Discovery of Novel KRAS-PDEδ Inhibitors by Fragment-Based Drug

Mar 6, 2018 - To tackle these challenges, herein, novel, highly potent KRAS-PDEδ inhibitors were identified by fragment-based drug design, providing ...
1 downloads 0 Views 3MB Size
Download PDF
Brief Article Cite This: J. Med. Chem. 2018, 61, 2604−2610

pubs.acs.org/jmc

Discovery of Novel KRAS-PDEδ Inhibitors by Fragment-Based Drug Design Long Chen,† Chunlin Zhuang,† Junjie Lu,† Yan Jiang, and Chunquan Sheng* School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China S Supporting Information *

ABSTRACT: Targeting KRAS-PDEδ protein−protein interactions with small molecules represents a promising opportunity for developing novel antitumor agents. However, current KRAS-PDEδ inhibitors are limited by poor cellular antitumor potency and the druggability of the target remains to be validated by new inhibitors. To tackle these challenges, herein, novel, highly potent KRAS-PDEδ inhibitors were identified by fragment-based drug design, providing promising lead compounds or chemical probes for investigating the biological functions and druggability of KRAS-PDEδ interaction.

■

INTRODUCTION Oncogenic KRAS signaling is an important antitumor pathway.1,2 The KRAS protein is often mutated in different kinds of cancers and especially in a large proportion (90%) of pancreatic cancers.3,4 Targeting KRAS signaling is becoming an important field in anticancer drug discovery and has achieved great success.5−9 Recently, inhibition of mammalian KRAS-PDEδ protein−protein interaction (PPI) by small molecules has been seen as a promising opportunity for the discovery of novel antitumor agents.10−16 PDEδ, also named PDE6D, determines the KRAS dynamic distribution in the cell. 17−19 The farnesylated KRAS protein is solubilized after binding with PDEδ, which enhances KRAS diffusion throughout the cell.7,18,20,21 After being discharged from the PDEδ binding pocket, the farnesylated KRAS is trapped by the recycling endosome and then relocalized to the plasma membrane by vesicular transport. Aberrant oncogenic signaling ultimately results from the high concentration of KRAS at the plasma membrane.18,22 Recently, Waldmann’s group reported pioneering works in the discovery of small-molecule KRAS-PDEδ inhibitors (Figure 1), including benzimidazole inhibitor deltarasin (1),10 pyrazolopyridazinone inhibitor deltazinone (2),11,14 and bis(sulfonamide) inhibitor deltasonamide (3).16 Compound 1 was the first PDEδ inhibitor with nanomolar binding affinity; however, it was a nonselective inhibitor and showed apparent cytotoxicity.10 Inhibitors 2 and 3 showed better selectivity toward PDEδ than inhibitor 1, but they were limited by poor cellular antitumor potency.15 Previously, our group reported novel bis-quinazolinone 4 and quinazolinone-pyrazolopyridazinone 5 as inhibitors based on a © 2018 American Chemical Society

Figure 1. Chemical structures of representative KRAS-PDEδ inhibitors.

virtual screening and structural biology-guided drug design.13 Compound 5 exhibited excellent binding affinity (KD = 2 ± 0.5 nM) and moderate antitumor activity (IC50 = 18.6 ± 1.3 μM) against Capan-1 pancreatic cancer cells.13 Nevertheless, its cellular potency needs improvement. Additionally, a new class Received: January 14, 2018 Published: March 6, 2018 2604

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610

Journal of Medicinal Chemistry

Brief Article

molecular docking and intermolecular distance measurements, fragment-linking was proposed at different sites on the two inhibitors. First, the distance from the benzene ring of benzimidazole inhibitor 7 to the nitrogen atom of the amide in quinazolinone inhibitor 6 is 5.3 Å (Figure 2B). Second, the distance between the benzene ring of inhibitor 6 to the imidazole nitrogen atom of inhibitor 7 is 5.0 Å (Figure 2D). These distances were both suitable for using an ether linker between the two methylenes. Thus, two series of compounds were designed and docked into the PDEδ protein. As shown in Figure 2E,F, two representative compounds (8a and 9a) could insert well into the binding pocket and form hydrogen bonds with Arg61 and Tyr149, mimicking the binding modes of the unlinked fragment-like inhibitors 6 and 7 (Supporting Information (SI), Figure S1A,S1B). Although the predicted orientation of the benzimidazole group in compounds 9a and 7 was flipped (SI, Figure S1B,S1C), the hydrogen-bonding interaction with Tyr149 was retained. Biological Evaluations, Structure−Activity Relationship and Binding Modes. Initially, fragment-like PDEδ inhibitors 6 and 7 were linked by an ethyl ether (−(CH2)2O−) linker, and pyridine/benzene replacement was used to improve the water solubility. On the basis of these structural features, compound 8a was synthesized and assayed. Compared to inhibitors 6 and 7, compound 8a (KD = 9.0 ± 2.3 nM) showed improved PDEδ inhibitory activity and its activity was comparable to that of compound 2 (KD = 8 ± 3 nM). When substituents were introduced at the 6 position of the quinazolinone scaffold, the resulting compounds (8b−8d, KD range 4.5−11.6 nM) showed superior or comparable binding affinities to that of compound 8a. In contrast, the activities of the 7-substituted derivatives (8e−8g) were slightly lower than that of 8a. Among the prepared compounds, compound 8b, with a 6-fluoro substituent, had the best PDEδ inhibitory activity (KD = 4.5 ± 1.1 nM). However, compounds 8a−8g showed no cellular antitumor activity toward Capan-1 pancreatic cancer cells (Table 1). The ligand efficiency (LE)29 of the target compounds was also calculated (SI, Table S1). Moderate LE of the fragment-like inhibitors 6 (LE=

