Specific Inhibition of Bacterial Î²-Glucuronidase by ... - ACS Publications

Article Cite This: J. Med. Chem. 2017, 60, 9222-9238

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Specific Inhibition of Bacterial β‑Glucuronidase by Pyrazolo[4,3‑c]quinoline Derivatives via a pH-Dependent Manner To Suppress Chemotherapy-Induced Intestinal Toxicity Kai-Wen Cheng,†,△ Chih-Hua Tseng,‡,§,∥,⊥,△ Chia-Ning Yang,# Cherng-Chyi Tzeng,∥,∇ Ta-Chun Cheng,○ Yu-Lin Leu,◆ Yu-Chung Chuang,# Jaw-Yuan Wang,¶,∞ Yun-Chi Lu,● Yeh-Long Chen,*,⊥,∇ and Tian-Lu Cheng*,†,○,●,◇,× †

Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan § Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan ∥ Research Center for Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung 807, Taiwan ⊥ Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan # Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan ∇ Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan ○ Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung 807, Taiwan ◆ Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan City 717, Tainan ¶ Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan ∞ Division of Gastroenterology and General Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan ● Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan ◇ Department of Biomedical and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan × Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, 807, Taiwan ‡

S Supporting Information *

ABSTRACT: The direct inhibition of bacterial β-glucuronidase (βG) activity is expected to reduce the reactivation of glucuronideconjugated drugs in the intestine, thereby reducing drug toxicity. In this study, we report on the effects of pyrazolo[4,3-c] quinolines acting as a new class of bacterial βG-specific inhibitors in a pH-dependent manner. Refinement of this chemotype for establishing structure−activity relationship resulted in the identification of potential leads. Notably, the oral administration of 3-amino-4-(4-fluorophenylamino)-1H-pyrazolo[4,3-c]quinoline (42) combined with chemotherapeutic CPT-11 treatment prevented CPT-11-induced serious diarrhea while maintaining the antitumor efficacy in tumor-bearing mice. Importantly, the inhibitory effects of 42 to E. coli βG was reduced as the pH decreased due to the various surface charges of the active pocket of the enzyme, which may make their combination more favorable at neutral pH. These results demonstrate novel insights into the potent bacterial βG-specific inhibitor that would allow this inhibitor to be used for the purpose of reducing drug toxicity.

Received: July 18, 2017 Published: November 9, 2017 © 2017 American Chemical Society

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DOI: 10.1021/acs.jmedchem.7b00963 J. Med. Chem. 2017, 60, 9222−9238

Journal of Medicinal Chemistry

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occurrence of diarrhea in mice receiving CPT-11,18 but compound 2 also hindered the antitumor efficacy of CPT-11.19 More recently, a tricyclic antidepressant called amoxapine, which is known to act as a dopamine receptor antagonist, was found to reduce eβG activity.20 Previous study reported that oral administration of amoxapine suppressed CPT-11-induced diarrhea and reduced tumor growth in mice.19 Consequently, specific inhibition of bacterial βG is effective in preventing CPT-11-inuced intestinal toxicity. In this study, we present a new class of bacterial βG-specific inhibitors, pyrazolo[4,3-c]quinolines, for preventing CPT-11induced intestinal toxicity and also detail its inhibitory mechanism. More specifically, we first discovered 3-amino-4-anilino-1Hpyrazolo[4,3-c]quinoline (29) as potential eβG inhibitors by high-throughput screening (HTS) of our compound library (compounds 3−29, Table 1). Among these diversified small molecules, compound 29 was the most active and was more potent and selective than the previously reported βG inhibitors, compounds 1 and 2. Although several pyrazolo[4,3-c]quinoline derivatives have been reported,21 very little information is known about its biological activities. This motivated us to determine the structure−activity relationship (SAR) of the pyrazolo[4,3-c] quinolines for specifically inhibiting eβG versus hβG. We further determined the protective effects of the potential derivative against CPT-11-induced intestinal toxicity and the impact of it on the antitumor efficacy of CPT-11 in tumor-bearing mice. We also investigated the underlying mechanism for inhibitory selectivity of the derivative through molecular modeling. Finally, we also examined the inhibitory potency of the derivative under various pH and analyzed the surface charge of eβG to illustrate the difference.

INTRODUCTION Hepatic glucuronidation, known as phase II metabolism, is a major subprocess of the larger overall process of drug detoxification.1 Generally, drugs are conjugated with glucuronic acid to become inactive and also to increase their water solubility, and in turn, to facilitate their subsequent elimination from the body.2 However, when the pharmacologically inactive glucuronides enter the intestine via bile, they are reactivated by intestinal β-glucuronidase (βG) expressed by intestinal microflora, thus releasing the cytotoxic drugs and leading to intestinal injury.3 This mechanism has been suggested to be responsible for the cytotoxicity of a variety of pharmaceuticals, including chemotherapeutic CPT-11. CPT-11 (irinotecan) is a clinically approved chemotherapeutic drug used for the treatment of various cancers.4 However, diarrhea is one of its common side effects, occurring in up to 50−80% of the patients who take it as part of their chemotherapy regimen.5 Extensive studies have described bacterial βG in the intestine lumen as a key factor in activating SN-38 glucuronide (SN-38G), the inactive metabolite of CPT-11, thereby causing the release of the active metabolite SN-38 in the intestine.6 The accumulation of SN-38 in the intestinal lumen causes mucosal damage and toxicity to epithelial cells that directly induces dose-dependent diarrhea.7 Currently, supportive methods such as antidiarrhea agents are used to relieve diarrhea symptoms, but the intestinal damage caused by CPT-11 still persists.4 With the above points in mind, the reduction of bacterial βG activity is expected to reduce the level of SN-38 in the intestine, which would, in turn, alleviate intestinal toxicity. Although this concept has been understood for decades, there are no applicable βG inhibitors for clinical use. The bacterial βG is found in most E. coli strains that catalyze the hydrolysis of β-D-glucuronides and acquire glucuronide as carbon source. The E. coli βG (eβG) shares 50% amino acid sequence identity with highly conserved active sites to human (hβG).8 HβG is an important lysosomal enzyme for the degradation of glycosaminoglycan. As such, a deficiency in or reduced activity of hβG due to genetic mutations causes the accumulation of glycosaminoglycan in many tissues and organs, a condition known as MPS VII (or Sly syndrome).9 Therefore, it is important to specifically inhibit eβG but not to hβG. Moreover, it was reported that high levels of hβG are present in necrotic area around solid tumors.10 Relatedly, tumors with elevated human βG in the tumor microenvironment are more sensitive to CPT-11 treatments.11,12 The βG activity at the tumor site contributes the reactivation of local SN-38G to SN-38, as well as other glucuronide prodrugs.13,14 Concern remains that the lack of inhibitory specificity of the βG inhibitor may impair the antitumor response of CPT-11, thus limiting their clinical applications. Therefore, the inhibitory specificity of βG inhibitors is important. A potent and specific βG inhibitor that reduces CPT-11-induced intestinal toxicity and would not hamper the treatment efficacy of CPT-11 would be of great clinical value. In fact, in one previous study, the use of antibiotics to eliminate bacteria caused less βG activity in the luminal contents and markedly reduced the intestinal toxicity of CPT-11.4,15 However, antibiotics also kill the native gut flora, which may have a negative impact on a patient’s health.16 Several βG inhibitors have been developed to more precisely reduce the bacterial βG activity in the intestine. For example, feeding rats with a nonselective βG inhibitor, D-saccharic acid 1.4-lactone (1), was found to protect intestinal mucosa from CPT-11-induced injury.17 Wallace and colleagues identified a specific eβG inhibitor, 1-((6,8-dimethyl-2oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1(2-hydroxyethyl)thiourea (2), that was shown to decrease the

