Hydroxamic Acid Hybrids Targeting Both Histone

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Novel #-Carboline/Hydroxamic Acid Hybrids Targeting Both Histone Deacetylase and DNA Display High Anticancer Activity via Regulation of the P53 Signaling Pathway Yong Ling, Chenjun Xu, Lin Luo, Jingyi Cao, Jiao Feng, Yu Xue, Qing Zhu, Caoyun Ju, Fengzhi Li, Yihua Zhang, Yanan Zhang, and Xiang Ling J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01052 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 12, 2015

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Journal of Medicinal Chemistry

Novel β-Carboline/Hydroxamic Acid Hybrids Targeting Both Histone Deacetylase and DNA Display High Anticancer Activity via Regulation of the P53 Signaling Pathway Yong Ling,†,‡,§ ChenjunXu,†,§ Lin Luo,†,§ Jingyi Cao,†,§ Jiao Feng,† Yu Xue,† Qing Zhu,†,§ Caoyun Ju,†,§ Fengzhi Li,⊥ Yihua Zhang,‡ Yanan Zhang,†,§,* and Xiang Ling†,§,* †

School of Pharmacy, Nantong University, Nantong 226001, P. R. China

‡

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009,

PR China §Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target; Nantong University; Nantong 226001, P. R. China ⊥

Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New

York, USA KEYWORDS: β-Carboline alkaloids; Antitumor agents; Histone deacetylases inhibitors; DNA damage; p53 signaling pathway

ACS Paragon Plus Environment

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ABSTRACT: A novel series of hybrids from β-carboline and hydroxamic acid were designed and synthesized. Several compounds (5m, 11b-d, and 11h) not only exerted significant antiproliferation activity against four human colorectal cancer (CRC) cell lines, but also showed histone deacetylase inhibitory effects in vitro. The most potent compound 11c exhibited anticancer potencies seven-fold higher than SAHA. 11c triggered more significant cancer cell apoptosis than SAHA by cleavage of both PARP and caspase-3 in a dose dependent manner. Furthermore, 11c simultaneously increased the acetylation of histone H3 and α-tubulin, enhanced DNA damage markers histone H2AX phosphorylation and p-p53 (Ser15) expression, and activated p53 signaling pathway in HCT116 cells. Finally, 11c showed low acute toxicity to mice and inhibited the growth of implanted human CRC in mice more potently than SAHA. Together, 11c possessed potent antitumor activity and may be a promising candidate for the potential treatment of human CRC.

Introduction Colorectal cancer (CRC) is currently the third most common cancer worldwide and the second leading cause of cancer death in both men and women in the United States. Despite the advance in the diagnosis and treatment of CRC that has resulted in significant improvement on management of this malignancy, significant challenges remain in the treatment of CRC. In particular, many patients with advanced and metastatic tumors still succumb to the disease.1,2 Alternative and novel molecular-targeted therapeutic approaches are urgently needed. Traditionally, agents designed to hit single biological targets have been the main approach in therapeutics development. However, it becomes apparent that these 'magic bullet' drugs often have limited clinical utility and might not be the answer to treating complex illnesses such as cancer. This is because tumors normally harbor multiple misregulated growth and survival


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pathways, which can easily evolve during the drug treatment.3 Therefore, single drugs with multiple biological targets have increasingly attracted interest in cancer treatment. Natural products have been a rich source of compounds for drug discovery and the majority of anticancer drugs currently used in clinical are derived from natural product scaffolds such as paclitaxel and vincaleukoblastine. β-Carbolines are a class of naturally occurring alkaloids that contain a planar tricyclic pyrido-[3,4-b]indole ring structure, and are widely distributed in plants, marine creatures, insects and mammals, as well as human tissues and bodily fluids.4 The βcarboline alkaloids such as harmine and its derivatives have shown a variety of pharmacological activities, particularly antitumor effects (Figure 1).5,6 These compounds have been shown to inhibit cancer cell growth, and lead to apoptosis through multiple mechanisms such as inhibition of DNA topoisomerases I and II, CDK, PLK, and MAO.7-10 In particular, β-carbolines can intercalate with DNA, alter DNA replication fidelity, and influence enzymatic activities in DNA repair processes due to the presence of polycyclic aromatic planar pharmacophore, which is capable of stacking between DNA base pairs.11,12 In fact, several β-carbolines including harmane, norharman, and β-carboline–benzimidazole conjugates have been reported to intercalate into DNA leading to altered DNA replication fidelity or influencing enzymatic activities in DNArepair processes.11-13 Inhibition of histone deacetylases (HDACs) has emerged as a promising therapeutic approach to the treatment of cancer. It has been shown that HDAC inhibitors (HDACi) significantly suppress cancer cell proliferation, angiogenesis, and metastasis, and induce apoptosis through multiple mechanisms, including changes in gene expression and alterations of both histone and non-histone proteins such as p53 and heat shock protein-90.14-16 Several HDAC inhibitors have been approved to date. Despite promising results in the treatment of cutaneous T-cell lymphoma,


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the two US FDA approved HDACi’s, Vorinostat (SAHA)16 and romidepsin,17 have not been proven effective in clinical trials involving solid tumors.19-21 Other HDAC inhibitors that have recently been approved include belinostat (PXD101), panobinostat (LBH589), and chidamide (Epidaza), for the treatment of PTCL or multiple myeloma,[22-24] but their effects on solid tumors have not been examined. Interestingly, recent results from in vitro and in vivo studies have demonstrated that HDACi possess synergistic or additive antitumor effects in combination with several classes of anticancer drugs.25-27 For instance, several reports suggest a synergy between HDACi and DNA damage reagents cisplatin, topotecan or etoposide.3,28-30 Therefore, given the DNA damage effects of β-carbolines, the combination of histone deacetylase inhibitors and β-carboline alkaloids may represent a promising strategy to achieve increased anticancer efficacy (Figure 1). In this first proof-of-principle study, we aimed to simultaneously target multiple pathways by integrating the key structural elements of β-carboline alkaloids and HDACi into one single structure to form hybrids.


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Figure 1. Chemical structures of representative β-carboline derivatives, HDAC inhibitor SAHA, and designed β-carboline/hydroxamic acid hybrids 5a-o and 11a−j. HDAC inhibitors generally consist of three domains: a zinc-binding group (ZBG) that chelates with the zinc ion to form a complex; a cap group, generally a hydrophobic and aromatic group, linked to the ZBG that fits into the tubular pocket; a saturated or unsaturated linker domain, composed of linear or cyclic structures that connect the ZBG and the cap group.31 It has been demonstrated the cap group can be replaced with other moieties to generate HDACis with dual or multiple targets and several of these have displayed potential in preclinical studies.32,33 In fact, Kozikowski et al in 2012 reported a series of HDAC6 inhibitors based on the β- or γtetrahydrocarboline and SAHA combination that showed improved isoform-selectivity.34 In this study, we used β-carboline aromatic heterocyclic ring as a cap group, and designed novel hybrids by introducing the ZBG group (the hydroxamic acid fragment) into the carboxyl or uramido group of β-carboline via alkyl linkers (Figure 1). We herein report the synthesis and biological evaluation of a series of novel β-carboline/hydroxamic acid hybrids (5a-o, 11a−j), as well as the investigation on their anti-tumor mechanisms in multiple colon cancer cell lines.

Results and Discussion Chemistry. The synthetic route to compounds 5a-o is depicted in Scheme 1. The substituted β-carbolines 3a-c were prepared in a two-step sequence. First, the starting L-tryptophan 1 was converted to 1-substituted-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid 2a-c in a PictetSpengler reaction with the treatment of differently substituted aldehydes. Intermediates 2a-c were then oxidized by KMnO4 in DMF to afford compounds 3a-c, which were then reacted with


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NH2(CH2)nCOOMe in the presence of ethyl chloroformate to produce esters 4a-o. Finally, intermediates 4a-o were treated with NH2OK to give the target compounds 5a-o. Compounds 11a−j were obtained according to the procedures described in Scheme 2. Acid 3c was esterified to give methyl ester 6, which was then reacted with iodomethane in the presence of NaH to afford the N-methyl compound 7. The intermediate 6 or 7 was treated with hydrazine hydrate to form hydrazide derivatives 8a-b. The hydrazide group of 8a-b was converted to the acylazide group in the presence of NaNO2 to provide 9a-b, which were then reacted with NH2(CH2)nCOOMe to give 10a-j via Curtius rearrangement. Finally, ureas 10a-j were converted to hydroxamic acids 11a-j with the treatment of NH2OK in MeOH. The final products 5a-o and 11a-j were purified by column chromatography, and their structures were characterized by 1H NMR, MS, and HRMS.

Scheme 1. Reagents and conditions: (a) H+ or OH-, RCHO, reflux, 2-4 h, 81-86%; (b) KMnO4, DMF, reflux, 6 h, 60-70%; (c) ethyl chloroformate, N-methylmorpholine, NH2(CH2)nCOOMe, rt, 5-8h, 71-83%; (d) NH2OK, MeOH, rt, 10-15h, 68−77%.


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Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 0 °C ,1h, and then reflux 6 h, 90%; (b) CH3I, NaH, CH2Cl2, rt, 2-4 h, 81-86%; (c) NH2-NH2•H2O, CH3OH, 0 °C, 1h, 90-95%; (d) NaNO2, CH3OH, 0 °C, 6 h, 73-80%; (e) NH2(CH2)nCOOMe, toluene, reflux, 1h, 77-85%; (f) NH2OK, MeOH, 63-75%.

