3,5-Bis(3-alkylaminomethyl-4-hydroxybenzylidene ... - ACS Publications

Note. After this paper was published January 7, 2016, corrections were made to Scheme 3. The corrected version was reposted January 12, 2016...
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3,5-Bis(3-alkylaminomethyl-4-hydroxybenzylidene)-4-piperidones: A Novel Class of Potent Tumor-Selective Cytotoxins Subhas S. Karki,†,# Umashankar Das,*,† Naoki Umemura,‡ Hiroshi Sakagami,‡ Shoko Iwamoto,‡ Masami Kawase,§ Jan Balzarini,∥ Erik De Clercq,∥ Stephen G. Dimmock,⊥ and Jonathan R. Dimmock*,† †

Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan S7H 5C9, Canada ‡ Department of Diagnostic and Therapeutic Sciences, Division of Pharmacology, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0238, Japan § Faculty of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-Chu, Matsuyama, Ehime 790-8578, Japan ∥ Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium ⊥ Department of Finance, Nanyang Technological University, Singapore 639798, Singapore S Supporting Information *

ABSTRACT: Novel 4-piperidone derivatives 2a−f are disclosed as potent cytotoxins. Many of these compounds are more potent than the reference drug melphalan. The compounds in series 2, 4−7 display selective toxicities toward various neoplasms compared to some normal cells. 2a is one of the promising lead molecules that display >11-fold higher growth inhibiting potency than 5fluorouracil against human colon cancer cells. 2a induces apoptosis, DNA fragmentation, and cleavage of poly ADP-ribose polymerase.

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INTRODUCTION The major interest in this laboratory is the development of conjugated unsaturated ketones as candidate cytotoxins. α,βUnsaturated ketones possess a preferential or exclusive affinity for thiols in contrast to amino or hydroxyl groups1,2 which are present in nucleic acids. Hence conjugated unsaturated ketones should be free from the genotoxic effects of many alkylating agents used in cancer chemotherapy.3 Various studies revealed that chemosensitization of some tumor cells makes them more vulnerable to a second chemical attack than normal cells.4,5 Hence the current focus is on developing compounds containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore (ARCHC(R)COC(R)CHAR) which permits sequential alkylation of important cellular thiols such as the isozymes of glutathione S-transferase, thioredoxin reductase, and succinoxidase. These interactions can take place at one of the olefinic carbon atoms and subsequently with the remaining electrophilic center. A number of years ago, the impressive cytotoxicity of 1a toward human Molt4/C8 and CEM T-lymphocytes and murine L1210 leukemic cells was observed6 (Table 1) which rivals the potency of melphalan. However, while 1a (as the hydrochloride salt) demonstrated promising cytotoxic properties toward murine P388 cells, the in vivo result demonstrated only a marginal increase in the median survival time of mice.7 This result may have been due to a variety of factors including facile metabolism and excretion, plasma protein binding, and plasma half-life. In particular, the lipophilicity of 1a may have contributed to its virtual lack of potency. Hence, in order to reduce hydrophobicity, a decision was made to introduce © XXXX American Chemical Society

Scheme 1. Synthesis of Series 1−3

hydroxyl groups in the aryl rings of 1a, the Hansch π value of the aryl hydroxyl group being −0.67.8 The second structural feature incorporated into the aryl rings of 1a in this study is one or more alkylaminoalkyl groups with the consideration that the rate and extent of reaction of these molecules with cellular thiols will be influenced by the magnitude of the atomic charges on the olefinic carbon Received: November 3, 2015

A

DOI: 10.1021/acs.jmedchem.5b01706 J. Med. Chem. XXXX, XXX, XXX−XXX

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were aminomethylated to produce 4a,b and 5 (Scheme 2). 1H NMR spectroscopy revealed that 1b, 2a−f, 3, 4a,b, and 6 adopt

Table 1. Evaluation of 1−7 and Melphalan for Their Antiproliferative Activity against Human Molt 4/C8 and CEM T-Lymphocytes and Murine Leukemia L1210 Cells

Scheme 2. Synthesis of Series 4−7a

IC50 a (μM) compd

Molt 4/C8

CEM

L1210

1a 1b 2a 2b 2c 2d 2e 2f 3 4a 4b 5 6 7 melphalan

1.67 ± 0.15 5.02 ± 2.74 0.428 ± 0.01 0.402 ± 0.08 0.334 ± 0.05 0.400 ± 0.04 1.60 ± 0.18 0.883 ± 0.24 >500 4.05 ± 1.37 61.0 ± 20.1 7.70 ± 0.76 1.06 ± 0.65 3.69 ± 1.54 3.24 ± 0.56

