Novel Derivative of Bardoxolone Methyl Improves Safety for the

Article Cite This: J. Med. Chem. 2017, 60, 8847-8857

pubs.acs.org/jmc

Novel Derivative of Bardoxolone Methyl Improves Safety for the Treatment of Diabetic Nephropathy Zhangjian Huang,*,†,‡,# Yi Mou,†,‡,# Xiaojun Xu,*,†,‡ Di Zhao,†,§ Yisheng Lai,†,‡ Yuwen Xu,∥ Cen Chen,∥ Ping Li,† Sixun Peng,†,‡ Jide Tian,⊥ and Yihua Zhang*,†,‡ †

State Key Laboratory of Natural Medicines, ‡Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, and §Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, P.R. China ∥ Crystal Pharmatech Co., Ltd., Suzhou 215123, P.R. China ⊥ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095, United States S Supporting Information *

ABSTRACT: Currently, no effective and safe medicines are available to treat diabetic nephropathy (DN). Bardoxolone methyl (CDDO-Me) has displayed promising anti-DN activity as well as serious side effects in clinical trials, probably because the highly reactive α-cyano-α,β-unsaturated ketone (CUK) in ring A of CDDO-Me can covalently bind to thiol functionalities in many biomacromolecules. In this study, we designed and synthesized a γ-glutamyl transpeptidase (GGT)-based and CUK-modified derivative of CDDO-Me (2) to address this issue. 2 can be specifically cleaved by GGT, which is highly expressed in the kidney, to liberate CDDO-Me in situ. It should be noted that 2 exhibited anti-DN efficacy comparable to that of CDDO-Me with much less toxicity in cells and db/db mice, suggesting that its safety is better than CDDO-Me. Our findings not only reveal the therapeutic potential of 2 but also provide a strategy to optimize other synthetic molecules or natural products bearing a pharmacophore like CUK to achieve safer pharmaceutical drugs.

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NF-κB signaling.9 Given that oxidative stress and inflammation are major risk factors for the development of diabetic nephropathy (DN),10−12 CDDO-Me has already been clinically investigated for its anti-DN effects. Treatment with CDDO-Me in an amorphous spray-dried dispersion formulation initially improved the estimated glomerular filtration rate (eGFR) in patients with DN at stages 3b−4;13,14 however, CDDO-Me increased the hospitalization rate and mortality due to heart failure in patients with stage 4 DN in a phase III clinical trial,14,15 leading to the termination of the clinical trial. Currently, the mechanism underlying the detrimental adverse effects of CDDO-Me is still elusive. Notably, mitochondrial function is essential for maintaining myocardial tissue homeostasis, and many anticancer drugs such as doxorubicin, herceptin, and cisplatin directly or indirectly deteriorate cardiac mitochondrial function, leading to serious cardiotoxic effects.16 It was reported that a rapid and selective depletion of mitochondrial glutathione induced by CDDO-Me may be responsible, at least in part, for its cytotoxic effects.17 Furthermore, it is known that CDDO-Me is a potent antiangiogenesis agent,18 and impaired angiogenesis may bring about the transition from cardiac hypertrophy to heart failure.19 It is also known that diabetic patients with a higher risk

INTRODUCTION α,β-Unsaturated ketone is an important pharmacophore in many natural products and synthetic small molecules that reacts with a variety of thiols in biomacromolecules via Michael addition. These reactions may modulate signaling pathways and lead to various biological activities in vivo.1−3 Synthetic derivatives of the natural product oleanolic acid (OA),4 such as 2-cyano-3,12dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its ester (CDDO-Me, i.e., bardoxolone methyl) and amide (CDDO-Im) forms (Figure 1) with two α,β-unsaturated ketone moieties in rings A and C, respectively, display potent anti-inflammatory and antioxidant activities through activating the Kelch-like ECHassociated protein 1 (Keap1)−nuclear factor E2 related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway.5,6 It has been reported that α-cyano-substituted α,βunsaturated ketone (CUK) in ring A instead of α,β-unsaturated ketone in ring C of CDDO-Me attacks Cys151 in the BTB domain of Keap1 protein through Michael addition,7 resulting in the dissociation of Keap1 and Nrf2 and preventing the proteolysis of Nrf2. The free Nrf2 then translocates into the nucleus and binds to the ARE, inducing the expression of Nrf2 target genes, including NAD(P)H:quinoneoxidoreductase 1 (NQO1), glutathione-S-transferases (GSTs), and heme-oxygenase-1 (HO-1).7,8 Similarly, CDDO-Me also attacks Cys-179 of inhibitor of nuclear factor kappa-B kinase (IKK) and thus inhibits © 2017 American Chemical Society

Received: July 1, 2017 Published: October 10, 2017 8847

DOI: 10.1021/acs.jmedchem.7b00971 J. Med. Chem. 2017, 60, 8847−8857

Journal of Medicinal Chemistry

Article

Figure 1. Rationale for the design of a GGT-based and CUK-modified derivative of CDDO-Me (2).