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RESULTS Chemistry. To establish SAR of the pyrazolo[4,3-c]quinolines for inhibiting eβG activity, the derivatives 33−55 were synthesized according to Scheme 1. Reaction of the known 2,4-dichloro-3quinolinecarbonitrile (31a) with hydrazine produced 4-chloro1H-pyrazolo[4,3-c]quinolin-3-amine (32a) which was then treated with 2-chloroaniline in DMF to give compound 33 (2′-Cl) in a fairly good overall yield. Accordingly, the substituted anilino derivatives 34−47 were synthesized by the treatment of compounds 32a−e and substituted anilines. To optimize the inhibitory potency of the derivatives, structures of compounds 39 (4′-Cl) and 40 (4′-Me) were modified by the introduction of various substitutions at C-6, C-7, and C-8, respectively. Reaction of 6-chloro-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbonitrile (30b) with POCl3 gave 2,4,6-trichloro3-quinolinecarbonitrile (31b) which was then treated with hydrazine to give 4,8-dichloro-1H-pyrazolo[4,3-c]quinolin-3amine (32b) in a fairly good overall yield. Treatment of compound 32b with 4-chloroaniline and 4-methylaniline respectively afforded their respective 4-substituted anilino derivatives, compounds 48 (4′-Cl) and 49 (4′-Me). Accordingly, compounds 50−55 were prepared by the treatment of 32b−e with substituted anilines. Biological Evaluation. Isolated Targets. To identify a potent bacterial βG specific inhibitor, compounds 33−55 were compared for their inhibitory potency by in vitro βG activity assays which were conducted with purified eβG and hβG enzymes. As can be seen in Table 2, we found that pyrazolo[4,3-c] quinoline derivatives are potential eβG inhibitors, exhibiting varied IC50 value against eβG from 14.4 nM to >10 000 nM. Certain derivatives with reduced IC50 value against eβG were more active compared to compounds 1 (IC50 > 10 000 nM) and 2 (IC50 = 846 nM). Notably, the two previously reported βG 9223



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Table 1. Chemical Structures of Identified Small Molecules and Their Inhibition of E. coli βG and Human βG Activity

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Table 1. continued

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Table 1. continued

a

Enzyme activity was determined with 10 μM compound.

Scheme 1. Chemical Structures and Synthesis of Pyrazolo[4, 3-c]quinolines

order of compounds 37 (3′-Me, >10 000 nM) > 34 (2′-Me, 673.3 nM) > 40 (4′-Me, 37.3 nM). Conversely, derivative contains a hydrophilic substituent such as hydroxyl group which at the para position of the anilino moiety had a similar IC50 with the same substituent at different positions. Moreover, the properties of substituents at the para position also determine the inhibitory potency. Interestingly, while several derivatives such as compounds 42 (4′-F), 43 (4′-CF3), 46 (4′-COOH), and 47 (4′-COMe) showed modest inhibitory activity, compounds 41

inhibitors displayed distinct inhibitory character and potency. Compound 1 was more active in inhibiting hβG activity, whereas compound 2 was able to inhibit eβG activity with relative high potency. Importantly, the position of substituent at the anilino moiety directs the inhibitory potency against eβG. As shown in Table 2, the IC50 value against eβG decreased in the order of compounds 36 (3′-Cl, 307.8 nM) > 33 (2′-Cl, 281.4 nM) > 39 (4′-Cl, 34.8 nM). The same phenomenon was observed for the methylanilino counterparts that displayed decreased IC50 in the 9226



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Table 2. In Vitro Inhibitor Efficacy of the Pyrazolo[4,3-c]quinolines against E. coli βG and Human βG Enzyme

a

IC50 value against eβG enzyme activity was determined with compound at the concentration from 0.64 nM to 10 000 nM. Error represents SD, n = 3 biological replicates. bEnzyme activity was determined with 10 μM compound. Error represents SD, n = 3 biological replicates.

To examine whether substitution at quinoline moiety can increase the inhibitory potency of the pyrazolo[4,3-c]quinolines in inhibiting eβG, we selected the two most active compounds, 39 (4′-Cl, IC50 = 34.8 nM) and 40 (4′-Me, IC50 = 37.3 nM), further modified by introducing various substituents at C-6, C-7, and C-8. For 4-chloroanilino derivatives, the eβG inhibitory

(4′-OH), 44 (4′-NH2), and 45 (4′-NO2) had low inhibitory activity. These results indicate the importance of a hydrophobic substituent at the para position of the aniline moiety for effectively inhibiting eβG, and a hydrophilic substituent or substituents at the other position of the aniline moiety may decrease the inhibitory activity of the derivatives. 9227



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49 (8-Cl, 4′-Me), 50 (8-Br, 4′-Cl), 51 (8-Br, 4′-Me), and 53 (7-F, 4′-Me) exhibited >80% inhibition at 50 μM. In contrast, compounds 1 and 2 exhibited no such inhibitory activity. We further conducted bacterial cytotoxicity assays to confirm the effects of the inhibitors on bacterial growth. Among the abovementioned eight compounds, only compounds 40 (4′-Me) and 42 (4′-F) remained nonlethal to E. coli while the others exhibited toxicity at 100 μM after 6 h of incubation (Figure 1D). Therefore, compounds 40 (4′-Me) and 42 (4′-F) that exhibited relatively effective inhibition of the endogenous eβG activity with no toxicity at high concentration were selected for further study. Mice Efficacy. To determine whether the pyrazolo[4,3-c] quinolines inhibit intestinal βG in mice, compounds 40 (4′-Me) and 42 (4′-F) were orally administered to mice for 5 consecutive days, respectively, and the intestinal βG activity of the mice was detected by the use of fluorescein di-β-D-glucuronide (FDGlcU) as previously described,22 which is hydrolyzed to fluorescein by

activity increased in the order of 54 (6-Me, IC50 = 238.2 nM) < 48 (8-Cl, IC50 = 55.6 nM) < 52 (7-F, IC50 = 29.6 nM) < 50 (8-Br, IC50 = 18.0 nM), indicating that the inhibitory potency was enhanced by introducing an F group at C-7 or a Br group at C-8. Conversely, for 4-methylanilino counterparts, 49 (8-Cl, IC50 = 14.4 nM) and 55 (6-Me, IC50 = 24.7 nM) had lower IC50 compared to the parental compound 40. Importantly, most derivatives showed poor inhibition against hβG except for compounds 33, 38, and 44, which displayed >20% inhibition against hβG at a concentration of 10 μM. Bacteria. To further assess whether the pyrazolo[4,3-c] quinolines inhibit endogenous βG activity in living intact bacterial cells, we used easily manipulated E. coli cells with high βG activity. We selected the potential compounds (IC50 against eβG of 85%) to evaluate for inhibition of endogenous βG activity. Figure 1C demonstrates the effective inhibition of endogenous eβG activity. Of the 13 compounds, compounds 39 (4′-Cl), 40 (4′-Me), 42 (4′-F), 48 (8-Cl, 4′-Cl),

Figure 1. Specific inhibition of E. coli βG by pyrazolo[4, 3-c]quinolines. The potential compounds were evaluated based on their selective inhibition for purified (A) eβG enzyme activity versus (B) hβG. The βG activity was determined by hydrolysis of 4-nitrophenyl β-D-glucuronide (pNPG). Data are displayed as percent of βG activity compared with the control treated with 10% DMSO. (C) Endogenous eβG activity was tested at 50 μM, 10 μM, and 2 μM of each compound in cultured E. coli. Data are displayed as percent of eβG activity compared with the control treated with 1% DMSO. (D) Bacterial cytotoxicity was tested at 100 μM and 10 μM of each compound for 6 h of incubation in E. coli. Cell viability was displayed as percent of cell growth (OD at 600 nm) compared with the control treated with 1% DMSO. Error bars, mean ± SD, n = 3 biological replicates. 9228



