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Article

Populene D Analogues: Design, Concise Synthesis and Antiproliferative Activity

by
Kachi R. Kishore Kumar Reddy
1,
Giovanna B. Longato
2,
João E. de Carvalho
2,
Ana L. T. G. Ruiz
2,* and
Luiz F. Silva, Jr.
1,*
1
Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CP 26077, CEP São Paulo 05513-970, SP, Brazil
2
Divisão de Farmacologia e Toxicologia, Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA), UNICAMP, CP6171, Campinas 13083-970, SP, Brazil
*
Authors to whom correspondence should be addressed.
Molecules 2012, 17(8), 9621-9630; https://doi.org/10.3390/molecules17089621
Submission received: 15 June 2012 / Revised: 2 August 2012 / Accepted: 3 August 2012 / Published: 10 August 2012
(This article belongs to the Section Organic Chemistry)
Browse Figures
Versions Notes

Abstract

:
An efficient and concise synthesis of nine populene D analogues was performed using an iodine-catalyzed Prins cyclization as the key transformation. The antiproliferative activity of these new pyrans against several cancer cell lines was then investigated. Among them, an isochromene with moderate activity (mean logGI50 = 0.91) was found. Additionally, compounds with selectivity toward the tumor cell lines NCI-ADR/RES, OVCAR-3, and HT29 were discovered.

1. Introduction

Natural products have always played an important role in drug discovery [1,2,3,4,5]. Although it is not always possible to understand the exact function of the secondary metabolites isolated from natural sources, they are often used as inspiration for new drugs, particularly in the area of cancer [6,7,8,9]. The pyran subunit can be frequently recognized in the structure of numerous natural and synthetic compounds with remarkable biological and pharmacological properties [10,11,12,13]. Populene D (Figure 1) is a natural sesquiterpenoid recently isolated from Thespesia populnea, which possesses strong inhibitory activity against several human cancer cell lines: cervical cancer (HeLa, IC50 = 1.85 µg/mL) and breast cancer (MCF-7, IC50 = 0.95 µg/mL) [14]. Populene D is also active against colon cancer (HT-29, IC50 = 2.37 µg/mL), oral cancer (KB, IC50 = 3.10 µg/mL) and has antibacterial properties (B. subtilis, MIC = 4.69 µg/mL) [14].
Molecules 17 09621 g001 550
Figure 1. Structure of populene D.
Figure 1. Structure of populene D.
Molecules 17 09621 g001
We envisioned that populene D analogues could be synthesized by the iodine-catalyzed Prins cyclization of homoallylic alcohols and acetone (Scheme 1) [15]. Considering that several homoallylic alcohols can be easily prepared from commercially available tetralones [16], this route could provide access to analogues of a natural compound with potential anticancer activity. Herein, we describe our first results regarding the concise synthesis of nine isochromenes and their antiproliferative activity against several tumor cell lines.
Molecules 17 09621 g002 550
Scheme 1. Retrosynthesis for populene D analogues.
Scheme 1. Retrosynthesis for populene D analogues.
Molecules 17 09621 g002

2. Results and Discussion

2.1. Synthesis of Populene D Analogues

The homoallylic alcohols 1a–d were prepared in three steps from commercially available 1-tetralones as previously reported [16]. The Prins cyclization of the homoallylic alcohols 1a–d and acetone in the presence of iodine gave the methoxy substituted populene D analogues 2a–d in 73–88% yield (Table 1, entries 1–4). The treatment of 2a–c with sodium ethanethiolate [17,18] gave hydroxy substituted populene D analogues 3a–c in 71–82% yield (Table 2, entries 1–3). Under analogous conditions, the dimethoxy substrate 2d gave the mono deprotected compound 3d in 42% yield (entry 4). The structure of 3d was assigned by NMR analysis, including HMBC experiments. The double deprotection to obtain 3e was achieved using excess of sodium ethanethiolate and longer reaction time (entry 5). In summary, nine new populene D analogues were efficiently prepared using as key reaction a Prins cyclization and fully characterized.
Table
Table 1. Iodine-catalyzed Prins cyclization.
Table 1. Iodine-catalyzed Prins cyclization.
Molecules 17 09621 i003
Entry Homoallylic alcohols 1a–d Products 2a–e (yield)
1 Molecules 17 09621 i004 Molecules 17 09621 i005
2 Molecules 17 09621 i006 Molecules 17 09621 i007
3 Molecules 17 09621 i008 Molecules 17 09621 i009
4 Molecules 17 09621 i010 Molecules 17 09621 i011
Table
Table 2. Cleavage of the ether moiety.
Table 2. Cleavage of the ether moiety.
Molecules 17 09621 i012
EntrySubstrates 2a–dProducts 3a–e (yield)
12a Molecules 17 09621 i013
22b Molecules 17 09621 i014
32c Molecules 17 09621 i015
42d Molecules 17 09621 i016
52d Molecules 17 09621 i017
a using excess of sodium ethanethiolate and longer reaction time.

