2015 年 63 巻 11 号 p. 882-889
Novel tranylcypromine/hydroxylcinnamic acid hybrids 15a, b, and 19a–l were designed and synthesized by connecting tranylcypromine with hydroxylcinnamic acid, and their biological activities were evaluated. The in vitro assay of their inhibitory activities against lysine-specific demethylase 1 (LSD1) showed that most of the target compounds displayed high potency with IC50 values ranging from submicromolar to single-digit micromolar levels. In particular, compound 19l had robust, selective LSD1 inhibitory activity, which was obviously higher than the inhibitory activity against homologues monoamine oxidase-A (MAO-A) and MAO-B, respectively. Furthermore, the most potent compound 19l selectively inhibited cancer cell but not nontumor colon cell proliferation in vitro. In addition, compound 19l also dose-dependently increased the expression of H3K4me2 at the cellular level. Our findings suggest that tranylcypromine/hydroxylcinnamic acid hybrids as LSD1 inhibitors may hold great promise as therapeutic agents for the treatment of human cancers.
Epigenetic disregulation often leads to the aberrant gene expression programs characteristic of cancer.1,2) Transcriptional regulation through chromatin modification is reversible and dynamic such that enzymes implicated in the disregulation of chromatin represent a new class of protein targets for drug development. Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that belongs to the amine oxidase protein superfamily, which oxidatively cleaves methyl groups of histone H3 at lysine 4 (H3K4me1 and H3K4me2) and lysine 9 (H3K9me1 and H3K9me2) through flavin adenine dinucleotide (FAD)-dependent enzymatic oxidation, and catalyzes methylated lysine substrates to generate hydrogen peroxide and formaldehyde as byproducts3–5) (Fig. 1).
LSD1 is also able to demethylate non-histone substrates, such as the tumor suppressor p53 and the cell cycle and apoptosis regulator E2F1.6,7) Furthermore, LSD1 is often overexpressed in various cancer cells and tissues: neuroblastoma, hepatocarcinomas, colon cancer, breast cancer, gastric cancer, and bladder cancer cells.8–12) LSD1 inhibition decreased expression of target genes in these cancers. Therefore, LSD1 has been considered an important and attractive target for the treatment of cancer.13,14) LSD1 inhibitors are of interest not only as tools to elucidate the biological functions of the enzyme, but also as promising therapeutic agents.
To date, a number of prior LSD1 inhibitors have been reported including monoamine oxidase (MAO) inhibitors such as tranylcypromine (1, PCPA, Fig. 2), pargyline (2), and derivatives thereof (3–6), polyamines (7), peptides (8), as well as guanidine derivatives (9).4,15–20) Among these LSD1 inhibitors, PCPA is a classical MAO inhibitor which has been well studied, and biological studies of PCPA have uncovered the important roles of LSD1 in several cancer diseases.21) PCPA inhibits LSD1 involving an oxidative cyclopropylamine ring-opening reaction through a single-electron transfer mechanism.22) However, currently PCPA and its derivatives display poor selectivity, low potency, or weak antitumor activity, Therefore, identification of novel potent reversible LSD1 inhibitors is essential to further elucidate LSD1’s role in cancer and identify whether or not effective inhibition targeting LSD1 is a viable therapeutic strategy.
As part of our ongoing effort to discover novel anticancer agents, we were inspired by the fact that hydroxylcinnamic acids, such as ferulic acid and p-hydroxycinnamic acid, are known as phenolic compounds occurring in natural plant product, and their derivatives displayed selective antiproliferative activity against some types of cancer cells.23–25) On the basis of the aforementioned information, we conjugated tranylcypromine with hydroxylcinnamic acid to afford lead compounds 15a, b, and different amino acid as the linker connected with tranylcypromine and hydroxylcinnamic acid to design novel hybrids 19a–l. We hypothesized that the novel types of tranylcypromine/hydroxylcinnamic acid hybrids will efficaciously develop inhibitory effect of LSD1 and selectively inhibit tumor cell proliferation. Therefore, a total of twenty target compounds (15a, b, 19a–l) were designed and synthesized, and their in vitro antitumor effects were investigated. Herein, the synthesis and preliminary biological evaluation of these compounds were reported.
The synthetic route to target compounds 15a, b and 19a–l was depicted in Chart 1. Firstly, the alkyl amine of 4-(2-aminocyclopropyl)aniline scaffold 10 was protected by treatment with di-t-butyl-dicarbonate to generate compound 11, and the phenolic hydroxy of 4-hydroxycinnamic acid 12a or ferulic acid 12b was also protected by etherification with chloromethyl methyl ether (MOM-Cl) to give 13a and b. Then compound 11 was respectively reacted with 13a and b in the presence of 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to gain amidated products 14a and b, which was followed treated with saturated HCl in dry AcOEt to obtain targeted compounds 15a and b. In addition, compounds 13a and b were also reacted with different substituted methyl aminoacetates to offer intermediates 16a–l, which was further hydrolyzed with NaOH solution to get compounds 17a–l. Then target compounds 19a–l were prepared from intermediates 17a–l and 11 according to the similar synthetic method of 15a and b. The final products 15a, b, and 19a–l were purified by column chromatography, and their structures were characterized by IR, 1H-NMR, MS, and elemental analyses. All compounds were of >95% purity determined by HPLC.