of KRAS-PDEδ inhibitors is highly desirable to validate the druggability of the target. Fragment-based drug design (FBDD) is emerging as a powerful tool in drug discovery.23−27 In this study, novel KRAS-PDEδ inhibitors were identified using a FBDD strategy based on the co-crystal complexes of fragmentlike inhibitors with PDEδ.10,11,13,16 Interestingly, several new inhibitors showed improved cellular antitumor activities, which makes them promising lead compounds or chemical probes for investigating the biological functions and druggability of KRASPDEδ interactions.

■

RESULTS AND DISCUSSION FBDD of KRAS-PDEδ Inhibitors. Recently, our group identified quinazolinone fragment hit 6 (Figure 2) as a novel

Table 1. Chemical Structures, PDEδ Binding Affinities, and Cellular Level Activities of Compounds 8a−g

Figure 2. Design of novel KRAS-PDEδ inhibitors by a computational FBDD strategy. (A) Co-crystal structure of quinazolinone fragment 6 with PDEδ protein (PDB 5X73). (B) First fragment-linking strategy. (C) Co-crystal structure of benzimidazole fragment 7 with PDEδ protein (PDB 4JV6). (D) the second fragment-linking strategy. (E) Proposed binding mode of compound 8a with PDEδ protein. (F) Proposed binding mode of compound 9a with PDEδ protein. Yellow dashed lines represent hydrogen-bonding interactions. Red dashed lines represent intermolecular distance measurements.

KRAS-PDEδ inhibitor (KD = 467 ± 65 nM) using a structurebased virtual screening (SBVS).13 The crystal structure of the complex of PDEδ with fragment 6 (PDB 5X73) indicated that the two molecules of inhibitor 6 bound to Arg61 and Tyr149 in the active site mainly through hydrogen-bonding interactions (Figure 2A). Similarly, Waldmann’s work revealed that two molecules of benzimidazole fragment 7 (KD = 165 ± 23 nM, Figure 2) could also bind in the two active sites (Figure 2C, PDB 4JV6).10 Inspired by these binding modes, computational FBDD28 was used to rationally design novel PDEδ inhibitors by linking the two fragment-like inhibitors. On the basis of

compd

R

8a 8b 8c 8d 8e 8f 8g 2 5

H 6-F 6-Cl 6-CH3 7-F 7-Cl 7-OCH3

PDEδa (KD, nM)

Capan-1b (IC50, μM)

± ± ± ± ± ± ± ± ±

>100 >100 >100 >100 >100 >100 >100 48 ± 6 18.6 ± 1.3

9.0 4.5 11.6 9.3 12.1 16.0 9.8 8 2

2.3 1.1 2.6 2.5 3.6 3.4 2.2 3 0.5

Determined by fluorescence anisotropy assay. bDetermined by the CCK8 method. a

2605

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610

Journal of Medicinal Chemistry

Brief Article

0.08) and 7 (LE= 0.20) was observed. After fragment linking, compounds 8a−g showed improved LE (LE range: 0.23−0.31). When the position of the fragment linker was changed to the quinazolinone N3-phenyl group and the imidazole nitrogen atom, the resulting compounds (9a−9c) generally showed decreased binding affinity (KD range 146−192 nM), low LE (0.10−0.13, SI, Table S1), and no in vitro antitumor activity (Table 2). The removal of the benzene ring from the Table 2. Structures, PDEδ Binding Affinities, and Cellular Level Activities of Compounds 9a−i Designed by the Fragment-Linking Strategy

Figure 3. Proposed binding mode of compounds 10g (A) and 11b (B) with PDEδ.

compd

Ar

R

9a 9b 9c 9d 9e 9f 9g 9h 9i 2 5

2-F-Ph 2-F-Ph pyridin-2-yl 2-F-Ph 2-F-Ph 2-F-Ph pyridin-2-yl pyridin-2-yl pyridin-2-yl

Ph CH3 Ph Ph CH3 H Ph CH3 H

PDEδa (KD, nM)

Capan-1b (IC50, μM)