100 80

Inhibition (%)

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HCT116 SW620 LOVO

60 40 20 0

Concentrations (25 µM)


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Figure 2. Inhibition of cell proliferation (%) of target compounds 5a-o against HCT116, SW620, and LOVO cell lines after incubation for 48 h at a concentration of 25 µM. Data are expressed as means ± SD of each compound from three separate measurements. In vitro biological evaluations. Compounds 5a-o were preliminarily screened at 25 µM for their cancer cell growth inhibitory activity against three human CRC cell lines (HCT116, SW620, and LOVO) in the MTT assay. These compounds were designed to evaluate the substitutions-activity relationships at the C1 position of the β-carbolines. SAHA, the FDA approved HDAC inhibitor, was used as a reference compound. As shown in Figure 2, SAHA significantly inhibited the proliferation of the cells. Interestingly, compounds 5a-e with R = H as in norhamane and 5f-j with R = Me as in harmane all showed limted antiproliferative activities. β-Carbolines (5k-o) with a p-methoxyphenyl group at the C1 positions as present in the previously reported β-carboline-benzimidazole conjugates markedly inhibited all three CRC cells at 25 µM, showing comparable or even greater inhibition than SAHA of the growth of all three CRC cells. These results indicate that a p-methoxyphenyl group is preferred for activity at the C1 position. Table 1. Compounds 5a-o and 11a-j and their HDAC inhibition nuclear extract Compd.

R

n

ClogP

SAHA

-

-

0.86

harmine

-

-

5a

H

1

-0.23

6.17 ± 0.73

5b

H

2

-0.06

3.23 ± 0.41

5c

H

3

0.16

1.56 ± 0.34

(IC50a, µM) 0.56 ± 0.06 >10


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a

5d

H

4

0.46

1.43 ± 0.23

5e

H

5

0.69

1.81 ± 0.30

5f

Me

1

0.23

5.37 ± 0.75

5g

Me

2

0.40

2.45 ± 0.36

5h

Me

3

0.62

1.21 ± 0.25

5i

Me

4

0.92

1.26 ± 0.18

5j

Me

5

1.15

1.43 ± 0.22

5k

p-MeOPh

1

1.77

3.02 ± 0.59

5l

p-MeOPh

2

1.94

1.31 ± 0.35

5m

p-MeOPh

3

2.15

0.67 ± 0.08

5n

p-MeOPh

4

2.45

0.81 ± 0.09

5o

p-MeOPh

5

2.68

1.17 ± 0.22

11a

H

1

2.87

2.24 ± 0.35

11b

H

2

3.03

0.43 ± 0.06

11c

H

3

3.25

0.27 ± 0.05

11d

H

4

3.55

0.35 ± 0.05

11e

H

5

3.78

0.82 ± 0.13

11f

Me

1

3.14

3.16 ± 0.37

11g

Me

2

3.30

1.33 ± 0.20

11h

Me

3

3.52

0.51 ± 0.06

11i

Me

4

3.82

0.86 ± 0.11

11j

Me

5

4.05

1.05 ± 0.16

The data are expressed as the mean ± SD of three independent experiments data.

Compounds 5a-o were also assayed for their inhibitory activity against HeLa cell nuclear extract which is a rich source of HDACs.35 The calculated lipophilicity (ClogP) and IC50 values of compounds 5a-o against HDAC are listed in Table 2. Compounds with one to two methylene


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units as the linker showed weak enzymatic inhibition (5a, 5b, 5f, 5g, 5k, and 5l). The HDAC inhibitory activities increased for compounds with linker bearing three to five methylene units. This trend is the same in all three series (5a-e, 5f-j, and 5k-o), independent of the C1substitution. This clearly shows that the linker length is highly important for HDAC activity. However, the substitutions on β-carboline C1-positions also played a role in the HDAC inhibitory activities, albeit less pronounced. Compounds 5k–o with the p-methoxyphenyl group were more potent than the corresponding compounds in the R = H (5a–e) or R = Me (5f–j) series. It should be noted that the ClogP showed the same trend across the three series (e.g. 5c, 5h and 5m are 0.16, 0.62, and 2.15, respectively). Among all the compounds, 5m-n showed similar IC50 values as SAHA in the nuclear extract. These results suggest that a three (n = 3) or four (n = 4) carbon linker is preferred for HDAC activity, similar to the antiproliferation activities of these compounds. Compounds 5k-o were then further tested and the dose-response curves against four colon cancer cell lines (HCT116, SW620, SW480, and LOVO) were obtained. In these cell lines, these compounds all inhibited cell proliferation at significantly greater potency than the β-carboline compound harmine, and were comparable to SAHA (Table 2). Similarly, a clear dependency of the potency on the linker length is present during this series, where the antiproliferative potency initially increased and then deceased with the elongation of the linker. Compounds 5m-n with 3 or 4 carbon linkers (n = 3 or 4) were the more potent compounds in the series. Table 2. IC50 values of compounds 5k-o and 11a-j against four CRC cell linesa In vitro antiproliferative activity (IC50a, µM) Compd. HCT116

SW620

LOVO


SW480

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SAHA

5.53 ± 0.68

4.32 ± 0.45

6.75 ± 0.58

3.97 ± 0.52

harmine

46.7 ± 3.92

42.8 ± 4.31

53.2 ± 4.35

NDb

6

63.4 ± 6.75

71.7 ± 6.82

76.5 ± 8.16

ND

5k

>12.5

6.92 ± 0.59

>12.5

10.5 ± 0.93

5l

8.64 ± 0.77

6.73 ± 0.71

9.01 ± 0.84

11.2 ± 0.85

5m

2.72 ± 0.36

4.46 ± 0.52

3.77 ± 0.39

4.65 ± 0.56

5n

3.35 ± 0.42

2.53 ± 0.35

5.28 ± 0.48

4.43 ± 0.40

5o

7.16 ± 0.73

8.06 ± 0.68

7.90 ± 0.65

9.83 ± 0.86

11a

8.52 ± 0.90

7.12 ± 0.63

9.36 ± 1.02

10.1 ± 0.94

11b

3.08 ± 0.41

2.69 ± 0.30

3.61 ± 0.45

3.11 ± 0.33

11c

0.83 ± 0.18

0.94 ± 0.15

1.63 ± 0.31

1.16 ± 0.28

11d

0.99 ± 0.26

2.02 ± 0.29

2.17 ± 0.26

1.73 ± 0.30

11e

4.84 ± 0.45

6.25 ± 0.70

7.34 ± 0.65

5.09 ± 0.60

11f

9.30 ± 0.73

8.36 ± 0.75

>12.5

9.81 ± 1.03

11g

4.27 ± 0.50

5.02 ± 0.49

5.66 ± 0.60

4.40 ± 0.38

11h

1.33 ± 0.17

1.93 ± 0.22

3.02 ± 0.35

3.15 ± 0.41

11i

3.87 ± 0.43

5.09 ± 0.48

6.24 ± 0.66

5.80 ± 0.47

11j

8.76 ± 0.91

9.53 ± 0.80

>12.5

>12.5

a

The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data are expressed as the mean ± SD of three independent experiments data. bND: not detected. We next introduced structural modifications at other positions of the β-carboline ring to further investigate the SAR. Given the importance of the p-methoxyphenyl group at the C1 position, it was retained in these new analogs 11a-j. The SAR studies focused on the C3 and N9 positions. First, the amide group at the C3 position was replaced with a urea group to afford hybrids 11a-e (Scheme 2). The corresponding N9 methylated compounds (11f-j) were also prepared. The results were shown in Table 2. Similar to the 5 series, most of compounds 11a-j exhibited good


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antitumor activities that were significantly greater than the parent compound 6 and harmine. The antiproliferation activities of 11a-e containing urea group were slightly higher than those of 5k-o with amide group at the C3 positions of β-carboline in 4 CRC cells, suggesting the urea group can be tolerated for antiproliferation activity. Compounds 11c-d and 11h (IC50s = 0.83~3.15 µM), which have linkers comprised of 3-4 carbon units, showed antiproliferative activity greater than SAHA against all cancer cells in the series, confirming the linker length effect observed in the 5 series. In particular, compound 11c (IC50 = 0.83 µM) displayed the highest antiproliferative activity in all compounds, with IC50 value nearly seven-fold lower than SAHA (IC50 = 5.53 µM) in HCT116 cells. The methylation of the N9 position seems to have little impact on the potency as the two series (11a-e vs. 11f-j) showed similar potency against both the CRC cells and nuclear extract. The IC50 values of compounds 11a-j against HDAC in HeLa cell nuclear extract are listed in Table 1. Similar to the antiproliferative activity, active compounds 11b-e and 11h displayed significant HDAC inhibitory activities with IC50 values of 0.27-0.51 µM in Hela nuclear extract, comparable or lower than SAHA (IC50 = 0.56 µM). Interestingly, while 5m (IC50= 0.67 µM) and 11h (IC50 = 0.51 µM) showed HDAC inhibition potencies similar to SAHA, the cell growth inhibitions induced by these two compounds are 2-4 times stronger than SAHA in HCT116 cells. Furthermore, the HDAC inhibition induced by 11c (IC50 = 0.27 µM) was only two times higher than the HDAC inhibition induced by SAHA (IC50 = 0.56 µM), but the growth inhibition of 11c was nearly 7 times greater than that induced by SAHA in HCT116 cells. These results suggest that the cytotoxicity induced by these β-carboline/hydroxamic acid hybrids may be the result of both the active β-carboline ring and HDAC inhibition.


