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Article

New 1H-Benzo[f]indazole-4,9-diones Conjugated with C-Protected Amino Acids and Other Derivatives: Synthesis and in Vitro Antiproliferative Evaluation

by
Aurora Molinari
1,*,
Alfonso Oliva
1,*,
Marlene Arismendi-Macuer
1,
Leda Guzmán
1,
Mauricio Fuentealba
1,
Marcela Knox
1,
Raúl Vinet
2,3 and
Arturo San Feliciano
4
1
Instituto de Química, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile
2
Facultad de Farmacia, Universidad de Valparaíso, Valparaíso 2360102, Chile
3
Centro Regional de Estudios en Alimentos y Salud (CREAS), Valparaíso 2362696, Chile
4
Facultad de Farmacia, Departamento de Química Farmacéutica, CIETUS, IBSAL, Universidad de Salamanca, Salamanca 37007, Spain
*
Authors to whom correspondence should be addressed.
Molecules 2015, 20(12), 21924-21938; https://doi.org/10.3390/molecules201219809
Submission received: 4 November 2015 / Revised: 30 November 2015 / Accepted: 1 December 2015 / Published: 8 December 2015
(This article belongs to the Special Issue Design and Synthesis of Novel Conjugated and Non Conjugated Small Molecules)
Browse Figures
Versions Notes

Abstract

:
1H-Benzo[f]indazole-4,9-dione derivatives conjugated with C-protected amino acids (glycine, l-alanine, l-phenylalanine and l-glutamic acid) 6a–l were prepared by chemically modifying the prenyl substituent of 3-methyl-7-(4-methylpent-3-enyl)-1H-benzo[f]indazole-4,9-dione 2 through epoxidation, degradative oxidation, oxidation and N-acyl condensation reactions. The chemical structures of the synthesized compounds were elucidated by analyzing their IR, 1H-NMR and 13C-NMR spectral data together with elemental analysis for carbon, hydrogen and nitrogen. The preliminary in vitro antiproliferative activity of the synthesized derivatives was evaluated on KATO-III and MCF-7 cell lines using a cell proliferation assay. The majority of the derivatives exhibited significant antiproliferative activity with IC50 values ranging from 25.5 to 432.5 μM. These results suggest that 1H-benzo[f]indazole-4,9-dione derivatives are promising molecules to be researched for developing new anticancer agents.

Graphical Abstract

1. Introduction

A considerable number of naturally occurring and synthetic compounds that contain a 1,4-quinone moiety have been investigated for antitumor activity [1,2,3,4,5]. These compounds generate a quinone/hydroquinone one-electron redox process that inhibits mitochondrial electron transport and decouples oxidative phosphorylation [5]. Additionally, they act as topoisomerase inhibitors via DNA intercalation or as alkylating agents that add across both strands of the double helix, thereby leading to cancer cell death. Furthermore, it has been suggested that the quinone-induced inhibition of cancer cell growth can be attributed to the generation of reactive oxygen species (ROS) after redox cycling [1,2,3,4,5,6,7]. 1H-Indazolediones are nitrogen-containing heterocyclic 1,4-quinones that possess interesting chemical and biological properties, including antitumor activities against Ehrlich ascites carcinoma growth in male CF1 mice, P-388 lymphocytic leukemia in male BDF1 mice and L1210 murine leukemia cells [8,9,10,11,12,13,14,15,16,17,18]. In the literature, there are various synthetic methods for preparing 1H-indazoles [8], whereas 1H-indazole-4,7 and 4,9-diones are synthesized via the 1,3-dipolar cycloaddition of diazomethanes to 1,4-quinones [10,19,20,21,22]. We have recently reported the synthesis of unsubstituted and N-substituted 3-methyl-7-(4-methylpent-3-enyl)-1H-benzo[f]indazole-4,9-diones 2 from the reaction of 2-acetyl-6-(4-methylpent-3-enyl)-1,4-naphthoquinone 1 with hydrazine or substituted hydrazines [23]. The objective of this work was to continue previous research on the design and synthesis of new potentially cytotoxic 1,4-naphthoquinone compounds [24,25] while taking into account the enhanced cytotoxic effect that has been observed in drugs or compounds conjugated with amino acids [26,27]. Therefore, we prepared twenty one new 1H-benzo[f]indazole-4,9-dione compounds conjugated with glycine and the l-type amino acids alanine, phenylalanine, and glutamic acid 6a–l, as well as the epoxides 3a–c, aldehydes 4a–c and carboxylic acids 5a–c, by chemically modifying the prenyl 7-(4-methylpent-3-enyl) substituent of 2. Additionally, we evaluated the antiproliferative activity of these new compounds on KATO-III and MCF-7 cell lines of human gastric cancer and human breast cancer, respectively.