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in the saline group, whereas CPT-11 treatment induced the destruction of both mucosal villi and crypt structures in the intestine. Notably, severe damage was observed in the ileum and colon of the CPT-11-treated group. However, this CPT-11induced damage was dramatically decreased in mice fed with compound 42. These findings demonstrate the protective effect of compound 42 for reducing the intestinal mucosa injury caused by CPT-11. To test whether the inhibition of intestinal βG by compound 42 eliminates CPT-11-induced diarrhea while maintaining the therapeutic efficacy of CPT-11, the administration of CPT-11 in combination with compound 42 was conducted in tumorbearing mice. The co-treatment with compound 42 resulted in less diarrhea over the course of the entire study and significantly suppressed the severity of diarrhea on day 18 (P = 0.0362, two-way ANOVA) (Figure 4A). By day 18, half of the mice in the CPT-11 group exhibited severe diarrhea. In contrast, no severe diarrhea was exhibited by the mice that received compound 42. Moreover, all the mice in the CPT-11 group had diarrhea, whereas only 50% of the mice in the compound 42 group had diarrhea (Figure 4B). In addition, CPT-11 treatment significantly reduced tumor growth in the absence and presence of compound 42 compared to the control group (P < 0.0001, two-way ANOVA, Figure 4C), indicating that compound 42 had no effects on the antitumor efficacy of CPT-11. The systemic metabolism of CPT-11, as measured by CPT-11-induced weight loss, is not affected (Figure 4D). Taken together, these findings indicate that co-treatment with compound 42 suppressed CPT-11-induced diarrheas while maintaining CPT-11 treatment efficacy. Molecular Ducking. To further determine the inhibitory mechanism by which compound 42 inhibits eβG but not hβG, a series of molecular modeling, using molecular docking, molecular dynamics simulation, and MMPBSA-based binding free energy calculations, has been conducted. The crystal structure of eβG (PDB code 3LPF)18 revealed that it is a homotetramer and contains two neighboring monomers in the active site. As indicated in Figure 5A, eβG possesses two catalytic residues, E413 and E504, and a unique bacterial loop (360−367). Our docking model suggested that in the eβG-42 complex, compound 42 is bound to the entrance of the active site and interacting with one monomer (Figure 5B). A hydrogen bond is formed between the NH2 group of compound 42 and the catalytic residue E413 (with a separation distance = 2.69 Å), indicating the primary contact between compound 42 and eβG for potent inhibition of eβG (Figure 5C). In addition, a hydrogen bond is formed between the F of compound 42 with W549 of eβG (with a separation distance of 3.20 Å). Compound 42 also maintains hydrophobic interactions with the catalytic residue E504 and its surrounding residues D163, V446, M447, Y468, Y472, R562, and K568 in the active site. Importantly, two resides S360 and L361 in the bacterial loop also make hydrophobic contact with compound 42 (Figure 5C). On the other hand, the binding mode of compound 42 in hβG predicted by our molecular docking also showed similar pose except the lack of bacterial loop in hβG (Figure 5D). The binding mode prediction is in line with the MMPBSA-based binding free energy calculation listed in Table 3 where the binding energy in the eβG-42 complex (−40.91 kcal/mol) was better than in the hβG-42 complex (−26.99 kcal/mol). Collectively, these results show that compound 42 binds to the active site and interacts with the bacterial loop of eβG, illustrating the selectively inhibitory ability of compound 42 against eβG from the aspect of molecular modeling.

Figure 2. Reduced intestinal βG activity by pyrazolo[4,3-c]quinoline derivatives in mice. Mice were administered two selected compounds 40 (4′-Me) and 42 (4′-F) and control DDW. (A) Noninvasive optical imaging of intestinal βG activity was performed at 2 h after oral administration of βG probe. βG activity was determined by the presence of fluorescein. (B) Average radiance in the intestine is shown. (C) Mice feces were collected in order to test the βG activity. βG activity was determined through the presence of pNP. Error bars, mean ± SD, n = 6 biological replicates. P values were determined by a two-tailed Student’s t test: (∗∗∗) P < 0.0005; (∗∗) P < 0.005; (∗) P < 0.05.

bacterial βG. Reduced fluorescence in the intestine was observed in the inhibitor-treated groups (Figure 2A). A further analysis of the average radiance in the intestine indicated that compounds 40 and 42 significantly suppressed the intestinal βG activity by ∼40% and by ∼75% compared to the control group (P = 0.0358 and P = 0.0004, t test), respectively (Figure 2B). We also analyzed the fecal βG activity of each group and found that the mice fed with 40 or 42 had significantly reduced βG activity in their feces compared to the control group (P = 0.0144 and P = 0.0032, t test) (Figure 2C). These results indicate the effective inhibition of intestinal βG by the two inhibitors, with compound 42 showing more potent inhibition efficacy in mice. Mice Side Effects. To examine the ability of compound 42 to protect against the intestinal toxicity caused by CPT-11, compound 42 was orally administered (3 mg kg−1 day−1) to mice along with 10 consecutive days of intraperitoneal CPT-11 (50 mg kg−1 day−1) injections. After that, we performed histological examinations of the intestinal morphology of CPT-11treated mice. Figure 3 displays that tightly arrayed epithelial cells and a healthy glandular structure were exhibited by the mice 9229



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Figure 3. Suppression of CPT-11-induced intestinal damage by compound 42 in mice. Mice were treated with compound 42 or control DDW on day −2. Daily intraperitoneal injection of saline or CPT-11 was given from day 0. Intestine tissues were analyzed after 10 consecutive days of CPT-11 treatment. Intestinal sections from jejunum, ileum, and colon were stained with H&E. Destruction of both mucosal villi and crypt structures (arrows) was observed in the intestines of CPT-11/DDW mice, whereas a better morphology was seen in CPT-11/compound 42 mice. n = 3−5 mice per group. Scale bar, 100 μm.

Figure 4. Protective effects of compound 42 against diarrhea and antitumor activity of CPT-11 in tumor-bearing mice. Mice were inoculated with CT26 cells on day −8. Either compound 42 or control DDW was orally administered throughout the experimental period (day −2 onward). Mice were given daily intraperitoneal injections of either CPT-11 or saline starting on day 0. (A) Diarrhea was scored every other day. Data represent average scores. (B) Percentage of each diarrhea severity level on day 18. (C) Tumor growth and (D) weight were estimated every other day. Error bars, mean ± SD, n = 6 mice per group. P values were determined by two-way ANOVA: (∗) P < 0.05, (∗∗∗∗) P < 0.0001. n.s., not significant.

pH-Dependent Inhibition. Considering that eβG exhibits optimal activity at neutral pH condition,23 the inhibitory efficacy of compound 42 to eβG under different pH levels was also investigated. Figure 6A shows that the inhibitory effects of compound 42 to eβG were reduced while the pH decreased. Compound 42 shows a higher inhibitory activity at a concentration of 10 μM, approximately ∼90% inhibition at pH 6.5 and 7.5, whereas its inhibitory activity decreased to ∼80% and ∼50% inhibition at pH 6.0 and pH 5.5, respectively, indicating that compound 42 has optimal inhibition against eβG at the range from slightly acidic to neutral (pH 6.5−7.0) in the colon.

We hypothesized that this pH-dependent inhibition may be due to the electrostatic potential character of the active pocket of eβG varying at different pH conditions. Thus, we further analyzed the electrostatic surfaces of compound 42 and the active pocket of eβG to characterize the interplay between ligand and binding sites in terms of pH-dependent electrostatic properties. As indicated in Figure 6B, some of the amino acid residues in the binding pocket become protonated and turn the surface electropositive from pH 7.0 to 5.5. In the meantime, the two nitro atoms N-2 and N-5 of compound 42 also are protonated, as indicated Figure 6C. Accordingly, compound 42 is not favorable by eβG at 9230



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Figure 5. Species-specific inhibitory mechanism of compound 42 against E. coli βG. (A) The initial structure of eβG (PDB code 3LPF): monomer 1 (olive), monomer 3 (blue), and the bacterial loop 360−367 (red). E413 and E504 are the two catalytic residues of eβG. (B) Predicted binding mode and (C) 2D representation of interactions of compound 42 (yellow) in the active site of eβG (olive) after 1 ns MD simulations. (D) Predicted binding mode of compound 42 in the active site of hβG (olive) (PDB code 3HN3). E451 and E540 are the catalytic resides of hβG. The olive dashed lines indicate hydrogen-bonding interactions.

Table 3. Binding Free Energies of Compound 42 to E. coli βG and Human βG Modelsa model

ΔGvdW

ΔGele

ΔGpolar,sol

ΔGnopolar,sol

ΔGMM

ΔGsol

ΔGbinding

eβG hβG

−34.26 −29.44

−43.83 −32.18

41.71 38.36

−4.53 −3.72

−78.10 −61.62

37.18 34.63

−40.91 −26.99

a

All the energies are in kcal/mol. Snapshots extracted from the last 0.2 ns MD simulation were submitted to MMPBSA.py for the free energy calculation.

the bacterial growth. The oral administration of compound 42 to mice reduced intestinal βG activity and CPT-11-induced intestinal damage. We also demonstrated that compound 42 prevented CPT-11-induced serious diarrhea while exhibiting no impacts on the therapeutic efficacy of CPT-11. Moreover, the computer modeling revealed the inhibition selectivity of compound 42 against eβG by binding to the active site and interacting with the bacterial loop. Compound 42 also displays a pH-dependent inhibition which is influenced by protonation of compound 42 and surface charge of the active pocket of eβG at different pH conditions. Compound 42 is a potent and specific eβG inhibitor that binds to the active pocket of eβG by directly contacting with the two catalytic residues E413 and E504 and also a portion of the bacterial loop at residues S360 and L361. Importantly, the unique 17-residue bacterial loop which is absent in hβG is essential for full activity of eβG activity and specific inhibition24 and thus serves as an target site for in silico screening for eβG-specific inhibitor.25

low pH condition; thus the inhibitory efficacy of compound 42 decreased. Additionally, it is worth mentioning that the binding pockets present in electrostatic potential surface at pH = 7.0 for eβG are mixed with electroneutral and electronegative spots (Figure 6B), whereas for hβG there are electropositive spots (Figure 6D), implying the reluctance of compound 42 to associate with hβG. These results show a new characteristic of an eβG inhibitor, in which compound 42 inhibited eβG in a pH-dependent manner that is influenced by the surface charge, making the combination more favorable at slightly acidic to neutral condition in the colon.

