English

• 1.   Introduction

  Dithiocarbamate (DTC) is one of the simplest organic sulfur compounds. Because it is an organic synthetic intermediate, DTC can be used to synthesize trifluoromethylamines, heterocyclic compounds, and tetrathiafulvalene superconducting materials. DTC is also used as a sewage additive and an additive for the rubber industry. DTC-based fungicides have been used in agriculture for decades.^{[1, 2]}

  Due to the unique structures and properties of DTC compounds, DTC has been used as a pharmacophore in the field of drug discovery.^{[1, 2]} Disulfiram (DSF) was first used as a rubber additive, and was approved for the treatment of chronic alcoholism as an irreversible aldehyde dehydrogenase inhibitor after 1948.^[3] DSF has also been found to exhibit anti-tuberculosis, anti-tumor, anti-leishmanial, and antiplatelet aggregation activities.^[4] DTC derivatives display a wide range of antioxidant, ⁵ antiviral, ^[6] antibacterial, ^[7] and other activities.^[8] In recent years, DTC derivatives have attracted increasing attention as potential antitumor compounds.^{[1, 2]} As shown in Figure 1, many different substituents have been used in the structural modification of DTC derivatives with antitumor activity. DTC derivatives not only exhibit good antiproliferative activity against tumor cell lines, ^[9] but also show strong inhibitory activities against carbonic anhydrase, ^[10] tubulin, ^[11] histone demethylase, ^[12] epidermal growth factor receptor, ^[13] sirtuin 1 (SIRT1), ^[14] topoisomerase Ⅱ, ^[15] indoleamine 2, 3-dioxygenase, ^[16] proteasomes, ^[17] and other biological targets.

  Figure 1

  

  Figure 1.  Reported DTC derivatives with antiproliferative activity

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  Quinazolinone, ^[18] benzoxazinone, ^[19] and coumarin^{[20, 21]} heterocycles serve as the backbones of many natural products and are present in the structures of many bioactive molecules. Quinazolinone, benzoxazinone, and coumarin derivatives have been reported to act on protein kinase, dihydrofolate reductase, chymotrypsin, serine protease, elastase, methyltransferase, cyclooxygenase, herpes simplex virus, opioid receptor, tubulin, and other targets, thus showing anti-tumor, anti-inflammatory, antihypertensive, anti-malarial, antibacterial, and other biological activities.

  In our previous studies, DTC was merged into the 2-position, 3-position, and 6-position of the chromone ring with a flexible methylene linker to generate the lead compound 1 (Figure 2). The most promising lead compounds were 3-chloro-4-oxo-4H-chromen-2-yl)methyl piperdine- 1-carbodithioate and 6-chloro-4-oxo-4H-chromen-3-yl)- methylpiperidine-1-carbodithioate. These exhibited broad- spectrum inhibitory activities against all six tested human cancer cell lines were successfully discovered.^[22] In addition, we synthesized a series of chromone, aurone, coumarin, quinazolinone, and other flavonoids and their analogs that showed good antiproliferative activity.^{[23, 24]} Based on our previous experiences with chromone-DTC derivatives and other sulfur-containing flavonoid derivatives, and using a strategy of bioisosterism, quinazolinone, benzoxazinone, and coumarin moieties were merged with DTC pharmacophore via a flexible methylene linker. We then conducted structure-activity relationship (SAR) studies on their antiproliferative activity to identify novel lead compounds.

  Figure 2

  

  Figure 2.  Design of diversified fused heterocyclic derivatives bearing a dithiocarbamate unit

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  1.   Results and discussion

  1.1   Chemistry

  Using a previously reported synthetic method for DTC- chromone derivatives as a reference, ^{[22, 23]} the key halomethyl-substituted fused heterocyclic intermediates were first synthesized based on the optimized reported routes (Scheme 1). The same 2-aminobenzoic acid intermediate (M-0) was used as the starting material to undergo condensation with formamide, condensation with formaldehyde, and chlorination to successfully yield a 3-(chloro- methyl)quinazolin-4-one intermediate (M-3).^[25] The amidation of M-0 with 2-chloroacetyl chloride and intramolecular esterification produced 2-chloromethyl benzo[d][1, 3]oxazin-4-one (M-5).^[26]

  Scheme 1

  

  Scheme 1.  Synthesis of 2-chloromethyl quinazolin-4-one (M-3) and 2-chloromethyl benzo[d][1, 3]oxazin-4-one (M-5)

  Reagents and conditions: (a) formamide, 130~160 ℃; (b) HCHO, 1, 4- dioxane, 80 ℃; (c) SOCl₂, 1, 4-dioxane, r.t.; (d) 2-chloroacetyl chloride, r.t.; (e) HOAc, 100 ℃

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  As shown in Eq. 1, the diversified different 4-cholo- romethyl-coumarin intermediates (M-7) were obtained from the subsequent condensation-cyclization reaction of ethyl 4-chloroacetoacetate and substituted phenol (M-6).^[27] The DTC derivatives bearing quinazolinone, benzoxazinone, and coumarin moieties were synthesized via a one- pot three-component reaction.^[28] In the presence of potassium phosphate as base, a subsequent three-component reaction of the corresponding amine, carbon disulfide and the halomethyl-substituted fused heterocyclic intermediates M-3/M-5/M-7 afforded target compounds 2, 3 and 4, which had the advantages of good yields, short reaction times, mild conditions and ready isolation of the products. All target compounds were confirmed by ¹H NMR, ¹³C NMR, EI-MS and elemental analysis (EA).

  The structural characteristics of the representative compound 2h were investigated using X-ray single crystal diffraction (Figure 3).^[29] In the crystal structure, the quinazolinone ring and the DTC unit (N3-C10-S1-S2) all showed good co-planarity with maximum deviation at the 4-position carbonyl carbon atom (0.045 Å) and thiocarbonyl carbon atom (0.005 Å). The dihedral angle between the quinazolinone plane and the DTC plane was 68.92°, which was significantly larger than the corresponding dihedral angle of the 3-DTC-chromone derivative (36.61°).