2. Results and Discussion

2.1. Chemistry

The new derivatives were prepared using 1H-benzo[f]indazole-4,9-diones 2 (R = -H; -CH2CH2OH; -CH2CH2OAc) as starting substrates, which were conveniently obtained through a direct cyclization reaction of 2-acetyl-6-(4-methylpent-3-enyl)-1,4-naphthoquinone 1 with hydrazines, triethylamine and catalytic glacial acetic acid [23]. The first step of this reaction could be (i) the conjugate addition of the hydrazine to the 1,4-naphthoquinone unit to afford the 3-substituted 1,4-hydroquinone compound 1’ or (ii) the condensation reaction between the acetyl group and hydrazine to form the corresponding hydrazone 1T’.
To rationalize the possible products, we performed full geometry optimizations of the structures using preliminary density functional theory (DFT) calculations (see the Experimental Section). With the aim of exploring the reactants and possible products of the first step of this reaction from a thermodynamics perspective, we subtracted the total bonding energies of the products from the total bonding energies of the reactants. The comparison between these energy changes indicated that the formation of 1’ is 4-fold more favorable than the formation of 1T’ (Scheme 1). Moreover, the HOMO-LUMO gaps of compounds 1’ and 1T’ were 2.28 eV and 1.39 eV, respectively, which indicates that the most stable derivative is compound 1’ (Figure 1). These results suggest that cyclization must initially occur by the conjugate addition to afford the 3-substituted 1,4-hydroquinone compound 1’, but a complete transition state (TS) search and intrinsic reaction coordinate (IRC) study are needed to confirm this hypothesis. For this reason, our research group is currently working to obtain a full reaction pathway for all reaction steps.
Molecules 20 19809 g002 550
Scheme 1. Comparison between reaction energies of 1 to 1’ and 1T’.
Scheme 1. Comparison between reaction energies of 1 to 1’ and 1T’.
Molecules 20 19809 g002
Molecules 20 19809 g001 550
Figure 1. Molecular orbital diagrams of compounds 1T’ and 1’. The HOMO energies have been arbitrarily set to zero for clarity.
Figure 1. Molecular orbital diagrams of compounds 1T’ and 1’. The HOMO energies have been arbitrarily set to zero for clarity.
Molecules 20 19809 g001
The second step of the reaction is the cyclization of the 3-substituted 1,4-hydroquinone compound 1’ by the nucleophilic addition/elimination reaction between the amino and carbonyl groups of 1’ to afford the fused pyrazolo-1,4-naphthohydroquinone compound 1’’. Finally, this compound is oxidized to the 1,4-naphthoquinone 2 by the initial 2-acetyl-1,4-naphthoquinone 1 (Scheme 2), a step that is supported by the isolation of 2-acetyl-6-(4-methylpent-3-enyl)-1,4-naphthohydroquinone as a by-product [10,28].
Molecules 20 19809 g003 550
Scheme 2. Cyclization pathway for the N-substituted 1H-benzo[f]indazole-4,9-diones 2.
Scheme 2. Cyclization pathway for the N-substituted 1H-benzo[f]indazole-4,9-diones 2.
Molecules 20 19809 g003
To synthesize the new 1H-benzo[f]indazole-4,9-diones conjugated with C-protected amino acids 6a–l, we followed the synthetic pathway shown in Scheme 3. The epoxidation of the double bond in the 7-(4-methylpent-3-enyl) group of 2a–c to afford oxiranyl compounds 3a–c was accomplished with m-chloroperoxybenzoic acid (mCPBA), and treatment of these compounds with periodic acid afforded the aldehydes 4a–c [29]. Oxidation of these aldehydes to the carboxylic acids 5a–c was performed with sodium chlorite in the presence of a catalytic amount of 2-methyl-2-butene. The reactivity of the carboxylic group was then enhanced through the in situ formation of the mixed anhydride with ethyl chloroformate followed by the addition of the corresponding methyl ester of glycine, l-alanine, l-phenylalanine and l-glutamic acid [25]. In all of these synthesized compounds, the l-configuration must be retained in the amino acid unit. The physical and analytical data of the compounds are presented in the experimental section along with the IR, 1H and 13C spectroscopic data; chemical shifts are reported according to the carbon numbering of compounds 2 in Scheme 3.
Molecules 20 19809 g004 550
Scheme 3. Synthetic pathway for the new conjugated derivatives 6a–l. (a) mCPBA, CH2Cl2, NaHCO3, rt, 4 h; (b) H5IO6, THF, H2O, rt, 2 h; (c) NaClO2, NaH2PO4, H2O, t-BuOH, 2-methyl-2-butene, rt, 27 h; (d) EtOCOCl, Et3N, THF, 20 min, 0 °C, R1CH(NH2)CO2Me, rt, 16 h.
Scheme 3. Synthetic pathway for the new conjugated derivatives 6a–l. (a) mCPBA, CH2Cl2, NaHCO3, rt, 4 h; (b) H5IO6, THF, H2O, rt, 2 h; (c) NaClO2, NaH2PO4, H2O, t-BuOH, 2-methyl-2-butene, rt, 27 h; (d) EtOCOCl, Et3N, THF, 20 min, 0 °C, R1CH(NH2)CO2Me, rt, 16 h.
Molecules 20 19809 g004
The common features from the spectral data of compounds 6a–l are closely related to those previously reported for the starting compounds 2a–c [23], and they are as follows:
-
In some cases, their IR spectra show two carbonyl-quinone absorptions at approximately 1680 and 1670 cm−1, but the latter absorption is primarily observed.
-
In the 1H spectra, the singlet of the C-10 methyl group appears at approximately 2.60 to 2.80 ppm, the coupled methylene groups of C-11 and C-12 carbons show triplets or multiplets between 2.50 and 3.00 ppm (J = 7.3–8.0 Hz), and the coupled aromatic hydrogen of carbon C-5, C-6 and C-8 are observed as doublets of doublets and two doublets at 7.70 to 8.10 ppm (J = 7.6 and 1.6 Hz).
-
The 13C-NMR spectra contain signals for carbonyl-quinone C-4 and C-9 carbon atoms at 170 to 180 ppm.

2.2. Biological Assay

The antiproliferative activity of the synthesized compounds was assessed on KATO-III and MCF-7 cell lines using a CellTiter 96® AQueous One Solution Proliferation Assay (MTS) from Promega (Madison, WI, USA) with doxorubicin as a control. The results were expressed as the concentration determining 50% inhibition of cell proliferation (IC50).
Table 1 and Table 2 show the IC50 values for the antiproliferative activity obtained for each derivative tested in KATO-III and MCF-7 cell lines, respectively. Each column in Table 1 and Table 2 contains the IC50 values for derivatives belonging to Series-I, -II and -III, respectively.
The antiproliferative activity determined in KATO-III cell lines for Series-I, -II and -III of 1H-benzo[f]indazole-4,9-dione-based derivatives ranged from 60.3 (2a) to 326.6 μM (5a), 25.5 (2b) to 401.8 μM (3b), and 33.0 (2c) to 324.3 (3c), respectively (Table 1). Similarly, the antiproliferative activity assayed in MCF-7 cell lines for Series-I, -II and -III 1H-benzo[f]indazole-4,9-dione-based derivatives ranged from 63.2 (2a) to 432.5 μM (3a), 27.5 (2b) to 415.9 μM (3b), and 29.4 (2c) to 389.9 μM (3c), respectively (Table 2).
By comparing Table 1 and Table 2 is possible to observe the similarity between the patterns generated from the IC50 values obtained from Series-I, -II and -III derivatives. Moreover, these patterns were very similar in both cell models.
Table
Table 1. In vitro antiproliferative activities of 1H-benzo[f]indazole-4,9-dione-based derivatives expressed as IC50 values obtained in KATO-III cell line.
Table 1. In vitro antiproliferative activities of 1H-benzo[f]indazole-4,9-dione-based derivatives expressed as IC50 values obtained in KATO-III cell line.
Series-ISeries-IISeries-III
IC50 (CI 95%) μMIC50 (CI 95%) μMIC50 (CI 95%) μM
ppp
2a2b2a
60.3 (18.9–192.4)25.5 (9.4–69.1)33.0 (8.0–136.5)
C: NA, R: NAC: NA, R: NSC: NA, R: NS
3a3b3c
313.3 (110.4–889.3)401.8 (166.8–967.8)324.3 (132.9–791.3)
C: ***, R: NAC: ****, R: NSC: ****, R: NS
4a4b4c
99.5 (50.1–197.7)63.0 (22.4–176.9)60.5 (29.8–123.1)
C: ***, R: NAC: NS, R: NSC: NS, R: NS
5a5b5c
326.6 (167.9–635.4)337.3 (192.3–591.5)162.6 (70.6–374.2)
C: ***, R: NAC: ****, R: NSC **, R: NS
6a6e6i
230.7 (82.9–642.2)310.5 (132.5–727.4)208.0 (92.5–467.6)
C: **, R: NAC: ****, R: NSC: ***, R: NS
6b6f6j
114.8 (57.7–228.6)43.5 (15.9–118.4)54.1 (13.6–215.1)
C: NS, R: NAC: NS, R: *C: NS, R: NS
6c6g6k
126.8 (41.3–389.4)37.6 (11.5–123.4)34.9 (16.7–72.9)
C: NS, R: NAC: NS, R: *C: NS, R: **
6d6h6l
111.7 (35.3–353.1)52.8 (16.8–166.7)109.6 (32.5–370.4)
C: NS, R: NAC: NS, R: NSC: NS, R: NS
The results are presented as means and 95% confidence intervals (CI 95%) for three independent experiments. C and R indicate column and row, respectively. *, **, *** and **** indicate significant differences at p < 0.05, 0.01, 0.001 and 0.0001, respectively. NA and NS indicate not available and not significant, respectively. Doxorubicin exhibited an IC50 of 4.0 μM (0.9–17.4) μM in KATO-III cell line.
Table
Table 2. In vitro antiproliferative activities of 1H-benzo[f]indazole-4,9-dione-based derivatives expressed as IC50 values obtained in MCF-7 cell line.
Table 2. In vitro antiproliferative activities of 1H-benzo[f]indazole-4,9-dione-based derivatives expressed as IC50 values obtained in MCF-7 cell line.
Series-ISeries-IISeries-III
IC50 (CI 95%) μMIC50 (CI 95%) μMIC50 (CI 95%) μM
ppp
2a2b2c
63.2 (24.8–161.5)27.5 (71.1–106.8)29.4 (14.1–61.3)
C: NA, R: NAC: NA, R: NSC: NA, R: NS
3a3b3c
432.5 (167.8–1115.1)415.9 (202.0–856.2)389.9 (222.2–684.4)
C: ****, R: NAC: ****, R: NSC: ****, R: NS
4a4b4c
123.6 (39.6–385.7)43.4 (18.6–101.1)33.0 (10.8–100.7)
C: NS, R: NAC: NS, R: *C: NS, R: **
5a5b5c
372.4 (185.1–749.2)335.0 (186.7–601.1)244.9 (95.0–631.0)
C: ***, R: NAC: ****, R: NSC: ****, R: NS
6a6e6i
413.0 (138.2–1234.7)291.1 (155.1–546.2)255.3 (115.8–562.8)
C: ****, R: NAC: ****, R: NSC: ****, R: NS
6b6f6j
94.2 (44.8–197.9)62.7 (17.0–231.2)52.6 (24.2–114.2)
C: NS, R: NAC: NS, R: NSC: NS, R: NS
6c6g6k
154.9 (59.2–405.2)39.0 (11.6–131.1)35.4 (8.4–149.5)
C: NS, R: NAC: NS, R: **C: NS, R: ***
6d6h6l
143.9 (60.3–343.5)87.9 (45.0–171.7)99.8 (52.2–190.6)
C: NS, R: NAC: NS, R: NSC: NS, R: NS
The results are presented as means and 95% confidence intervals (CI 95%) for three independent experiments. C and R indicate column and row, respectively. *, **, *** and **** indicate significant differences at p < 0.05, 0.01, 0.001 and 0.0001, respectively. NA and NS indicate not available and not significant, respectively. Doxorubicin exhibited an IC50 of 0.3 μM (0.3–1.3) μM in MCF-7 cell line.
A closer analysis using a two-way ANOVA test followed by a Dunnett’s multiple comparison post-test showed that the most promising derivatives were compounds 2 (i.e., 2a, 2b and 2c), compounds 4 (i.e., 4a, 4b and 4c) and derivatives conjugated with l-alanine (i.e., 6b, 6f and 6j), l-phenylalanine (i.e., 6c, 6g and 6k) and l-glutamic acid (i.e., 6d, 6h and 6l). Additionally, the statistical analysis shows that the compounds of Series-II and -III have better IC50 values compared to compounds of Series-I.