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Table 3. The HDAC inhibitory activities of compounds 5m, 11b-d, and 11h. IC50a (µM) Compd.

a

HDAC1

HDAC6

HDAC8

SAHA

0.19 ± 0.03

0.076 ± 0.01

0.33 ± 0.05

5m

0.13 ± 0.02

0.061 ± 0.008

0.39 ± 0.05

11b

0.11 ± 0.02

0.36 ± 0.05

0.43 ± 0.05

11c

0.026 ± 0.004

0.034 ± 0.005

0.16 ± 0.03

11d

0.079 ± 0.008

0.093 ± 0.01

0.20 ± 0.03

11h

0.24 ± 0.03

0.15 ± 0.02

0.57 ± 0.07

Data are expressed as mean ± SD of three independent experiments.

Encouraged by the inhibitory activity of this series of compounds against the HeLa cell nuclear extract which mainly contains HDAC1 and HDAC2, we further tested a selected group of compounds (5m, 11b-d, and 11h) for their in vitro inhibitory activities against several HDAC enzymes. SAHA was used as the positive control. The potencies of these compounds were obtained by measuring the ﬂuorescent-based HDAC biochemical activity using recombinant human HDAC1, HDAC6, and HDAC8 enzymes and the IC50 values are summarized in Table 3. Most of the compounds had high inhibitory potencies against all three enzymes. In particular, compound 11c showed IC50 values against HDAC1 and HDAC6 of 0.026 µM and 0.034 µM respectively, which were five to six-fold lower than that against HDAC8. The potency of 11c against HDAC1 was seven-fold higher than SAHA. Since compound 11c showed the highest potency in inhibition of both cell growth and HDAC, it was advanced for further evaluation. Acetylation of histone H3 and α-tubulin. Given that the inhibition of HDACs by hybrid 11c enhanced the tumor cell antiproliferative activity, the HDAC inhibitory effects of 11c on the


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levels of acetylation of histone H3 and α-tubulin were determined by immunoblotting assays using β-actin as a negative control (Figure 3). HCT116 cells were incubated with the vehicle alone, SAHA, or 11c (0.3, 0.8, and 2.4 µM). Compared with the control group, compound 11c demonstrated the ability to increase the expression of acetyl-histone H3 and acetyl-α-tubulin in a dose dependent manner. Level of acetyl-histone H3 in all 11c treated groups (0.3, 0.8, and 2.4 uM) were higher than the values from the 5.0 µM SAHA treated group, which was consistent with the results from the HDAC fluorimetric activity assay.

Figure 3. Immunoblot analysis of the expression of the acetylation for histone H3 and α-tubulin in vitro. (A) HCT116 cells were treated with 11c and SAHA for 48 h at the indicated concentrations. Cell lysates were prepared and subjected to SDS-PAGE and immunoblot analysis using anti-acetyl-histone H3, anti-acetyl-α-tubulin, and anti-β-actin antibodies, respectively. βActin was used as the loading control. (B) Quantitative analysis. The relative levels of Ac-H3 and Ac-α-tubulin used to control β-actin were determined by densimetric scanning. The data are expressed as means ± SD of three separate experiments. *P< 0.01 vs control. Further analysis revealed that treatment with increased dose of 11c had no significant effect on the survival of non-tumor IEC-18 cells under 5.0 µM while the same treatment induced the majority of HCT116 cell death (Figure 4). Moreover, 11c displayed significant growth inhibitory activities with GI50 values of 1.17 µM in HCT116 cells, nearly five-fold lower than 11c in IEC-


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18 cells (GI50 = 5.46 µM). These results suggest that 11c has selective antiproliferation activity against tumor cells. This effect was more significant than SAHA (GI50 = 4.64 µM in HCT116 and GI50 = 9.95 µM in IEC-18) under the same treatment conditions.

Cell viability (% of control)

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120

HCT116 11c

100

IEC18 11c HCT116 SAHA

80

IEC18 SAHA

60 40 20 0 0

1

2 3 4 Drug concentrations (µM)

5

Figure 4. Inhibitory effects of 11c and SAHA on the proliferation of HCT116 and IEC-18 cells. Cells were incubated with the indicated concentrations of tested compounds for 48 h. Cell proliferation was assessed using the MTT assay. Data are means + SD of the inhibition (%) from three independent experiments.

Compound 11c Induces Apoptosis in HCT116 Cells. To determine whether the inhibitory effects of 11c on colon cancer cellular proliferation are accompanied by enhanced cancer cell apoptosis, FITC-Annexin V/Propidium Iodide (PI) staining and flow cytometry assay was performed and the percentages of apoptotic cells were determined. HCT116 cells were incubated with different concentrations of 11c or SAHA for 48h. As shown in Figure 5, 11c treated HCT116 cells exhibited a dose-dependent increase (P < 0.01) of apoptosis by 21.6%, 41.3%, and 73.9% at 0.1 µM, 1.0 µM, and 5.0 µM, respectively. The 73.9% induction of HCT116 cell


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apoptosis with incubation with 5.0 µM of 11c was significantly higher than SAHA at 34.3% apoptotic cells at the same concentration. To further investigate the apoptosis induction of 11c, we examined the expression of apoptotic proteins Bax, Bcl-2, and the cleavage states of caspase-3 and PARP, in response to 11c treatment (Figure 6). Sub-confluent HCT116 colon cancer cells were treated with or without 11c for 48h, and then lysed and analyzed by western blot. β-Actin expression was used as an internal control. It was revealed that treatment with 11c dramatically increased the relative levels of pro-apoptotic Bax expression, but reduced the levels of anti-apoptotic Bcl-2 expression (Figure 6A and 6B) in a dose-dependent manner. Furthermore, compound 11c resulted in more significant cleavage of both PARP and caspase-3 than the control group in Figure 6C. Importantly, 11c treatment also induced more cleavage of PARP and caspase-3 than the SAHA treated group. Taken together, these results confirm that 11c treatment induced apoptosis in HCT116 cells.

Figure 5. Compound 11c induces HCT116 cell apoptosis in vitro. HCT116 cells were incubated with the indicated concentrations of 11c, or SAHA (5.0 µM) for 48 h, and the cells were stained


16

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with FITC-Annexin V/PI, followed by flow cytometry analysis. (A) Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means ± SD of the percentages of apoptotic cells from three independent experiments. *P< 0.01 vs control.

Figure 6. The expression of Bax, Bcl-2 (A), cleaved Caspase 3, PARP (C), and β-actin was examined by western blot analysis. HCT116 cells were incubated with, or without, 11c and SAHA at the indicated concentrations for 48 h and the levels of protein expression were detected using specific antibodies. Data shown are representative images of each protein for three separate experiments. (B) Quantitative analysis of Bax, Bcl-2; (D) Quantitative analysis of cleaved Caspase 3 and PARP. The relative levels of each protein compared to control β-actin were determined by densimetric scanning. Data are expressed as means ± SD from three separate experiments. *P < 0.01 vs respective control.


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Figure 7. Immunoblot analysis of the expression of DNA damage and DNA-damage response p53 signaling pathway events in vitro. (A) and (C) HCT116 cells were treated with vehicle (control), different doses of 11c, 11h, harmine, or SAHA were homogenized, and their lysates were subjected to immunoblot analysis using anti-H2AXS139ph, antiphospho-p53 (ser15), antip53, anti-p21, and anti-β-actin antibodies, respectively. β-Actin was used as the control. (B) and (D) Quantitative analysis. The relative levels of each signaling event to control β-actin were determined by densimetric scanning. The data are expressed as means ± SD from three separate experiments. *P < 0.01 vs respective control. Compound 11c induces DNA damage and activates the p53 signaling pathway. It is known that the planar structured β-carbolines can bind to DNA and induce DNA damage.11,12 To verify whether the anti-cancer activities of these β-carboline/hydroxamic acid hybrids were


18

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partly from DNA damage, we examined the DNA damage extent and the change of the DNAdamage response p53 signaling pathway caused by 11c in CRC cells. In the present study, we used histone H2AX phosphorylation and phosphor-p53, or p-p53 (Ser15) as DNA damage markers. HCT116 cells were treated with vehicle, or 11c, or 11h for 12 h, using harmine and SAHA as positive controls. H2AXS139ph and p-p53 (ser15) were then detected using western blot analysis. As shown in Figure 7A and 7C, treatment with 11c or 11h dose-dependently increased the levels of H2AX phosphorylation in HCT116 cells. Importantly, H2AXS139ph level in both 1.0 µM and 5.0 µM 11c or 11h treatment groups was significantly higher than the same concentrations within the SAHA group. Since 11h and SAHA showed similar potency in HDAC inhibition, these results clearly indicate that the increased level likely resulted from greater DNA damage induced in the 11h treated cells. Phospho-H2AX level in 20 µM harmine treatment group was similar to 1.0 µM 11h group, and was significantly lower than 5.0 µM 11h group. Phosphorylation of p53 (Ser15) has been known to be the result of DNA damage induced by treatment with cisplatin, isomerase I or II inhibitors.11 Ser 15, a common site for a variety of kinases such as ATM/ATR/DNA-PK, is activated during the DNA repair pathway. It has been well documented that HDACi induced DNA damage is associated with p53 phosphorylation at threonine 18, but not p-p53 (ser15).36 Interestingly, p-p53 (ser15) can only be detected in the 11c treatment group, but none in the SAHA treatment group (Figure 7A). Since β-carboline complex induced DNA damage was companied significantly with p53 activation and p-p53 (ser15), these results clearly indicated that both the key structural elements of HDACi and β-carboline alkaloids are involved in DNA damage induced by 11c and 11h treatment.