2.2. Antiproliferative Activity of Populene D Analogues

The in vitro antiproliferative activity of populene analogues 2a–e and 3a–e was investigated toward nine human tumor cell lines [CNS (U251), breast (MCF-7), ovarian (NCI-ADR/RES, OVCAR-03), renal (786-0), non-small cell lung (NCI-H460), prostate (PC-3), colon (HT-29) and leukemia (k-562)] and one human normal cell line (HaCat, human keratinocytes). The populene D analogues were tested at concentrations between 0.25 and 250 µg/mL and doxorubicin (DOX 0.025–25 µg/mL) was used as positive control. Two effective concentrations, eliciting 50% growth inhibition (GI50) and total growth inhibition (TGI), were determined after 48 h-cell treatment (Table 3 and Table 4). To analyze the GI50 parameter, mean log GI50 was calculated by conversion of the GI50 values for each tumor cell line tested (not including the normal cell line HaCat) against a test compound and then these values were averaged. According to National Cancer Institute (NCI/EUA), if mean logGI50 < 1.50 a tested compound could be considered as active and might be classified as of weak (1.1 < mean logGI50 < 1.5), moderate (0 < mean logGI50 < 1.1) or potent (mean logGI50 < 0) activity [19]. Using these criteria, phenol 3e (mean logGI50 = 0.91) was the most active populene analogue synthetized, presenting a moderate cytostatic activity with low selective against human cell lines evaluated. On the other hand, heterocycles 3c and 3d presented similar general weak cytostatic effect (mean log GI50 = 1.15 and 1.16, respectively), but compound 3c showed a high selectivity to the ovarian cell line OVCAR-3 (GI50 = 1.8 µg/mL), whereas pyran 3d was selective for the ovarian expressing multidrug resistance phenotype (NCI/ADR-RES, GI50 = 5.2 µg/mL) and to colon (HT29, GI50 = 6.0 µg/mL) cell lines. Isochromenes 3a and 3b were considered inactive (mean logGI50 > 2.32 and > 2.00, respectively). The methoxy derivatives 2a–c presented a weak cytostatic activity (mean logGI50 from 1.12 to 1.23), whereas compound 2d was inactive. However, the following differences in selectivity were found for these ethers: (i) pyran 2a is more selective to leukemia (K562, GI50 = 3.1 µg/mL); (ii) compound 2b was more active against colon (HT29, GI50 = 6.6 µg/mL) cell line; (iii) heterocycle 2e presented a slight selectivity to glioma (U251, GI50 = 6.3 µg/mL) cell line.
In summary, these results suggest that the hydroxyl/methoxyl pattern of substitution has an important influence in the antiproliferative activity. Moreover, the dihydroxy-substituted populene D analogue 3e is the most active compound in the 2a–d and 3a–e series. Considering the monosubstituted compounds, the conversion of the methoxy (2a and 2b) into a hydroxy (3a and 3b) group resulted in lower activity, whereas the substitution from 2c to 3c led to a slightly increase in the cytostatic activity. Among the disubstituted compounds, the conversion of the methoxy (2d) to hydroxy (3d and 3e) increased the activity.
The results obtained for the TGI parameter confirm the GI50 evaluation, indicating moderate activity for populene analogues 2a–2c and 3c–3e (Table 4). The evaluation for the normal human cell lines (HaCat) shows GI50 and TGI values on the same order of magnitude than for the tumor cell lines, suggesting that populene D analogues may present in vivo toxicity, similar to the known chemotherapic drugs.