Reaction conditions and reagents: a) Di-t-butyl-dicarbonate, 1 N NaOH, rt, 10 h, 56%; b) MOMCl, MeCN, NaH, rt, 3 h, 67–71%; c) EDCI, DMAP, CH2Cl2, rt, 5–8 h, 65–77%; d) Saturated HCl in dry AcOEt, rt, 6–10 h, 88–94%; e) Different substituted methyl aminoacetate, EDCI, DMAP, CH2Cl2, rt, 5–10 h, 63–72%; f) 1 N NaOH, MeOH, rt, 1–2 h, 86–93%.
All the compounds synthesized in this study were examined in vitro for their inhibitory effect on LSD1 activity with a commercialized assay kit. LSD1 inhibitor 4 was used as a positive control. The inhibitory activity results were shown in Table 1. With the exception of compounds 19b, d, e, and k, most of the compounds exhibited moderate to good potency with IC50 values ranging from submicromolar to single-digit micromolar. Among them, compounds 19f, i, and l (IC50s=0.93–2.1 µM) were more potent than or comparable to positive control 4 (IC50=1.8 µM). Particularly, compound 19l showed the most potent activity to LSD1, which was nearly 2 times higher than that of 4.
 | |||
|---|---|---|---|
| Compd | R | R1 | LSD1 inhibitory activity (IC50a), µM) |
| 4 | — | — | 1.8 |
| 15a | H | — | 7.2 |
| 15b | OCH3 | — | 4.9 |
| 19a | H | H | 6.6 |
| 19b | H | CH2 | >10 |
| 19c | H | CH2Ph | 3.2 |
| 19d | H | CH(CH3)2 | >10 |
| 19e | H | CH(CH3)CH2CH3 | >10 |
| 19f | H |  | 2.1 |
| 19g | OCH3 | H | 4.5 |
| 19h | OCH3 | CH2 | 7.1 |
| 19i | OCH3 | CH2Ph | 1.4 |
| 19j | OCH3 | CH(CH3)2 | 8.9 |
| 19k | OCH3 | CH(CH3)CH2CH3 | >10 |
| 19l | OCH3 |  | 0.93 |
a) Data are expressed as the mean IC50 value from the dose–response curves of at least three independent experiments.
As LSD1 belongs to the MAO family, the highly potent LSD1 inhibitors 19f, i, and l were chosen to assay its homologies MAO-A and MAO-B to assess their selectivity profiles using commercially available kits. As shown in Table 2, the inhibitory effects of compounds 19f, i, and l on MAO-A were nearly 2–4 fold less than those against LSD1, and also less potent than the positive control 4 against MAO-A. In addition, all the compounds exhibited no significant inhibitory effects on MAO-B activities.
| Compd | IC50a) (µM) | ||
|---|---|---|---|
| LSD1 | MAO-A | MAO-B | |
| 4 | 1.8 | 2.3 | >10 |
| 19f | 2.1 | 4.1 | >10 |
| 19i | 1.4 | 3.7 | >10 |
| 19l | 0.93 | 3.6 | >10 |
a) The data are the mean values of IC50 from at least three independent experiments.
Next, in order to explore their antitumor activity, active compounds 19f, i, and l were further evaluated their antiproliferative effects against three human cancer cells HGC-27 (human gastric cancer cells), HCT-116 (human colon carcinoma cells), and LP-1 (human myeloma cells) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in vitro, and 4 was used as a positive control. The results (shown in Table 3) illustrated the IC50 values of active compounds against each tumor cell line. All the tested compounds showed significant antiproliferative activities, which were similar or stronger anticancer activities than positive control 4. Among these compounds, compound 19l (IC50s=5.3–13.4 µM) exhibited higher antiproliferative activities than the other target compounds against three tumor cells, and its IC50 value against HCT-116 cells was nearly 3-fold less than those of 4 (IC50=15.1 µM).
| Compd | Growth inhibition (IC50a), µM) | ||
|---|---|---|---|
| HGC-27 cells | HCT116 cells | LP-1 cells | |
| 4 | 12.8 | 15.1 | 16.3 |
| 19f | 13.5 | 11.8 | 14.5 |
| 19i | 7.9 | 8.8 | 15.2 |
| 19l | 8.1 | 5.3 | 13.4 |
a) The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data are the mean values of IC50 from at least three independent experiments.
Given that these active hybrids showing strong LSD1 and antitumor inhibitory effects, we wonder to know whether those compounds were toxic to the normal cells. So the cytotoxicities of 19l with the highest anticancer activities were further determined for human normal cell CCD841 and human colon carcinoma cells HCT116 by MTT assay. As shown in Fig. 3, treatment with 20 µM 19l promoted above 85% of the inhibition in HCT-116 cells, while the same treatment had little significant effect on the survival of CCD-841 cells (less than 25% of inhibition). Apparently, hybrid 19l had selective cytotoxicity to human cancer cells in vitro.
