomer (R-9s) gave subnanomolar potency, which was approximately 19 times more potent than the corresponding S enantiomer (S-9s). Further extending the phenyl ring by inserting a methylene group (9w) resulted in a drop in potency. Notably, all compounds exhibited excellent selectivity for Bcl-2 over Bcl-xL based on their biochemical and/or cellular potency. Optimization of the Linker. Although R-9s had a subnanomolar IC50 in the Bcl-2 WT biochemical assay, it was still not potent enough to achieve good in vivo PD (data not shown) and has the potential for drug−drug interactions (DDIs) due to its obvious cytochrome P450 2C9 (CYP2C9) inhibition (IC50 = 0.4 μM). As mentioned previously, the linker is highly important for adjusting the pose of the left headgroup to fit favorably into the P2 pocket by regulating the distance and direction. Therefore, starting with 9s, we screened various linkers to identify a suitable and optimized pose of the inhibitor into the P2 pocket, which could theoretically lead to a boost in potency. As highlighted in Table 2, a series of compounds with diverse linker motifs were designed and https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX C synthesized. Initially, F substitution on the linker phenyl ring, as in compounds 10a and 10b, did not obviously influence the potency, while Cl substitution (10c) abolished biological activity, which may be attributed to a clash with protein by aNumber of determinations = 1; ND, not determined. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX D twisting the angle of two phenyl groups. Replacement of the phenyl linker with a pyridine ring (10d) slightly improved the biochemical activity, but its cellular potency decreased, as observed in compound 9k, indicating that lipophilicity is needed to obtain good cellular potency. Increasing the flexibility and distance by inserting one or two methylene groups on the left and/or right side of the phenyl ring, as well as tuning the direction by changing the substituted position on the phenyl ring or the ring size (10e−10j), generally showed varying degrees of reduced potency, among which compound 10h was 4-fold less potent than 9r (8.1 nM vs 2.1 nM) in the biochemical assay. To further regulate the flexibility and distance, compound 10k, bearing a relatively rigid azetidine ring, was designed, but its biochemical potency decreased to 24 nM. Compared to the flat phenyl ring, a saturated or semi- saturated ring system may orient the compound in a more suitable direction to interact with the P2 pocket. Thus, compounds with cyclohexane (10l), cyclohexene (10m), and piperidine (10n) were prepared, and they indeed showed improved biochemical potency (IC50 ∼ 1 nM) and moderate cellular potency, as expected. In light of these promising aNumber of determinations = 1; ND, not determined. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX E results, we then synthesized compounds with an extra flexible methylene group (10o−10q), among which compound 10q displayed further improved potency to 0.27 nM in the Bcl-2 biochemical assay and 48 nM in the RS4;11 cell viability assay. The aforementioned structure−activity relationship (SAR) results further confirmed that the interaction between the P2 pocket of the Bcl-2 protein and inhibitors is highly sensitive to the flexibility, length, and direction of the linker and inspired us to further modify its configuration. Consequently, a more rigid spiro linker, 7-azaspiro[3.5]nonane, was introduced to generate compound 10r with a relatively fixed pose. Surprisingly, the biochemical potency significantly improved to 0.049 nM, and the cellular potency increased to 5.8 nM. Similar to 9s, we also prepared two enantiomers of 10r from commercially available starting materials with known absolute configurations. Notably, the S enantiomer (S-10r) was ∼31- fold more potent than the corresponding R enantiomer (R10r) in the biochemical assay (0.039 nM vs 1.2 nM), which was contrary to the previous trend that the R enantiomer (R9s) was ∼19-fold more potent than the corresponding S enantiomer (S-9s) in the case of the phenyl linker (0.62 nM vs 12 nM). In addition, compound 10s, utilizing a shorter 2- azaspiro[3.3]heptane linker, also demonstrated excellent Bcl-2 inhibition, although the potency was slightly lower than that of compound 10r. We next tried to insert a methylene group between the spiro linker and pyrrolidine ring in compounds 10t and 10u, but these modifications led to an obvious decrease in potency in both the biochemical and cellular assays, as did the replacement of the spiro cyclic linker with other ring systems, such as 4-cyclobutylpiperidine (10v), 1,3′- biazetidine (10w), and octahydrocyclopenta[c]pyrrole (10x). A comprehensive screening of various spiro linkers was conducted afterward;32 however, the 7-azaspiro[3.5]nonane in 10r was still the preferred linker. Analysis of the Binding Mode. To better understand the effect of the linker and the binding mode of the newly developed Bcl-2 inhibitors, X-ray cocrystal structures of compounds S-9c and S-10r in the Bcl-2 protein were solved. Based on the overlay of the cocrystal structures with venetoclax (Figure 4a,b), the azaindole and nitrobenzene groups bind to the P4 pocket of Bcl-2 similarly. The nitrobenzene group forms face-to-face π−π stacking interactions with Tyr202, and the azaindole group forms a T-shape stacking interaction with Phe104. Additionally, the acidic sulfonamide forms a hydrogen bond with Asn143. Although the benzene linker in S-9c shares a relatively similar binding conformation with the piperazine linker of venetoclax, the chlorobenzene group displayed a different binding pose in which the chlorobenzene occupies the position of the dimethylcyclohexene ring of venetoclax and the chloro substituent points to the inner side of the P2 pocket (Figure 4a). Moreover, the unique 7-azaspiro[3.5]nonane linker in S-10r moves toward the α4 helix compared to the piperidine linker in venetoclax (Figure 4b) and leads to the cyclopropylphenyl pyrrolidine shifting away from the P2 pocket side. Thus, the cyclopropyl-modified phenyl ring forms the desired shallow and broad interaction with the P2 pocket of Bcl-2 as designed. The center phenyl ring between the cyclopropyl and pyrrolidine groups binds shallowly and forms a sulfonyl−π interaction with the side chain of Met115 (Figure 4c), which contributes to the improved selectivity over Bcl-xL (Leu rather than Met). The cyclopropyl of S-10r induces the P2 residues to adopt different conformations, including Asp111, Phe112, and Met115, which creates an extra subpocket. These extra hydrophobic interactions contribute to the better potency of S10r. In addition, the cocrystal structure also highlights the water bridge between the basic pyrrolidine nitrogen atom and backbone carbonyls of Val133, Glu136, and Leu137. Given that the molecular basis of the acquired resistance of the G101V mutant for venetoclax29 is the steric clash between the deeply inserted chlorobenzene with the key residue Glu152 pushed into the base of the P2 pocket by the bulkier valine, S10r can theoretically mitigate this repulsion by forming shallow and broad interactions with the P2 pocket of the Bcl-2 protein and thus maintain binding affinity to the G101V mutant. This hypothesis has been proven by the remarkable improvement in the biochemical potency of S-10r for Bcl-2 G101V compared to venetoclax (1.3 nM vs 25 nM, Table 3), which was further proven by the cocrystal structure of Bcl-2 G101V with the final clinical candidate, BGB-11417.33 Further Modification of the P2 Binding Motif with S10r. Inspired by the promising biological activity of S-10r to both WT Bcl-2 and the G101V mutant, we optimized the binding moiety with the P2 pocket to further improve the potency against G101V (Table 3). Considering that the determined SAR with the spiro linker may differ from that with https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX F the phenyl linker, we revisited the most favorable position on the phenyl ring by walking a Cl atom around (11a−11c), and the o-Cl analogue still presented the highest potency for both WT Bcl-2 and the G101V mutant. Replacing the Cl with the larger Br (11d) resulted in slightly higher potency. A quick screening of alkyl substituents (11e−11i) showed SARs similar to the cases with phenyl linker (9o−9v in Table 2), in which relatively small alkyl groups were compatible and the isopropyl analogue 11g had the highest potency. Next, we evaluated the tolerability of heteroatoms at the ortho position, and generally, oxygen (11j−11m) and nitrogen (11n and 11o) were well tolerated in the biochemical assay but not in the cellular assay, except for the N,N-dimethylamino group (11n), which showed comparable potency to S-10r and 11g. To identify the optimal ortho substituent among cyclopropyl (S-10r), isopropyl (11g), and N,N-dimethylamino (11n), we further compared their liver microsomal (LM) stabilities and CYP inhibition profiles, as shown in Table 4. The isopropyl analogue 11g stood out for its more balanced LM stability in terms of all evaluated species and the lowest CLint in humans in addition to the weakest inhibition of CYP2C9, although these properties remained to be improved in further optimization. Optimization of the Right Tail of 11g. To reduce the risk of DDIs and further improve the potency for both WT Bcl-2 and the G101V mutant, various right tail segments were screened based on the lead compound 11g (Table 5). Other oxa fused ring systems (12a and 12b), methylpiperidine moiety (12c), and difluorocyclohexane moiety (12d) gave slightly lower biochemical and/or cellular potency. When the oxygen of the tetrahydropyran in 11g was placed out of the ring with an additional methyl group, we surprisingly found that the trans isomer 12e, also known as BGB-11417, displayed an improved IC50 against Bcl-2 WT and G101V to 0.019 and 0.34 nM in the biochemical assay, respectively, while the cis isomer 12f provided lower potency for G101V. Notably, introduction of the tertiary hydroxy group reduces the DDI risk due to its noninhibition of the five major CYP enzymes: 1A2, 2C9, 2C19, 2D6, and 3A4. Further methylation of the hydroxy group of 12e to generate 12g slightly diminished the potency. When 1,4-dioxane was incorporated as the right tail, we interestingly found that the S enantiomer (12h) did not show obvious inhibition of CYP2C9, while the R enantiomer (12i) moderately inhibited CYP2C9 (IC50 = 5.44 μM); however, they presented similar potencies. Next, we tried to fine-tune the flexibility and configuration of the right tail via cyclization of the side chain on the nitrophenyl ring (12j− 12m); unfortunately, neither the five- nor six-membered fused rings gave better results relative to 12e. Therefore, 12e was selected for further optimization of the top azaindole, but the preliminary SAR study demonstrated that the current oxygenlinked 7-azaindole was still optimal.32 As illustrated in our paper recently published in Blood,33 we further confirmed that 12e maintained a strong interaction with the G101V mutant with a surface plasmon resonance (SPR) assay (KD = 0.24 nM), while the KD of venetoclax for the mutant dropped dramatically to 29 nM. The superiority of 12e to venetoclax was also demonstrated by its significantly higher potency in the pairwise Bcl-2:BIM disruption assay and aNumber of determinations = 1. bNumber of determinations = 6. cNumber of determinations = 3. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX G cell viability assay in RS4;11 G101V KI (knock-in) cells and remarkably better PD and efficacy in RS4;11 Bcl-2 G101V xenografts. The robust interactions of 12e with other mutations (e.g., D103Y, V156D, A113G, and R129L) on SPR assay suggest 12e may cover venetoclax resistance mutations. Besides the data published in Blood, we also compared 12e with other clinical Bcl-2 selective inhibitors including venetoclax, S65487, lisaftoclax, and lacutoclax in both biochemical assay and cellular assay (Table 6). In the WT Bcl-2 biochemical assay, 12e showed 7−63-fold more potent than the other clinical Bcl-2 inhibitors, which was consistent with the 8−21-fold greater potency in the RS4;11 cell viability assay. Furthermore, 12e exhibited 16−168-fold more potent in the Bcl-2 G101V mutant biochemical assay and 16−27-fold more potent in Bcl-2 G101V knock-in the RS4;11 cell viability assay than the other clinical Bcl-2 inhibitors, suggesting that 12e might also be effective for the patients with Bcl-2 G101V mutation. In Vitro Druglike Properties of the Novel Bcl-2 Inhibitors 12e and 12h. The druglike properties of lead compounds 12e and 12h were assessed as summarized in Table 7. In addition to the low DDI risk indicated by the aNumber of determinations = 1. bNumber of determinations = 6. cNumber of determinations = 3; ND, not determined. aHead-to-head data; number of determinations = 6. bNumber of determinations = 3. aAbbreviations: AUCinf, area under the concentration−time curve from time 0 to infinity; AUClast, AUC from time 0 to the last quantifiable concentration; CLint, intrinsic clearance; CL, clearance; Cmax, maximum plasma concentration; F%, absolute oral bioavailability; iv, intravenous; NA, not applicable; po, oral administration; T1/2, elimination half-life; PK, pharmacokinetics; Tmax, time to reach Cmax; and Vss, volume of distribution at steady state. PK data are presented as the mean value of three animals. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX H noninhibition of various CYP enzymes as discussed previously, 12e also demonstrated good in vitro liver microsomal stability in all four common species, among which the CLint in mice remained the highest, and 12h exhibited slightly better human LM stability but worse rat and mouse LM stability. The pharmacokinetics (PK) in animals were then further studied. The dose for intravenous (iv) injection was 1 mg/kg in both mice and dogs, and the dose for oral administration (po) was 10 mg/kg in mice and 5 mg/kg in dogs. Overall, both 12e and 12h exhibited good and comparable PK profiles in both mice and dogs. Thanks to the lower intrinsic clearance from dogs than from mice (28.9 vs 48.7 for 12e; 28.5 vs 81.4 for 12h), the in vivo clearance from dogs was much lower than that from mice. Consequently, the Cmax and AUC were much higher in dogs than in mice, as was the bioavailability for oral administration. In Vivo PD and Efficacy Studies. Oral administration of single doses of compounds 12e and 12h demonstrated robust induction of cleaved caspase-3 expression in tumors due to the outstanding in vitro potency as well as the acceptable exposure in both plasma and tumors, as shown in Figure 5. The proapoptotic activities of compounds 12e and 12h were apparently higher than that of venetoclax at 4 and 8 h posttreatment. It is not surprising to observe a faster descent of the PD effect of either compound at 24 h, which is related to the relatively sharper reduction in tumor exposure compared to venetoclax. Compared to 12h, compound 12e presented slightly better performance in terms of caspase activation with a higher AUCPD 0−24 h (p = 0.0873). This result is reasonable and related to the higher exposure of compound 12e in the plasma and tumor as well as the slightly better in vitro potency described above. The in vivo efficacies of compounds 12e and 12h were then evaluated in RS4;11-bearing NCG mice and compared with that of venetoclax. Daily oral gavage of both compounds at 7.5 and 25 mg/kg was well tolerated and exhibited significant antitumor activity. As shown in Figure 6, although all three compounds showed good efficacy at 25 mg/kg, the great advantage of compound 12e was exhibited at the lower dose of 7.5 mg/kg. Every individual animal treated with 7.5 mg/kg compound 12e showed complete tumor regression, with a mean tumor growth inhibition (TGI) value of 103% at day 21 posttreatment, while venetoclax displayed only moderate tumor growth inhibition (TGI of 86%). A prime reason for the insufficient efficacy of venetoclax in a phase I clinical trial in patients with non-Hodgkin lymphoma (NHL)34 may be its moderate potency, which was suggested by an exposure− efficacy correlation revealed from logistic regression analysis of the probability of response performance, although the result was not statistically significant.35 With significantly improved potency, compound 12e was also tested in venetoclaxinsensitive models and showed remarkably better PD and efficacy than venetoclax in DLBCL (Toledo) xenograft models and RS4;11 Bcl-2 G101V xenograft models.33 These results suggest that 12e is highly effective in treating hematological cancers, including certain indications of NHL, and potentially overcomes the Bcl-2 G101V mutation and possibly other mutations as well. ■ CHEMISTRY The synthetic routes for compounds 9a−9i and 9k−9w are outlined in Scheme 1. Buchwald−Hartwig reaction of secondary amines 13a−13v, which were commercially available or readily prepared using literature procedures, with 4-bromoiodobenzene gave rise to 14a−14v in the presence of Pd2(dba)3, BINAP, and t-BuOK. Suzuki−Miyaura reactions of https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX I 14a−14v with Pd(dppf)Cl2 as catalyst afforded key intermediates 15a−15v. The tert-butyl ester groups in 15a−15v were removed by TFA in DCM to give carboxylic acids 16a− 16v. Final condensation reactions of 16a−16v with 3-nitro-4( ( ( t e t r a h y d r o - 2H - p y r a n - 4 - y l )m e t h y l ) am i n o ) - benzenesulfonamide were performed in the presence of HATU or EDCI to deliver the desired products 9a−9i and 9k−9w. The synthetic routes for compounds 10r, 11a−11o, and 12a−12m are outlined in Scheme 2. Reductive amination of 17 with different secondary amines 18a−18p, which were commercially available or readily prepared using literature procedures, in the presence of NaBH(OAc)3 gave rise to 19a− 19p. Compounds 19a−19p were hydrolyzed in the presence of NaOH solution to give acids 20a−20p. Condensation reactions of 20a−20p with the corresponding sulfonamides were performed in the presence of HATU or EDCI to deliver the desired products 10r, 11a−11o, and 12a−12m. For the compounds in Table 2, the synthetic routes were similar to those in Scheme 1 and 2,32 and please refer to the Supporting Information for more details. ■ CONCLUSION In summary, we developed a highly potent and selective Bcl-2 inhibitor, BGB-11417 (12e, sonrotoclax), based on the rational design to form a shallow and broad interaction with the P2 pocket of Bcl-2 protein. Our initial efforts identified that a novel fragment of phenylpyrrolidine with an alkyl substitution at an ortho position can interact shallowly and broadly with the P2 pocket as expected, revealed by the cocrystal structures with Bcl-2. The following screening of the linker led to a rigid spiro linker, 7-azaspiro[3.5]nonane, which provided more favorable interactions with the P2 pocket of Bcl-2 protein and thus presented a boost of potency. Guided by structure−property relationship study to improve the druglike properties, such as CYP2C9 inhibition and in vivo PD/efficacy, comprehensive optimization resulted in BGB-11417 with (1r,4r)-4-(aminomethyl)-1-methylcyclohexan-1-ol moiety in the right tail part and 7-azaindole in the top part. BGB-11417 showed superior in vitro activity than venetoclax on the inhibition of both WT Bcl-2 and the G101V mutant, as well as other mutations, with better selectivity over Bcl-xL. The superiority of BGB-11417 against venetoclax was further demonstrated by its significantly greater efficacy in xenograft mouse models. Currently, BGB11417 has shown an excellent safety profile in phase I/II clinical trials and entered phase III to further evaluate its efficacy and safety as monotherapy and combination treatment in CLL, NHL, and other patients of hematologic malignancy. ■ EXPERIMENTAL METHODS Chemistry. All solvents and chemical used were reagent grade. Unless indicated otherwise, the reactions set forth below were performed under a positive pressure of nitrogen or argon or with a drying tube in anhydrous solvents; the reaction flasks were fitted with rubber septa for the introduction of substrates and reagents via syringe; and glassware was oven-dried and/or heat-dried. 1H NMR spectra were recorded on a Bruker spectrometer operating at 400 MHz. 1H NMR spectra were obtained using CDCl3, CD3OD, D2O, and DMSO-d6 as solvents and tetramethylsilane (0.00 ppm) or residual solvent (CDCl3, 7.26 ppm; CD3OD, 3.31 ppm; D2O, 4.79 Scheme 1. General Syntheses of Compounds 9a−9i and 9k−9wa aReagents and conditions: (a) 4-bromoiodobenzene, Pd2(dba)3, BINAP, t-BuOK; (b) Pd(dppf)Cl2, Cs2CO3 or K2CO3, toluene or 1,4-dioxane; (c) TFA, DCM; (d) 3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)benzenesulfonamide, HATU or EDCI, TEA, DMAP, DCM. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX J ppm; DMSO-d6, 2.50 ppm) as the reference standard. Coupling constants, when given, are reported in hertz (Hz). Preparative HPLC was conducted on a column (150 × 21.2 mm i.d., 5 μm, Gemini NXC18) at a flow rate of 20 mL/min and injection volume of 2 mL, at room temperature, and with UV detection at 214 and 254 nm. The purities of all final compounds were determined to be above 95% by high performance liquid chromatography (HPLC) or LC−MS, except for 9a, 9k, 9r, 9q, 9t, 9w, 10r, 11f, 11g, 11h, 11k, and 12l (88.6− 94.9% purity). HPLC conditions were as follows: Gemini C18 column at room temperature, 4.6 cm × 150 cm, 5 μm, 10−90% acetonitrile (0.05% TFA)/water (0.05% TFA), 10 min run, flow rate 1 mL/min, and UV detection (λ = 214 nm, 254 and 280 nm). LC− MS conditions were as follows: LC−MS spectrometer (Agilent 1260) detector, MWD (190−400 nm); mass detector, 6120 SQ; mobile phase A, acetonitrile with 0.1% formic acid; mobile phase B, water with 0.1% formic acid; column, Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7 μm; gradient method, 5−95% B in 1.5 min, 95% B, 0.5 min, 95− 5% B, 2.0−2.1 min, 5% B, 2.1−3.0 min; flow rate, 1.8 mL/min. Accurate mass was determined by analysis of samples on a calibrated Waters Acquity UPLC Xevo G2 QToF-MS. General Procedure for the Preparation of 9a−9w and 10a− 10k, Exemplified by 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(2-phenylpyrrolidin-1-yl)-[1,1′-biphenyl]4-carboxamide (9e). Step 1. To a solution of 2-phenylpyrrolidine (588 mg, 4.0 mmol), 1-bromo-4-iodobenzene (1.1 g, 16.0 mmol), BINAP (497 mg, 0.8 mmol), and t-BuOK (1.2 g, 12.0 mmol) in toluene (25 mL) was added Pd2(dba)3 (366 mg, 0.40 mmol). The resulting mixture was bubbled with N2 for 5 min and then stirred overnight at 90 °C. The reaction was cooled to ambient temperature, the organic solvent was washed with water and brine in sequence, dried over anhydrous Na2SO4, and concentrated, and the residue was purified by column chromatography on silica gel (100−200 mesh; eluent, petroleum ether:EtOAc from 20:1 to 5:1) to give 14e as a colorless oil (750 mg, 62.0%). MS (ESI, m/z) [M + H]+: 302.0, 304.1. Step 2. To a mixture of 1-(4-bromophenyl)-2-phenylpyrrolidine 14e (525 mg, 1.74 mmol) in 1,4-dioxane (50 mL) and H2O (10 mL) was added tert-butyl 2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (985 mg, 2.26 mmol), Pd(dppf)Cl2 (128 mg, 0.17 mmol), and K2CO3 (480 mg, 3.48 mmol); the mixture was stirred at 90 °C under N2 atmosphere for 16 h. The reaction was cooled to ambient temperature and concentrated under reduced pressure. EtOAc (20 mL) was added to the residue, the organic phase was washed with water (5.0 mL) and brine (5.0 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (100−200 mesh; eluent, petroleum ether:EtOAc from 9:1 to 1:1) to give 15e as a white foamed solid (530 mg, 57.4%). MS (ESI, m/z) [M + H]+ 532.3. Step 3. To a solution of tert-butyl-3-((1H-pyrrolo[2,3-b]pyridin-5yl)oxy)-4′-(2-phenylpyrrolidin-1-yl)-[1,1′-biphenyl]-4-carboxylate 15e (531 mg, 1.00 mmol) in DCM (25 mL) was added trifluoroacetic acid (5.0 mL). The reaction mixture was stirred at ambient temperature for 16 h. Then the solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (100−200 mesh; eluent, 5.0% methanol in DCM) to give 16e as a white foamed solid (400 mg, 84.2%). MS (ESI, m/z) [M + H]+: 476.2. Step 4. To a solution of 3-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′(2-phenylpyrrolidin-1-yl)-[1,1′-biphenyl]-4-carboxylic acid 16e (95 mg, 0.20 mmol) in DCM (25 mL) was added HATU (114 mg, 0.30 mmol) and Et3N (0.20 mL). The mixture was stirred at ambient temperature for 30 min, and then 3-nitro-4-(((tetrahydro-2H-pyran4-yl)methyl)amino)benzenesulfonamide (126 mg, 0.40 mmol) was added. The mixture was stirred at ambient temperature for 16 h. The reaction solution was washed with water (10 mL), dried over Scheme 2. General Syntheses of Compounds 10r, 11a−11o, and 12a−12ma aReagents and conditions: (a) NaBH(OAc)3, DCM; (b) 3 N NaOH, MeOH, THF; (c) sulfonamide intermediate, HATU or EDCI, TEA, DMAP, DCM. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX K anhydrous Na2SO4, and then concentrated in a vacuum. The residue was purified by prep-HPLC to give 9e as a yellow solid (31 mg, 20.0%). 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.67 (s, 1H), 8.62−8.48 (m, 2H), 8.03 (s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.59−7.46 (m, 3H), 7.33−7.25 (m, 5H), 7.20−7.14 (m, 3H), 7.08 (d, J = 8.0 Hz, 1H), 6.89 (s, 1H), 6.42 (d, J = 8.8 Hz, 2H), 6.37 (s, 1H), 4.77 (d, J = 7.2 Hz, 1H), 3.89−3.79 (m, 2H), 3.72−3.62 (m, 1H), 3.40−3.20 (m, 54H), 2.42−2.30 (m, 1H), 2.00−1.83 (m, 3H), 1.82− 1.72 (m, 1H), 1.65−1.52 (m, 2H), 1.38−1.14 (m, 2H). MS (ESI, m/ z) [M + H]+: 773.3. HPLC purity: 99.6% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(4-chlorophenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9a). Yield: 14.6%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.70 (s, 1H), 8.65−8.57 (m, 1H), 8.56 (d, J = 2.2 Hz, 1H), 8.05 (d, J = 2.