Scheme 1. Synthesis of Compound 2a

Reaction conditions and reagents: (a) EEDQ, CH2Cl2, rt for 12 h; (b) PBr3, anhydrous THF, 0 °C for 0.5 h; (c) K2CO3, DMF, 0 °C for 12 h; (d) NHEt2, anhydrous CH2Cl2; (e) plasma or liver homogenate.

a

of heart dysfunction may be more sensitive to CDDO-Me.20,21 Accordingly, although a phase II clinical trial of CDDO-Me for the treatment of DN has been reinitiated in Japan,22 the cardiac safety of CDDO-Me remains a serious concern. Since CDDO-Me interacts with 577 cellular proteins, as revealed by a proteomic analysis,23 the side effects of CDDO-Me may be attributed to its highly reactive CUK moiety in ring A, which reacts with thiol(s) in other proteins besides Keap1 and IKKβ through Michael addition. Accordingly, we envisioned that rational structural modification of CUK in CDDO-Me might reduce its off-target effects. However, the ordinary, simple modification of α,β-unsaturated ketone compounds24,25 is not suitable for use with CDDO-Me. The major issue is that the 1,4addition reaction of CUK results in a reversible product.26,27 Indeed, we have found that the 1,4-addition product, enol 1 (Figure 1), obtained by reaction of the CUK with sodium hydroxide was easily converted back to CDDO-Me through a 1,4-elimination reaction. Nevertheless, it has been reported that the enol, generated from the 1,4-addition of a CUK-containing

compound to a Grignard reagent, could be etherified by a substituted chlorosilane to generate a stable siloxane compound.28 These successful conversions of CUK to the enol and then to the siloxane suggested the possibility of designing a new derivative of CDDO-Me by a similar structural modification of its CUK. Considering the fact that γ-glutamyl transpeptidase (GGT) is expressed mainly in the kidney and has been utilized for GGT-based renal targeting drugs,29−36 we designed a GGTbased and CUK-modified derivative of CDDO-Me (2) by coupling enol 1 with 4-glutamylbenzyl bromide, and we hypothesized that 2 would be relatively stable in circulation and other organs but could be specifically cleaved by GGT in the kidney, followed by 1,6-elimination37 to generate intermediate 1 in situ, which may subsequently produce CDDO-Me via 1,4elimination, as depicted in Figure 1. To verify this hypothesis, we synthesized compound 2, investigated its stability, pharmacokinetics, and tissue distribution, and compared its therapeutic efficacy and safety with those of CDDO-Me in vitro and in vivo. 8848



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Figure 2. Stability of 2 in various media. 2 was incubated in (A) PBS (pH = 7.4), (B) PBS (pH = 7.4) with (1 U/mL) GGT, (C) PBS (pH = 7.4) with (1 U/mL) GGT and (2 U/mL) esterase from porcine liver, (D) rat kidney homogenate, (E) rat plasma, and (F) rat liver homogenate. The conversion of 2 to 8 or further to CDDO-Me was measured by HPLC. Results in panels B−F are expressed as the mean ± SD from three individual experiments.

Figure 3. Compound 2 activates Nrf2 signaling. (A) HEK293 cells were transfected with an ARE-dependent luciferase reporter plasmid and subsequently treated in triplicate with CDDO-Me or 2 at the indicated concentrations for 48 h. Luciferase activity was measured to assess Nrf2 activity. HEK293 cells were treated in triplicate with 2 (200, 1000 nM) or CDDO-Me (200, 1000 nM) for 48 h, and the relative levels of Nrf 2 (B), HO-1 (C), and NQO1 (D) mRNA transcripts were analyzed by quantitative RT-PCR. Furthermore, the relative levels of nuclear and cytosolic Nrf2 (E), the ratio of nuclear to cytosolic Nrf2 (G), HO-1 (F, H), and cytoplasmic NQO1 (F, I) expression were analyzed by western blot assay. Results are representative images and expressed as the mean ± SD of each group from three individual experiments. *P < 0.05, **P < 0.01.

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Figure 4. Effect of 2 on NF-κB signaling. (A) HEK293 cells were transfected with an NF-κB driven luciferase reporter plasmid, and the effects of 2 on TNF-α induced NF-κB activity were determined. Furthermore, HEK293 cells were pretreated with TNF-α (10 ng/mL) for 6 h and then incubated in triplicate with 2 (200, 1000 nM) or CDDO-Me (200, 1000 nM) for 24 h. The relative levels of iNOS (B), COX2 (C), MCP-1 (D), and TNF-α (E) mRNAs to the control GAPDH mRNA in HEK293 cells were detected by quantitative RT-PCR, whereas the relative levels of nuclear and cytosolic p65 (F, G) and the expression of NF-κB target genes iNOS (H, I) and COX2 (H, J) in HEK293 cells were measured by western blot assay. Results are representative images or expressed as the mean ± SD of each group of cells from three individual experiments. *P < 0.05, **P < 0.01.