These cells were incubated with the indicated concentrations of 19l for 48 h. Cell proliferation was assessed using the MTT assay. Data are mean±S.D. of the inhibition (%) from three independent experiments.
To get insight into the preliminarily molecular mechanism underlying the LSD1 inhibition on 19l-treated cells, the effects of 19l on the methylation levels of LSD1 substrates H3K4 were analyzed by Western blot analysis in HCT116 cells. The expression of H3K4 methylation and histone H3 were examined using specific antibodies, which were incubated with the vehicle alone (dimethyl sulfoxide (DMSO)), 19l (1.0 or 5.0 µM), or 4 (5.0 µM). As can be seen in Fig. 4, after 48 h treatment with 19l, there was a dose-dependent increase in the H3K4me2 expression, which was slightly stronger than that of 4 at the same concentration of 5.0 µM. However, there were no significant reproducible changes in H3K4me1, H3K4me3, and histone H3. These results validated that compound 19l can specifically inhibit LSD1 activity at the cellular level in HCT116 cells.
The total levels of histone H3 were used as loading control. HCT116 cells were incubated with or without 19l, or 4 at the indicated concentrations for 48 h and the levels of protein expression were detected using H3 methylation specific antibodies. Data shown are representative images of each protein for three separate experiments.
Analysis of SAR revealed that the inhibitory activity of tranylcypromine/ferulic acid hybrids 19a–f were more potent against LSD1 than that of tranylcypromine/p-hydroxycinnamic acid hybrids 19g–l, which suggested that the electron-donating substitutions (OCH3) on the ferulic acid derivatives may contribute to their inhibitory activities to these molecules. Secondary, hybrids with amino acid linker such as glycine, phenylalanine, or proline, displayed slightly stronger inhibitory effects on LSD1 activity than hybrids 15a and b without linker. Among these amino acid linkers, different amino acid fragments connected between tranylcypromine and hydroxycinnamic acids were also crucial for their LSD1 inhibitory activities in vitro. These hybrid molecules linked with phenylalanine or proline, particularly for proline, exhibited the outstanding inhibitory activities to LSD1 while the hybrids linked with valine or isoleucine showed relatively weak activities.
In summary, two series of novel hybrid molecules 15a, b, and 19a–l were designed and synthesized by coupling the carboxyl group of hydroxylcinnamic acid with tranylcypromine, and their in vitro biological activities were evaluated. It was discovered that most of target compounds showed promising LSD1 inhibitory activities, particularly for 19f, i, and l, which had significant LSD1 inhibitory and antiproliferative activities comparable to or slightly stronger than positive control 4 against human carcinoma cells. Furthermore, compound 19l exhibited significant selectivity for LSD1 over its homologies MAO-A and MAO-B. In addition, compound 19l also could dose-dependently increase the level of H3K4me2 in HCT116 cells. Therefore, our novel work could serve as a foundation for further exploration of novel LSD1 inhibitors for the treatment of cancer.
Melting points were determined on a RDCSY-I capillary apparatus and were uncorrected. The compounds synthesized were purified by column chromatography using silica gel (200–300 mesh) except for recrystallization and TLC using silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical Company, China). Infrared (IR) spectra (KBr) were recorded on a Nicolet Impact 410 instrument (KBr pellet). 1H-NMR spectra were recorded with a Bruker Avance 300 MHz spectrometer at 300 K, using tetramethylsilane (TMS) as an internal standard. MS spectra were recorded on a Mariner Mass Spectrum (electrospray ionization (ESI)). Element analysis was performed on an Eager 300 instrument. 4-(2-Aminocyclopropyl)aniline 10 and compounds 12a and b were commercially available. All solvents were reagent grade and, when necessary, were purified and dried by standards methods. 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.
tert-Butyl (2-(4-Aminophenyl)cyclopropyl)carbamate (11)To a solution of 10 (3.0 g, 20 mmol) and 1 M NaOH (15 mL) in acetone (30 mL) at 0°C was added Boc2O (4.4 g, 20 mmol) by slow dropwise. The solution was allowed to warm to room temperature and stirred for 10 h, and then extracted with AcOEt (30×3 mL). The organic extracts were combined, dried with anhydrous sodium sulfate, filtered, and concentrated to afford 2.8 g of 11 in 56% yield. MS (ESI) m/z=249 [M+H]+.
(E)-3-(4-(Methoxymethoxy)phenyl)acrylic Acid (13a)4-Hydroxycinnamic acid 12a (1.64 g, 10 mmol) was dissolved in acetonitrile (20 mL), and then NaH (0.36 g, 15 mmol) was added at 0°C. The mixture was stirred for 10 min and then added MOMCl (0.96 g, 12 mmol) in acetonitrile (5 mL) and stirred for another 3 h. After the reaction was completed, the solvent was removed under reduced pressure. The crude residue was dissolved in water and washed with ether (10×3 mL). The water layer was acidified with 1 M HCl to pH 4, and the precipitation was collected by filtration to give 13a, yeild 71%. MS (ESI) m/z=209 [M+H]+.
(E)-3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylic Acid (13b)The title compound was obtained from ferulic acid 12b and MOMCl according to the synthetic procedure of 13a in yield 67%. MS (ESI) m/z=239 [M+H]+.