3. Experimental Section

3.1. Chemistry

3.1.1. General

All reactions were performed using reagents and solvents purchased from commercial sources and purified by standard procedures as necessary. Starting N-substituted 1H-benzo[f]indazole-4,9-diones 2a–c were synthesized according to a previously described procedure [23]. IR spectra were recorded on a Perkin Elmer FT IR 1600 spectrophotometer (Norwalk, CN, USA) as a film over NaCl discs. NMR spectra were recorded on a Bruker Avance 400 Digital NMR spectrometer (Bruker/Analytic, Karlsruhe, Germany) operating at 400.13 MHz for 1H and 100.62 MHz for 13C in CDCl3, acetone-d6 or DMSO-d6 with internal TMS as a reference. Chemical shifts (δ) were expressed in ppm, followed by multiplicity and coupling constant (J) in Hz. Elemental analyses of C, H and N were performed using a Perkin Elmer 2400 Series II CHN Elemental Analyzer (Perkin Elmer Inc., Waltham, MA 02451, USA). The reaction progress was monitored by thin layer chromatography with Silica gel 60 F254 (0.25 mm thick, Merck, Darmstadt, Germany) aluminum sheets, whereas column chromatographies were performed on Silica gel 60 (230–400 mesh, Merck) using solvent mixtures with variable proportions as eluents. Melting points were determined on a Stuart SMP 10 apparatus (Stone, Staffordshire, UK), and they were not corrected.

3.1.2. General Procedure for the Preparation of 7-[2-(3,3-Dimethyloxiranyl)-ethyl]-3-methyl-1H-benzo[f]indazole-4,9-diones 3a–c

The compounds were synthesized by mCPBA (9.5 mmol) epoxidation of the 1H-benzo[f]indazole-4,9-dione 2a–c (9.5 mmol) and 1.34 g of NaHCO3 in CH2Cl2 (250 mL) at rt for 2 h under agitation. The crude epoxide was purified by column chromatography with n-hexane/ethyl acetate as the eluent.
7-[2-(3,3-Dimethyloxiranyl)-ethyl]-3-methyl-1H-benzo[f]indazole-4,9-dione (3a): This compound was prepared following the general procedure from 3-methyl-7-(4-methylpent-3-enyl)-1H-benzo[f]indazole-4,9-dione 2a. Light orange solid purified with 1:1 hexane/ethyl acetate, 84% yield, m.p. 134–136 °C; IR (NaCl, ν/cm−1) 3140 (NH), 1668 (C=O). 1H-NMR (CDCl3) δ 1.16 (s, 3H, CH3, H16), 1.28 (s, 3H, CH3, H15), 1.83–1.90 (m, 2H, CH2, H12), 2.75 (s, 3H, CH3, H10), 2.80 (t, J = 7.4 Hz, 1H, CH, H13), 2.87 (t, J = 7.4 Hz, 2H, CH2, H11), 7.97 (dd, J1 = 7.9 Hz, J2 = 1.6 Hz, 1H, CH, H6), 8.06 (d, J = 1.6 Hz, 1H, CH, H8), 8.15 (d, J = 7.9 Hz, 1H, CH, H5), 13.9 (s, 1H, NH, R). 13C-NMR (CDCl3) δ 11.7, 18.6, 24.7, 30.1, 32.8, 58.7, 63.5, 118.2, 127.0, 127.5, 128.2, 129.7, 130.2, 133.5, 134.0, 147.8, 178.3, 180.1. Elemental analysis calcd for C18H18N2O3: C 69;77; H 5.84; N 9.03; found: C 67.99; H 5.88; N 8.94.
7-[2-(3,3-Dimethyloxiranyl)-ethyl]-1-(2-hydroxyethyl)3-3-methyl-1H-benzo[f]indazole-4,9-dione (3b): This compound was prepared following the general procedure from 1-(2-hydroxy-ethyl)-3-methyl-7-(4-methylpent-3-enyl)-1H-benzo[f]indazole-4,9-dione 2b. Brown solid purified with 1:1 hexane/ethyl acetate, 74% yield, m.p. 62–64 °C; IR (NaCl, ν/cm−1) 3330 (OH), 1670, 1658 (C=O). 1H-NMR (CDCl3) δ 1.05 (s, 3H, CH3, H16), 1.26 (s, 3H, CH3, H15), 1.72–1.84 (m, 2H, CH2, H12), 2.44 (s, 3H, CH3, H10), 2.73 (t, J = 6.4 Hz, 1H, CH, H13), 2.84 (t, J = 6.4 Hz, 2H, CH2, H11), 3.77 (t, J = 5.8 Hz, 2 H, CH2N, R), 4.55 (t, J = 5.8 Hz, 2 H, CH2O, R), 4.90 (s, 1H, OH, R), 7.67 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.86 (d, J = 1.6 Hz, 1H, CH, H8), 7.93 (d, J = 8.0 Hz, 1H, CH, H5). 13C-NMR (CDCl3) δ 13.0, 18.7, 24.8, 29.9, 32.3, 53.6, 58.1, 60.0, 62.7, 119.5, 126.7, 126.8, 132.0, 133.5, 134.8, 138.3, 148.1, 148.2, 175.7, 179.8. Elemental analysis calcd for C20H22N2O4: C 67.78; H 6.26; N 7.90; found: C 67.85; H 6.31; N 7.95.
2-{7-[2-(3,3-Dimethyloxiranyl)-ethyl]-3-methyl-4,9-dioxo-4,9-dihydro-benzo-[f]indazol-1-yl}-ethyl acetate (3c): This compound was prepared following the general procedure from 1-(2-acetoxyethyl)-3-methyl-7-(4-methylpent-3-enyl)-1H-benzo[f]indazole-4,9-dione 2c. Yellow solid purified with 2:1 hexane/ethyl acetate, 64% yield, m.p. 104–106 °C; IR (NaCl, ν/cm−1) 1744, 1669 (C=O). 1H-NMR (CDCl3) δ 1.50 (s, 3H, CH3, H16), 1.63 (s, 3H, CH3, H15), 1.98 (s, 3H, CH3, R), 1.89–1.94 (m, 2 H, CH2, H12), 2.61 (s, 3H, CH3, H10), 2.77 (t, J = 6.2Hz, 1H, CH, H13), 2.92 (t, J = 6.5 Hz, 2H, CH2, H11), 4.53 (t, J = 5.3 Hz, 2 H, CH2N, R), 4.88 (t, J = 5.3 Hz, 2 H, CH2O, R), 7.59 (dd, J1 = 7.9 Hz, J2 = 1.6 Hz, 1H, CH, H6), 8.01 (d, J = 1.6 Hz, 1H, CH, H8), 8.14 (d, J = 7.9 Hz, 1H, CH, H5). 13C-NMR (CDCl3) δ 13.1, 18.7, 20.7, 24.7, 30.2, 32.9, 50.3, 58.3, 62.3, 63.4, 120.0, 126.7, 127.3, 132.4, 133.4, 133.5, 134.5, 147.7, 149.6, 170.5, 176.3, 180.1. Elemental analysis calcd for C22H24N2O5: C 66.65; H 6.10; N 7.07; found: C 66.60; H 6.15; N 7.14.