1.70 ± 0.02 13.2 ± 9.20 0.640 ± 0.10 0.530 ± 0.12 0.384 ± 0.14 0.390 ± 0.14 1.67 ± 0.35 1.14 ± 0.24 >500 4.00 ± 1.93 62.1 ± 20.2 7.14 ± 2.82 6.96 ± 2.85 3.38 ± 0.32 2.47 ± 0.21

7.96 ± 0.11 96.8 ± 34.2 1.28 ± 0.18 1.19 ± 0.45 0.774 ± 0.28 0.867 ± 0.25 7.78 ± 1.74 2.00 ± 0.54 >500 7.84 ± 0.33 165 ± 49 13.4 ± 0.3 10.7 ± 3.4 7.74 ± 3.49 2.13 ± 0.02

a

(i) 4-OHC 6 H4 CHO, acetic acid, dry HCl; (ii) N,N,N′,N′tetramethylenediamine (2.2 mol equiv), acetonitrile; (iii) N,N,N′,N′tetramethylenediamine (8 mol equiv), acetonitrile

Scheme 3. Preparation of Intermediates 8 and 9a

a

Compound concentration required to inhibit tumor cell proliferation by 50%.

atoms. For example, in the case of the dimethylaminomethyl group, the Hammett σm value is 0.00 which when protonated [CH2(+)NH(CH3)2] becomes 0.40.9 The pH of a number of tumors is lower than nonmalignant cells,10 and under these circumstances there will be a greater percentage of charged ions in neoplastic tissues that will lower the electron density on the olefinic carbon atoms and toxic effects may well occur preferentially in tumors. These considerations led to the decision to prepare the compounds in series 2−5. In addition to the reaction of cellular thiols with the olefinic carbon atoms of these compounds, the alkylaminoalkyl groups could react at contiguous binding sites thereby enhancing potency. For example, the methylene and methyl groups are available to form van der Waals bonds with different cellular constituents. In addition, the nitrogen atoms of the dialkylaminoalkyl group may form hydrogen bonds and when charged produce ionic bonds with anionic groups at a binding site. Hence the nature of the alkylaminoalkyl group may influence the IC50 values significantly. The objectives of the current investigation are to develop novel potent cytotoxins displaying tumor-specific properties and to investigate the mode of actions of a promising lead compound. In addition, the aspiration was also made to determine various physicochemical properties that may influence the cytotoxicity to gain some insight for further designing of potent cytotoxic agents with improved tumorselective toxicities.

a

(i) Paraformaldehyde (1 mol equiv), amine (1 mol equiv), methanol; (ii) ether, dry HCl; (iii) CH2N+(CH3)2Cl−, acetonitrile.

the E configuration, since the olefinic protons absorb in the region 7.74−7.92 ppm.11 The coupling constants of the olefinic hydrogen atoms in 5 and 7 revealed the E stereochemistry of this molecule. The molecular volumes, torsion angles θA and θB, and log P values of series 1−7 are presented in Table 2. Table 2. Some Physicochemical Constants of Series 1−7

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RESULTS The preparation of 1−7 was accomplished as follows. Acidic catalysis between 4-piperidone and the appropriate aryl aldehydes afforded the dienones in series 1−3 (Scheme 1). As indicated in Scheme 3, the aryl aldehydes 8 required in the syntheses of 2a−f were synthesized by a Mannich reaction between 4-hydroxybenzaldehyde, paraformaldehyde, and the appropriate amine while 9 required in the preparation of 3 was synthesized using dimethylaminomethylene chloride. Condensation between 4-hydroxybenzaldehyde and cyclohexanone or acetone led to the formation of 6 and 7, respectively, which

compd

molecular volume (Å3)

θA (deg)

θB (deg)

log P

1a 1b 2a 2b 2c 2d 2e 2f 3 4a 4b 5 6 7

264.43 280.47 405.88 473.09 452.37 485.97 470.34 511.06 531.30 410.28 535.70 370.72 284.87 245.30

125.04 129.00 127.17 115.84 126.58 117.77 115.28 117.62 110.91 127.77 116.12 164.55 134.46 175.75

54.26 50.65 51.50 56.97 51.05 56.57 64.66 59.01 85.96 50.83 91.85 14.68 46.40 6.03

3.36 2.40 2.79 4.30 3.60 4.61 2.49 2.58 1.84 4.41 3.45 3.62 4.01 3.23

Series 1−7 were screened against human Molt4/C8 and CEM T-lymphocytes as well and murine L1210 leukemia cells. The results are portrayed in Table 1. The cytotoxic potencies of 2a−f, 3, 4a,b, and 5−7 were assessed against human HSC-2, HSC-3, and HSC-4 oral squamous cell carcinomas and human HL-60 promyelocytic leukemic cells. In addition, these compounds were assayed against human HGF gingival fibroblasts, HPC pulp cells, and HPLF periodontal ligament fibroblasts which are nonmalignant cells. These results are summarized in Table 3. A representative lead cytotoxin 2a B

DOI: 10.1021/acs.jmedchem.5b01706 J. Med. Chem. XXXX, XXX, XXX−XXX

caused apoptosis in HSC-2 and HL-60 cells as revealed by it causing internucleosomal DNA fragmentation (Figure 2) and activating caspases 3 and 7 (Figure 3). In addition, 2a cleaved poly ADP-ribose polymerase 1 (PARP1) as indicated in Figure 4.