(E)-N-(4-(2-Aminocyclopropyl)phenyl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride (15a)A mixture of compounds 11 (0.25 g, 1.01 mmol) and 13a (0.2 g, 0.96 mmol), EDCI (0.27 g, 1.44 mmol) and catalytic amounts of DMAP in CH2Cl2 (10 mL) was stirred at room temperature overnight. Then 20 mL of CH2Cl2 was added and the mixture was washed with water (30 mL×3) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to afford the crude product, which was then purified by column chromatography to give compound 14a. Then compound 14a was dissolved in 15 mL of saturated HCl in dry AcOEt and the reaction was stirred at room temperature for 8 h. The solvent was evaporated in vacuo to give compound 15a, yield 61%. Analytical data for 15a: IR (KBr, cm−1): 3465, 3225, 2926, 1712, 1629, 1585, 1241, 1192; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.29 (br s, 1H, PhNHCO), 8.34 (br s, 3H, NH3Cl), 7.70 (d, J=6.9 Hz, 2H, ArH), 7.59 (d, 2H, J=6.9 Hz, Ar-H), 7.40 (d, 1H, J=16.2 Hz, ArCH=), 7.13–7.18 (m, 4H, Ar-H), 6.55 (d, 1H, J=16.2 Hz, CH=CH-Ar), 2.78 (m, 1H, CHN), 2.27 (m, 1H, PhCH), 1.35 (m, 1H, CHH cyclopropane), 1.23 (m, 1H, CHH cyclopropane); MS (ESI) m/z=330 [M]+. Anal. Calcd for C18H19ClN2O2: C, 65.35; H, 5.79; N, 8.47. Found: C, 65.28; H, 5.92; N, 8.41.
(E)-N-(4-(2-Aminocyclopropyl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide Hydrochloride (15b)The title compound was obtained from compounds 11 and 13b according to the synthetic procedure of 15a in yield 57%. Analytical data for 15b: IR (KBr, cm−1): 3448, 3220, 2933, 1710, 1618, 1570, 1245, 1183; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.21 (br s, 1H, PhNHCO), 8.31 (br s, 3H, NH3Cl), 7.68 (d, 2H, J=6.9 Hz, ArH), 7.41 (d, 1H, J=16.2 Hz, ArCH=), 7.13–7.22 (m, 5H, Ar-H), 6.61 (d, 1H, J=16.2 Hz, CH=CH-Ar), 2.75 (m, 1H, CHN), 2.28 (m, 1H, PhCH), 1.33 (m, 1H, CHH cyclopropane), 1.19 (m, 1H, CHH cyclopropane); MS (ESI) m/z=360 [M]+. Anal. Calcd for C19H21ClN2O3·H2O: C, 60.24; H, 6.12; N, 7.39. Found: C, 60.08; H, 6.27; N, 7.31.
(E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)acetic Acid (17a)To a solution of 13a (0.2 g, 0.96 mmol) in dry CH2Cl2 (10 mL), methyl 2-aminoacetate (0.10 g, 1.12 mmol), EDCI (0.27 g, 1.44 mmol) and catalytic amounts of DMAP was added and the mixture was stirred at room temperature overnight. Then 20 mL of CH2Cl2 was added and the mixture was washed with water (30 mL×3) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated in vacuo, and the crude product was purified by column chromatography to give 16a, which was then dissolved in 5 mL methanol containing 1.5 mL 2 M NaOH. The reaction was stirred and refluxed for 3 h, and then cooled. The solvent was evaporated in vacuo, the residue was neutralized to pH=5 with 1 M HCl. The precipitate was filtered, washed with water, and dried in vacuum to afford 17a in yield 69%. MS (ESI) m/z=266 [M+H]+.
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)propanoic Acid (17b)The title compound was obtained from 13a and (S)-methyl 2-aminopropanoate according to the synthetic procedure of 17a in yield 67%. MS (ESI) m/z=280 [M+H]+.
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)-3-phenylpropanoic Acid (17c)The title compound was obtained from 13a and (S)-methyl 2-amino-3-phenylpropanoate according to the synthetic procedure of 17a in yield 66%. MS (ESI) m/z=356 [M+H]+.
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)-3-methylbutanoic Acid (17d)The title compound was obtained from 13a and (S)-methyl 2-amino-3-methylbutanoate according to the synthetic procedure of 17a in yield 66%. MS (ESI) m/z=368 [M+H]+.
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)-3-methylpentanoic Acid (17e)The title compound was obtained from 13a and (2S,3R)-methyl 2-amino-3-methylpentanoate according to the synthetic procedure of 17a in yield 63%. MS (ESI) m/z=322 [M+H]+.
(S,E)-1-(3-(4-(Methoxymethoxy)phenyl)acryloyl)pyrrolidine-2-carboxylic Acid (17f)The title compound was obtained from 13a and (S)-methyl pyrrolidine-2-carboxylate according to the synthetic procedure of 17a in yield 61%. MS (ESI) m/z=306 [M+H]+.