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P21 is a tumor suppressor gene activated by the p53 gene. As shown in Figure 7A and 7C, after both drug treatments, p21 expression level was significantly upregulated compared to vehicle control group. Moreover, both 11c and 11h dramatically activated the expression of p21 in HCT116 cells at increased concentrations, and p21 expression level was much higher in the 11c or 11h treatment group than in the SAHA treatment group, consistent with the hypothesis that p21 induction by the hybrids 11c or 11h treatment involves in both HDAC inhibition and βcarboline alkaloids DNA binding.

Figure 8. (A) The expression profile of p53 in shRNA control and shRNA p53 HCT116 cell lines by western blot in vitro. β-Actin was used as control. (B) Quantitative analysis. (C and D) Effects of SAHA or 11c treatment on the shRNA control and shRNA p53 HCT116 colorectal cancer cell growth for 48 h at a wide range of concentrations. The data are expressed as means ± SD of three duplicate experiments. *P < 0.01 vs respective control. Antitumor activity of compound 11c involves the p53 pathway. To determine whether the p53 pathway is involved in the antitumor activity of compound 11c, we examined the effects of 11c on cell growth and cell death with shRNA control and shRNA p53 HCT116 colorectal cancer cells in parallel. It has been reported that HDACi not only increased p53 activation but


20

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also increased p53 acetylation to inhibit cancer cell growth. As shown in Figure 8A, shRNA significantly inhibited HCT116 p53 expression. Compared with the shRNA control group, 11c induced 10% to 40% less growth inhibition across a wide range of concentrations in the shRNA p53 group (Figure 8C), which was more sensitive than SAHA treatments (Figure 8B). This is consistent with a previous report that p53 activation contributed to HDACi drug response.36 In vivo anti-cancer activity of 11c. To evaluate the safety of 11c in vivo, groups of ICR mice were injected intraperitoneally with different doses of 11c or vehicle control, respectively. The survival of mice was monitored up to 14 days after injection. Only three mice that had been treated with 11c at the highest dose (585.9 mg/kg) survived (shown in Table 4). In contrast, injection with 11c at the lowest dose (240 mg/kg) did not cause any death and abnormality in eating, drinking, body weight, and activity throughout the observation period. As a result, the median lethal dose (LD50) value of 11c was calculated to be 471.7 mg/kg for this strain of mice. Table 4. Acute toxicity of 11c in mice Dose

Number of dead mice

(mg/kg)

Number of mice

5-14d

Total death

Survival (%) on day 14

1h

4h

1d

2d

3d

4d

585.9

10

0

0

1

1

2

2

1

7

30

468.8

10

0

0

1

2

1

1

0

5

50

375.0

10

0

0

0

0

1

1

1

3

70

300.0

10

0

0

0

0

0

1

0

1

90

240.0

10

0

0

0

0

0

0

0

0

100


21


Control

1800

11c (45 mg/kg, ip) 11c (90 mg/kg, ip)

1500

Mean Tumor Volume (mm3)

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11c (90 mg/kg, po) SAHA (90 mg/kg, po) 1200

900

600

** * *

300

* * * *

* * *# *

* * *

#

*

0 1

4

7

10

13

16

19

22

Days

Figure 9. Inhibitory effects of 11c on the growth of an implanted HCT116 xenograft in nude mice. BALB/c nude mice were subcutaneously inoculated with HCT116 cells. After establishment of solid tumor, the mice were randomized and treated with solvent control, SAHA, or 11c at the indicated doses, respectively. The growth of tumors was measured longitudinally. Data are expressed as means (SD of tumor volumes at each time point for each group of mice (n = 6 per group)). *P < 0.01 vs control group, #P < 0.05 vs SAHA group. Next, to evaluate the in vivo antitumor activity of 11c, we established a BALB/c nude mice model which was inoculated subcutaneously with HCT116 cells. After the establishment of solid tumor, BALB/c nude mice were randomly intraperitoneally or orally administered with vehicle, 11c, and SAHA, respectively. The changes in tumor size and body weight were monitored twice per week over 22 days. There was no statistical difference in body weight change among the three groups of mice. A steady tumor growth was observed with the vehicle treated group. In contrast, both intravenous and oral 11c treatment significantly reduced the volumes of implanted colon tumors. As shown in Figure 9, while oral treatment with the same dose of 11c initially exhibited almost identical anti-tumor activity as the SAHA treatment, 11c produced greater


22

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tumor reduction at the end of the treatment (days 19 and 22). The tumor weight (0.53 ± 0.12 g) in mice treated with oral 11c at 90 mg/kg were reduced by 61.6% compared to the vehicle treated controls (1.38 ± 0.35 g) and the tumor weight was 0.62 ± 0.13 g in SAHA treated group at the same dosing. According to the preliminary pharmacokinetic profiling of 11c in SD rats (Supporting Information Table S1), there were no significant differences in the pharmacokinetics between intraperitoneal and oral administration. Together, our data clearly demonstrated that 11c had potent antitumor activity against the growth of implanted human colon tumors in vivo.

Conclusions In summary, our group has successfully designed, synthesized and evaluated a series of novel β-carboline/hydroxamic acid hybrids that have the key structures of β-carbolines and HDACi. Our in vitro and in vivo studies of these compounds have resulted in several significant findings. Firstly, β-carboline/hydroxamic acid hybrids (5m, 11b-d, and 11h) with three or four-carbon alkyl linkers exhibited greater potency in antiproliferative activity than their analogs and the FDA approved clinical chemotherapeutic agent SAHA. Cytotoxicity experiments in vitro demonstrated that hybrids with p-methoxyphenyl substituted β-carboline exhibited higher cytotoxic potency than other hybrids with methyl or hydrogen substituted β-carboline. Secondly, treatment of CRC cells with 11c, demonstrated a significant induction of cell apoptosis and significant cleavage of both PARP and caspase-3 in vitro. Thirdly, our study demonstrated that the potent anticancer activity of 11c is related to HDAC inhibition, DNA damage, and activation of the p53 signaling pathway. Finally, the most potent compound 11c displayed low acute toxicity and significant growth inhibition of cancer cells in vivo. These results demonstrated the great promise of β-carboline/hydroxamic acid hybrids with multiple anticancer activity. Our


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findings may assist the design and development of more hybrids as novel anticancer agents. Additional structural refinement and biological evaluations are ongoing in our lab and the results will be reported in due course.

Experimental section General procedures. 1H NMR spectra were recorded with a Bruker Avance 300 MHz spectrometer at 300 K, using TMS as an internal standard. MS spectra were recorded on a Mariner Mass Spectrum (ESI). High resolution mass spectra were recorded using an Agilent Technologies LC/MSD TOF. All compounds were routinely checked by TLC and 1H NMR. TLCs and preparative thin-layer chromatography were performed on silica gel GF/UV 254, and the chromatograms were conducted on silica gel (200–300 mesh, Merck) and visualized under UV light at 254 and 365 nm. All solvents were reagent grade and, when necessary, were purified and dried by standards methods. L-tryptophan 1 and different substituted aldehydes were commercially available. Compounds 3a-c, 4a-c, and 6 were synthesized according literatures.7,11,12 Solutions after reactions and extractions were concentrated using a rotary evaporator operating at a reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. The high-performance liquid chromatography (HPLC) analysis conditions: Column: Shimadzu C18 (150mm×4.6mm×5µm); Mobile phase: Methanol: aqueous solution = 65: 35; Wavelength: 254 nm; Rate: 1 mL/min; Temperature: 30 °C; Pressure: 85-142 kgf. All compounds were of >95% purity determined by HPLC. General procedure for the preparation of 4a-o. To a solution of 3a-c (1.0 mmol) in 5 mL anhydrous THF at 0 °C was added N-methylmorpholine (0.1 g, 1.0 mmol) and ethyl carbonochloridate (0.11 g, 1.0 mmol). After 0.5h, the mixture was added dropwise to a solution


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of NH2(CH2)nCOOMe(1.0 mmol) and Et3N (1.0 mmol) in 5 mL anhydrous THF, and the reaction was then stirred at room temperature for 5-8h. After the reaction, the resulting mixture was poured into ice-water, and extracted with ethyl acetate (30 mL × 3). The organic phase was combined and washed with water and brine, then dried with anhydrous sodium sulfate. After filtration, the filtrate was collected and concentrated in vacuo. The resulting residue was purified by column chromatography (EtOAc-PE =1:1-4:1, v/v as the eluate) to afford compound 4a-o. Methyl 2-(9H-pyrido[3,4-b]indole-3-carboxamido)acetate (4a). The title compound was obtained starting from 3a, ethyl carbonochloridate, and NH2CH2COOMe as an off-white solid, yield: 81%. MS (ESI) m/z =284 [M+H]+. Methyl 3-(9H-pyrido[3,4-b]indole-3-carboxamido)propanoate (4b). The title compound was obtained starting from 3a, ethyl carbonochloridate, and NH2(CH2)2COOMe as an off-white solid, yield: 78%. MS (ESI) m/z =298 [M+H]+. Methyl 4-(9H-pyrido[3,4-b]indole-3-carboxamido)butanoate (4c). The title compound was obtained starting from 3a, ethyl carbonochloridate, and NH2(CH2)3COOMe as an off-white solid, yield: 77%. MS (ESI) m/z =312 [M+H]+. Methyl 5-(9H-pyrido[3,4-b]indole-3-carboxamido)pentanoate (4d). The title compound was obtained starting from 3a, ethyl carbonochloridate, and NH2(CH2)4COOMe as an off-white solid, yield: 73%. MS (ESI) m/z =326 [M+H]+. Methyl 6-(9H-pyrido[3,4-b]indole-3-carboxamido)hexanoate (4e). The title compound was obtained starting from 3a, ethyl carbonochloridate, andNH2(CH2)5COOMe as an off-white solid, yield: 71%. MS (ESI) m/z =340 [M+H]+.