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RESULTS Synthesis of Compound 2. N-Fmoc glutamic acid monomethyl ester 3 was condensed with 4-aminophenylmethanol 4 in the presence of 2-ethoxy-1(2H)-quinoline carboxylic acid ethyl ester (EEDQ) to give amide 5. Bromination of 5 in the presence of phosphorus tribromide furnished benzyl bromide 6. Meanwhile, CDDO-Me was treated with NaOH in DMF to offer enol 1 (Figure 1). Without further purification, 1 was etherified with 6 to give enol ether 7. Finally, removal of the N-protective group in 7 provided target compound 2. Compound 8 could be generated by hydrolysis of 2 in the presence of plasma or liver homogenate (Scheme 1). Absolute Configuration of C1 in 2. As mentioned above, the 1,4-addition of sodium hydroxide to the 1,2-double bond in CDDO-Me generates enol 1 (Figure 1) with chirality at C1. Since the methyl group with a large amount of steric hindrance at C10 is present on the plane of ring A, the OH− should theoretically attack C1 (ortho to C10) under the plane, finally generating C1 with an R configuration in compounds 1, 2, 7, and 8. Initially, we tried to prepare a single crystal of compound 2 to confirm this, but we failed after several attempts over period of months. Eventually, analogue 9 [(1-hydroxyl-2-cyano-3-benzyloxy)-12-oxo-oleana-2(3),9(11)-dien-28-oic acid methyl ester, CCDC1471081, see the Supporting Information] with the same

configuration of C1 as that of 2 was synthesized and analyzed by X-ray diffraction. The absolute configuration of C1 in 9 was successfully assigned as R, thus establishing the R configuration of C1 in compound 2. 2 Has Improved Solubility Compared to CDDO-Me. Because the introduction of both hydroxyl and glutamic acid moieties at C1 and C3 in ring A of CDDO-Me, respectively, may improve aqueous solubility, we determined the solubility of 2 by using a well-established HPLC method.38 We found that its solubility was 0.0089 mg/mL in saline, which increased significantly to 12.23 mg/mL in mixed solvents of saline with 1% 1,3-propylene glycol and 1% Tween 80. In contrast, CDDOMe could not be dissolved in these solvents. Release of CDDO-Me from 2 Promoted by GGT in Vitro. Next, we investigated the stability of 2 in different medium by HPLC. After incubating 2 in PBS (pH = 7.4) at 37 °C for 8 h, around 20% of it was turned over (Figure 2A) with no CDDOMe detected. To determine whether 2 could release CDDO-Me in the presence of GGT, incubation of 2 in Krebs buffer (pH = 7.4) with GGT (1 U/mL) at 37 °C for 8 h resulted in around 54% of 2 converted to CDDO-Me (Figure 2B). Subsequently, incubation of 2 with GGT (1 U/mL) and a porcine liver esterase (2 U/mL) under the same conditions increased the conversion of 2 to CDDO-Me up to 88% (Figure 2C). We 8850



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Figure 5. Treatment with 2 significantly mitigates DN-related kidney damage in mice. The mice were treated with CDDO-Me (0.65 mg/kg, the same molar ratio as 1 mg/kg 2) or 2 (1 or 2 mg/kg) for 12 weeks. At the 11th week, the 24 h urine volumes (A) and levels of urinary albumin (B) and creatinine (C) in individual mice were measured before the mice were sacrificed, and the urine albumin to creatinine ratio (D) in individual mice was calculated. Furthermore, the kidney sections (5 μm) were stained with hematoxylin−eosin, Masson’s trichrome, and periodic acid Schiff (E, magnification ×400). The ultrathin kidney sections were subjected to TEM analysis (F−J). The thickness of the GBM in individual mice was measured, and a total of 30 locations for each mouse were calculated (K). Data are representative images and expressed as the mean ± SD of individual groups of mice (n = 6).

reasoned that GGT might preferentially recognize 8 with a free glutamine acid moiety, which was produced via hydrolysis of 2 by the esterase to generate CDDO-Me. Furthermore, incubation of 2 with rat kidney homogenates at 37 °C for 8 h caused 66% of 2

to be converted to CDDO-Me (Figure 2D). In addition, we found that over 60% of 2 in rat plasma was converted into 8, and only 4.2% was hydrolyzed to CDDO-Me (Figure 2E); 32% of 2 was converted to CDDO-Me in the liver homogenates (Figure 8851



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Figure 6. (A) Plasma concentration−time profiles of 2 and CDDO-Me. After intravenous administration of 2 (10 mg/kg) or CDDO-Me (10 mg/kg) to rats, blood samples were taken from the suborbital vein at 0, 2, 5, 15, 30, and 60 min and 1.5, 2, 4, 6, 8, 12, and 24 h and analyzed by LC-MS/MS. (B−D) Tissue distribution study of 2 and CDDO-Me. Individual rats were injected intraperitoneally with CDDO-Me (0.04 mmol, 20 mg/kg) or 2 (0.04 mmol, 30 mg/kg), and their tissue (including heart, kidney, and liver) samples were collected longitudinally and analyzed by LC-MS/MS. (B) Tissue distribution of CDDO-Me, (C) tissue distribution of 2, and (D) generation of CDDO-Me from 2 measured by LC-MS/MS.