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.64−7.48 (m, 3H), 7.38− 7.24 (m, 5H), 7.18 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 9.2 Hz, 1H), 6.89 (s, 1H), 6.46−6.34 (m, 3H), 4.78 (d, J = 7.2 Hz, 1H), 3.90−3.78 (m, 2H), 3.72−3.62 (m, 1H), 3.32−3.18 (m, 5H), 2.42−2.30 (m, 1H), 2.00−1.82 (m, 3H), 1.81−1.71 (m, 1H), 1.64−1.54 (m, 2H), 1.34− 1.16 (m, 2H). MS (ESI, m/z) [M + H]+: 807.1. HPLC purity: 93.3% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(3-chlorophenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9b). Yield: 37.9%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.70 (s, 1H), 8.64−8.58 (m, 1H), 8.57 (s, 1H), 8.05 (s, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.59 (s, 1H), 7.57−7.46 (m, 2H), 7.40−7.17 (m, 6H), 7.13 (d, J = 8.4 Hz, 2H), 6.90 (s, 1H), 6.43 (d, J = 8.4 Hz, 2H), 6.38 (s, 1H), 4.79 (d, J = 7.6 Hz, 1H), 3.90−3.78 (m, 2H), 3.75−3.64 (m, 1H), 3.31−3.18 (m, 5H), 2.42−2.28 (m, 1H), 2.02−1.72 (m, 4H), 1.66−1.54 (m, 2H), 1.32−1.16 (m, 2H). MS (ESI, m/z) [M + H]+: 807.1. HPLC purity: 98.1% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(2-chlorophenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9c). Yield: 8.9%, yellow solid. Compound 9c was separated two enantiomeric stereoisomers, S-9c (peak 1, S enantiomer, retention time at 39.4 min in chiral analysis, the absolute configuration was confirmed by cocrystal structure of Bcl-2) and R-9c (peak 2, R enantiomer, retention time at 42.4 min in chiral analysis), by chiral prep-HPLC (column, CHIRALPAK IG; column size, 2 cm × 25 cm; mobile phase, CO2:[DCM:EtOH (0.1% DEA) = 1:2] = 55:45; flow rate, 40 mL/min; wavelength, UV 220 nm). 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.70 (s, 1H), 8.65−8.55 (m, 2H), 8.06−8.02 (m, 1H), 7.83 (d, J = 9.4 Hz, 1H), 7.63−7.57 (m, 1H), 7.56−7.49 (m, 2H), 7.49−7.43 (m, 1H), 7.37−7.27 (m, 3H), 7.27−7.16 (m, 2H), 7.16−7.10 (m, 1H), 7.03−6.97 (m, 1H), 6.92−6.87 (m, 1H), 6.40− 6.30 (m, 3H), 4.97 (d, J = 7.2 Hz, 1H), 3.88−3.80 (m, 2H), 3.78− 3.70 (m, 1H), 3.31−3.19 (m, 5H), 2.43−2.35 (m, 1H), 2.06−1.94 (m, 1H), 1.93−1.75 (m, 3H), 1.65−1.55 (m, 2H), 1.33−1.15 (m, 2H). MS (ESI, m/z) [M + H]+: 807.1. HPLC purity (9c): 97.6% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(2-chloro-6fluorophenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9d). Yield: 1.5%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.70 (s, 1H), 8.64−8.58 (m, 1H), 8.56 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 2.2 Hz, 1H), 7.85−7.79 (m, 1H), 7.58 (d, J = 2.2 Hz, 1H), 7.54−7.46 (m, 2H), 7.35−7.24 (m, 5H), 7.15−7.05 (m, 2H), 6.90 (s, 1H), 6.40−6.30 (m, 3H), 5.26−5.20 (m, 1H), 3.87− 3.78 (m, 2H), 3.55−3.50 (m, 1H), 3.30−3.15 (m, 5H), 2.35−2.30 (m, 1H), 2.10−1.92 (m, 4H), 1.90−1.80 (m, 1H), 1.63−1.55 (m, 2H), 1.33−1.15 (m, 2H). MS (ESI, m/z) [M + H]+: 825.1. HPLC purity: 96.1% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-methyl-2-phenylpyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9f). Yield: 22.2%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.70 (s, 1H), 8.66−8.58 (m, 1H), 8.57 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.84−7.78 (m,, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.54− 7.46 (m, 2H), 7.35−7.24 (m, 3H), 7.24−7.10 (m, 6H), 6.87 (s, 1H), 6.40−6.36 (m, 1H), 6.32 (d, J = 8.8 Hz, 1H), 3.90−3.78 (m, 2H), 3.64−3.52 (m, 2H), 3.30−3.20 (m, 4H), 2.18−2.06 (m, 1H), 2.00− 1.91 (m, 2H), 1.90−1.78 (m, 2H), 1.71 (s, 3H), 1.65−1.54 (m, 2H),1.30−1.20 (m, 2H). MS (ESI, m/z) [M + H]+: 787.1. HPLC purity: 99.1% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(2-phenylazetidin-1-yl)-[1,1′-biphenyl]-4-carboxamide (9g). Yield: 4.9%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.38 (d, J = 1.6 Hz, 1H), 8.36−8.28 (m, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.66− 7.58 (m, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.45−7.40 (m, 1H), 7.29 (d, J = 2.4 Hz, 1H), 7.26−7.18 (m, 2H), 7.16−7.08 (m, 3H), 7.05 (d, J = 7.2 Hz, 2H), 6.85−6.73 (m, 3H), 6.54 (d, J = 8.4 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 6.07 (s, 1H), 4.15−4.07 (m, 1H), 3.90−3.78 (m, 2H), 3.30−3.12 (m, 5H), 3.06−2.94 (m, 1H), 2.06−1.94 (m, 1H), 1.94− 1.76 (m, 2H), 1.66−1.54 (m, 2H), 1.32−1.16 (m, 3H). MS (ESI, m/ z) [M + H]+: 758.8. HPLC purity: 98.4% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(2-phenylpiperidin-1-yl)-[1,1′-biphenyl]-4-carboxamide (9h). Yield: 31.8%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 11.63 (s, 1H), 8.53−8.43 (m, 2H), 8.05−7.97 (m, 1H), 7.80−7.70 (m, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.54−7.44 (m, 2H), 7.35−7.27 (m, 3H), 7.25−7.17 (m, 4H), 7.15−7.07 (m, 1H), 7.05−6.93 (m, 1H), 6.93−6.82 (m, 3H), 6.37−6.32 (m, 1H), 4.85−4.73 (m, 1H), 3.90− 3.78 (m, 2H), 3.46−3.36 (m, 1H), 3.32−3.18 (m, 5H), 1.98−1.80 (m, 3H), 1.76−1.55 (m, 4H), 1.55−1.38 (m, 2H), 1.30−1.20 (m, 2H). MS (ESI, m/z) [M + H]+: 787.1. HPLC purity: 95.2% (214 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(3-phenylpyrrolidin-1-yl)-[1,1′-biphenyl]-4-carboxamide (9i). Yield: 24.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.72 (s, 1H), 8.65−8.59 (m, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.09 (d, J = 2.8 Hz, 1H), 7.87−7.80 (m, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.54−7.50 (m, 1H), 7.44−7.34 (m, 3H), 7.34−7.28 (m, 4H), 7.26−7.18 (m, 1H), 7.14 (d, J = 9.2 Hz, 1H), 6.93 (d, J = 1.2 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 6.42−6.38 (m, 1H), 3.90−3.80 (m, 2H), 3.74−3.64 (m, 1H), 3.55−3.40 (m, 2H), 3.40−3.20 (m, 5H), 2.42−2.30 (m, 1H), 2.14−2.00 (m, 1H), 1.95−1.80 (m, 1H), 1.66− 1.54 (m, 2H), 1.34−1.16 (m, 2H). MS (ESI, m/z) [M + H]+: 773.1. HPLC purity: 96.0% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(1-phenylpyrrolidin-2-yl)-[1,1′-biphenyl]-4-carboxamide (9j). Yield: 26.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 11.73 (s, 1H), 8.64−8.59 (m, 1H), 8.57 (d, J = 2.0 Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.84 (dd, J = 9.0, 2.4 Hz, 1H), 7.65−7.57 (m, 2H), 7.54− 7.49 (m, 1H), 7.48−7.40 (m, 3H), 7.22 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 9.6 Hz, 1H), 7.06−6.96 (m, 3H), 6.54−6.47 (m, 1H), 6.41−6.34 (m, 3H), 4.76−4.68 (m, 1H), 3.90−3.78 (m, 2H), 3.70−3.60 (m, 1H), 3.30−3.18 (m, 5H), 2.42−2.28 (m, 1H), 2.00−1.80 (m, 2H), 1.80−1.70 (m, 1H), 1.64−1.54 (m, 2H), 1.32−1.16 (m, 2H). MS (ESI, m/z) [M + H]+: 773.1. HPLC purity: 98.7% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(2-(pyridin-3yl)pyrrolidin-1-yl)-[1,1′-biphenyl]-4-carboxamide (9k). Yield: 4.5%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 11.72 (s, 1H), 8.68−8.52 (m, 4H), 8.06 (d, J = 1.6 Hz, 1H), 8.03−7.96 (m, 1H), 7.86−7.80 (m, 1H), 7.73−7.64 (m, 1H), 7.61 (d, J = 2.0 Hz, 1H), 7.58−7.48 (m, 2H), 7.38−7.26 (m, 3H), 7.14 (d, J = 9.2 Hz, 1H), 6.87 (s, 1H), 6.47 (d, J = 8.8 Hz, 2H), 6.42−6.33 (m, 1H), 5.00−4.93 (m, 1H), 3.90−3.80 (m, 2H), 3.79−3.69 (m, 1H), 3.43− 3.33 (m, 1H), 3.32−3.18 (m, 4H), 2.46−2.38 (m, 1H), 2.06−1.94 (m, 1H), 1.94−1.80 (m, 2H), 1.65−1.52 (m, 2H), 1.34−1.18 (m, 2H). MS (ESI, m/z) [M + H]+: 774.2. HPLC purity: 94.6% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-cyclohexylpyrrolidin1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9l). Yield: 20.0%, https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX L yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.64 (s, 1H), 8.57−8.42 (m, 2H), 8.03 (s, 1H), 7.80−7.64 (m, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.55−7.44 (m, 2H), 7.36−7.34 (m, 3H), 6.92 (s, 1H), 6.57 (d, J = 8.4 Hz, 2H), 6.37 (s, 1H), 3.88−3.80 (m, 2H), 3.65−3.58 (m, 1H), 3.45−3.36 (m, 1H), 3.30−3.20 (m, 4H), 3.10− 3.03 (m, 1H), 1.90−1.82 (m, 2H), 1.72−1.55 (m, 7H), 1.47−1.41 (m, 1H), 1.40−1.32 (m, 1H), 1.30−1.23 (m, 3H), 1.20−1.10 (m, 1H), 1.06−0.95 (m, 3H), 0.95−0.85 (m, 2H). MS (ESI, m/z) [M + H]+: 779.2. HPLC purity: 96.4% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(2-fluorophenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9m). Yield: 7.7%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 11.67 (s, 1H), 8.60−8.46 (m, 2H), 8.03 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.54−7.45 (m, 3H), 7.36−7.28 (m, 3H), 7.28−7.15 (m, 2H), 7.10−7.01 (m, 2H), 7.00−6.94 (m, 1H), 6.90 (s, 1H), 6.42 (d, J = 8.4 Hz, 2H), 6.36 (s, 1H), 4.97 (d, J = 7.6 Hz, 1H), 3.90−3.78 (m, 2H), 3.74−3.62 (m, 1H), 3.35−3.20 (m, 5H), 2.45−2.30 (m, 1H), 2.05− 1.95 (m, 1H), 1.94−1.78 (m, 2H), 1.65−1.54 (m, 2H), 1.32−1.15 (m, 2H). MS (ESI, m/z) [M + H]+: 791.1. HPLC purity: 99.2% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-([1,1′-biphenyl]-2yl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9n). Yield: 19.6%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.62 (s, 1H), 8.60−8.35 (m, 2H), 8.04−7.94 (m, 1H), 7.80−7.65 (m, 1H), 7.56−7.46 (m, 4H), 7.46−7.36 (m, 4H), 7.32− 7.23 (m, 5H), 7.22−7.16 (m, 2H), 7.12−7.06 (m, 1H), 6.90 (s, 1H), 6.36−6.30 (m, 3H), 4.64−4.58 (m, 1H), 3.87−3.80 (m, 2H), 3.74− 3.66 (m, 1H), 3.32−3.20 (m, 5H), 2.18−2.06 (m, 1H), 2.05−1.95 (m, 2H), 1.93−1.85 (m, 1H), 1.84−1.74 (m, 1H), 1.64−1.54 (m, 2H), 1.30−1.20 (m, 2H). MS (ESI, m/z) [M + H]+: 849.1. HPLC purity: 100% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4′-(2-(o-tolyl)pyrrolidin-1-yl)-[1,1′-biphenyl]-4-carboxamide (9o). Yield: 13.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.39 (d, J = 1.2 Hz, 1H), 8.37−8.30 (m, 1H), 7.95 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 7.09 (dd, J = 7.2, 7.2 Hz, 1H), 7.02 (dd, J = 7.2, 7.2 Hz, 1H), 6.92−6.79 (m, 3H), 6.34 (d, J = 8.6 Hz, 2H), 6.29 (d, J = 2.8 Hz, 1H), 4.86 (d, J = 7.6 Hz, 1H), 3.88− 3.78 (m, 2H), 3.76−3.64 (m, 1H), 3.34−3.14 (m, 5H), 2.45−2.35 (m, 4H), 2.03−1.93 (m, 1H), 1.93−1.80 (m, 2H), 1.76−1.66 (m, 1H), 1.64−1.54 (m, 2H), 1.32−1.12 (m, 2H). MS (ESI, m/z) [M + H]+: 787.1. HPLC purity: 97.8% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(2-ethylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9p). Yield: 29.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.69 (s, 1H), 8.65−8.53 (m, 2H), 8.04 (d, J = 2.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.60−7.46 (m, 3H), 7.36−7.26 (m, 3H), 7.22 (d, J = 7.6 Hz, 1H), 7.17−7.07 (m, 2H), 7.01 (dd, J = 7.4, 7.4 Hz, 1H), 6.89 (s, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.41−6.32 (m, 3H), 4.91 (d, J = 7.6 Hz, 1H), 3.88−3.79 (m, 2H), 3.75−3.65 (m, 1H), 3.42−3.35 (m, 1H), 3.31−3.18 (m, 4H), 2.86−2.74 (m, 1H), 2.74−2.62 (m, 1H), 2.46−2.36 (m, 1H), 2.03−1.92 (m, 2H), 1.92−1.80 (m, 2H), 1.75− 1.65 (m, 1H), 1.64−1.54 (m,2H), 1.33−1.17 (m, 5H). MS (ESI, m/ z) [M + H]+: 801.2. HPLC purity: 99.0% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(2-isopropylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9q). Yield: 15.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.69 (s, 1H), 8.64−8.54 (m, 2H), 8.04 (d, J = 2.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.62−7.46 (m, 3H), 7.38−7.26 (m, 4H), 7.20−7.06 (m, 2H), 7.01−6.95 (m, 1H), 6.91 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.42−6.30 (m, 3H), 4.98 (d, J = 8.4 Hz, 1H), 3.88−3.80 (m, 2H), 3.75−3.65 (m, 1H), 3.40−3.20 (m, 5H), 2.45−2.38 (m, 1H), 2.02− 1.92 (m, 2H), 1.90−1.80 (m, 1H), 1.74−1.66 (m, 1H), 1.63−1.55 (m, 2H), 1.32−1.16 (m, 8H). MS (ESI, m/z) [M + H]+: 815.2. HPLC purity: 94.4% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-4′-(2-(2-(tert-butyl)phenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9r). Yield: 6.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.67 (s, 1H), 8.65−8.45 (m, 2H), 8.01 (s, 1H), 7.83− 7.70 (m, 1H), 7.55−7.46 (m, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.35− 7.25 (m, 3H), 7.20−7.10 (m, 2H), 7.07−7.00 (m, 2H), 6.92 (s, 1H), 6.69−6.62 (m, 1H), 6.41 (d, J = 8.4 Hz, 2H), 6.38−6.32 (m, 1H), 5.25 (d, J = 8.0 Hz, 1H), 3.88−3.80 (m, 2H), 3.76−3.68 (m, 1H), 3.48−3.23 (m, 5H), 2.03−1.93 (m, 2H), 1.92−1.83 (m, 1H), 1.82− 1.72 (m, 1H), 1.64−1.56 (m, 2H), 1.50 (s, 9H), 1.35−1.25 (m, 2H). MS (ESI, m/z) [M + H]+: 828.8. HPLC purity: 94.9% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9s). Yield: 38.7%, yellow solid. Compound 9s was separated into two enantiomeric stereoisomers, R-9s (peak 1, R enantiomer, retention time at 10.4 min in chiral analysis) and S-9s (peak 2, S, retention time at 11.4 min in chiral analysis), by chiral prep-HPLC (column, CHIRAL ART Cellulose-SB; column size, 2 cm × 25 cm; mobile phase, (hexane:DCM = 5:1)(0.1% FA):EtOH = 50:50; flow rate, 20 mL/min; wavelength, UV 220 nm). The two enantiomers of 9s were confirmed and synthesized from commercially available starting materials with known absolute configurations. 1H NMR (400 MHz, CDCl3) δ 10.31 (s, 1H), 9.14 (s, 1H), 8.93 (d, J = 2.0 Hz, 1H), 8.57−8.50 (m, 1H), 8.24 (d, J = 2.2 Hz, 1H), 8.19 (dd, J = 9.1, 1.5 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 2.2 Hz, 1H), 7.45−7.41 (m, 1H), 7.30−7.27 (m, 1H), 7.20 (d, J = 8.6 Hz, 2H), 7.15−7.08 (m, 1H), 7.06−6.97 (m, 2H), 6.95−6.90 (m, 2H), 6.81 (s, 1H), 6.55−6.50 (m, 1H), 6.36 (d, J = 8.6 Hz, 2H), 5.20 (d, J = 8.0 Hz, 1H), 4.08−3.98 (m, 2H), 3.77−3.67 (m, 1H), 3.49−3.37 (m, 3H), 3.32−3.23 (m, 2H), 2.49−2.35 (m, 1H), 2.04−1.91 (m, 5H), 1.78−1.69 (m, 2H), 1.50−1.37 (m, 2H), 1.06−0.94 (m, 2H), 0.86− 0.79 (m, 1H), 0.73−0.66 (m, 1H). MS (ESI, m/z) [M + H]+: 813.1. HPLC purity (9s): 95.4% (214 nm). HPLC purity (R-9s): 98.4%. HPLC purity (S-9s): 98.3%. 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclobutylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9t). Yield: 2.2%, yellow solid. 