  Figure 3

  

  Figure 3.  X-ray structure of 2h

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  1.2   Pharmacology

  The in vitro anti-proliferative activities of the synthesized compounds 2, 3 and 4 were tested in six cancer cell lines, including HCCLM-7 (hepatoma carcinoma cells), Hela (cervical carcinoma cells), MDA-MB-435S (mammary adenocarcinoma cells), SW-480 (colon carcinoma cells), Hep-2 (laryngocarcinoma cells), and MCF-7 (mam- mary adenocarcinoma cells). Cell proliferative activity was assayed using the MTT method.^[30] The results, expressed as IC₅₀, are summarized in Table 1.

  Table 1

  

  Table 1.  Antiproliferative activity of the synthesized derivatives

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  Compd.	X/R1~R4	Amine unit	Antiproliferative activity [IC50/(µmol·L-1)]
  			HCCLM-7	Hela	MDA-MB-435s	SW-480	Hep-2	MCF-7
  2a	H	A1	> 25ar	> 25	> 25	> 25	21.4	13.5
  2b	H	A2	> 25	> 25	> 25	> 25	> 25	> 25
  2c	H	A3	9.5	> 25	> 25	11.8	22.5	> 25
  2d	H	A4	> 25	> 25	> 25	> 25	> 25	> 25
  2e	H	A5	31.7	> 25	> 25	16.3	19.9	> 25
  2f	H	A6	> 25	> 25	> 25	> 25	> 25	> 25
  3a	H	A2	> 25	> 25	9.1	> 25	21.9	5.0
  3b	H	A3	13.5	9.5	8.4	16.0	3.5	7.8
  3c	H	A4	8.5	21.7	6.3	21.8	3.3	15.1
  3d	H	A7	> 25	> 25	16.6	> 25	23.0	8.9
  4a	7-OH	A4	3.5	13.2	4.5	11.5	7.8	3.0
  4b	7-OH	A6	> 25	> 25	> 25	> 25	> 25	> 25
  4c	6-Cl-7-OH	A2	13.4	37.4	18.8	8.7	> 30	> 20
  4d	6-Cl-7-OH	A4	8.0	12.3	7.0	11.6	6.7	3.7
  4e	6-Cl-7-OH	A6	21.0	16.2	> 25	> 25	> 25	> 25
  4f	7-OH-8-Me	A2	14.4	> 25	10.4	13.4	> 25	> 25
  4g	7-OH-8-Me	A4	13.4	2.5	8.6	15.3	7.8	16.4
  4h	7-OH-8-Me	A5	8.5	> 25	> 25	13.3	20.3	32.0
  4i	5, 7-(OH)2	A2	> 25	> 25	> 25	> 25	> 25	> 25
  4j	7, 8-(OH)2	A4	> 25	> 25	> 25	> 25	> 25	> 25
  4k	[7, 8]benzo	A2	> 25	> 25	> 25	> 25	> 25	> 25
  4l	[7, 8]benzo	A4	> 25	> 25	> 25	22.9	7.8	> 25
  5-FU			18.6	128.7	14.5	91	44	8.1

  In the series of quinazolinone-DTC derivatives (2), only compounds 2a, 2c, and 2e exhibited potent antiproliferative activity against some cell lines. For example, the activities of 2a, 2c, and 2e against Hep-2 cells were more potent than that of 5-fluorouracil (5-FU). The activity of 2c against MCF-7 cells was slightly better than that of 5-FU. The activities of 2c and 2e against SW-480 cells were significantly better than that of 5-FU (5.5-7.7 times). The activity of 2a against MCF-7 cells was comparable to that of 5-FU. We proposed that the activity of quinazolinone-3-DTC derivatives was significantly weaker than that of the chromone-3-DTC derivatives because the atom connecting the DTC unit and fused heterocyclic moieties changed from C to N, and the molecular orientation was significantly changed (as in the previous analysis of the single crystal structure).

  The benzoxazinone-DTC series were designed based on the SAR of the quinazolinone-DTC series. The connecting atom of DTC and the fused heterocycle moieties changed back to C, and the connection position was shifted to the 2-position corresponding to the chromone. The results of activity screening showed that the benzoxazinone-DTC series 3 exhibited more potent activity than the quinazolinone-DTC series 2. Compound 3a displayed better activity against MDA-MB-435s, Hep-2, and MCF-7 cells than 5-FU. The IC₅₀ of compound 3b against MCF-7 cells was as low as 5.0 µmol·L^-1, and the activities of compound 3b against Hela and Hep-2 cells were more than 10-fold as potent as those of 5-FU. Compound 3c displayed a slightly weaker activity against MCF-7 cells, stronger activity against the other five cell lines, and 13-times higher activity against Hep-2 cells than 5-FU. When the amine moiety was N-methyl aniline with a large steric effect, the activity was decreased significantly.