phosphorylation and degradation.39 To determine whether 2 has anti-inflammatory activity, HEK293 cells were transfected with an NF-κB luciferase reporter plasmid to test the impact of 2 on TNF-α stimulated NF-κB activation and luciferase activity. Treatment with TNF-α alone significantly increased the luciferase activity in HEK293 cells, whereas treatment with either 2 or CDDO-Me significantly reduced TNF-α induced luciferase activity (Figure 4A). Similarly, treatment with TNF-α alone significantly increased the relative levels of iNOS, COX2, MCP-1, and TNF-α mRNA transcripts, whereas treatment with 2 or CDDO-Me significantly decreased the TNF-α induced upregulation of NF-κB target genes such as iNOS, COX2, and MCP-1 (Figure 4B−E). In addition, treatment with 2 or CDDOMe significantly mitigated the TNF-α induced increase in the ratio of nuclear to cytosolic p65, as well as the expression of NFκB target genes in HEK293 cells (Figure 4F−J). In conclusion, 2 exhibited antioxidant and anti-inflammatory activities in HEK293 cells, comparable to those of CDDO-Me. 2 Displays Anti-DN Effects Comparable to CDDO-Me. To examine the effect of 2 on DN,40 db/db mice at 8 weeks of age were randomized and treated intraperitoneally with vehicle, 2 (1 or 2 mg/kg), or CDDO-Me (0.65 mg/kg) three times per week for 12 consecutive weeks. We found that there was no significant difference in the amount of food and water consumed among the different groups of mice (Table S10). Furthermore, the vehicletreated mice lost about 15% of their body weight by the end of experiment. Treatment with CDDO-Me significantly increased the body weight of the mice by 11.3%, but treatment with either dose of 2 did not significantly change the body weight of the mice. In comparison with the DN control mice, treatment with 2 or CDDO-Me significantly reduced the levels of plasma urea nitrogen (BUN) and uric acid (UA) and also slightly decreased the levels of plasma creatinine in mice. Treatment with 2 or

2F). As shown in Figure 2B−D, 8 might be easily transferred to CDDO-Me in the presence of GGT, a combination of GGT and esterase, and rat kidney homogenate. Collectively, 2 was relatively stable in aqueous buffers, plasma, and liver homogenates, whereas it was labile, liberating CDDO-Me through the action of GGT in kidney homogenate. 2 Activates Keap1−Nrf2−ARE Signaling More Strongly than CDDO-Me. It is known that CDDO-Me can activate the Keap1−Nrf2−ARE signaling axis and exhibit a potent cytoprotective effect.7 Additionally, 2 was found to be converted to CDDO-Me in the presence of GGT, as well as in rat kidney homogenate, plasma, and liver homogenate (Figure 2B−F, respectively). It was therefore of interest to determine whether 2 possesses the ability to activate Nrf2 signaling. Human embryonic kidney 293 (HEK293) cells were transfected with a Nrf2/ARE luciferase reporter and then treated with 2 or CDDOMe. Treatment with 2 stimulated the activity of Nrf2 in HEK293 cells, which was similar to treatment with CDDO-Me (Figure 3A). Next, the effect of 2 on Nrf2 activation was assessed by quantitative RT-PCR and western blot assays. Treatment with 2 or CDDO-Me did not change the relative levels of the Nrf 2 mRNA transcript (Figure 3B), but it significantly increased the relative levels of HO-1 and NQO1 mRNA transcripts in HEK293 cells (Figure 3C,D). Furthermore, treatment with 2 or CDDOMe significantly increased the ratio of nuclear to cytosolic Nrf2 protein and the relative levels of HO-1 and NQO1 in HEK293 cells. Interestingly, the effect of 2 on stimulating Nrf2 signaling was stronger than that of CDDO-Me and tended to be dosedependent (Figure 3E−I). 2 Exhibits Antioxidant and Anti-Inflammatory Activities Comparable to CDDO-Me. As mentioned previously, CDDO-Me can inhibit inflammation and NF-κB activation by inhibiting IκBα kinase β (IKKβ) activity to prevent IκB 8852



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Table 1. Plasma Pharmacokinetic Parameters after Intravenous Administration of 2 and CDDO-Me at a Dose of 10 mg/kg to Ratsa 2 CDDO-Me a

AUC0−t (μg·h/L)

AUC0−∞ (μg·h/L)

t1/2 (h)

CL (L/h/kg)

Vss (L/kg)

Cmax (μg/L)

509.461 ± 169.171 2057.201 ± 369.88

531.346 ± 178.19 2179.198 ± 370.487

1.307 ± 0.602 4.413 ± 2.372

20.398 ± 7.19 4.683 ± 0.838

35.066 ± 12.377 29.07 ± 14.552

3020 ± 1361.653 5016.667 ± 2631.812

Mean ± SD, n = 3.