Table
Table 3. Antiproliferative activity (GI50, μg/mL) of 2a–d and 3a–e on human cell lines.
Table 3. Antiproliferative activity (GI50, μg/mL) of 2a–d and 3a–e on human cell lines.
Cell lines aDoxorubicine b2a2b2c2d3a3b3c3d3e
U2510.06225.024.625.0>250>25046.327.823.119.6
MCF-70.2125.023.225.038.8>250107.824.724.65.5
NCI-ADR/RES1.325.026.825.0100.7>250>25025.05.25.7
786-00.0425.025.525.045.837.336.824.624.15.6
NCI-H460<0.025N.T.N.T.N.T.N.T.>25041.424.245.117.4
PC-30.2725.027.025.071.4>250>25024.5N.T.N.T.
OVCAR-30.2625.024.525.0193.4>250>2501.824.33.9
HT-290.2225.06.625.023.1>25078.422.96.019.5
K-5620.43.19.625.084.6>25041.28.7N.T.N.T.
HaCat0.2325.026.125.0>250>250>2503.64.24.2
Mean logGI50c, d−0.78 P1.17 W1.18 W1.21 W>1.70 I>2.31 I>1.95 I1.22 W1.24 W0.95 M
N.T.: not tested. a Tumor cell lines: U251—glioma; MCF-7—mammary; NCI-ADR/RES—drug resistant ovary; 786-0—kidney; NCI-H460—lung; NCI-PC-3—prostate; OVCAR-3—ovary; HT-29 colon; K562—leukemia. Normal cell lines: HaCat—immortalized keratinocytes; b Positive control; c Mean logGI50: The GI50 value for each tumor cell line tested (not including the normal cell line HaCat) against a test compound is converted to its logGI50 value and these logGI50 values are averaged; d NCI’s criteria [19]: I: mean logGI50 > 1.5 = inactive; W, weak activity: 1.1 < mean logGI50 < 1.5; M, moderate activity: 0 < mean logGI50 < 1.1; P, potent activity: mean log GI50 < 0.
Table
Table 4. Total growth inhibition (TGI, μg/mL) of 2a–d and 3a–e on human cell lines.
Table 4. Total growth inhibition (TGI, μg/mL) of 2a–d and 3a–e on human cell lines.
Cell lines aDoxorubicine b2a2b2c2d3a3b3c3d3e
U2511.152.053.540.0>250>250>25067.945.928.4
MCF-78.851.346.945.3>250>250>25045.058.241.6
NCI-ADR/RES25.083.5104.466.3>250>250>250199.537.6>250
786-00.6250.644.036.1>250>250>25043.047.819.4
NCI-H4600.025N.T.N.T.N.T.N.T.>250>25045.9>250>250
PC-34.464.641.469.2>250>250>25051.8N.T.N.T.
OVCAR-33.952.356.267.0>250>250>25031.569.934.4
HT-2925.039.133.041.8>250>250>25053.332.339.0
K-56225.0>250>250128.1>250>250>25039.2N.T.N.T.
HaCat0.6766.954.432.0>250>250>250>250160.7>250
N.T.: not tested; a Tumor cell lines: U251—glioma; MCF-7—mammary; NCI-ADR/RES—drug resistant ovary; 786-0—kidney; NCI-H460—lung; NCI-PC-3—prostate; OVCAR-3—ovary; HT-29 colon; K562—leukemia. Normal cell lines: HaCat—immortalized keratinocytes. b Positive control.

3. Experimental

3.1. General

All commercially available reagents were used without further purification unless otherwise noted. CH2Cl2 and DMF were freshly distilled over CaH2. TLC analyses were performed in silica gel plates, using UV and/or p-anisaldehyde solution for visualization. Flash column chromatography was performed using silica gel 200–400 mesh. Melting points are uncorrected. All NMR analyses were recorded using CDCl3 as solvent and TMS as internal pattern in Bruker (AC200) or Varian (INOVA300) spectrometers. IR spectra were measured on a Perkin-Elmer 1750-FT. HRMS analysis were performed on a Bruker Daltonics Microtof Eletrospray. Melting points were recorded on a Buchi R-535 apparatus and are uncorrected. The homoallylic alcohols 1a–c were prepared in three steps from commercially available 1-tetralones as previously reported [16].

