Development of Potent NEDD8-Activating Enzyme Inhibitors Bearing a Pyrimidotriazole Scaffold

Chaodong Xiong,¶ Lina Zhou,¶ Jing Tan, Shanshan Song, Xubin Bao, Ning Zhang, Huaqian Ding, Jiannan Zhao, Jin-Xue He,* Ze-Hong Miao,* and Ao Zhang*

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*sı Supporting Informat■


The ubiquitin (Ub)-proteasome system (UPS) modulates the homeostasis of a broad range of intracellular proteins that possess essential cell functions, including survival, proliferation, apoptosis, and differentiation.1−5 The UPS pathway is executed by iterative covalent linking of the small protein ubiquitin to the substrate protein through a cascade of three different enzymes (E1, E2, and E3) to initiate the proteasomal degradation.1 It is reported that the ubiquitylation pathway is involved in the degradation of more than 80% of intracellular proteins and dysregulation of UPS can cause many diseases,including cancer, diabetes, and neurodegenerative diseases.6 Unfortunately, though the UPS pathway has been extensively exploited as drug targets, drugs targeting the three enzymes (E1, E2, and E3) remain premature with critical challenge.6 Bortezomib,7 a drug targeting the downstream kinase 26S proteasome also caused many toXic side effects due to its poor selectivity in substrate degradation.

In addition to the ubiquitin proteins, ubiquitin-like (Ubl) proteins,9 such as neural precursor cell-expressed developmen- tally downregulated 8 (NEDD8) and many others, have also been found as homologous pathways involved in the conjugation and turnover of many functional proteins. Like ubiquitylation, NEDD8-engagement of proteins (neddyla- tion)9 is also a post-translational modification and catalyzed by three key enzymes successively, including the NEDD8- activating enzyme E1 (NAE; a heterodimer of AppBp1 and UBA3), the conjugating enzyme E2 (UBC12 and UBE2F), and the substrate-recruiting E3 ligase (e.g., RBX1/2 or DCN1). The culminating evidence indicated that the neddylation pathway is involved in the modulation of many biological mechanisms and dysregulation of this process can lead to tumorigenesis.10−17 Meanwhile, overactive neddylation has been reported in a variety of malignancies.18−20 Because NAE is involved in the first step of the neddylation pathway and is the only activation enzyme of this process, it has been proposed as an emergent target for the development of anticancer drugs. Many strategies have been exploited either to target the different binding domains of NAE21−23 or to disrupt its protein−protein interactions with E2 or other coen- zymes,24,25 but most of them are in a premature state.

Because NEDD8 protein forms a NEDD8-adenosine 5′- monophosphate (AMP) intermediate under the catalysis of NAE during the initial activation process, structurally similar AMP mimics generally have capacity of binding with NAE in high potency.26 Compounds 1−327−29 are the representatives of this class with compound 1 showing the optimal combination with both high potency and selectivity against NAE. This compound exhibits good antitumor efficacy in a variety of tumor Xenograft models, including hematologic malignancies30−32 and several solid tumors.19,33−36 Intrigu- ingly, the reported phase I clinical results showed that the single-agent of compound 1 (pevonedistat) only exhibited an overall response rate (ORR) of 17% in acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS).37 In addition, compound 1 has been used in combination with azacitidine in phase Ib and showed higher responses (ORR was 50%) in patients with AML.38 In a phase II clinical trial, the combination showed promising activity and acceptable toXicity in patients with higher-risk MDS as well.39 Currently, more single-agent trials for various tumors are carried out and a few phase III trials in combination with chemotherapies are also initiated (

Figure 1. Represented AMP mimics as NAE inhibitors. Scaffold hopping refers to the search for active compounds containing different core structures.

The exact reason for the relatively modest ORR in the reported clinical 1 trials with the single-agent of compound 1 is unknown, but more investigations with structurally different inhibitors in various tumor types are needed. In this regard, we recently conducted a structural modification of the clinical compound 1 by structural hopping on the central deazapurine bicyclic framework (shown in red in the X-ray crystal structure, Figure 1), followed by further optimization of the solvent interaction region (shown in blue in the X-ray crystal structure, Figure 1), leading to a new compound 26 bearing a pyrimidotriazole scaffold (Figure 1). This new compound showed comparable potency to that of compound 1, but with improved overall PK properties. Herein, we report the design, synthesis, and pharmacological evaluation of this compound.


Structure-Based Drug Design. The cocrystal structure26 of the compound 1 NEDD8 NAE complex (PDB: 3GZN) (Figure 1) showed that the ribose 3′-OH of compound 1 forms a H-bonding with the residues of Asp100 and Lys124 in the NAE enzyme. One of the sulfamate oXygens forms a H- bonding with the backbone amide NH of Gly79, and the sulfamoyl group forms a covalent adduct with the carbonyl moiety of Gly76 at the end of the NEDD8 protein. Meanwhile, the N1 and N6 on the deazapurine ring form a H-bonding with the backbone amide NH of Ile148 and the carbonyl of Gln149, respectively. The 2,3-dihydroindene fragment occupies a “gate- keeper” region of the NAE enzyme. The structural analysis clearly indicates that there is no direct contact around the pyrroles C2 and C3 of the central deazapurine ring (Figure 1, marked in red). Compared to AMP and its analogue 2 both bearing a purine core, compound 1 and its analogue 3 are structurally different by containing a deazapurine (pyrrolo[2,3- d]pyrimidine) core. All these compounds have high potency to bind NAE but with different selectivity, indicating that the N6- substituent interacting at the gate-keeper region is a determinant factor for NAE selectivity over other Ub E1 and Ubl E1. In this regard, we first conducted a scaffold-hopping approach to screen a small set of compounds bearing different central cores with both the sulfamoyl group and the 2,3- dihydroindene component intact.