CC50 values are the concentrations of the compounds required to kill 50% of the cells. bSI refers to the selectivity index, which is obtained by dividing the average CC50 of the nonmalignant cells by the CC50 of a specific neoplastic cell line. cPSE refers to the potency selectivity expression, which is the product of the reciprocal of the average CC50 toward the four tumor cell lines and the average SI multiplied by 100.

3.56 >3.28 9.03 >31.2 3.42 47.3 3.38 >2.11 8.49 >105 4.21 150 2.97 >1.74 6.46 >3.23 3.35 6.56 2.29 >3.57 7.07 >4.00 3.04 8.40 5.60 >5.71 14.1 >12.5 3.09 24.1

6.84 8.68 7.45 7.32 6.05 8.65

0.57 ± 0.02 0.40 ± 0.02 0.38 ± 0.01 0.66 ± 0.06 0.60 ± 0.10 0.69 ± 0.08 >200 5.3 ± 0.33 35 ± 8.2 2.1 ± 0.18 16 ± 6.7 5.3 ± 0.07 8.7 ± 4.2 2a 2b 2c 2d 2e 2f 3 4a 4b 5 6 7 melphalan

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DISCUSSION Series 1−7 were evaluated in the Molt4/C8 and CEM bioassays to ascertain if these dienones are toxic to human transformed cells. The use of the L1210 screen was undertaken, since a number of drugs used in cancer chemotherapy are toxic to this cell line,12 and hence this assay may reveal novel candidate anticancer agents. The biodata are presented in Table 1. The introduction of a hydroxyl group into 1a led to 1b whereby potency was retained but reduced. However, the introduction of one alkylaminoalkyl substituent into each of the aryl rings of 1b led to 2a−f with substantially greater cytotoxic potencies than 1b in all three bioassays. Thus, series 2, in which 61% of the IC50 values are submicromolar, is clearly a novel cluster of potent cytotoxins. Of particular interest are 2a−d which have average IC50 values in the three bioassays of 0.78, 0.71, 0.50, and 0.55 μM, respectively. The placement of two dimethylaminomethyl groups into each of the aryl rings of 1b led to 3 with IC50 values of >500 μM. This result is very surprising; e.g., the difference in potency between 2a and 3 in the Molt4/C8 screen is >1160-fold. The contribution of the piperidinyl and cyclohexyl rings or the absence of a scaffold was addressed. The order of potencies (average IC50 in μM in parentheses) are 7 (4.94) > 6 (6.24) > 1b (38.3); i.e., no scaffold > cyclohexyl analog > piperidone. On the other hand, 2a (0.78) > 4a (5.30) > 5 (9.41), revealing the opposite order of potencies. The introduction of a second dimethylaminomethyl group of 4a leading to 4b lowers potency considerably as was noted when comparing the IC50 values of 2a and 3. The importance of the biodata generated on some of the compounds in series 1−7 was reinforced when comparing their potencies with that of melphalan which is an alkylating agent used in cancer chemotherapy. The following compounds have statistically significant lower IC50 values than melphalan (fold increases in potency in parentheses). In the Molt4/C8 assay, 1a (1.94), 2a (7.54), 2b (8.10), 2c (9.82), 2d (8.10), 2e (2.03), 2f (3.68), and 6 (3.06) are more toxic to the T-lymphocytes than melphalan while 1b, 4a, and 7 are equipotent. The results in the CEM screen revealed increased potency by 1a (1.45), 2a (3.86), 2b (4.66), 2c (6.50), 2d (6.33), 2e (1.48), 2f (2.17), and 4a is equipotent with melphalan. In the L1210 test, 2a (1.66), 2b (1.79), 2c (2.77), and 2d (2.45) are more potent than the established drug while 2f has the same potency. Of particular interest is the observation that in 89% of the comparisons made between the IC50 values of 2a−f and melphalan, the dienones in series 2 are more potent. A question was evaluated whether the relative potencies of series 1−7 are the same toward each cell line. Should this be the case, then the chemical and physicochemical properties of these molecules influence cytotoxic potencies in a similar manner, and it is likely therefore that each of the dienones acts by the same biochemical mechanisms. In this case one is justified in taking the average IC50 in seeking correlations between potencies and one or more physicochemical parameters. Kendall’s coefficient of concordance W is a test for assessing similarity of rankings,13 and the derivation of the equation used in determining W is presented in the Supporting

a

38 >4.36 251 >96 70.1 282

518 679 603 353 532 396

3.90 3.47 2.83 4.83 3.63 5.97 >200 29.7 >200 29.7 >200 16.4 210 1.2 ± 0.16 0.84 ± 0.04 0.77 ± 0.01 1.4 ± 0.09 1.0 ± 0.15 1.7 ± 0.06 >200 13 ± 0.90 56 ± 42 4.2 ± 0.20 50 ± 3.9 5.4 ± 0.12 25 ± 7.7