3.2. General Procedure for Prins Cyclizations

To a stirred solution of 1a–d (1.0 mmol) and (CH3)2CO (1.2 mmol) in CH2Cl2 (5 mL), was added I2 (0.050 mmol). After 2 h, Na2SO3 (0.60 mmol) and H2O (10 mL) were added. The aqueous phase was extracted with AcOEt (3 × 5 mL). The combined organic phase was washed with brine (5 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (5% AcOEt in hexanes), affording 2a–d.
4,5,6-Tetrahydro-7-methoxy-1,4,4-trimethyl-1H-benzo[f]isochromene (2a). Yield: 80%. White solid; mp: 65–67 °C; IR (film): 3073, 2969, 1574, 1470, 1460, 784 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.23 (d, J = 6.8 Hz, 3H), 1.32 (s, 3H), 1.42 (s, 3H), 2.05–2.25 (m, 2H), 2.48–2.66 (m, 2H), 2.84–2.99 (m, 1H), 3.65 (dd, J = 2.4, 11.1 Hz, 1H), 3.84 (s, 3H), 3.93 (dd, J = 3.3, 11.1 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 7.19 (dd, J = 7.8, 8.2 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.2, 20.4, 23.9, 24.1, 27.9, 29.2, 55.5, 65.8, 74.8, 109.0, 115.3, 123.6, 126.4, 129.6, 135.2, 138.6, 155.9; LRMS m/z (rel. int.): 258 (M+, 9.1), 244 (13), 243 (100), 227 (15), 105 (35), 77 (53), 43 (66); HRMS [ESI(+)] calcd. for [C17H22O2+ H]+ 259.1693, found 259.1679.
2,4,5,6-Tetrahydro-8-methoxy-1,4,4-trimethyl-1H-benzo[f]isochromene (2b). Yield: 82%. White solid; mp: 89.5–91.3 °C; IR (film): 3005, 2970, 1610, 1570, 1492, 1427, 670, 612 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.21 (d, J = 6.8 Hz, 3H), 1.29 (s, 3H), 1.39 (s, 3H), 203–2.15 (m, 2H), 2.56–2.72 (m, 3H), 3.62 (dd, J = 2.2, 11.1 Hz, 1H), 3.78 (s, 3H), 3.90 (dd, J = 3.3, 11.1 Hz, 1H), 6.69–6.75 (m, 2H), 7.16 (d, J = 8.3 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.0, 24.0, 24.3, 27.9, 28.9, 29.0, 55.0, 65.6, 74.6, 110.9, 113.4, 123.3, 126.9, 129.3, 135.7, 137.4, 157.9; LRMS m/z (rel. int.): 258 (M+, 9.9), 244 (13), 243 (100); Elemental analysis calcd. for [C17H22O2] C 79.03, H 8.58, found C 78.61, H 8.37.
2,4,5,6-Tetrahydro-9-methoxy-1,4,4-trimethyl-1H-benzo[f]isochromene (2c). Yield: 73%. Colorless viscous oil; IR (film): 3076, 2969, 1605, 1573, 1496, 1461, 835, 803 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.23 (d, J = 6.8 Hz, 3H), 1.30 (s, 3H), 1.40 (s, 3H), 2.03–2.16 (m, 2H), 2.59–2.69 (m, 3H), 3.63 (dd, J = 2.4, 11.2 Hz, 1H), 3.79 (s, 3H), 3.91 (dd, J = 3.2, 11.2 Hz, 1H), 6.67 (dd, J = 2.4, 8.1 Hz, 1H), 6.84 (d, J = 2.4 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.0, 24.1, 24.9, 27.7, 27.8, 28.9, 55.2, 65.7, 74.8, 109.3, 110.3, 127.8, 128.0, 129.7, 135.1, 139.2, 158.4; LRMS m/z (rel. int.): 258 (M+, 11.5), 244 (14), 243 (100); HRMS [ESI(+)] calcd. for [C17H22O2+ H]+ 259.1693, found 259.1699.
2,4,5,6-Tetrahydro-8,9-dimethoxy-1,4,4-trimethyl-1H-benzo[f]isochromene (2d). Yield: 81%. White solid; mp: 115–117 °C; IR (film): 2968, 2931, 1512, 1238, 1203, 856, 806 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.24 (d, J = 6.8 Hz, 3H), 1.30 (s, 3H), 1.40 (s, 3H), 2.04–2.17 (m, 2H), 2.58–2.70 (m, 3H), 3.64 (dd, J = 2.2, 11.2 Hz, 1H), 3.88 (s, 3H), 3.90–3.96 (m, 1H), 6.69 (s, 1H), 6.83 (s, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.1, 24.1, 24.7, 28.0,28.3, 29.1, 55.9, 56.2, 65.6, 74.8, 106.9, 111.2, 126.7, 128.5, 129.2, 136.4, 147.2, 147.3; LRMS m/z (rel. int.): 288 (M+, 22), 274 (17), 273 (100); CH analysis calcd. for [C18H24O3] C 74.97, H 8.39, found C 74.77, H 8.75.