1H NMR (CDCl3) δ 10.24 (s, 1H), 9.53 (s, 1H), 8.92 (s, 1H), 8.60−8.45 (m, 1H), 8.28−8.15 (m, 2H), 8.08 (d, J = 8.4 Hz, 1H), 7.79 (s, 1H), 7.50−7.46 (m, 1H), 7.36−7.26 (m, 2H), 7.24−7.14 (m, 3H), 7.06−6.98 (m, 1H), 6.96− 6.89 (m, 2H), 6.79 (s, 1H), 6.60−6.51 (m, 1H), 6.34 (d, J = 8.4 Hz, 2H), 4.86 (d, J = 8.0 Hz, 1H), 4.09−3.96 (m, 2H), 3.82−3.63 (m, 2H), 3.46−3.38 (m, 2H), 3.31−3.21 (m, 2H), 2.40−2.18 (m, 2H), 2.05−1.80 (m, 5H), 1.78−1.66 (m, 3H), 1.50−1.39 (m, 2H), 1.35− 1.20 (m, 3H). MS (ESI, m/z) [M + H]+: 826.8. HPLC purity: 92.7% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclopentylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9u). Yield: 17.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.70 (s, 1H), 8.64−8.52 (m, 2H), 8.04 (d, J = 2.0 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.60−7.46 (m, 3H), 7.36−7.26 (m, 4H), 7.18−7.08 (m, 2H), 6.98 (m, 1H), 6.91 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.40−6.30 (m, 3H), 5.02 (d, J = 7.6 Hz, 1H), 3.88−3.80 (m, 2H), 3.74−3.66 (m, 1H), 3.31−3.20 (m, 5H), 2.45−2.42 (m, 1H), 2.21−2.13 (m, 1H), 2.02−1.92 (m, 2H), 1.92− 1.76 (m, 4H), 1.76−1.63 (m, 4H), 1.62−1.56 (m, 3H), 1.29−1.20 (m, 3H). MS (ESI, m/z) [M + H]+: 841.2. HPLC purity: 96.7% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclohexylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9v). Yield: 14.5%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 11.68 (s, 1H), 8.65−8.53 (m, 2H), 8.04 (s, 1H), 7.85−7.77 (m, 1H), 7.62−7.54 (m, 1H), 7.54−7.46 (m, 2H), 7.35−7.28 (m, 4H), 7.18−7.08 (m, 2H), 7.01−6.95 (m, 1H), 6.91 (s, https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX M 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.40−6.31 (m, 3H), 4.97 (d, J = 8.8 Hz, 1H), 3.88−3.80 (m, 2H), 3.74−3.66 (m, 1H), 3.42−3.35 (m, 1H), 3.30−3.21 (m, 4H), 2.01−1.95 (m, 2H), 1.91−1.77 (m, 5H), 1.75−1.65 (m, 3H), 1.62−1.55 (m, 3H), 1.48−1.38 (m, 3H), 1.30− 1.25 (m, 4H). MS (ESI, m/z) [M + H]+: 854.8. HPLC purity: 96.6% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclopropylbenzyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (9w). Yield: 11.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 11.72 (s, 1H), 8.65−8.54 (m, 2H), 8.08 (s, 1H), 7.84 (d, J = 8.0 Hz 1H), 7.66−7.60 (m, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.55−7.49 (m, 1H), 7.44−7.36 (m, 3H), 7.25−7.17 (m, 1H), 7.14−7.07 (m, 3H), 6.96−6.90 (m, 2H), 6.66 (d, J = 7.2 Hz, 2H), 6.43−6.38 (m, 1H), 4.16−4.08 (m, 1H), 3.90−3.80 (m, 2H), 3.45−3.35 (m, 1H), 3.30−3.21 (m, 3H), 3.18−3.06 (m, 1H), 2.78− 2.68 (m, 1H), 2.08−1.95 (m, 3H), 1.95−1.82 (m, 2H), 1.80−1.70 (m, 1H), 1.65−1.55 (m, 2H), 1.50−1.40 (m, 1H), 1.35−1.25 (m, 3H), 0.98−0.90 (m, 2H), 0.88−0.82 (s, 2H). MS (ESI, m/z) [M + H]+: 826.8. HPLC purity: 90.3% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-3′-fluoro-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (10a). Yield: 14.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 11.66 (s, 1H), 8.66−8.44 (m, 2H), 8.01 (d, J = 1.2 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.58−7.44 (m, 3H), 7.36 (d, J = 8.0 Hz, 1H), 7.25 (m, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.09−7.04 (m, 2H), 7.02−6.93 (m, 3H), 6.51−6.38 (m, 1H), 6.36 (s, 1H), 5.37 (m, 1H), 4.01−3.90 (m, 1H), 3.88−3.78 (m, 2H), 3.57− 3.49 (m, 1H), 3.30−3.24 (m, 5H), 2.46−2.40 (m, 1H), 2.09−2.01 (m, 1H), 1.96−1.82 (m, 3H), 1.78−1.68 (m, 1H), 1.65−1.55 (m, 2H), 1.31−1.16 (m, 2H), 1.05−0.89 (m, 3H), 0.78−0.62 (m, 2H). MS (ESI, m/z) [M + H]+: 830.8. HPLC purity: 100% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-2′-fluoro-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (10b). Yield: 16.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 11.72 (s, 1H), 8.65−8.55 (m, 2H), 8.05 (d, J = 1.6 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.57−7.48 (m, 2H), 7.21 (d, J = 8.0 Hz, 1H),7.18−7.06 (m, 3H), 7.05−6.97 (m, 2H), 6.86−6.75 (m, 2H), 6.39 (s, 1H), 6.22− 6.06 (m, 2H), 5.18 (d, J = 8.0 Hz, 1H), 3.87−3.80 (m, 2H), 3.74− 3.65 (m, 1H), 3.34−3.23 (m, 5H), 2.47−2.35 (m, 1H), 2.09−2.01 (m, 1H), 2.01−1.93 (m, 1H), 1.90−1.77 (m, 2H), 1.65−1.52 (m, 2H), 1.29−1.17 (m, 2H), 1.08−0.90 (m, 3H), 0.81−0.72 (m, 1H), 0.70−0.60 (m, 1H). MS (ESI, m/z) [M + H]+: 830.8. HPLC purity: 100% (254 nm). 3-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-2′-chloro-4′-(2-(2cyclopropylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (10c). Yield: 1.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 11.70 (s, 1H), 8.62−8.52 (m, 2H), 8.05− 7.98 (m, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.61 (s, 1H), 7.54−7.46 (m, 2H), 7.15−7.05 (m, 3H), 7.05−6.97 (m, 3H), 6.86 (d, J = 7.6 Hz, 1H), 6.66 (s, 1H), 6.40−6.34 (m, 2H), 6.29 (d, J = 8.4 Hz, 1H), 5.18 (d, J = 7.6 Hz, 1H), 3.87−3.79 (m, 2H), 3.74−3.66 (m, 1H), 3.40− 3.34 (m, 1H), 3.30−3.20 (m, 4H), 2.46−2.36 (m, 1H), 2.09−1.95 (m, 2H), 1.94−1.78 (m, 3H), 1.63−1.53 (m, 2H), 1.30−1.17 (m, 2H), 1.06−0.89 (m, 2H), 0.85−0.75 (m, 1H), 0.68−0.60 (m, 1H). MS (ESI, m/z) [M + H]+: 846.7. HPLC purity: 97.7% (254 nm). 2 - ( ( 1H -Py r r o l o [ 2 , 3 - b ]p y r i d i n - 5 - y l ) o x y ) - 4 - ( 5 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)pyridin-2-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10d). Yield: 2.7%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 11.75 (s, 1H), 8.65−8.53 (m, 2H), 8.06 (d, J = 2.0 Hz, 1H), 7.85 (d, J = 9.2 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.67−7.57 (m, 3H), 7.55−7.50 (m, 2H), 7.35 (s, 1H), 7.17−7.07 (m, 2H), 7.05−6.96 (m, 2H), 6.83 (d, J = 7.2 Hz, 1H), 6.65 (dd, J = 8.8, 2.0 Hz, 1H), 6.44−6.34 (m, 1H), 5.25 (d, J = 8.0 Hz, 1H), 3.88−3.80 (m, 2H), 3.80−3.72 (m, 1H), 3.47−3.39 (m, 1H), 3.32−3.22 (m, 4H), 2.45−2.41 (m, 1H), 2.11−1.96 (m, 2H), 1.96−1.80 (m, 3H), 1.64− 1.54 (m, 2H), 1.30−1.15 (m, 2H), 1.04−0.91 (m, 2H), 0.78−0.66 (m, 2H). MS (ESI, m/z) [M + H]+: 813.8. HPLC purity: 98.4% (254 nm). 3 - ( ( 1H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]4-carboxamide (10e). Yield: 15.2%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 11.70 (s, 1H), 8.62−8.48 (m, 2H), 8.08−8.02 (m, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.69−7.56 (m, 3H), 7.55−7.38 (m, 4H), 7.32 (d, J = 7.6 Hz, 2H), 7.24−7.16 (m, 1H), 7.15−7.04 (m, 2H), 7.02−6.90 (m, 2H), 6.39 (s, 1H), 4.00−3.88 (m, 1H), 3.88−3.78 (m, 2H), 3.76−3.62 (m, 1H), 3.32−3.20 (m, 5H), 3.14−2.90 (m, 2H), 2.36−2.23 (m, 1H), 2.20−1.98 (m, 2H), 1.92− 1.82 (m, 1H), 1.82−1.78 (m, 1H), 1.65−1.55 (m, 2H), 1.54−1.42 (m, 1H), 1.33−1.17 (m, 3H), 0.98−0.80 (m, 2H), 0.72−0.62 (m, 1H), 0.61−0.50 (m, 1H). MS (ESI, m/z) [M + H]+: 827.2. HPLC purity: 96.1% (254 nm). 2 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 - ( 5 - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)furan-2-yl)-N-((3-nitro-4(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10f). Yield: 22.8%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H), 11.77 (s, 1H), 8.66−8.52 (m, 2H), 8.08 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.69−7.52 (m, 3H), 7.46−7.38 (m, 2H), 7.16−7.07 (m, 1H), 7.07−6.99 (m, 1H), 6.97 (s, 1H), 6.93 (d, J = 2.8 Hz, 1H), 6.91−6.80 (m, 2H), 6.45−6.39 (m, 1H), 6.38−6.24 (m, 1H), 3.90−3.79 (m, 3H), 3.68−3.52 (m,1H), 3.33−3.23 (m, 5H), 3.13−2.91 (m, 2H), 2.32−2.15 (m, 1H), 1.98−1.82 (m, 2H), 1.78−1.64 (m, 1H), 1.64−1.54 (m, 2H), 1.50−1.35 (m, 1H), 1.33− 1.17 (m, 3H), 0.89−0.76 (m, 2H), 0.61−0.44 (m, 2H). MS (ESI, m/ z) [M + H]+: 816.8. HPLC purity: 95.6% (254 nm). 3 - ( ( 1H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 3 ′ - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1'-biphenyl]4-carboxamide (10g). Yield: 12.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 11.71 (s, 1H), 8.60−8.50 (m, 2H), 8.08 (s, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.67−7.60 (m, 2H),7.58−7.48 (m, 2H), 7.44−7.32 (m, 4H), 7.31−7.24 (m, 1H), 7.17−7.02 (m, 3H), 7.00−6.89 (m, 2H), 6.39 (s, 1H), 4.00−3.88 (m, 1H), 3.88− 3.78 (m, 2H), 3.76−3.60 (m, 1H), 3.31−3.17 (m, 5H), 3.16−3.03 (m, 1H), 3.03−2.84 (m, 1H), 2.32−2.16 (m, 1H), 2.06−1.94 (m, 1H), 1.94−1.80 (m, 2H), 1.79−1.65 (m, 1H), 1.64−1.54 (m, 2H), 1.32−1.17 (m, 2H), 0.94−0.79 (m, 2H), 0.68−0.48 (m, 2H). MS (ESI, m/z) [M + H]+: 826.8. HPLC purity: 99.1% (254 nm). (S)-3-((1H-Pyrrolo[2,3-b]pyridin-5-yl )oxy)-4 ′- (2-(2-(2- cyclopropylphenyl)pyrrolidin-1-yl)ethyl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]4-carboxamide (10h). Yield: 42.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 11.72 (s, 1H), 9.92 (s, 1H), 8.65− 8.50 (m, 2H), 8.06 (s, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.80−7.70 (m, 1H), 7.65−7.56 (m, 2H), 7.55−7.49 (m, 1H), 7.48−7.39 (m, 3H), 7.36−7.25 (m, 2H), 7.22−7.15 (m, 2H), 7.15−7.05 (m, 2H), 6.97 (s, 1H), 6.39 (s, 1H), 5.15−4.95 (m, 1H), 3.90−3.70 (m, 3H), 3.29− 3.21 (m, 5H), 3.15−3.13 (m, 1H), 3.13−2.90 (m, 1H), 2.90−2.80 (m, 1H), 2.24−2.08 (m, 2H), 2.08−1.94 (m, 2H), 1.92−1.80 (m, 1H), 1.64−1.54 (m, 2H), 1.30−1.16 (m, 3H), 0.98−0.80 (m, 4H), 0.69−0.55 (m, 2H). MS (ESI, m/z) [M + H]+: 840.8. HPLC purity: 98.4% (254 nm). 2 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 - ( 4 - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)benzyl)-N-((3-nitro-4(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10i). Yield: 37.8%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 11.66 (s, 1H), 8.60−8.46 (m, 2H), 7.97 (s, 1H), 7.77 (d, J = 8.8 Hz), 7.63 (d, J = 7.6 Hz, 1H), 7.55−7.42 (m, 3H), 7.25−7.18 (m, 1H), 7.18−7.10 (m, 3H), 7.10−7.00 (m, 3H), 7.00−6.90 (m, 2H), 6.68 (s, 1H), 6.36 (s, 1H), 4.08−3.78 (m, 5H), 3.78−3.60 (m, 1H), 3.30−3.16 (m, 5H), 3.16−2.86 (m, 2H), 2.40− 2.24 (m, 1H), 2.10−1.93 (m, 1H), 1.92−1.70 (m, 3H), 1.62−1.50 (m, 2H), 1.30−1.14 (m, 3H), 0.96−0.82 (m, 2H), 0.72−0.62 (m, 1H), 0.62−0.52 (m, 1H). MS (ESI, m/z) [M + H]+: 840.8. HPLC purity: 99.1% (254 nm). https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX N (R)-2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (4- (2- (2- cyclopropylphenyl)pyrrolidin-1-yl)benzyl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10j). Yield: 5.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 11.71 (s, 1H), 8.63−8.56 (m, 1H), 8.53 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.80 (dd, J = 9.0, 2.2 Hz, 1H), 7.54−7.48 (m, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.14−7.06 (m, 2H), 7.04−6.97 (m, 2H), 6.92−6.85 (m, 2H), 6.81 (d, J = 8.4 Hz, 2H), 6.63 (s, 1H), 6.38−6.33 (m, 1H), 6.20 (d, J = 8.0 Hz, 2H), 5.08 (d, J = 8.4 Hz, 1H), 3.88−3.80 (m, 2H), 3.70−3.61 (m, 3H), 3.34−3.20 (m, 6H), 2.47−2.32 (m, 1H), 2.10−1.92 (m, 2H), 1.92−1.75 (m, 2H), 1.63− 1.54 (m, 2H), 1.31−1.16 (m, 3H), 1.05−0.90 (m, 2H), 0.81−0.75 (m, 1H), 0.69−0.61 (m, 1H). MS (ESI, m/z) [M + H]+: 826.8. HPLC purity: 95.1% (254 nm). 3 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 ′ - ( 3 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)azetidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-[1,1′-biphenyl]-4-carboxamide (10k). Yield: 30.8%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 11.70 (s, 1H), 8.68−8.50 (m, 2H), 8.08−8.02 (m, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.63−7.45 (m, 4H), 7.40−7.26 (m, 3H), 7.20−7.04 (m, 3H), 7.00−6.88 (m, 2H), 6.42−6.36 (m, 1H), 6.32 (d, J = 8.2 Hz, 2H), 4.15−4.00 (m, 1H), 3.89−3.77 (m, 3H), 3.74−3.67 (m, 1H), 3.66−3.56 (m, 2H), 3.31− 3.20 (m, 5H), 3.06−2.93 (m, 2H), 2.32−2.19 (m, 1H), 2.05−1.95 (m, 2H), 1.90−1.76 (m, 2H), 1.64−1.56 (m, 2H), 1.50−1.40 (m, 1H), 1.32−1.26 (m, 2H), 0.92−0.82 (m, 2H), 0.71−0.62 (m, 1H), 0.56−0.45 (m, 1H). MS (ESI, m/z) [M + H]+: 867.8. HPLC purity: 98.1% (254 nm). 2 - ( ( 1H -Py r r o l o [ 2 , 3 - b ]p y r i d i n - 5 - y l ) o x y ) - 4 - ( 4 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)cyclohexyl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10l). Yield: 12.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.65 (s, 1H), 8.60−8.40 (s, 2H), 7.95 (s, 1H), 7.82− 7.64 (m, 2H), 7.54−7.38 (m, 3H), 7.34−7.24 (m, 1H), 7.16−7.00 (m, 2H), 6.98−6.86 (m, 2H), 6.59 (s, 1H), 6.42−6.32 (m, 1H), 5.20−5.06 (m, 1H), 3.88−3.78 (m, 2H), 3.70−3.56 (m, 1H), 3.28− 3.05 (m, 5H), 3.06−2.90 (m, 1H), 2.44−2.30 (m, 2H), 2.15−1.95 (m, 4H), 1.92−1.80 (m, 2H), 1.77−1.64 (m, 2H), 1.63−1.54 (m, 3H), 1.53−1.38 (m, 2H), 1.35−1.25 (m, 3H), 1.00−0.80 (m, 2H), 0.77−0.65 (m, 1H), 0.65−0.50 (m, 1H). MS (ESI, m/z) [M + H]+ 819.2. HPLC purity: 96.9% (254 nm). 3 - ( ( 1 H - P y r r o l o [ 2 , 3 - b ] p y r i d i n - 5 - y l ) o x y ) - 4 ′ - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-N-((3-nitro-4-(((tetrahydro-2Hpyran-4-yl)methyl)amino)phenyl)sulfonyl)-2′,3′,4′,5′-tetrahydro[1,1′-biphenyl]-4-carboxamide (10m). Yield: 1.