  Because the benzoxazinone ring may have chemical and metabolic instability, we merged the coumarin moiety that is widely used in drug discovery with DTC pharmacophore. In addition, coumarin scaffolds can facilitate development of fluorescent probes, favoring subsequent study on the mechanism of action. Due to the convenience of synthesis and structural diversity, the connection site was shifted to the 4-position of the coumarin ring to further explore the SAR of the fused heterocycle-DTC derivatives. The activity tests showed that some of the compounds had excellent antiproliferative activity. When a 7-hydroxyl substitution was made on the coumarin ring and the amine moiety was a piperidine ring, the activities of compound 4a against all six tested cell lines were better than those of 5-FU (2.7~9.8 times). In particular, the IC₅₀ values of 4a against HCCLM-7 and MCF-7 cells were as low as 3.5 and 3.0 µmol·L^-1, respectively, and compound 4a was the most active compound. When the amine moiety was a phenylpiperazine ring with large steric hindrance, the activity of compound 4b was lost. When a 6-chloro-7-hy- droxyl group was introduced to the coumarin ring, SAR analysis of the amine moiety showed that piperidine was better than diisopropylamine and was significantly better than phenylpiperazine. Piperidine ring-containing compounds 4d and 4a had comparable activity, and showed better activities against all six tested cell lines than 5-FU (1.1~9.5 times). Compound 4c with a diisopropylamine substitution exhibited significantly decreased activity and essentially no activity against Hep-2 and MCF-7 cells. Meanwhile, compound 4e with a phenylpiperazine substitution only exhibited good inhibitory activity against HCCLM-7 and Hela cells. When the 7-hydroxy-8-methyl group was introduced to the coumarin ring, SAR analysis of the amine moieties showed that the activities were in the order of piperidine > diisopropylamine > morpholine ring. Compound 4g containing the same piperidine ring exhibited excellent activity and showed better activities against five cell lines (excluding MCF-7 cells) than 5-FU, better activities against HCCLM-7 and MCF-7 cells than compound 4a, and comparable activity against MDA-MB- 435s, SW-480, and Hep-2 cells. Notably, for Hela cells the IC₅₀ value was as low as 2.5 µmol·L^-1, which was 50 times as much as that of 5-FU and 4.3 times as much as that of compound 4a. Compound 4f with a diisopropylamine substitution only had good inhibitory activity against HCCLM-7, MDA-MB-435s, and SW-480 cells. Compound 4h with a morpholine substitution only exhibited significant inhibitory activity against HCCLM-7, SW-480, and Hep-2 cells. Other than the hydroxyl group at the 7-position, the introduction of an additional hydroxyl group at either the 5-position or 8-position of the coumarin ring could cause the activity of the compounds 4i and 4j to be lost. When another benzene ring was re-fused at the 7, 8-position of the coumarin ring, compound 4l only showed good inhibitory activity against SW-480 and Hep-2 cells, which might be due to the steric hindrance of the skeleton.

  2.   Conclusions

  Based on the bioisosterism and substructure splicing strategy, DTC units were merged with fused heterocyclic moieties-quinazolinone, benzoxazinone, and coumarin-via a flexible methylene linker. A library of target compounds with abundant structural types was conveniently constructed using a one-pot three-component reaction. These compounds produced good yields and functioned quickly under mild conditions, and the desired products were readily isolated. The results of MTT screening showed that the benzoxazinone-DTC derivative 3b and coumarin-DTC derivatives 4a and 4d showed more potent antiproliferative activities against six tested cell lines (IC₅₀: 3.5~13.5 µmol·L^-1) than the control drug 5-FU (IC₅₀: 8.1~128.7 µmol·L^-1). The activity of some compounds was more than 10-fold more potent than that of 5-FU (e.g., the IC₅₀ values of compounds 3b, 4d, and 4g against Hela cells were between 2.5 and 12.3 µmol·L^-1; the IC₅₀ of compounds 4c against SW-480 cells was 8.7 µmol·L^-1; the IC₅₀ values of compounds 3b and 3c against Hep-2 cells were between 3.3 and 3.5 µmol·L^-1). The results indicated that DTC derivatives bearing fused heterocyclic moieties could be served as lead for further development of new antitumor active compounds.

  3.   Experimental section

  3.1   General methods

  ¹H NMR spectra were recorded at 400 MHz, in CDCl₃ or DMSO-d₆ solution on a Mercury-Plus400 spectrometer. Chemical shifts were recorded with TMS as the internal reference. MS spectra were determined with a Tracems 2000 organic mass spectrometer. Melting points were measured on a Buchi B-545 melting point apparatus and uncorrected. Element analyses were measured on a Vario ELIII CHNSO elemental analyzer. All commercially available solvents and reagents were used as supplied by Acros Organics, unless otherwise stated. The silica gel (200~300 mesh) for flash column chromatography was purchased from Qingdao Marine Chemical Factory in China.

  3.2   General procedure for synthesis of compounds 2~4

  The intermediates M3, M5 and M7 were synthesized according to the previously reported methods.^[25~27] To a solution of amine (1 mmol) in DMF (2 mL) was added dropwise carbon disulfide (2 mmol) and anhydrous potassium phosphate (1 mmol). The resulted mixture was stirred at room temperature for 30 min. Then chlorinated fused heterocyclic intermediates M3, M5 or M7 (1 mmol) was added by one-portion and stirring was continued. After completion of the reaction (monitored by TLC), the mixture was diluted with ice-cold water (20 mL) and the precipitate was filtered, and recrystallized from ethanol to give the target compounds 2, 3 and 4.

  (4-Oxoquinazolin-3(4H)-yl)methyl diethylcarbamodithioate (2a): 84% yield. m.p. 131~133 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.27 (t, J＝7.2 Hz, 6H, CH₃), 3.73 (q, J＝7.2 Hz, 2H, CH₂), 4.03 (q, J＝7.2 Hz, 2H, CH₂), 6.00 (s, 2H, CH₂), 7.53 (t, J＝7.6 Hz, 1H, ArH), 7.72~7.77 (m, 2H, ArH), 8.31 (t, J＝8.0 Hz, 1H, ArH), 8.81 (s, 1 H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.53, 159.93, 147.46, 146.90, 134.74, 127.32, 127.10, 125.95, 121.13, 51.32, 49.54, 46.90, 12.38, 11.20; MS (70 eV, EI) m/z (%): 308.1 (M+H⁺, 100), 330.1 (M+Na⁺, 7). Anal. calcd for C₁₄H₁₇N₃OS₂: C 54.69, H 5.57, N 13.67; found C 54.51, H 5.70, N 13.53.