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DISCUSSION We have discovered certain pyrazolo[4,3-c]quinoline derivatives as potential eβG-specific inhibitors. One of the derivatives, compound 42, exhibited specific inhibition against eβG enzyme activity but not to hβG. Compound 42 also effectively reduces endogenous βG activity in living E. coli without causing harm to 9231



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Figure 6. pH-dependent inhibition of eβG by compound 42. (A) Compound 42 was evaluated for the inhibition effects to eβG at pH 5.5, 6.0, 6.5, and 7.0, respectively. Data are displayed as percent of βG activity compared with the control treated with 10% DMSO at each pH condition. Error bars, mean ± SD, n = 3 biological replicates. (B) Electrical potential surface of eβG active pocket at pH 5.5−7.0. Surface charges are shown from +5kT/e in blue and −5kT/e per electron in red. Red indicates negative charges, white is neutral, and blue represents positively charged. The white dotted line shows the active pocket of eβG. (C) Protonation of compound 42. Arrows indicate the two protonation sites. (D) Electrical potential surface of hβG active pocket at pH 7.0.

An ideal bacterial βG-specific inhibitor must maintain its inhibitory ability in living bacteria while also being nontoxic to said bacteria. The use of various antibiotics has shown promise in reducing CPT-11-induced intestinal toxicity in preclinical and clinical studies.4,5 The removal of intestinal microflora through the use of antibiotics also results in decreased βG levels in the intestine. However, it also has several drawbacks, including its effects on intestinal metabolism26 and a resulting imbalance of intestinal microflora, that lead to an increase in the chance of infection by pathogenic bacteria such as C. dif f icile and enterohemorrhagic E. coli and fungal infection.4 In addition, the use of antibiotics may increase the prevalence of subdominant bacterial species with higher βG activity and, thus, exacerbate intestinal toxicity.27 Recent studies have highlighted the crucial impact of intestinal microflora on the modulation of immune activation and improvements to the antitumor efficacy of chemotherapy and immunotherapy.28,29 Thus, unlike antibiotics, compound 42 that has no effect on intestinal microflora growth may have more potential clinical implications. It is important to reduce the toxicity of drugs while also maintaining their therapeutic efficacy. Concerns may be raised that bacterial βG-specific inhibitors could influence CPT-11 metabolism and result in a reduction of its antitumor efficacy. However, such an effect would depend on the level of SN-38 in plasma instead of that in the intestine. In studies involving rats, the use of antibiotics only decreased SN-38 levels in the intestinal contents, whereas the levels of CPT-11, SN-38, and SN-38G in the serum were not altered.30,31 The same phenomena were observed in colorectal cancer patients in that oral neomycin ameliorated diarrhea but had no effect on systemic exposure to CPT-11 and its metabolites.32 Thus, reducing SN-38 in the intestine through the use of a bacterial βG-specific inhibitor, such as compound 42, is not believed to impair the therapeutic efficacy of CPT-11. Indeed, we found that the therapeutic efficacy of CPT-11 was

Although we did not apply all the studied compounds for docking simulation, based on the aforementioned binding mode predicted for compound 42 in eβG, conclusions regarding SAR were made. Most studied compounds in this class displayed relatively potent inhibition against eβG than hβG which may due to a similar binding mode, but the derivatives showed various inhibitory character against eβG. For example, compound 45 (4′-NO2) with a poor IC50 value of 9200 nM against eβG in comparison with compound 42 (4′-F, 136.5 nM), may experience a steric hindrance between the bulky NO2 group and W549 and accordingly has a lower chance to be accommodated by eβG. Likewise, the hydrophobic interactions between the anilino moiety of compound 42 (4′-F) and the surrounding residues of eβG are likely to be altered by changing the substituent to ortho or meta position from the para position of the anilino moiety, and hence compound 37 (3′-Me) with IC50 > 10 000 nM against eβG may undergo a collision with either the side with E504 or the side with D163, Y472, R562, and K568, depending on the orientation of the anilino moiety. Although the binding mode of compound 42 in hβG is very similar to that in eβG, additional contacts with the bacterial loop in eβG may provide more contact to secure the bound compound 42. Roberts and colleagues indicated that the bacterial loop is a flexible structure that is capable of conforming to the presence of different bound inhibitors.24 Moreover, the distinct electrostatic potential character between eβG and hβG is also the key for selective inhibition of compound 42. Because compound 42 links with two negatively charged glutamate residues (E413 and E504 in eβG; E451 and E540 in hβG) in the binding site by two amino hydrogen atoms, the electropositive surface in hβG may repel the electropositive end of compound 42 away from the catalytic residues E451 and E540. Collectively, these modeling results illustrate that compound 42 is capable of specific eβG inhibition. 9232



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E. coli, (4) the capacity to reduce chemical-induced intestinal toxicity, (5) the capacity to maintain drug efficacy, and (6) a new characteristic of action based on pH-dependent surface charge inhibition. Considering these advantages, compound 42 is a potent bacterial βG-specific inhibitor with great potential to combine with various drugs in order to reduce their intestinal toxicity while also helping to prevent colon tumorigenesis caused by chemical carcinogens.

not altered in the presence of compound 42 (Figure 4C). The inhibition of intestinal βG by compound 42 may only prevent the activation of SN-38G to SN38 in the intestine, but the systemic metabolism of CPT-11, as measured by CPT-11induced weight loss, is not affected (Figure 4D). However, further pharmacokinetics research is required to verify the effects of compound 42 on CPT-11 and its metabolites. Hepatic glucuronidation is an essential metabolic mechanism for many drug classes. It is an important route for the detoxification of compounds bearing COOH, OH, NH, and SH groups.33 However, the hydrolysis of the glucuronide conjugates by bacterial βG in the intestine is likely to damage the intestine. In addition to its role in CPT-11-induced diarrhea, bacterial βG in the distal small intestine is also a determinant factor of enteropathy caused by nonsteroidal anti-inflammatory (NSAID) agents.34,35 Inhibiting intestinal βG activity by using compound 42 thus seems to constitute a mechanism-based strategy for reducing intestinal toxicity and damage due to the reactivation of glucuronide drugs in the intestine. Moreover, approximately one-tenth of the top 200 prescribed drugs in the U.S. are metabolized by glucuronidation.36 Drugs such as anticancer chemotherapy drugs (etoposide, epirubicin, and flavopiridol), anticancer targeted drugs (sorafenib), NSAID agents (indomethacin, diclofenac, and naproxen), and antivirals (zidovudine) are found to be metabolized through hepatic glucuronidation.37 Thus, a bacterial βG-specific inhibitor such as compound 42 could be used widely in combination with various drugs as a means of reducing their intestinal toxicity. The glucuronidation-mechanism-based strategy of inhibiting bacterial βG through the use of compound 42 in the intestine could potentially be applied in preventing colon cancer. A few studies have indicated a correlation between intestinal βG and colon cancer.38 For example, Kim and Jin found that the βG activity in the feces of colon cancer patients was 12.1 times higher than that in the feces of healthy controls.39 In another study, the presence of intestinal βG increased the genotoxicity of a common food-borne carcinogen in rat colon by about 3 times.40 Similarly, it has been demonstrated that a diet associated with a significant decrease in intestinal βG activity reduced the incidence of carcinogen-induced aberrant crypt foci (ACF) by 59% in rats.41 It seems that bacterial βG also liberates carcinogens that have been processed via hepatic glucuronidation and thus contributes to the exposure of the intestine to free carcinogens. Hence, inhibiting bacterial βG may prevent the local release of carcinogens in the intestine and thus reduce colon carcinogenesis. For example, luteolin administration was found to significantly decrease colon cancer incidence as well as the βG activity in carcinogen-treated rats.42 Mice treated with synthetic precursors of compound 1 at concentrations of 0.5% and 2% exhibited significant reductions of carcinogen-induced ACF of 48.6% and 55.3%, respectively.43 Therefore, it is widely accepted that bacterial βG inhibitors may act as colon cancer chemoprevention agents. Compound 42, being a potent and bacterial βG-specific inhibitor, could thus potentially be used as a protective agent in preventing precancerous lesions caused by carcinogens.