indicated that CDDO-Me was distributed preferably in the kidney (4.52 μg/g, 90 min), but was present in lower amounts in the liver (1.55 μg/g, 90 min) and heart (1.58 μg/g, 90 min) (Figure 6A,B). In contrast, 2 was predominantly distributed in the liver (6.04 μg/g, 90 min), but was present in lower amounts in the heart (1.31 μg/g, 90 min) and kidney (1.23 μg/g, 10 min; 1.0 μg/g, 30 min; 0.8 μg/g, 90 min) (Figure 6C). Importantly, after administration of 2, CDDO-Me liberated from 2 was detected exclusively in the kidney (0.04 μg/g, 10 min; 0.09 μg/g, 30 min; and 0.18 μg/g, 90 min) (Figure 6D). Although the amount of CDDO-Me detected in the kidney of 2-treated rats was relatively lower than that of CDDO-Me-treated rats, the ratio of CDDO-Me to 2 in the former case increased over 90 min (from 3.3 to 9% and then to 22.5%). Given that CDDO-Me reportedly exhibits potent anti-inflammatory and antioxidant activities at nanomolar levels,42 it would be expected that 2 would exert anti-DN activity at such low concentrations of CDDO-Me generated from 2 in the kidney. On the other hand, at higher concentrations (micromolar to millimolar), CDDO-Me could generate potent toxicity toward cancer and noncancer cells.43 Accordingly, we believe that the gradual generation of CDDOMe with lower concentrations from 2 in the kidney where GGT is highly expressed as well as the lack of CDDO-Me existing in the heart 90 min after the administration of 2 may be beneficial for the safety of 2, especially cardiac safety. 2 Is Less Cytotoxic and Relatively Safer than CDDO-Me. To determine the cytotoxicity of 2, cardiomyocyte H9C2 and HEK293 cells were treated with or without 2 or CDDO-Me for 24 h. The cytotoxicity of each compound was measured by MTT assay, and the IC50 values were calculated (Table S11). Compound 2 was less toxic toward HEK293 cells than CDDO-Me (IC50: 56.5 vs 2.2 μM). Similarly, 2 had less toxicity against H9C2 cells than CDDO-Me (IC50: 112.5 vs 5.2 μM). The lower cytotoxicity of 2 may be attributed to its slow release of CDDO-Me by GGT in both types of cells, suggesting that 2 may be relatively safer than CDDO-Me. The human ether-a-go-go-related gene (hERG) encodes the α subunit of a potassium ion channel (Kv11.1), and inhibition of this channel can result in long QT syndrome, a fetal disorder associated with severe adverse effects of drugs.44 Accordingly, we tested the potential effects of 2 and CDDO-Me on hERG channel activity by a patch-clamp assay in Chinese hamster ovary (CHO) cells that stably express hERG. Both 2 and CDDO-Me had very low hERG inhibitory activity (IC50 > 200 μM), and treatment with 2 at 200 μM inhibited hERG activity by only 36.09%, which was significantly lower than the 45.72% inhibition observed by the same dose of CDDO-Me (Figure S5). These results suggest that 2 may display less inhibition of hERG than CDDO-Me. To evaluate toxicity in vivo, we first performed an acute toxicity test. We found that intraperitoneal treatment with CDDO-Me up to 600 mg/kg or 2 up to 1000 mg/kg daily for 7 days resulted in a LD50 value of 2 (886 mg/kg), which was over 2fold higher than that of CDDO-Me (425 mg/kg) in ICR mice. Further TUNEL analysis indicated that a high frequency of apoptotic cells was detected in the cardiac tissue from aged db/db