  (4-Oxoquinazolin-3(4H)-yl)methyl diisopropylcarbamo- dithioate (2b): 82% yield. m.p. 123~125 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.06~1.50 (m, 12H, CH₃), 4.01~4.35 (m, 1H, i-Pr-CH), 4.75~4.94 (m, 1H, i-Pr-CH), 6.02 (s, 2H, CH₂), 7.51 (t, J＝7.6 Hz, 1H, ArH), 7.71~7.77 (m, 2H, ArH), 8.31 (t, J＝8.0 Hz, 1H, ArH), 8.80 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.0, 159.98, 147.45, 147.06, 134.72, 127.30, 127.15, 125.96, 121.15, 50.40, 19.60, 19.10; MS (70 eV, EI) m/z (%): 336.2 (M+H⁺, 100), 358.2 (M+Na⁺, 9). Anal. calcd for C₁₆H₂₁N₃OS₂: C 57.28, H 6.31, N 12.53; found C 57.35, H 6.46, N 12.63.

  (4-Oxoquinazolin-3(4H)-yl)methyl pyrrolidine-1-carbo- dithioate (2c): 70% yield. m.p. 130~132 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.97~2.00 (m, 2H, CH₂), 2.04~2.07 (m, 2H, CH₂), 3.64 (t, J＝6.8 Hz, 2H, CH₂), 3.93 (t, J＝6.8 Hz, 2H, CH₂), 5.98 (s, 2H, CH₂), 7.51 (t, J＝8.0 Hz, 1H, ArH), 7.72~7.79 (m, 2H, ArH), 8.31 (t, J＝7.6 Hz, 1H, ArH), 8.82 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 188.24, 159.93, 147.47, 146.89, 134.78, 127.36, 127.18, 125.93, 121.13, 55.47, 50.75, 25.53, 23.62; MS (70 eV, EI) m/z (%): 306.2 (M+H⁺, 92), 328.2 (M+Na⁺, 100). Anal. calcd for C₁₄H₁₅N₃OS₂: C 55.06, H 4.95, N 13.76; found C 55.23, H 4.87, N 13.65.

  (4-Oxoquinazolin-3(4H)-yl)methyl piperidine-1-carbo- dithioate (2d): 76% yield. m.p. 119~122 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.50~1.70 (m, 6H, CH₂), 3.80~3.90 (m, 2H, CH₂), 4.19~4.29 (m, 2H, CH₂), 6.01 (s, 2H, CH₂), 7.51 (t, J＝7.6 Hz, 1H, ArH), 7.71~7.77 (m, 2H, ArH), 8.31 (t, J＝7.6 Hz, 1H, ArH), 8.80 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.12, 159.92, 147.47, 146.93, 134.74, 127.32, 127.27, 125.94, 121.14, 52.76, 51.32, 25.79, 23.34; MS (70 eV, EI) m/z (%): 320.2 (M+H⁺, 100), 342.2 (M+Na⁺, 10). Anal. calcd for C₁₅H₁₇N₃OS₂: C 56.40, H 5.36, N 13.15; found C 56.21, H 5.53, N 13.08.

  (4-Oxoquinazolin-3(4H)-yl)methyl morpholine-4-carbo- dithioate (2e): 78% yield. m.p. 137~139 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 3.60~3.74 (m, 4 H, CH₂), 3.85~3.96 (m, 2H, CH₂), 4.15~4.26 (m, 2H, CH₂), 5.99 (s, 2H, CH₂), 7.52 (t, J＝7.6 Hz, 1H, ArH), 7.72~7.78 (m, 2H, ArH), 8.31 (t, J＝8.0 Hz, 1H, ArH), 8.76 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 193.06, 159.94, 147.46, 146.95, 134.77, 127.34, 127.21, 125.93, 121.13, 65.53, 51.50, 51.05; MS (70 eV, EI) m/z (%): 322.1 (M+H⁺, 100), 344.1 (M+Na⁺, 12). Anal. calcd for C₁₄H₁₅- N₃O₂S₂: C 52.32, H 4.70, N 13.07; found C 52.12, H 4.87, N 13.21.

  (4-Oxoquinazolin-3(4H)-yl)methyl 4-phenylpiperazine- 1-carbodithioate (2f): 80% yield. 167~169 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 3.22~3.36 (m, 4 H, CH₂), 3.97~4.08 (m, 2H, CH₂), 4.33~4.43 (m, 2H, CH₂), 6.01 (s, 2H, CH₂), 6.91~6.94 (m, 3H, ArH), 7.26~7.31 (m, 2H, ArH), 7.51 (t, J＝7.6 Hz, 1H, ArH), 7.71~7.78 (m, 2H, ArH), 8.31 (t, J＝7.6 Hz, 1H, ArH), 8.76 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 192.65, 159.95, 149.86, 147.47, 146.98, 134.81, 128.99, 127.37, 125.95, 121.14, 119.20, 115.37, 51.20, 49.58, 47.39; MS (70 eV, EI) m/z (%): 397.2 (M+H⁺, 100), 419.2 (M+Na⁺, 53). Anal. calcd for C₂₀H₂₀N₄OS₂: C 60.58, H 5.08, N 14.13; found C 60.37, H 5.23, N 14.00.

  (8-Chloro-4-oxoquinazolin-3(4H)-yl)methyl diisopro- pylcarbamodithioate (2g): 84% yield. m.p. 128~130 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.08~1.65 (m, 12H, CH₃), 4.09~4.28 (m, 1H, i-Pr-CH), 4.60~4.85 (m, 1H, i-Pr-CH), 6.03 (s, 2H, CH₂), 7.51 (d, J＝8.0 Hz, 1H, ArH), 7.85 (t, J＝8.0 Hz, 1H, ArH), 8.23 (d, J＝8.0 Hz, 1H, ArH), 8.95 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 192.02, 159.96, 148.50, 144.41, 135.33, 131.35, 128.24, 125.67, 123.37, 51.40, 19.70, 19.57; MS (70 eV, EI) m/z (%): 370.2 (M+H⁺, 100), 392.1 (M+ Na⁺, 73). Anal. calcd for C₁₆H₂₀ClN₃OS₂: C 51.95, H 5.45, N 11.36; found C 51.805, H 5.62, N 11.42.