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EXPERIMENTAL SECTION

Chemistry. General. TLC: precoated (0.2 mm) silica gel 60 F254 plates from EM Laboratories, Inc.; detection by UV light (254 nm). Mp: Electrothermal IA9100 digital melting-point apparatus; uncorrected. 1H and 13C NMR spectra: Varian-Unity-400 spectrometer at 400 and 100 MHz or Varian-Gemini-200 spectrometer at 200 and 50 MHz, chemical shifts in ppm with SiMe4 as an internal standard (=0 ppm), coupling constants J in Hz. Elemental analyses were carried out on a Heraeus CHN-O-Rapid elemental analyzer, and results were within ±0.4% of calculated values. The purity of all target compounds used in the biophysical and biological studies was ≥95%. N4-(2-Chlorophenyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (33). Yield: 52%. Mp: 243−244 °C. UV λmax nm (log ε): 293 (4.28), 239 (4.52), 213 (4.52) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.46 (br s, 2H, NH2), 7.43−7.47 (m, 1H, Ar-H), 7.52−7.59 (m, 3H, Ar-H), 7.71−7.75 (m, 2H, Ar-H), 7.79 (d, 1H, J = 8.0 Hz, Ar-H), 8.17 (d, 1H, J = 7.6 Hz, Ar-H), 8.30 (br s, 1H, NH), 11.45 (br s, 1H, NH). N4-(o-Tolyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (34). Yield: 31%. Mp: 228−230 °C. UV λmax nm (log ε): 295 (4.27), 243 (4.63), 241 (4.63), 217 (4.55) in MeOH. 1H NMR (400 MHz, DMSO-d6): 2.31 (s, 3H, Me), 5.48 (br s, 2H, NH2), 7.02−7.05 (m, 1H, Ar-H), 7.23−7.27 (m, 3H, Ar-H), 7.45 (br s, 1H, Ar-H), 7.55 (d, 1H, J = 7.6 Hz, Ar-H), 8.05 (br s, 2H, Ar-H), 8.31 (br s, 1H, NH), 12.96 (br s, 1H, NH). Anal. Calcd for C17H15N5·0.2H2O: C, 69.68; H, 5.30; N, 23.90. Found: C, 69.95; H, 5.17; N, 23.70. 3-Amino-4-(2-hydroxyphenylamino)-1H-pyrazolo[4,3-c]quinoline (35). Yield 64%. Mp: 156−157 °C. UV λmax nm (log ε): 302 (4.39), 240 (4.63), 225 (4.61) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.51 (br s, 2H, NH2), 6.84−6.99 (m, 3H, Ar-H), 7.29−7.32 (m, 1H, Ar-H), 7.50−7.53 (m, 1H, Ar-H), 7.60 (d, 1H, J = 7.2 Hz, Ar-H), 8.06 (d, 2H, J = 7.6 Hz, Ar-H), 8.43 (s, 1H, NH), 11.22 (br s, 1H, OH), 13.06 (s, 1H, NH). Anal. Calcd for C16H13N5O·0.5H2O: C 63.98, H 4.71, N 23.32. Found: C 63.66, H 4.65, N 23.33. N4-(3-Chlorophenyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (36). Yield: 57%. Mp: 227−229 °C. UV λmax nm (log ε): 306 (4.33), 242 (4.53), 216 (4.55) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.66 (br s, 2H, NH2), 7.05 (dd, 1H, J = 8.0, 1.6 Hz, Ar-H), 7.34−7.39 (m, 2H, Ar-H), 7.54 (m, 1H, Ar-H), 7.67 (d, 1H, J = 7.6 Hz, Ar-H), 7.89 (d, 1H, J = 7.2 Hz, Ar-H), 8.09 (d, 1H, J = 7.6 Hz, Ar-H), 8.27 (s, 1H, Ar-H), 8.39 (br s, 1H, NH), 12.97 (br s, 1H, NH). Anal. Calcd for C16H12ClN5: C, 62.04; H, 3.90; N, 22.61. Found: C, 61.91; H, 3.88; N, 22.40. N4-(m-Tolyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (37). Yield: 50%. Mp: 281−282 °C. UV λmax nm (log ε): 305 (4.31), 241 (4.51), 218 (4.52), 214 (4.52) in MeOH. 1H NMR (400 MHz, DMSO-d6): 2.39 (s, 3H, Me), 7.23 (d, 1H, J = 7.6 Hz, Ar-H), 7.38−7.47 (m, 4H, Ar-H), 7.57 (dd, 1H, J = 8.0 Hz, Ar-H), 7.78 (d, 1H, J = 8.0 Hz, Ar-H), 8.16 (d, 1H, J = 6.4 Hz, Ar-H), 10.88 (br s, 1H, NH), 11.70 (br s, 1H, NH). Anal. Calcd for C17H15N5·1.1HCl: C, 61.95; H, 4.93; N, 21.25. Found: C, 61.95; H, 4.67; N, 21.18. 3-Amino-4-(3-hydroxyphenylamino)-1H-pyrazolo[4,3-c]quinoline (38). Yield 84%. Mp: 319−321 °C. UV λmax nm (log ε): 235 (4.50), 225 (4.50) in MeOH. 1H NMR (400 MHz, DMSO-d6): 6.82 (d, 1H, J = 7.6 Hz, Ar-H), 6.98 (d, 2H, J = 6.8 Hz, Ar-H),7.30−7.34 (m, 1H, Ar-H), 7.42−7.46 (m, 1H, Ar-H), 7.54−7.58 (m, 1H, Ar-H), 7.78 (d, 1H, J = 7.6 Hz, Ar-H), 8.15 (s, 1H, Ar-H), 9.95 (s, 1H, NH), 10.81 (br s, 1H, OH), 11.78 (br s, 1H, NH). Anal. Calcd for C16H13N5O· 1.25HCl: C 57.03, H 4.27, N 20.79. Found: C 56.95, H 4.62, N 21.02. 3-Amino-4-(4-chlorophenylamino)-1H-pyrazolo[4,3-c]quinoline (39). Yield 75%. Mp: 246−248 °C. UV λmax nm (log ε): 305 (4.40), 242 (4.57), 215 (4.53), 213 (4.54) in MeOH. 1H NMR (400 MHz,

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CONCLUSION The results of this study demonstrate that the pyrazolo[4,3-c] quinoline derivative, compound 42, as a bacterial βG-specific inhibitor is useful for reducing chemical-induced intestinal toxicity. Collectively, compound 42, possesses several advantages, including (1) a facile, high-yielding synthetic process, (2) high specificity and potency against eβG, (3) low cytotoxicity to 9233