(E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylamido)acetic Acid (17g)The title compound was obtained from 13b and methyl 2-aminoacetate according to the synthetic procedure of 17a in yield 65%. MS (ESI) m/z=296 [M+H]+.
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylamido)propanoic Acid (17h)The title compound was obtained from 13b and (S)-methyl 2-aminopropanoate according to the synthetic procedure of 17a in yield 67%. MS (ESI) m/z=310 [M+H]+.
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylamido)-3-phenylpropanoic Acid (17i)The title compound was obtained from 13b and (S)-methyl 2-amino-3-phenylpropanoate according to the synthetic procedure of 17a in yield 63%. MS (ESI) m/z=386 [M+H]+.
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylamido)-3-methylbutanoic Acid (17j)The title compound was obtained from 13b and (S)-methyl 2-amino-3-methylbutanoate according to the synthetic procedure of 17a in yield 60%. MS (ESI) m/z=338 [M+H]+.
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylamido)-3-methylpentanoic Acid (17k)The title compound was obtained from 13b and (2S,3R)-methyl 2-amino-3-methylpentanoate according to the synthetic procedure of 17a in yield 62%. MS (ESI) m/z=352 [M+H]+.
(S,E)-1-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acryloyl)pyrrolidine-2-carboxylic Acid (17l)The title compound was obtained from 13b and (S)-methyl pyrrolidine-2-carboxylate according to the synthetic procedure of 17a in yield 57%. MS (ESI) m/z=336 [M+H]+.
(E)-N-(2-((4-(2-Aminocyclopropyl)phenyl)amino)-2-oxoethyl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride (19a)The title compound was obtained from compounds 11 and 17a according to the synthetic procedure of 15a in yield 59%. Analytical data for 19a: IR (KBr, cm−1): 3432, 2941, 1725, 1620, 1577, 1265, 1187; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.31 (br s, 1H, PhNHCO), 10.16 (br s, 1H, NHCO), 8.33 (br s, 3H, NH3Cl), 7.71 (d, J=6.9 Hz, 2H, ArH), 7.59 (d, 2H, J=6.9 Hz, Ar-H), 7.41 (d, 1H, J=16.2 Hz, ArCH=), 7.15–7.19 (m, 4H, Ar-H), 6.57 (d, 1H, J=16.2 Hz, CH=CH-Ar), 3.73 (m, 2H, NCH2), 2.78 (m, 1H, CHN), 2.29 (m, 1H, PhCH), 1.36 (m, 1H, CHH cyclopropane), 1.22 (m, 1H, CHH cyclopropane); MS (ESI) m/z=387 [M]+. Anal. Calcd for C20H22ClN3O3: C, 61.93; H, 5.72; N, 10.83. Found: C, 61.76; H, 5.85; N, 10.69.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-1-oxopropan-2-yl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride (19b)The title compound was obtained from compounds 11 and 17b according to the synthetic procedure of 15a in yield 55%. Analytical data for 19b: IR (KBr, cm−1): 3440, 3236, 2942, 1718, 1620, 1573, 1258, 1176; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.27 (br s, 1H, PhNHCO), 10.11 (br s, 1H, PhNHCO), 8.36 (br s, 3H, NH3Cl), 7.71 (d, J=6.9 Hz, 2H, ArH), 7.61 (d, 2H, J=6.9 Hz, Ar-H), 7.41 (d, 1H, J=16.2 Hz, ArCH=), 7.15-7.19 (m, 4H, Ar-H), 6.56 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.75 (m, 1H, NCHCO), 2.80 (m, 1H, CHN), 2.28 (m, 1H, PhCH), 1.35-1.38 (m, 4H, CHH cyclopropane, NCHCH3), 1.23 (m, 1H, CHH cyclopropane); MS (ESI) m/z=401 [M]+. Anal. Calcd for C21H24ClN3O3: C, 62.76; H, 6.02; N, 10.46. Found: C, 62.58; H, 6.23; N, 10.35.