± ± ± ± ± ± ± ± ± ± ±

>100 >100 >100 >100 >100 >100 52 ± 2.5 >100 >100 48 ± 6 18.6 ± 1.3

168 146 192 29 53 80 22 166 170 8 2

25 22 35 11 19 15 8 33 28 3 0.5

expected, when methyl and cycloalkyl groups were introduced, the resulting compounds (11a and 11b, respectively) showed improved PDEδ inhibitory activities (KD = 37 ± 22 nM and KD = 38 ± 17 nM, respectively) (Table 3). In particular, compound 11b showed good antitumor activity (Capan-1, IC50 = 8.8 ± 2.4 μM), and it was more potent than compound 2 (Capan-1, IC50 = 48 ± 6 μM) and quinazolinone inhibitor 5 Table 3. Structures, PDEδ Binding Affinities, and Cellular Level Activities of Compounds 10a−g and 11a−e

Determined by fluorescence anisotropy assay. bDetermined by the CCK8 method. a

benzimidazole scaffold increased the binding affinity for PDEδ (compounds 9d−9i, KD range 22−170 nM). Also, compounds 9d, 9e, and 9g showed improved LE (0.22−0.30, SI, Table S1). The phenyl substitutions on the imidazole ring were important for the inhibitory activity. For example, methyl derivative 9e and unsubstituted derivative 9f were less potent than the corresponding compounds with the phenyl substituents. To improve the water solubility, further pyridine/ benzene replacement provided compounds 9g−9i, which showed similar activities. Among 9g−9i, compound 9i possessed moderate inhibitory activity against Capan-1 cells (IC50 = 52 ± 2.5 μM), which is comparable to that of compound 2 (IC50 = 48 ± 6 μM). Inspired by our previously identified quinazolinone inhibitors (4 and 5),13 the phenyl group in compound 9f was replaced by a piperidinyl group. Interestingly, resulting compound 10a showed improved cellular activity (Capan-1, IC50 = 34 ± 3.4 μM). However, further introduction of substituents on the quinazolinone scaffold did not have positive effects on the cytotoxicity. Subsequently, molecular docking studies indicated that compound 10g could insert well into the two binding pockets of PDEδ and form hydrogen bonds with both Arg61 and Glu88 (Figure 3A). Notably, there was a small hydrophobic pocket around the quinazolinone NH and no hydrogen-bonding interaction was observed, suggesting that the incorporation of a suitable hydrophobic group improved the binding affinity. Thus, as

Determined by fluorescence anisotropy assay. bDetermined by the CCK8 method. a

2606

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610

Journal of Medicinal Chemistry

Brief Article

(Capan-1, IC50 = 18.6 ± 1.3 μM). When bulkier groups, namely cyclobutyl (11c), cyclopentyl (11d), and cyclohexyl (11e), were introduced, their PDEδ inhibitory activities and cytotoxicities were lower probably due to steric hindrance. Quantitative activity−activity relationships (QAAR)30 analysis indicated that there was good positive correlation (R2 = 0.62, SI, Figure S2) between the inhibitory activity against PDEδ and Capan-1 cells. Furthermore, the binding mode of compounds 10g and 11b was investigated by molecular docking studies. As shown in Figure 3B, the binding mode of compound 11b was similar to that of compound 10g. The cyclopropyl group of 11b formed additional hydrophobic interactions with Ile129, Val145, and Leu147. Finally, compound 11b had a good combination of PDEδ binding affinity (KD = 38 ± 17 nM) and cytotoxicity (IC50 = 8.8 ± 2.4 μM) and was therefore subjected to further biological evaluations. Compound 11b Inhibited the Phosphorylation of Akt and Erk in Capan-1 Cells. The RAS family regulates the MAPK and PI3K-Akt-mTOR pathways4 and influences cell growth, proliferation, and differentiation.6 Thus, the effects of the most potent compound (11b) on the phosphorylation of extracellular signal-regulated kinase (Erk) and protein kinase B/ Akt were evaluated. The epidermal growth factor (EGF) was used to induce the expression of MAPK and Akt.13 Phosphorylation levels of Erk and Akt were determined using Capan-1 cells unstimulated and stimulated with EGF and then treated with compounds 11b or 2 for 1 h (Figure 4). When unstimulated cells were treated with compound 2 (10 μM), no differences were observed in Akt and Erk phosphorylation. When compound 11b (10 μM) was used, Akt phosphorylation was downregulated, but there was no effect on Erk phosphorylation. At 20 μM, compound 2 significantly decreased Akt phosphorylation and slightly decreased Erk phosphorylation. The phosphorylation of both Akt and Erk was dramatically reduced by treatment with compound 11b at the same dose. When EGF-stimulated cells were treated with compound 2, there was no change in the phosphorylation of Akt, but the phosphorylation of Erk decreased in a dosedependent manner. In contrast, treatment with compound 11b significantly downregulated the phosphorylation of both Akt and Erk. Compound 11b Induced Apoptosis and Distribution of Endogenous RAS to Endomembrane in Capan-1 Cells. The cell apoptosis assay indicated that compound 2 (50 μM) induced apoptosis at a rate of 20.50% (Figure 5A,B). In contrast, compound 11b induced apoptosis in 17.95% and 36.40% of the cells at 10 and 25 μM, respectively. 11b induced apoptosis in a dose-dependent manner and was more potent than compound 2 even at a lower dose. Immunofluorescence staining was used to evaluate the RAS distribution in Capan-1 cells (Figure 5C). Compound 11b induced the distribution of endogenous RAS to the endomembrane, and compound 2 was less potent at the same concentration (20 μM).

