(Z)-1-Aryl-3-arylamino-2-propen-1-ones, Highly Active Stimulators of Tubulin Polymerization: Synthesis, Structure Activity Relationship (SAR), Tubulin Polymerization and Cell Growth Inhibition Studies

Abstract

Tubulin, the major structural component of microtubules, is a target for the development of anticancer agents. A series of (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) were synthesized and evaluated for anti-proliferative activity in cell based assay. The most active compound (Z)-1-(2- bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)-prop-2-en-1-one (10ae) was tested in 20 tumor cell lines including multidrug resistant phenotype and was found to induce apoptosis in all these cell lines with similar GI₅₀ values. Flow cytometry studies showed that 10ae arrested the cells in G2/M phase of cell cycle. In addition to G2/M block, these compounds caused microtubule stabilization like paclitaxel and induced apoptosis via activation of the caspase family. The observations made in this investigation demonstrate that (Z)-1-Aryl-3- arylamino-2-propen-1-one (10) represents a new class of microtubule – stabilizing agents.

Introduction

Tubulin containing structures, such as microtubules are important for the formation of the mitotic spindle during the process of mitosis. They play a critical role in cell growth, division and cytoskeletal organization of normal and tumor cells as well as being implicated in motility, shape and intracellular transport.¹ Microtubules are hollow tubes of α- and β-tubulin heterodimers that polymerize parallel to a cylindrical axis. Tubulin binding molecules interfere with the dynamic instability of microtubules and thereby disrupt microtubules inducing cell cycle arrest in the M-phase, forming abnormal mitotic spindles resulting in the apoptotic cell death.² A number of natural compounds (see chart 1) such as paclitaxel (A), epothilone (B), vinblastine (C), combretastatin A-4 (CA4) (D), dolastatin 10 (G), and colchicine (H), attack microtubules by interfering with the dynamics of tubulin polymerization and depolymerization, resulting mitotic arrest.³ However, many clinically useful chemotherapy drugs face substantial limitations, such as drug resistance, high systemic toxicity, complex syntheses, and isolation procedures. This has encouraged scientists to develop new antimitotic agents. Recent studies have reported compounds targeting the colchicine-binding domain of β-tubulins that can act as vasculardisrupting agents, rapidly depolymerizing microtubules of newly formed vasculature to block the blood supply to tumors,⁴ while leaving blood supply to healthy cells intact. These compounds include drug candidates Combretastatin A-4P (CA4P) (E), Oxi4503, R, R₁ = OPO₃Na₂ (F), ZD6126 (I) which are shown in chart 1.

Chart 1.

Examples of Tubulin Interacting Agents

The encouraging antivascular activity of compound E has stimulated significant interest in exploring new antitublin agents. A variety of synthetic small molecules have been reported as inhibitors of polymerization, which compete with the colchicine-binding site of tubulin.⁵ Structurally, they involve various heteroaromatic cores, for instance including the benzothiophene,⁶ benzofuran,⁷ imidazole,⁸ thiozole,⁹ and oxadiazoline,¹⁰ moieties. A number of indole-based compounds, for example 2-aroylindoles,¹¹ 3-aroylindoles,¹² 3-aroyl-2- phenylindoles,⁷ 3-arylthioindoles-2-carboxylate,¹³ and indolyl-3-glyoxamides¹⁴ have shown strong anti-proliferative and tubulin depolymerizing activity.

Compounds like paclitaxel promote tubulin polymerization into microtubules and block microtubule dynamics, leading to abnormal mitosis and subsequent apoptosis.^15,16 Paclitaxel was the first microtubule-stabilizing agents (MSA) discovered.¹⁷ It promotes the in vitro polymerization of tublin in the presence of guanosine triphosphate (GTP), which is normally required for microtubules assembly, and in cells, paclitaxel induces microtubule stabilization leading to the formation of characteristic microtubule bundles.¹⁸

Three non-taxoid compounds have also been shown to stabilize microtubules. The macrolides Epothilone A and Epothilone B (B) from the bacterium Sorangium cellulosum,¹⁹ and Discodermolide (J) from the marine sponge Discodermia dissolute,²⁰ share with Paclitaxel the ability to arrest cells in mitosis, cause formation of bundles of intracellular microtubules in nonmitotic cells, and induces the formation of hyperstable tubulin polymers.

A large number of small molecules have been described in the literature which inhibit tubulin polymerization in vitro and in vivo resulting in the arrest of the cells in mitosis, ultimately leading to apoptosis. So far only two reports have described small synthetic molecules that stimulate tubulin polymerization and microtubule stabilization.^21,22 In this paper, we describe the synthesis, structure activity relationship (SAR) and mode of action of a new series of (Z)-1-Aryl- 3-arylamino-2-propen-1-one (10) that stimulate tubulin polymerization and stabilizes microtubules..

Chemistry

The synthesis of target compounds (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) is illustrated in Scheme 1. Arylaldehydes 1, 2, 3, 6 were treated with ethynylmagnesium bromide in dry tetrahydrofuran (THF) at 0 °C to room temperature, produces 1-aryl-2-propyn-1-ol (7). Oxidation of 7 with 2-iodoxybenzoic acid (IBX) in the presence of dimethyl sulfoxide (DMSO) gave 1-aryl-2-propyn-1-one (8). Condensation of 8 with aryl amines (9) in ethanol at room temperature resulted in the formation of (Z)-1-aryl-3-arylamino-2-propen-1-one (10) in good yields.

Scheme 1.

Synthesis of (Z)- 1-Aryl-3-arylamino-2-propen-1-one from 1-aryl-2-propyn-1-one^a

^aReagents and conditions: (a) Ethynylmagnesium bromide, THF, 0 °C-rt, 8–9 h, 60–70 %; (b) 2-iodoxybenzoic acid, DMSO, rt, 5 h, 70–72 %; (c) ethanol, rt, 4 h, 60–82 %. H₂N Y Y = Aryl or Heteroaryl

Some of the substituted arylaldehydes (2, 3, 6), which are not commercially available were synthesized as shown in the schemes 2, 3 and 4. Chlorination of 3,4,5-trimethoxybenzaldehyde (1c) with sulfuryl chloride (SO₂Cl₂) in the presence of dichloromethane (CH₂Cl₂), afforded 2- chloro-3,4,5-trimethoxybenzaldehyde, 2 (Scheme 2). Similarly the corresponding bromo compound 3 was obtained according to methods reported in literature.²³ Bromination of 3,4,5-trimethoxybenzaldehyde (1c), with N-Bromosuccinimide (NBS) in chloroform at reflux temperature for 3 h, afforded 2-bromo-3,4,5-trimethoxybenzaldehyde, 3 (scheme 3). Whereas, the 2-nitro-3,4,5-trimethoxybenzaldehyde, 6 resulted by the oxidation of (3,4,5-trimethoxy-2-nit-rophenyl)methanol (5) with manganese dioxide (MnO₂) in the presence of chloroform (CHCl₃) at room temperature for 48 h. 5, which in turn was obtained by reduction of methyl 3,4,5- trimethoxy-2-nitrobenzoate (4) with diisobutylaluminum hydride (DIBAH) in the presence of toluene at −50 °C (scheme 4).

Scheme 2.

Synthesis of 2-Chloro-3,4,5-trimethoxybenzaaldehyde^a

^aReagents and Condions: (a) Sulfuryl chloride, dichloromethane, rt, 2 h, 60 %.

Scheme 3.

Synthesis of 2-Bromo-3,4,5-trimethoxybenzaldehyde^a

^aReagents and conditions: (a) NBS, chloroform, reflux, 3 h, 97 %.

Scheme 4.

Synthesis of 2-Nitro-3,4,5-trimethoxybenzaldehyde^a

^aReagents and Condions: (a) Anhydrous toluene, diisobutylaluminum hydride, −50 °C, 30 min, 55 %; (b) chloroform, manganese dioxide, rt, 48 h, 63 %.

Structure-Activity Relationships (SAR)