3.1.3. General Procedure for the Preparation of N-Substituted 3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)-propanal 4a–c

These compounds were synthesized by the degradative oxidation of epoxides 3a–c (0.31 mmol) dissolved in THF (10 mL) with H5IO6 (0.140 g, 0.61 mmol) in H2O (3 mL) stirred 1 h at r.t. After diluting with diethyl ether (20 mL), the organic phase was washed with a 5% aqueous solution of Na2S2O7 (4 × 10 mL) and 5% Na2CO3 (10 mL). The products were purified by column chromatography with n-hexane/ethyl acetate as the eluent.
3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)-propanal (4a): This compound was prepared following the general procedure from epoxide 3a. Yellow solid purified with 1:4 hexane/ethyl acetate, 72% yield, m.p. 248–250 °C; IR (NaCl, ν/cm−1) 3198 (NH), 1720, 1666 (C=O). 1H-NMR (DMSO-d6) δ 2.56 (s, 3H, CH3, H10), 2.88 (t, J = 7.2 Hz, 2H, CH2, H12), 3.00 (t, J = 7.2 Hz, 2H, CH2, H11), 7.69 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1 H, CH, H6), 7.98 (d, J = 1.6 Hz, 1H, CH, H8), 8.10 (d, J = 7.8 Hz, 1H, CH, H5), 9.71 (s, 1H, H13), 13.7 (s, 1H, NH, R). 13C-NMR (DMSO-d6) δ 11.6, 28.6, 45.1, 116.6, 128.2, 129.9, 131.9, 133.6, 134.1, 142.3, 142.6, 145.1, 171.9, 180.5, 201.1. Elemental analysis calcd for C15H12N2O3: C 67.16; H 4.51; N 10.44; found: C 68.03; H 4.30; N 10.53.
3-[1-(2-Hydroxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)-propanal (4b): This compound was prepared following the general procedure from epoxide 3b. Yellow orange solid purified with 1:1 hexane/ethyl acetate, 96% yield, m.p. 141–143 °C; IR (NaCl, ν/cm−1) 3382 (OH), 1722, 1662 (C=O). 1H-NMR (CDCl3) δ 2.52 (s, 3H, CH3 H10), 2.55 (t, J = 7.4 Hz, 2H, CH2, H12), 3.07 (t, J = 7.4 Hz, 2H, CH2, H11), 3.84 (t, J = 5.7 Hz, 2H, CH2N, R), 4.64 (t, J = 5.7 Hz, 2H, CH2O, R), 4.96 (s, 1H, OH, R), 7.75 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.98 (d, J = 1.6 Hz, 1H, CH, H8), 8.01 (d, J = 7.8 Hz, 1H, CH, H5), 9.71 (s, 1H, H13). 13C-NMR (CDCl3) δ 13.1, 27.7, 44.4, 53.6, 60.0, 119.5, 126.7, 126.8, 132.1, 133.6, 134.8, 138.4, 146.8, 148.1, 175.4, 179.8, 202.4. Elemental analysis calcd for C17H16N2O4: C 65.38; H 5.16; N 8.97; found: C 65.41; H 5.19; N 9.00.
2-[3-Methyl-4,9-dioxo-7(3-oxopropyl)-4,9-dihydro-benzo[f]indazol-1-yl]-ethyl acetate (4c): This compound was prepared from epoxide 3c following the general procedure. Yellow solid purified with 1:1 hexane/ethyl acetate, 98% yield, m.p. 90–91 °C; IR (NaCl, ν/cm−1) 1732, 1718, 1666 (C=O). 1H-NMR (CDCl3) δ 1.97 (s, 3H, CH3, R), 2.61 (s, 3H, CH3, H10), 2.90 (t, J = 7.4 Hz, 2H, CH2, H12), 3.10 (t, J = 7.4 Hz, 2H, CH2, H11), 4.53 (t, J = 5.2 Hz, 2H, CH2N, R), 4.87 (t, J = 5.2 Hz, 2H, CH2O, R), 7.59 (dd, J1 = 7.9 Hz, J2 = 1.7 Hz, 1H, CH, H6), 7.99 (d, J = 7.9 Hz, 1H, CH, H8), 8.09 (d, J = 1.7 Hz, 1H, CH, H5), 9.80 (s, 1H, H13). 13C-NMR (CDCl3) δ 13.1, 20.7, 27.9, 44.4, 50.2, 62.2, 120.2, 126.5, 127.4, 132.6, 133.6, 133.8, 134.5, 146.7, 149.6, 175.6, 176.2, 180.0, 200.2. Elemental analysis calcd for C19H18N2O5: C 64.40; H 5.11; N 7.90; found: C 64.35; H 5.09; N 7.94.