3.25 4.13 3.68 3.45 3.63 3.51

1.1 ± 0.03 0.94 ± 0.07 0.88 ± 0.01 1.6 ± 0.04 1.1 ± 0.09 1.8 ± 0.10 >200 10 ± 0.35 115 ± 44 4.6 ± 0.09 62 ± 20 4.9 ± 0.07 32 ± 8.8

3.55 3.69 3.22 3.02 3.30 3.32

0.75 ± 0.09 0.84 ± 0.00 0.87 ± 0.02 1.29 ± 0.13 0.71 ± 0.07 1.1 ± 0.01 >200 8.8 ± 0.55 95 ± 1.2 3.5 ± 1.2 1.9 ± 0.21 3.9 ± 0.23 1.4 ± 1.2

5.20 4.13 3.25 3.74 5.11 5.43

0.91 0.76 0.73 1.24 0.85 1.32 >200 9.28 75.3 3.60 32.5 4.88 16.8

4.71 5.16 4.40 4.38 4.52 5.23

4.0 ± 0.03 3.2 ± 0.44 2.9 ± 0.12 5.3 ± 3.2 3.7 ± 1.7 7.5 ± 0.72 >200 35 ± 1.4 >200 35 ± 2.3 >200 24 ± 0.49 161 ± 27

3.0 ± 0.08 3.5 ± 0.05 2.4 ± 0.67 3.7 ± 0.02 3.4 ± 0.09 4.8 ± 1.0 >200 18 ± 0.25 >200 24 ± 1.9 >200 9.2 ± 0.06 269 ± 153

4.7 ± 0.07 3.7 ± 0.12 3.2 ± 0.43 5.5 ± 1.4 3.8 ± 0.66 5.6 ± 1.4 >200 36 ± 1.7 >200 30 ± 3.8 >200 16 ± 1.1 199 ± 60

PSEc av CC50 HPLF HPC av CC50 SIb HL-60 SIb HSC-4 SIb HSC-3 SIb HSC-2 compd

human tumor cell line, CC50 (μM)a

Table 3. Evaluation of 2a−f, 3, 4a,b, and 5−7 against Human Neoplastic and Nonmalignant Cell Lines

av SI

HGF

human nonmalignant cell, CC50 (μM)a

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.5b01706 J. Med. Chem. XXXX, XXX, XXX−XXX

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However, the rise in lipophilicity may prove to be detrimental when in vivo studies are undertaken. Series 2−7 were also assessed against HSC-2, HSC-3, HSC4, and HL-60 neoplasms. These results are portrayed in Table 3. In general, the relative cytotoxic potencies are similar to the biodata presented in Table 1. Thus, 2a−f have the lowest CC50 values, the dienone 3 is by far the least toxic compound, while 4a is more potent than 4b and 6. In regard to series 2, 58% of the CC50 values are submicromolar and these compounds are all substantially more potent than melphalan toward HSC-2, HSC-3, and HSC-4 cells; e.g., 2c is 36 times more potent than the reference drug in the HSC-4 bioassay. The unsaturated ketones in series 2−7 were examined for their toxic effects toward HGF, HPC, and HPLF nonmalignant cells to discover whether selective toxicity toward neoplasms is demonstrated. The results are indicated in Table 3. Under clinical conditions, a tumor will be surrounded by different types of normal cells. Hence selectivity index (SI) values were calculated by comparing the CC50 values of a compound against a particular neoplastic cell line with the average CC50 values toward the three nonmalignant cells. All of the compounds demonstrated tumor-selectivity. The highest average SI in excess of 31 was demonstrated by 6, and in particular its selective toxicity for HL-60 cells is noteworthy. A promising lead compound should demonstrate significant potency toward neoplasms coupled with greater toxicity toward tumors than normal cells. To identify such molecules, the potency selectivity expression (PSE) values for series 2, 4−7 were determined which are the products of the reciprocal of the average CC50 of the compound multiplied by the average SI and 100. The PSE values are listed in Table 3. All of the compounds in series 2 have excellent PSE values and are considerably greater than the value obtained for melphalan. In addition, the PSE of 5 is encouraging. To investigate some of the mechanisms whereby a representative compound 2a exerts its cytotoxic effects, the following experimentation was undertaken. First, 2a causes internucleosomal DNA fragmentation in HSC-2 and HL-60 cells as indicated in Figure 2 which reveals that 2a causes