  (8-Chloro-4-oxoquinazolin-3(4H)-yl)methyl piperidine- 1-carbodithioate (2h): 86% yield. m.p. 130~132 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.55~1.73 (m, 6H, CH₂), 3.82~3.95 (m, 2H, CH₂), 4.20~4.31 (m, 2H, CH₂), 6.01 (s, 2H, CH₂), 7.43 (d, J＝8.0 Hz, 1H, ArH), 7.85 (t, J＝7.8 Hz, 1H, ArH), 8.23 (d, J＝8.0 Hz, 1H, ArH), 8.96 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 190.92, 159.39, 147.84, 143.92, 134.84, 130.86, 127.75, 125.13, 122.86, 52.74, 51.44, 25.81, 25.10, 23.34; MS (70 eV, EI) m/z (%): 354.1 (M+H⁺, 60), 376.2 (M+Na⁺, 100). Anal. calcd for C₁₅H₁₆ClN₃OS₂: C 50.91, H 4.56, N 11.87; found C 50.76, H 4.62, N 11.90.

  (8-Chloro-4-oxoquinazolin-3(4H)-yl)methyl morpholine-4-carbodithioate (2i): 78% yield. m.p. 151~153 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 3.58~3.75 (m, 4H, CH₂), 3.85~3.96 (m, 2H, CH₂), 4.15~4.36 (m, 2H, CH₂), 5.99 (s, 2H, CH₂), 7.44 (d, J＝8.0 Hz, 1H, ArH), 7.85 (t, J＝7.8 Hz, 1H, ArH), 8.23 (d, J＝8.0 Hz, 1H, ArH), 8.91 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 192.87, 159.41, 147.89, 143.93, 134.88, 130.86, 127.79, 125.14, 122.87, 65.53, 51.15, 50.51; MS (70 eV, EI) m/z (%): 356.1 (M+H⁺, 55), 378.1 (M+Na⁺, 100). Anal. calcd for C₁₄H₁₄ClN₃O₂S₂: C 47.25, H 3.97, N 11.81; found C 47.33, H 4.13, N 11.79.

  (8-Chloro-4-oxoquinazolin-3(4H)-yl)methyl 4-phenylpi- perazine-1-carbodithioate (2j): 80% yield. m.p. 178~180 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 3.15~3.40 (m, 4H, CH₂), 3.91~4.08 (m, 2H, CH₂), 4.25~4.45 (m, 2H, CH₂), 6.01 (s, 2H, CH₂), 6.92~6.95 (m, 3H, ArH), 7.26~7.30 (m, 2H, ArH), 7.44 (d, J＝8.0 Hz, 1H, ArH), 7.85 (t, J＝8.8 Hz, 1H, ArH), 8.23 (d, J＝8.0 Hz, 1H, ArH), 8.92 (s, 1H, 2-CH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 192.99, 159.92, 150.34, 148.36, 144.42, 135.36, 131.37, 129.48, 128.26, 125.63, 123.37, 119.70, 115.85, 51.81, 50.11, 47.90; MS (70 eV, EI) m/z (%): 430.3 (M+H⁺, 100). Anal. calcd for C₂₀H₁₉ClN₄OS₂: C 55.74, H 4.44, N 13.00; found C 55.58, H 4.60, N 13.12.

  (4-oxo-4H-Benzo[d][1, 3]oxazin-2-yl)methyl diisoprop- ylcarbamodithioate (3a): 78% yield. 131~133 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.01~1.75 (m, 14 H, i-Pr), 4.64 (s, 2H, CH₂), 7.52 (t, J＝7.2 Hz, 1H, ArH), 7.61 (d, J＝8.0 Hz, 1H, ArH), 7.80 (t, J＝7.8 Hz, 1H, ArH), 8.19 (d, J＝7.8 Hz, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.10, 158.83, 158.50, 145.77, 136.97, 128.72, 127.98, 126.42, 116.37, 55.40, 39.27, 19.36; MS (70 eV, EI) m/z (%): 337.2 (M+H⁺, 100), 359.2 (M+Na⁺, 7). Anal. calcd for C₁₆H₂₀N₂O₂S₂: C 57.11, H 5.99, N 8.33; found C 57.03, H 5.78, N 8.40.

  (4-oxo-4H-Benzo[d][1, 3]oxazin-2-yl)methyl pyrrolidi- ne-1-carbodithioate (3b): 82% yield. m.p. 120~122 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 2.01 (q, 2H, CH₂), 2.13 (q, 2H, CH₂), 3.74 (t, J＝6.8 Hz, 2H, CH₂), 3.94 (t, J＝6.8 Hz, 2H, CH₂), 4.67 (s, 2H, CH₂), 7.52 (t, J＝7.6 Hz, 1H, ArH), 7.62 (d, J＝8.0 Hz, 1H, ArH), 7.80 (t, J＝7.8 Hz, 1H, ArH), 8.19 (d, J＝7.8 Hz, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 188.96, 158.80, 158.40, 145.70, 136.98, 128.77, 128.00, 126.45, 116.39, 55.49, 25.66, 23.75; MS (70 eV, EI) m/z (%): 307.1 (M+H⁺, 100), 327.1 (M+ Na⁺, 15). Anal. calcd for C₁₄H₁₄N₂O₂S₂: C 54.88, H 4.61, N 9.14; found C 54.95, H 4.72, N 9.01.

  (4-oxo-4H-Benzo[d][1, 3]oxazin-2-yl)methyl piperidine- 1-carbodithioate (3c): 76% yield. m.p. 140~142 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.52~1.73 (m, 6H, CH₂), 3.87~4.02 (m, 2H, CH₂), 4.10~4.28 (m, 2H, CH₂), 4.65 (s, 2H, CH₂), 7.52 (t, J＝7.8 Hz, 1H, ArH), 7.62 (d, J＝8.0 Hz, 1H, ArH), 7.80 (t, J＝8.0 Hz, 1H, ArH), 8.19 (d, J＝8.0 Hz, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.87, 158.80, 158.41, 145.73, 136.98, 128.75, 127.99, 126.44, 116.36, 52.92, 51.31, 25.83, 23.43; MS (70 eV, EI) m/z (%): 321.2 (M+H⁺, 100). Anal. calcd for C₁₅H₁₆N₂- O₂S₂: C 56.22, H 5.03, N 8.74; found C 56.03, H 5.31, N 8.60.