Structure−Activity Relationship Study. As shown in Table 1, compound 4 bearing a pyrimidotriazole core structure
showed an IC50 value of 0.21 nM against NAE, which is equally potent to that of the reference compound 1 (IC50 = 0.26 nM). However, compound 5 containing a pyrazolopyridine core completely abolished the activity. Replacement of the deazapurine core of 1 with a simple pyrimidine moiety led to compound 6 showing much reduced potency with an IC50 value of 219 nM, indicating the importance of the central bicyclic core structure. Because compound 4 is slightly more potent than the reference compound 1, and in view of the well acceptance of triazole compounds in drug discovery, we selected this compound as a new lead NAE inhibitor for further structural optimization with the central pyrimidotria- zole core intact.As shown in Table 2, an array of substituted alkylamino compound 13 obtained by replacing the 2,3-dihydro-1H- indene fragment in compound 4 with a simple n-hexyl group21 retained moderate potency by showing an IC50 value of 21.1 nM, but the cyclopentyl analogue 14 completely lost the NAE activity. Compound 15 bearing a 4-methoXycarbonyl cyclo- pent-2-ene component showed a moderate potency of 80.9 nM. Replacing the 2,3-dihydro-1H-indene fragment of compound 4 with other bicyclic heterocycles afforded compounds 16−18. Unfortunately, all these compounds lost activity against NAE. These results indicate that the pyrimidin- 4-yl C4 substituent in pyrimidotriazole 4 can tolerate limited structural variations and the rigid structure of 2,3-dihydro-1H- indene framework remains the most advantageous to interact with NAE.

In view of the structural similarity between compounds 1 and 4, we speculate that both compounds might interact with NAE in a similar mode. A look back of the crystal structure of compound 1 NEDD8 NAE complex indicated that there is a large space around the binding domain of the 2,3-dihydro- 1H-indene fragment (Figure 1, marked in red). Therefore, we decided to test compounds bearing water-soluble groups at the C2-position of the 2,3-dihydro-1H-indene fragment that yielded compounds 19−23. As shown in Table 3, the groups were applied to replace the 2,3-dihydro-1H-inden-1-hydroXyl-substituted compound 19 showed good potency amino component as the C4 substituent of the pyrimidine ring within the pyrimidotriazole core. It was found that opening the five-membered 2,3-dihydro-1H-indene framework and intro- ducing a fluoro-substituent yielded compound 7, which retained moderate potency against NAE with an IC50 value of 6.11 nM, 29-fold less potent than that of compound 4. Further replacement of the phenyl with a furyl or thienyl moiety generated compounds 8 and 9 showing more reduced potency with IC50 values of 30.6 and 15.9 nM, respectively. EXtension of the linker yielding compounds 10−12 further reduced the potency against NAE (81−149 nM). Interestingly,against NAE with an IC50 value of 2.28 nM, 10-fold less potent than compound 4. Much reduced potency was observed for the dimethylamino substituted compound 20 (207 nM). Interestingly, the etheric analogue 21 also showed good potency with an IC50 value of 2.28 nM, but the amido analogue 22 exhibited much reduced potency with an IC50 value of 170 nM. Notably, the trans-isomeric amino-amido analogue 23 was inactive by showing an IC50 value greater than 10 μM.

Because the introduction of hydrophilic substituents on the 2,3-dihydro-1H-indene fragment did not afford compounds with higher potency than the lead compound 4, we decided to further explore the electronic and steric tolerance by introducing more bulky spiropiperidine framework at the C3- position of the 2,3-dihydro-1H-indene fragment. As shown in Table 4, the unsubstituted spiropiperidine compound 24 and the N-methyl substituted spiropiperidine 25 displayed modest potency against NAE with IC50 values of 66.9 and 187 nM, respectively. However, carbamate analogues 26−34 all showed significantly increased potency. Most of these compounds exhibited subnanomolar potency, among which, carbamates 26 and 34 were the most potent with IC50 values of 0.55 and 0.57 nM, respectively, although they were still 2-fold less potent than compound 4, but not statistically different. In order to increase aqueous solubility, an additional water-soluble group was introduced to afford compounds 32−33, showing reduced and distinct potency with IC50 values of 120 and 9.98 nM, respectively. Insertion of a methylene unit to the carbamate 26 yielded compound 35, showing 10-fold reduced potency with an IC50 value of 5.77 nM. Notably, the N-tert-butyl urea compound 36 showed an extremely high potency against NAE with an IC50 value of 0.02 nM, which is 10-fold more potent than that of compound 4. These results confirmed that the insertion of bulky spiropiperidine moieties to the 2,3-dihydro-1H-indene component was well-tolerated and both the steric and electronic characters of the N-substituent on the spiropiperidine motif played an important role in the activity against NAE.