3.3. General Procedure for the Deprotections of 3a–d

Under an inert atmosphere of N2, NaH (17.5 mmol, 60% in mineral oil) was washed with anhydrous hexanes (2 × 10 mL). After a few minutes, anhydrous DMF (5 mL) was added. To this mixture was slowly added a solution of EtSH (12.6 mmol) in anhydrous DMF (0.4 mL) at 0 °C and the resulting yellow gray solution was stirred for 20 min at rt. A solution of 2a–d (0.39 mmol) in DMF (1 mL) was then added dropwise and the resulting mixture was stirred for 5 h at 140 °C, becoming slightly brown. The mixture was cooled to the rt and a saturated solution of NH4Cl (5 mL) was added. The mixture was extracted with Et2O (3 × 10 mL) and the organic phase was washed with H2O (5 mL), with brine (5 mL), and dried over anhydrous MgSO4. The solvent was removed under reduced pressure and the resulting brown oil was purified by flash chromatography (30% AcOEt in hexanes), affording 3a–d.
1,4,4-Trimethyl-1,4,5,6-tetrahydro-2H-benzo[f]isochromen-7-ol (3a). Yield: 82%. White solid; mp: 139.2–140.3 °C; IR (film): 3289, 2971, 2932, 1578, 1468, 916, 833 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.22 (d, J = 6.8 Hz, 3H), 1.32 (s, 3H), 1.42 (s, 3H), 2.05–2.25 (m, 2H), 2.49–2.65 (m, 2H), 2.80–2.91 (m, 1H), 3.64 (dd, J = 2.3, 11.1 Hz, 1H), 3.92 (dd, J = 3.3, 11.1 Hz, 1H), 6.67 (d, J = 0.6, 7.9 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 7.09 (dd, J = 7.8, 8.0 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.2, 20.5, 23.8, 24.1, 27.9, 29.2, 65.8, 74.8, 113.8, 115.6, 121.2, 126.6, 129.9, 135.6, 138.5, 151.9; LRMS m/z (rel. int.): 244 (M+, 9.0), 230 (13), 229 (100); CH analysis calcd. for [C16H21O2] C 78.65, H 8.25, found C 78.45, H 8.00.
1,4,4-Trimethyl-1,4,5,6-tetrahydro-2H-benzo[f]isochromen-8-ol (3b). Yield: 79%. White solid; mp: 199–201 °C; IR (film): 3350, 2968, 2932, 1605, 1502, 1440, 849, 834 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.22 (d, J = 6.8 Hz, 3H), 1.31 (s, 3H), 1.40 (s, 3H), 2.08–2.16 (m, 2H), 2.57–2.71 (m, 3H), 3.64 (dd, J = 2.3, 11.2 Hz, 1H), 3.93 (dd, J = 3.2, 11.2 Hz, 1H),5.13 (s, 1H), 6.64–6.70 (m, 2H), 7.12 (d, J = 8.1 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.1, 24.1, 24.4, 28.0, 28.9, 29.0, 65.7, 74.9, 112.7, 114.7, 123.7, 127.1, 129.4, 135.8, 137.9, 154.1; LRMS m/z (rel. int.): 244 (M+, 9.8), 230 (15), 229 (100); CH analysis calcd. for [C16H21O2] C 78.65, H 8.25, found C 78.19, H 8.04.