After the synthesis of these compounds, their in vitro cytotoxicity was assessed using two different human tumor cell lines derived from human prostate (DU145) and leukemic (K562) cancers. The results of the study are presented in Table 1. These studies reveal that the cytotoxicity of the 1-aryl-3-arylamino-2-propen-1-ones (10) is totally dependent on the nature and position of the substituents present on the two aromatic rings. For the purpose of structure activity relationship, we have selected few compounds from a library of enaminones synthesized in our laboratory. The cytotoxicity data (Table 1 and data not shown) clearly shows that the molecules are inactive when both aromatic rings are mono substituted (10a). A moderate cytoxicity was observed when benzoyl aromatic ring was tri substituted at 3^rd, 4^th and 5^th positions with methoxy groups and anilino ring with a methoxy at 2^nd position (10k). A fivefold increase in the activity was observed in 10k when the 2-methoxy group on the anilino ring was relocated to 4^th position (10l). When the pattern of substitution is reversed by keeping 3,4,5- trimethoxy groups on anilino ring and 4-methoxy group on the benzoyl ring (10b), the tumor cellular toxicity is reduced by more than 100 folds. These results show that the molecules with three methoxy groups on the benzoyl ring are more cytotoxic towards tumor cells than the compounds with three methoxy groups on the anilino ring. Once the benzoyl ring was identified as a correct moiety for tri substitution then we looked at the position of the methoxy groups on the ring in modulating the cytotoxicity of the molecule. To analyze the role of the position of the methoxy groups, we have made a number of compounds with 2,4,6-trimethoxy substitutions on the benzoyl ring (10c, 10d, 10e, 10f, 10g and 10h). All the compounds with 2,4,6-trimethoxy benzoyl substituted lost the cytotoxicity effect on the tumor cells compared to the corresponding 3,4,5-trisubstituted benzoyl compounds. It clearly shows that 10l, 10m and 10o which are 3,4,5 trimethoxy benzoyl are 50 fold more active than 10c and 10e which are 2,4,6-trimethoxy benzoyl enaminones. This confirms that the benzoyl ring of the enaminones presented in Table 1 require three methoxy groups at 3^rd,4^th and 5^th position to attain maximum cytotoxicity towards cancer cells. To analyze the effect of additional substituents on the aniline aromatic ring, we have synthesized a number of analogues containing 3-hydroxy-4-methoxy (10p), 3-amino-4-methoxy (10q), 3-fluro-4-methoxy (10r), 3-chloro-4-methoxy (10s) and 2-chloro-5-hydroxy (10t) groups; the cytotoxicity analyses of these analogues showed that the compounds with hydroxy and amino substituents at third position (10p, 10q) exhibited the best activity in the series compared to the halo substituents (10r, 10s, and 10t). These compounds 10p and 10q are 5 fold more active than 10l. In an attempt to further enhance the activity of the molecule, we have replaced aryl anilines with heteroaryl anilines. The replacement of heteroaryl anilines did not improve the cytotoxicity of the molecules (10w, 10x, 10y, 10z, 10aa and 10ab). After assigning 3^rd,4^th and 5^th positions to methoxy groups on benzoyl ring for optimum activity, we looked for the effect of a substituent at the ortho position of the benzoyl ring. It is clear from the cytotoxicity data of 10ac, 10ad, 10ae, 10af, 10ag and 10ah, the inclusion of a halo or a nitro group at the ortho-position moderately improved the activity of the molecules compared to the corresponding ortho-unsubstituted compounds. The SAR analysis of these compounds also shows that replacement of a methoxy (10k and 10l), hydroxy (10i and 10j) or chloro (10m) or trifluoro (10o) atoms or groups in these compounds with trimethoxy groups at 3,4,5 (10v) or 2,4,6 (10u) positions of anilino ring lead to the total loss of activity.

Table 1.

In vitro cytotoxicity of (Z)-1-Aryl-3-arylamino-2-propen-1-one 10

Comp.	X	Y	IC50 (μM)
			DU145	K562
10a	4-OCH3	4-OCH3-C6H4	75	100
10b	4-OCH3	3,4,5-(OCH3)3-C6H2	50	75
10c	2,4,6-(OCH3)3	4-OCH3-C6H4	75	75
10d	2,4,6-(OCH3)3	2-OH-C6H4	25	75
10e	2,4,6-(OCH3)3	4-Cl-C6H4	25	25
10f	2,4,6-(OCH3)3	2,4-(OCH3)2-C6H3	75	75
10g	2,4,6-(OCH3)3	2,4,6-(OCH3)3-C6H2	75	50
10h	2,4,6-(OCH3)3	3,4,5-(OCH3)3- C6H2	35	75
10i	3,4,5-(OCH3)3	2-OH-C6H4	0.25	0.5
10j	3,4,5-(OCH3)3	3-OH-C6H4	0.75	0.5
10k	3,4,5-(OCH3)3	2-OCH3- C6H4	2.5	2.5
10l	3,4,5-(OCH3)3	4-OCH3- C6H4	0.5	0.5
10m	3,4,5-(OCH3)3	4-Cl- C6H4	0.5	0.25
10n	3,4,5-(OCH3)3	4-OCF3- C6H4	3	20
10o	3,4,5-(OCH3)3	4-CF3- C6H4	0.5	0.5
10p	3,4,5-(OCH3)3	3-OH,4-OCH3- C6H3	0.1	0.1
10q	3,4,5-(OCH3)3	3-NH2,4-OCH3-C6H3	0.1	0.25
10r	3,4,5-(OCH3)3	3-F,4-OCH3- C6H3	0.25	0.25
10s	3,4,5-(OCH3)3	3-Cl,4-OCH3-C6H3	0.2	0.25
10t	3,4,5-(OCH3)3	2-Cl,5-OH-C6H3	0.75	2.5
10u	3,4,5-(OCH3)3	2,4,6-(OCH3)3-C6H2	75	50
10v	3,4,5-(OCH3)3	3,4,5-(OCH3)3-C6H2	40	25
10w	3,4,5-(OCH3)3	5-Indolyl	0.2	0.5
10x	3,4,5-(OCH3)3	6-Indolyl	0.1	0.2
10y	3,4,5-(OCH3)3	7-Indolyl	2.5	2.5
10z	3,4,5-(OCH3)3	4-Indazole	0.75	2.5
10aa	3,4,5-(OCH3)3	3-Quinoline	0.5	0.75
10ab	3,4,5-(OCH3)3	2-CH3,5-Indolyl	0.6	0.75
10ac	2-Br,3,4,5-(OCH3)3	2-OH- C6H4	0.25	0.2
10ad	2-Br,3,4,5-(OCH3)3	4-OCH3-C6H4	0.2	0.25
10ae	2-Br,3,4,5-(OCH3)3	3-OH,4-OCH3-C6H3	0.06	0.1
10af	2-Br,3,4,5-(OCH3)3	5-Indolyl	0.15	0.2
10ag	2-Cl,3,4,5-(OCH3)3	3-OH,4-OCH3-C6H3	0.2	0.25
10ah	2-NO2,3,4,5-(OCH3)3	4-OCH3-C6H4	0.6	0.6

Standard Deviation for IC50 values were all within +/− 0.05 μM

Biological Results and Discussions

In vitro anti-tumor effects of 10ae compound

We next tested the activity of the most active compound (10ae) listed in Table 1 against different human tumor cell lines and surprisingly, they were found to induce apoptosis of all of these cell lines with similar GI₅₀ values (selected data shown in Table 2). The wide range of cell killing across multiple tumor types suggests that this compound is inducing tumor cell killing by inhibiting an intrinsically important process of tumor cell division.

Table 2.

Evaluation of 10ae against a panel of Human Tumor Cell lines

Cell Line	Tumor Type	10ae (μM)
BT20	Breast (ER−)	0.75
MCF-7	Breast (ER+)	0.4
SK-BR-3	Breast (ER−)	0.3
BT474	Breast (ER+)	0.1
DU145	Prostate (AR−)	0.06
MIA-Paca2	Pancreatic	0.8
A549	NSCLC	0.9
Hela	Cervical	0.2
COLO-320	Colo-rectal	0.15
Panc-1	Pancreatic	0.2
K562	CML	0.1
SK-MEL-28	Melanoma	0.2
HTB-126	Breast Carcinoma	0.1
Caco-2	Colo-rectal	0.25
Raji	Burkitt’s Lymphoma (B-cell)	0.2
SNU-5	Gastric Carcinoma	0.25
N87	Gastric Carcinoma	0.3
HCT-15	Colo-rectal	0.04
MES-SA	Sarcoma	0.25
MES-SA/DX5	Resistant Sarcoma	0.50

Standard Deviation for IC50 values were all within +/− 0.025 μM

10ae compound is also active against drug resistant tumor cell line

Development of resistance to classical chemotherapeutic agents is widely observed in patients who have not responded or have relapsed after first round of therapy and is the primary cause of treatment failure. Many microtubulin poisons such as paclitaxel, vincristine, and vinblastine are substrates for the multidrug resistant family members. To further investigate the activity of these compounds against MDR positive tumor types, we determined the IC₅₀ value of 10ae using a classical MDR positive cell line. The results shown in Table 2 show a 96 h dose response of the uterine sarcoma cell line MES-SA and the multidrug resistant sub-line MES-SA/DX5²⁴ treated with 10ae. This cell line has been shown to express high levels of P-glycoprotein and is resistant to a number of drugs including doxorubicin, paclitaxel, vincristine, vinblastine, etoposide, mitoxantrone, dactinomycin, and daunorubucin. The activity of our compound was then compared to the activity of paclitaxel (MDR sensitive drug). Our results show that the parental cell line was very sensitive to paclitaxel (IC₅₀ 4 nM) but the MDR positive sub-line was greater than 100 fold resistant (IC₅₀ 750 nM). When the two cell lines were treated with 10ae, both the parental and the MDR positive cell lines were almost equally sensitive to the cell killing activity of the compound.

Effects of 10ae on cell cycle progression of tumor cells

Treatment of cancer cells with 10ae results in a time dependent rounding of the cells indicative of mitotic arrest.²⁵ In order to study the cell cycle effects of 10ae treatment we ran a series of experiments by fluorescence-activated cell sorting (FACS) and signal transduction studies using human prostate cancer cells. DU145 cells which are androgen negative prostate cancer cells were treated with increasing concentrations of 10ae for 24 h. Since these cells were one of the more sensitive cell lines tested, and since DU145 cells have a very predictable cell cycle time making them suitable for cell cycle analysis, these cells were used for the following experiment. ^26–28 Cell cycle analysis performed by FACS analysis revealed that the cells accumulated in G2/M phase of the cell cycle in a concentration-dependent manner. Results presented in Figure 1 show that treatment with 0.25 μM of 10ae resulted in the accumulation of tumor cells in the G2/M phase of the cell cycle with a majority of cells showing G2/M arrest, when treated with 0.5 μM concentration of the compound. The FACS analysis determined that the treated cells had greater than 2N amount of DNA but did not differentiate between G₂ and mitotic phases of the cell cycle. Based on these results we next studied the effects of 10ae on the phosphorylation status of proteins known to be important markers of mitotic arrest and spindle checkpoint activation. Hela cells derived from a human cervical tumor which have been extensively studied as a model cell line for mitotic inhibitors²⁹ were treated with 1 μM concentration of 10ae for various time points and the pattern of expression of Histone H3 and its phosphorylated mitotic (ser10) form were analyzed. Phosphorylation of Histone H3 (ser10) is a widely accepted indicator of mitotic arrest as well as a diagnostic tool for the mitotic index of tumors.^30–33 As shown in figure 2, increased levels of phospho-histone H3 were observed within 6 h after treatment with maximal accumulation at 24 h (121 maximum fold increase). Phosphorylation of BCL-2 has also been known to be a marker for mitosis,³⁴ and this protein became phosphorylated at the same time as histone H3 (88 fold). Concurrently to the accumulation of phospho-histone H3, the protein levels of both cyclin B1 (2.5 fold) and PLK-1(2.5 fold) increased after 6 h of treatment. The expression of these proteins is known to be closely linked to progression through mitosis.^35–37 Phosphorylation of BubR1 is a useful marker of mitotic arrest due to the activation of the spindle checkpoint,^38,39 so we next analyzed the phosphorylation status of this protein. Similar to histone H3 and Bcl-2, BubR1 (2.95 fold) became phosphorylated at 6 h following treatment of cancer cells with 10ae. Taken together, these data strongly suggest that 10ae treatment induces a mitotic arrest, with the activation of spindle checkpoint within 6 h of treatment.