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride (19c)The title compound was obtained from compounds 11 and 17c according to the synthetic procedure of 15a in yield 51%. Analytical data for 19c: IR (KBr, cm−1): 3455, 3228, 1726, 1622, 1573, 1246, 1182; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.13 (br s, 1H, NHCO), 8.36 (br s, 3H, NH3Cl), 7.69 (d, 2H, J=6.9 Hz, ArH), 7.58 (d, 2H, J=6.9 Hz, Ar-H), 7.41 (d, 1H, J=16.2 Hz, ArCH=), 7.22–7.28 (m, 3H, Ar-H), 7.15–7.18 (m, 6H, Ar-H), 6.60 (d, 1H, J=16.2 Hz, CH=CH-Ar), 5.11 (m, 1H, NCHCO), 3.09 (d, 2H, J=6.0 Hz, PhCH2), 2.77 (m, 1H, CHN), 2.29 (m, 1H, PhCH), 1.36 (m, 1H, CHH cyclopropane), 1.21 (m, 1H, CHH cyclopropane); MS (ESI) m/z=477 [M]+. Anal. Calcd for C27H28ClN3O3·3H2O: C, 60.95; H, 6.44; N, 7.90. Found: C, 60.78; H, 6.58; N, 7.79.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-hydroxyphenyl)acrylamido)-3-methylbutanamide Hydrochloride (19d)The title compound was obtained from compounds 11 and 17d according to the synthetic procedure of 15a in yield 53%. Analytical data for 19d: IR (KBr, cm−1): 3446, 3234, 2938, 1724, 1617, 1575, 1252, 1181; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.15 (br s, 1H, NHCO), 8.19 (br s, 3H, NH3Cl), 7.66 (d, 2H, J=6.9 Hz, ArH), 7.57 (m, 2H, Ar-H), 7.39 (d, 1H, J=16.2 Hz, ArCH=), 7.13–7.17 (m, 4H, Ar-H), 6.58 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.65 (m, 1H, NCHCO), 2.73 (m, 1H, CHN), 2.66 (m, 1H, NCHCH), 2.27 (m, 1H, PhCH), 1.35 (m, 1H, CHH cyclopropane), 1.17 (m, 1H, CHH cyclopropane), 0.93 (d, 6H, J=7.5 Hz, 2×CH3); MS (ESI) m/z=429 [M]+. Anal. Calcd for C23H28ClN3O3: C, 64.25; H, 6.56; N, 9.77. Found: C, 64.06; H, 6.68; N, 9.62.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-hydroxyphenyl)acrylamido)-3-methylpentanamide Hydrochloride (19e)The title compound was obtained from compounds 11 and 17e according to the synthetic procedure of 15a in yield 53%. Analytical data for 19e: IR (KBr, cm−1): 3471, 3242, 2940, 1728, 1621, 1569, 1250, 1186; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.25 (br s, 1H, NHCO), 8.28 (br s, 3H, NH3Cl), 7.70 (d, J=6.9 Hz, 2H, ArH), 7.59 (m, 2H, Ar-H), 7.40 (d, 1H, J=16.2 Hz, ArCH=), 7.13–7.18 (m, 4H, Ar-H), 6.56 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.55 (m, 1H, NCHCO), 2.76 (m, 1H, CHN), 2.28 (m, 1H, PhCH), 2.17 (m, 1H, NCHCH), 1.36 (m, 1H, CHH cyclopropane), 1.15–1.21 (m, 4H, CHH cyclopropane, CH3), 0.92 (m, 3H, CH3); MS (ESI) m/z=443 [M]+. Anal. Calcd for C24H30ClN3O3·0.5H2O: C, 63.64; H, 6.90; N, 9.28. Found: C, 63.78; H, 7.02; N, 9.36.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-1-((E)-3-(4-hydroxyphenyl)acryloyl)pyrrolidine-2-carboxamide Hydrochloride (19f)The title compound was obtained from compounds 11 and 17f according to the synthetic procedure of 15a in yield 48%. Analytical data for 19f: IR (KBr, cm−1): 3457, 3234, 2918, 1719, 1621, 1568, 1255, 1178; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.36 (br s, 1H, PhNHCO), 8.37 (br s, 3H, NH3Cl), 7.72 (d, J=6.9 Hz, 2H, ArH), 7.61 (d, 2H, J=6.9 Hz, Ar-H), 7.42 (d, 1H, J=16.2 Hz, ArCH=), 7.17–7.19 (m, 4H, Ar-H), 6.56 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.46 (m, 1H, NCHCO), 3.48 (m, 2H, NCH2), 2.81 (m, 1H, CHN), 2.30 (m, 1H, PhCH), 2.03 (m, 2H, NCHCH2), 1.68 (m, 2H, NCH2CH2), 1.38 (m, 1H, CHH cyclopropane), 1.24 (m, 1H, CHH cyclopropane); MS (ESI) m/z=427 [M]+. Anal. Calcd for C23H26ClN3O3: C, 64.55; H, 6.12; N, 9.82. Found: C, 64.46; H, 6.29; N, 9.71.