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 11.68 (s, 1H), 8.62− 8.45 (m, 2H), 7.98 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.66−7.56 (m, 1H), 7.56−7.43 (m, 3H), 7.35−7.22 (m, 1H), 7.18−6.90 (m, 4H), 6.73 (s, 1H), 6.37 (s, 1H), 6.02−5.80 (m, 1H),5.35−5.05 (m, 1H), 3.88−3.78 (m, 2H), 3.75−3.58 (m, 1H), 3.54−34.40 (m, 1H), 3.30− 3.20 (m, 5H), 2.46−2.37 (m, 1H), 2.36−2.24 (m, 2H), 2.24−2.08 (m, 2H), 2.06−1.94 (m, 2H), 1.92−1.80 (m, 2H), 1.78−1.66 (m, 1H), 1.64−1.54 (m, 2H), 1.50−1.40 (m, 1H), 1.32−1.18 (m, 3H), 1.00−0.86 (m, 2H), 0.76−0.64 (m, 1H), 0.63−0.50 (m, 1H). MS (ESI, m/z) [M + H]+: 817.2. HPLC purity: 96.2% (254 nm). General Procedure for the Preparation of 10n, 10p−10x, 11a−11o, and 12a−12m, Exemplified by 2-((1H-Pyrrolo[2,3b]pyridin-5-yl)oxy)-N-((4-((((1r,4r)-4-hydroxy-4-methyl cyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-4-(2-((S)-2(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7yl)benzamide (12e). Step 1. To a mixture of (S)-2-(2isopropylphenyl)pyrrolidine hydrochloride 18g (120 g, 0.54 mol; please see the synthesis in Supporting Information) in DCM (2.20 L) was added methyl 2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4-(2-oxo7-azaspiro[3.5]nonan-7-yl)benzoate 17 (218 g, 0.51 mol), and NaBH(OAc)3 (216 g, 1.02 mol) was added in portions while the temperature of the mixture was kept below 30 °C. Then the reaction mixture was stirred at ambient temperature until the starting material was totally consumed. The pH of the mixture was adjusted to 4−5 with diluted HCl (0.5 N in water). The separated organic phase was washed with water (600 mL × 2), sat. aq NaHCO3 (600 mL × 2), and sat. aq NaCl (600 mL × 2), dried over anhydrous Na2SO4, and concentrated to give the crude product 19g (256 g, 82.1%) as an offwhite solid, which was directly used in the next step without further purification. MS (ESI, m/z) [M + H]+: 579.0. Step 2. To a solution of methyl (S)-2-((1H-pyrrolo[2,3-b]pyridin5-yl)oxy)-4-(2-(2-(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzoate 19g (105 g, 181.70 mmol) in THF (525 mL) and MeOH (525 mL) was added a solution of NaOH (72.7 g, 1.80 mol) in water (525 mL), and the reaction solution was stirred at ambient temperature until the starting material was totally consumed. The mixture was concentrated under reduced pressure to remove the organic solvent, and water (3.50 L) was added to the residue. The solution was adjusted to pH 5−6 with 3 N HCl and then filtered. The solid was washed with water (100 mL) and dried in vacuo to give 20g (102 g, 99%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 7.95 (d, J = 1.6 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 6.8 Hz, 1H), 7.44 (s, 1H), 7.38−7.31 (m, 1H), 7.24−7.17 (m, 1H), 7.16−7.05 (m, 2H), 6.68 (d, J = 8.8 Hz, 1H), 6.38−6.26 (m, 2H), 3.68−3.56 (m, 1H), 3.33−3.27 (m, 1H), 3.13−3.08 (m, 1H), 3.07−3.01 (m, 3H), 2.99−2.93 (m, 2H), 2.39−2.27 (m, 1H), 2.22− 2.10 (m, 1H), 1.81−1.64 (m, 4H), 1.56−1.48 (m, 1H), 1.48−1.40 (m, 3H), 1.40−1.32 (m, 3H), 1.16 (d, J = 6.4 Hz, 6H). MS (ESI, m/ z) [M + H]+: 565.1. Step 3. To a mixture of (S)-2-((1H-pyrrolo[2,3-b]pyridin-5yl)oxy)-4-(2-(2-(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzoic acid 20g (44.0 g, 78.00 mmol) in DCM (880 mL) were added Et3N (15.7 g, 156.00 mmol), EDCI (19.4 g, 101.0 mmol), DMAP (19.0 g, 156.00 mmol), and 4-((((1r,4r)-4-hydroxy-4methylcyclohexyl)-methyl)amino)-3-nitrobenzenesulfonamide (26.8 g, 78.00 mmol), and the mixture was stirred at ambient temperature for 16 h. After the starting material was totally consumed, the reaction mixture was heated to 35 °C and TMEDA (17.2 g, 195 mmol) was added. The mixture was stirred for 12 h; then it was cooled to ambient temperature and washed with 10% aq AcOH (300 mL× 2) and sat. aq NaHCO3 (300 mL × 2). The organic layer was concentrated to about 90 mL under reduced pressure, and silica gel (100−200 mesh, 22.0 g) was added. The mixture was stirred at ambient temperature for 2 h and filtered. EA (180 mL) was added into the filtrate, and the mixture was concentrated to remove DCM. The resulting solution was heated to reflux and stirred for 5 h. After it was cooled to ambient temperature, the mixture was filtered and the cake was washed with EA (180 mL) and dried in a vacuum to give 12e (48 g, 69.5%) as a yellow solid. 12e (S, trans; retention time 24.1 min) and enantiomer (R, trans; retention time 22.3 min) were tested in chiral analysis conditions (column, ChiralPak IC; column size, 4.6 × 250 mm; mobile phase, MeOH (0.6% DEA + 0.5% FA):CAN (0.6% DEA + 0.5% FA) = 80:20 (v/v); flow rate, 0.4 mL/min; wavelength, UV 284 nm), which was used to test chiral purity. 1H NMR (400 MHz, CDCl3) δ 10.27 (s, 1H), 8.87 (d, J = 2.3 Hz, 1H), 8.50 (t, J = 5.3 Hz, 1H), 8.20 (d, J = 2.4 Hz, 1H), 8.13 (dd, J = 9.3, 2.3 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.56 (d, J = 7.1 Hz, 1H), 7.49−7.45 (m, 1H), 7.21−7.08 (m, 3H), 6.87 (d, J = 9.3 Hz, 1H), 6.56−6.50 (m, 2H), 5.97 (d, J = 2.1 Hz, 1H), 3.66−3.56 (m, 1H), 3.32−3.21 (m, 3H), 3.19−3.10 (m, 1H), 3.08−2.86 (m, 5H), 2.36−2.26 (m, 1H), 2.24−2.10 (m, 1H), 1.95− 1.81 (m, 3H), 1.81−1.71 (m, 6H), 1.66−1.56 (m, 1H), 1.55−1.47 (m, 3H), 1.46−1.34 (m, 5H), 1.30−1.26 (m, 3H), 1.26−1.22 (m, 2H), 1.22−1.14 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 162.1, 159.5, 155.6, 147.9, 146.3, 145.9, 145.1, 141.3, 136.3, 135.5, 133.8,
130.6, 129.1, 127.4 (2C), 126.5, 125.5, 125.1, 124.7, 120.7 (2C),
113.7, 109.1, 108.0, 101.3, 100.5, 70.5, 62.5, 52.8, 50.5, 48.5, 44.4,
44.2, 38.9 (2C), 38.4, 37.3, 36.2, 35.4 (2C), 35.2, 31.7, 28.0, 27.9 (2C), 26.0, 24.1, 24.0, 22.7. HRMS (ESI) calcd for C49H60N7O7S+ [M + H]+: 890.4269; found, 890.4267. FTIR (neat): 3307, 3108, 2920, 2854, 1666, 1603, 1552, 1465, 1433, 1256, 1225, 991, 968, 663 cm−1. UV (nm): 260, 285, 430. [α]D25 −13.9 (c 1.0, THF:CH3CN:H2O = 20:64:16 (v/v/v)). HPLC purity: 95.9% (254 nm). The structure was further confirmed by single crystal of BGB-11417 EtOAc solvate in Supporting Information. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX O 2 - ( ( 1H -Py r r o l o [ 2 , 3 - b ]p y r i d i n - 5 - y l ) o x y ) - 4 - ( 4 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)piperidin-1-yl)-N-((3-nitro-4(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10n). Yield: 9.2%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 11.43 (s, 1H), 8.65−8.45 (m, 2H), 8.02 (s, 1H), 7.83−7.70 (m, 1H), 7.60−7.40 (m, 4H), 7.20−6.98 (m, 3H), 6.95−6.85 (m, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.37 (s, 1H), 6.18 (s, 1H), 4.34−4.20 (m, 1H), 3.90−3.80 (m, 2H), 3.70−3.50 (m, 2H), 3.31−3.19 (m, 5H), 3.16−3.04 (m, 1H), 3.04−2.88 (m, 1H), 2.70− 2.54 (m, 2H), 2.30−2.15 (m, 1H), 2.05−1.93 (m, 1H), 1.93−1.80 (m, 1H), 1.79−1.65 (m, 2H), 1.65−1.57 (m, 2H), 1.50−1.35 (m, 3H), 1.32−1.11 (m, 4H), 0.95−0.80 (m, 2H), 0.70−0.60 (m, 1H), 0.55−0.45 (m, 1H). MS (ESI, m/z) [M + H]+: 819.8. HPLC purity: 97.0% (254 nm). 3- ( (1H-Pyr ro lo [2 ,3 -b ]pyr id in-5-y l )oxy ) -4 ′ - ( ( (S ) -2 - (2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-4-carboxamide (10o). Yield: 25.7%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 11.70 (s, 1H), 8.62−8.45 (m, 2H), 8.01 (s, 1H), 7.85−7.72 (m, 1H), 7.70−7.40 (m, 4H), 7.32−7.17 (m, 1H), 7.15−6.80 (m, 4H), 6.74−6.61 (m, 1H), 6.39 (s, 1H), 6.10−5.89 (m, 1H), 3.94−3.66 (m, 3H), 3.53−3.40 (m, 1H), 3.30−3.17 (m, 5H), 3.16−2.96 (m, 1H), 2.90−2.70 (m, 1H), 2.27−1.96 (m, 7H), 1.93−1.73 (m, 3H), 1.68−1.55 (m, 3H), 1.31− 1.13 (m, 3H), 1.06−1.01 (m, 1H), 0.96−0.80 (m, 2H), 0.72−0.57 (m, 1H), 0.56−0.42 (m, 1H). MS (ESI, m/z) [M + H]+: 830.8. HPLC purity: 100% (254 nm). 2 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 - ( 3 - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)azetidin-1-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10p). Yield: 6.3%, yellow solid. 1H NMR (400 MHz, CDCl3) δ 10.11 (s, 1H), 9.14 (s, 1H), 8.91−8.85 (m, 1H), 8.57−8.46 (m, 1H), 8.24−8.09 (m, 2H), 7.90 (d, J = 8.8 Hz, 1H), 7.68 (s, 1H), 7.50−7.40 (m, 2H), 7.16−7.05 (m, 2H), 6.97−6.87 (m, 2H), 6.58−6.50 (m, 1H), 6.01 (d, J = 8.4 Hz, 1H), 5.42−5.32 (m, 1H), 4.07−3.98 (m, 2H), 3.86−3.71 (m, 2H), 3.49−3.38 (m, 3H), 3.37−3.30 (m, 2H), 3.29−3.23 (m, 2H), 3.22−3.14 (m, 1H), 2.80− 2.60 (m, 2H), 2.39−2.27 (m, 1H), 2.27−2.11 (m, 2H), 2.04−1.90 (m, 2H), 1.82−1.67 (m, 3H), 1.50−1.35 (m, 3H), 0.93−0.81 (m, 2H), 0.73−0.62 (m, 1H), 0.56−0.46 (m, 1H). MS (ESI, m/z) [M + H]+: 805.8. HPLC purity: 99.3% (254 nm). 2 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 - ( 4 - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)piperidin-1-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10q). Yield: 19.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.38 (s, 1H), 8.65−8.50 (m, 2H), 8.04 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.58−7.36 (m, 4H), 7.17−7.01 (m, 3H), 6.97−6.84 (m, 1H), 6.70−6.60 (m, 1H), 6.42−6.34 (m, 1H), 6.15 (s, 1H), 3.90−3.80 (m, 2H), 3.80−3.69 (m, 1H), 3.65−3.48 (m, 3H), 3.30−3.19 (m, 5H), 2.74−2.59 (m, 2H), 2.30−2.06 (m, 3H), 2.06−1.84 (m, 4H), 1.84−1.74 (m, 1H), 1.70− 1.56 (m, 3H), 1.53−1.38 (m, 2H), 1.32−1.19 (m, 2H), 0.98−0.80 (m, 2H), 0.68−0.58 (m, 1H), 0.54−0.43 (m, 1H). MS (ESI, m/z) [M + H]+: 833.8. HPLC purity: 98.4% (254 nm). 2 - ( ( 1H -Py r r o l o [ 2 , 3 - b ]p y r i d i n - 5 - y l ) o x y ) - 4 - ( 2 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10r). Yield: 17.1%, yellow solid. S-10r and R10r were synthesized by using the chiral material of 2- (cyclopropyphenyl)pyrrolidine similar as the synthesis of 18g; please see the Supporting Information. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.44 (s, 1H), 10.12 (s, 1H), 8.67−8.52 (m, 2H), 8.03 (s, 1H), 7.85−7.70 (m, 2H), 7.55−7.45 (m, 3H), 7.36−7.24 (m, 2H), 7.15−7.01 (m, 2H), 6.68 (d, J = 8.4 Hz, 1H), 6.38 (s, 1H), 6.17 (s, 1H), 5.05−4.92 (m, 1H), 3.95−3.80 (m, 3H), 3.73−3.60 (m, 1H), 3.30−3.15 (m, 5H), 3.10−3.03 (m, 2H), 3.02−2.90 (m, 3H), 2.20− 1.97 (m, 6H), 1.94−1.84 (m, 1H), 1.67−1.50 (m, 4H), 1.48−1.33 (m, 5H), 1.32−1.22 (m, 2H), 1.04−0.90 (m, 2H), 0.72−0.58 (m, 2H). MS (ESI, m/z) [M + H]+: 859.8. HPLC purity: 94.1% (254 nm). 2 - ( ( 1H -Py r r o l o [ 2 , 3 - b ]p y r i d i n - 5 - y l ) o x y ) - 4 - ( 6 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-2-azaspiro[3.3]heptan-2-yl)-N((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10s). Yield: 7.2%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 11.34 (br s, 1H), 8.68−8.60 (m, 1H), 8.58 (s, 1H), 8.08−8.02 (m, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.81−7.65 (m, 1H), 7.61 (s, 1H), 7.55−7.50 (m, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.34−7.20 (m, 2H), 7.16 (d, J = 9.2 Hz, 1H), 7.11−6.97 (m, 1H), 6.41 (s, 1H), 6.07 (d, J = 8.8 Hz, 1H), 5.52−5.46 (m, 1H), 5.05−4.80 (m, 1H), 3.90−3.80 (m, 2H), 3.75−3.54 (m, 5H), 3.31− 3.20 (m, 5H), 3.19−3.10 (m, 1H), 2.44−2.38 (m, 1H), 2.15−1.96 (m, 4H), 1.91−1.76 (m, 3H), 1.66−1.56 (m, 2H), 1.32−1.20 (m, 4H), 0.99−0.88 (m, 2H), 0.70−0.58 (m, 2H). MS (ESI, m/z) [M + H]+: 832.8. HPLC purity: 98.0% (254 nm). 2 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 - ( 6 - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)-2-azaspiro[3.3]heptan2-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10t). Yield: 13.9%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 11.41 (s, 1H), 8.68− 8.56 (m, 2H), 8.08 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.78−7.63 (m, 2H), 7.58−7.52 (m, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.38−7.10 (m, 3H), 7.04−6.92 (m, 1H), 6.44 (s, 1H), 6.07 (d, J = 8.4 Hz, 1H), 5.45 (s, 1H), 5.05−4.80 (m, 1H), 3.88−3.82 (m, 2H), 3.74−3.68 (m,, 2H), 3.57−3.47 (m, 1H), 3.43−3.34 (m, 1H), 3.31−3.16 (m, 5H), 3.12−2.95 (m, 1H), 2.85−2.70 (m, 1H), 2.45−2.30 (m, 1H), 2.25− 2.12 (m, 3H), 2.06−1.95 (m, 2H), 1.95−1.82 (m, 2H), 1.75−1.65 (m, 1H), 1.65−1.55 (m, 2H), 1.34−1.20 (m, 4H), 1.00−0.85 (m, 2H), 0.68−0.48 (m, 2H). MS (ESI, m/ze) [M + H]+: 845.9. HPLC purity: 98.7% (254 nm). 2 - ( ( 1H -Py r ro lo [2 , 3 -b ]py r i d in -5 - y l ) oxy ) - 4 - ( 2 - ( ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)methyl)-7-azaspiro[3.5]nonan-7yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10u). Yield: 5.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 11.44 (s, 1H), 8.67− 8.52 (m, 2H), 8.05 (s, 1H), 7.84−7.77 (m, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.56−7.46 (m, 3H), 7.33−7.27 (m, 1H), 7.16−7.06 (m, 2H), 6.73−6.67 (m, 1H), 6.42−6.36 (m, 1H), 6.18 (s, 1H), 4.04−3.96 (m, 1H), 3.89−3.80 (m, 2H), 3.78−3.64 (m, 1H), 3.31−3.21 (m, 5H), 3.12−3.04 (m, 3H), 3.01−2.94 (m, 3H), 2.20−2.10 (m, 1H), 2.06− 1.96 (m, 3H), 1.92−1.78 (m, 4H), 1.65−1.57 (m, 2H), 1.51−1.44 (m, 3H), 1.37−1.26 (m, 6H), 0.99−0.89 (m, 2H), 0.72−0.59 (m, 2H). MS (ESI, m/z) [M + H]+: 873.9. HPLC purity: 97.8% (254 nm). 2- ( (1H-Pyrro lo[2 ,3-b]pyr id in-5-y l )oxy) -4- (4- (3- (2- (2- cyclopropylphenyl)pyrrolidin-1-yl)cyclobutyl)piperidin-1-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10v). Yield: 9.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 11.41 (s, 1H), 9.52 (s, 1H), 8.66− 8.54 (m, 2H), 8.08−8.00 (m, 1H), 7.80 (d, J = 9.2 Hz, 1H), 7.62− 7.46 (m, 3H), 7.34−7.25 (m, 2H), 7.16−7.03 (m, 2H), 6.67 (d, J = 9.2 Hz, 1H), 6.42−6.35 (m, 1H), 6.15 (s, 1H), 5.07−4.96 (m, 1H), 3.90−3.80 (m, 2H), 3.75−3.50 (m, 5H), 3.35−3.15 (m, 5H), 3.05− 2.90 (m, 1H), 2.70−2.55 (m, 3H), 2.25−1.96 (m, 5H), 1.95−1.75 (m, 2H), 1.70−1.55 (m, 3H), 1.52−1.42 (m, 1H), 1.35−1.15 (m, 4H), 1.02−0.91 (m, 2H), 0.90−0.78 (m, 2H), 0.73−0.58 (m, 2H). MS (ESI, m/z) [M + H]+: 873.9. HPLC purity: 96.6% (254 nm). 2 - ( ( 1H -Py r r o l o [ 2 , 3 - b ]p y r i d i n - 5 - y l ) o x y ) - 4 - ( 3 - ( 2 - ( 2 - cyclopropylphenyl)pyrrolidin-1-yl)-[1,3′-biazetidin]-1′-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10w). Yield: 17.