Figure 1.

10ae treatment of cancer cells results in G2/M arrest. DU145, human prostate cancer cells were treated for 24 h with increasing concentrations of 10ae, fixed and stained with propidium iodide and analyzed for their DNA content by flow cytometry. G1 (2N) S-Phase (>2N<4N) and G2/M (4N) cells are indicated solid blue, striped, and solid red, respectively. 10ae treated cells become arrested in G2/M in a dose responsive manner. DU145 cells were plated at a cell density of 1.0×10⁶ cells per 100mm dish. N=3

Figure 2.

10ae treatment induces mitotic arrest. Hela cells were treated with 1 μM 10ae and cells were harvested for western blotting at the indicted times. The blot was sequentially hybridized with antibodies specific for phospho-histone H3, histone H3, cyclin B1, PLK-1, Bub- R1 and GAPDH was used as a loading control. Phosphorylation of histone H3, BubR1 and Bcl- 2 and the accumulation of mitotic specific proteins (cyclin B1 and PLK-1) indicates that 10ae treatment induces a mitotic arrest with cells starting to accumulate in mitosis by 6 h following treatment. Primary antibody dilutions were 1:1000 for all westerns, and 1:10,000 for secondary antibodies. N=3. The numbers below the panel represents the fold increase, calculated as described in materials and methods, of the respective protein, or phospho-protein.

Compound 10ae induces apoptosis in tumor cells

Tumor cells treated with 10ae accumulated in the G2/M phase of the cell cycle in a dosedependent manner and appeared to be unable to exit from this phase, leading to the activation of apoptotic pathways as judged by PARP [Poly(ADP-ribose) polymerase-1] cleavage⁴⁰ which is a marker for caspase activation (Figure 3).

Figure 3.

10ae treatment activates caspase-3 and induces apoptosis. Hela cells were treated with 1.0 μM 10ae for the indicated times and protein lysates were resolved by SDS-PAGE and analyzed for caspase-3 activation by western blotting using anti-PARP specific antibodies. PARP cleavage was observed starting at 24 h. This data indicates that the apoptosis pathway has been activated. Primary antibody dilutions were 1:1000 for all westerns, and 1:10,000 for secondary antibodies. N=3

Compound 10ae kills tumor cells by stabilizing microtubules

The polymerization of microtubules from purified tubulin can be monitored in vitro by measuring an increase in light scattering. This in vitro experiment removes complicating factors, such as microtubule-associated proteins (MAPs), which might be part of a putative target that leads to disruption of microtubules as observed with microscopy. To test our hypothesis that the target of (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) is tubulin, and not a MAP, we monitored the polymerization of tubulin after treatment with 10ag, 10ah and 10ae (Figure 4). In this experiment, paclitaxel, a microtubule stabilizer, enhanced the rate of tubulin polymerization, while vincristine, a microtubule destabilizer, prevented the polymerization of tubulin. Similar to paclitaxel, 10ag, 10ah and 10ae stabilized tubulin polymerization at 1 μM. Compound 10ae was most potent in cytotoxicity assay compared to the other analogues and also from the fig. 4, 10ae is superior to paclitaxel in that it causes more microtubule assembly at the same concentration (1 μM). Thus, the enaminone based compounds stabilize the formation of microtubules in vitro.

Figure 4.

MAP-rich tubulin from bovine brain had been pre-incubated with various drugs (1 μM final) or vehicle dimethyl sulfoxide at room temperature for 5 min before guanosine triphosphate (GTP) was added to start the tubulin polymerization reactions. The reaction was monitored at various time points at OD350nm at 37 °C. Tubulin polymerization enhancer (Taxol) and inhibitor (Vincristine) were included as controls.

Compound 10ae treatment induces abnormal mitotic spindle development

Since 10ae was found to alter tubulin in vitro polymerization kinetics, we next examined the mitotic spindle apparatus in cancer cells treated with 10ae. Hela cells were treated with 10ae for 18 hours and fixed and stained for alpha-tubulin. Figure 5 shows that Hela cells became arrested in mitosis and the mitotic cells had abnormal mitotic spindles. When cells were treated with the 0.25 μM of 10ae, near the GI50 value, the mitotic cells displayed an atypical bipolar metaphase plate in which the tubulin appeared bundled at the poles and some chromosomes were not attached aligned at the metaphase plate (see arrows). When the cells were treated with higher concentrations of 10ae, the mitotic cells acquired multiple tubulin bundles (multipolar) without chromosomes aligned at the metaphase plate with more dispersion of the chromosomes. We compared the mitotic spindle phenotype to cells treated with paclitaxel, tubulin stabilizer, and to cells treated with vincristine, a tubulin depolymerizer. As shown in figure 5, 10ae treatment is unique in its effect on mitotic spindle formation, in that although the mitotic cells multipolar which is similar to paclitaxel, the intensity of the tubulin staining is lower and the the chromosome spreads are different. At lower concentrations, 10ae was similar to paclitaxel in that unbound chromosomes were observed outside the metaphase plate while higher concentrations resulted in a more dispersed chromosome spread with 3 or more tubulin bundles. When one compares 10ae to vincristine treated cells, it is clear that 10ae is not destabilizing tubulin since we do not observe complete loss of tubulin staining.

Figure 5.

10ae treatment results in abnormal microtubule spindle development. Hela cells were treated with 0.25 μM or 0.5 μM of 10ae, 0.005 μM paclitaxel, 0.005 μM vincristine or DMSO for 18 h and fixed and stained with anti-α-tubulin-FITC specific antibodies (green) followed by DAPI to visualize chromosomes (blue). 10ae and paclitaxel treatment resulted in abnormal mitotic cells with chromosomes not aligned at the metaphase plate (see arrows) without complete loss of tubulin polymers as observed in cells treated with vincristine. Cell density was 1.0 × 10⁶ cells per 100mm dish containing 8 coverslips each.

The effects of 10ae treatment on tubulin stability can also be examined by analysis of the tubulin organization of non-mitotic interphase cells. Figure 6 shows that treatment with high concentrations of 10ae did not result in an overall disruption of cytoplasmic microtubule organization as evident in cells treated vincristine. The microtubule organization appeared to be somewhat shortened throughout the cytoplasm, but the overall protofilament structure was still intact. Paclitaxel treatment resulted in bundling of the microtubule fibers near the nucleus, this was not observed with 10ae treatment. This data suggests that 10ae treatment does not depolymerize tubulin but interferes with microtubule spindle development during mitosis without altering cytoplasmic microtubules resulting in a mitotic block and cell death.

Figure 6.

10ae treatment does not alter cytoplasmic microtubules. High concentrations of 10ae (0.5 μM) did not result in bundling of the cytoplasmic microtubules near as seen with paclitaxel (0.005 μM) treatment (arrow) nor did 10ae treatment depolymerize cytoplasmic microtubules as observed in cells treated with vincristine (0.005 μM). Cell density was 1.0 × 10⁶ cells per 100mm dish containing 8 coverslips each.

Conclusion

In this communication, we describe the synthesis of a series of (Z)-1-Aryl-3-arylamino-2- propen-1-ones (10) molecules, which induce apoptotic death of a wide variety of human tumor cell lines at nanomolar concentrations by promoting tubulin polymerization and stabilizing microtubules. This is a first report that shows a group of small synthetic molecules (10) behave like paclitaxel and epothilones in tumor cell killing in nano molar range by promoting tubulin polymerization and microtubule stabilization in in vitro. Our studies show that the cytotoxic activity of the (Z)-1-Aryl-3-arylamino-2-propen-1-ones (10) is completely dependent on the nature and position of the substituents on the two aromatic rings. These structure function studies show that a molecule with an aniline ring having 3-hydroxy, 4-methoxy groups and a benzoyl ring with a bromine atom at ortho and methoxy groups at 3, 4 and 5-positions (10ae) showed optimum biological activity. Biological evaluation of the activity of 10ae shows that this compound is highly active against a wide variety of human tumor cell lines including those that are resistant to the activity of many of the currently used chemotherapeutic agents.