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8-Chloro-N4-(4-chlorophenyl)-1H-pyrazolo[4,3-c]quinoline-3,4diamine (48). Yield: 33%. Mp: 270−272 °C. UV λmax nm (log ε): 237 (4.60), 226 (4.62) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.72 (br s, 2H, NH2), 7.40 (d, 2H, J = 8.8 Hz, Ar-H), 7.51 (d, 1H, J = 8.4 Hz, Ar-H), 7.64 (d, 1H, J = 8.4 Hz, Ar-H), 8.00 (d, 2H, J = 8.0 Hz, Ar-H), 8.15 (s, 1H, Ar-H), 8.41 (s, 1H, NH), 12.98 (br s, 1H, NH). Anal. Calcd for C16H11Cl2N5·1.3HCl: C, 49.04; H, 3.17; N, 17.88. Found: C, 48.90; H, 3.47; N, 17.77. 8-Chloro-N4-(p-tolyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (49). Yield: 46%. Mp: 261 °C. UV λmax nm (log ε): 236 (4.58), 223 (4.60) in MeOH. 1H NMR (400 MHz, DMSO-d6): 2.30 (s, 3H, Me), 5.67 (br s, 2H, NH2), 7.16 (d, 2H, J = 8.0 Hz, Ar-H), 7.49 (d, 1H, J = 7.6 Hz, Ar-H), 7.60 (d, 1H, J = 8.8 Hz, Ar-H), 7.80 (d, 2H, J = 8.0 Hz, Ar-H), 8.13 (s, 1H, Ar-H), 8.23 (s, 1H, NH), 12.95 (br s, 1H, NH). Anal. Calcd for C17H14ClN5·1.3HCl: C, 54.45; H, 4.14; N, 18.68. Found: C, 54.24; H, 4.36; N, 18.38. 8-Bromo-N4-(4-chlorophenyl)-1H-pyrazolo[4,3-c]quinoline-3,4diamine (50). Yield: 50%. Mp: 264 °C (dec). UV λmax nm (log ε): 237 (4.65), 225 (4.61) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.71 (br s, 2H, NH2), 7.39 (d, 2H, J = 8.8 Hz, Ar-H), 7.56−7.63 (m, 2H, Ar-H), 8.00 (d, 2H, J = 8.8 Hz, Ar-H), 8.30 (s, 1H, Ar-H), 8.40 (s, 1H, NH), 12.95 (br s, 1H, NH). 8-Bromo-N4-(p-tolyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (51). Yield: 73%. Mp: 276 °C. UV λmax nm (log ε): 311 (4.43), 244 (4.57) 224 (4.59) in MeOH. 1H NMR (400 MHz, DMSO): 2.30 (s, 3H, Me), 5.68 (br s, 2H, NH2), 7.17 (d, 2H, J = 8.4 Hz, Ar-H), 7.52−7.61 (m, 2H, Ar-H), 7.79 (d, 2H, J = 6.8 Hz, Ar-H), 8.26 (br s, 2H, Ar-H and NH), 12.96 (br s, 1H, NH). N4-(4-Chlorophenyl)-7-fluoro-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (52). Yield: 52%. Mp: 254 °C (dec). UV λmax nm (log ε): 307 (4.45), 243 (4.60), 215 (4.59) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.70 (br s, 2H, NH2), 7.21 (br m, 1H, Ar-H), 7.35−7.42 (m, 3H, Ar-H), 8.00 (d, 2H, J = 8.4 Hz, Ar-H), 8.09−8.14 (m, 1H, Ar-H), 8.42 (br s, 1H, NH), 12.97 (br s, 1H, NH). Anal. Calcd for C16H11ClFN5: C, 58.63; H, 3.38; N, 21.37. Found: C, 58.72; H, 3.45; N, 21.45. 7-Fluoro-N4-(p-tolyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (53). Yield: 81%. Mp: 246−247 °C. UV λmax nm (log ε): 234 (4.56), 220 (4.58) in MeOH. 1H NMR (400 MHz, DMSO-d6): 2.30 (s, 3H, Me), 5.68 (br s, 2H, NH2), 7.14−7.17 (m, 3H, Ar-H), 7.29 (dd, 1H, J = 11.2, 2.4 Hz, Ar-H), 7.80 (d, 2H, J = 8.0 Hz, Ar-H), 8.08 (dd, 1H, J = 8.4, 6.8 Hz, Ar-H), 8.22 (br s, 1H, NH), 12.85 (br s, 1H, NH). Anal. Calcd for C17H14FN5·1HCl: C, 59.37; H, 4.40; N, 20.37. Found: C, 59.31; H, 4.27; N, 20.36. N4-(4-Chlorophenyl)-6-methyl-1H-pyrazolo[4,3-c]quinoline-3,4diamine (54). Yield: 83%. Mp: 249−250 °C. UV λmax nm (log ε): 310 (4.48), 258 (4.58), 251 (4.58), 245 (4.59), 211 (4.53) in MeOH. 1 H NMR (400 MHz, DMSO-d6): 2.61 (s, 3H, Me), 5.67 (br s, 2H, NH2), 7.20−7.24 (m, 1H, Ar-H), 7.39−7.43 (m, 3H, Ar-H), 7.94 (d, 1H, J = 7.6 Hz, Ar-H), 8.11 (d, 2H, J = 8.8 Hz, Ar-H), 8.37 (br s, 1H, NH), 12.90 (br s, 1H, NH). 6-Methyl-N4-(p-tolyl)-1H-pyrazolo[4,3-c]quinoline-3,4-diamine (55). Yield: 81%. Mp: 270−271 °C. UV λmax nm (log ε): 309 (4.44), 258 (4.57), 213 (4.53) in MeOH. 1H NMR (400 MHz, DMSO-d6): 2.29 (s, 3H, Me), 2.60 (s, 3H, Me), 5.65 (br s, 2H, NH2), 7.15−7.19 (m, 3H, Ar-H), 7.39 (d, 1H, J = 6.8 Hz, Ar-H), 7.90−7.97 (m, 3H, Ar-H), 8.18 (s, 1H, NH), 12.85 (br s, 1H, NH). Anal. Calcd for C18H17N5: C, 71.67; H, 5.65; N, 23.09. Found: C, 71.21; H, 5.69; N, 23.00. Compounds and CPT-11. For in vitro assays, compound 1 (D-saccharic acid 1,4-lactone monohydrate; Sigma-Aldrich number S0375), compound 2 (β-glucuronidas inhibitor, Calbiochem; Merck number 347423), and our inhibitor compounds were dissolved in DMSO to 10 mM as stock and diluted to various concentrations. For in vivo studies, inhibitor compounds were dissolved in DDW to a final concentration of 0.3 mg/mL by sonication. CPT-11 (irinotecan hydrochloride; Sigma-Aldrich number I1406) as a hydrochloride salt (>97% HPLC purified grade) was dissolved in DDW to a final concentration of 10 mg/mL and heated to 70−80 °C prior to use. Cells and Mice. The CT26 cell line, a murine Balb/c colon adenocarcinoma, was maintained in Dulbecco’s minimal essential medium (DMEM) (Sigma-Aldrich) supplemented with 10% bovine