Figure 4. (A) Phosphorylation levels of Erk and Akt using oncogenic KRAS-dependent Capan-1 cells that were unstimulated or stimulated with EGF (125 ng/mL, 5 min). From top to bottom: phosphorylated Akt on S473 (p-Akt), total level of Akt1 (t-Akt1), phosphorylated Erk on Thr202 and Tyr204 (p-Erk), total level of Erk (t-Erk), and loading control (GAPDH). (B) Gray intensity analysis of the Western blots in which the quantification of p-Erk/t-Erk ± SEM (left) and p-Akt/t-Akt ± SEM (right) was normalized to the EGF-stimulated control (0.1% DMSO). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. EGF, epidermal growth factor.

endomembranes. The lack of cellular potency is the major limitation of current KRAS-PDEδ inhibitors, and 11b showed more potent antitumor activity than pyrazolopyridazinone and quinazolinone inhibitors. Thus, this molecule is a promising lead compound for investigating the biological functions and druggability of KRAS-PDEδ PPI. Further lead optimization studies are in progress.

■

EXPERIMENTAL SECTION

General. All the reagents and solvents were analytically pure and were used as received from the vendors. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). Silica gel chromatography was carried out on 300−400 mesh gel. The anhydrous solvents and reagents were dried by routine protocols. NMR spectra were recorded on a Bruker Avance 600 spectrometer (Bruker Company, Germany) using TMS as an internal standard and CDCl3 or DMSO-d6 as the solvents. The chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. The melting points were measured on a WRR digital melting point apparatus (Shanghai Jingke Instruments, China). The mass spectra were recorded on an API-3000 LC-MS mass spectrometer. The purities of the compounds were determined by

■

CONCLUSION In summary, novel quinazolinone-imidazole KRAS-PDEδ inhibitors were successfully identified by FBDD. All the target compounds showed nanomolar inhibitory activity toward PDEδ, confirming the power of FBDD in drug discovery. Compound 11b had a good combination of binding affinity and antitumor activity. In Capan-1 cells, 11b significantly induced apoptosis, downregulated EGF-induced Erk and Akt phosphorylation, and induced the distribution of endogenous RAS to the 2607