1,4,4-Trimethyl-1,4,5,6-tetrahydro-2H-benzo[f]isochromen-9-ol (3c). Yield: 71%, White solid; mp: 160–162 °C; IR (film): 3235, 2962, 2943, 1615, 1572, 1497, 834, 809 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.21 (d, J = 6.8 Hz, 3H), 1.35 (s, 3H), 1.45 (s, 3H), 2.17–2.08 (m, 2H), 2.69–2.55 (m, 3H), 3.65 (dd, J = 11.1, 2.4 Hz, 1H), 3.93 (dd, J = 11.1, 3.3 Hz, 1H), 6.64 (dd, J = 7.8, 2.4 Hz, 1H), 6.79 (d, J = 2.4 Hz, 1H), 6.98 (d, J = 7.8 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.0, 24.2, 24.9, 27.69, 27.74, 28.9, 65.6, 75.4, 109,9, 112.8, 127.5, 128.1, 129.6, 135.1, 138.8, 154.6; LRMS m/z (rel. int.): 244 (M+, 9.3), 230 (15), 229 (100); HRMS [ESI(+)] calcd. for [C16H20O2+ Na]+ 267.1355, found. 267.1359.
8-Methoxy-1,4,4-trimethyl-1,4,5,6-tetrahydro-2H-benzo[f]isochromen-9-ol (3d). Yield: 42%. Pale yellow solid; mp: 141–143 °C; IR: 3396, 2967, 2924, 1509, 869, 809 cm−1; 1H-NMR (200 MHz, CDCl3) δ: 1.23 (d, J =7.2 Hz, 3H), 1.30 (s, 3H), 1.40 (s, 3H), 2.05–2.14 (m, 2H), 2.56–2.66 (m, 3H), 3.66 (dd, J = 2.2, 11.1 Hz, 1H), 3.89 (s, 3H), 3.89–3.96 (m, 1H), 5.60 (br, 1H), 6.69 (d, J = 12.2 Hz, 1H), 6.83 (d, J = 18.2 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ: 18.1, 24.1, 24.7, 28.0, 28.1, 29.2, 56.3, 65.7, 74.9, 106.1, 109.5, 110.5, 114.1, 126.4, 129.3, 129.4, 136.3, 144.0, 144.9; LRMS m/z (rel. int.): 274 (15.8), 260 (13.1), 259 (100); HRMS [ESI(+)] calcd. for [C17H22O3+ H]+ 275.1642, found 275.1648.
1,4,4-Trimethyl-1,4,5,6-tetrahydro-2H-benzo[f]isochromene-8,9-diol (3e). The reaction was performed following the general procedure of Section 4.3, but using NaH (23 mmol), EtSH (18 mmol), 2d (0.144, 0.500 mmol). Yield: 76%. Pale yellow solid; mp: 122–123 °C; IR (film): 3605, 3533, 2876, 2828, 1619, 1601, 959, 939, 896, 687 cm−1; 1H-NMR (300 MHz, CDCl3) δ: 1.18 (d, J = 6.8 Hz, 3H), 1.32 (s, 3H), 1.42 (s, 1H), 2.08–2.13 (m, 2H), 2.55–2.62 (m, 3H), 3.64 (dd, J = 2.4, 11.2 Hz, 1H), 3.92 (dd, J = 3.2, 11.2 Hz, 1H), 3.67 (br, 2H), 6.67 (s, 1H), 6.81 (s, 1H); 13C-NMR (75 MHz, CDCl3) δ: 18.1, 24.1, 24.7, 28.0, 28.1, 29.1, 65.7, 110.3, 114.7, 127.2, 128.8, 129.3, 136.2, 141.9, 142.0; LRMS m/z (rel. int.):260 (M+, 10), 246 (13), 245 (100); HRMS [ESI(+)] calcd. for [C16H19O3+ Na]+ 283.1305, found 283.1316.