CDDO-Me significantly reduced the 24 h urine volumes and the levels of urinary albumin, whereas it increased the levels of urinary creatinine and reduced the urine albumin to creatinine ratio (UACR) (Figure 5A−D). These results indicate that the anti-DN effect of 2 was comparable to that of CDDO-Me. Histological examination revealed moderate and severe fibrosis, reduced numbers of intraglomerular mesangial cells, and enlarged mesangial area with thickened capillary walls in the glomeruli of the model group of mice (Figure 5E). In contrast, treatment with either 2 or CDDO-Me significantly reduced the pathogenic changes in the glomeruli of mice. Further transmission electron microscopy (TEM) analysis revealed that fused podocytes with a loss of foot processes and increased mesangial expansion and glomerular basement membrane (GBM) thickening, the hallmarks of DN,41 were detected in the kidney glomeruli of the model group of mice, whereas treatment with 2 or CDDO-Me improved the glomerular injury in mice (Figure 5F−J). Quantitative analysis indicated that treatment with either 2 or CDDO-Me significantly mitigated the DN-related GBM thickness in the kidney of the mice (Figure 5K). Collectively, treatment with 2 significantly improved the kidney function in db/db mice, similar to that with CDDO-Me. Pharmacokinetics of 2 in Rats. The pharmacokinetics of 2 was then evaluated in rats and compared with CDDO-Me. After intravenous administration of 2 (10 mg/kg) or CDDO-Me (10 mg/kg) to rats, blood samples were taken from the suborbital vein at 0, 2, 5, 15, 30, and 60 min and 1.5, 2, 4, 6, 8, 12, and 24 h and analyzed by LC-MS/MS. The corresponding plasma concentration−time profiles of 2 and CDDO-Me are shown in Figure 6A, and pharmacokinetic parameters are given in Table 1. AUC0−∞ (area under the concentration−time curve from time zero to infinity), t1/2 (half-life), and Cmax (maximum plasma concentration) of 2 were 531.3 ± 178.2 μg·h/L, 1.3 ± 0.6 h, and 3020 ± 1361.7 μg/L, and for CDDO-Me, they were 2179.2 ± 370.5 μg·h/L, 4.4 ± 2.4 h, and 5016.7 ± 2631.8 μg/L, respectively. The CL (clearance rates) of 2 and CDDO-Me was 20.4 ± 7.2 and 4.7 ± 0.8 L/h/kg, respectively, and Vss (distribution volumes) for 2 and CDDO-Me was 35.1 ± 12.4 and 29.1 ± 14.6 L/kg, respectively. Considering the fact that 2 could be rapidly converted to CDDO-Me and 8 in the presence of GGT as well as in rat kidney homogenate and plasma (Figure 2B−E) and that 8 as a precursor of CDDO-Me with a free glutamic acid moiety could be preferably recognized by GGT and quickly transferred to CDDO-Me (Figure 2B−D), it would be expected that 2 would possess a faster metabolic behavior relative to CDDO-Me, leading to the lower values of AUC0‑∞, t1/2, and Cmax of 2 compared to those of CDDO-Me. Liberation of CDDO-Me from 2 in the Kidney of Rats. On the basis of the above pharmacokinetics results and the potent anti-DN effect of 2, the tissue distributions of 2 and CDDO-Me were investigated. Individual rats were injected intraperitoneally with CDDO-Me (0.04 mmol, 20 mg/kg) or 2 (0.04 mmol, 30 mg/kg), and tissue (including heart, kidney, and liver) samples were collected longitudinally. LC-MS/MS analysis 8853



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Figure 7. Compound 2 is relatively safer than CDDO-Me. Control C57BL/6 mice (A) or 8 week old db/db mice were randomized and treated intraperitoneally with vehicle control (B), CDDO-Me (0.65 mg/kg, C), or 2 (1 mg/kg, D; 2 mg/kg, E) for 12 weeks. The apoptotic cells in the cardiac tissues were determined by TUNEL assay, and the percentage of apoptotic cardiomyocytes was calculated from three sections of each mouse (n = 6 per group, F). Data are representative images (magnification ×200) or expressed as the mean ± SD of each group from two separate experiments.

aged db/db mice. These results strongly suggest that 2 may be a relatively safer agent for intervention of DN. Importantly, this CUK-modifying strategy may be valuable for other pharmacological applications of CDDO-Me to avoid offtarget effects. Given that CDDO-Me possesses potent anticancer activity,53,54 4-boronic acid pinacol ester-substituted benzyl55,56 may be employed to etherify the 3-hydroxy of 1 to generate a ROS-inducible compound, which can be oxidized in cancer cells where ROS abundantly exists,57 to produce a 4-hydroxyl benzyl moiety, initiating a 1,6-elimination reaction to form 1. Subsequently, 1 may undergo 1,4-elimination to liberate CDDO-Me, exerting anticancer activity. In addition to CDDO-Me, this CUK-modifying strategy can be extended to other active synthetic molecules or natural products bearing a Michael acceptor similar to CUK to avoid the off-target effects associated with these compounds.

mice, and treatment with 2, but not with CDDO-Me, significantly reduced the percentage of apoptotic cardiomyocytes (Figure 7A−F). These results further indicated that treatment with 2, but not CDDO-Me, protected from long-term hyperglycemia-mediated cardiomyocyte apoptosis in aged db/db mice.

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DISCUSSION Long-term hyperglycemia is associated with the development of complications such as DN. Approximately, 20−40% of diabetic patients eventually develop DN.45 Control of hyperglycemia and hypertension can help slow the pathogenic process of DN, and a great number of drugs have already been developed for the treatment of DN in recent decades. However, the clinical efficacy and safety of these drugs are unsatisfactory, particularly in patients with later stage DN.46−49 Therefore, new drugs with an improved efficacy and safety profile are urgently needed for the treatment of patients with DN.50 Our previous studies focused on the anti-inflammation and anticancer properties of CDDO-Me and its derivatives.43,51,52 In this study, we presented a novel strategy to rationally mask the CUK moiety of CDDO-Me in the non-target organs and tissues, leading to a CUK-modified CDDO-Me derivative (2), which could be specifically cleaved into CDDO-Me in the kidney by GGT. Compound 2 displayed better water solubility than CDDO-Me due to the introduction of hydroxyl and amine moieties to C1 and C3 in ring A of CDDO-Me, respectively. Interestingly, 2 exhibited comparable anti-DN efficacy to CDDO-Me. In db/db mice, 2 decreased proteinuria, inhibited glomerular sclerosis, and mitigated DN-related GBM thickness and podocyte damage. Importantly, 2 has obvious advantages in term of safety as compared with CDDO-Me. The cytotoxicity of 2 against H9C2 and HEK293 cells was much less than that of CDDO-Me. In ICR mice, the LD50 value of 2 was over 2-fold higher than that of CDDO-Me. Treatment with CDDO-Me greatly increased the frequency of apoptotic cardiomyocytes; in sharp contrast, treatment with 2 significantly mitigated long-term hyperglycemia-induced spontaneous cardiomyocyte apoptosis in