Information section S4. If the ranking for the Molt4/C8, CEM, and L1210 screens are the same, then W is 1; conversely if there is no agreement, then W is zero. In the case of series 1−7, Kendall’s coefficient of concordance is 0.9687 (P = 0.0003), revealing that the relative potency rankings are the same in the three cytotoxicity screens. In light of these encouraging biodata, attempts were made to discern whether one or more physicochemical properties of the dienones in series 1−7 influence cytotoxic potencies. These compounds are designed as multitargeted thiol alkylators; i.e., the potencies exhibited are believed to be dependent, inter alia, on the shapes and hydrophobicity of the compounds. In regard to the topography of the dienones, the molecular volumes control the alignments at different binding sites at least to some extent. Furthermore, on occasion, the size of the torsion angles between aryl rings and adjacent unsaturated groups has influenced the magnitude of different bioactivities.14 Hence the molecular volumes and the torsion angles θA and θB (as indicated in the Figure 1) were determined. The hydro-

Figure 1. Designation of torsion angles θA and θB.

phobicity of the molecules in series 1−7 will influence the rate and extent of the penetration of the compounds via the tumor cell membranes. Furthermore, in the consideration of taking promising compounds forward for in vivo evaluation, log P values of 0−3 are considered optimal in terms of absorption from the gastrointestinal tract.15 The molecular volumes, θA, θB, and log P of 1−7 are presented in Table 2. Some specific comments on the data in Table 2 are as follows. The dienones 2a−d with submicromolar average IC50 have molecular volumes in the range 406−486 Å3, suggesting that analogs with similar properties may possess good cytotoxic potencies. One may also note that the two least potent compounds 3 and 4b have the highest molecular volumes. The torsion angles θA and θB reveal that the aryl rings A and B in 1− 7 are oriented in different directions; i.e., the rings are not parallel with each other. The insertion of one alkylaminomethyl group into the aryl rings of 1b leading to 2a−f brought about a buttressing effect; i.e., the group in the 3 position of the aryl ring exerts a steric effect on the adjacent ortho proton which increases the torsion angles. The insertion of a second dimethylaminomethyl group into 2a led to 3 with substantially greater torsion angles which may be related to the average IC50 of 3 being >500 μM. A similar rise in torsion angles was noted with the increased aryl substitution in the analogs 4b > 4a > 6 and 5 > 7. Compounds with log P values in the range 0−3 are 1b, 2a,e,f, and 3. Bearing in mind the average IC50 values, one can identify 2a in particular as being an attractive candidate for in vivo experimentation. In addition to these specific comments, a quantitative estimate was made as to whether the average IC50 values of 1, 2, 4−7 are correlated with the molecular volumes, θA, θB, and log P. Linear, logarithmic, and semilogarithmic plots were made. Trends to a positive correlation between the IC50 and θB and a negative correlation with log P were observed (p < 0.1). Hence in terms of increasing potencies, compounds with smaller θB and increased hydrophobicity are indicated.

Figure 2. Effect of 2a on DNA fragmentation in HSC-2 and HL-60 cells. The cells were incubated with varying concentrations of 2a for 6 and 24 h. As a positive control, cells were irradiated with UV light for 1 min. Treated and control cells were then incubated for 3 h in regular culture media, after which time the DNA was prepared and applied to 2% agarose gel electrophoresis. M is a 100 bp DNA ladder marker.

apoptosis in these cell lines.16 Second, caspases play important roles in the process of apoptosis.17 In particular caspases 3 and 7 may be activated by the mitochondrial and death receptor pathways. The results in Figure 3 reveal that substantial activation of these caspases takes place in HSC-2 cells. Third, PARP1 is activated when DNA breaks, since its principal action is the sensing and repair of DNA single-strand breaks.18 Thus, compounds that cleave PARP1 may be useful agents in cancer D

DOI: 10.1021/acs.jmedchem.5b01706 J. Med. Chem. XXXX, XXX, XXX−XXX

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Table 4. Evaluation of 2a against a Subpanel of Human Colon Cancer Cells

Figure 3. Activation of caspases 3 and 7 by 2a in HSC-2 cells.

cell line

IC50 a (μM)

TGI a (μM)

CC50 a (μM)

COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 av for 2a av for 5-fluorouracil

1.05 1.70 1.20 2.19 0.89 1.00 1.26 1.26 14.8

2.24 3.24 2.57 10.5 2.29 2.19 2.69 3.09 >1862

4.90 6.17 5.50 39.8 5.50 4.68 5.89 7.25 >2512

a

Concentrations required to inhibit the growth of the cells by 50% (IC50), to inhibit growth completely (TGI), and to kill 50% of the cells (CC50).

chemotherapy. The dienone 2a cleaved PARP1 in HSC-2 and HL-60 cells as indicated in Figure 4. In summary, the cytotoxic effects of 2a are due to apoptosis as evidenced by DNA fragmentation and activation of caspases 3 and 7 in addition to its ability to inhibit PARP1.

their cytotoxic potencies, selective toxicities, and PSE values, 2a−c emerge as the lead preclinical drug candidates.