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610

Journal of Medicinal Chemistry

Brief Article

dried, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM/MeOH = 100:1 to 100:5) to give target molecules 9a−9i. General Procedure C: Synthesis of Compounds 10. As described in Scheme S3 in the SI, a mixture of m33−m39 (0.66 mmol) and 20% Pd(OH)2 in MeOH (10 mL) was stirred under a H2 atmosphere at room temperature overnight. Then the mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to afford the crude product without purification. Then, the crude product was dissolved in acetonitrile (15 mL). The solution was combined with m25 (300 mg, 1.32 mmol), K2CO3 (364 mg, 2.64 mmol), and KI (11 mg, 0.07 mmol) and refluxed for 12 h. The mixture was poured into water (50 mL) and extracted with EtOAc (2 × 30 mL). The combined organic phases were dried and then concentrated under reduced pressure. The crude product was purified by column chromatography (DCM/MeOH = 40:1) to afford 10a−10g. General Procedure D: Synthesis of Compounds 11. As described in Scheme S3 in the SI, to a solution of 10g (0.22 g, 0.42 mmol) in DMF (4 mL) was added NaH (60%, 30 mg, 0.63 mmol) slowly at 0 °C over 0.5 h. Then, the brominated alkane (0.84 mmol) in DMF (1 mL) was added dropwise. The reaction mixture was stirred at room temperature for another 2 h and was then poured into water (50 mL) and extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with water (2 × 30 mL) and brine (30 mL), dried, and then concentrated under reduced pressure. The crude product was purified by column chromatography (hexane/EtOAc = 2:1) to afford compounds 11a−11e (45%−63% yield). 2-(2-Fluorophenyl)-3-(2-(4-(1-(pyridin-4-ylmethyl)-1H-benzo-[d]imidazole-2-yl)phenoxy)ethyl)-2,3-dihydroquinazolin-4(1H)-one (8a). Yellow solid, yield 53.1%, mp 154−159 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.45 (d, 2H, J = 4.39 Hz), 7.84 (t, 2H, J = 8.78 Hz), 7.56 (d, 2H, J = 8.38 Hz), 7.39 (d, 1H, J = 7.98 Hz), 7.30 (s, 2H), 7.17− 7.24 (m, 5H), 7.06−7.08 (m, 1H), 6.94−6.97 (m, 4H), 6.64−6.68 (m, 2H), 6.31 (s, 1H), 5.56 (s, 2H), 4.17−4.20 (m, 1H), 4.09−4.14 (m, 2H), 3.24−3.28 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 164.65, 162.22, 161.17, 160.59, 155.08, 151.91, 147.90, 147.88, 144.55, 137.72, 135.38, 132.41, 132.36, 132.29, 129.58, 129.49, 129.31, 129.23, 126.36, 124.49, 124.18, 124.12, 123.03, 121.03, 119.18, 117.92, 117.78, 116.55, 116.29, 116.16, 112.60, 67.72, 67.61, 48.47, 45.53. MS (ESI+): m/z calcd for C35H28FN5O2 569.22, found 570.69 [M + H]+. 2-(2-Fluorophenyl)-3-(4-(2-(2-phenyl-1H-benzo[d]imidazol-1-yl)ethoxy)phenyl)-2,3-dihydroquinazolin-4(1H)-one (9a). Pale solid, yield 33.6%, mp 185−191 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.97 (d, 1H, J = 8.6 Hz), 7.89 (d, 2H, J = 7.1 Hz), 7.77 (d, 1H, J = 7.7 Hz), 7.70 (d, 1H, J = 8.1 Hz), 7.64−7.65 (m, 3H), 7.40−7.50 (m, 4H), 7.25−7.31 (m, 2H), 7.08−7.12 (m, 2H), 7.06 (d, 2H, J = 9.1 Hz), 6.71−6.74 (m, 2H), 6.68 (d, 2H, J = 8.7 Hz), 6.40 (s, 1H), 4.75 (t, 2H, J = 4.8 Hz), 4.29 (t, 2H, J = 4.8 Hz). 13C NMR (150 MHz, DMSO-d6) δ 163.53, 161.33, 159.69, 157.18, 153.61, 147.75, 135.13, 135.01, 134.64, 132.52, 131.97, 131.92, 131.29, 130.27, 129.67, 129.44, 129.10, 128.57, 128.48, 125.77, 125.65, 118.80, 117.96, 117.10, 116.96, 115.86, 115.82, 115.53, 114.00, 69.18, 66.93, 45.82. MS (ESI+): m/z calcd for C35H27FN4O2 554.21, found 555.58 [M + H]+. 6-Fluoro-2-(2-fluorophenyl)-3-(1-(3-(2-phenyl-1H-imidazol-1-yl)propyl)piperidin-4-yl)-2,3-dihydroquinazolin-4(1H)-one (10g). White solid, yield 37.2%, mp 172−174 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.57−7.58 (m, 2H), 7.33−7.41 (m, 5H), 7.26−7.29 (m, 3H), 7.05−7.15 (m, 3H), 6.97 (d, 1H, J = 1.0 Hz), 6.70 (dd, 1H, J = 4.6, 8.8 Hz), 6.07 (d, 1H, J = 3.4 Hz), 4.27−4.32 (m, 1H), 4.03 (t, 2H, J = 7.1 Hz), 2.73 (d, 1H, J = 10.7 Hz), 2.57 (d, 1H, J = 10.7 Hz), 2.05−2.13 (m, 2H), 1.85 (t, 1H, J = 10.5 Hz), 1.68−1.77 (m, 4H), 1.52−1.54 (m, 1H), 1.26 (d, 1H, J = 12.0 Hz), 1.05−1.12 (m, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 161.62, 159.95, 158.33, 156.18, 154.63, 147.07, 142.18, 131.68, 130.84, 130.79, 129.45, 129.36, 129.01, 128.76, 128.66, 128.38, 127.75, 124.65, 121.84, 121.21, 121.05, 117.07, 117.03, 116.44, 116.30, 113.25, 113.09, 61.06, 54.12, 53.10, 52.63, 51.92, 44.47, 29.80, 29.73, 28.17. MS (ESI+): m/z calcd for C31H31F2N5O 527.25, found 528.77 [M + H]+. 1-(Cyclopropylmethyl)-6-fluoro-2-(2-fluorophenyl)-3-(1-(3-(2phenyl-1H-imidazol-1-yl)propyl)piperidin-4-yl)-2,3-dihydro-quina-