Experimental Section

Chemistry: General Methods

All reagents and solvents were obtained from commercial suppliers and used without further purification unless otherwise stated. Solvents were dried using standard procedures and reactions requiring anhydrous conditions were performed under N₂ atmosphere. Reactions were monitored by Thin Layer Chromatography (TLC) on preloaded silica gel F254 plates (Sigma-Aldrich) with a UV indicator. Column chromatography was performed with Merck 70– 230 mesh silica gel 60 A. Yields were of purified product and were not optimized. Melting points were determined using an Electro thermal Mel-Temp 3.0 micro melting point apparatus and are uncorrected. ¹H NMR spectra were obtained with a Bruker AM 300 and 400 MHz spectrometer. The chemical shifts are reported in parts per million (δ) downfield using tetramethylsilane (SiMe₄) as internal standard. Spin multiplicities are given as s (singlet), d (doublet), dd (double doublet) brs (broad singlet), m (multiplet), and q (quartet). Coupling constants (J values) were measured in hertz (Hz). All LC/MS data were gathered on an Agilent 1200 LC with Agilent 6410 triple quadrupole mass spectrometer detectors. The compound solution was infused into the electrospray ionization source operating positive and negative modes in methanol:water: TFA (50:50:0.1% v/v) at 0.4ml/min. The sample cone (declustering) voltage was set at 100 V. The instrument was externally calibrated for the mass range m/z 100 to m/z 1000. The purity of the final compounds was determined by HPLC and is 95% or higher unless specified otherwise.

Synthesis of 2-Chloro-3,4,5-trimethoxybenzaldehyde (2)

3,4,5-trimethoxybenzaldehyde 1c (1.0 g, 5 mmol) was dissolved in 25 mL of dichloromethane and sulfuryl chloride (0.49 mL, 6.1 mmol) was added and stirred at 10 °C for 2h. After reaction was completed (checked by TLC) the solvent was concentrated under vacuum and washed with hexane to get pure 2-chloro-3,4,5- trimethoxybenzaldehyde 2 with 60% yield. Semi solid; ¹H NMR (CDCl₃, 300 MHz): δ 3.91 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃), 7.27 (s, 1H, Ar-H), 10.39 (s, 1H, H-C=O). MS found (M+H)⁺ (m/z): 231.20, Cacld for C₁₀H₁₁ClO₄ m/z: 230.03.

Synthesis of 2-Bromo-3,4,5-trimethoxybenzaldehyde (3)

To a solution of 3,4,5-trimethoxy benzaldehyde 1c (5.0 g, 25.4 mmol) in 50 mL chloroform (CHCl₃), was added Nbromosuccinimide (5.44 g, 30.5 mmol). The solution was heated at reflux temperature for 3 h. After the reaction was completed by TLC reaction mixture was brought to room temperature, the solution was washed with water and extracted with diethyl ether (Et₂O). The combined extracts were dried with anhydrous Na₂SO₄ and concentrated. The crude bromobenzaldehyde was recrystallized from hexanes and Et₂O to get pure white solid with 97% yield. mp 69–70 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.90 (s, 3H, OCH₃), 3.92 (s, 6H, OCH₃), 7.30 (s, 1H, Ar-H), 10.15 (s, 1H, H-C=O). MS found (M+H)⁺ (m/z): 275.10, Cacld for C₁₀H₁₁BrO₄ m/z: 273.98.

Synthesis of 3,4,5-Trimethoxy-2-nitrobenzaldehyde (6)

Step 1: Synthesis of (3,4,5-Trimethoxy- 2-nitrophenyl)methanol (5)

Methyl 3,4,5-trimethoxy-2-nitrobenzoate 4 (1.5 g, 3.69 mmol) was dissolved in 20 mL of anhydrous toluene, cooled to −50 °C and treated with a solution of diisobutylaluminum hydride (1.0 M solution in hexane, 4.6 mL, 4.6 mmol) over a period of 10 min. After 30 min the reaction was quenched with 10 mL of methanol and brought to room temperature. The resulting reaction mixture was diluted with water and extracted with ethyl acetate. The extract was dried over anhydrous Na₂SO₄ and concentrated under vacuum to give (3,4,5-Trimethoxy-2-nitrophenyl)methanol 5 which was purified by column chromatography (silica gel, 30% ethyl acetate in hexane, R_f = 0.33) as brown solid with 55% yield. mp 59–60 °C; ¹H NMR (CDCl₃, 300 MHz): δ 2.34 (brs, 1H, OH), 3.90 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 6.85 (s, 1H, Ar-H). MS found (M+H)⁺ (m/z): 244.10, Cacld for C₁₀H₁₃NO₆ m/z: 243.07.

Step 2: Synthesis of 3,4,5-Trimethoxy-2-nitrobenzaldehyde (6)

The compound 5 (1.0 g, 4.1 mmol) was dissolved in 50 mL chloroform to which MnO₂ (2.14 g, 24.6 mmol) was added and stirred for overnight and additional portion of MnO₂ (0.53 g, 6.15 mmol) was added and stirred for 12 h. The solids were removed by filtration through a celite pad and washed with chloroform. The chloroform was evaporated in vacuo to get pure compound 6 as brown solid with 63% yield. mp 64–65 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.91 (s, 3H, OCH₃), 3.93 (s, 6H, OCH₃), 7.15 (s, 1H, Ar-H), 9.80 (s, 1H, H-C=O). MS found (M+H)⁺ (m/z): 242.20, Cacld for C₁₀H₁₁NO₆ m/z: 241.06.

General procedure for the synthesis of 1-aryl-2-propyn-1-ol (7)

A solution of aldehyde 1– 6 (5 mmol) in dry tetrahydrofuran (THF) was added to a stirred solution of ethynylmagnesium bromide in THF (0.5 M solution, 7.5 mmol) at 0 °C. The solution was stirred at 0 °C temperature for 2 h then warmed to room temperature and stirred for another 6 -7 h. Saturated aqueous ammonium chloride solution 5 mL was added, the mixture was evaporated in vacuo and partitioned between ethyl acetate and saturated ammonium chloride solution. The organic layer was washed with brine, dried over anhydrous Na₂SO₄ and evaporated in vacuo to get pure compounds and were used for next step without further purification.

1-(4-Methoxyphenyl)prop-2-yn-1-ol (7a)

Starting from 4-methoxybenzaldehyde 1a, 70% of 7a was obtained according to the method described in general procedure 7. liquid; ¹H NMR (CDCl₃, 400 MHz): δ 2.65 (d, 1H, J = 2.2 Hz, ≡−H), 3.79 (s, 3H, OCH₃), 5.37-5-40 (m, 1H, H-COH), 6.87–6.90 (m, 2H, Ar-H), 7.43–7.47 (m, 2H, Ar-H). MS found (M-H₂O+H)⁺ (m/z): 145.20, Cacld for C₁₀H₁₀O₂ m/z: 162.07.

1-(2,4,6-Trimethoxyphenyl)prop-2-yn-1-ol (7b)

Starting from 2,4,6-trimethoxybenzaldehyde 1b, 65% of 7b was obtained as brown solid according to the method described in general procedure 7. mp 119–120 °C; ¹H NMR (CDCl₃, 400 MHz): δ 2.42 (d, 1H, J = 2.3 Hz, ≡−H), 3.80 (s, 3H, OCH₃), 3.86 (s, 6H, OCH₃), 5.80-5-84 (m, 1H, H-C-OH), 6.14 (s, 2H, Ar-H). MS found (M-H₂O+H)⁺ (m/z): 205.30, Cacld for C₁₂H₁₄O₄ m/z: 222.09.

1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-ol (7c)

Starting from 3,4,5-trimethoxybenzaldehyde 1c, 65% of 7c was obtained as brown solid according to the method described in general procedure 7. mp 51–52 °C; ¹H NMR (CDCl₃, 400 MHz): δ 2.62 (d, 1H, J = 2.1 Hz, ≡−H), 3.83 (s, 3H, OCH₃), 3.86 (s, 6H, OCH₃), 5.39-5-54 (m, 1H, H-C-OH), 6.77 (s, 2H, Ar-H). MS found (M-H₂O+H)⁺ (m/z): 205.20, Cacld for C₁₂H₁₄O₄ m/z: 222.09.

1-(2-Chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-ol (7d)

Starting from 2-chloro-3,4,5-trimethoxybenzaldehyde 1d, 70% of 7d was obtained as brown solid according to the method described in general procedure 7. mp 65–66 °C; ¹H NMR (CDCl₃, 400 MHz): δ 2.66 (d, 1H, J = 2.4 Hz, ≡−H), 3.89 (s, 3H, OCH₃), 3.90 (s, 6H, OCH₃), 5.79-5-80 (m, 1H, H-C-OH), 7.14 (s, 1H, Ar-H). MS found (M-H₂O+H)⁺ (m/z): 239.10, Cacld for C₁₂H₁₃ClO₄ m/z: 256.05.

1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-ol (7e)

Starting from 2-bromo-3,4,5-trimethoxybenzaldehyde 1e, 70% of 7e was obtained as brown solid according to the method described in general procedure 7. mp 59–60 °C; ¹H NMR (CDCl₃, 400 MHz): δ 2.66 (d, 1H, J = 2.1 Hz, ≡−H), 3.88 (s, 3H, OCH₃), 3.89 (s, 6H, OCH₃), 5.79-5-80 (m, 1H, H-C-OH), 7.18 (s, 1H, Ar-H). MS found (M-H₂O+H)⁺ (m/z): 283.20, Cacld for C₁₂H₁₃BrO₄ m/z: 300.00.

1-(3,4,5-Trimethoxy-2-nitrophenyl)prop-2-yn-1-ol (7f)

Starting from 2-nitro-3,4,5-trimethoxybenzaldehyde 1f, 60% of 7f was obtained as brown solid according to the method described in general procedure 7. mp 75–76 °C; ¹H NMR (CDCl₃, 400 MHz): δ 2.50 (d, 1H, J = 2.2 Hz, ≡−H), 3.72 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 5.42–5.42 (m, 1H, HC- OH), 6.91 (s, 1H, Ar-H). MS found (M-H₂O+H)⁺ (m/z): 250.20, Cacld for C₁₂H₁₃NO₆ m/z: 267.07.