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Methyl

2-(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamido)acetate

Page 26 of 48

(4f).

The

title

compound was obtained starting from 3b, ethyl carbonochloridate, and NH2CH2COOMe as an off-white solid, yield: 80%. MS (ESI) m/z =298 [M+H]+. Methyl 3-(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamido)propanoate (4g). The title compound was obtained starting from 3b, ethyl carbonochloridate, and NH2(CH2)2COOMe as an off-white solid, yield: 79%. MS (ESI) m/z =312 [M+H]+. Methyl 4-(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamido)butanoate (4h). The title compound was obtained starting from 3b, ethyl carbonochloridate, and NH2(CH2)3COOMe as an off-white solid, yield: 77%. MS (ESI) m/z =326 [M+H]+. Methyl 5-(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamido)pentanoate (4i). The title compound was obtained starting from 3b, ethyl carbonochloridate, and NH2(CH2)4COOMe as an off-white solid, yield: 73%. MS (ESI) m/z =340 [M+H]+. Methyl 6-(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamido)hexanoate (4j). The title compound was obtained starting from 3b, ethyl carbonochloridate, and NH2(CH2)5COOMe as an off-white solid, yield: 75%. MS (ESI) m/z =354 [M+H]+. Methyl 2-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamido)acetate (4k). The title compound was obtained starting from 3c, ethyl carbonochloridate, and NH2CH2COOMe as an off-white solid, yield: 83%. MS (ESI) m/z =390 [M+H]+. Methyl 3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamido)propanoate (4l). The title compound was obtained starting from 3c, ethyl carbonochloridate, and NH2(CH2)2COOMe as an off-white solid, yield: 80%. MS (ESI) m/z =404 [M+H]+.


26

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Methyl 4-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamido)butanoate (4m). The title compound was obtained starting from 3c, ethyl carbonochloridate, and NH2(CH2)3COOMe as an off-white solid, yield: 77%. MS (ESI) m/z =418 [M+H]+. Methyl 5-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamido)pentanoate (4n). The title compound was obtained starting from 3c, ethyl carbonochloridate, and NH2(CH2)4COOMe as an off-white solid, yield: 74%. MS (ESI) m/z =432 [M+H]+. Methyl 6-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamido)hexanoate (4o). The title compound was obtained starting from 3c, ethyl carbonochloridate, and NH2(CH2)5COOMe as an off-white solid, yield: 75%. MS (ESI) m/z =446 [M+H]+.

General procedure for the preparation of 5a-o. To a solution of 4a-o (0.4 mmol) in 5 mL of anhydrous methanol, was added a solution of NH2OK (0.09 g, 4 mmol) in 3 mL of anhydrous methanol. The mixture was stirred for 10-15 h and the solvent was evaporated under vacuum. The residue was diluted with saturated NH4Cl aqueous solution, and then extracted with ethyl acetate (8 mL × 5). The organic layers were combined, washed with brine (10 mL), dried over anhydrous Na2SO4 and evaporated with the residue being purified by column chromatography (eluting with EA followed by 20:1 CHCl3/MeOH followed by 10:1 CHCl3/MeOH) on silica gel to yield 5a-o as off-white solids (68-77%). N-(2-(Hydroxyamino)-2-oxoethyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5a). The title compound was obtained starting from 4a and NH2OK as an off-white solid, yield: 73%. Analytical data for 5a: 1H NMR (DMSO-d6, 300 MHz): δ11.35 (s, 1H, NH), 10.12 (s, 1H, NH), 8.88 (s, 1H, Ar-H), 8.71 (s, 1H, Ar-H), 8.23-8.29 (m, 2H, CONH, Ar-H), 7.68 (m, 1H, Ar-H),


27


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Page 28 of 48

7.33 (m, 1H, Ar-H), 4.27 (m, 2H, NCH2). MS (ESI) m/z =285 [M+H]+.HRMS (ESI): m/z calcd for C14H13N4O3: 285.0988; found: 285.0997 [M+H]+. N-(3-(Hydroxyamino)-3-oxopropyl)-9H-pyrido[3,4-b]indole-3-carboxamide (5b). The title compound was obtained starting from 4b and NH2OK as an off-white solid, yield: 77%. Analytical data for 5b: 1H NMR (DMSO-d6, 300 MHz): δ11.20 (s, 1H, NH), 9.03 (s, 1H, NH), 8.85 (s, 1H, Ar-H), 8.73 (s, 1H, Ar-H), 8.27 (m, 1H, CONH), 8.16(m, 1H, Ar-H), 7.70(m, 1H, Ar-H), 7.56 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 3.65 (m, 2H, NCH2), 2.71 (m, 2H, CH2C=O). MS (ESI) m/z =299 [M+H]+. HRMS (ESI): m/z calcd for C15H15N4O3:299.1144; found: 299.1152 [M+H]+. N-(4-(Hydroxyamino)-4-oxobutyl)-9H-pyrido[3,4-b]indole-3-carboxamide (5c). The title compound was obtained starting from 4c and NH2OK as an off-white solid, yield: 72%. Analytical data for 5c: 1H NMR (DMSO-d6, 300 MHz): δ11.08 (s, 1H, NH), 9.16 (s, 1H, NH), 8.89 (s, 1H, Ar-H), 8.70 (s, 1H, Ar-H), 8.28 (m, 1H, CONH), 8.20 (m, 1H, Ar-H), 7.71 (m, 1H, Ar-H), 7.60 (m, 1H, Ar-H), 7.36 (m, 1H, Ar-H), 3.48 (m, 2H, NCH2), 2.63 (m, 2H, CH2CO), 1.91 (m, 2H, CH2CH2CH2). MS (ESI) m/z =313 [M+H]+. HRMS (ESI): m/z calcd for C16H17N4O3:313.1301; found: 313.1310 [M+H]+. N-(5-(Hydroxyamino)-5-oxopentyl)-9H-pyrido[3,4-b]indole-3-carboxamide (5d). The title compound was obtained starting from 4d and NH2OK as an off-white solid, yield: 73%. Analytical data for 5d: 1H NMR (DMSO-d6, 300 MHz): δ10.15 (s, 1H, NH), 8.87 (s, 1H, Ar-H), 8.70 (s, 1H, Ar-H), 8.27 (m, 1H, CONH), 8.20 (m, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.38 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 3.45 (m, 2H, NCH2), 2.36 (m, 2H, CH2C=O), 1.81-1.85 (m, 4H, CH2CH2CH2CH2). MS (ESI) m/z = 327 [M+H]+. HRMS (ESI): m/z calcd for C17H19N4O3: 327.1457; found: 327.1446 [M+H]+.


28

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N-(6-(Hydroxyamino)-6-oxohexyl)-9H-pyrido[3,4-b]indole-3-carboxamide (5e). The title compound was obtained starting from 4e and NH2OK as an off-white solid, yield: 68%. Analytical data for 5e: 1H NMR (DMSO-d6, 300 MHz): δ11.41 (s, 1H, NH), 8.89 (s, 1H, Ar-H), 8.71 (s, 1H, Ar-H), 8.21-8.27 (m, 1H, CONH, Ar-H), 7.61 (m, 1H, Ar-H), 7.39 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 3.41 (m, 2H, NCH2), 2.35 (m, 2H, CH2C=O), 1.72-1.77 (m, 4H, NCH2CH2CH2CH2), 1.50 (m, 2H, NCH2CH2CH2). MS (ESI) m/z =341 [M+H]+. HRMS (ESI): m/z calcd for C18H21N4O3:341.1614; found: 341.1622 [M+H]+. N-(2-(Hydroxyamino)-2-oxoethyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (5f). The title compound was obtained starting from 4f and NH2OK as an off-white solid, yield: 71%. Analytical data for 5f:1H NMR (DMSO-d6, 300 MHz): δ9.12 (s, 1H, NH), 8.75 (m, 2H, Ar-H, CONH), 8.28 (m, 1H, Ar-H), 8.10 (m, 1H, Ar-H), 7.56 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 4.24 (m, 2H, CH2), 2.91 (s, 3H, CH3). MS (ESI) m/z =299 [M+H]+. HRMS (ESI): m/z calcd for C15H14N4O3:299.1144; found: 299.1156 [M+H]+. N-(3-(Hydroxyamino)-3-oxopropyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (5g). The title compound was obtained starting from 4g and NH2OK as an off-white solid, yield: 69%. Analytical data for 5g: 1H NMR (DMSO-d6, 300 MHz): δ9.01 (s, 1H, NH), 8.72 (s, 1H, Ar-H), 8.29-8.41 (m, 2H, CONH, Ar-H), 8.07 (m, 1H, Ar-H), 7.59 (m, H, Ar-H), 7.28 (m, 1H, Ar-H), 3.62 (m, 2H, NCH2), 2.89 (s, 3H, CH3),2.73 (m, 2H, CH2CO). MS (ESI) m/z =313 [M+H]+. HRMS (ESI): m/z calcd for C16H17N4O3:313.1301; found: 313.1311 [M+H]+. N-(4-(Hydroxyamino)-4-oxobutyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (5h). The title compound was obtained starting from 4h and NH2OK as an off-white solid, yield: 75%. Analytical data for 5h: 1H NMR (DMSO-d6, 300 MHz): δ11.68 (m, 1H, NH), 8.76 (s, 1H, Ar-H), 8.69 (m, 1H, CONH), 8.27 (m, 1H, Ar-H), 8.08 (m, 1H, Ar-H), 7.60 (m, H, Ar-H), 7.28 (m, 1H,


29


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Ar-H), 3.77 (m, 2H, NCH2), 2.92 (s, 3H, CH3), 2.67 (m, 2H, CH2CO), 2.01 (s, 1H, OH), 1.91 (m, 2H, NCH2CH2CH2). MS (ESI) m/z = 327 [M+H]+. HRMS (ESI): m/z calcd for C17H19N4O3: 327.1457; found: 327.1465 [M+H]+. N-(5-(Hydroxyamino)-5-oxopentyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (5i). The title compound was obtained starting from 4i and NH2OK as an off-white solid, yield: 72%. Analytical data for 5i: 1H NMR (DMSO-d6, 300 MHz): δ9.56 (s, 1H, NH), 8.70 (s, 1H, Ar-H), 8.65 (m, 1H, CONH), 8.30 (m, 1H, Ar-H), 8.09 (m, 1H, Ar-H), 7.61 (m, H, Ar-H), 7.28 (m, 1H, Ar-H), 3.55 (m, 2H, NCH2), 2.89 (s, 3H, CH3),2.29 (m, 2H, CH2CO), 1.80-1.84 (m, 4H, NCH2CH2CH2).