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CONCLUSIONS 3,5-Bis(3-alkylaminomethyl-4-hydroxybenzylidene)-4-piperidones 2a−f are novel potent cytotoxins that demonstrate promising tumor-selectivity.23 In general, these compounds have substantially lower IC50 and CC50 toward neoplasms than melphalan. One of the promising lead compounds 2a induces apoptosis, causes DNA fragmentation and cleavage of PARP1 in HSC-2 and HL-60 cells, and demonstrates potent cytotoxicity toward a wide range of human tumor cells. Structural features that may contribute to cytotoxic potencies are the torsion angles between the aryl rings and the adjacent olefinic groups and the log P values. 2a−c are promising lead molecules possessing suitable druglikeness properties and therefore warrant further development.

Figure 4. Cleavage of PARP1 by 2a in HSC-2 and HL-60 cells.

A further issue to be resolved is whether these dienones likely display cytotoxic properties toward other human cancer cell lines. Hence a representative compound 2a was evaluated against 59 human neoplasms from nine different groups of tumors (average IC50 of the subpanel in μM in parentheses), namely, leukemic (1.17), colon (1.26), breast (1.38), prostate (1.45), central nervous system (1.66), renal (1.70), melanotic (2.04), non-small-cell lung (2.19), and ovarian (2.34) cancers.19 The average IC50 values of the subpanels reveal that the greatest toxicity is to leukemic and colon cancers. However, an examination of the mean graphs20 reveals that the lowest concentrations of 2a that cause total growth inhibition (TGI) and are required to kill 50% of the neoplasms (CC50) are against colon cancer cells. In other words, malignant colon cancer cells are more sensitive to 2a than other clusters of tumor cell lines. The evaluation of 2a toward the seven colon cancer cell lines employed in the screen is presented in Table 4. The data reveal that in general low micromolar concentrations of 2a inhibit the growth of colon cancers and exert lethal effects on these neoplasms. Furthermore, 5-fluorouracil (5-FU) is a drug used in treating colon cancers. The results in Table 4 reveal the significantly greater potency of 2a than 5-FU; for example, the average TGI of 2a is >600 times lower than the concentration required by the established anticancer drug. These data clearly reveal 2a as a lead compound and it is likely that closely related analogs have the potential to display promising antineoplastic effects toward a wide range of tumors. Finally, an assessment of the druglikeness properties of 2−5 was made based on the guidelines by Lipinski et al.21 and Verber et al.22 All compounds meet the required druglikeness criteria, which further enhances their potential for development (data supplied in Supporting Information). On the basis of

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EXPERIMENTAL SECTION

General. 4-Piperidone hydrochloride hydrate and 4-hydroxybenzaldehyde were purchased from Sigma-Aldrich. All other chemicals were analytical grade and obtained from commercial suppliers. The purity of all the synthesized compounds was >95% as ascertained by elemental analysis and 1H NMR. Elemental analyses were undertaken using an Elementer CHNS analyzer, and the measured values for C, H, and N were within ±0.4% of the theoretical values. The 500 MHz 1H NMR spectra were obtained using a Bruker Avance AMX 500 FT spectrometer equipped with a BBO probe. Chemical shifts are reported in ppm. Melting points in °C were determined using a Gallenkamp apparatus and are uncorrected. Synthesis of Series 1. The preparation of 1a has been described previously.6 3,5-Bis(4-hydroxybenzylidene)-4-piperidone Hydrochloride (1b). 1b was prepared from 4-piperidone hydrochloride monohydrate (0.005 mol) and 4-hydroxybenzaldehyde (0.01 mol) using the same procedure for preparing 1a. Yield, 85%; mp >300 °C; 1H NMR (DMSO-d6) 10.29 (s, 2H, 2 × OH), 9.50 (brs, 2H, NH+), 7.78 (s, 2H, 2 × CH), 7.38 (d, 2H, Ar-H), 6.91 (d, 2H, Ar-H), 4.38 (s, 4H, 2 × NCH2). Found C, 65.24; H, 5.48; N, 4.14%. Anal. (C19H18C1NO3· 0.25H2O) requires C, 65.46; H, 5.12; N, 4.02%. Synthesis of 2a−f. The compounds were prepared by the following general procedure. Dry hydrogen chloride gas was passed into a suspension of 4-piperidone hydrochloride monohydrate (0.003 mol) and 3-alkylaminomethyl-4-hydroxybenzaldehyde hydrochloride (8, 0.006 mol) in glacial acetic acid (50 mL) and stirred overnight at room temperature. The solid precipitated was filtered, washed with acetone, dried, and recrystallized from ethanol. Data for a representative compound 3,5-bis(3′-dimethylaminomethyl-4′-hydroxybenzylidene)-4-piperidone trihydrochloride (2a) are as follows. Yield, E