3.1.4. General Procedure for the Preparation of N-Substituted 3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)-propanoic Acids 5a–c

These compounds were prepared via the oxidation of aldehydes 4a–c (0.33 mmol) in t-BuOH (9 mL) with NaClO2 (0.5 mL aqueous solution 25%), NaH2PO4 (0.4 mL aqueous solution 5%) and catalytic 2-methyl-2-butene (0.2 mL) at r.t. for 72 h. After acid work-up with 2 M HCl and extraction with ethyl acetate (3 × 10 mL) and CH2Cl2 (10 mL), the products were purified by column chromatography with hexane/ethyl acetate 2:1 as the eluent.
3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)-propanoic acid (5a): Following the general procedure, this compound was obtained from aldehyde 4a. White solid, 98% yield, m.p. 288–290 °C; IR (NaCl, ν/cm−1) 3410 (OH), 3204 (NH), 1704, 1667 (C=O). 1H-NMR (DMSO-d6) δ 2.52 (s, 3H, CH3, H10), 2.62 (t, J = 7.4 Hz, 2H, CH2, H12), 2.98 (t, J = 7.4 Hz, 2H, CH2, H11), 7.71 (dd, J1 = 8.2 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.93 (d, J = 1.6 Hz, 1H, CH, H8), 7.99 (d, J = 8.2 Hz, 1H, CH, H5), 12.23 (s, broad, 1H, H13), 14.20 (s, 1H, NH, R). 13C-NMR (DMSO-d6) δ 13.9, 30.1, 34.3, 114.1, 117.9, 126.5, 134.0, 134.2, 147.3, 173.4, 179.6. Elemental analysis calcd for C15H12N2O4: C 63.37; H 4.25; N 9.85; found: C 63.41; H 4.30; N 9.65.
3-[1-(2-Hydroxyethyl)3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoic acid (5b): Following the general procedure, this compound was obtained from aldehyde 4b. Yellow solid, 64% yield, m.p. 218–219 °C; IR (NaCl, ν/cm−1) 3331 (OH), 1700, 1664 (C=O). 1H-NMR (DMSO-d6) δ 2.50 (s, 3H, CH3, H10), 2.63 (t, J = 7.4 Hz, 2H, CH2, H12), 2.98 (t, J = 7.4 Hz, 2H, CH2, H11), 3.77 (t, J = 5.5 Hz, 2H, CH2N, R), 4.58 (t, J = 5.5 Hz, 2H, CH2O, R), 4.90 (s, 1H, OH, R), 7.71 (dd, J1 = 7.9 Hz, J2 = 1.5 Hz, 1H, CH, H6), 7.90 (d, J = 1.5 Hz, 1H, CH, H8), 8.00 (d, J = 1.5 Hz, 1H, CH, H5), 12.30 (s, broad, 1H, H13). 13C-NMR (DMSO-d6) δ 13.1, 30.5, 34.7, 53.6, 60.0, 119.5, 126.7, 126.8, 132.1, 133.6, 134.8, 147.8, 148.1, 173.5, 175.8, 179.9. Elemental analysis calcd for C17H16N2O5: C 62.19; H 4.91; N 8.53; found: C 62.14; H 4.86; N 8.48.
3-[1-(2-Acetoxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoic acid (5c): Following the general procedure, this compound was obtained from aldehyde 4c. Grey solid, 48% yield, m.p. 186–187 °C; IR (NaCl, ν/cm−1) 3400 (OH), 1742, 1705, 1670, 1664 (C=O). 1H-NMR (DMSO-d6) δ 1.90 (s, 3H, CH3, R), 2.50 (s, 3H, CH3, H10), 2.64, (t, J = 7.4 Hz, 2H, CH2, H12), 3.00 (t, J = 7.4 Hz, 2H, CH2, H11), 4.45 (t, J = 5.2 Hz, 2H, CH2N, R), 4.79 (t, J = 5.2 Hz, 2H, CH2O, R), 7.74 (dd, J1 = 7.9 Hz, J2 = 1.4 Hz, 1H, H6), 7.97 (d, J = 1.4 Hz, 1H, CH, H8), 8.00 (d, J = 7.9 Hz, 1H, CH, H5), 12.05 (s, broad, 1H, H13). 13C-NMR (DMSO-d6) δ 12.7, 20.4, 30.1, 34.3, 49.8, 61.8, 119.3, 126.3, 126.5, 131.7, 133.1, 138.1, 148.0, 170.0, 173.4, 174.5, 179.5. Elemental analysis calcd for C19H18N2O6: C 61.62; H 4.96; N 7.50; Found: C 61.55; H 4.90; N 7.60.

3.1.5. General Procedure for the Preparation of [3-(3-Methyl-4,9-dioxo-4,9-dhydro-1H-benzo[f]indazol-7-yl)propanoylamino]-methyl Ester 6a–l