Figure 2. Kinase selectivity of compounds 1 and 26 in the DiscoveRX KINOMEScan screening platform. The assays were performed at a single 1 μM concentration. Upper panel: TREEspot interaction maps for compounds 1 and 26 in 468 kinase targets. Lower panel: S-scores of compounds 1 and 26 with percent control numbers less than 35, 10, and 1, respectively. S-score (S = Number of hits/Number of assays) is a quantitative measure of compound selectivity.

Cell Proliferation of Selected New NAE Inhibitors. To investigate the inhibitory effects of the new NAE inhibitors on the proliferation of cancer cells, several high-potency compounds with IC50 values less than 20 nM were selected for cellular assay against the HCT-116 cell. As shown in Table 5, all these compounds showed moderate to good inhibitory activity against the growth of HCT-116 cells. Among these, nine compounds (4, 19, 21, 26−29, 31, and 34) showed IC50 values less than 150 nM, and three compounds 4, 19, and 26 were similarly potent compared to the reference compound 1, showing IC50 values less than 50 nM. It was somewhat disappointing that the highly biochemically potent compound 36 only showed moderate cell growth inhibition, compared to compound 1 (151 vs 33.89 nM), likely due to the general poor PK properties and low aqueous solubility of the urea structure in compound 36.

hERG Inhibition and Calculated Physicochemical

Properties of Selected Compounds. To quickly glimpse the overall safety and drug-likeness of these new NAE inhibitors, we tested the four potent compounds 4, 19, 26, and 31 that possess cellular potency less than 100 nM for their inhibition on the hERG channel and calculated their physicochemical properties through ADMET lab software ( As shown in Table 5, although the new NAE inhibitors have a high loading of N-atoms, none of them showed inhibition on the hERG with IC50 values greater than 40 μM, indicating their optimal cardiac safety profile. Meanwhile, these compounds showed a higher polar surface area (tPSA) and improved aqueous solubility potential (LogS), compared to the reference compound 1. In view of the high biochemical and cellular potencies, as well as the slightly better predicted physicochemical properties, compound 26 was selected for further study.

Pharmacokinetic Parameters of Compound 26. To investigate the developability of compound 26, the pharmaco- kinetic properties of this compound in Sprague−Dawley (SD) rats were profiled. The reference compound 1 was evaluated as well as a comparison. As shown in Table 6, after intravenous injection (iv) at a dose of 1 mg/kg, compound 26 has lower systemic plasma clearance (CL = 16.5 vs 29.7 mL/min/kg), a longer half-life (T1/2 = 1.36 vs 0.71 h), and two-fold higher plasma exposure (AUClast = 1328 vs 563 h·ng/mL), compared to the reference 1. The overall improved PK parameters of compound 26 encouraged further evaluation.

Kinase Selectivity Assays of Compounds 26. Because our new NAE inhibitor 26 bears more N-atoms than the reference compound 1, off-targets might be a concern. To exclude the off-target liability, we evaluated the kinase selectivity of both compounds 1 and 26 through the KINOMEScan screening against a panel of 468 kinases and their mutants at 1 μM concentration.40 As shown in Figure 2, compound 1 inhibited only one kinase TAOK1 at a concentration of 1 μM and the inhibitory effect is as low as 35% [S-score (35) = 0.002], otherwise both compounds showed high selectivity against NAE without significant inhibition against all the tested kinases and their mutants [S- score (10) and S-score (1) = 0] (see Supporting Information for details).

Compound 26 Potently Inhibited Both NAE and UAE Activity In Vitro. Compound 26 was further evaluated using mechanism-based in vitro assays. As shown in Figure 3, compound 26 exhibited comparable inhibitory potency against NAE relative to the reference 1 with IC50 values of 0.55 and 0.26 nM, respectively (Figure 3a). At much higher concentrations, compound 26 was found to inhibit the related ubiquitin-activating enzyme (UAE) with an IC50 value of 66.84 nM (Figure 3b). As shown in Figure 3c, a concentration- dependent reduction of the intensity of Ubc12-NEDD8 bands (as well as UBA3-NEDD8 and Cul1-NEDD8) was observed and the formation of Ubc12-NEDD8 was evidently inhibited at 0.15 μM. These results indicated that compound 26 is indeed an effective NAE inhibitor and suppresses NAE-mediated Ubc12-NEDD8 conjugation in cell-based assays.

Figure 3. NAE and UAE dual inhibition by compound 26. (a,b) The dose−response relationship of compound 26 for inhibition of NAE (a) and UAE (b) assayed by HTRF. Data are expressed as the mean ± SD from three independent experiments. (c) Compound 26 inhibited neddylation in the cellular-based assay. Whole-cell lysates were prepared from HCT-116 treated with compound 26 or the positive control for 4 h, as the concentration showed, and analyzed by western blotting. (d) Inhibition of compound 26 for the E1 enzymes NAE, UAE, and SAE. The activity of NAE, UAE, and SAE was assessed by immunoblot measuring of the thioester levels of Ubc12- NEDD8, UbcH10-Ub, and Ubc9-SUMO, respectively.