MS

(ESI)

m/z

=341

[M+H]+.

HRMS

(ESI):

m/z

calcd

for

C18H21N4O3:341.1614; found: 341.1623 [M+H]+. N-(6-(Hydroxyamino)-6-oxohexyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (5j). The title compound was obtained starting from 4j and NH2OK to afford as an off-white solid, yield: 75%. Analytical data for 5j: 1H NMR (DMSO-d6, 300 MHz): δ10.30 (s, 1H, NH), 8.75 (s, 1H, Ar-H), 8.37-8.43 (m, 2H, CONH, Ar-H), 8.11 (m, 1H, Ar-H), 7.62 (m, H, Ar-H), 7.30 (m, 1H, Ar-H), 3.51 (m, 2H, NCH2), 2.89 (s, 3H, CH3),2.30 (m, 2H, CH2CO), 1.71-1.76 (m, 4H, NCH2CH2CH2CH2), 1.48 (m, 4H, NCH2CH2CH2). MS (ESI) m/z = 355 [M+H]+. HRMS (ESI): m/z calcd for C19H23N4O3:355.1770; found: 355.1759 [M+H]+. N-(2-(Hydroxyamino)-2-oxoethyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3carboxamide (5k). The title compound was obtained starting from 4k and NH2OK as an offwhite solid, yield: 77%. Analytical data for 5k: 1H NMR (DMSO-d6, 300 MHz): δ9.91 (s, 1H, NH), 8.71 (m, 2H, Ar-H, CONH), 8.59 (m, 1H, Ar-H), 8.15 (d, 1H, J = 7.5 Hz, Ar-H), 7.75 (d, 2H, J = 7.5 Hz, Ar-H), 7.63 (m, 1H, Ar-H), 7.41 (m, 1H, Ar-H), 7.02 (d, 2H, J = 7.5 Hz, Ar-H),


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4.21 (m, 2H, NCH2), 3.85 (s, 3H, OCH3). MS (ESI) m/z =391 [M+H]+. HRMS (ESI): m/z calcd for C21H19N4O4:391.1406; found: 391.1415 [M+H]+. N-(3-(Hydroxyamino)-3-oxopropyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3carboxamide (5l). The title compound was obtained starting from 4l and NH2OK as an off-white solid, yield: 71%. Analytical data for 5l: 1H NMR (DMSO-d6, 300 MHz): δ10.13 (s, 1H, NH), 8.71 (s, 1H, Ar-H), 8.42 (m, 2H, Ar-H, CONH), 8.18 (d, 2H, J = 7.5 Hz, Ar-H), 7.74 (m, 1H, ArH), 7.61 (m, 1H, Ar-H), 7.48 (m, 1H, Ar-H), 7.09 (d, 2H, J = 7.5 Hz, Ar-H), 3.89 (s, 3H, OCH3), 3.67 (m, 2H, NCH2), 2.74 (m, 2H, CH2CO). MS (ESI) m/z =405 [M+H]+. HRMS (ESI): m/z calcd for C22H21N4O4:405.1563; found: 405.1575 [M+H]+. N-(4-(Hydroxyamino)-4-oxobutyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3carboxamide (5m). The title compound was obtained starting from4m and NH2OK as an offwhite solid, yield: 72%. Analytical data for 5m: 1H NMR (DMSO-d6, 300 MHz): δ11.02 (s, 1H, NH), 9.46 (s, 1H, NH), 8.75 (s, 1H, Ar-H), 8.46 (m, 2H, Ar-H, CONH), 8.19 (d, 2H, J = 7.5 Hz, Ar-H), 7.84 (m, 1H, Ar-H), 7.65 (m, 1H, Ar-H), 7.47 (m, 1H, Ar-H), 7.06 (d, 2H, J = 7.5 Hz, ArH), 3.89 (s, 3H, OCH3), 3.48 (m, 2H, NCH2), 2.32 (m, 2H, CH2CO), 1.89 (m, 2H, NCH2CH2). MS (ESI) m/z =419 [M+H]+. HRMS (ESI): m/z calcd for C23H23N4O4:419.1719; found: 419.1731 [M+H]+. N-(5-(Hydroxyamino)-5-oxopentyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3carboxamide (5n). The title compound was obtained starting from 4n and NH2OK as an offwhite solid, yield: 69%. Analytical data for 5n: 1H NMR (DMSO-d6, 300 MHz): δ11.45 (s, 1H, NH), 10.26 (s, 1H, NH), 8.70 (s, 1H, Ar-H), 8.25 (m, 1H, CONH), 8.13-8.17 (m, 2H, Ar-H), 7.86 (d, 2H, J = 7.5 Hz, Ar-H), 7.66 (m, 1H, Ar-H), 7.46 (m, 1H, Ar-H), 7.07 (d, 2H, J = 7.5 Hz, ArH), 3.89 (s, 3H, OCH3), 3.55 (m, 2H, NCH2), 2.29 (m, 2H, CH2CO), 1.98 (m, 1H, OH), 1.72-


31


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1.77 (m, 4H, CH2CH2CH2CH2). MS (ESI) m/z =433 [M+H]+. HRMS (ESI): m/z calcd for C24H25N4O4: 433.1876; found: 433.1887 [M+H]+. N-(6-(Hydroxyamino)-6-oxohexyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3carboxamide (5o). The title compound was obtained starting from 4o and NH2OK as an offwhite solid, yield: 70%. Analytical data for 5o: 1H NMR (DMSO-d6, 300 MHz): δ11.20 (s, 1H, NH), 8.72 (s, 1H, Ar-H), 8.14-8.25 (m, 3H, Ar-H,CONH), 7.86 (d, 2H, J = 8.76 Hz, Ar-H), 7.66 (m, 1H, Ar-H), 7.48 (m, 1H, Ar-H), 7.07 (d, 2H, J = 7.5 Hz, Ar-H), 3.89 (s, 3H, OCH3), 3.53 (m, 2H, NCH2), 2.39 (m, 2H, CH2CO), 1.70 (m, 4H, 2 × NCH2CH2), 1.49 (m, 2H, CH2(CH2CH2)2); MS (ESI) m/z = 447 [M+H]+. HRMS (ESI): m/z calcd for C25H27N4O4: 447.2032; found: 447.2043 [M+H]+. Methyl 1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate(7). To 3c (1.82 g, 10 mmol) in anhydrous DMF (25 ml) was slowly added 60%NaH (0.6 g, 15 mmol). The mixture was stirred for 0.5h, and then iodomethane (1 ml, 15 mmol) were slowly added into the mixture. The mixture was stirred at rt for 1 h. The resulting mixture was poured into water (50 ml), and extracted with ethyl acetate (30 mL × 2). The organic phase was washed with water and brine, then dried over anhydrous Na2SO4, and concentrated. The residue 7 was directly used in the next reaction without further purification. General procedure for the preparation of 9a-b. A solution of 6 or 7 (3 mmol) and 85% hydrazine hydrate (5 ml) in 30 ml methanol was refluxed for 8 h. The resulting mixture was cooled, and the precipitate was collected by filtration, washed with methanol, and then dried in vacuum to obtain 8a-b, which was poured into 50 ml water and dissolved by concentrated hydrochloric acid (6.5 ml). The yellow solution was cooled to 0 °C before the addition of a solution of NaNO2 (0.7 g, 10 mmol) in 10 ml water and continually stirred for 40 min at 0 °C.


32

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After the reaction, the mixture was basified with saturated sodium hydrogen carbonate aqueous, and the precipitate was collected by filtration, washed with water, and dried under vacuum to yield an off-white solid 9a or 9b. These compounds were easy to decompose in the air and were used without further purification in the following steps. 1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonyl azide (9a). The title compound was obtained starting from 6, hydrazine hydrate, and NaNO2 as an off-white solid, yield: 73%. MS (ESI) m/z =344 [M+H]+. 1-(4-Methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indole-3-carbonyl azide (9b). The title compound was obtained starting from 7, hydrazine hydrate, and NaNO2 as an off-white solid, yield: 80%. MS (ESI) m/z =358 [M+H]+. General procedure for the preparation of 10a-j. A solution of 9a or 9b (0.5mmol) and NH2(CH2)nCOOMe (0.75mmol) in anhydrous toluene (20 ml) was refluxed for 2 h. After the reaction, the mixture was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatographyon silica gel to afford 10a-j as the yellowish solid. Methyl 2-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)acetate (10a). The title compound was obtained starting from 9a and NH2CH2COOMe as an off-white solid, yield: 82%. MS (ESI) m/z = 405 [M+H]+. Methyl 3-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)propanoate (10b). The title compound was obtained starting from 9a and NH2(CH2)2COOMe as an off-white solid, yield: 83%. MS (ESI) m/z =419 [M+H]+. Methyl

4-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)butanoate

(10c).