General procedure for the synthesis of 1-aryl-2-propyn-1-one (8)

A solution of 2-iodoxybenzoic acid (IBX) (12.5 mmol) in dimethyl sulfoxide (DMSO) (90 mL) was stirred for 5 min at room temperature until to get homogeneous. A solution of secondary alcohol 7 (5 mmol) in DMSO (5 mL) was added and the mixture was stirred for 5 h. Water (10 mL) was added and the mixture was stirred at room temperature for 10 min, cooled in ice and partitioned between water and ethyl acetate. The mixture was filtered through celite and the aqueous layer was further extracted with ethyl acetate. The organic extracts were combined, washed with water followed by saturated sodium bicarbonate solutions and brine, dried over sodium sulfate and evaporated in vacuo to get the pure product and was used for next step without further purification.

1-(4-Methoxyphenyl)prop-2-yn-1-one (8a)

Starting from 1-(4-Methoxyphenyl)prop-2-yn- 1-ol 7a, 72% of 8a was obtained as pale yellow solid according to the method described in above mentioned general procedure 8. mp 81–82 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.36 (s, 1H, ≡−H), 3.88 (s, 3H, OCH₃), 6.94–6.96 (m, 2H, Ar-H), 8.11–8.13 (m, 2H, Ar-H). MS found (M+H)⁺ (m/z): 161.30, Cacld for C₁₀H₈O₂ m/z: 160.05.

1-(2,4,6-Trimethoxyphenyl)prop-2-yn-1-one (8b)

Starting from 1-(2,4,6-Trimethoxyphenyl) prop-2-yn-1-ol 7b, 70% of 8b was obtained as pale yellow solid according to the method described in above mentioned general procedure 8. mp 83–84 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.14 (s, 1H, ≡−H), 3.76 (s, 6H, OCH₃), 3.77 (s, 3H, OCH₃), 6.03 (s, 2H, Ar-H). MS found (M+H)⁺ (m/z): 221.20, Cacld for C₁₂H₁₂O₄ m/z: 220.07.

1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one (8c)

Starting from 1-(3,4,5-Trimethoxyphenyl) prop-2-yn-1-ol 7c, 70% of 8c was obtained as pale yellow solid according to the method described in above mentioned general procedure 8. mp 123–124 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.35 (s, 1H, ≡−H), 3.86 (s, 3H, OCH₃), 3.88 (s, 6H, OCH₃), 7.36 (s, 2H, Ar-H). MS found (M+H)⁺ (m/z): 221.20, Cacld for C₁₂H₁₂O₄ m/z: 220.07.

1-(2-Chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (8d)

Starting from 1-(2-Chloro- 3,4,5-trimethoxyphenyl)prop-2-yn-1-ol 7d, 70% of 8d was obtained as brown solid according to the method described in above mentioned general procedure 8. mp 84–85 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.50 (s, 1H, ≡−H), 3.91 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃), 7.44 (s, 1H, Ar-H). MS found (M+H)⁺ (m/z): 255.20, Cacld for C₁₂H₁₁ClO₄ m/z: 254.03.

1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (8e)

Starting from 1-(2-Bromo-3,4,5- trimethoxyphenyl)prop-2-yn-1-ol 7e, 70% of 8e was obtained as brown solid according to the method described in above mentioned general procedure 8. mp 80–81 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.50 (s, 1H, ≡−H), 3.90 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.98 (s, 3H, OCH₃), 7.47 (s, 1H, Ar-H). MS found (M+H)⁺ (m/z): 298.90, Cacld for C₁₂H₁₁BrO₄ m/z: 297.98.

1-(3,4,5-Trimethox-2-nitroyphenyl)prop-2-yn-1-one (8f)

Starting from 1-(3,4,5- trimethoxy-2-nitrophenyl)prop-2-yn-1-ol 7f, 70% of 8f was obtained as brown solid according to the method described in above mentioned general procedure 8. mp 87–88 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.42 (s, 1H, ≡−H), 3.88 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 7.39 (s, 1H, Ar-H). MS found (M+H)⁺ (m/z): 266.30, Cacld for C₁₂H₁₁NO₆ m/z: 265.06.

General procedure for the synthesis of (Z)-1-Aryl-3-arylamino-2-propen-1-one (10)

The ethynylketone 8 (5 mmol), was dissolved in absolute ethanol and arylamine (5 mmol) was added. The reaction was stirred for 4 h at room temperature. After the completion of reaction (checked by TLC), the reaction mixture was diluted with water and filtered the product. The crude product was recrystallized from methanol to get pure compound 10.

(Z)-1-(4-Methoxyphenyl)-3-(4-methoxyphenylamino)prop-2-en-1-one (10a)

Starting from 1-(4-methoxyphenyl)prop-2-yn-1-one 8a and 4-methoxyphenylamine, 76% of 10a was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.71 (30% ethyl acetate in hexane). mp 187–188 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.80 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 5.94 (d, 1H, J = 7.8 Hz, COCH=), 6.88–6.95 (m, 4H, Ar-H), 7.02–7.05 (m, 2H, Ar- H), 7.37–7.42 (dd, 1H, J = 7.8, 12.3 Hz, =CH-NH), 7.90–7.93 (m, 2H, Ar-H), 12.06 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 284.20, Cacld for C₁₇H₁₇NO₃ m/z: 283.12.

(Z)-1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenylamino)prop-2-en-1-one (10b)

Starting from 1-(4-methoxyphenyl)prop-2-yn-1-one 8a and 3,4,5-trimethoxyphenylamine, 74% of 10b was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.43 (30% ethyl acetate in hexane). mp 100–101 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.82 (s, 3H, OCH₃), 3.87 (s, 9H, OCH₃), 5.98 (d, 1H, J = 12.9 Hz, COCH=), 6.31 (s, 2H, Ar-H), 6.94–6.99 (m, 2H, Ar-H), 7.39–7.44 (dd, 1H, J = 7.8, 12.3 Hz, =CH-NH), 7.91–7.94 (m, 2H Ar-H), 12.12 (d, 1H, J = 12.0 Hz, NH). MS found (M+H)⁺ (m/z): 344.20, Calcd for C₁₉H₂₁NO₅ m/z: 343.14.

(Z)-3-(4-Methoxyphenylamino)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (10c)

Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 4-methoxyphenylamine, 77% of 10c was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.28 (30% ethyl acetate in hexane). mp 130–131 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.80 (s, 6H, OCH₃), 3.82 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 5.52 (d, 1H, J = 7.7 Hz, COCH=), 6.14 (s, 2H, Ar-H), 6.95–6.98 (m, 2H, Ar-H), 7.20–7.26 (m, 1H, =CH-NH & 2H, Ar-H), 11.86 (d, 1H, J = 12.2 Hz, NH). MS found (M+H)⁺ (m/z): 344.20, Calcd for C₁₉H₂₁NO₅ m/z: 343.14.

(Z)-3-(2-Hydroxyphenylamino)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (10d)

Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 2-aminophenol, 69% of 10d was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.10 (30% ethyl acetate in hexane). mp 164–165 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.47 (s, 6H, OCH₃), 3.56 (s, 3H, OCH₃), 5.27 (d, 1H, J = 7.4 Hz, COCH=), 5.85 (s, 2H, Ar-H), 6.43–6.54 (m, 3H, Ar- H), 6.80–6.82 (m, 1H, Ar-H), 7.08–7.13 (dd, 1H, J = 7.6, 12.8 Hz, =CH-NH), 11.85 (d, 1H, J = 12.2 Hz, NH). MS found (M+H)⁺ (m/z): 330.20, Calcd for C₁₈H₁₉N₂O₅ m/z: 329.13.

(Z)-3-(4-Chlorophenylamino)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (10e)

Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 4-chlorophenylamine, 72% of 10e was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.26 (30% ethyl acetate in hexane). mp 180–181 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.80 (s, 6H, OCH₃), 3.84 (s, 3H, OCH₃), 5.54 (d, 1H, J = 7.8 Hz, COCH=), 6.13 (s, 2H, Ar-H), 6.97–7.00 (m, 2H, Ar- H), 7.23–7.28 (m, 1H, =CH-NH & 2H, Ar-H), 11.85 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 348.20, Calcd for C₁₈H₁₈ClNO₄ m/z: 347.09.

(Z)-3-(2,4-Dimethoxyphenylamino)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (10f)

Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 2,4-dimethoxyphenylamine, 70% of 10f was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.10 (30% ethyl acetate in hexane). mp 105–106 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.79 (s, 6H, OCH₃), 3.84 (s, 6H, OCH₃), 3.89 (s, 3H, OCH₃), 5.47 (d, 1H, J = 7.6 Hz, COCH=), 6.12 (s, 2H, Ar-H), 6.44–6.50 (m, 2H, Ar-H), 7.03 (d, 1H, J = 8.6 Hz, Ar-H), 7.27–7.33 (dd, 1H, J = 7.6, 12.8 Hz, =CH-NH), 11.90 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 374.30, Calcd for C₂₀H₂₃NO₆ m/z: 373.15.

(Z)-1-(2,4,6-Trimethoxyphenyl)-3-(2,4,6-trimethoxyphenylamino)prop-2-en-1-one (10g)

Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 2,4,6-trimethoxyphenylamine, 80% of 10g was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.06 (30% ethyl acetate in hexane). mp 129–130 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.78 (s, 6H, OCH₃), 3.80 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.86 (s, 6H, OCH3), 5.35 (d, 1H, J = 7.5 Hz, COCH=), 6.12 (s, 2H, Ar-H), 6.17 (s, 2H, Ar-H), 7.85–7.90 (dd, 1H, J = 7.5, 12.7 Hz, =CHNH), 12.00 (d, 1H, J = 12.2 Hz, NH). MS found (M+H)⁺ (m/z): 404.20, Calcd for C₂₁H₂₅NO₇ m/z: 403.16.