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CONCLUSIONS

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EXPERIMENTAL SECTION

In summary, we developed a novel GGT-based and CUKmodifying strategy by rationally masking the highly reactive pharmacophore CUK in CDDO-Me. The achieved compound 2 exhibited comparable activity against DN but much less cardiotoxicity than CDDO-Me. These findings together with the high sequence homology between rodent and human GGT58 suggest that compound 2 may be a relatively safer and valuable candidate for the treatment of DN. In addition, we provide a new strategy to optimize other molecules with a pharmacophore like CUK to obtain safer pharmaceutical drugs.

The purity of all tested compounds was characterized by HPLC analysis (LC-10A HPLC system consisting of LC-10ATvp pumps and a SPD10Avp UV detector). Individual compounds with a purity of >95% were used for subsequent experiments. Synthesis of 2 [1-Hydroxyl-2-cyano-3-(4-(methyl-L-glutaminate-N5-yl)-benzyloxy)-12-oxo-oleana-2(3),9(11)-dien-28-oic Acid Methyl Ester]. HNEt2 (4 mL) was added to a solution of 7 (720 mg, 0.72 mmol) in CH2Cl2 (10 mL). The mixture was stirred at ambient temperature for 30 min before it was neutralized with ice-cold saturated 8854



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aqueous NaHCO3 (10 mL) and further diluted with water (20 mL). The resulting opaque solution was extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with saturated aqueous NaHCO3 and brine (20 mL × 3) and were then dried over anhydrous Na2SO4. The residue was purified by flash column chromatography (CH2Cl2/ CH3OH, 2/1) to give 2 as an amorphous solid (396 mg, 71%). mp: 158−160 °C; 1H NMR (300 MHz, CD3OD): δ 7.59 (s, 2H, ArH), 7.38 (s, 2H, ArH), 5.87 (s, 1H, CDDO C11H), 5.40−5.44 (m, 2H, benzylCH2), 4.45 (s, 1H, CDDO C1H), 3.72 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.50−3.58 (m, 1H, Glu-α-CH), 2.22−2.38 (m, 2H, Glu-γCH2), 1.28 (s, 3H), 1.16 (s, 6H), 1.06 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.89 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 202.1, 179.9, 178.7, 176.9, 176.2, 172.5, 140.6, 132.9, 132.8, 130.1, 129.4, 126.3, 121.1, 88.5, 75.2, 75.0, 53.8, 53.5, 52.4, 51.0, 46.7, 45.8, 43.8, 42.4, 41.8, 37.2, 35.4, 34.0, 33.7, 33.3, 33.0, 31.9, 31.5, 30.8, 29.4, 28.7, 24.7, 24.3, 23.8, 23.5, 21.6, 20.8, 19.4; IR (neat, cm−1): 2978 (s), 2945 (s), 2208 (s), 1720 (s), 1662 (s), 1521 (s), 1219 (m), 1064 (m); ESI-MS (m/z): 772.5 [M + H]+; HRMS (m/z): [M + H]+ calculated C45H61N3O8Na 772.4537, found 772.4551, PPM error 1.8. Stability of 2. Compound 2 (100 μM) was incubated in triplicate in Krebs buffer (37 °C, pH 7.4) in the presence or absence of GGT (1 U/ mL, MBS173093, MyBioSource) and the glutamyl acceptor Gly-Gly (5 mM, Energy Chemical). After hydrolysis by GGT, the reactions were diluted with two volumes of acetonitrile and centrifuged to remove the enzyme, followed by HPLC analysis. Similarly, the stability of 2 was assessed in Krebs buffer (37 °C, pH 7.4) with the addition of esterase from porcine liver (2 U/mL, E3019, Sigma-Aldrich), GGT (1 U/mL), and Gly-Gly (5 mM). The plasma samples were prepared from three Wistar rats, and their kidney and liver tissue homogenates were also prepared in PBS. Subsequently, 2 (100 μM) was mixed with the plasma or homogenates, and the amount of 8 was detected using HPLC at intervals of 60 min for 8 h. Cell Culture, Luciferease Reporter Assay, Quantitative RTPCR, Western Blot. HEK293 and H9C2 cells were from ATCC and maintained in high-glucose DMEM supplemented with 10% FBS (Hyclone). HEK-293 cells were transfected with a plasmid encoding NF-κB-controlled luciferase expression (Promega) and treated in triplicate with 2 or CDDO-Me for 48 h. Subsequently, the luciferase activity in each group of cells was measured using the dual-luciferase reporter assay system, according to the manufacturer’s instruction. Furthermore, HEK293 cells were pretreated with or without TNF-α (10 ng/mL) for 6 h and then treated in triplicate with or without 2 or CDDO-Me for 48 h. Total RNA was extracted from individual groups of cells with TRIzol (Invitrogen) and reverse transcribed into cDNA using iScript reverse transcriptase (Bio-Rad). The levels of target gene mRNA transcripts relative to that of control GAPDH were determined by quantitative RT-PCR using specific primers (Table S8) and SsoFast EvaGreen supermix (Bio-Rad) in a Roche LightCycler 96 system. The data were analyzed by the 2−ΔΔCt method. In addition, the relative levels of Nrf2, TBP, HO-1, NQO1, β-actin, iNOS, COX2, and nuclear and cytosolic NF-κB p65 in individual groups of cells were determined by western blot assays using specific antibodies (Cell Signaling Technology). Animal Experiments. All animal experiments were conducted according to the guidelines approved by the Ministry of Health of the People’s Republic of China (Document No. 55, 2001) as well as by the Science and Technology Department of Jiangsu Province (SYXK (SU) 2016-0011). Male db/db mice at 8 weeks of age were obtained from the National Resource Center of Model Mice, Nanjing, and housed in a specific pathogen-free facility with normal chow and filtered water ad libitum. The mice were randomized and treated intraperitoneally with vehicle (5% Tween-80 in 0.9% saline, the model group), 2 (1 or 2 mg/ kg, n = 6 per group), or CDDO-Me (0.65 mg/kg) three times per week for 12 consecutive weeks. Their body weight and water and food consumption were measured every other day. Eleven weeks after drug administration, the 24 h urine samples were collected from individual mice for analysis of total volumes. Subsequently, blood samples from individual mice were obtained. The mice were sacrificed, and their kidneys, hearts, livers, and other major organs were collected. The levels of urine albumin and creatinine in individual mice were measured. The