  (4-oxo-4H-Benzo[d][1, 3]oxazin-2-yl)methyl methyl- (phenyl)carbamodithioate (3d): 79% yield. m.p. 140~142 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 3.79 (s, 3H, CH₃), 4.51 (s, 2H, CH₂), 7.31 (d, J＝6.8 Hz, 2H, ArH), 7.45~7.51 (m, 4H, ArH), 7.58 (d, J＝8.0 Hz, 1H, ArH), 7.78 (t, J＝7.2 Hz, 1H, ArH), 8.17 (d, J＝8.0 Hz, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 195.71, 158.76, 158.15, 145.64, 143.96, 136.91, 129.90, 129.31, 128.75, 127.95, 126.94, 126.45, 116.39, 56.01, 46.30; MS (70 eV, EI) m/z (%): 343.2 (M+H⁺, 100), 356.1 (M+Na⁺, 21). Anal. calcd for C₁₇H₁₄N₂O₂S₂: C 59.63, H 4.12, N 8.18; found C 59.40, H 4.31, N 8.06.

  (6-Chloro-4-oxo-4H-chromen-3-yl)methyl piperidine-1- carbodithioate (4a): 76% yield. m.p. 164~168 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.55~1.70 (m, 6H, CH₂), 3.86~3.98 (m, 2H, CH₂), 4.17~4.30 (m, 2H, CH₂), 4.76 (s, 2H, CH₂), 6.31 (s, 1H, 3-CH), 6.75 (s, 1H, ArH), 6.83 (d, J＝9.2 Hz, 1H, ArH), 7.71 (d, J＝8.8 Hz, 1H, ArH), 10.59 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.73, 161.37, 160.02, 155.06, 151.44, 126.28, 113.03, 110.53, 102.45, 53.13, 51.16, 25.84, 23.44; MS (70 eV, EI) m/z (%): 336.1 (M+H⁺, 100). Anal. calcd for C₁₆H₁₇NO₃S₂: C 57.29, H 5.11, N 4.18; found C 57.12, H 5.31, N 4.23.

  (7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl 4-phenylpi- perazine-1-carbodithioate (4b): 79% yield. m.p. 196~198 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 3.22~3.35 (m, 4H, CH₂), 4.00~4.15 (m, 2H, CH₂), 4.30~4.45 (m, 2H, CH₂), 4.95 (s, 2H, CH₂), 6.17 (s, 1H, 3-CH), 6.21~6.28 (m, 2H, ArH), 6.81 (t, J＝7.2 Hz, 1H, ArH), 6.93 (d, J＝8.0 Hz, 2H, ArH), 7.24 (t, J＝7.6 Hz, 2H, ArH), 10.43 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 194.40, 162.25, 161.35, 159.92, 157.19, 152.26, 149.93, 128.99, 119.18, 115.40, 109.09, 100.60, 99.28, 94.68, 50.95, 49.28, 47.48; MS (70 eV, EI) m/z (%): 413.2 (M+H⁺, 100). Anal. calcd for C₂₁H₂₀N₂O₃S₂: C 61.14, H 4.89, N 6.79; found C 61.03, H 4.70, N 6.85.

  (6-Chloro-7-hydroxy-2-oxo-2H-chromen-4-yl)methyl diisopropyl-carbamodithioate (4c): 80% yield. m.p. 156~158 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.89~1.47 (m, 12H, CH₃), 4.06~4.24 (m, 1H, i-Pr-CH), 4.92 (s, 2H, CH₂), 6.45 (s, 1H, 3-CH), 6.99 (s, 1H, ArH), 7.03 (s, 1H, ArH), 11.32 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 190.83, 159.58, 156.46, 153.22, 151.05, 125.86, 117.01, 111.74, 111.29, 103.55, 56.00, 19.32, 18.51; MS (70 eV, EI) m/z (%): 386.2 (M+H⁺, 100), 408.1 (M+Na⁺, 6). Anal. calcd for C₁₇H₂₀ClNO₃S₂: C 52.91, H 5.22, N 3.63; found C 52.76, H 5.43, N 3.48.

  (6-Chloro-7-hydroxy-2-oxo-2H-chromen-4-yl)methyl piperidine-1-carbodithioate (4d): 84% yield. m.p. 171~173 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.56~1.71 (m, 6H, CH₂), 3..80~3.93 (m, 2H, CH₂), 4.12~4.24 (m, 2H, CH₂), 4.80 (s, 2H, CH₂), 6.36 (s, 1H, 3-CH), 6.92 (s, 1H, ArH), 7.01 (s, 1H, ArH), 11.48 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 193.73, 160.18, 159.16, 152.99, 151.45, 122.85, 111.86, 111.04, 110.48, 110.19, 65.53, 51.82, 50.36, 36.17; MS (70 eV, EI) m/z (%): 370.2 (M+H⁺, 100). Anal. calcd for C₁₆H₁₆ClNO₃S₂: C 51.95, H 4.36, N 3.79; found C 51.81, H 4.54, N 3.93.