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 11.19 (s, 1H), 8.65−8.55 (m, 2H), 8.06 (d, J = 2.0 Hz, 1H), 7.87−7.79 (m, 1H), 7.61 (d, J = 2.0 Hz, 1H), 7.55−7.41 (m, 3H), 7.19−7.01 (m, 3H), 6.95−6.85 (m, 1H), 6.44−6.36 (m, 1H), 6.12 (d, J = 8.8 Hz, 1H), 5.57 (s, 1H), 3.88−3.80 (m, 2H), 3.78−3.68 (m, 2H), 3.63−3.53 (m, 1H), 3.52−3.42 (m, 3H), 3.32−3.20 (m, 8H), 3.19−3.03 (m, 2H), 2.97−2.78 (m, 1H), 2.29−2.17 (m, 1H), 2.08−1.98 (m, 1H), 1.95−1.72 (m, 3H), 1.64− 1.55 (m, 2H), 1.32−1.20 (m, 3H), 0.91−0.81 (m, 2H), 0.68−0.58 (m, 1H), 0.55−0.43 (m, 1H). MS (ESI, m/z) [M + H]+: 846.9. HPLC purity: 98.2% (254 nm). https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX P 2-( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (5- ( (S) -2- (2- cyclopropylphenyl)pyrrolidin-1-yl)hexahydrocyclopenta[c]pyrrol2(1H)-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (10x). Yield: 13.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 11.28 (s, 1H), 8.67− 8.56 (m, 2H), 8.08 (s, 1H), 7.88−7.74 (m, 2H), 7.62 (s, 1H), 7.56− 7.48 (m, 2H), 7.32−7.24 (m, 2H), 7.16 (d, J = 9.6 Hz, 1H), 7.11− 7.04 (m, 1H), 6.45−6.38 (m, 1H), 6.34 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H), 5.11−4.98 (m, 1H), 3.90−3.80 (m, 2H), 3.76−3.62 (m, 2H), 3.32−3.12 (m, 5H), 3.10−3.00 (m, 2H), 2.99−2.88 (m, 2H), 2.27− 2.16 (m, 1H), 2.15−2.02 (m, 3H), 2.00−1.82 (m, 3H), 1.75−1.57 (m, 4H), 1.33−1.23 (m, 4H), 1.14−1.03 (m 1H), 0.98−0.89 (m, 2H), 0.60−0.60 (m, 2H). MS (ESI, m/z) [M + H]+: 845.8. HPLC purity: 97.3% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- chlorophenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11a). Yield: 18.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 11.45 (s, 1H), 8.65−8.59 (m, 1H), 8.56 (d, J = 2.0 Hz, 1H), 8.10−7.98 (m, 2H), 7.79 (dd, J = 9.2, 2.0 Hz, 1H), 7.56−7.31 (m, 6H), 7.11 (d, J = 9.2 Hz, 1H), 6.72−6.65 (m, 1H), 6.40−6.36 (m, 1H), 6.20−6.15 (m, 1H), 3.90−3.80 (m, 2H), 3.41−3.34 (m, 3H), 3.30−3.21 (m, 3H), 3.14−3.03 (m, 5H), 3.00− 2.92 (m, 1H), 2.22−2.02 (m, 3H), 1.96−1.82 (m, 1H), 1.65−1.56 (m, 3H), 1.54−1.32 (m, 4H), 1.26−1.22 (m, 3H), 1.21−1.19 (m, 3H). MS (ESI, m/z) [M + H]+: 853.8. HPLC purity: 99.7% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (3- chlorophenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11b). Yield: 23.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.45 (s, 1H), 8.66−8.52 (m, 2H), 8.08−7.98 (m, 1H), 7.86−7.74 (m, 1H), 7.62−7.55 (m, 1H), 7.54− 7.45 (m, 4H), 7.45−7.29 (m, 2H), 7.17−7.05 (m, 1H), 6.69 (d, J = 8.8 Hz, 1H), 6.42−6.35 (m, 1H), 6.22−6.11 (m, 1H), 4.54−4.43 (m, 1H), 4.41−4.10 (m, 1H), 3.90−3.78 (m, 2H), 3.31−3.21 (m, 5H), 3.11−3.00 (m, 2H), 2.99−2.87 (m, 3H), 2.41−2.23 (m, 1H), 2.16− 1.96 (m, 3H), 1.96−1.80 (m, 2H), 1.69−1.53 (m, 3H), 1.51−1.32 (m, 5H), 1.31−1.18 (m, 3H). MS (ESI, m/z) [M + H]+: 853.8. HPLC purity: 99.7% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (4- chlorophenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11c). Yield: 19.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.65−8.56 (m, 1H), 8.54 (d, J = 2.0 Hz, 1H), 8.02 (d, J = 2.8 Hz, 1H), 7.78 (dd, J = 9.2, 2.0 Hz, 1H), 7.66−7.55 (m, 2H), 7.54−7.43 (m, 5H), 7.08 (d, J = 9.2 Hz, 1H), 6.68 (dd, J = 9.0, 2.0 Hz, 1H), 6.39 (dd, J = 3.2, 1.6 Hz, 1H), 6.17 (d, J = 2.0 Hz, 1H), 4.35−4.15 (m, 1H), 3.89−3.79 (m, 2H), 3.76−3.66 (m, 1H), 3.34−3.19 (m, 5H), 3.13−3.00 (m, 2H), 2.99−2.88 (m, 3H), 2.40−2.26 (m, 1H), 2.13−1.93 (m, 5H), 1.92−1.82 (m, 1H), 1.65−1.52 (m, 3H), 1.47−1.31 (m, 5H), 1.29−1.19 (m, 2H). MS (ESI, m/z) [M + H]+: 853.8. HPLC purity: 99.7% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- bromophenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11d). Yield: 12.6%, yellow solid. 1H NMR (DMSO-d6) δ 11.80−11.40 (m, 2H), 8.64−8.57 (m, 1H), 8.55 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.82−7.74 (m, 1H), 7.64−7.56 (m, 1H), 7.53−7.45 (m, 3H), 7.45−7.37 (m, 1H), 7.31−7.18 (m, 1H), 7.10 (d, J = 9.2 Hz, 1H), 6.67 (dd, J = 9.2, 2.0 Hz, 1H), 6.40− 6.34 (m, 1H), 6.17 (d, J = 1.6 Hz, 1H), 4.75−4.30 (m, 1H), 3.89− 3.79 (m, 2H), 3.70−3.45 (m, 1H), 3.34−3.20 (m, 5H), 3.12−2.83 (m, 5H), 2.43−2.27 (m, 1H), 2.12−1.76 (m, 5H), 1.66−1.52 (m, 3H), 1.52−1.31 (m, 5H), 1.30−1.19 (m, 3H). MS (ESI, m/z) [M + H]+: 897.7 and 899.7. HPLC purity: 100% (254 nm). (S)-2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(2-(2-(otolyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzamide (11e). Yield: 62.9%, yellow solid. 1H NMR (DMSO-d6) δ 11.69 (s, 1H), 11.54 (s, 1H), 8.66−8.58 (m, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.92−7.76 (m, 1H), 7.54−7.44 (m, 3H), 7.30−7.16 (m, 3H), 7.10 (d, J = 9.2 Hz, 1H), 6.68 (d, J = 9.0, 1.8 Hz, 1H), 6.38 (dd, J = 3.2, 1.8 Hz, 1H), 6.17 (d, J = 1.6 Hz, 1H), 4.65−4.40 (m, 1H), 3.89−3.79 (m, 2H), 3.70−3.55 (m, 1H), 3.33−3.22 (m, 5H), 3.14−2.98 (m, 3H), 2.98−2.89 (m, 2H), 2.45−2.29 (m, 4H), 2.18− 1.96 (m, 5H), 1.92−1.83 (m, 1H), 1.66−1.56 (m, 2H), 1.52−1.32 (m, 6H), 1.31−1.17 (m, 2H). MS (ESI, m/z) [M + H]+: 833.9. HPLC purity: 96.4% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- ethylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11f). Yield: 27.4%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.45 (s, 1H), 10.06 (s, 1H), 8.65−8.58 (m, 1H), 8.56 (d, J = 1.6 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.82−7.72 (m, 2H), 7.54−7.44 (m, 3H), 7.35−7.27 (m, 2H), 7.27−7.17 (m, 1H), 7.11 (d, J = 9.2 Hz, 1H), 6.72−6.63 (m, 1H), 6.40−6.35 (m, 1H), 6.17 (s, 1H), 4.68−4.56 (m, 1H), 3.90−3.80 (m, 2H), 3.72−3.60 (m, 1H), 3.30−3.21 (m, 3H), 3.15−3.00 (m, 4H), 2.99−2.88 (m, 2H), 2.83−2.72 (m, 1H), 2.70−2.58 (m, 1H), 2.20− 2.04 (m, 4H), 2.03−1.94 (m, 2H), 1.91−1.82 (m, 1H), 1.65−1.56 (m, 2H), 1.50−1.39 (m, 3H), 1.38−1.20 (m, 6H), 1.12 (t, J = 7.4 Hz, 3H). MS (ESI, m/z) [M + H]+: 847.9. HPLC purity: 94.8% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11g). Yield: 6.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.44 (s, 1H), 9.90−9.60 (m, 1H), 8.66−8.48 (m, 2H), 8.03 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.70−7.63 (m, 1H), 7.55−7.46 (m, 3H), 7.41−7.25 (m, 2H), 7.16−7.05 (m, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.38 (s, 1H), 6.17 (s, 1H), 4.85−4.70 (m, 1H), 3.92−3.80 (m, 2H), 3.72−3.60 (m, 1H), 3.30−3.21 (m, 4H), 3.12−3.00 (m, 3H), 2.98−2.89 (m, 3H), 2.22−1.96 (m, 4H), 1.95−1.83 (m, 1H), 1.76−1.55 (m, 2H), 1.50−1.20 (m, 11H), 1.20− 1.02 (m, 4H). MS (ESI, m/z) [M + H]+: 861.9. HPLC purity: 93.4% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- isobutylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11h). Yield: 7.6%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.70−11.30 (m, 2H), 9.70−9.50 (m, 1H), 8.55− 8.26 (m, 2H), 8.02−7.86 (m, 1H), 7.71−7.50 (m, 3H), 7.49−7.36 (m, 1H), 7.29−6.92 (m, 4H), 6.70−6.56 (m, 2H), 6.37−6.26 (m, 1H), 6.25−6.15 (m, 1H), 3.90−3.78 (m, 2H), 3.60−3.46 (m, 1H), 3.28−3.21 (m, 4H), 3.14−3.05 (m, 1H), 3.03−2.93 (m, 3H), 2.92− 2.84 (m, 3H), 2.32−2.23 (m, 1H), 2.20−2.08 (m, 1H), 2.03−1.94 (m, 3H), 1.90−1.80 (m, 1H), 1.80−1.68 (m, 3H), 1.67−1.55 (m, 3H), 1.51−1.40 (m, 4H), 1.38−1.27 (m, 5H), 0.91−0.79 (m, 6H). MS (ESI, m/z) [M + H]+: 875.9. HPLC purity: 88.6% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- cyclobutylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11i). Yield: 20.6%, yellow solid. 1H NMR (DMSO-d6) δ 11.68 (s, 1H), 11.43 (s, 1H), 8.64−8.50 (m, 2H), 8.02 (s, 1H), 7.90−7.70 (m, 2H), 7.55−7.43 (m, 3H), 7.37−7.17 (m, 3H), 7.08 (d, J = 8.8 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.37 (s, 1H), 6.17 (s, 1H), 4.61−4.33 (m, 1H), 3.91−3.80 (m, 2H), 3.79−3.69 (m, 1H), 3.66−3.46 (m, 1H), 3.31−3.21 (m, 5H), 3.10−2.98 (m, 2H), 2.97− 2.86 (m, 2H), 2.38−2.22 (m, 3H), 2.21−2.12 (m, 1H), 2.12−1.84 (m, 7H), 1.82−1.72 (m, 1H), 1.66−1.55 (m, 2H), 1.47−1.18 (m, 10H). MS (ESI, m/e) [M + H]+: 873.8. HPLC purity: 98.2% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- methoxyphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11j). Yield: 6.5%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 11.46 (s, 1H), 9.38 (s, 1H), 8.67− 8.52 (m, 2H), 8.08−8.00 (m, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.57− 7.36 (m, 4H), 7.16−6.96 (m, 3H), 6.71 (d, J = 8.8 Hz, 1H), 6.41− 6.35 (m, 1H), 6.19 (s, 1H), 4.73−4.56 (m, 1H), 3.93−3.76 (m, 6H), 3.59−3.44 (m, 1H), 3.34−3.20 (m, 5H), 3.18−3.05 (m, 2H), 3.03− https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX Q 2.92 (m, 2H), 2.37−2.24 (m, 1H), 2.20−2.05 (m, 3H), 2.03−1.94 (m, 2H), 1.93−1.74 (m, 2H), 1.66−1.56 (m, 2H), 1.53−1.42 (m, 2H), 1.42−1.34 (m, 2H), 1.32−1.24 (m, 3H). MS (ESI, m/z) [M + H]+: 849.9. HPLC purity: 97.6% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- ethoxyphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((3nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11k). Yield: 37.2%, yellow solid. 1H NMR (DMSO-d6) δ 11.68 (s, 1H), 11.45 (s, 1H), 9.97 (s, 1H), 8.68−8.45 (m, 2H), 8.02 (s, 1H), 7.76 (d, J = 7.2 Hz, 1H), 7.65−7.43 (m, 4H), 7.43−7.27 (m, 1H), 7.17−6.92 (m, 3H), 6.69 (d, J = 8.0 Hz, 1H), 6.37 (s, 1H), 6.20 (s, 1H), 4.80−4.55 (m, 1H), 4.20−4.00 (m, 2H), 3.94−3.74 (m, 2H), 3.60−3.42 (m, 1H), 3.33−3.20 (m, 5H), 3.15− 3.03 (m, 3H), 3.02−2.86 (m, 2H), 2.36−2.25 (m, 1H), 2.20−1.96 (m, 4H), 1.93−1.83 (m, 1H), 1.82−1.70 (m, 1H), 1.67−1.56 (m, 2H), 1.56−1.32 (m, 8H), 1.32−1.16 (m, 3H). MS (ESI, m/z) [M + H]+: 863.8. HPLC purity: 93.8% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- isopropoxyphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11l). Yield: 37.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.46 (s, 1H), 10.06 (s, 1H), 8.67−8.52 (m, 2H), 8.03 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.67− 7.57(m, 1H), 7.55−7.44 (m, 3H), 7.43−7.30 (m, 1H), 7.18−7.03 (m, 2H), 7.02−6.92 (m, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.38 (s, 1H), 6.19 (s, 1H), 4.82−4.58 (m, 2H), 3.92−3.74 (m, 3H), 3.62−3.47 (m, 1H), 3.32−3.22 (m, 5H), 3.20−3.03 (m, 3H), 3.03−2.90 (m, 2H), 2.38− 2.24 (m, 1H), 2.20−2.00 (m, 4H), 1.95−1.82 (m, 1H), 1.78−1.68 (m, 1H), 1.66−1.57 (m, 2H), 1.56−1.36 (m, 5H), 1.35−1.15 (m, 9H). MS (ESI, m/z) [M + H]+: 877.9. HPLC purity: 99.1% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- (methoxymethyl)phenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11m). Yield: 14.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.44 (s, 1H), 8.68− 8.46 (m, 2H), 8.02 (s, 1H), 7.92−7.72 (m, 2H), 7.61−7.41 (m, 3H), 7.40−7.32 (m, 1H), 7.28−7.17 (m, 1H), 7.16−7.04 (m, 1H), 6.67 (d, J = 7.2 Hz, 1H), 6.37 (s, 1H), 6.17 (s, 1H), 4.60−4.39 (m, 3H), 3.88−3.80 (m, 2H), 3.72−3.60 (m, 1H), 3.31−3.22 (m, 6H), 3.10− 2.90 (m, 7H), 2.18−2.04 (m, 2H), 2.04−1.95 (m, 3H), 1.93−1.82 (m, 2H), 1.81−1.68 (m, 1H), 1.66−1.56 (m, 3H), 1.50−1.26 (m, 6H). MS (ESI, m/z) [M + H]+: 863.8. HPLC purity: 98.2% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- (dimethylamino)phenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (11n). Yield: 68.8%, yellow solid. 1H NMR (DMSO-d6) δ 11.70 (s, 1H), 11.45 (s, 1H), 10.17 (s, 1H), 8.67−8.59 (m, 1H), 8.56 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.82−7.76 (m, 1H), 7.75−7.68 (m, 1H), 7.54−7.45 (m, 3H), 7.42−7.34 (m, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.24−7.16 (m, 1H), 7.11 (d, J = 8.8 Hz, 1H), 6.73−6.64 (m, 1H), 6.40−6.36 (m, 1H), 6.21−6.11 (m, 1H), 4.99−4.83 (m, 1H), 3.91−3.80 (m, 2H), 3.80− 3.67 (m, 1H), 3.67−3.51 (m, 1H), 3.32−3.22 (m, 4H), 3.20−3.12 (m, 1H), 3.12−3.01 (m, 2H), 3.00−2.90 (m, 2H), 2.60 (s, 6H), 2.48−2.40 (m, 1H), 2.20−1.96 (m, 5H), 1.95−1.81 (m, 1H), 1.65− 1.57 (m, 2H), 1.57−1.48 (m, 1H), 1.47−1.33 (m, 4H), 1.33−1.18 (m, 3H). MS (ESI, m/z) [M + H]+: 862.9. HPLC purity: 100% (254 nm). (S)-2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(2-(2-(2(pyrrolidin-1-yl)phenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzamide (11o). Yield: 9.6%, yellow solid. 1H NMR (DMSO-d6) δ 11.70 (s, 1H), 11.46 (s, 1H), 8.67−8.58 (m, 1H), 8.55 (s, 1H), 8.03 (s, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.74−7.59 (m, 1H), 7.57−7.43 (m, 3H), 7.37−7.27 (m, 1H), 7.22−7.04 (m, 3H), 6.69 (d, J = 8.8 Hz, 1H), 6.38 (s, 1H), 6.18 (s, 1H), 4.88−4.68 (m, 1H), 3.89−3.80 (m, 2H), 3.78−3.65 (m, 1H), 3.62−3.50 (s, 1H), 3.31−3.19 (m, 6H), 3.16−3.10 (m, 1H), 3.09−3.01 (m, 2H), 3.00−2.89 (m, 3H), 2.86− 2.78 (m, 2H), 2.21−2.07 (m, 3H), 2.02−1.80 (m, 7H), 1.64−1.58 (m, 2H), 1.56−1.49 (m, 1H), 1.46−1.24 (m, 8H). MS (ESI, m/z) [M + H]+: 888.8. HPLC purity: 96.7% (254 nm). 2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((4-(((3-oxabicyclo[3.1.0]hexan-6-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-4-(2-((S)2-(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzamide (12a). Yield: 59.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.44 (s, 1H), 9.75−9.52 (m, 1H), 8.66− 8.58 (m, 1H), 8.56 (s, 1H), 8.03 (s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.64−7.55 (m, 1H), 7.53−7.44 (m, 3H), 7.41−7.23 (m, 3H), 7.09 (d, J = 8.8 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 6.38 (s, 1H), 6.17 (s, 1H), 4.88−4.66 (m, 1H), 4.00−3.80 (m, 1H), 3.76−3.70 (m, 2H), 3.59− 3.51 (m, 2H), 3.32−3.22 (m, 3H), 3.21−3.11 (m, 1H), 3.09−2.99 (m, 2H), 2.46−2.36 (m, 1H), 2.22−1.92 (m, 5H), 1.74−1.68 (m, 2H), 1.50−1.32 (m, 5H), 1.29−1.20 (m, 5H), 1.14−1.02 (m, 4H). MS (ESI, m/z) [M + H]+: 859.9. HPLC purity: 97.8% (254 nm). 