levels of plasma BUN, creatinine, and uric acid in individual mice were analyzed. Histology. The collected tissues were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. The kidney tissue sections (5 μm) were stained with hematoxylin−eosin (HE), periodic acid Schiff (PAS), or Masson’s trichrome. The frequency of cardiomyocytes in the heart tissue sections (5 μm) was determined by a terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) assay using a specific kit, according to manufactory’s instructions (Beyotime). Some kidney tissues from each group were fixed with 3% glutaraldehyde, and ultramicrotome sections (80 nm) were prepared (Leica EM UC7) and examined in a JEM-1011 electron microscope (JEOL, Inc., Peabody, MA). The thickness of the glomerular basement membrane (GBM) in 30 different locations randomly selected from three sections of each kidney was measured using ImageJ software. The average thickness of the GBM in individual mice was calculated. Statistical Analysis. Data are expressed as the mean ± SD of each group, and differences among groups were analyzed by one-way ANOVA and Tukey’s post hoc test. The difference between two groups was determined by two-tailed Student’s t-tests. All statistical analyses were performed using GraphPad Prism 5.0. A P-value < 0.05 was considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00971. Synthetic and experimental details; 1H NMR, 13C NMR, and HRMS spectra of new compounds; crystallographic data for 9; and additional biological data of 2 (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.H.). *E-mail: [email protected] (X.X.). *E-mail: [email protected] (Y.Z.). ORCID

Zhangjian Huang: 0000-0001-6409-8535 Yihua Zhang: 0000-0003-2378-7064 Author Contributions #

Z.H. and Y.M. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (nos. 21372261, 81673305, and 21472244) and Jiangsu Province Funds for Distinguished Young Scientists (BK20160033). Part of the work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Program for New Century Excellent Talents in University (NCET-13-1033), and Jiangsu Shuang Chuang team.

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ABBREVIATIONS USED DN, diabetic nephropathy; CUK, α-cyano-α,β-unsaturated ketone; OA, oleanolic acid; CDDO, 2-cyano-3,12-dioxooleana1,9(11)-dien-28-oic acid; Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein; ARE, antioxidant response element; NQO1, NAD(P)H:quinone oxidoreductase-1; GSTs, glutathione-S-transferases; HO-1, 8855



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heme-oxygenase-1; eGFR, estimated glomerular filtration rate; GGT, γ-glutamyl transpeptidase; EEDQ, 2-ethoxy-1(2H)-quinoline carboxylic acid ethyl ester; AUC0−∞, area under the concentration−time curve from time zero to infinity; t1/2, halflife; Cmax, maximum plasma concentration; CL, clearance rate; Vss, distribution volume; IKKβ, IκBα kinase β; BUN, plasma urea nitrogen; UA, uric acid; UACR, urine albumin to creatinine ratio; TEM, transmission electron microscopy; GBM, glomerular basement membrane; hERG, human ether-a-go-go-related gene; SDD, spray dried dispersion; PFA, paraformaldehyde; HE, hematoxylin−eosin; PAS, periodic acid Schiff

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