(Z)-1-(2,4,6-Trimethoxyphenyl)-3-(3,4,5-trimethoxyphenylamino)prop-2-en-1-one (10h)

Starting from 1-(2,4,6-trimethoxyphenyl)prop-2-yn-1-one 8b and 3,4,5-trimethoxyphenylamine, 75% of 10h was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.08 (30% ethyl acetate in hexane). mp 177–178 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.82- 3.90 (m, 18H, OCH₃), 5.55 (d, 1H, J = 7.6 Hz, COCH=), 6.19 (s, 2H, Ar-H), 6.40 (s, 2H, Ar-H), 7.30–7.37 (dd, 1H, J = 7.6, 12.8 Hz, =CH-NH), 11.98 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 404.20, Calcd for C₂₁H₂₅NO₇ m/z: 403.16.

(Z)-3-(2-Hydroxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10i)

Starting from 1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one 8c and 2-aminophenol, 72% of 10i was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.22 (30% ethyl acetate in hexane). mp 190–191 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.89 (s, 3H, OCH₃), 3.93 (s, 6H, OCH₃), 6.02 (d, 1H, J = 7.7 Hz, COCH=), 6.91 (s, 2H, Ar-H), 7.18–7.26 (m, 4H, Ar- H), 7.57–7.62 (dd, 1H, J = 7.7, 12.8 Hz, =CH-NH), 12.35 (d, 1H, J = 12.4 Hz, NH). MS found (M+H)⁺ (m/z): 330.20, Calcd for C₁₈H₁₉N₂O₅ m/z: 329.13.

(Z)-3-(3-Hydroxyyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10j)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 3-aminophenol, 70% of 10j was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.64 (50% ethyl acetate in hexane). mp 120–121 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.74 (s, 3H, OCH₃), 3.76 (s, 6H, OCH₃), 5.36 (s, 1H, OH), 5.80 (d, 1H, J = 7.8 Hz, COCH=), 6.38–6.51 (m, 3H, Ar- H), 7.01–7.05 (m, 3H, Ar-H), 7.28–7.33 (dd, 1H, J = 8.0, 12.3 Hz, =CH-NH), 11.88 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 330.10, Calcd for C₁₈H₁₉NO₅ m/z: 329.13.

(Z)-3-(2-Methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10k)

Starting from 1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one 8c and 2-methoxyphenylamine, 81% of 10k was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.46 (30% ethyl acetate in hexane). mp 118–119 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.84 (s, 3H, OCH₃), 3.86 (s, 6H, OCH₃), 3.91 (s, 3H, OCH₃), 5.94 (d, 1H, J = 7.8 Hz, COCH=), 6.85–6.97 (m, 4H Ar-H), 7.16 (s, 2H, Ar-H), 7.44–7.49 (dd, 1H, J = 7.6, 12.6 Hz, =CH-NH), 12.10 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 344.20, Calcd for C₁₉H₂₁NO₅ m/z: 343.14.

(Z)-3-(4-Methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10l)

Starting from 1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one 8c and 4-methoxyaniline, 82% of 10l was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.40 (30% ethyl acetate in hexane). mp 107–108 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.81 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.94 (s, 6H, OCH₃), 5.93 (d, 1H, J = 7.8 Hz, COCH=), 6.89–6.92 (m, 2H, Ar-H), 7.04–7.07 (m, 2H, Ar-H), 7.20 (s, 2H, Ar-H), 7.41–7.48 (dd, 1H, J = 7.8, 12.3 Hz, =CHNH), 12.18 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 344.20, Calcd for C₁₉H₂₁NO₅ m/z: 343.14.

(Z)-3-(4-Chlorophenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10m)

Starting from 1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one 8c and 4-chlorophenylamine, 72% of 10m was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.80 (50% ethyl acetate in hexane). mp 147–148 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.92 (s, 3H, OCH₃), 3.94 (s, 6H, OCH₃), 6.01 (d, 1H, J = 8.1 Hz, COCH=), 7.02–7.05 (m, 2H, Ar-H), 7.20 (s, 2H, Ar-H), 7.30–7.33 (m, 2H, Ar-H), 7.42–7.49 (dd, 1H, J = 7.8, 12.3 Hz, =CH-NH), 12.13 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 348.10, Calcd for C₁₈H₁₈ClNO₄ m/z: 347.09.

(Z)-3-(4-Trifluoromethoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10n)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 4-trifluoromethoxyphenylamine 75% of 10n was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.80 (50% ethyl acetate in hexane). mp 107–108 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.92 (s, 3H, OCH₃), 3.94 (s, 6H, OCH₃), 6.02 (d, 1H, J = 7.8 Hz, COCH=), 7.08–7.12 (m, 2H, Ar-H), 7.20–7.24 (m, 4H, Ar-H), 7.42–7.47 (dd, 1H, J = 8.1, 12.3 Hz, =CH-NH), 12.15 (d, 1H, J = 12.0 Hz, NH). MS found (M+H)+ (m/z): 398.10, Calcd for C₁₉H₁₈F₃NO₅ m/z: 397.11.

(Z)-3-(4-Trifluoromethylphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10o)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 4-trifluoromethylphenylamine, 68% of 10o was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.80 (50% ethyl acetate in hexane). mp 92–93 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.86 (s, 3H, OCH₃), 3.87 (s, 6H, OCH₃), 6.01 (d, 1H, J = 8.0 Hz, COCH=), 7.10 (d, 2H, J = 8.4 Hz, Ar-H), 7.15 (s, 2H, Ar-H), 7.43–7.48 (dd, 1H, J = 8.0, 12.0 Hz, =CH-NH), 7.58 (d, 2H, J = 8.5 Hz, Ar- H), 12.05 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)+ (m/z): 382.20, Calcd for C₁₉H₁₈F₃NO₄ m/z: 381.12.

(Z)-3-(3-Hydroxy-4-methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10p)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 2-methoxy-5- aminophenol, 70% of 10p was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.26 (30% ethyl acetate in hexane). mp 142–143 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.59 (s, 3H, OCH₃), 3.62 (s, 3H, OCH₃), 3.65 (s, 6H, OCH₃), 5.64 (d, 1H, J = 7.7 Hz, COCH=), 6.28–6.31(m, 1H, Ar-H), 6.46 (d, 1H, J = 2.6 Hz, Ar-H), 6.53 (d, 1H, J = 8.6 Hz, Ar- H), 6.91 (s, 2H, Ar-H), 7.10–7.15 (dd, 1H, J = 7.7, 12.3 Hz, =CH-NH), 11.80 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 360.10, Calcd for C₁₉H₂₁NO₆ m/z: 359.14.

Synthesis of (Z)-3-(3-Amino-4-methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)-prop-2- en-1-one (10q)

Step 1: Synthesis of (Z)-3-(4-nitro-3-methoxyphenylamino)-1-(3,4,5- trimethoxyphenyl)prop-2-en-1-one

The ethynylketone 8c (1.94 g, 5 mmol), was dissolved in absolute ethanol and 4-methoxy-3-nitrophenylamine (0.84 g, 5mmol) was added. The reaction was stirred for 4 h at room temperature. After the completion of reaction by TLC, the reaction mixture was diluted with water and filtered the product. The crude product was recrystallized from methanol to get pure compound with 74% yield as yellow solid, R_f = 0.40 (30% ethyl acetate in hexane). mp 115–116 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.93 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 3.98 (s, 6H, OCH₃), 6.05 (d, 1H, J = 7.8 Hz, COCH=), 7.13 (d, 1H, J = 9 Hz, Ar-H), 7.21 (s, 2H, Ar-H), 7.28–7.30 (m, 2H, Ar-H), 7.42–7.46 (dd, 1H, J = 7.8, 12.6 Hz, =CH-NH), 12.22 (d, 1H, J = 11.7 Hz, NH). MS found (M+H)⁺ (m/z): 389.20, Calcd for C₁₉H₂₀N₂O₇ m/z: 388.13.

Step 2: Synthesis of (Z)-3-(3-Amino-4-methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)- prop-2-en-1-one (10q)

The above nitro compound (2.0 g, 5.11 mmol) obtained from step1 was dissolved in acetone: water (2:1 ratio) 100 mL and heated to 50 °C for 30 min. After completion of the reaction by TLC, the contents were cooled to room temperature and reaction mixture was extracted with ethyl acetate. The organic extract was washed with water, brine solution and dried over anhydrous Na₂SO₄ and concentrated under vacuum to get crude product. The pure product 10q was obtained by flash chromatography, R_f = 0.22 (30% ethyl acetate in hexane). Yield: 69%; Yellow solid, mp 98–99 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.65 (s, 3H, OCH₃), 3.72 (s, 3H, OCH₃), 3.73–3.79 (m, 6H, OCH₃ & 2H, NH₂), 5.70 (d, 1H, J = 7.7 Hz, COCH=), 6.25–6.30 (m, 2H, Ar-H), 6.55 (d, 1H, J = 8.4 Hz, Ar-H), 7.00 (s, 2H, Ar-H), 7.19–7.24 (dd, 1H, J = 7.7, 12.5 Hz, =CH-NH), (d, 1H, J = 12.0 Hz, NH). MS found (M+H)⁺ (m/z): 359.20, Calcd for C₁₉H₂₂N₂O₅ m/z: 358.15.