Figure 5. (A) Apoptosis in the Capan-1 cell line induced by 48 h of treatment with compound 2 (50 μM) and compound 11b (10 and 25 μM). (B) Immunostaining of Capan-1 with antipan-RAS (red) and anti-PDEδ (green) after treatment with various compounds for 2 h (2, 20 μM; 11b, 20 μM). The vehicle control for the above experiments was 0.1% DMSO. (***) p < 0.001. HPLC (Agilent 1260), and all final compounds exhibited purities greater than 95%. General Procedure A: Synthesis of Compounds 8. As described in Scheme S1 in the SI, intermediate m3 (190 mg, 0.52 mmol) and 10% Pd-C were combined in MeOH (10 mL) under a H2 atmosphere and stirred at room temperature overnight. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to afford crude product m4, which was used directly in the next step without purification. Crude m4 was dissolved in DMF (5 mL) and treated with HBTU (300 mg, 0.78 mmol), TEA (160 mg, 1.56 mmol), and the substituted 2-aminobenzoic acid (0.52 mmol) at room temperature for 12 h. The mixture was poured into water (100 mL) to precipitate the product as a white solid. The solid was isolated by filtration, dissolved in acetic acid (5 mL), and then treated with 2fluorobenzaldehyde (130 mg, 1.04 mmol) under reflux for 4 h. The mixture was poured into water (50 mL) and extracted with EtOAc (3 × 30 mL). The combined organic phases were dried and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM/MeOH = 100:2) to give target compounds 8a−8g. General Procedure B: Synthesis of Compounds 9. As described in Scheme S2 in the SI, a solution of m12 or m13 (0.6 mmol), the corresponding intermediate from m19−m23 (0.6 mmol), and triphenylphosphine (0.32 g, 1.2 mmol) were stirred in dry toluene (10 mL) at 0 °C under a nitrogen atmosphere. TMAD was added to this mixture (0.21 g, 1.2 mmol) in dry toluene (5 mL) over a period of 5 min. The resulting suspension was heated for 48 h at 65 °C. The solvent was evaporated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with saturated sodium bicarbonate solution (2 × 30 mL). The aqueous phase was reextracted with EtOAc (30 mL). The combined organic phases were 2608

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610

Journal of Medicinal Chemistry

Brief Article

zolin-4(1H)-one (11b). White solid, yield 50.8%, mp 135−139 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.57 (d, 2H, J = 6.9 Hz), 7.50 (dd, 1H, J = 3.1, 8.9 Hz), 7.30−7.38 (m, 5H), 7.24 (dd, 1H, J = 8.2, 10.2 Hz), 7.16−7.19 (m, 1H), 7.03−7.05 (m, 2H), 6.97 (s, 1H), 6.90−6.92 (m, 1H), 6.13 (s, 1H), 4.27−4.31 (m, 1H), 3.99−4.06 (m, 2H), 3.42−3.45 (m, 1H), 3.15−3.19 (m, 1H), 2.79 (d, 1H, J = 9.1 Hz), 2.61 (d, 1H, J = 9.1 Hz), 2.11−2.16 (m, 2H), 1.84−1.89 (m, 2H), 1.73−1.78 (m, 3H), 1.59 (d, 1H, J = 8.4 Hz), 1.24−1.28 (m, 1H), 1.09−1.18 (m, 2H), 0.44−0.58 (m, 2H), 0.27−0.30 (m, 1H), 0.16−0.19 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 161.37, 160.19, 158.56, 157.07, 155.50, 146.98, 142.02, 131.54, 130.86, 130.80, 128.96, 128.74, 128.64, 128.29, 127.99, 127.85, 127.76, 124.59, 121.89, 121.47, 121.42, 120.92, 120.76, 119.76, 119.71, 116.43, 116.29, 113.48, 113.32, 66.44, 56.27, 54.24, 53.00, 52.75, 52.22, 44.57, 29.72, 29.59, 29.42, 28.11. MS (ESI+): m/z calcd for [M + H]+ C35H37F2N5O: 582.30, found: 582.55.