A solution containing 0.37 mmol of carboxylic acids 5a–c, 0.041 g (0.407 mmol, 56 μL) of triethylamine and 0.044 g (0.407 mmol, 38 μL) of ethyl chloroformate in 12 mL of dry THF was stirred for 20 min at 0 °C. After the addition of 0.407 mmol of the protected l-amino acid (Gly, Ala, Phe, and Glu), the mixture was stirred 16 h at r.t. After filtration over Celite-545 and evaporation of the solvent, the residue was dissolved in 70 mL of ethyl acetate. The organic solution was extracted with 40 mL of a 5% NaHCO3 solution and water (40 mL). After drying with Na2SO4, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography with chloroform/acetone 7:3 as the eluent.
[3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)propanoylamino]-methyl acetate (6a): This compound was prepared following the general procedure from carboxylic acid 5a and glycine methyl ester hydrochloride. White solid, 80% yield, m.p. 213–215 °C; IR (NaCl, ν/cm−1) 3219 (NH), 1750, 1668, 1640 (C=O). 1H-NMR (DMSO-d6) δ 2.53 (t, J = 7.6 Hz, 2H, CH2, H12), 2.57 (s, 3H, CH3, H10), 2.99 (t, J = 7.6 Hz, 2H, CH2, H11), 3.59 (s, 3H, CH3O), 3.81 (d, J = 5.8 Hz, 2H, CH2), 7.70 (dd, J1 = 7.9 Hz, J2 = 1.5 Hz, 1H, CH, H6), 7.96 (d, J = 1.5 Hz, 1H, CH, H8), 7.99 (d, J = 7.9 Hz, 1H, CH, H5), 8.35 (d, J = 5.0 Hz, 1H, NH), 14.26 (s, 1H, NH, R). 13C-NMR (DMSO-d6) δ 11.0, 30.7, 35.7, 40.5, 51.6, 114.1, 117.8, 126.4, 126.5, 132.8, 134.0, 147.5, 170.8, 171.5, 179.6. Elemental analysis calcd for C18H17N3O5: C 60.84; H 4.82; N 11.82; found: C 60.85; H 4.87; N 11.90.
2-[3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)propanoylamino]-methyl propanoate (6b): This compound was prepared following the general procedure from carboxylic acid 5a and l-alanine methyl ester hydrochloride. Yellow solid, 49% yield, m.p. 246–248 °C; IR (NaCl, ν/cm−1) 3221 (NH), 1743, 1667, 1645 (C=O). 1H-NMR (DMSO-d6) δ 1.21 (d, J = 7.3 Hz, 3H, CH3, R1), 2.49 (t, J = 7.4 Hz, 2H, CH2, H12), 2.57 (s, 3H, CH3, H10), 2.95 (t, J = 7.4 Hz, 2H, CH2, H11), 3.57 (s, 3H, CH3O), 4.20 (q, J = 7.3 Hz, 1H, CH), 7.69 (dd, J1 = 8.0 Hz; J2 = 1.6 Hz, 1H, CH, H6), 7.96 (d, J = 1.6 Hz, 1H, CH, H8), 8.01 (d, J = 8.0 Hz, 1H, CH, H5), 8.34 (d, J = 7.0 Hz, 1H, NH), 14.10 (s, 1H, NH, R). 13C-NMR (DMSO-d6) δ 10.8, 17.0, 30.7, 42.7, 51.7, 117.8, 126.3, 126.4, 126.6, 126.7, 134.0, 134.2, 147.3, 170.8, 173.0, 179.0. Elemental analysis calcd for C19H19N3O5: C 61.78; H 5.18; N 11.38; found: C 61.85; H 5.20; N 11.43.
2-[3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)propanoylamino]-methyl-3-phenyl propanoate (6c): This compound was prepared following the general procedure from carboxylic acid 5a and l-phenylalanine methyl ester hydrochloride. Brown solid, 98% yield, m.p. 215–217 °C; IR (NaCl, ν/cm−1) 3219 (NH), 1774, 1691, 1667 (C=O). 1H-NMR (CDCl3) δ 2.66 (t, J = 7.9 Hz, 2H, CH2, H12), 2.85 (s, 3H, CH3, H10), 3.06–3.12 (m, 4H, 2 CH2, H11, R1), 3.75 (s, 3H, CH3O), 4.13 (q, J = 7.2 Hz, 1H, CH), 7.17–7.60 (m, 8H, CH, aromatic), 8.11 (s, broad, 1H, NH), 13.96 (s, 1H, NH, R). 13C-NMR (CDCl3) δ 14.1, 26.5, 37.5, 40.9, 51.9, 114.5, 126.2, 126.8, 127.1, 128.5, 129.2, 133.0, 137.1, 171.0 173.0, 175.3. Elemental analysis calcd for C25H23N3O5: C 67.41; H 5.20; N 9.40; found: C 67.35; H 5.25; N 9.60.
2-[3-(3-Methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)propanoylamino]-dimethyl pentane-dioate (6d): This compound was prepared following the general procedure from carboxylic acid 5a and l-glutamic acid dimethyl ester hydrochloride. White solid, 38% yield, m.p. 222–224 °C; IR (NaCl, ν/cm−1) 3226 (NH), 1746, 1691, 1669 (C=O). 1H-NMR (DMSO-d6) δ 1.70–1.90 (m, 2H, CH2, R1), 2.00–2.35 (m, 2H, CH2, R1), 2.52 (t, J = 7.3 Hz, 2H, CH2, H12), 2.57 (s, 3H, CH3, H10), 2.98 (t, J = 7.3 Hz, 2H, CH2, H11), 3.46 (s, 3H, CH3O), 3.57 (s, 3H, CH3O), 4.10–4.30 (m, 1H, CH), 7.68 (dd, J1 = 7.9 Hz, J2 = 1.5 Hz, 1H, CH, H6), 7.92 (d, J = 1.5 Hz, 1H, CH, H8), 7.99 (d, J = 7.9 Hz, 1H, CH, H5), 8.29 (d, J = 7.7 Hz, NH), 14.24 (s, 1H, NH, R). 13C-NMR (DMSO-d6) δ 11.0, 26.0, 29.3, 30.8, 35.7, 50.8, 51.2, 51.8, 114.1, 117.8, 126.4, 126.5, 132.7, 133.5, 133.9, 134.2, 147.4, 171.1, 172.0, 172.4, 179.6. Elemental analysis calcd for C22H23N3O7: C 59.85; H 5.25; N 9.52; found: C 60.01; H 5.56; N 9.63.
{3-[1-(2-Hydroxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoylamino}-methyl acetate (6e): This compound was prepared following the general procedure from carboxylic acid 5b and glycine methyl ester hydrochloride. White solid, 80% yield, m.p. 188–190 °C; IR (NaCl, ν/cm−1) 3325 (broad, NH, OH), 1762, 1742, 1671, 1651 (C=O). 1H-NMR (DMSO-d6) δ 2.50 (s, 3H, CH3, H10), 2.53 (t, J= 7.5 Hz, 2H, CH2, H12), 2.99 (t, J = 7.5 Hz, 2H, CH2, H11), 3.60 (s, 3H, CH3O), 3.77 (d, J= 5.6 Hz, 2H, CH2), 3.81 (t, J = 5.6 Hz, 2H, CH2, CH2N, R), 4.61 (t, J = 5.6 Hz, 2H, CH2O, R), 4.92 (s, 1H, OH, R), 7.71 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.93 (d, J = 1.6 Hz, 1H, CH, H8), 7.97 (J = 8.0 Hz, 1H, CH, H5), 8.38 (t, J = 5.6 Hz, 1H, NH). 13C-NMR (DMSO-d6) δ 12.8, 30.4, 30.7, 35.7, 40.5, 51.6, 53.3, 59.6, 119.2, 124.9, 126.4, 126.5, 131.7 133.3, 134.5, 138.1, 147.8, 170.3, 171.5, 175.5, 179.6. Elemental analysis calcd for C20H21N3O6: C 60.15; H 5.30; N 10.52; found: C 60.19; H 5.34; N 10.53.
2-{3-[1-(2-Hydroxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoylamino}-methyl propanoate (6f): This compound was prepared following the general procedure from carboxylic acid 5b and l-alanine methyl ester hydrochloride. Yellow solid, 49% yield, m.p. 181–182 °C; IR (NaCl, ν/cm−1) 3317 (broad, NH, OH), 1729, 1663, 1647 (C=O). 1H-NMR (DMSO-d6) δ 1.22 (d, J = 7.2 Hz, 3H, CH3, R1); 2.50 (s, 3H, CH3, H10), 2.52 (t, J = 7.2 Hz, 2H, CH2, H12), 2.98 (t, J = 7.2 Hz, 2H, CH2, H11), 3.59 (s, 3H, CH3O), 3.79 (t, J = 5.6 Hz, 2H, CH2N, R), 4.24–4.27 (m, 1H, CH), 4.61 (t, J = 5.6 Hz, 2H, CH2O, R), 4.92 (s, 1H, OH), 7.70 (dd, J1 = 7.8, J2 = 1.5 Hz, 1H, CH, H6), 7.93 (d, J= 1.5 Hz, 1H, CH, H8), 7.97 (d, J = 7.8 Hz, 1H, CH, H5), 8.35 (d, J = 7.2 Hz, 1H, NH). 13C-NMR (DMSO-d6) δ 12.8, 17.0, 30.7, 35.7, 47.4, 51.7, 53.2, 59.6, 119.1, 126.4, 131.7, 133.2, 134.5, 138.1, 147.7, 170.8, 173.1, 175.5, 179.6. Elemental analysis calcd for C21H23N3O6: C 61.01; H 5.61; N 10.16; found: C 61.05; H 5.66; N 10.21.