(Z)-3-(3-Fluoro-4-methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10r)

Starting from 1-(3,4,5-trimethoxyphenyl)-propynone 8c and 3-fluoro-4-methoxyaniline, 68% of 10r was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.40 (30% ethyl acetate in hexane). mp 110–111 °C; ¹H NMR (CDCl₃, 400 MHz): δ 4.05 (s, 3H, OCH₃), 4.08 (s, 3H, OCH₃), 4.10 (s, 6H, OCH₃), 6.13 (d, 1H, J = 7.8 Hz, COCH=), 6.98- 7.14 (m, 3H, Ar-H), 7.36 (s, 2H, Ar-H), 7.51–7.56 (dd, 1H, J = 7.8, 12.2 Hz, =CH-NH), 12.0 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 362.20, Calcd for C₁₉H₂₀FNO₅ m/z: 361.13.

(Z)-3-(3-Chloro-4-methoxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10s)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 3-chloro-4-methoxyphenylamine, 70% of 10s was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.66 (50% ethyl acetate in hexane). mp 133–134 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.90 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.94 (s, 6H, OCH₃), 5.96 (d, 1H, J = 7.8 Hz, COCH=), 6.90–6.99 (m, 2H, Ar-H), 7.17 (d, 1H, J = 2.5 Hz, Ar-H), 7.19 (s, 2H, Ar-H), 7.35–7.42 (dd, 1H, J = 7.6, 12.6 Hz, =CH-NH), 12.15 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 378.20, Calcd for C₁₉H₂₀ClNO₅ m/z: 377.10.

(Z)-3-(2-Chloro-5-hydroxyphenylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10t)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 3-amino-4-chlorophenol, 73% of 10t was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.56 (50% ethyl acetate in hexane). mp 99–100 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.87 (s, 3H, OCH₃), 3.88 (s, 6H, OCH₃), 6.02 (d, 1H, J = 8.0 Hz, COCH=), 6.40–6.42 (m, 1H, Ar-H), 6.67 (d, 1H, J = 2.1 Hz, Ar-H), 7.16 (s, 2H, Ar-H), 7.34–7.36 (m, 1H, =CH-NH & 1H, Ar-H), 12.20 (d, 1H, J = 12.2 Hz, NH). MS found (M+H)⁺ (m/z): 364.10, Calcd for C₁₈H₁₈ClNO₅ m/z: 363.09.

(Z)-1-(3,4,5-Trimethoxyphenyl)-3-(2,4,6-trimethoxyphenylamino)prop-2-en-1-one (10u)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 2,4,6-trimethoxyphenylamine, 80% of 10u was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.20 (30% ethyl acetate in hexane). mp 132–133 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.78 (s, 3H, OCH₃), 3.86 (s, 6H, OCH₃), 3.87 (s, 3H, OCH₃), 3.89 (s, 6H, OCH₃), 5.81 (d, 1H, J = 7.7 Hz, COCH=), 6.16 (s, 2H, Ar-H), 7.17 (s, 2H, Ar-H), 7.89–7.94 (dd, 1H, J = 7.6, 12.7 Hz, =CHNH), 12.05 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 404.10, Calcd for C₂₁H₂₅NO₇ m/z: 403.16.

(Z)-1-(3,4,5-Trimethoxyphenyl)-3-(3,4,5-trimethoxyphenylamino)prop-2-en-1-one (10v)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 3,4,5-trimethoxyphenylamine, 78% of 10v was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.22 (30% ethyl acetate in hexane). mp 181–182 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.75 (s, 3H, OCH₃), 3.80 (s, 6H, OCH₃), 3.84 (s, 3H, OCH₃), 3.87 (s, 6H, OCH₃), 5.90 (d, 1H, J = 7.8 Hz, COCH=), 6.25 (s, 2H, Ar-H), 7.13 (s, 2H, Ar-H), 7.36–7.41 (dd, 1H, J = 7.8, 12.2 Hz, =CHNH), 12.20 (d, 1H, J = 12.2 Hz, NH); MS found (M+H)⁺ (m/z): 404.20, Calcd for C₂₁H₂₅NO₇ m/z: 403.16.

(Z)-3-(1H-Indole-5-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10w)

Starting from 1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one 8c and 5-aminoindole, 65% of 10w was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.24 (30% ethyl acetate in hexane). mp 174–175 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.98 (s, 3H, OCH₃), 4.01 (s, 6H, OCH₃), 6.01 (d, 1H, J = 7.6 Hz, COCH=), 6.60 (s, 1H, Ar-H), 7.07 (d, 1H, J = 6.7 Hz, Ar-H), 7.29–7.45 (m, 5H, Ar-H), 7.62–7.67 (dd, 1H, J = 7.6, 12.8 Hz, =CH-NH), 8.29 (bs, 1H, NH), 12.38 (d, 1H, J = 12.1 Hz, NH). MS found (M+H)⁺ (m/z): 353.20, Calcd for C₂₀H₂₀N₂O₄ m/z: 352.14.

(Z)-3-(1H-Indole-6-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10x)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 6-aminoindole, 69% of 10x was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.60 (30% ethyl acetate in hexane). mp 131–132 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.86 (s, 3H, OCH₃), 3.88 (s, 6H, OCH₃), 6.07 (d, 1H, J = 8.1 Hz COCH=), 7.16 (s, 2H, Ar-H), 7.19 (s, 1H, Ar-H), 7.48–7.60 (m, 1H, =CH-NH & 1H, Ar-H), 7.68–7.71 (m, 2H, Ar-H), 8.00 (d, 1H, J = 8.4 Hz, Ar- H), 8.72 (brs, 1H, NH), 12.26 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 353.20, Calcd for C₂₀H₂₀N₂O₄ m/z: 352.14.

(Z)-3-(1H-Indole-7-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10y)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 7-aminoindole, 66% of 10y was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.82 (50% ethyl acetate in hexane). mp 86–87 °C; ¹H NMR (CDCl₃, 400 MHz): δ 3.86 (s, 3H, OCH₃), 3.87 (s, 6H, OCH₃), 6.01 (d, 1H, J = 7.4 Hz, COCH=), 6.55 (s, 1H, Ar-H), 6.96–7.09 (m, 3H, Ar-H), 7.15 (s, 2H, Ar-H), 7.40 (d, 1H, J = 7.6 Hz, Ar-H), 7.57–7.62 (dd, 1H, J = 7.8, 12.3 Hz, =CHNH), 8.75 (s, 1H, NH), 12.45 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 353.30, Calcd for C₂₀H₂₀N₂O₄ m/z: 352.14.

(Z)-3-(1H-Indazole-5-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10z)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 4-aminoindazole, 65% of 10z was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.16 (50% ethyl acetate in hexane). mp 160–161 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.92 (s, 3H, OCH₃), 3.95 (s, 6H, OCH₃), 6.00 (d, 1H, J = 7.8 Hz, COCH=), 7.20 (s, 2H, Ar-H), 7.46–7.60 (m, 1H, =CH & 4H, Ar-H), 8.06 (s, 1H, NH), 12.31 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 354.20, Calcd for C₁₉H₁₉N₃O₄ m/z: 353.14.

(Z)-3-(Quinolin-3-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10aa)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 3-aminoquinoline, 64% of 10aa was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.36 (50% ethyl acetate in hexane). mp 120–121 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.92 (s, 3H, OCH₃), 3.95 (s, 6H, OCH₃), 5.96 (d, 1H, J = 7.8 Hz, COCH=), 6.52–6.54 (m, 1H, Ar-H), 6.94–6.97 (m, 1H, Ar-H), 7.13 (brs, 1H, Ar-H), 7.17–7.19 (m, 1H, Ar-H), 7.22 (s, 2H, Ar-H), 7.55–7.62 (m, 1H, =CH-NH & 1H, Ar-H), 8.26 (brs, 1H, Ar-H), 12.35 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 365.10, Calcd for C₂₁H₂₀N₂O₄ m/z: 364.14.

(Z)-3-(2-Methyl-1H-indole-5-ylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10ab)

Starting from 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8c and 5-amino-2- methylindole, 65% of 10ab was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.56 (50% ethyl acetate in hexane). mp 176–177 °C; ¹H NMR (CDCl₃, 400 MHz): δ 2.39 (s, 3H, CH₃), 3.85 (s, 3H, OCH₃), 3.88 (s, 6H, OCH₃), 5.86 (d, 1H, J = 7.6 Hz, COCH=), 6.13 (s, 1H, Ar-H), 6.83–6.85 (m, 1H, Ar-H), 7.16 (s, 2H, Ar-H), 7.17–7.20 (m, 2H, Ar-H), 7.48–7.53 (dd, 1H, J = 7.6, 12.5 Hz, =CH-NH), 7.87 (s, 1H, NH), 12.25 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 367.20, Calcd for C₂₁H₂₂N₂O₄ m/z: 366.16.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-(2-hydroxyphenylamino)prop-2-en-1-one (10ac)

Starting from 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 2-aminophenol, 70% of 10ac was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.66 (50% ethyl acetate in hexane). mp 189–190 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.85 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 5.77 (d, 1H, J = 7.5 Hz, COCH=), 6.78–6.80 (m, 1H, Ar-H), 6.87–6.90 (m, 2H, Ar-H), 6.94 (s, 1H, Ar-H), 7.17–7.20 (m, 1H, Ar-H), 7.56–7.63 (dd, 1H, J = 7.8, 13.2 Hz, =CH-NH), 12.33 (d, 1H, J = 13.2 Hz, NH). MS found (M+H)⁺ (m/z): 408.10, Calcd for C₁₈H₁₈BrNO₅ m/z: 407.04.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-(4-methoxyphenylamino)prop-2-en-1-one (10ad)

Starting from 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 4-methoxyphenylamine, 68% of 10ad was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.74 (50% ethyl acetate in hexane). mp 70–71 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.81 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 5.63 (d, 1H, J = 7.5 Hz, COCH=), 6.86 (s, 1H, Ar-H), 6.89–6.93 (m, 2H, Ar-H), 7.05–7.09 (m, 2H, Ar- H), 7.37–7.44 (dd, 1H, J = 7.5, 12.6 Hz, =CH-NH), 11.94 (d, 1H, J = 12.6 Hz, NH). MS found (M+H)⁺ (m/z): 424.10, Calcd for C₁₉H₂₀BrNO₅ m/z: 421.05.