To determine the probability of compound 26 suppressing other E1 isoforms, a dose−response experiment was performed to measure levels of E2-UBL thioester products by western blotting. As shown in Figure 3d, there was no appreciable suppression of the formation of Ubc9-SUMO even at 10 μM of compound 26, suggesting no off-target effect on SAE (SUMO activation enzyme E1). However, in line with enzymatic activity, compound 26 suppressed UbcH10-Ub formation at 3.3 μM, suggesting its modest inhibition against UAE. Taken together, through enzyme-based and cell-based assays, compound 26 was validated as a high-potency NAE and modest potency UAE dual inhibitor.

Compound 26 Inhibited CRL Substrates’ Turnover and Elicited DNA Damage Responses. Because inhibition of NAE can result in inactivation of Cullin-RING ligases (CRLs), leading to the accumulation of CRL substrates,27 therefore, we tested these effects as well. It was found that treatment of compound 26 inhibited neddylation and increased CRL substrates CDT1, p27, p21, and p-IκBα (Ser32/36) in a concentration-dependent manner, which led to DNA damage (γ-H2AX) accumulation and checkpoint activation (p-Chk1) (Figure 4).

Compound 26 Inhibited the Proliferation of Various Cancer Cells. To assess the overall antiproliferative activity of compound 26 and identify the most sensitive cancer cells for in vivo study, a panel of cell lines, including leukemia, colon, and prostate cancer, were treated with increasing concentrations of compound 26 for 72 h. The results showed that the growth was effectively inhibited in all the cell lines tested, with IC50 values ranging between 33.9 and 482 nM (Table 7). The proliferation inhibition observed for compound 26 was similar to that for the reference compound 1 (IC50: 36.25−484 nM). Notably, compound 26 exerted the most potent anticancer effects against HCT-116 colon cancer cells with an IC50 value of 33.9 nM. Meanwhile, slightly lower but still high potency was observed in the leukemia MV-4-11 cells with an IC50 value of 106 nM. On the basis of these results, the two cell lines were selected for in vivo studies.

Figure 4. Inhibitory effects of compound 26 on the neddylation and DNA damage pathway. HCT-116 and HuTu80 cancer cells were treated with indicated drugs for 24 h. Whole-cell lysates were immunoblotted for Cul1- or Ubc12-NEDD8, CRL substrates, including CDT1, p27, p21, p-IκBα (Ser32/36), and DNA damage markers γH2AX and p-Chk1(Ser317).

Figure 5. Cell cycle arrest and apoptosis induced by compound 26. (a) Cell cycle arrest was analyzed by PI-staining-based flow cytometry after HCT-116 was treated with the indicated drug for 24 h. (b,c) Compound 26 induced apoptosis through caspase-3/7 activation. HCT-116 cells were treated with the indicated drugs for 48 h and then subjected to Caspase-Glo 3/7 assays (b) or western blotting (c). Error bars represent the SEM for at least three independent experiments. Data are analyzed by one-way ANOVA, **, p < 0.01, ***, p < 0.001. Compound 26 Induced Cell Cycle Arrest and Apoptosis in HCT-116 Cells. To clarify the anticancer mechanism of compound 26 in the most sensitive HCT-116 cells, it was treated at a lower concentration for 24 h. As shown in Figure 5, an increase in the G2/M phase was observed. However, higher concentrations led to cell accumulation in the S phase, accompanied by an increase of the polyploidy population (DNA content > 4N) (Figure 5a). Consistently, a luminescent assay that measures caspase-3 and -7 activities indicated that compound 26 promoted cell apoptosis through activating caspase-3/7 (Figure 5b). Furthermore, treatment with compound 26 increased the expression of cleaved caspase-3, -7, and PARP1 (Figure 5c). Collectively, these results suggest that compound 26 suppresses cell proliferation through cell cycle arrest and apoptosis.

Compound 26 Exhibited Potent Antitumor Efficacy

In Vivo in Xenograft Models. Based on the promising in vitro potency of the new NAE inhibitor 26, we further evaluated its antitumor activity in vivo in nude mice xenografts from human HCT-116 and MV-4-11 cancer cells. The animals were injected subcutaneously with the vehicle, positive control compound 1, or compound 26 for 18−21 days. A previous study has established the optimal dosing paradigm of compound 1 to achieve significant antitumor efficacy with good tolerance.27 Therefore, we followed this schedule as suggested for compound 1 in the HCT-116 mice: 60 mg/kg twice daily (BID) with 5 on/5 off/8 on. In our preliminary study, the maximum tolerated single-dose for compound 26 was determined as 150 mg/kg, with 130 mg/kg being well- tolerated (data not shown) in this models. Therefore, compound 26 was given at either 65 mg/kg or 130 mg/kg once daily (QD) consecutively. In the MV-4-11 mice, the same dosing paradigm (BID) was used for both compounds. As shown in Figure 6a,b, both compounds 26- and 1-treated mice showed significant suppression of tumor growth compared to vehicle-treated mice in a dose-dependent manner (compound 26 at 130 mg/kg, QD: T/C = 0.20 or 0.19; compound 1 at 60 mg/kg, BID: T/C = 0.36 or 0.10). At these doses, no significant change of the body weight was observed, indicating there was no apparent toXicity upon the treatments in both Xenograft models. In general, both compounds showed similar high antitumor efficacy in these models but compound 26 has a better safety profile with a convenient dosing schedule.