2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((4-(((hexahydro-1Hcyclopenta[c]furan-5-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-4(2-((S)-2-(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan7-yl)benzamide (12b). Yield: 13.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H), 11.40 (s, 1H), 8.65−8.40 (m, 2H), 8.00 (s, 1H), 7.84−7.75 (m, 1H), 7.58−7.42 (m, 4H), 7.29−6.96 (m, 4H), 6.66 (d, J = 8.8 Hz, 1H), 6.36 (s, 1H), 6.17 (s, 1H), 3.75−3.67 (m, 2H), 3.44−3.36 (m, 2H), 3.30−3.24 (m, 1H), 3.23−3.13 (m, 3H), 3.05−2.98 (m, 3H), 2.97−2.90 (m, 3H), 2.21−2.06 (m, 3H), 2.02−1.95 (m, 1H), 1.93−1.84 (m, 2H), 1.82−1.62 (m, 3H), 1.55− 1.45 (m, 4H), 1.43−1.29 (m, 6H), 1.21−1.08 (m, 7H). MS (ESI, m/ z) [M + H]+: 887.9. HPLC purity: 97.0% (254 nm). (S) -2- ( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- (2- (2- isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((4(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)benzamide (12c). Yield: 13.5%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.47−8.35 (m, 2H), 7.95−7.87 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 6.8 Hz, 1H), 7.45−7.39 (m, 1H), 7.27 (s, 1H), 7.22−7.17 (m, 1H), 7.16− 7.07 (m, 2H), 6.91−6.81 (m, 1H), 6.61 (d, J = 7.2 Hz, 1H), 6.30 (s, 1H), 6.21 (s, 1H), 3.66−3.58 (m, 1H), 3.30−3.15 (m, 5H), 3.12− 3.02 (m, 2H), 3.02−2.93 (m, 3H), 2.92−2.82 (m, 2H), 2.70−2.65 (m, 1H), 2.62−2.55 (m, 3H), 2.36−2.30 (m, 1H), 2.21−2.11 (m, 1H), 2.05−1.93 (m, 1H), 1.86−1.70 (m, 6H), 1.51−1.33 (m, 9H), 1.19−1.13 (m, 6H). MS (ESI, m/z) [M + H]+: 874.9. HPLC purity: 98.9% (254 nm). (S)-2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((4-(((4,4difluorocyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-4-(2-(2(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzamide (12d). Yield: 37.9%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.44 (s, 1H), 8.68−8.61 (m, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.98−7.83 (m, 1H), 7.79 (dd, J = 8.6, 1.8 Hz, 1H), 7.54−7.44 (m, 3H), 7.37−7.21 (m, 3H), 7.11 (d, J = 9.2 Hz, 1H), 6.67 (dd, J = 9.0, 2.0 Hz, 1H), 6.40− 6.35 (m, 1H), 6.20−6.14 (m, 1H), 4.84−4.60 (m, 1H), 3.92−3.72 (m, 1H), 3.72−3.56 (m, 1H), 3.32−3.22 (m, 2H), 3.14−2.99 (m, 3H), 2.97−2.90 (m, 2H), 2.46−2.30 (m, 1H), 2.24−1.94 (m, 7H), 1.88−1.68 (m, 5H), 1.48−1.30 (m, 6H), 1.30−1.18 (m, 6H), 1.11 (d, J = 6.8 Hz, 3H). MS (ESI, m/z) [M + H]+: 896.9. HPLC purity: 98.0% (254 nm). 2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((4-((((1s,4s)-4-hydroxy4-methylcyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-4-(2((S)-2-(2-isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7yl)benzamide (12f). Yield: 49.8%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.58−8.48 (m, 2H), 8.00 (d, J = 2.4 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.55−7.40 (m, 4H), 7.28−7.09 (m, 3H), 6.98 (d, J = 9.2 Hz, 1H), 6.65 (dd, J = 8.8, 2.0 Hz, 1H), 6.35 (dd, J = 3.0, 1.8 Hz, 1H), 6.17 (d, J = 1.6 Hz, 1H), 3.95 (s, 1H), 3.32−3.16 (m, 5H), 3.06−2.96 (m, 2H), 2.95−2.86 (m, 2H), 2.31− 2.14 (m, 1H), 1.91−1.66 (m, 4H), 1.59−1.44 (m, 7H), 1.43−1.29 (m, 7H), 1.29−1.19 (m, 3H), 1.17 (d, J = 6.8 Hz, 3H), 1.13 (d, J = 6.8 Hz, 3H), 1.07 (s, 3H). MS (ESI, m/z) [M + H]+: 890.1. HPLC purity: 99.4% (254 nm). 2-( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- ( (S) -2- (2- isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((4((((1r,4r)-4-methoxy-4-methylcyclohexyl)methyl)amino)-3nitrophenyl)sulfonyl)benzamide (12g). Yield: 16.3%, yellow solid. https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX R 1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 11.43 (s, 1H), 8.62−8.51 (m, 2H), 8.05−7.99 (m, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.54−7.42 (m, 3H), 7.39−7.16 (m, 3H), 7.07 (d, J = 9.6 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.40−6.34 (m, 1H), 6.17 (s, 1H), 4.86−4.58 (m, 1H), 3.89−3.72 (m, 1H), 3.71−3.55 (m, 1H), 3.32−3.25 (m, 3H), 3.09 (s, 3H), 3.08−2.98 (m, 2H), 2.97−2.88 (m, 2H), 2.41− 2.28 (m, 1H), 2.18−1.92 (m, 4H), 1.74−1.56 (m, 6H), 1.45−1.33 (m, 6H), 1.27−1.18 (m, 6H), 1.17−1.07 (m, 8H). MS (ESI, m/z) [M + H]+: 903.9. HPLC purity: 96.8% (254 nm). N-((4-((((S)-1,4-Dioxan-2-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4-(2-((S)-2-(2isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzamide (12h). Yield: 1.8%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 11.46 (s, 1H), 10.35−10.05 (m, 1H), 8.63−8.50 (m, 2H), 8.02 (d, J = 2.0 Hz, 1H), 7.90−7.76 (m, 2H), 7.54−7.42 (m, 3H), 7.39−7.22 (m, 3H), 7.10 (d, J = 9.6 Hz, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.41−6.32 (m, 1H), 6.18 (s, 1H), 4.82−4.67 (m, 1H), 3.89−3.74 (m, 3H), 3.72−3.55 (m, 3H), 3.53−3.44 (m, 2H), 3.43−3.36 (m, 1H), 3.31−3.20 (m, 1H), 3.17−3.10 (m, 1H), 3.09−3.00 (m, 2H), 3.00−2.86 (m, 2H), 2.46−2.34 (m, 1H), 2.22− 1.90 (m, 5H), 1.52−1.28 (m, 7H), 1.24 (d, J = 6.4 Hz, 3H), 1.11 (d, J = 6.4 Hz, 3H). MS (ESI, m/z) [M + H]+: 863.9. HPLC purity: 99.1% (254 nm). N-((4-((((R)-1,4-Dioxan-2-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4-(2-((S)-2-(2isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)benzamide (12i). Yield: 23.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 11.47 (s, 1H), 10.50−10.30 (m, 1H), 8.63−8.50 (m, 2H), 8.02 (d, J = 2.0 Hz, 1H), 7.90−7.76 (m, 2H), 7.54−7.42 (m, 3H), 7.39−7.22 (m, 3H), 7.10 (d, J = 9.6 Hz, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.41−6.32 (m, 1H), 6.18 (s, 1H), 4.82−4.67 (m, 1H), 3.89−3.74 (m, 4H), 3.72−3.55 (m, 3H), 3.53−3.44 (m, 2H), 3.43−3.36 (m, 1H), 3.31−3.20 (m, 1H), 3.17−3.10 (m, 1H), 3.10−3.00 (m, 2H), 2.98−2.86 (m, 2H), 2.46−2.34 (m, 1H), 2.22− 1.90 (m, 5H), 1.52−1.28 (m, 7H), 1.24 (d, J = 6.4 Hz, 3H), 1.11 (d, J = 6.4 Hz, 3H). MS (ESI, m/z) [M + H]+: 863.9. HPLC purity: 100% (254 nm). 2-( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- ( (S) -2- (2- isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((2(morpholinomethyl)-7-nitroindolin-5-yl)sulfonyl)benzamide (12j). Yield: 14.1%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 11.38 (s, 1H), 8.47 (s, 1H), 8.23 (s, 1H), 8.06−8.01 (m, 1H), 7.94−7.76 (m, 1H), 7.54−7.44 (m, 4H), 7.38−7.20 (m, 3H), 6.72−6.63 (m, 1H), 6.40−6.33 (m, 1H), 6.17 (s, 1H), 4.85−4.65 (m, 1H), 4.40−4.25 (m, 1H), 3.93−3.77 (m, 1H), 3.72−4.47 (m, 4H), 3.31−3.21 (m, 1H), 3.21−3.07 (m, 2H), 3.07−2.99 (m, 2H), 2.98− 2.87 (m, 2H), 2.87−2.75 (m, 2H), 2.65−2.55 (m, 1H), 2.47−2.30 (m, 5H), 2.20−1.95 (m, 5H), 1.50−1.30 (m, 7H), 1.28−1.17 (m, 6H), 1.11 (d, J = 6.4 Hz, 3H). MS (ESI, m/z) [M + H]+: 889.0. HPLC purity: 96.1% (254 nm). 2-( (1H-Pyrrolo[2 ,3-b]pyr id in-5-y l )oxy)-4- (2- ( (S) -2- (2- isopropylphenyl)pyrrolidin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-N-((5nitro-3-(tetrahydro-2H-pyran-4-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)sulfonyl)benzamide (12k). Yield: 24.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.76 (s, 1H), 8.14 (s, 1H), 7.99 (s, 1H), 7.56−7.49 (m, 2H), 7.48−7.40 (m, 2H), 7.30 (d, J = 1.6 Hz, 1H), 7.27−7.10 (m, 3H), 6.69−6.61 (m, 1H), 6.35 (s, 1H), 6.20−6.13 (m, 1H), 4.34−4.25 (m, 1H), 4.04−3.97 (m, 1H), 3.90− 3.80 (m, 2H), 3.53−3.44 (m, 1H), 3.32−3.14 (m, 6H), 3.09−2.97 (m, 2H), 2.97−2.87 (m, 2H), 2.31−2.09 (m,1H), 1.94−1.69 (m, 5H), 1.68−1.54 (m, 3H), 1.53−1.46 (m, 1H), 1.45−1.22 (m, 8H), 1.18 (d, J = 6.4 Hz, 3H), 1.14 (d, J = 6.4 Hz, 3H). MS (ESI, m/z) [M + H]+: 889.9. HPLC purity: 97.6% (254 nm). 2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-(((3R)-3-(4-hydroxy-4methylcyclohexyl)-5-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-7yl)sulfonyl)-4-(2-((S)-2-(2-isopropylphenyl)pyrrolidin-1-yl)-7azaspiro[3.5]nonan-7-yl)benzamide (12l). Yield: 2.0%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 11.35 (s, 1H), 8.73 (s, 1H), 8.13 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.62−7.37 (m, 4H), 7.34−7.00 (m, 4H), 6.65 (d, J = 8.0 Hz, 1H), 6.35 (s, 1H), 6.16 (s, 1H), 4.28−4.19 (m, 1H), 4.10−4.01 (m, 1H), 4.00−3.90 (m, 1H), 3.54−3.43 (m, 2H), 3.31−3.21 (m, 2H), 3.07−2.97 (m, 2H), 2.95− 2.85 (m, 2H), 2.35−2.13 (m, 1H), 2.00−1.67 (m, 4H), 1.60−1.30 (m, 13H), 1.29−1.10 (m, 10H), 1.09−1.01 (m, 3H). MS (ESI, m/z) [M + H]+: 917.9. HPLC purity: 90.7% (254 nm). 2-((1H-Pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-(((3R)-3-(4-hydroxy-4methylcyclohexyl)-5-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-7yl)sulfonyl)-4-(2-((S)-2-(2-isopropylphenyl)pyrrolidin-1-yl)-7azaspiro[3.5]nonan-7-yl)benzamide (12m). Yield: 7.7%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 11.42 (s, 1H), 8.79 (s, 1H), 8.18 (d, J = 1.6 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.94−7.72 (m, 1H), 7.54−7.44 (m, 3H), 7.40−7.20 (m, 4H), 6.72− 6.62 (m, 1H), 6.39−6.33 (m, 1H), 6.19−6.13 (m, 1H), 4.85−4.65 (m, 1H), 4.31−4.21 (m, 2H), 4.10−4.01 (m, 1H), 3.92−3.76 (m, 1H), 3.73−3.58 (m, 1H), 3.57−3.49 (m, 1H), 3.30−3.22 (m, 1H), 3.08−2.98 (m, 2H), 2.97−2.88 (m, 2H), 2.45−2.31 (m, 1H), 2.18− 1.96 (m, 4H), 1.73−1.62 (m, 2H), 1.60−1.48 (m, 3H), 1.45−1.30 (m, 7H), 1.30−1.19 (m, 8H), 1.14−1.06 (m, 6H). MS (ESI, m/z) [M + H]+: 917.9. HPLC purity: 97.3% (254 nm). Determination of Bcl-2 Crystal Structure: Protein Expression and Purification. Human Bcl-2 (residues 6−34, 92−207) gene was cloned into a pet28a vector with His6-SUMO tag and Ulp cleavage site. The protein was expressed in E. coli BL21 (DE3) grown in LB media. Protein expression was induced with 0.1 mM IPTG for 16 h at 16 °C. The His6-SUMO tagged protein was purified with Ni-NTA beads (Qiagen) in 20 mM Tris pH 8.0 supplemented with 300 mM NaCl and 5 mM β-mercaptoethanol. The SUMO tag was removed overnight at 4 °C with Ulp1 protease. The protein was further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 75 column (GE Healthcare). Crystallization, Data Collection, and Structure Determination. Purified Bcl-2 was concentrated to 10 mg/mL in a buffer containing 20 mM Tris pH 8.0 and 150 mM NaCl. The protein was incubated with 1 mM compounds on ice for 30 min before crystallization. The crystals were obtained at 20 °C by the hanging drop vapor diffusion method by mixing 1 μL of protein solution with 1 μL of reservoir. Specifically, Bcl-2:S-9c crystals were obtained from a reservoir containing 0.1 M ammonium acetate, 0.1 M bis-tris pH 5.5, 17% PEG 10000; Bcl-2:S-10r crystals were obtained from 0.2 M sodium chloride, 0.1 M bis-tris pH 5.5, and 25% PEG 3350. The crystals were cryoprotected with the addition of 20% glycerol before being flashfrozen in liquid nitrogen. Diffraction data were collected at beamlines BL02U1 at the Shanghai Synchrotron Radiation Facility. An XDS package was used for indexing, scaling, and data reduction.36 The structures were determined by the Phaser-MR program in Phenix37 using Bcl-2 (PDB ID: 4MAN)38 as the search model. Model building was performed using Coot,39 and the structures were refined with Phenix Refine. The X-ray diffraction and structure refinement statistics are summarized in Table S1. Biochemical Competitive Binding Assay. The disruption of the Bcl-2:BAK complex was tested at room temperature using TR-FRET methodology. Compounds (0−1 μM) were preincubated with Histagged Bcl-2 or Bcl-2 G101V protein for 30 min. The BAK-derived peptide (Ac-GQVGRQLAIIGDK (FITC) INR-amide) and the detection reagents were then added to plates and incubated for another 60 min. The TR-FRET signals (excitation at 337 nm, emission at 490 nm/520 nm) were recorded on the BMG PHERAstar FSX reader (BMG Labtech, Ortenberg, Germany), and the IC50 of each compound was determined by fitting the inhibition percentage of the protein−ligand interaction at different compound concentrations using the four-parameter logistic model in Dotmatics (Dotmatics, Boston, MA). Similar methods were used for Bcl-xL with the exception that the compound concentrations ranged from 0 to 10 μM. Surface Plasmon Resonance (SPR). The binding kinetics of BGB11417 and venetoclax were measured in an SPR assay using Biacore 8K (Cytiva, Marlborough, MA) at room temperature. The experiments were performed in an HBS-N buffer containing 10 mM HEPES pH 7.4, 250 mM NaCl, 50 μM EDTA, 0.1% tween 20, and 1% DMSO. Briefly, His-tagged Bcl-2 (1.33 μg/mL) or His-tagged Bcl-2 G101V (1.56 μg/mL) was captured using an NTA sensor chip (Cytiva, Marlborough, MA). BGB-11417 (0−40 nM) or venetoclax https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX S (0−800 nM) was flowed over the chip with 240-s injections and 800-s dissociations at a flow rate of 50 μL/min. The binding KD (equilibrium binding constant) was best fitted using a 1:1 binding site kinetic model. Cell Viability Assay. RS4;11 and MOLT-4 were obtained from ATCC (Manassas, VA). RS4;11 and MOLT-4 cells were grown in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1% penicillin−streptomycin (Thermo Fisher Scientific, Waltham, MA). RS4;11 overexpressing different Bcl-2 mutants were generated by lenti-vectors transduction. In brief, the coding sequences (CDS) of different Bcl-2 mutants were inserted into pWPI plasmid. Transfection