3.4. Antiproliferative Assays

Human tumor cell lines [U251 (glioma), MCF-7 (breast), NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance), 786–0 (renal), NCI-H460 (lung, non-small cells), PC-3 (prostate), OVCAR-03 (ovarian) and HT-29 (colon)] were kindly provided by Frederick Cancer Research & Development Center–National Cancer Institute–Frederick, MA, USA. HaCat cell line (immortalized human keratinocytes) was kindly donated by Dr. Ricardo Della Coletta, FOP–Unicamp. Stock cultures were grown in 5 mL of RPMI 1640 (GIBCO BRL, Life Technologies) supplemented with 5% of fetal bovine serum. Penicillin:streptomycin (1,000 μg mL−1:1,000 UI mL‑1, 1 mL L−1) was added to the experimental cultures.
Cells in 96-well plates (100 μL cells/well) were exposed to various concentrations of compounds 2a–d and 3a–d diluted in DMSO (0.25, 2.5, 25 and 250 μg/mL) at 37 °C, 5% of CO2 for 48 h. The final concentration of DMSO did not affect the cell viability. Afterwards cells were fixed with 50% trichloroacetic acid and cell proliferation determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B assay[20]. Doxorubicin (0.025–25 mg/mL) was used as positive control. Three measurements were obtained at the beginning of incubation (time zero, T0) and 48 h post-incubation for compound-free (C) and tested (T) cells. Cell proliferation was determined according to the equation 100 × [(T − T0)/C − T0], for T0 < T ≤ C, and 100 × [(T − T0)/T0], for T ≤ T0 and a concentration-response curve for each cell line was plotted and, from these curves, GI50 (concentration causing 50% growth inhibition) and TGI (concentration that promotes total growth inhibition) were determined by means of non-linear regression analysis using software ORIGIN 8.0 (OriginLab Corporation) [20,21] The average activity (mean of log GI50) of each compound tested was also determined using MS Excel software. Compounds were regarded as inactive (mean > 1.5), weakly (1.1 < mean < 1.5), moderately (0 < mean < 1.1) or potently (mean < 0) active on basis of the NCI criteria for the mean of logGI50[19].

4. Conclusions

In conclusion, the efficient synthesis of nine new populene D analogues 2a–d and 3a–e was performed using an iodine catalyzed Prins cyclization as key transformation. The antiproliferative activity of these compounds against several cancer cell lines was investigated. This evaluation demonstrated that isochromene 3e is the most active compound on the cell lines used. Although the populene D analogues do not display potent antiproliferative activity, we think this work might inspire the discovery of new highly active isochromenes.

Supplementary Materials

Supplementary materials can be accessed at: https://www.mdpi.com/1420-3049/17/8/9621/s1.

Acknowledgments

We thank CAPES, CNPq, and FAPESP for financial support.

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MDPI and ACS Style

Reddy, K.R.K.K.; Longato, G.B.; Carvalho, J.E.d.; Ruiz, A.L.T.G.; Silva, L.F., Jr. Populene D Analogues: Design, Concise Synthesis and Antiproliferative Activity. Molecules 2012, 17, 9621-9630. https://doi.org/10.3390/molecules17089621

AMA Style

Reddy KRKK, Longato GB, Carvalho JEd, Ruiz ALTG, Silva LF Jr. Populene D Analogues: Design, Concise Synthesis and Antiproliferative Activity. Molecules. 2012; 17(8):9621-9630. https://doi.org/10.3390/molecules17089621

Chicago/Turabian Style

Reddy, Kachi R. Kishore Kumar, Giovanna B. Longato, João E. de Carvalho, Ana L. T. G. Ruiz, and Luiz F. Silva, Jr. 2012. "Populene D Analogues: Design, Concise Synthesis and Antiproliferative Activity" Molecules 17, no. 8: 9621-9630. https://doi.org/10.3390/molecules17089621

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