DMSO-d6): 5.62 (br s, 2H, NH2), 7.26−7.30 (m, 1H, Ar-H), 7.35 (d, 2H, J = 8.8 Hz, Ar-H), 7.46−7.50 (m, 1H, Ar-H), 7.61 (d, 1H, J = 8.0 Hz, Ar-H), 7.99−8.05 (m, 3H, Ar-H), 8.31 (br s, 1H, NH), 12.92 (br s, 1H, NH). Anal. Calcd for C16H12ClN5·0.25H2O: C 61.13, H 4.02, N 22.29. Found: C 61.45, H 3.86, N 22.67. 3-Amino-4-(4-methylphenylamino)-1H-pyrazolo[4,3-c]quinoline (40). Yield 81%. Mp: 292−294 °C (lit. 296−298 °C).28 UV λmax nm (log ε): 236 (4.47), 217 (4.49) in MeOH. 1H NMR (400 MHz, DMSO-d6): 2.30 (s, 3H, Me), 5.62 (br s, 2H, NH2), 7.16 (d, 2H, J = 8.4 Hz, Ar-H), 7.26−7.29 (m, 1H, Ar-H), 7.47−7.51 (m, 1H, Ar-H), 7.60 (d, 1H, J = 8.4 Hz, Ar-H), 7.84 (d, 2H, J = 8.4 Hz, Ar-H), 8.05 (d, 1H, J = 8.0 Hz), 8.15 (s, 1H, NH), 12.89 (br s, 1H, NH). Anal. Calcd for C17H15N5O·0.25H2O: C 69.48, H 5.33, N 23.84. Found: C 69.12, H 5.14, N 23.86. 3-Amino-4-(4-hydroxyphenylamino)-1H-pyrazolo[4,3-c]quinoline (41). Yield 82%. Mp: 347−349 °C (dec). UV λmax nm (log ε): 230 (4.61) in MeOH. 1H NMR (400 MHz, DMSO-d6): 6.96 (d, 2H, J = 8.8 Hz, Ar-H), 7.36 (d, 2H, J = 8.4 Hz, Ar-H), 7.39−7.43 (m, 1H, Ar-H), 7.51−7.56 (m, 1H, Ar-H), 7.79−7.81 (m, 1H, Ar-H), 8.14 (m, 1H, Ar-H), 9.96 (br s, 1H, NH), 10.59 (br s, 1H, OH), 12.21 (br s, 1H, NH). Anal. Calcd for C16H13N5O·2.0H2O: C 58.70, H 5.24, N 21.40. Found: C 58.47, H 5.24, N 21.70. 3-Amino-4-(4-fluorophenylamino)-1H-pyrazolo[4,3-c]quinoline (42). Yield 79%. Mp: 266−268 °C. UV λmax nm (log ε): 236 (4.52), 218 (4.49) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.64 (br s, 2H, NH2), 7.16−7.21 (m, 2H, Ar-H), 7.27−7.30 (m, 1H, Ar-H), 7.47−7.51 (m, 1H, Ar-H), 7.60 (d, 1H, J = 8.0 Hz, Ar-H), 7.96−7.99 (m, 2H, Ar-H), 7.61 (d, 1H, J = 7.6 Hz, Ar-H), 8.25 (br s, 1H, NH), 12.91 (br s, 1H, NH). Anal. Calcd for C16H12FN5: C 65.52, H 4.12, N 23.88. Found: C 65.52, H 4.39, N 23.63. 3-Amino-4-(4-trifluoromethylphenylamino)-1H-pyrazolo[4,3-c]quinoline (43). Yield 75%. Mp: 251−252 °C. UV λmax nm (log ε): 308 (4.51), 241 (4.67), 212 (4.57) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.68 (br s, 2H, NH2), 7.34−7.38 (m, 1H, Ar-H), 7.53−7.57 (m, 1H, Ar-H), 7.68−7.72 (m, 3H, Ar-H), 8.40 (d, 1H, J = 7.6 Hz, Ar-H), 8.20 (d, 2H, J = 8.8 Hz, Ar-H), 8.61 (br s, 1H, NH), 13.00 (br s, 1H, NH). Anal. Calcd for C17H12F3N5: C 59.48, H 3.52, N 20.40. Found: C 59.55, H 3.57, N 20.26. 3-Amino-4-(4-aminophenylamino)-1H-pyrazolo[4,3-c]quinoline (44). Yield 61%. Mp: 355−356 °C. UV λmax nm (log ε): 251 (4.51), 232 (4.59) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.57 (br s, 2H, NH2), 6.35 (br s, 2H, NH), 6.71 (d, 2H, J = 8.8 Hz, Ar-H), 7.17 (d, 2H, J = 8.0 Hz, Ar-H), 7.39−7.51 (m, 3H, Ar-H), 7.77−7.81 (m, 1H, Ar-H), 8.08 (br s, 1H, NH), 10.41 (br s, 1H, NH), 10.98 (br s, 1H, NH), 12.96 (br s, 1H, NH). Anal. Calcd for C16H14N6·2.0H2O·1.25HCl: C 51.65, H 5.23, N 22.60. Found: C 51.78, H 5.37, N 22.61. 3-Amino-4-(4-nitrophenylamino)-1H-pyrazolo[4,3-c]quinoline (45). Yield 36%. Mp: 303−305 °C (dec). UV λmax nm (log ε): 369 (4.29), 243 (4.51), 216 (4.52), 214 (4.52) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.73 (br s, 2H, NH2), 7.40−7.43 (m, 1H, Ar-H), 7.58−7.61 (m, 1H, Ar-H), 7.78 (d, 1H, J = 7.6 Hz, Ar-H), 8.14 (d, 1H, J = 8.0 Hz, Ar-H), 8.25 (s, 4H, Ar-H), 9.10 (br s, 1H, NH), 13.08 (br s, 1H, NH). Anal. Calcd for C16H12N6O2·0.3HCl: C 58.01, H 3.73, N 25.37. Found: C 57.72, H 4.03, N 24.98. 4-(3-Amino-1H-pyrazolo[4,3-c]quinolin-4-ylamino)benzoic Acid (46). Yield 76%. Mp: 362−364 °C. UV λmax nm (log ε): 316 (4.45), 241 (4.62), 238 (4.62), 225 (4.61), 221 (4.60) in MeOH. 1H NMR (400 MHz, DMSO-d6): 5.65 (br s, 2H, NH2), 7.47−7.51 (m, 1H, Ar-H), 7.59−7.63 (m, 1H, Ar-H), 7.74−7.78 (m, 3H, Ar-H), 8.07 (d, 2H, J = 8.4 Hz, Ar-H), 8.19 (d, 1H, J = 8.0 Hz, Ar-H), 11.21 (br s, 1H, NH), 12.17 (br s, 1H, NH), 12.95 (br s, 1H, COOH). Anal. Calcd for C17H13N5O2·HCl: C 57.39, H 3.97, N 19.68. Found: C 57.45, H 3.84, N 19.76. 3-Amino-4-(4-hydroxyphenylamino)-1H-pyrazolo[4,3-c]quinoline (47). Yield 82%. Mp: 347−349 °C (dec). UV λmax nm (log ε): 346 (4.59), 334 (4.58), 247 (4.65), 216 (4.57) in MeOH. 1H NMR (400 MHz, DMSO-d6): 6.96 (d, 2H, J = 8.8 Hz, Ar-H), 7.36 (d, 2H, J = 8.4 Hz, Ar-H), 7.39−7.43 (m, 1H, Ar-H), 7.51−7.56 (m, 1H, Ar-H), 7.79−7.81 (m, 1H, Ar-H), 8.14 (m, 1H, Ar-H), 9.96 (br s, 1H, NH), 10.59 (br s, 1H, OH), 12.21 (br s, 1H, NH). Anal. Calcd for C16H13N5O· 2.0H2O: C 58.70, H 5.24, N 21.40. Found: C 58.47, H 5.24, N 21.70. 9234



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In Vivo Modeling of CPT-11 Efficacy. CT26 cells (1 × 106 cells in 0.05 mL of PBS) were subcutaneously injected into the right flank region of the mice. Tumor volumes were estimated using the formula length × width × height × 0.52. Solid tumors were allowed to reach a volume of 10−50 mm3 (which took approximately ∼9 days), with the day at which this range was achieved then being defined as day 0. Compound 42 (3 mg kg−1 day−1) or DDW was then delivered daily by oral gavage starting on day 0. CPT-11 (50 mg kg−1 day−1) or saline was injected intraperitoneally on a daily basis starting on day 2. The mice were examined daily for signs of diarrhea, and their tumor volume and body weight were monitored every 2 days. Statistical analysis was performed using GraphPad Prism 6. Two-way ANOVA was used to test the significance of differences in diarrhea and tumor growth. Molecular Docking. The molecular structure of eβG was extracted from the Protein Data Bank (PDB) database (PDB code 3LPF).18 The structure of hβG (tetramer structure; PDB code 3HN3) was used as a template to build the tetramer structure of eβG. The two monomers of structures of eβG which comprise the ligand-binding site were kept as a receptor in the following molecular docking experiments. The docking studies were performed using AutoDock 4.2.45 The Lamarckian genetic algorithm (LGA) was selected to perform the molecular docking studies. All the rotatable bonds in compound 42 were assigned to be rotatable in order to optimize the interaction with the receptor. A box with the dimensions of 126 × 126 × 126 and grid points with grid spacing of 0.15 Å was applied. The center coordinate of the grid box was chosen to be located near the E413 of eβG. The final docked 300 conformations were clustered based on a tolerance of 1 Å all-atom rmsd from the lowest-energy structure. The docked conformations were ranked into clusters based on the binding energy, and the most favorable binding conformation with the lowest free energies was selected as the binding pose for the follow-up molecular dynamics simulations. Molecular Dynamics Simulations. The molecular dynamics simulations were performed using the AMBER 11.0 software package46 with gaff47 and ff99SB force fields48 to compare the binding modes and selectivity of compound 42 in eβG and hβG at pH = 7. The force-field parameters for the ligand were generated using the general AMBER force field by employing the Antechamber program.49 The partial atomic charges for the ligand atoms were assigned using the AM-BCC protocol50 after electrostatic potential calculations at the HF/6-31G* level. All hydrogen atoms of the three proteins were assigned using the LEaP module in consideration of ionizable residues set at their default protonation states at a neutral pH value. Each complex was immersed in a cubic box of the TIP3P water model.51 The size of the box was set such that the distance between the atoms in the studied complex and the wall was greater than 10 Å. Fifty sodium ions were added to neutralize the compound-eβG and compound-hβG complexes. The solvated system was energy-minimized through three stages, with each stage employing 5000 steps of the steepest descent algorithm and 5000 steps of the conjugate-gradient algorithm with a nonbonded cutoff of 8.0 Å. At stage 1, the protein and compound were restrained, enabling the added TIP3P water molecules to adjust to their proper orientations. At stage 2, the protein backbone was restrained to enable the amino acid side chains to find a superior way of accommodating compound. At stage 3, the entire solvated system was minimized without any restraint. The molecular dynamics simulations were performed according to the standard protocol, which consists of gradual heating, density, equilibration, and production procedures in the isothermal isobaric ensemble (NPT, P = 1 atm and T = 300 K) MD. A minimized solvated system was used as the starting structure for subsequent MD simulations. In the 500 ps heating procedure, the system was gradually heated from 0 to 300 K for 250 ps, then was followed by a density at 300 K for 500 ps and was then subjected to constant equilibration at 300 K for 500 ps. After the equilibration procedure, the system underwent a 1 ns production procedure for conformation collection. The time step was set at 2 fs. Snapshots were taken at 1 ps intervals to record the conformation trajectory during the production MD stimulation. An 8 Å cutoff was applied to treat nonbonding interactions, such as short-range electrostatics and van der Waals interactions, and the particle-mesh Ewald method was applied to treat long-range electrostatic interactions.52 The SHAKE algorithm53 was used to limit all bonds containing hydrogen