  (6-Chloro-7-hydroxy-2-oxo-2H-chromen-4-yl)methyl 4-phenyl-piperazine-1-carbodithioate (4e): 78% yield. m.p. 206~208 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 3.20~3.40 (m, 4H, CH₂), 4.02~4.16 (m, 2H, CH₂), 4.32~4.46 (m, 2H, CH₂), 5.10 (s, 2H, CH₂), 6.45 (s, 1H, 3-CH), 6.82 (t, J＝7.2 Hz, 1H, ArH), 6.92 (d, J＝8.0 Hz, 2H, ArH), 6.97 (s, 1H, ArH), 7.11 (s, 1H, ArH), 7.24 (t, J＝7.2 Hz, 2H, ArH), 11.08 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 193.74, 160.38, 154.95, 153.04, 151.31, 150.44, 149.93, 131.18, 119.73, 115.91, 115.35, 110.91, 110.41, 104.13, 51.95, 50.02, 48.05; MS (70 eV, EI) m/z (%): 447.1 (M+H⁺, 100), 469.1 (M+Na⁺, 12). Anal. calcd for C₂₁H₁₉ClN₂O₃S₂: C 56.43, H 4.28, N 6.27; found C 56.56, H 4.35, N 6.20.

  (7-Hydroxy-8-methyl-2-oxo-2H-chromen-4-yl)methyl diisopropyl-carbamodithioate (4f): 85% yield. m.p. 146~148 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.20~1.37 (m, 12H, CH₃), 2.32 (s, 3H, CH₃), 4.20~4.32 (m, 1H, i-Pr-CH), 4.70 (s, 2H, CH₂), 4.94 (s, 1H, i-Pr-CH), 6.42 (s, 1H, 3-CH), 6.86 (d, J＝8.8 Hz, 1H, ArH), 7.50 (d, J＝8.8 Hz, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 193.53, 159.88, 152.53, 144.04, 134.14, 132.59, 128.38, 118.51, 116.39, 56.06, 30.92, 25.00, 24.59; MS (70 eV, EI) m/z (%): 366.2 (M+H⁺, 100). Anal. calcd for C₁₈H₂₃NO₃S₂: C 59.15, H 6.34, N 3.83; found C 59.01, H 6.52, N 3.74.

  (7-Hydroxy-8-methyl-2-oxo-2H-chromen-4-yl)methyl piperidine-1-carbodithioate (4g): 72% yield. m.p. 167~169 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.53~1.70 (m, 6H, CH₂), 2.16 (s, 3H, CH₃), 3.85~3.97 (m, 2H, CH₂), 4.16~4.28 (m, 2H, CH₂), 4.76 (s, 2H, CH₂), 6.31 (s, 1H, 3-CH), 6.88 (d, J＝8.8 Hz, 1H, ArH), 7.56 (d, J＝8.8 Hz, 1H, ArH), 10.53 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.77, 160.16, 159.13, 152.98, 151.69, 122.85, 111.83, 111.01, 110.50, 110.11, 53.11, 51.15, 25.85, 23.44; MS (70 eV, EI) m/z (%): 350.2 (M+H⁺, 86), 372.3 (M+Na⁺, 9). Anal. calcd for C₁₇H₁₉NO₃S₂: C 58.43, H 5.48, N 4.01; found C 58.14, H 5.67, N 4.21.

  (7-Hydroxy-8-methyl-2-oxo-2H-chromen-4-yl)methyl morpholine-4-carbodithioate (4h): 80% yield. m.p. 150~153 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 2.16 (s, 3H, CH₃), 3.55~3.68 (m, 4H, CH₂), 3.85~3.97 (m, 2H, CH₂), 4.10~4.24 (m, 2H, CH₂), 4.78 (s, 2H, CH₂), 6.32 (s, 1H, 3-CH), 6.89 (d, J＝8.8 Hz, 1H, ArH), 7.56 (d, J＝8.8 Hz, 1H, ArH), 10.54 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 191.61, 159.63, 156.63, 153.27, 150.98, 125.83, 117.10, 111.54, 111.21, 103.58, 53.29, 51.18, 25.20, 23.44; MS (70 eV, EI) m/z (%): 352.2 (M+H⁺, 100). Anal. calcd for C₁₆H₁₇NO₄S₂: C 54.68, H 4.88, N 3.99; found C 54.81, H 4.72, N 3.80.

  (5, 7-Dihydroxy-2-oxo-2H-chromen-4-yl)methyl diisopropylcarbamodithioate (4i): 76% yield. m.p. 178~181 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.29~1.53 (m, 12H, CH₃), 4.02~4.32 (m, 1H, i-Pr-CH), 4.89 (s, 2H, CH₂), 5.76 (b, 1H, i-Pr-CH), 6.12 (s, 1H, ArH), 6.21 (s, 1H, ArH), 6.27 (s, 1H, 3-CH), 10.41 (b, 1H, OH), 10.90 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 190.98, 161.30, 159.97, 157.27, 156.62, 152.63, 109.07, 100.77, 99.25, 94.79, 55.00, 19.30, 17.98; MS (70 eV, EI) m/z (%): 368.2 (M+H⁺, 100). Anal. calcd for C₁₇H₂₁ClNO₄S₂: C 55.56, H 5.76, N 3.81; found C 55.71, H 5.65, N 3.69.

  (7, 8-Dihydroxy-2-oxo-2H-chromen-4-yl)methyl piperidine-1-carbodithioate (4j): 80% yield. m.p. 167~169 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.30~1.66 (m, 6H, CH₂), 3.82~3.96 (m, 2H, CH₂), 4.10~34 (m, 2H, CH₂), 4.66 (s, 2H, CH₂), 6.38 (s, 1H, 3-CH), 6.85 (d, J＝8.4 Hz, 1H, ArH), 7.31 (d, J＝8.8 Hz, 1H, ArH), 9.49 (b, 1H, OH), 10.17 (b, 1H, OH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.89, 159.91, 155.98, 1550.53, 134.83, 128.44, 128.00, 124.58, 122.11, 121.34, 52.08, 49.36, 25.33, 19.74; MS (70 eV, EI) m/z (%): 352.2 (M+H⁺, 100). Anal. calcd for C₁₆H₁₇NO₄S₂: C 54.68, H 4.88, N 3.99; found C 54.42, H 4.99, N 3.90.