The title compound was obtained starting from 9a and NH2(CH2)3COOMe as an off-white solid, yield: 79%. MS (ESI) m/z =433 [M+H]+.


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Methyl 5-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)pentanoate (10d). The title compound was obtained starting from 9a and NH2(CH2)4COOMe as an off-white solid, yield: 77%. MS (ESI) m/z =447 [M+H]+. Methyl

6-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)hexanoate

(10e).

The title compound was obtained starting from 9a and NH2(CH2)5COOMe as an off-white solid, yield: 77%. MS (ESI) m/z =461 [M+H]+. Methyl

2-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido)acetate

(10f). The title compound was obtained starting from 9b and NH2CH2COOMe as an off-white solid, yield: 85%. MS (ESI) m/z =419 [M+H]+. Methyl

3-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido)

propanoate (10g). The title compound was obtained starting from 9b and NH2(CH2)2COOMe as an off-white solid, yield: 82%. MS (ESI) m/z = 433 [M+H]+. Methyl 4-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido)butanoate (10h). The title compound was obtained starting from 9b and NH2(CH2)3COOMe as an off-white solid, yield: 81%. MS (ESI) m/z = 447 [M+H]+. Methyl

5-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido)

pentanoate (10i). The title compound was obtained starting from 9b and NH2(CH2)4COOMe as an off-white solid, yield: 78%. MS (ESI) m/z = 461 [M+H]+. Methyl 6-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido)hexanoate (10j). The title compound was obtained starting from 9b and NH2(CH2)5COOMe as an off-white solid, yield: 79%. MS (ESI) m/z =475 [M+H]+. General procedure for the preparation of 11a-j. To a solution of 10a-j (0.2 mmol) in 3 mL of anhydrous methanol, was added a solution of NH2OK (0.05 g, 2 mmol) in 3 mL of anhydrous


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methanol. The mixture was stirred for 10-15 h and the solvent was evaporated under vacuum. The residue was acidified with 2 mol/L HCl to pH = 7, then extracted with ethyl acetate (10 mL × 3). The organic layers were combined, washed with brine (10 mL × 3), dried over anhydrous Na2SO4 and evaporated with the residue being purified by column chromatography on silica gel, yield 11a-j as an off-white solid (42-55%).

N-Hydroxy-2-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3yl)ureido)acetamide(11a). The title compound was obtained starting from 10a and NH2OK as an off-white solid, yield: 68%. Analytical data for 11a: 1H NMR (DMSO-d6, 300 MHz): δ11.28 (s, 1H, NH), 8.73 (m, 1H, Ar-H), 8.40-8.47 (m, 3H, Ar-H, CONH), 8.16 (d, 2H, J =7.5 Hz, ArH), 7.68 (m, 1H, Ar-H), 7.60 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 7.13 (d, 2H, J = 7.5 Hz, Ar-H), 3.88 (s, 3H, OCH3), 4.33 (m, 2H, NCH2). MS (ESI) m/z =406 [M+H]+. HRMS (ESI): m/z calcd for C21H20N5O4: 406.1515; found: 403.1528 [M+H]+. N-Hydroxy-3-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)propanamide (11b). The title compound was obtained starting from 10b and NH2OK as an off-white solid, yield: 75%. Analytical data for 11b: 1H NMR (DMSO-d6, 300 MHz): δ9.40 (s, 1H, NH), 8.75 (m, 1H, Ar-H), 8.39-8.47 (m, 3H, Ar-H, CONH), 8.16 (d, 2H, J =7.5 Hz, Ar-H), 7.68 (m, 1H, Ar-H), 7.59 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 7.15 (d, 2H, J = 7.5 Hz, Ar-H), 3.89 (s, 3H, OCH3), 3.66 (m, 2H, NCH2), 2.72 (m, 2H, CH2). MS (ESI) m/z = 420 [M+H]+. HRMS (ESI): m/z calcd for C22H22N5O4: 420.1672; found: 420.1685 [M+H]+. N-Hydroxy-4-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)butanamide (11c). The title compound was obtained starting from 10c and NH2OK as an off-white solid, yield: 73%. Analytical data for 11c: 1H NMR (DMSO-d6, 300 MHz): δ11.81 (s, 1H, NH), 10.43


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(s, 1H, NH), 8.77 (m, 3H, Ar-H, 2 × CONH), 8.41 (d, 1H, J = 7.5 Hz, Ar-H), 8.17 (d, 2H, J =7.5 Hz, Ar-H), 7.71 (m, 1H, Ar-H), 7.61 (m, 1H, Ar-H), 7.33 (m, 1H, Ar-H), 7.19 (d, 2H, J = 7.5 Hz, Ar-H), 3.90 (s, 3H, OCH3), 3.40 (m, 2H, NCH2), 2.07 (m, 2H, CH2), 1.84 (m, 2H, CH2). MS (ESI) m/z =434 [M+H]+. HRMS (ESI): m/z calcd for C23H24N5O4: 434.1828; found: 434.1841 [M+H]+. N-Hydroxy-5-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)pentanamide (11d). The title compound was obtained starting from 10d and NH2OK as an off-white solid, yield: 66%. Analytical data for 11d: 1H NMR (DMSO-d6, 300 MHz): δ11.63 (s, 1H, NH), 10.52 (s, 1H, NH), 8.72 (m, 1H, Ar-H), 8.42 (m, 1H, Ar-H), 8.27 (m, 2H, CONH), 8.16 (d, 2H, J =7.5 Hz, Ar-H), 7.69 (m, 1H, Ar-H), 7.60 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 7.17 (d, 2H, J = 7.5 Hz, Ar-H), 3.89 (s, 3H, OCH3), 3.41 (m, 2H, NCH2), 2.27 (m, 2H, CH2), 1.73-1.78 (m, 4H, CH2CH2CH2CH2). MS (ESI) m/z =448 [M+H]+. HRMS (ESI): m/z calcd for C24H26N5O4: 448.1985; found: 448.1996 [M+H]+. N-Hydroxy-6-(3-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)ureido)hexanamide (11e). The title compound was obtained starting from 10e and NH2OK as an off-white solid, yield: 63%. Analytical data for 11e: 1H NMR (DMSO-d6, 300 MHz): δ11.20 (s, 1H, NH), 10.36 (s, 1H, NH), 8.79 (m, 1H, Ar-H), 8.40-8.45 (m, 3H, Ar-H, 2 × CONH), 8.17 (d, 2H, J =7.5 Hz, Ar-H), 7.73 (m, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 7.18 (d, 2H, J = 7.5 Hz, ArH), 3.89 (s, 3H, OCH3), 3.43 (m, 2H, NCH2), 2.28 (m, 2H, CH2), 1.70-1.73 (m, 4H, NCH2CH2CH2CH2), 1.49 (m, 2H, NCH2CH2CH2). MS (ESI) m/z =462 [M+H]+. HRMS (ESI): m/z calcd for C25H28N5O4: 462.2141; found: 462.2156 [M+H]+. N-Hydroxy-2-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido) acetamide (11f). The title compound was obtained starting from 10f and NH2OK as an off-white


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solid, yield: 75%. Analytical data for 11f: 1H NMR (DMSO-d6, 300 MHz): δ8.71 (m, 1H, Ar-H), 8.42-8.48 (m, 3H, Ar-H, CONH), 8.15 (d, 2H, J =7.5 Hz, Ar-H), 7.67 (m, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.31 (m, 1H, Ar-H), 7.13 (d, 2H, J = 7.5 Hz, Ar-H), 3.87 (s, 3H, OCH3), 4.30 (m, 2H, NCH2), 3.56 (s, 3H, NCH3). MS (ESI) m/z =420 [M+H]+. HRMS (ESI): m/z calcd for C22H22N5O4: 420.1672; found: 420.1689 [M+H]+. N-Hydroxy-3-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido) propanamide(11g). The title compound was obtained starting from10g and NH2OK as an offwhite solid, yield: 71%. Analytical data for 11g: 1H NMR (DMSO-d6, 300 MHz): δ9.07 (s, 1H, NH), 8.70 (s, 1H, Ar-H), 8.39-8.45 (m, 3H, Ar-H, CONH), 8.14 (d, 2H, J = 7.5 Hz, Ar-H), 7.68 (m, 1H, Ar-H), 7.59 (m, 1H, Ar-H), 7.47 (m, 1H, Ar-H), 7.12 (d, 2H, J = 7.5 Hz, Ar-H), 3.87 (s, 3H, OCH3), 3.55-3.63 (m, 5H, NCH3, NCH2), 2.69 (m, 2H, CH2CO). MS (ESI) m/z =434 [M+H]+. HRMS (ESI): m/z calcd for C23H24N5O4: 434.1828; found: 434.1844 [M+H]+. N-Hydroxy-4-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido) butanamide (11h). The title compound was obtained starting from 10h and NH2OK as an offwhite solid, yield: 72%. Analytical data for 11h: 1H NMR (DMSO-d6, 300 MHz): δ10.36 (s, 1H, NH), 8.76 (m, 1H, Ar-H), 8.43-8.51 (m, 3H, Ar-H, 2 × CONH), 8.17 (d, 2H, J =7.5 Hz, Ar-H), 7.70 (m, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.31 (m, 1H, Ar-H), 7.18 (d, 2H, J = 7.5 Hz, Ar-H), 3.90 (s, 3H, OCH3), 3.59 (s, 3H, NCH3), 3.43 (m, 2H, NCH2), 2.26 (m, 2H, CH2), 1.83 (m, 2H, NCH2CH2). MS (ESI) m/z =448 [M+H]+. HRMS (ESI): m/z calcd for C24H26N5O4: 448.1985; found: 448.2002 [M+H]+. N-Hydroxy-5-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido) pentanamide(11i). The title compound was obtained starting from 10i and NH2OK as an offwhite solid, yield: 66%. Analytical data for 11i: 1H NMR (DMSO-d6, 300 MHz): δ9.61 (s, 1H,