■

Bastiaens, P. I. An Acylation Cycle Regulates Localization and Activity of Palmitoylated Ras Isoforms. Science 2005, 307, 1746−1752. (4) Malumbres, M.; Barbacid, M. RAS Oncogenes: the First 30 Years. Nat. Rev. Cancer 2003, 3, 459−465. (5) Maurer, T.; Garrenton, L. S.; Oh, A.; Pitts, K.; Anderson, D. J.; Skelton, N. J.; Fauber, B. P.; Pan, B.; Malek, S.; Stokoe, D.; Ludlam, M. J.; Bowman, K. K.; Wu, J.; Giannetti, A. M.; Starovasnik, M. A.; Mellman, I.; Jackson, P. K.; Rudolph, J.; Wang, W.; Fang, G. SmallMolecule Ligands Bind to A Distinct Pocket in Ras and Inhibit SOSMediated Nucleotide Exchange Activity. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 5299−5304. (6) Berndt, N.; Hamilton, A. D.; Sebti, S. M. Targeting Protein Prenylation for Cancer Therapy. Nat. Rev. Cancer 2011, 11, 775−791. (7) Ismail, S. A.; Chen, Y. X.; Rusinova, A.; Chandra, A.; Bierbaum, M.; Gremer, L.; Triola, G.; Waldmann, H.; Bastiaens, P. I.; Wittinghofer, A. Arl2-GTP and Arl3-GTP Regulate a GDI-like Transport System for Farnesylated Cargo. Nat. Chem. Biol. 2011, 7, 942−949. (8) Bournet, B.; Buscail, C.; Muscari, F.; Cordelier, P.; Buscail, L. Targeting KRAS for Diagnosis, Prognosis, and Treatment of Pancreatic Cancer: Hopes and Realities. Eur. J. Cancer 2016, 54, 75−83. (9) Sun, Q.; Burke, J. P.; Phan, J.; Burns, M. C.; Olejniczak, E. T.; Waterson, A. G.; Lee, T.; Rossanese, O. W.; Fesik, S. W. Discovery of Small Molecules that Bind to K-Ras and Inhibit Sos-Mediated Activation. Angew. Chem., Int. Ed. 2012, 51, 6140−6143. (10) Zimmermann, G.; Papke, B.; Ismail, S.; Vartak, N.; Chandra, A.; Hoffmann, M.; Hahn, S. A.; Triola, G.; Wittinghofer, A.; Bastiaens, P. I.; Waldmann, H. Small Molecule Inhibition of the KRAS-PDEdelta Interaction Impairs Oncogenic KRAS Signalling. Nature 2013, 497, 638−642. (11) Papke, B.; Murarka, S.; Vogel, H. A.; Martin-Gago, P.; Kovacevic, M.; Truxius, D. C.; Fansa, E. K.; Ismail, S.; Zimmermann, G.; Heinelt, K.; Schultz-Fademrecht, C.; Al Saabi, A.; Baumann, M.; Nussbaumer, P.; Wittinghofer, A.; Waldmann, H.; Bastiaens, P. I. Identification of Pyrazolopyridazinones as PDEdelta Inhibitors. Nat. Commun. 2016, 7, 11360. (12) Zimmermann, G.; Schultz-Fademrecht, C.; Kuchler, P.; Murarka, S.; Ismail, S.; Triola, G.; Nussbaumer, P.; Wittinghofer, A.; Waldmann, H. Structure Guided Design and Kinetic Analysis of Highly Potent Benzimidazole Inhibitors Targeting the PDEdelta Prenyl Binding Site. J. Med. Chem. 2014, 57, 5435−5448. (13) Jiang, Y.; Zhuang, C.; Chen, L.; Lu, J.; Dong, G.; Miao, Z.; Zhang, W.; Li, J.; Sheng, C. Structural Biology-Inspired Discovery of Novel KRAS-PDEdelta Inhibitors. J. Med. Chem. 2017, 60, 9400− 9406. (14) Murarka, S.; Martin-Gago, P.; Schultz-Fademrecht, C.; Al Saabi, A.; Baumann, M.; Fansa, E. K.; Ismail, S.; Nussbaumer, P.; Wittinghofer, A.; Waldmann, H. Development of Pyridazinone Chemotypes Targeting the PDEdelta Prenyl Binding Site. Chem. Eur. J. 2017, 23, 6083−6093. (15) Martin-Gago, P.; Fansa, E. K.; Wittinghofer, A.; Waldmann, H. Structure-Based Development of PDEdelta Inhibitors. Biol. Chem. 2017, 398, 535−545. (16) Martin-Gago, P.; Fansa, E. K.; Klein, C. H.; Murarka, S.; Janning, P.; Schurmann, M.; Metz, M.; Ismail, S.; Schultz-Fademrecht, C.; Baumann, M.; Bastiaens, P. I.; Wittinghofer, A.; Waldmann, H. A PDE6delta-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2. Angew. Chem., Int. Ed. 2017, 56, 2423−2428. (17) Nikolova, S.; Guenther, A.; Savai, R.; Weissmann, N.; Ghofrani, H. A.; Konigshoff, M.; Eickelberg, O.; Klepetko, W.; Voswinckel, R.; Seeger, W.; Grimminger, F.; Schermuly, R. T.; Pullamsetti, S. S. Phosphodiesterase 6 Subunits are Expressed and Altered in Idiopathic Pulmonary Fibrosis. Respir. Res. 2010, 11, 146. (18) Chandra, A.; Grecco, H. E.; Pisupati, V.; Perera, D.; Cassidy, L.; Skoulidis, F.; Ismail, S. A.; Hedberg, C.; Hanzal-Bayer, M.; Venkitaraman, A. R.; Wittinghofer, A.; Bastiaens, P. I. The GDI-Like

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00057. Synthetic protocols, detailed experimental procedures, QAAR graph, LE calculation, and 1H and 13C NMR spectra of target compounds (PDF) SMILES molecular formula strings (CSV)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: 86-21-81871239. E-mail: [email protected]. ORCID

Chunlin Zhuang: 0000-0002-0569-5708 Chunquan Sheng: 0000-0001-9489-804X Author Contributions †

L.C., C.Z., and J.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grants 21738002 and 81725020 to C.S.) and the “Young Elite Scientists Sponsorship” from the China Association for Science and Technology (C.Z.).