(Z)1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)-prop-2- en-1-one (10ae)

Starting from 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 2- methoxy-5-aminophenol, 65% of 10ae was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.60 (50% ethyl acetate in hexane). mp 150–151 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.81 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.91 (s, H, OCH₃), 3.92 (3H, OCH₃) 5.64 (d, 1H, J = 7.5 Hz, COCH=), 5.73 (brs, 1H, OH), 6.58–6.62 (m, 1H, Ar-H), 6.76–6.84 (m, 2H, Ar-H), 6.86 (s, 1H, Ar-H), 7.34–7.41 (dd, 1H, J = 7.5, 12.6 Hz, =CH-NH), 11.86 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 438.10, Calcd for C₁₉H₂₀BrNO₆ m/z: 437.05.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-(1H-indol-5-ylamino)prop-2-en-1-one (10af)

Starting from 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8e and 5-aminoindole, 64% of 10af was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.70 (50% ethyl acetate in hexane). mp 109–110 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.78 (s, 3H, OCH₃), 3.83 (s, 6H, OCH₃), 5.54 (d, 1H, J = 7.5 Hz, COCH=), 6.42 (s, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 6.87–7.91 (m, 1H, Ar-H), 7.13–7.18 (m, 1H, Ar-H), 7.25–7.30 (m, 2H, Ar-H), 7.42–7.49 (dd, 1H, J = 7.6, 12.7 Hz, =CH-NH), 8.50 (s, 1H, NH), 12.00 (d, 1H, J = 12.6 Hz, NH). MS found (M+H)⁺ (m/z): 431.10, Calcd for C₂₀H₁₉BrN₂O₄ m/z: 430.05.

(Z)-1-(2-Chloro-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)prop-2- en-1-one (10ag)

Starting from 1-(2-chloro-3,4,5-trimethoxyphenyl)prop-2-yn-1-one 8d and 2- methoxy-5-aminophenol, 65% of 10ag was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.60 (50% ethyl acetate in hexane). mp 102–103 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.89 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 3.93 (s, H, OCH₃), 3.94 (3H, OCH₃) 5.73 (d, 1H, J = 7.5 Hz, COCH=), 5.76 (brs, 1H, OH), 6.59–6.63 (m, 1H, Ar-H), 6.76–6.85 (m, 2H, Ar-H), 6.86 (s, 1H, Ar-H), 7.35–7.42 (dd, 1H, J = 7.5, 12.6 Hz, =CH-NH), 11.93 (d, 1H, J = 12.3 Hz, NH). MS found (M+H)⁺ (m/z): 394.20, Calcd for C₁₉H₂₀ClNO₆ m/z: 393.10.

(Z)-3-(4-Methoxyphenylamino)-1-(3,4,5-trimethoxy-2-nitrophenyl)prop-2-en-1-one (10ah)

Starting from 1-(3,4,5-Trimethox-2-nitroyphenyl)prop-2-yn-1-one 8f and 4-methoxyphenylamine, 60% of 10ah was obtained as yellow solid according to above mentioned general procedure 10. R_f = 0.68 (50% ethyl acetate in hexane). mp 132–133 °C; ¹H NMR (CDCl₃, 300 MHz): δ 3.82 (s, 3H, OCH₃), 3.96 (s, 6H, OCH₃), 4.00 (s, H, OCH₃), 5.63 (d, 1H, J = 7.5 Hz, COCH=), 6.89–6.92 (m, 2H, Ar-H), 6.97 (s, 1H, Ar-H), 7.04–7.07 (m, 2H, Ar-H), 7.39-7.45 (dd, 1H, J = 7.5, 12.6 Hz, =CH-NH), 11.93 (d, 1H, J = 12.9 Hz, NH). MS found (M+H)⁺ (m/z): 389.10, Calcd for C₁₉H₂₀N₂O₇ m/z: 388.13.

Biology

Tissue Culture and Reagents

Paclitaxel was purchased from Sigma. Cell lines were purchased from ATCC. Cell lines were routinely grown in DMEM or RPM1 (CellGro) supplemented with 10% fetal bovine serum (CellGeneration, CO) and 1 unit/mL penicillinstreptomycin (Gibco).

Cytotoxicity Assay

We have tested a number of tumor cell lines using a dose response end point assay system. The cells were grown in either DMEM or RPMI supplemented with 10% fetal bovine serum and 1unit/mL Penicillin-Streptomycin solution. The tumor cells were plated into 6 well dishes at a cell density of 1.0 × 10⁵ cells/mL/well and compounds were added 24 h later at various concentrations. Cell counts were determined from duplicate wells after 96 h of treatment. The total number of viable cells was determined by trypan blue exclusion.

Flow Cytometry

Cancer cell line DU145 (human prostate tumor) cells, were grown in DMEM (Cellgro) supplemented with 10% fetal bovine serum and 1 unit/mL penicillinstreptomycin. The cells were plated onto 100 mm² dishes at a cell density of 1.0 × 10⁶ cells/dish, and 24 h later, they were treated with 2.5 μM of the compound. The cells were harvested 24, 48 and 72 h after treatment. The cells were removed from the plate by trypsin digestion and combined with the non-attached cells found in the medium. The cell pellets were washed in phosphate buffered saline (PBS), and fixed in ice cold 70% ethanol for at least 24 h. The fixed cells were then washed with room temperature PBS and stained with propidium iodide (50 mg/mL) and RNase A (0.5 mg) for 30 min at 37 °C. The stained cells were then analyzed on a Becton-Dickinson (BD) (FACScan) flow cytometer and the data analyzed by cell cycle analysis software (Modfit, BD).

Western Blotting Analysis

Hela cells were harvested at the indicated times and cell pellets were frozen on dry ice before lysis. Cell pellets were lysed (25 mM HEPES, 0.1% Triton-X100, 300 mM NaCl, 20 mM β-glycerophosphate, 0.5 mM DTT, 1.0 mM EDTA, 1.5 mM MgCl₂, pH 7.5, 0.2 mM sodium orthovanadate, and protease inhibitors). 100 μg of each lysate was resolved by 10%-SDS-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose filter paper and hybridized to the following antibodies: phosphospecific histone H3 (Upstate;cat#05-806), histone H3 (upstate;cat#07-690), bcl-2 (BD Biosciences;cat#610558), bub R1 (BD Biosciences;cat#612503), plk-1(Santa Cruz; sc-17783), cyclin B1(Santa Cruz;sc- 594), PARP (BD Biosciences;cat#556362 and GAPDH (Fitzgerald;cat#RDI-TRK5G4-6C5). Following the primary hybridization, the blots were then treated with secondary antibodies conjugated to infrared dyes (IRDye 800 or IRDye 680) and analyzed on an infrared scanning system (Odyssey, Li-Cor Biosciences, NE). All hybridization and washing conditions were performed as specified by the manufacturer. Quantitation of the bands was performed using Odyssey software. Fold increase was determined by first normalizing each band against GAPDH and then dividing that number by the value of the normalized control.

PARP Western

DU145 cells were plated at a density of 3.0 × 10⁶ cells per 150 mm² plate and treated 24 h later with either DMSO or 10ae. The cells were collected at the indicated time points and cell pellets were frozen. The frozen cell pellets were lysed in 1% NP40/PBS lysis buffer containing protease inhibitors. Equal amounts of total cellular protein was then resolved on a 10%-SDS-polyacrylamide gel. The gels were transferred onto nitrocellulose paper (S/S), hybridized with anti-PARP antibodies (BD) and developed using ECL (Perkin-Elmer, MA) solution.

Tubulin Polymerization

MAP-rich tubulin form bovine brain (Cytoskeleton), resuspended in PEM [80 mM PIPES, (pH 6.9), 0.5 mM EGTA, 2 mM MgCl₂], was pre-incubated with drug or its vehicle dimethyl sulfoxide at room temperature for 5 min. PEG containing GTP was added to start the tubulin polymerization reaction. The reaction was monitored by a spectrophotometer at OD350nm at 37 °C. The final concentrations of tubulin and GTP were 1.15 mg/mL and 4 mM, respectively.

Tubulin Staining

Hela cells were grown on glass coverslips and treated with 10ae (0.25 μM, 0.5 uM), paclitaxel (0.005 μM) or vincristine (0.005 μM) for 18 h. The coverslips were washed in phosphate buffered saline and fixed in freshly prepared 4% paraformaldehyde. The cells were blocked and permeabilized and stained with 1:400 dilution of anti-α-tubulin conjugated with FITC (Sigma) followed by staining with DAPI (0.5 ug/mL). The coverslips were mounted onto glass slides using Prolong Gold Antifade, (Molecular Probes/InVitrogen, CA) solution and visualized using an Olympus IX-71 inverted fluorescent microscope at 100x magnification. The cells were photographed using the DP-71 camera system using two filters followed by merging and representative merged images are shown.

ABBREVIATIONS USED

MSA

microtubule-stabilizing agents

GTP

guanosine triphosphate

FACs

fluorescence-activated cell sorting

PLK-1

Polo-like kinase-1

Bcl-2

B-cell lymphoma-2

MAPS

microtubule-associated proteins

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

PARP

Poly(ADP-ribose)polymerase