Figure 6. In vivo activities of compound 26 in cell-derived Xenografts. Nude mice bearing subcutaneous Xenografts derived from HCT-116
(a) or MV-4-11 (b) were injected subcutaneously daily with 65 or 130 mg/kg compound 26, or twice daily with 60 mg/kg compound 1, respectively. 5 on/5 off/8 on represents 5 days treatment followed by 5 days free of treatment and 8 days treatment in an 18 days study. Relative tumor volume and body weight were separately plotted over the treatment time. Mean tumor volumes ± SEM are shown. n = 6 mice per group. Data are analyzed by Student t-test, *, p < 0.05; **, p < 0.01; and ***, p < 0.001. ■ CHEMISTRY The synthesis of compounds 4 and 6−23 bearing a pyrimidotriazole core is outlined in Scheme 1. The key amino-diol intermediate 37 was prepared according to the reported process.41 A subsequent reaction of 37 with 5-amino- 4,6-dichloropyrimidine afforded compound 38 in 73% yield. Cyclization of compound 38 with isoamyl nitrite delivered unstable intermediate 39, which was then subjected to aromatic nucleophilic substitution immediately with (S)- (+)-1-aminoindane hydrochloride in the presence of N, N- diisopropylethylamine (DIPEA) to provide compound 40a in 98% yields over two steps. Subsequent sulfamoylation of 40a with chlorosulfonamide afforded the pyrimidotriazole core compound 4 in 17% yield. Similarly, aromatic nucleophilic substitution of the intermediate 39 by various commercially available primary amines provided compounds 40a-k and 40n- r in 25−98% yields over two steps. Alternatively, a Pd-catalyzed Buchwald−Hartwig coupling of 39 with different aromatic primary amines produced 40l-m in 40−52% yields over two steps. Meanwhile, the reaction of the intermediate 39 with 4,5,6-trifluoropyrimidine through a microwave irradiation provided compound 41 in 46% yield. Aromatic nucleophilic substitution of pyrimidine 41 with (S)-(+)-1-aminoindane hydrochloride in the presence of DIPEA provided compound 42 in 25% yield. Finally, synthesis of target compounds 6−23 was achieved from 42 and 40b-r by using chlorosulfonamide as the sulfonylation reagent. The preparation of compound 5 bearing a pyrazolopyridine core is shown in Scheme 2. Compound 4342 reacted with 4- chloro-1H-pyrazolo[3,4-b]pyridine via the Mitsunobu reaction to afford compound 44. Subsequent O-Bn deprotection with BCl3 provided the diol intermediate 45 in 48% yield over two steps. Substitution of the pyrazolopyridinyl chloride 45 with (S)-(+)-1-aminoindane under microwave irradiation delivered compound 46 in 45% yield. Treatment of 46 with chlorosulfonamide generated pyrazolopyridine 5 in 15% yield. The synthesis of compounds 24-36 is described in Scheme 3. The chiral amine 4743 was treated with intermediate 39 to provide compound 48, which was sulfamoylated with chlorosulfonamide to yield compound 31 in 7.1% yield over two steps. Subsequent N-Boc deprotection of 31 with 4 M HCl in methanol afforded compound 24 in 76.2% yield. Treatment of compound 24 with CH3I or tert-butylisocyanate afforded compounds 25 and 36 in 29.6 and 19.5% yields, respectively. Meanwhile, acylation of compound 24 with various commercially available chloroformate afforded com- pounds 26−29 and 34−35 in 32.7−44.2% yields. Alternatively, treatment of amine 24 with commercially available 4-nitrophenyl carbonate produced compounds 30 and 33 in 23.5 and 26.5% yields, respectively. It is of note that compound 32 was prepared in 23% overall yield through the reaction of compound 24 with 2,2-dimethyl-1,3-dioXan-5-yl (4- nitrophenyl)carbonate, followed by hydroXyl deprotection with 2 M HCl (aq). ■ CONCLUSIONS In order to develop structurally different high-potency NEDD8-activating enzyme (NAE) inhibitors, we conducted a structural hopping strategy by first investigating the structural variations of the central deazapurine bicyclic framework of the clinical compound 1, followed by focused optimization of the solvent interaction region, leading to identification of a new NAE inhibitor 26 bearing a pyrimidotriazole scaffold. This new inhibitor 26 has IC50 values of 0.55 and 36.2 nM, respectively, from the biochemical and HCT-116 cell assays. Compound 26 also exhibited slightly less-potent activity against UAE with an IC50 value of 66 nM. Treatment with compound 26 was found to inhibit the neddylation pathway and increase CRL substrates. Compared to its prototype compound 1, compound 26 displayed obviously higher plasma concentrations and better PK parameters in mice. In vivo, compound 26 showed significant antitumor efficacy and a good safety profile in both the HCT-116 and MV-4-11 Xenograft models. Collectively, compound 26 is a new high-potency NAE inhibitor with improved PK profiles and worthy of further profiling. ■ EXPERIMENTAL SECTION Chemistry General. All reactions were performed in glassware containing a Teflon-coated stir bar. Commercially available solvents and reagents were obtained from Adamas, Accela, Aikonchem, Sinopharm Chemical, Energy Chemical, Aladdin, Innochem, Macklin, Meryer, Leyan, MCE, Bidepharm, Sigma-Aldrich, Alfa Aesar, J&K, and TCI, and used without further purification. 