(E)-N-(2-((4-(2-Aminocyclopropyl)phenyl)amino)-2-oxoethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide Hydrochloride (19g)The title compound was obtained from compounds 11 and 17g according to the synthetic procedure of 15a in yield 56%. Analytical data for 19g: IR (KBr, cm−1): 3477, 3248, 2935, 1726, 1615, 1568, 1250, 1172; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.35 (br s, 1H, PhNHCO), 10.17 (br s, 1H, NHCO), 8.39 (br s, 3H, NH3Cl), 7.77 (d, 2H, J=6.9 Hz, ArH), 7.43 (d, 1H, J=16.2 Hz, ArCH=), 7.13–7.22 (m, 5H, Ar-H), 6.65 (d, 1H, J=16.2 Hz, CH=CH-Ar), 3.76 (m, 2H, NCH2), 2.83 (m, 1H, CHN), 2.32 (m, 1H, PhCH), 1.38 (m, 1H, CHH cyclopropane), 1.25 (m, 1H, CHH cyclopropane); MS (ESI) m/z=417 [M]+. Anal. Calcd for C21H24ClN3O4: C, 60.36; H, 5.79; N, 10.06. Found: C, 60.17; H, 5.93; N, 9.92.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-1-oxopropan-2-yl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride (19h)The title compound was obtained from compounds 11 and 17h according to the synthetic procedure of 15a in yield 51%. Analytical data for 19h: IR (KBr, cm−1): 3468, 3232, 1715, 1622, 1565, 1253, 1184; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.14 (br s, 1H, NHCO), 8.34 (br s, 3H, NH3Cl), 7.72 (d, J=6.9 Hz, 2H, ArH), 7.60 (m, 2H, Ar-H), 7.41 (d, 1H, J=16.2 Hz, ArCH=), 7.11–7.18 (m, 5H, Ar-H), 6.58 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.77 (m, 1H, NCHCO), 2.83 (m, 1H, CHN), 2.29 (m, 1H, PhCH), 1.36–1.42 (m, 4H, CHH cyclopropane, NCHCH3), 1.23 (m, 1H, CHH cyclopropane); MS (ESI) m/z=431 [M]+. Anal. Calcd for C22H26ClN3O4·0.5H2O: C, 59.93; H, 6.17; N, 9.53. Found: C, 60.04; H, 6.36; N, 9.60.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride (19i)The title compound was obtained from compounds 11 and 17i according to the synthetic procedure of 15a in yield 52%. Analytical data for 19i: IR (KBr, cm−1): 3435, 3218, 1732, 1618, 1576, 1252, 1171; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.11 (br s, 1H, NHCO), 8.30 (br s, 3H, NH3Cl), 7.70 (d, 2H, J=6.9 Hz, ArH), 7.39 (d, 1H, J=16.2 Hz, ArCH=), 7.23–7.28 (m, 3H, Ar-H), 7.12–7.19 (m, 7H, Ar-H), 6.59 (d, 1H, J=16.2 Hz, CH=CH-Ar), 5.09 (m, 1H, NCHCO), 3.10 (d, 2H, J=6.0 Hz, PhCH2), 2.73 (m, 1H, CHN), 2.27 (m, 1H, PhCH), 1.34 (m, 1H, CHH cyclopropane), 1.15 (m, 1H, CHH cyclopropane); MS (ESI) m/z=507 [M]+. Anal. Calcd for C28H30ClN3O4: C, 66.20; H, 5.95; N, 8.27. Found: C, 66.08; H, 6.04; N, 8.11.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-hydroxyphenyl)acrylamido)-3-methylbutanamide Hydrochloride (19j)The title compound was obtained from compounds 11 and 17j according to the synthetic procedure of 15a in yield 55%. Analytical data for 19j: IR (KBr, cm−1): 3459, 3228, 1716, 1621, 1568, 1246, 1178; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.28 (br s, 1H, PhNHCO), 8.33 (br s, 3H, NH3Cl), 7.70 (d, J=6.9 Hz, 2H, ArH), 7.59 (d, 2H, J=6.9 Hz, Ar-H), 7.39 (d, 1H, J=16.2 Hz, ArCH=), 7.12–7.18 (m, 5H, Ar-H), 6.56 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.67 (m, 1H, NCHCO), 2.81 (m, 1H, CHN), 2.69 (m, 1H, NCHCH), 2.30 (m, 1H, PhCH), 1.36 (m, 1H, CHH cyclopropane), 1.21 (m, 1H, CHH cyclopropane), 0.95 (d, 6H, J=7.5 Hz, 2×CH3); MS (ESI) m/z=459 [M]+. Anal. Calcd for C24H30ClN3O4: C, 62.67; H, 6.57; N, 9.14. Found: C, 62.53; H, 6.75; N, 9.04.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-hydroxyphenyl)acrylamido)-3-methylpentanamide Hydrochloride (19k)The title compound was obtained from compounds 11 and 17k according to the synthetic procedure of 15a in yield 50%. Analytical data for 19k: IR (KBr, cm−1): 3472, 3244, 2934, 1720, 1616, 1570, 1245, 1181; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.20 (br s, 1H, PhNHCO), 8.28 (br s, 3H, NH3Cl), 7.67 (d, 2H, J=6.9 Hz, ArH), 7.38 (d, 1H, J=16.2 Hz, ArCH=), 7.10–7.19 (m, 5H, Ar-H), 6.58 (m, 1H, CH=CH-Ar), 2.71 (m, 1H, CHN), 4.53 (m, 1H, NCHCO), 2.26 (m, 1H, PhCH), 2.15 (m, 1H, NCHCH), 1.35 (m, 1H, CHH cyclopropane), 1.13–1.18 (m, 4H, CHH cyclopropane, CH3), 0.91 (m, 3H, CH3); MS (ESI) m/z=473 [M]+. Anal. Calcd for C25H32ClN3O4·2H2O: C, 58.87; H, 7.11; N, 8.24. Found: C, 58.68; H, 7.23; N, 8.15.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-1-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)pyrrolidine-2-carboxamide Hydrochloride (19l)The title compound was obtained from compounds 11 and 17l according to the synthetic procedure of 15a in yield 47%. Analytical data for 19l: IR (KBr, cm−1): 3465, 3246, 2911, 1716, 1625, 1560, 1252, 1172; 1H-NMR (DMSO-d6, 300 MHz, δ ppm) 10.24 (br s, 1H, PhNHCO), 8.35 (br s, 3H, NH3Cl), 7.76 (d, 2H, J=6.9 Hz, ArH), 7.41 (d, 1H, J=16.2 Hz, ArCH=), 7.13–7.22 (m, 5H, Ar-H), 6.63 (d, 1H, J=16.2 Hz, CH=CH-Ar), 4.45 (m, 1H, NCHCO), 3.46 (m, 2H, NCH2), 2.80 (m, 1H, CHN), 2.31 (m, 1H, PhCH), 2.04 (m, 2H, NCHCH2), 1.68 (m, 2H, NCH2CH2), 1.38 (m, 1H, CHH cyclopropane), 1.23 (m, 1H, CHH cyclopropane); MS (ESI) m/z=457 [M]+. Anal. Calcd for C24H28ClN3O4: C, 62.95; H, 6.16; N, 9.18. Found: C, 63.06; H, 6.24; N, 9.01.