■

ABBREVIATIONS USED FBDD, fragment-based drug design; PPI, protein−protein interaction; SBVS, structure-based virtual screening; LE, ligand efficiency; Erk, extracellular signal-regulated kinase; EGF, epidermal growth factor; HBTU, O-benzotriazole-N,N,N′,N′tetra-methyl-uronium-hexafluorophosphate; TMAD, N,N,N′,N′-tetramethyldiazene-1,2-dicarboxamide; QAAR, quantitative activity−activity relationships

■

REFERENCES

(1) Brunsveld, L.; Kuhlmann, J.; Alexandrov, K.; Wittinghofer, A.; Goody, R. S.; Waldmann, H. Lipidated Ras and Rab Peptides and Proteins–Synthesis, Structure, and Function. Angew. Chem., Int. Ed. 2006, 45, 6622−6646. (2) Gelb, M. H.; Brunsveld, L.; Hrycyna, C. A.; Michaelis, S.; Tamanoi, F.; Van Voorhis, W. C.; Waldmann, H. Therapeutic Intervention Based on Protein Prenylation and Associated Modifications. Nat. Chem. Biol. 2006, 2, 518−528. (3) Rocks, O.; Peyker, A.; Kahms, M.; Verveer, P. J.; Koerner, C.; Lumbierres, M.; Kuhlmann, J.; Waldmann, H.; Wittinghofer, A.; 2609

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610

Journal of Medicinal Chemistry

Brief Article

Solubilizing Factor PDEdelta Sustains the Spatial Organization and Signalling of Ras Family Proteins. Nat. Cell Biol. 2012, 14, 148−158. (19) Nancy, V.; Callebaut, I.; El Marjou, A.; de Gunzburg, J. The Delta Subunit of Retinal Rod cGMP Phosphodiesterase Regulates the Membrane Association of Ras and Rap GTPases. J. Biol. Chem. 2002, 277, 15076−15084. (20) Zhang, H.; Liu, X. H.; Zhang, K.; Chen, C. K.; Frederick, J. M.; Prestwich, G. D.; Baehr, W. Photoreceptor cGMP Phosphodiesterase Delta Subunit (PDEdelta) Functions as a Prenyl-Binding Protein. J. Biol. Chem. 2004, 279, 407−413. (21) Chen, Y. X.; Koch, S.; Uhlenbrock, K.; Weise, K.; Das, D.; Gremer, L.; Brunsveld, L.; Wittinghofer, A.; Winter, R.; Triola, G.; Waldmann, H. Synthesis of the Rheb and K-Ras4B GTPases. Angew. Chem., Int. Ed. 2010, 49, 6090−6095. (22) Schmick, M.; Vartak, N.; Papke, B.; Kovacevic, M.; Truxius, D. C.; Rossmannek, L.; Bastiaens, P. I. KRas Localizes to the Plasma Membrane by Spatial Cycles of Solubilization, Trapping and Vesicular Transport. Cell 2014, 157, 459−471. (23) Erlanson, D. A.; Fesik, S. W.; Hubbard, R. E.; Jahnke, W.; Jhoti, H. Twenty Years on: the Impact of Fragments on Drug Discovery. Nat. Rev. Drug Discovery 2016, 15, 605−619. (24) Speck-Planche, A.; Cordeiro, M. N. D. S. Fragment-Based in Silico Modeling of Multi-Target Inhibitors against Breast CancerRelated Proteins. Mol. Diversity 2017, 21, 511−523. (25) Speck-Planche, A.; Dias Soeiro Cordeiro, M. N. Speeding up Early Drug Discovery in Antiviral Research: A Fragment-Based in Silico Approach for the Design of Virtual Anti-Hepatitis C Leads. ACS Comb. Sci. 2017, 19, 501−512. (26) Speck-Planche, A.; Cordeiro, M. N. D. S. De Novo Computational Design of Compounds Virtually Displaying Potent Antibacterial Activity and Desirable in Vitro ADMET Profiles. Med. Chem. Res. 2017, 26, 2345−2356. (27) Kleandrova, V. V.; Speck Planche, A. Multitasking Model for Computer-Aided Design and Virtual Screening of Compounds with High Anti-HIV Activity and Desirable ADMET Properties. In MultiScale Approaches in Drug Discovery: From Empirical Knowledge to in Silico Experiments and Back, 1st ed.; Speck-Planche, A., Ed.; Elsevier: Oxford, UK, 2017; Chapter 3, pp 55−81. (28) Sheng, C.; Zhang, W. Fragment Informatics and Computational Fragment-Based Drug Design: An Overview and Update. Med. Res. Rev. 2013, 33, 554−598. (29) Schultes, S.; de Graaf, C.; Haaksma, E. E.J.; de Esch, I. J. P.; Leurs, R.; Krämer, O. Ligand Efficiency as A Guide in Fragment Hit Selection and Optimization. Drug Discovery Today: Technol. 2010, 7, e157−e162. (30) Sangion, A.; Gramatica, P. Ecotoxicity Interspecies QAAR Models from Daphnia Toxicity of Pharmaceuticals and Personal Care Products. SAR QSAR Environ. Res. 2016, 27, 781−798.

2610

DOI: 10.1021/acs.jmedchem.8b00057 J. Med. Chem. 2018, 61, 2604−2610