1H and 13C NMR spectra were recorded on a Varian Mercury 300, 400, or 500 NMR spectrometer and referenced to CD3OD. Chemical shifts (δ) were reported in ppm downfield from an internal tetramethylsilane standard. High-resolution mass spectrometry (HRMS) analysis was recorded at an anionizing voltage of 70 eV on a Finnigan/MAT95 spectrometer. Flash column chromatography on silica gel (200−300 mesh) was used for the routine purification of reaction products. All reactions were monitored by thin-layer chromatography (TLC) on silica gel plates (15 mm × 50 mm) and spots were visualized under UV light at 254 nm. Optical rotations were measured on a JASCO p- 2000 using a 1dm polarimeter tube at 589 nm. HPLC analysis was conducted for all bioassayed compounds on an Agilent Technologies 1260 series LC system (Eclipse XDB-CN, 4.6 mm × 150 mm, 5 μM, MeOH/H2O, 25 °C) with two ultraviolet wavelengths (UV 254 and 210 nm). The purities of these compounds were above 95%. Scheme 1. Synthesis of Compounds 4, 7−23a aReagents and conditions: (a) 5-amino-4,6-dichloropyrimidine, Et3N, n-butanol, 120 °C, overnight, and 73%; (b) 1-butyl nitrite or isoamyl nitrite, 4 Å, CH3CN, 70 °C, and 1−2 h; (c) for preparation of 40a-k and 40n-r and 42: primary amine DIPEA, CH3CN, rt (or 90 °C, MW, for 40k), 6−8 h, and 25−98% over two steps; (d) for preparation of 40l-m: primary amine Pd2(dba)3, Cs2CO3, Xantphos, DMF, N2, 100 °C, 4 h, and 40−52% over two steps; (e) NH2SO2Cl, pyridine, CH3CN, 0 °C, 30 min−2 h, and 8.9−25.2%; (f) 4,5,6-trifluoropyrimidine, DIPEA, n-butanol, MW, 120 °C, 1 h, and 46%. Synthetic Procedures. Compounds 1, 37, 43, and 47 were prepared according to reported literature procedures.41−44 The absolute structure of the synthetic new compounds was confirmed by the X-ray crystals of an analogue of this series (see Supporting Information).((1S,2S,4R)-4-(7-(((S)-2,3-Dihydro-1H-inden-1-yl)amino)-3H- [1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-hydroxycyclopentyl)methyl Sulfamate (4). To a solution of 37 (120 mg, 1.2 mmol) in n-butanol (5 mL) were added 5-amino-4,6-dichloropyrimidine (295 mg, 1.8 mmol) and Et N (0.34 mL, 2.4 mmol). The miXture was refluXed To a solution of 38 (129 mg, 0.5 mmol) in CH3CN (5 mL) was added isoamyl nitrite (135 μL, 1 mmol). The miXture was stirred at 70 °C for 1 h. The reaction was monitored by TLC. Upon completion, the reaction miXture was concentrated in vacuo to give the unstable intermediate 39, which was then dissolved in anhydrous CH3CN. (S)-(+)-1-Aminoindane hydrochloride (110.5 mg, 0.65 mmol) and DIPEA (248 μL, 1.5 mmol) were added to the solution of 39 in anhydrous CH3CN. The miXture was stirred at rt for 6−8 h. The miXture was concentrated in vacuo and the residue was purified Reagents and conditions: (a) 39, DIPEA, CH3CN, rt, 6−8 h, and 54.4%; (b) NH2SO2Cl, pyridine, CH3CN, 0 °C, 30 min−2 h, and 13%; (c) 4 M HCl, MeOH, rt, overnight, and 76.2%; (d) for 25: CH3I, DMF, rt, 15 min, and 29.6%; for 26−29 and 34−35: corresponding chloroformate, DIPEA, DMF, rt, 30 min, and 32.7−44.2%; for 30 and 33: corresponding 4-nitrophenyl carbonate, DIPEA, DMF, rt, 1−2 h, and 23.5−26.5%; for 36: tert-butylisocyanate, DIPEA, DMF, rt, 1 h, and 19.5%; (e) for 32: 2,2-dimethyl-1,3-dioXan-5-yl (4-nitrophenyl) carbonate, DIPEA, DMF, rt, 2 h, then 2 M HCl (aq), 2 h, and 23%. To a solution of compound 40a (180 mg, 0.49 mmol) in anhydrous CH3CN (5 mL) at 0 °C was added a solution of chlorosulfonamide (68 mg, 0.59 mmol) in CH3CN and pyridine (118 μL, 1.47 mmol). The miXture was stirred for 30 min−2 h, and additional chlorosulfonamide (0.3 equiv) was added until the amount of water and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/NH3 7.0 M solution in CH3OH from 100:1 to 20:1) to give a crude product, which was further purified using reverse-phase HPLC to afford compound 4 (38 mg, 17%) as a white solid. 