  (2-oxo-2H-Benzo[h]chromen-4-yl)methyl diisopropylcarbamodithioate (4k): 82% yield. m.p. 135~137 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.05~1.60 (m, 12H, CH₃), 4.01~4.25 (m, 1H, i-Pr-CH), 4.84 (s, 2H, CH₂), 6.66 (s, 1H, 3-CH), 7.63~7.66 (m, 2H, ArH), 7.71~7.77 (m, 2H, ArH), 7.87~7.90 (m, 1H, ArH), 8.56~8.59 (m, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 185.5, 159.89, 152.66, 150.52, 134.81, 129.35, 128.42, 127.99, 124.57, 122.75, 122.10, 121.33, 114.96, 114.38, 55.40, 39.27, 19.80; MS (70 eV, EI) m/z (%): 386.2 (M+H⁺, 100), 408.2 (M+ Na⁺, 16). Anal. calcd for C₂₁H₂₃NO₂S₂: C 65.42, H 6.01, N 3.63; found C 65.32, H 6.13, N 3.55.

  (2-oxo-2H-Benzo[h]chromen-4-yl)methyl piperidine-1- carbodithioate (4l): 80% yield. m.p. 132~134 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 1.50~1.73 (m, 6H, CH₂), 3.82~3.94 (m, 2H, CH₂), 4.19~4.32 (m, 2H, CH₂), 4.85 (s, 2H, CH₂), 6.67 (s, 1H, 3-CH), 7.63~7.66 (m, 2H, ArH), 7.70~7.76 (m, 2H, ArH), 7.86~7.90 (m, 1H, ArH), 8.55~8.58 (m, 1H, ArH); ¹³C NMR (100 MHz, DMSO- d₆) δ: 185.5, 159.89, 152.66, 150.52, 134.81, 129.35, 128.42, 127.99, 124.57, 122.75, 122.10, 121.33, 114.96, 114.38, 55.40, 39.27, 19.80; MS (70 eV, EI) m/z (%): 386.2 (M+H⁺, 100), 408.2 (M+Na⁺, 16). Anal. calcd for C₂₀H₁₉NO₂S₂: C 65.01, H 5.18, N 3.79; found C 64.89, H 5.32, N 3.85.

  3.3   Pharmacology

  Cells were seeded in 96-well microplates at 8000 cells/well. The test compounds were added, and cells were incubated for 72 h.^[30] Then, 20 µL (0.5 mg/mL, final concentration) of MTT (Sigma, USA) was added to each well, and cells were incubated for 4 h. MTT was converted to a blue formazan product by mitochondrial succinate dehydrogenase. This product was eluted from cells by addition of 150 mL of DMSO. Absorbance at 570 nm was determined by a NJ-2300 microplate spectrophotometer. For each assay, three different experiments were performed in triplicate. The results were statistically evaluated with the Student's t-test and presented as means±SEM.

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  Figure 1  Reported DTC derivatives with antiproliferative activity

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  Figure 2  Design of diversified fused heterocyclic derivatives bearing a dithiocarbamate unit

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  Scheme 1  Synthesis of 2-chloromethyl quinazolin-4-one (M-3) and 2-chloromethyl benzo[d][1, 3]oxazin-4-one (M-5)

  Reagents and conditions: (a) formamide, 130~160 ℃; (b) HCHO, 1, 4- dioxane, 80 ℃; (c) SOCl₂, 1, 4-dioxane, r.t.; (d) 2-chloroacetyl chloride, r.t.; (e) HOAc, 100 ℃

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  Figure 3  X-ray structure of 2h

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  Table 1.  Antiproliferative activity of the synthesized derivatives

  Compd.	X/R1~R4	Amine unit	Antiproliferative activity [IC50/(µmol·L-1)]
  			HCCLM-7	Hela	MDA-MB-435s	SW-480	Hep-2	MCF-7
  2a	H	A1	> 25ar	> 25	> 25	> 25	21.4	13.5
  2b	H	A2	> 25	> 25	> 25	> 25	> 25	> 25
  2c	H	A3	9.5	> 25	> 25	11.8	22.5	> 25
  2d	H	A4	> 25	> 25	> 25	> 25	> 25	> 25
  2e	H	A5	31.7	> 25	> 25	16.3	19.9	> 25
  2f	H	A6	> 25	> 25	> 25	> 25	> 25	> 25
  3a	H	A2	> 25	> 25	9.1	> 25	21.9	5.0
  3b	H	A3	13.5	9.5	8.4	16.0	3.5	7.8
  3c	H	A4	8.5	21.7	6.3	21.8	3.3	15.1
  3d	H	A7	> 25	> 25	16.6	> 25	23.0	8.9
  4a	7-OH	A4	3.5	13.2	4.5	11.5	7.8	3.0
  4b	7-OH	A6	> 25	> 25	> 25	> 25	> 25	> 25
  4c	6-Cl-7-OH	A2	13.4	37.4	18.8	8.7	> 30	> 20
  4d	6-Cl-7-OH	A4	8.0	12.3	7.0	11.6	6.7	3.7
  4e	6-Cl-7-OH	A6	21.0	16.2	> 25	> 25	> 25	> 25
  4f	7-OH-8-Me	A2	14.4	> 25	10.4	13.4	> 25	> 25
  4g	7-OH-8-Me	A4	13.4	2.5	8.6	15.3	7.8	16.4
  4h	7-OH-8-Me	A5	8.5	> 25	> 25	13.3	20.3	32.0
  4i	5, 7-(OH)2	A2	> 25	> 25	> 25	> 25	> 25	> 25
  4j	7, 8-(OH)2	A4	> 25	> 25	> 25	> 25	> 25	> 25
  4k	[7, 8]benzo	A2	> 25	> 25	> 25	> 25	> 25	> 25
  4l	[7, 8]benzo	A4	> 25	> 25	> 25	22.9	7.8	> 25
  5-FU			18.6	128.7	14.5	91	44	8.1

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