of the plasmid together with lentivirus packing plasmids pMD2.G and psPAX2 was performed on HEK293T cells. The culture medium containing virus was collected and used to infect RS4;11 cells, and GFP expressing cells were enriched by FACS. The growth inhibitory activities of compounds were measured using CellTiter-Glo luminescent cell viability assays (Promega, Madison, WI). Cells were treated with stepwise increasing concentrations of compounds for 2 days. An equal volume of CellTiter-Glo reagent was added to the cell culture medium in each well, and the solution was mixed on an orbital shaker for 2 min to allow cell lysis. The samples were incubated for 10 min to generate stabilized luminescent signal applied to quantify the amount of ATP and thus determine the number of metabolically active cells. The luminescent signal was measured on the PHERAstar FSX reader (BMG Labtech, Ortenberg, Germany). The IC50’s of compounds were determined by fitting the cell viability inhibition at different compound concentrations using the four-parameter logistic model in GraphPad Prism software. In Vitro ADME and Pharmacokinetics Studies. For metabolic stability, liver microsomes from different species are incubated with the test compound at 37 °C in the presence of NADPH. Samples are removed at the appropriate time points, and the disappearance of test compound is monitored by LC−MS/MS to estimate an in vitro intrinsic clearance (CLint). For cytochrome P450 (CYP) inhibition, isoform-specific substrates are incubated with human liver microsomes and a range of test compound concentrations (typically 0.1−30 μM). At the end of the incubation, the formation of metabolite is monitored by LC−MS/MS at each of the test compound concentrations. A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC50 value. Mice and dogs were used to evaluate the in vivo pharmacokinetic property of the test compounds. The animals (n = 3 per group) were orally or intravenously administered. The test compounds were dissolved with formulation of 30% PEG-400/60% Phosal 50 PG/10% ethanol for po dosing or 10% dimethylacetamide (DMA)/10% Solutol HS15/80% water for iv dosing. After the administration, blood samples were collected from all animals at designated time points. Plasma samples were obtained via centrifugation, and sample cleanup was conducted by protein precipitation with acetonitrile. Compound concentrations in plasma were quantified using a liquid chromatography with tandem mass spectrometry (LC−MS/MS) method. The in vivo pharmacokinetic studies were carried out at Shanghai ChemPartner Co. Ltd. (Shanghai, China). RS4;11 Tumor Xenografts. RS4;11 tumor cells (1 × 107/mouse) were implanted sc in the right flanks of the female NCG mice purchased from GemPharmatech Co., Ltd. When tumors reached approximately 170 mm3 in the efficacy study or 650 mm3 in the PD/ PK study (on day 11 and day 24 after inoculation, respectively), the mice were randomly divided into indicated groups (n = 10 per group for efficacy and n = 4 per group for PD/PK) based on tumor volume and body weight and treated with compounds for indicated days. All compounds were formulated for oral dosing in 60% (v/v) Phosal 50 PG, 30% (v/v) PEG-400, and 10% (v/v) ethyl alcohol. Mice were treated once or once daily (qd) via oral gavage (po) at 10 mL/kg with the doses. Tumor volume was measured twice weekly in two dimensions using a caliper. Body weights were recorded twice weekly. Mice were monitored daily for clinical signs of toxicity throughout the study. PD/PK Study. At indicated time points after single dosing of compounds, blood and tumors were removed and immediately snapfrozen. Total proteins were extracted from the tumors and subjected to ELISA detection of tumor cleaved caspase-3 (Ser 29) levels performed according to the instruction of the human caspase-3 (Ser 29) simple step ELISA kit (Abcam, Cambridge, U.K.). For the PK study. Plasma was collected by centrifuge at 5600 rpm for 7 min. The tumor samples were processed by adding ice-cold 50% methanol/ water solution to the precut and chopped tumor tissue and then homogenized by an MP FastPrep-24 tissue homogenizer. The proteins in plasma and tumor samples were precipitated using acetonitrile containing an internal standard and then removed by centrifugation at 13 000 rpm for 8 min. Bioanalysis was conducted at 3D BioOptima Co., Ltd. (Suzhou, China). The compound levels in plasma and tumor were determined by LC−MS/MS (Thermo, Waltham, MA). Animal Care and Handling. All experiments were conducted based on the protocols approved by the Animal Care and Use Committee of BeiGene according to the guidelines of the Chinese Association for Laboratory Animal Sciences. ■ ASSOCIATED CONTENT
Data Availability Statement
The authors will release the atomic coordinates for S-9c and S10r upon publication of this drug annotation. *sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00027. Cocrystal of S-9c and S-10r with Bcl-2; single crystal structure determination; general synthetic procedure of some intermediates and 10m and analytical spectrum (PDF) Molecular formula strings list (CSV) Crystallographic information file for BGB-11417 (CIF)
Accession Codes
The crystallographic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID: 8HTR for S-9c and 8HTS for S-10r). ■ AUTHOR INFORMATION
Corresponding Authors
Yunhang Guo − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China; orcid.org/0000-0003-3980-0673; Email: yunhang.guo@beigene.com Zhiwei Wang − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China; orcid.org/0000-0001-8792-9573; Email: zhiwei.wang@beigene.com
Authors
Hai Xue − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Nan Hu − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Ye Liu − Department of Molecular Science, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Hanzi Sun − Department of Molecular Science, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX T Desheng Yu − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Ling Qin − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Gongyin Shi − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Fan Wang − Department of DMPK-BA, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Lei Xin − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Weihua Sun − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Fan Zhang − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Xiaomin Song − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Shuran Li − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Qiang Wei − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Ying Guo − Department of Molecular Science, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Yong Li − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Xiaoxin Liu − Department of Molecular Science, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Shuaishuai Chen − Department of Discovery Biology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Taichang Zhang − Department of DMPK-BA, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Yue Wu − Department of DMPK-BA, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Dan Su − Department of DMPK-BA, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Yutong Zhu − Department of Discovery Biology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Aiying Xu − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Haipeng Xu − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Shasha Yang − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Zhijun Zheng − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Junhua Liu − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Xuefei Yang − Department of Discovery Biology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Xi Yuan − Department of Discovery Biology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Yuan Hong − Department of Molecular Science, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Xuebing Sun − Department of Molecular Science, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Yin Guo − Department of Discovery Biology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Changyou Zhou − Department of Medicinal Chemistry, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China; orcid.org/0000-0001-5214-8291 Xuesong Liu − Department of Discovery Biology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Lai Wang − Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd., Beijing 102206, People’s Republic of China Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.4c00027
Author Contributions
#Yunhang Guo and Hai Xue contributed equally to this work.
Notes
The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The authors acknowledge the contributions to this work of all past and present members of the Bcl-2 team. We thank all supporting functions, including the compound management team and analytical team, etc. We also would like to thank Jiuyang Liu, Xin Li, Qin Wang, Zhu Mei, and Xudong Luan for their great help. ■ ABBREVIATIONS USED AcOH, acetic acid; AML, acute myelogenous leukemia; Bcl-2, B - c e l l l ymphoma -2 ; B INAP , r a c em i c - 2 , 2 ′ - b i s - (diphenylphosphino)-1,1′-binaphthyl; binding KD, equilibrium binding constant; CLL, chronic lymphocytic leukemia; Cs2CO3, cesium carbonate; CYP, cytochrome P450; DCM, dichloromethane; DDIs, drug−drug interactions; DEA, diethanolamine; DLBCL, diffuse large B-cell lymphoma; DMAP, 4- dimethylaminopyridine; DMSO, dimethyl sulfoxide; EDCI, 1- ethyl-3-(3-(dimethylamino)propyl)carbodiimide; EDTA, ethylene diamine tetraacetic acid; ESI, electrospray ionization; EtOH, ethanol; FDA, U.S. Food and Drug Administration; G101V, Gly101Val; HATU, 2-(7-azabenzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate; HPLC, high performance liquid chromatography; iv, intravenous; K2CO3, potassium carbonate; KI, knock-in; LM, liver microsome; MM, multiple myeloma; mPFS, median progression-free survival; MS, mass spectrum; NaBH(OAc)3, sodium triacetoxyborohydride; NaCl, sodium chloride; NaHCO3, sodium bicarbonate; NaOH, sodium hydroxide; Na2SO4, sodium https://doi.org/10.1021/acs.jmedchem.4c00027 J. Med. Chem. XXXX, XXX, XXX−XXX U sulfate; NHL, non-Hodgkin lymphoma; NMR, nuclear magnetic resonance spectroscopy; N2, nitrogen; ORR, overall response rate; PD, pharmacodynamics; Pd(dppf)Cl2, [1,1′- bis(diphenylphosphino)ferrocene]dichloropalladium(II); Pd2(dba)3, tris(dibenzylideneacetone)dipalladium; PE, petroleum ether; PK, pharmacokinetics; po, oral administration; qd, once daily; SAR, structure−activity relationship; SPR, surface plasmon resonance; TEA, triethylamine; TGI, tumor growth inhibition; t-BuOK, potassium tert-butylate; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMEDA, N,N,N′,N′tetramethylethylenediamine; WT, wild type ■ REFERENCES (1) Czabotar, P. E.; Lessene, G.; Strasser, A.; Adams, J. M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat. Rev. Mol. Cell. Biol. 2014, 15, 49−63. (2) Hanahan, D.; Weinberg, R. A. The hallmarks of cancer. Cell 2000, 100, 57−70. (3) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646−674. (4) Youle, R. J.; Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell. Biol. 2008, 9, 47−59. (5) Adams, J. M.; Cory, S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 2007, 26, 1324−37. (6) Anderson, M. A.; Huang, D.; Roberts, A. Targeting BCL2 for the treatment of lymphoid malignancies. Semin Hematol 2014, 51, 219− 227. (7) Martin, L. A.; Dowsett, M. BCL-2: a new therapeutic target in estrogen receptor-positive breast cancer? Cancer Cell 2013, 24, 7−9. (8) Vaux, D. L.; Cory, S.; Adams, J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 1988, 335, 440−442. (9) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 1958−1962. (10) Roberts, A. W.; Huang, D. Targeting BCL2 with BH3 mimetics: basic science and clinical application of venetoclax in chronic Lymphocytic leukemia and related B cell malignancies. Clin. Pharmacol. Ther. 2017, 101, 89−98. (11) Oltersdorf, T.; Elmore, S.; Shoemaker, A.; et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005, 435, 677−681. (12) Wendt, M. D.; Shen, W.; Kunzer, A.; et al. Discovery and Structure-Activity Relationship of Antagonists of B-Cell Lymphoma 2 Family Proteins with Chemopotentiation Activity in Vitro and in Vivo. J. Med. Chem. 2006, 49, 1165−1181. (13) Bruncko, M.; Thorsten, K. O.; Belli, B. A.; et al. Studies Leading to Potent, Dual Inhibitors of Bcl-2 and Bcl-xL. J. Med. Chem. 2007, 50, 641−662. (14) Park, C.-M.; Bruncko, M.; Adickes, J.; et al. Discovery of an Orally Bioavailable Small Molecule Inhibitor of Prosurvival B-Cell Lymphoma 2 Proteins. J. Med. Chem. 2008, 51, 6902−6915. (15) Tse, C.; Shoemaker, A. R.; Adickes, J.; et al. ABT-263: A Potent and Orally Bioavailable Bcl-2 Family Inhibitor. Cancer Res. 2008, 68, 3421−3428. (16) Souers, A.; Leverson, J.; Boghaert, E.; et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 2013, 19, 202−210. (17) (a) Deeks, E. D. Venetoclax: first global approval. Drugs 2016, 76, 979−987. (b) AbbVie Announces US FDA Approval of VENCLEXTA® (venetoclax) as a Chemotherapy-Free Combination
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