1H NMR (500 MHz, methanol-d4): δ 8.39 (s, 0.74 H), 8.21 (s, 0.20 H), 7.20−7.30 (m, 3H), 7.14 (t, J = 7.3 Hz,1H), 6.54 (t, J = 7.4 Hz, 0.21 H), 5.97 (t, J = 7.5 Hz, 0.74 H), 5.64−5.55 (m, 1H), 4.59−4.54 (m, 1H), 4.40 (dd, J = 9.7, 7.6 Hz, 1H), 4.23 ((1S,2S,4R)-4-(4-(((S)-2,3-Dihydro-1H-inden-1-yl)amino)-1H- pyrazolo[3,4-b]pyridin-1-yl)-2-hydroxycyclopentyl)methyl Sulfa- mate (5). To a solution of compound 43 (1.87 g, 6 mmol) in THF (15 mL) at 0 °C under nitrogen were added 4-chloro-1H- pyrazolo[3,4-b]pyridine (920 mg, 6 mmol), PPh3 (3.14 g, 12 mmol), and DIAD (2.4 mL, 12 mmol). The miXture was stirred for 8 h and then diluted with ethyl acetate. The organic phase was washed with 1 M HCl (30 mL, ×3), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product 44 was obtained as a C17H19FN7O4S, 436.1209; found, 436.1205. [α]26.3 −5.00 (c 0.04,yellowish solid and used in the next step without further purification. To a solution of crude product 44 in DCM (50 mL) at −78 °C under nitrogen was added 1 M BCl3 in DCM (60 mL). The miXture was allowed to slowly warm up to 0 °C and stirred for 4 h. The reaction was quenched with MeOH/Et3N (1:1) and the miXture was stirred for 15 min and then concentrated in vacuo. The residue was purified by silica column chromatography (PE/EA from 10:1 to 1:1) to give compound 45 (384 mg, 48.1%). 1H NMR (400 MHz, methanol-d4): δ 8.20 (s, 1H), 7.95 (d, J = 6.4 Hz, 1H), 6.41 (d, J = 6.4 Hz, 1H), 5.48−5.39 (m, 1H), 4.56−4.50 (m, 1H), 3.81 (dd, J = 10.8,7.2 Hz, 1H), 3.67 (dd, J = 10.8, 6.5 Hz, 1H), 2.63−2.52 (m, 1H),2.50−2.39 (m, 1H), 2.34−2.25 (m, 1H), 2.23−2.11 (m, 1H), 2.11−2.01 (m, 1H). Compound 45 (384 mg, 1.44 mmol) was converted to the target compound 5 (44 mg, 6.89%) as a white solid through a procedure similar to the preparation of compound 4. 1H NMR (400 MHz, methanol-d4): δ 8.14 (s, 1H), 8.04 (d, J = 5.7 Hz, 1H), 7.29 (t, J = 7.7 Hz, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 6.44 (d, J = 5.6 Hz, 1H), 5.66−5.57 (m, 1H), 5.33 (t, J = 6.8 Hz, 1H), 4.54−4.50 (m, 1H), 4.37 (dd, J = 9.6, 7.7 Hz, 1H), 4.20 (dd, J = 9.6, 7.4 Hz,1H), 3.13−3.02 (m, 1H), 3.00−2.89 (m, 1H), 2.86−2.75 (m, 1H),2.70−2.59 (m, 1H), 2.47−2.37 (m, 1H), 2.35−2.26 (m, 1H), 2.25−2.14 (m, 1H), 2.14−1.99 (m, 2H). 13C NMR (126 MHz, methanol-d4): δ 152.26, 151.07, 150.51, 144.75, 144.45, 132.38, 129.07, 127.68,CH3OH).((1S,2S,4R)-4-(7-((Furan-2-ylmethyl)amino)-3H-[1,2,3]triazolo- [4,5-d]pyrimidin-3-yl)-2-hydroxycyclopentyl)methyl Sulfamate (8). White solid (21 mg, 10.5%). 1H NMR (400 MHz, methanol-d4): δ 8.38 (s, 0.70H), 8.25 (s, 0.14H), 7.43 (s, 1H), 6.34 (s, 2H), 5.66−5.54 (m, 1H), 5.27 (s, 0.36 H), 4.83 (s, 1.68 H), 4.60−4.53 (m, 1H),4.39 (dd, J = 9.7, 7.5 Hz, 1H), 4.22 (dd, J = 9.7, 7.3 Hz, 1H), 2.96−2.83 (m, 1H), 2.64−2.53 (m, 1H), 2.48−2.38 (m, 1H), 2.38−2.24 (m, 2H). 13C NMR (126 MHz, methanol-d4): δ 157.45, 155.96, 152.74, 149.26, 143.44, 126.31, 111.40, 108.55, 73.09, 70.44, 57.76,44.80, 42.97, 38.15, 34.35. ESI ([M − H]+) m/z: 408.4. HRMS (ESI): calcd for C15H18N7O5S, 408.1096; found, 408.1103. [α]26.3 +23.75 (c 0.08, CH3OH). Corresponding treatments as indicated. Tumor and body weight measurements were performed twice a week. All procedures for animal studies were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica. ASSOCIATED CONTENT *sı Supporting Information The Supporting Information is available free of charge at 1H and 13C spectra of all new compounds, HPLC of representative compounds, X-ray crystallographic data for analogues of compound 26, and KINOMEscan kinase selectivity profile for compounds 1 and 26 (PDF) In Vitro Enzyme Assays. Homogeneous time-resolved fluo- rescence (HTRF) assay was used to measure the in vitro activity of NAE or UAE as previously reported.27,45 Briefly, 10 μL of the reaction system containing 150 nM His-NEDD8, 80 nM GST-Ubc12, and 2 nM NAE recombinant enzyme (R&D systems, USA) in reaction buffer [50 mM N-(2-hydroXyethyl)piperazine-N′-ethanesulfonic acid (HEPES), 5 mM MgCl2, 20 μM ATP, 250 μM L-glutathione, 0.05% bovine serum albumin, and pH 7.5] was used to identify inhibitors of NAE. The reaction miXture was incubated at 27 °C for 2 h in a 384- well plate, and the reaction was stopped by stop/detection buffer [0.1 M HEPES, 20 mM ethylenediaminetetraacetic acid, 410 mM KF, 0.05% Tween 20, pH 7.5, 0.25 μg/mL MAb anti 6HIS-Eu and 2 μg/mL MAb anti GST-XL665 (CisBio International)]. After incubating overnight, the plate was read on the PerkinElmer EnVision using 330 nm excitation/620 nm emission and 330 nm excitation/665 nm emission. The inhibition rate (%) was calculated as: [1 − (HTRF value (compound)/HTRF value (control)] × 100%. The mean IC50 values were determined with the Logit method from three independent assays. A similar assay protocol was used to measure UAE enzymes. Western Blot Analysis. Immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis conducted under non- reducing conditions for E2-UBL thioesters or reducing conditions for other proteins