calf serum (GE Healthcare Life Science) and 1% penicillin− streptomycin (Thermo Fisher Scientific) at 37 °C in an atmosphere of 5% CO2. Healthy 6- to 8-week-old Balb/cJ mice (BALB/cByJNarl) were purchased from the National Laboratory Animal Center, Taiwan. The animal experiments were conducted as approved by the Kaohsiung Medical University Institutional Animal Care and Use Committee. In Vitro βG Activity Assay. Purified eβG and hβG proteins were obtained as described.25 The assay was performed in a 96-well plate. Each reaction consisted of 40 μL of purified βG (20 ng), 10 μL of compound (various concentrations), and 50 μL of 20 mM pNPG (Sigma-Aldrich number 1627). The βG and pNPG were prepared in assay buffer (PBS containing 10% DMSO and 0.05% BSA) at pH 7.5 and pH 4.5 for eβG and hβG, respectively. The βG was treated with each compound at 37 °C for 30 min and then sequentially incubated with pNPG for 1 h. Each reaction was immediately quenched by 5 μL of 2 N sodium hydroxide. For the pH-dependent assay, PBS in the assay buffer was replaced by different proportions of 0.2 M Na2HPO4 and 0.1 M citric acid. The βG activity was measured by detecting the presence of pNP at OD at 405 nm and was defined as percent of βG activity compared with the untreated control. The IC50 values were calculated from the concentration−response curve fit (four parameters) using GraphPad Prism 6. E. coli βG Activity Assay and Toxicity. BL21 (DE3) E. coli (Novagen) cells were grown for overnight incubation. The bacterial cells were then diluted to an OD at 600 nm of 0.1 in LB medium. This assay was similar to the in vitro assay. The reaction mixture contained 100 μL of bacteria, 40 μL of compound (various concentrations), 80 μL of LB medium, 80 μL of assay buffer, and 100 μL of 40 mM pNPG. The bacterial cells were treated with compound at 37 °C for 30 min and then sequentially incubated with pNPG for 1 h. The cells were then centrifuged at 6000g for 10 min. The resulting supernatant was collected and quenched by 5 μL of 2 N sodium hydroxide. The βG activity was measured by detecting the presence of pNP at OD at 405 nm and was defined as percent of βG activity compared with the untreated control. In the E. coli toxicity assay, each compound was added to the bacteria to a final concentration of 100 μM and incubated at 37 °C with shaking for 6 h. The number of bacterial cells was determined by detecting OD at 600 nm. Cell viability was defined as percent in the presence of compound compared with the untreated control. In Vivo Imaging of Intestinal βG Activity and Fecal βG Activity Assay. Mice were orally administered 3 mg/kg per day of each compound or DDW (200 μL) for 5 consecutive days. At 30 min after the fifth feeding, 500 μg/100 μL of FDGlcU (Thermo Fisher Scientific number 2915) was orally provided. The fluorescence intensity in the intestine was detected on an IVIS Spectrum in vivo imaging system (PerkinElmer) at 120 min after probe administration (λex:λem = 490 nm:525 nm). Fluorescence intensity in the intestine was analyzed by Living Image 4.2. For the fecal βG activity assay, naturally discharged and fresh stool was weighed and homogenized with the assay buffer (1:9 w/v). The reaction mixture contained 50 μL of fecal suspension, 50 μL of assay buffer, and 100 μL of 20 mM pNPG. The mixture was incubated at 37 °C for 1 h and was then centrifuged at 6000g for 10 min. The resulting supernatant was collected and mixed with 5 μL of 2 N sodium hydroxide. The βG activity was measured by detecting the presence of pNP at OD at 405 nm. The data were analyzed by t test using GraphPad Prism 6. In Vivo Modeling of CPT-11-Induced Intestinal Toxicity. Mice were orally administered compound 42 (3 mg/kg per day) or DDW (200 μL per day) starting 2 days before the administration of CPT-11. CPT-11 (50 mg/kg) (∼100 μL) or saline was injected intraperitoneally for 10 consecutive days. The mice were examined daily for signs of diarrhea. A grading index for diarrhea severity ranging from 0 to 3 was used,44 with 0 indicating solid stool (i.e., normal stool), 1 indicating slightly wet and soft stool (slight diarrhea), 2 indicating wet and unformed stool with moderate perianal staining of the coat (moderate diarrhea), and 3 indicating watery stool with severe perianal staining of the coat (severe diarrhea). On day 10 after sacrifice, jejunum, ileum, and colon samples were harvested and fixed in 10% formalin solution and paraffin-embedded. The prepared slides were stained with hematoxylin and eosin (H&E). 9235



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atoms to their equilibrium lengths. For structural and energetic analysis, we captured the trajectory in 1 ns (i.e., 1000 conformation snapshots) for each complex system. Binding Free Energy Calculations. MM/GBSA54 is a popular approach for investigating the energetic contribution to protein-small molecule binding affinity. To calculate the binding free energies of compound 42 binding with eβG and hβG, MM/GBSA calculation was applied to the snapshots extracted in 1 ns of the MD trajectories. The binding free energy was computed for each molecular species, including complexes, ligands, and proteins, as the difference

ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Science and Technology, Taipei, Taiwan (MOST 103-2320-B-037-011MY3, MOST 105-2320-B-037-007, MOST 105-2320-B-037-011, MOST 106-2320-B-037-015, MOST 106-2320-B-041-001, MOST 106-2632-B-037-003); the Taiwan Ministry of Health and Welfare (MOHW 106-TDU-B-212-144007) and Health and Welfare Surcharge of Tobacco Products; the Grant of Biosignature in Colorectal Cancers, Academia Sinica, Taiwan; and the Program for Translational Innovation of Biopharmaceutical DevelopmentTechnology Supporting Platform Axis. This study is also supported by Kaohsiung Medical University (KMU-DT106003, KMU-TP104E16, KMU-TP104E42, KMU-TP104H07, KMUTP104H08, KMU-TP105C00, KMU-TP105E17, KMUTP105E33, KMU-TP105H07, KMU-TP105H08); KMU-KMUH Co-Project of Key Research (KMU-DK106002, KMU-DK107001) from Kaohsiung Medical University

ΔG binding = GβG‐42 − [GβG + G42] where

Gmolecule = EMM + Gsolvation polar + Gsolvation nonpolar − TS

EMM = E internal + Eelectrostatic + EvdW

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and

ABBREVIATIONS USED ACF, aberrant crypt foci; βG, β-glucuronidase; eβG, E. coli β-glucuronidase; FDGlcU, fluorescein di-β-D-glucuronide; hβG, human β-glucuronidase; HTS, high-throughput screening; MPS VII, mucopolysaccharidosis type VII; NSAID, nonsteroidal anti-inflammatory; pNPG, 4-nitrophenyl β-D-glucuronide; SAR, structure−activity relationship; SN-38G, SN-38 glucuronide

Gsolvation nonpolar = γA + β ⟨...⟩ denotes the average for a set of structures extracted from a series of snapshots along an MD trajectory, and ΔGbinding is estimated from the contributions of gas-phase energies, i.e., ⟨EMM⟩, solvation free energies, including polar and nonpolar terms, and entropies. Regarding the gasphase energies, Einternal includes the bond, angle, and torsional energies, and Eelectostatic and EvdW represent the electrostatic and van der Waals energies, respectively. The polar solvation, Gsolvationpolar, is calculated using the generalized Born model.54 The nonpolar solvation term, Gsolvationnonpolar, is calculated with a value of 0.005 42 kcal mol−1 Å−2 for the surface tension proportionality constant γ and a value of 0.92 kcal mol−1 for the nonpolar free energy for a point solute β. The solvent accessible surface area, A, is varied according to the molecule and is calculated using a computer program. The entropy term, TS, arises from changes in the degrees of freedom, including the translational, rotational, and vibrational terms of the solute molecules, and is estimated using the classical statistical thermodynamics approach. Conformational entropy was not included in our approach because it is computationally expensive. Electrostatic Potential Surface. Electrostatic potential surfaces was modeling using the apo form eβG at pH 5.5−7.0 (PDB code 3LPF) and hβG (PDB code 3HN3) at pH 7.0 with the aid of the programs PDB2PQR55,56 and APBS.57

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REFERENCES

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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.7b00963.

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Article

Molecular formula strings and some data (CSV)

AUTHOR INFORMATION

Corresponding Authors

*Y.-L.C.: e-mail, [email protected]; phone, (+886) 7-3121101-2684; fax, (+886)7-3125339. *T.-L.C.: e-mail, [email protected]; phone, (+886) 7-3121101-2136-21; fax, (+886)7-3227508. ORCID

Chia-Ning Yang: 0000-0002-3969-720X Tian-Lu Cheng: 0000-0001-6424-4731 Author Contributions △

K.-W.C. and C.-H.T. contributed equally.

Notes

The authors declare no competing financial interest. 9236



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