DOI: 10.1021/acs.jmedchem.5b01706 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Brief Article

40%; mp 218−222 °C; 1H NMR (D2O) δ 7.87 (s, 2H, 2 × CH), 7.40 (m, 4H, Ar-H), 6.95 (m, 2H, Ar-H), 4.49 (s, 4H, 2 × NCH2), 4.23 (s, 4H, Ar-CH2-N), 2.77 (s, 12H, 4 × NCH3). Found C, 54.00; H, 6.79; N, 7.45%. Anal. (C25H34Cl3N3O3·1.5H2O) requires C, 53.77; H, 6.63; N, 7.52%. The data for 2b−f are in Supporting Information. Synthesis of 3,5-Bis(3′,5′-bis(dimethylaminomethyl)-4′-hydroxybenzylidene)-4-piperidone Pentahydrochloride (3). 3 was prepared from 4-piperidone hydrochloride monohydrate (0.003 mol) in the same way as 2a−f except 3,5-bis(dimethylaminomethyl)-4hydroxybenzaldehyde dihydrochloride (9, 0.006 mol) was used instead of 3-dimethylaminomethyl-4-hydroxybenzaldehyde. Yield, 65%; mp 205−209 °C; 1H NMR (D2O) δ 7.90 (s, 2H, 2 × CH), 7.50 (s, 4H, Ar-H), 4.55 (s, 4H, 2 × NCH2), 4.32 (s, 8H, 4 × Ar-CH2-N), 2.78 (s, 24H, 8 × NCH3). Found C, 49.89; H, 7.39; N, 9.32%. Anal. (C31H50Cl5N5O3·1.5H2O) requires C, 49.93; H, 7.11; N, 9.39%. Synthesis of 2,6-Bis(3′-dimethylaminomethyl-4′-hydroxybenzylidene)cyclohexanone (4a) and 2,6-Bis(4′-hydroxybenzylidene)cyclohexanone (6). Dry hydrogen chloride was passed into a mixture of cyclohexanone (0.003 mol) and 4hydroxybenzaldehyde (0.006 mol) in glacial acetic acid (50 mL) and stirred at room temperature overnight. The resultant precipitate was collected, dried, and recrystallized from aqueous ethanol to give 6. Yield, 80%; mp 277−278 °C (lit.,24 mp 279−282 °C). A mixture of 6 (0.003 mol), N,N,N′,N′-tetramethylmethylenediamine (0.0065 mol) in acetonitrile (50 mL) was refluxed overnight. The mixture was cooled to room temperature and the solid formed was filtered, washed with acetonitrile (2 × 5 mL, 10 °C), dried, and crystallized from acetonitrile to give 4a. Yield, 55%; mp 170−172 °C; 1 H NMR δ 7.74 (s, 2H, 2 × CH), 7.39 (d, 2H, Ar-H), 7.15 (s, 2H, Ar-H), 6.88 (d, 2H, Ar-H), 3.71 (s, 4H, 2 × Ar-CH2-N), 2.93 (t, 4H, 2 × CH2), 2.38 (s, 12H, 4 × NCH3), 1.83 (p, 2H, CH2). Found C, 73.91; H, 7.98; N, 6.75%. Anal. (C26H32N2O3) requires C, 74.26; H, 7.67; N, 6.66%. Synthesis of 2,6-Bis[3′,5′-bis(dimethylaminomethyl)-4′hydroxybenzylidene]cyclohexanone (4b). 4b was prepared in the same way as 4a from 6 using excess quantity of N,N,N′,N′tetramethylmethylenediamine (0.024 mol). Yield, 72%; mp 151−153 °C; 1H NMR (D2O) δ 7.73 (s, 2H, 2 × CH), 7.25 (s, 4H, Ar-H), 3.64 (s, 8H, 4 × Ar-CH2-N), 2.95 (t, 4H, 2 × CH2), 2.37 (s, 24H, 8 × NCH3), 1.84 (p, 2H, CH2). Found C, 71.74; H, 8.83; N, 10.51%. Anal. (C32H46N4O3) requires C 71.88; H, 8.67; N, 10.48%. Synthesis of 1,5-Bis(3′-dimethylaminomethyl-4′hydroxyphenyl)penta-1,4-dien-3-one (5) and 1,5-Bis(4′hydroxyphenyl)penta-1,4-dien-3-one (7). Dienone 7 was prepared from acetone (0.003 mol) and 4-hydroxybenzaldehyde (0.006 mol) by the same procedure as utilized in the synthesis of 6. Yield, 60%; mp 235−237 °C (lit.25 mp 238−239 °C); 1H NMR δ 10.07 (d, 2H, 2 × OH), 7.66 (m, 6H, 4 × Ar-H and 2 × CH), 7.11 (m, 2H, 2 × CH), 6.85 (m, 4H, Ar-H). Found C, 72.25; H, 7.65; N, 7.12%. Anal. (C23H28N2O3) requires C, 72.60; H, 7.42; N, 7.36%. A mixture of 7 (0.003 mol) and N,N,N′,N′-tetramethylmethylenediamine (0.012 mol) in acetonitrile (50 mL) was heated under reflux overnight. On cooling to room temperature, the precipitate was collected, washed with acetonitrile (2 × 10 mL, 10 °C), dried, and recrystallized from acetonitrile to give 5. Yield, 68%; mp 210−213 °C; 1 H NMR δ 7.65 (d, 2H, J = 15.9 Hz, 2 × CH), 7.55 (m, 4H, Ar-H), 7.11(d, 2H, J = 15.9 Hz, 2 × CH), 6.82−6.81 (d, 2H, J = 8.1 Hz, ArH), 3.65 (s, 4H, 2 × Ar-CH2-N), 2.29 (s, 12H, 4 × NCH3). Found C, 72.25; H, 7.65; N, 7.12%. Anal. (C23H28N2O3) requires C, 72.60; H, 7.42; N, 7.36%. Synthesis of Series 8. Aryl aldehydes 8a−f were prepared by the following general method. A solution of 4-hydroxybenzaldehyde (0.05 mol) in methanol (50 mL) was added to a preheated mixture of paraformaldehyde (0.05 mol) and the appropriate amine (0.05 mol) in methanol (50 mL) at 70 °C for 1 h. After the addition of 4hydroxybenzaldehyde, the mixture was heated at 70 °C for 2 h. After removal of the solvent, the residue was dissolved in ether and hydrogen chloride gas was passed through the solution. The resultant precipitate was collected, dried, and recrystallized from ethanol. The data for a representative compound 4-hydroxy-3-