2-{3-[1-(2-Hydroxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoylamino}-methyl-3-phenylpropanoate (6g): This compound was prepared following the general procedure from carboxylic acid 5b and l-phenylalanine methyl ester hydrochloride. Brown solid, 98% yield, m.p. 154–156 °C. IR (NaCl, ν/cm−1) 3309 (broad, NH, OH), 1743, 1659, 1644 (C=O). 1H-NMR (DMSO-d6) δ 2.44 (t, J = 7.4 Hz, 2H, CH2, H12), 2.48 (s, 3H, CH3, H10), 2.75–3.00 (m, 4H, CH2, CH2, H11, R1), 3.56 (s, 3H, CH3O); 3.77 (t, J = 5.6 Hz, 2H, CH2N, R), 4.43–4.45 (m, 1H, CH) 4.61 (t, J = 5.6 Hz, 2H, CH2O, R), 4.90 (s,1H, OH), 7.12–7.22 (m, 5H, CH, aromatic, R1), 7.60 (dd, J1 = 7.9 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.80 (d, J = 1.6 Hz, 1H, CH, H8), 7.94 (d, J = 7.9 Hz, 1H, CH, H5), 8.39 (d, J = 7.8 Hz, 1H, NH). 13C-NMR (DMSO-d6) δ 12.8, 30.7, 35.7, 36.7, 51.8, 53.3, 53.4, 59.6, 119.2, 126.4, 128.1, 128.9, 131.7, 133.2, 134.4, 137.1, 138.1, 147.7, 147.8, 171.0, 172.0, 175.5, 179.6. Elemental analysis calcd for C27H27N3O6: C 66.25; H 5.56; N 8.58; found: C 66.21; H 5.50; N 7.92.
2-{3-[1-(2-Hydroxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)propanoylamino]-dimethyl pentanedioate (6h): This compound was prepared following the general procedure from carboxylic acid 5b and l-glutamic acid dimethyl ester hydrochloride. Yellow solid, 44% yield, m.p. 160–162 °C. IR (NaCl, ν/cm−1) 3306 (broad, NH, OH), 1738, 1675, 1644 (C=O). 1H-NMR (DMSO-d6) δ 1.60–2.00 (m, 2H, CH2, R1), 2.12–2.19 (m, 2H, CH2, R1), 2.49 (s, 3H, CH3, H10), 2.52 (t, J = 7.6 Hz, 2H, CH2, H12), 2.96 (t, J = 7.6 Hz, 2H, CH2, H11), 3.47 (s, 3H, CH3O), 3.58 (s, 3H, CH3O), 3.78 (t, J = 5.7 Hz, 2H, CH2N, R), 4.23–4.25 (m, 1H, CH), 4.60 (t, J = 5.7 Hz, 2H, CH2O, R), 4.91(s,1H, OH), 7.68 (dd, J1 = 7.1 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.92 (d, J = 1.6 Hz, 1H, CH, H8), 7.97 (d, J = 7.1 Hz, 1H, CH, H5), 8.28 (d, J = 7.7 Hz, 1H, NH). 13C-NMR (DMSO-d6) δ 12.8, 26.0, 29.4, 30.8, 35.7, 50.9, 51.2, 51.8, 53.2, 59.6, 119.1, 126.4, 131.7, 133.2, 134.5, 138.1, 147.6, 147.7, 171.1, 172.0, 172.4, 175.5, 179.5. Elemental analysis calcd for C24H27N3O8: C 59.37; H 5.61; N 8.66; found: C 59.42; H 5.70; N 8.71.
{3-[1-(2-Acetoxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoylamino}-methyl acetate (6i): This compound was prepared following the general procedure from carboxylic acid 5c and glycine methyl ester hydrochloride. Yellow oil, 41% yield. IR (NaCl, ν/cm−1) 3330 (broad, NH), 1742, 1670, 1664, 1658 (C=O). 1H-NMR (CDCl3) δ 1.98 (s, 3H, CH3, R), 2.61 (s, 3H, CH3, H10), 2.65 (t, J = 7.4 Hz, 2H, CH2, H12), 3.14 (t, J = 7.4 Hz, 2H, CH2, H11), 3.75 (s, 3H, CH3O), 4.01 (d, J = 5.0 Hz, 2H, CH2), 4.53 (t, J = 5.2 Hz, 2H, CH2N, R), 4.87 (t, J = 5.2 Hz, 2H, CH2O, R), 6.06 (d, J = 5.0 Hz, 1H, NH), 7.62 (dd, J1 = 7.9 Hz, J2 = 1.6 Hz, 1H, CH, H6), 8.00 (d, J = 1.6 Hz, 1H, CH, H8), 8.12 (d, J = 7.9 Hz, 1H, CH, H5). 13C-NMR (CDCl3) δ 13.5, 21.1, 30.1, 31.6, 31.7, 37.1, 37.2, 41.7, 50.7, 52.8, 62.7, 120.7, 127.0, 127.8, 133.0, 134.1, 135.0, 138.7, 147.5, 148.8, 150.0, 170.3, 171.0, 171.1, 176.7, 180.5. Elemental analysis calcd for C22H23N3O7: C 59.85; H 5.25; N 9.52; found: C 59.91; H 5.32; N 9.85.
2-{3-[1-(2-Acetoxyethy)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoylamino}-methyl propanoate (6j): This compound was prepared following the general procedure from carboxylic acid 5c and l-alanine methyl ester hydrochloride. Amber solid, 56% yield, m.p. 130–132 °C. IR (NaCl, ν/cm−1) 3312 (broad, NH), 1744, 1670, 1654, 1648 (C=O). 1H-NMR (CDCl3) δ 1.31 (d, J = 7.1 Hz, 3H, CH3, R1), 1.91 (s, 3H, CH3, R), 2.50 (s, 3H, CH3, H10), 2.52 (t, J = 7.7 Hz, 2H, CH2, H12), 3.06 (t, J = 7.7 Hz, 2H, CH2, H11), 3.67 (s, 3H, CH3O,), 4.45 (t, J = 5.2 Hz, 2H, CH2N, R), 4.49 (q, J = 7.1 Hz, 1H, CH), 4.80 (t, J = 5.2 Hz, 2H, CH2O, R), 5.99 (d, J = 7.1 Hz, 1H, NH), 7.54 (dd, J1 = 7.9 Hz, J2 = 1.5 Hz, 1H, CH, H6), 7.93 (d, J = 1.5 Hz, 1H, CH, H8), 8.06 (d, J = 7.9 Hz, 1H, CH, H5). 13C-NMR (CDCl3) δ 13.5, 18.9, 21.1, 30.1, 31.6, 37.4, 48.4, 49.0, 50.7, 52.9, 125.5, 127.0, 127.8, 130.3, 132.2, 134.0, 135.0, 147.5, 150.0, 171.0, 171.5, 173.9, 176.7, 180.9. Elemental analysis calcd for C23H25N3O7: C 60.65; H 5.53; N 9.23; found: C 60.50; H 5.60; N 9.30.
2-{3-[1-(2-Acetoxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl]-propanoylamino}-methyl 3-phenylpropanoate (6k): This compound was prepared following the general procedure from carboxylic acid 5c and l-phenylalanine methyl ester hydrochloride. Yellow oil, 69% yield. IR (NaCl, ν/cm−1) 3302 (broad, NH), 1744, 1668, 1648 (C=O). 1H-NMR (CDCl3) δ 1.97 (s, 3H, CH3, R), 2.57 (t, J = 7.5 Hz, 2H, CH2, H12), 2.62 (s, 3H, CH3, H10), 3.07–3.10 (m, 4H, CH2, CH2, H11, R1), 3.72 (s, 3H, CH3O), 4.52 (t, J = 5.3 Hz, 2H, CH2N, R), 4.86–4.88 (m, 3H, CH, CH2O, R), 5.88 (d, J = 7.6 Hz, 1H, NH), 7.23–7.27 (m, 5H, aromatic CH, R1), 7.58 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H, CH, H6), 7.99 (d, J = 1.6 Hz, 1H, CH, H8), 8.12 (d, J = 8.0 Hz, 1H, CH, H5). 13C-NMR (CDCl3) δ 13.5, 21.1, 31.5, 37.4, 38.2, 50.7, 52.8, 53.4, 120.2, 127.0, 127.6, 127.8, 129.0, 129.6, 134.0, 135.0, 136.0, 147.4, 150.0, 171.0, 171.1, 172.3, 180.2. Elemental analysis calcd for C29H29N3O7: C 65.53; H 5.50; N 7.90; found: C 65.48; H 5.60; N 7.96.
2-{3-[1-(2-Acetoxyethyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indazol-7-yl)propanoylamino]-dimethyl pentanedioate (6l): This compound was prepared following the general procedure from carboxylic acid 5c and l-glutamic acid dimethyl ester hydrochloride. Yellow oil, 48% yield. IR (NaCl, ν/cm−1) 3330 (broad, NH), 1740, 1670, 1644 (C=O). 1H-NMR (CDCl3) δ 1.99 (s, 3H, CH3, R), 2.10–2.40 (m, 4H, CH2, CH2, R1), 2.60 (s, 3H, CH3, H10), 2.62 (t, J = 7.8 Hz, 2H, CH2, H12), 3.12 (t, J = 7.8 Hz, 2H, CH2, H11), 3.63 (s, 3H, CH3O), 3.76 (s, 3H, CH3O), 4.53 (t, J = 5.2 Hz, 2H, CH2N, R), 4.60 (d, J = 5.0 Hz, 1H, CH), 4.80 (t, J = 5.2 Hz, 2H, CH2O, R), 6.26 (d, J = 7.4 Hz, 1H, NH), 7.62 (dd, J1 = 7.9 Hz, J2 = 1.7 Hz, 1H, CH, H6), 8.01 (d, J = 1.7 Hz, 1H, CH, H8), 8.12 (d, J = 7.9 Hz, 1H, CH, H5). 13C-NMR (CDCl3) δ 13.5, 21.1, 27.6, 30.3, 31.6, 37.4, 50.7, 52.1, 52.3, 53.0, 122.0, 127.0, 127.8, 134.1, 135.0, 138.7, 147.4, 150.1, 171.0, 171.5, 172.6, 173.7, 175.0, 180.1. Elemental analysis calcd for C26H29N3O9: C 59.20; H 5.54; N 7.97; found: C 59.13; H 5.72; N 8.01.