Enzyme AssayLSD1 inhibition. The LSD1 screening biochemical assay kit was purchased from Cayman Chemical (700120). Test compounds were diluted to 20× the desired test concentration in 100% DMSO and 2.5 µL of the diluted drug sample was added to a black 96-well plate (Corning, cat. 3693). The LSD1 enzyme stock was diluted with assay buffer, and 40 µL of the diluted LSD1 enzyme was added to the appropriate wells. Substrate, consisting of horseradish peroxidase, H3K4me2 peptide corresponding to the first 21 amino acids of the N-terminal tail of histone H3, and 10-acetyl-3,7-dihydroxyphenoxazine was then added to wells. Resorufin was analyzed on an Envision plate reader with an excitation wavelength of 530 nm and an emission wavelength of 595 nm in order to evaluate the inhibition rate of the tested compound. The IC50 values were calculated using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA, U.S.A.).
MAO-A and MAO-B inhibition. MAO-A and MAO-B were purchased from Sigma (catalogue number M 7316 and M 7441). The MAO Glo Assay kit (Promega, catalogue number V1402) was used to measure the effect of inhibitors on MAO-A and MAO-B activity. The assay was performed at room temperature in 50 µL (25 µL reaction solution+25 µL detection reagent) in 96 well half area white plates (Corning, cat. 3693) on a Tecan Freedom EVO liquid handler (Tecan Group Ltd.). 50 nM MAO-A or 125 nM MAO-B were incubated with different inhibitor concentrations for 15 min at RT in MAO-A or MAO-B Buffer (MAO Glo Assay kit). Reaction was started with the addition of 40 mM MAO substrate for MAO-A or 14 mM for MAO-B. The reaction was stopped after 30 min with the detection reagent. Luminescence was measured after 20 min incubation in the dark using a microplate reader. All compounds were tested twice in duplicate and IC50 values were calculated using GraphPad Prism version 4.0 (GraphPad Software).
Cell CultureHGC-27, HCT-116, LP-1, or human normal cell line CCD841 cells were maintained in 10% fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco, Invitrogen), which were supplemented with 10% fetal calf serum (PAA, Austria) and antibiotics [100 IU/mL penicillin and 100 IU/mL streptomycin (Amresco)]. All of the cell lines were purchased from the Shanghai Institute of Cell Biology (Shanghai, China) and were grown at 37°C in a 5% CO2 atmosphere with medium changes every 2 d.
MTT AssayThe inhibitory effects on cell proliferation of test compounds were investigated by the MTT method. HGC-27, HCT-116, LP-1, or CCD841 cells at a final density of 1.0×104 cells/well were placed in 96-well cell plates overnight and treated with or without different concentrations of test compounds for various periods of time. During the last 4 h culture, the cells were exposed to MTT (5 mg/mL), and the resulting formazan crystals were dissolved in 150 µL of DMSO and measured using a spectrophotometer (Tecan) at a test wavelength of 570 nm. Experiments were conducted in triplicate. Inhibition rate (%)=[(Acontrol−Atreated)/Acontrol]×100%. The IC50 values were calculated using GraphPad Prism version 4.0 (GraphPad Software).
Western Blot AssayThe inhibitory activity of LSD1 was determined by Western blot assay with the total lysates by radio immunoprecipitation assay (RIPA) buffer (Sigma R0278) or histone purified with kit from Epigentek (OP-0006). After harvested and lyzed, the cell lysates (50 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% Bis-tris gel) and transferred onto nitrocellulose membranes. Concentration of whole cell lysates and histone extracts were determined using a Micro BCA Protein Assay Kit (Thermo Scientific 23235). We used 5% bovine serum albumin (BSA) to block the membrane in the case of H3K4me1/me2/me3 antibody and 5% fat-free milk for histone H3 antibody. The target proteins were probed with anti-H3K4me1, anti-H3K4me2, anti-H3K4me3, and anti-H3 antibodies (Biovision, 6864-25, 6865-25, 6866-25, and 6806-50), respectively. The bound antibodies were detected by horseradish peroxidase (HRP)-conjugated second antibodies and visualized using the enhanced chemiluminescent reagent.
The authors declare no conflict of interest.