(dimethylaminomethyl)benzaldehyde hydrochloride (8a) are as follows. Yield, 55%; mp 209−211 °C; 1H NMR (D2O) δ 9.67 (s, 1H, CHO), 7.88−7.85 (m, 2H, Ar-H), 7.06-7.04 (m, 1H, Ar-H), 4.29 (s, 2H, Ar-CH2-N), 2.79 (s, 6H, 2 × NCH3). The data for 8b−f are in Supporting Information. Synthesis of 9. A mixture of 4-hydroxybenzaldehyde (0.01 mol) and N,N-dimethyl-N-methyleneammonium chloride (0.025 mol) in dry acetonitrile (40 mL) was heated under reflux for 24 h. On cooling, the precipitate was collected, washed with acetonitrile (2 × 5 mL), and recrystallized from ether−ethanol to give 9. Yield, 60%; mp 231−232 °C (lit.26 mp 234−235 °C); 1H NMR (D2O) δ 9.32 (s, 1H, CHO), 7.66 (s, 2H, Ar-H), 3.97 (s, 4H, 2 × Ar-CH2-N), 2.61 (s, 12, 4 × NCH3).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01706. Synthetic procedures and compound characterization data for 2b−f and 8b−f, physicochemical determinations, Kendall’s coefficient of concordance, cytotoxic assays, mode of action studies (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*U.D.: phone, +1-306-966-6358; fax, +1-306-966-6377; e-mail, [email protected]. *J.R.D.: phone, +1-306-966-6331; fax, + 1-306-966-6377; email, [email protected]. Present Address #

S.S.K.: College of Pharmacy, KLE University, Rajajinagar, 2nd Block, Bangalore 560010, India. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Cancer Institute is thanked for screening representative compounds against a panel of 60 cancer cell lines. L. van Berckelaer of the Rega Institute, Belgium, kindly undertook the Molt4/C8, CEM, and L1210 bioassays. S.S.K. was a recipient of a BOYSCAST fellowship supported by the Department of Science & Technology (DST), India. Support was from Canadian Institutions of Health Research (CIHR) operating grant to J.R.D., Ministry of Education, Science, Sports and Culture Grant-in-Aid (No. 19592156) to H.S., and the Geconcerteerde Onderzoeksacties (GOA 05/19) for J.B.

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ABBREVIATIONS USED PARP1, poly ADP-ribose polymerase 1; SI, selective index; TGI, total growth inhibition; PSE, potency selectivity expression

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REFERENCES

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DOI: 10.1021/acs.jmedchem.5b01706 J. Med. Chem. XXXX, XXX, XXX−XXX