3.2. Computational Details

DFT calculations [30,31,32,33] were conducted using the Amsterdam Density Functional (ADF) program [34]. The Vosko-Wilk-Nusair parametrization [35] was used to treat electron correlation within the local density approximation (LDA). The numerical integration procedure applied for the calculation was developed by teVelde [33]. The standard ADF TZ2P basis set was used for all atoms. The frozen core approximation was used to treat core electrons at the following levels: C, 1s; N, 1s; and O, 1s [33]. Full geometry optimizations were performed on each complex using the analytical gradient method implemented by Verluis and Ziegler [36]. The geometries for all the model compounds discussed in the text were fully optimized and checked via analytical frequency calculations as either true minima (no imaginary values).

3.3. Antiproliferative Assay

KATO-III (human gastric cancer cell line) and MCF-7 (human breast adenocarcinoma cell line) cells were obtained from the American Type Culture Collection (ATCC). KATO-III and MCF-7 cells (2 × 103) were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were subcultured into fresh medium (100-mm-diameter plate dish) until a density of approximately 80% was obtained. Briefly, 2 × 103 cells were seeded in 96-well culture plates. After 24 h of incubation at 37 °C in a humidified 5% CO2 atmosphere, different concentrations (10−9 to 10−3 M) of 1H-benzo[f]indazole-4,9-dione-based derivatives were added. After 72 h of incubation, 20 μL of MTS (Promega, Madison, WI, USA) was added, and the wells were incubated for an additional 2 h at 37 °C. The absorbance at 490 nm was recorded using a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). Each variant of the experiment was performed in triplicate. To obtain IC50 values for each compound, dose-response curves were constructed in both KATO-III and MCF-7 cell lines. Doxorubicin was included in all evaluation to provide a reference of antiproliferative activity.

3.4. Statistical Analysis

Data are expressed as means ± CI 95% (95% confidence intervals) for three independent experiments. The concentration inducing a 50% decrease of cell proliferation (IC50) was performed using the four-parameters logistic fit—also known as “4PL”—supported by GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Statistical differences among means were assessed using a two-way ANOVA test followed by a Dunnett’s multiple comparison post-test. A p < 0.05 was taken as statistically significant.

4. Conclusions

In this study, we have synthesized three series of new 1H-benzo[f]indazole-4,9-dione-based derivatives containing oxiranyl, formyl, carboxylic and l- and C-protected N-aminoacidyl substituents attached to the side chain of the 1,4-naphthoquinone group in moderate to good yields. All compounds were characterized using spectroscopic techniques, namely, FT-IR, 1H-NMR, and 13C-NMR, and their data are in agreement with their proposed structures. DFT calculations provide the first insights into the reaction pathways; further investigations to clarify the entire reaction pathway are currently in progress. These families of compounds contain, in a single structure, a 1,4-quinone group fused to a pyrazolyl heterocyclic ring, substituents that are present individually in anticancer drugs such as doxorubicin, daunorubicin or in heterocyclic compounds with antitumoral properties. Moreover, they contain an amino acid group capable of orienting their transport into the cell organelles, where they could interfere with protein synthesis. Preliminary antiproliferative activity analyses showed that most of the derivatives presented some degree of activity. However, the derivatives 2, 4 and those conjugated with l-alanine, l-phenylalanine and l-glutamic acid, and especially those belonging to Series-II and -III, presented the highest activity, as indicated by their IC50 values. These results suggest that 1H-benzo[f]indazole-4,9-dione-based derivatives are promising compounds for the development of anticancer drugs.

Acknowledgments

This work was supported by the Comisión Nacional de Investigación Científica y Tecnológica CONICYT of Chile (Project FONDECYT 1100316) and by the Dirección de Investigación de la Vicerrectoría de Investigación y Estudios Avanzados de la Pontificia Universidad Católica de Valparaíso, Chile (Projects DI 125.780/2010-2013, DI 037.383/2014 and DI 037.437/2015). M. Arismendi-Macuer acknowledges Doctoral Fellowship 21090246 from CONICYT and the Postdoctoral Grant from Vicerrectoría de Investigación y Estudios Avanzados de la Pontificia Universidad Católica de Valparaíso, Chile. R. Vinet acknowledges Grant CREAS CONICYT-REGIONAL, GORE Región de Valparaíso, Chile [R12C1001].

Author Contributions

Conceived and designed the experiments: A.O. and A.M.; Performed the experiments: M.A.-M., A.M., A.O. and M.K.; Analyzed the data: A.S.F., L.G. and R.V.; Wrote the paper: A.O. and A.M.; Theoretical calculations: M.F., M.A.-M.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds 2a–c–6a–l are not available from the authors.

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Molinari, A.; Oliva, A.; Arismendi-Macuer, M.; Guzmán, L.; Fuentealba, M.; Knox, M.; Vinet, R.; San Feliciano, A. New 1H-Benzo[f]indazole-4,9-diones Conjugated with C-Protected Amino Acids and Other Derivatives: Synthesis and in Vitro Antiproliferative Evaluation. Molecules 2015, 20, 21924-21938. https://doi.org/10.3390/molecules201219809

AMA Style

Molinari A, Oliva A, Arismendi-Macuer M, Guzmán L, Fuentealba M, Knox M, Vinet R, San Feliciano A. New 1H-Benzo[f]indazole-4,9-diones Conjugated with C-Protected Amino Acids and Other Derivatives: Synthesis and in Vitro Antiproliferative Evaluation. Molecules. 2015; 20(12):21924-21938. https://doi.org/10.3390/molecules201219809

Chicago/Turabian Style

Molinari, Aurora, Alfonso Oliva, Marlene Arismendi-Macuer, Leda Guzmán, Mauricio Fuentealba, Marcela Knox, Raúl Vinet, and Arturo San Feliciano. 2015. "New 1H-Benzo[f]indazole-4,9-diones Conjugated with C-Protected Amino Acids and Other Derivatives: Synthesis and in Vitro Antiproliferative Evaluation" Molecules 20, no. 12: 21924-21938. https://doi.org/10.3390/molecules201219809

APA Style

Molinari, A., Oliva, A., Arismendi-Macuer, M., Guzmán, L., Fuentealba, M., Knox, M., Vinet, R., & San Feliciano, A. (2015). New 1H-Benzo[f]indazole-4,9-diones Conjugated with C-Protected Amino Acids and Other Derivatives: Synthesis and in Vitro Antiproliferative Evaluation. Molecules, 20(12), 21924-21938. https://doi.org/10.3390/molecules201219809

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