was carried out as previously described.27 Primary antibodies against Ubc12 (#5641), NEDD8 (#2754), UbcH10 (#14234), Ubc9 (#4786), CDT1 (#8064), p27 (#3688), p-IκBα (Ser32/36) (#9246), p-Chk1 (Ser317) (#2344), Chk1 (#2360), γ-H2AX (#2577), Caspase-3 (#9662), Cleaved Caspase-3 (#9661), Caspase-7 (#9492), cleaved Caspase-7 (#8438), and cleaved PARP (#5625) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Cul1 (sc-17775), UBA3 (sc-377272), PARP1 (sc-7150), and p21 (sc-271610) were purchased from Santa Cruz (Dallas, TX, USA). Antibody against GAPDH was purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies were diluted at 1:1000 with QuickBlock Western dilution reagent (Beyotime). The secondary antibodies (1:2000) including HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies used for Western blotting were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Cell Viability Assays. Cells viability was assessed using the sulforhodamine B (SRB) assays as previously described46 after cells cultured in 96-well plates were treated with the indicated drugs for 3 days.Cell Cycle Analysis. HCT-116 cells were incubated with the indicated drugs for 24 h and analyzed by PI staining-based flow cytometry as previously described46. Caspase-Glo 3/7 assays. HCT-116 cells were seeded into the 96-well plates and treated with indicated drugs for 48 h. Caspase-3/7 activities were assessed using the Caspase-Glo 3/7 Assay Reagent (Promega) according to the manufacturer′s protocol.In Vivo Anticancer Activity Experiments. HCT-116 and MV-4-11 Xenografts were established by inoculating 5 × 106 cells s.c. in nude mice, respectively. The efficacy study was initiated when tumors had reached approXimately 150 mm3.27 Tumor-bearing mice were randomly assigned to control and treatment groups. ■ AUTHOR INFORMATION Corresponding Authors Jin-xue He − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China; Phone: +86-21-50801068; Email: [email protected] Ze-Hong Miao − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China; Phone: +86-21-50806072; Email: [email protected] Ao Zhang − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; Pharm-X Center, School of Pharmacy and Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; University of Chinese Academy of Sciences, Beijing 100049, China; State Key Laboratory of Esophageal Cancer Prevention and Treatment, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, China; 0001-7205-9202; Phone: +86-21-34204020; Email: [email protected] Authors Chaodong Xiong − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; University of Chinese Academy of Sciences, Beijing 100049, China Lina Zhou − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China Jing Tan − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; University of Chinese Academy of Sciences, Beijing 100049, China Shanshan Song − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China Xubin Bao − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China Ning Zhang − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China Huaqian Ding − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China Jiannan Zhao − State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China; Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China Complete contact information is available at: Author Contributions ¶C.X. and L.Z. contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (grants 81773565, 82073875, 82073865, and 81703327). The start-up grants from Shanghai Jiao Tong University to the Research Laboratory of Medicinal Chemical Biology & Frontiers on Drug Discovery (AF1700037 and WF220217002) to the Platform on Target Identification of Innovative drugs (WH10117001) are also appreciated. ABBREVIATIONS:PARA Ub, ubiquitin; Ubl, ubiquitin-like; UPS, ubiquitin-proteasome system; NEDD8, neural precursor cell-expressed developmen- tally downregulated 8; NAE, NEDD8-activating enzyme; AMP, adenosine 5′-monophosphate; ORR, overall response rate; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; SAR, Structure−activity relationship; UAE, ubiq- uitin-activating enzyme; SAE, SUMO activation enzyme E1; CRLs, Cullin-RING ligases; T1/2, half-life; AUC, area under the plasma concentration−time curve; CL, clearance; Vss, volume of distribution; tPSA, topological polar surface area; HTRF, homogeneous time-resolved fluorescence; SRB, sulfo- rhodamine B REFERENCES (1) Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ. 2005, 12, 1191−1197. (2) Mocciaro, A.; Rape, M. Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control. J. Cell Sci. 2012, 125, 255− 263. (3) Orlowski, R. 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