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NH), 8.78 (m, 1H, Ar-H), 8.42-8.48 (m, 3H, Ar-H, CONH), 8.18 (d, 2H, J =7.5 Hz, Ar-H), 7.67 (m, 1H, Ar-H), 7.60 (m, 1H, Ar-H), 7.33 (m, 1H, Ar-H), 7.17 (d, 2H, J = 7.5 Hz, Ar-H), 3.90 (s, 3H, OCH3), 3.58 (s, 3H, NCH3), 3.46 (m, 2H, NCH2), 2.27 (m, 2H, CH2), 1.72-1.76 (m, 4H, NCH2CH2CH2). MS (ESI) m/z =462 [M+H]+. HRMS (ESI): m/z calcd for C25H28N5O4: 462.2141; found: 462.2128 [M+H]+. N-Hydroxy-6-(3-(1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)ureido) hexanamide(11j). The title compound was obtained starting from 10j and NH2OK as an offwhite solid, yield: 70%. Analytical data for 11j: 1H NMR (DMSO-d6, 300 MHz): δ9.07 (s, 1H, NH), 8.73 (m, 1H, Ar-H), 8.41-8.46 (m, 3H, Ar-H, CONH), 8.16 (d, 2H, J =7.5 Hz, Ar-H), 7.67 (m, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 7.15 (d, 2H, J = 7.5 Hz, Ar-H), 3.88 (s, 3H, OCH3), 3.55 (s, 3H, NCH3), 3.40 (m, 2H, NCH2), 2.26 (m, 2H, CH2), 1.70-1.75 (m, 4H, CH2CH2CH2CH2), 1.47 (m, 2H, NCH2CH2CH2). MS (ESI) m/z =476 [M+H]+. HRMS (ESI): m/z calcd for C26H30N5O4: 476.2298; found: 476.2312 [M+H]+. Cell Culture and Reagents. Human colon cancer cells (HCT116, SW620, Lovo, and SW480) and Hela cells were maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml of penicillin, and 0.1 µg/ml of streptomycin in a humid atmosphere incubator with 5% CO2 at 37 °C. All cell lines were originally from the Shanghai Institute of Cell Biology (Shanghai, China). Cells were routinely subcultured twice weekly. P53, p21, and PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), monoclonal anti-actin antibody, and goat peroxidase-conjugated anti-rabbit IgG antibody, and goat peroxidase-conjugated antimouse IgG antibody were purchased from Sigma-Aldrich (St. Louis, MO). FITC-Annexin V and PI are from (BioVision). The chemiluminescence (ECL) kit was purchased from Thermo Fisher


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Scientific (Rockford, IL). Antibodies such as acetyl-H3, acetyl-tubulin, Bax, Bcl-2, p-H2AX cleaved-caspase 3, and P-p53 (S15) are from Cell Signaling Technology (Danvers, MA). Cell viability assay. Anti-proliferative activities of synthesized compounds were evaluated in vitro against four human colon cancer cell lines (HCT116, SW620, LOVO, and SW480). Briefly, 100 µl of different colon cancer cells were plated in a 96-well flat bottom tissue culture plate at a density of 104 cells/ml, respectively, in DMEM medium and 10% fetal bovine serum and allowed to adhere overnight at 37°C in 5% CO2. The cells were treated by adding 100 µL of different compounds at various concentrations into the respective well. The reagent DMSO (0.1%) was used as a negative control. The cell viability assay (MTT assay) was carried out at 48 hours after drug treatment. The concentration, which inhibited 50% of cellular growth (IC50 value), was calculated by the following formula: Cell inhibition rate (%) = (1 − OD of treatment group/OD of control group) × 100%. The cytotoxicity potency of tested compounds on colon cancer cells was expressed as IC50 values or by a histogram (The bars are the mean ± SD). All the data were derived from three independent measurements. HDAC activity assay. HDAC activity assays were performed as previously reported.37 HeLa cell nuclear extract which is a rich source of HDACs was prepared using the EpiQuik nuclear extraction kit (OP-0002, Epigentek Group Inc). The HDAC activity was determined using the HDAC fluorimetric activity assay kit (Enzo Life Sciences Inc.) according to the manufacturer’s instructions. Briefly, HDAC enzyme solution (HeLa nuclear extract, HDAC1, HDAC6, or HDAC8) was incubated with test compounds at different concentrations in the presence of HDAC substrate (Boc-Lys (Ac)-AMC) at 37 °C for 60 min. Then the lysine developer was added to stop the reaction. After 30 min, the data was recorded in a fluorescence plate reader with excitation at 355 nm and emission at 460 nm. The HDAC activity was calculated as a


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percentage of activity compared with the control group. The concentration required for 50% inhibition (IC50) was calculated using the software GraphPad Prism (Version 4.03). Flow cytometry assay of cell apoptosis. HCT116 cells cells were cultured overnight and incubated in triplicate with different concentrations of 11c (0.1, 1.0, and 5.0 µM), SAHA (5.0 µM), or vehicle for 48 h. The cells were harvested and stained with FITC-Annexin V and PI at room temperature for 15 min. The percentage of apoptotic cells was determined by flow cytometry (Epics XL-MCL, Beckman Coulter, Indianapolis, USA) analysis. The FITC signal detector (FL1) and PI staining signal detector (FL3) were used to detect the cells with the flow cytometer (Ex = 488 nm; Em = 530 nm). Ten thousand cells were counted for three independent experiments. The data were analyzed using WinList 3D (version7.1) and the histogram was plotted using Excel 2010. Western Blot Analysis. HCT116 cells with or without harmine, 11c, or 11h treatment at indicated time and doses were washed with PBS and lysed on ice for 30 minutes in PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, and 20 mM leupeptin. The protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Up to 50 µg of total protein were separated onto an SDS-PAGE and transferred to polyvinylidenedifluoride membranes. After blocking with 5% fat free milk for 2 h, membranes were incubated overnight at 4 °C with a primary antibody in TBS-T and then reacted with a peroxidase-conjugated secondary antibody for 1 h. Immuno reactive proteins were detected with the ECL Western blotting Detection System. shRNA Knockdown. For p53 knockdown, lentiviruses encoding p53 shRNA or control shRNA were purchased from Open Biosystem. HCT116 cells were infected with p53 shRNA lentivirus or control shRNA lentivirus for 12 h. They were then cultured for 2 days. Positive


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clones expressing shRNAs were selected by puromycin (2 µg/ml). Immunoblot analysis was used to determine the expression levels of p53 in these cells. Maximum tolerated dose determination. All in vivo studies were approved by the Nantong University Animal Care and Use Committee. The maximum tolerated dose (MTD) of compound 11c in female ICR mice were determined by intraperitoneal injection of a single dose of 11c (585.9, 468.8, 374.0, 300, and 240 mg/kg) or vehicle control (n = 10 per group), respectively. The MTD was set below the dose that caused severe loss of body weight (>20% of original weight), or death of one or more animals of a dose group. MTD was defined as the highest dose that could be given resulting in no drug-related moribund state or death, while temporary body weight loss was within 20%. In Vivo Tumor Growth Inhibition. Female BALB/c nude mice at the age of 5 to 6-week-old were inoculated subcutaneously with 106 HCT116 cells. When tumor volumes reached 100 to 200 mm3, the mice were randomly administered with 11c, SAHA, and vehicle, respectively. The body weight of all animals was monitored throughout the study and animals were euthanized if they incurred 20 % weight loss between observations. Two axes (mm) of a tumor (L, longest axis; W, shortest axis) were measured with a Vernier caliper. Tumor volume (mm3) was calculated using a formula of “tumor volume = ½ (L×W2)”. Progression of tumors was monitored every 3 days up to 22 days posttreatment. At theend of the experiment, the mice were sacrificed, and their tumors were dissected out and weighed.

AUTHOR INFORMATION Corresponding Author


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*For X.L.: phone, +86-513-85051749; Email, [email protected]. *For Y.Z: phone, +86-513-85051892; Email, [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We gratefully acknowledge the financial support by the Natural Science Foundation of China (Grant Nos. 81302628 and 81473089), the Project of “Jiangsu Six Peaks of Talent” (2014SWYY-044), China Pharmaceutical University for the Open Project Program of State Key Laboratory of Natural Medicines (SKLNMKF201415), Jiangsu Government Scholarship for Overseas Studies (JS-2014-212), and also thank a project funded by the Priority Academic Programs Development of Jiangsu Higher Education Institutions (PAPD). ABBREVIATIONS CRC, Colorectal cancer; DMSO, dimethyl sulfoxide; ESI, electrospray ionization; HDAC, histone deacetylase; HPLC, highperformance liquid chromatography; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PE, petroleum ether; PI, propidium iodide; SAHA, Vorinostat; SD, standard deviation.

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Table of Contents Graphic


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Hydroxamic Acid Hybrids Targeting Both Histone

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