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Full Paper Antiproliferative and Antiviral Activity of Three Libraries of Adamantane Derivatives 1, Margareta Sohora1, Nikola Cindro1, Kata Mlinaric -Majerski1, Erik De Clercq2*, and Nikola Basaric 2 Jan Balzarini * 1 2
Department of Organic Chemistry and Biochemistry, Ruđer Boškovic Institute, Zagreb, Croatia Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium
Three libraries of adamantane derivatives were synthesized and evaluated for antiviral and antiproliferative activities against a broad variety of DNA and RNA viruses. Whereas none of the compounds exhibit antiviral activity at subtoxic concentrations, antiproliferative activity was found against murine leukemia cells (L1210), human T-lymphocyte cells (CEM), and cervix carcinoma cells (HeLa) for 4, 8, and 10. Keywords: Adamantanes / Antiproliferative activity / Antiviral activity / Phthalimides / Polycyclic compounds / Unnatural amino acids Received: September 11, 2013; Revised: November 15, 2013; Accepted: November 15, 2013 DOI 10.1002/ardp.201300345
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Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Introduction A number of different antiviral drugs exist nowadays in the market. However, combating viral diseases and finding appropriate drugs with minimal side effects remain a challenge in chemistry, pharmacy, and medicine. A major problem in antiviral therapy is the rapid mutations of viruses leading to development of drug resistance. Amantadine (1-aminoadamantane hydrochloride) and rimantadine [1-(2aminoethyl)adamantane hydrochloride] are widely used as anti-influenza agents that block M2 protein ion channel, which prevents fusion of the virus with the host-cell membrane and release of viral RNA into the cytoplasm [1]. The same compounds have been shown to exhibit inhibitory activity – although rather modest – against some other viruses such as HIV [2], hepatitis [3], and herpes simplex virus [4]. However, inappropriate use of these drugs in many countries contributed to an increasing number of resistant influenza viruses [5]. In the search of new drugs and novel mechanisms of action based on yet unknown intracellular targets, it is important to screen new libraries of molecules and develop Correspondence: Dr. Nikola Basari c, Department of Organic Chemistry and Biochemistry, Ruđer Boškovi c Institute, Bijeni cka Cesta 54, 10 000 Zagreb, Croatia. E-mail: [email protected] Fax: þ385 1 4680 195
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new structure–activity relationships (SAR). Herein, we report an investigation of the antiviral activity in vitro of three libraries of adamantane derivatives. In addition, the same libraries of compounds were also tested for antiproliferative activity. Recently, we have reported an investigation of antiproliferative activity of a series of adamantylphthalimides, where all investigated compounds exhibited higher activity than the clinically used drug thalidomide [6].
Materials and methods Structure design The first library of compounds (Fig. 1) is composed of adamantylphthalimides since adamantylphthalimides were previously reported to be endowed with anti-HIV activity [7]. Structural design of the phthalimide compounds is in connection with a pharmacophore phenytoin (PHT) that exhibits anticonvulsant activity. This membrane-reactive drug has been reported to inhibit HIV binding to CD4 positive lymphocytes [8], and reduced the CD4 receptor availability for ligand interaction [9]. It was also proposed that
*Additional correspondence: Prof. Erik De Clercq, E-mail: [email protected]; Prof. Jan Balzarini, E-mail: [email protected]
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Figure 1. Library 1: adamantylphthalimides.
PHT suppresses the influx of Ca2þ ions that occurs shortly after HIV infection [10]. Later, it was discovered that some of the HIV proteins may organize as sodium channel entities in planar lipid bilayers [11]. Screening indicated that the 4-aminophthalimide pharmacophore and N-(l-adamantyl) substitutions are required for their anti-HIV properties [7]. The structures in library 1 are 1- or 2-adamantyl derivatives ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wherein adamantane and phthalimide are connected or separated by an alkyl spacer of different length (compounds 1–8). Furthermore, structures were modified by substitution of the phthalimide by an amino or a nitro group (compounds 9–12), or by additional substitution of the adamantane moiety (compounds 13–15, 19, and 20), and a change of the polycyclic skeleton (compounds 16–18). www.archpharm.com
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The third library of compounds (Fig. 3) is composed of adamantane amino acids. Anticancer activity of cage amino acids and the corresponding peptides has recently been reported [13]. Moreover, it has been demonstrated that dipeptides with incorporated cage amino acids exhibit HIV protease inhibition [14].
Synthesis
Figure 2. Library 2: adamantyl aza-heterocyclic derivatives.
The second library (Fig. 2) is composed of different azaheterocyclic derivatives of adamantane. The design of compounds is based on reports of antiviral activity of a series of polycyclic amines and aza-heterocycles [12]. It was found that the size of the heterocyclic ring influences the activity against influenza A virus, with a higher activity with five- than with four- and three-membered rings [12]. On the other hand, substitution of the heterocylic nitrogen increases the anti-HIV activity [12]. Compounds in library 2 are derivatives of azepine (21–23) or pyrrolidine (24–26), that are also characterized by a different connectivity to the adamantane skeleton.
Compounds in library 1 were prepared according to the described procedures [6, 15]. Adamantylphthalimides 1–8 were prepared in a condensation reaction of phthalic anhydride and the corresponding 1- or 2-aminoalkyladamantane [15a]. Aminophthalimide derivatives 10 and 12 were obtained from the nitro derivatives 9 and 11, respectively, by reduction with Zn. Homo-, 16, and protoadamantanes, exo-17 and endo-18, were made by applying a Mitzunobu protocol, from phthalimide and the corresponding polycyclic alcohols [15b]. Compounds 19 and 20 were obtained in a photochemical reaction from carboxylic acid 13 in the presence of acrylonitrile or cyclohexenone, respectively [15c], whereas 15 was obtained as one of the products in the photochemical reaction of 2 [16]. Compounds in library 2 were obtained in photochemical reactions from the compounds in library 1 (21 from 6, 22 from 1, 23 from 3, 24 from 2, and 25 and 26 from 5) [16]. Amino acid 27, from library 3 was obtained by a base-hydrolysis of 22 [16a]. Amino acid derivatives 14, and 29 were synthesized from ester 30 in the condensation reaction with phthalic anhydride (Scheme 1). Base hydrolysis of ester 14 furnished diacid 28 (Scheme 2). Phthalimido acid 13 was also obtained in the condensation reaction of phthalic anhydride and 3-aminoadamantane-1-carboxylic acid [15c]. All chiral compounds were synthesized as racemates.
Antiviral activity assays The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK VZV strain 07–1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV)
Figure 3. Library 3: adamantane amino acid derivatives.
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Scheme 1. Synthesis of 29.
Scheme 2. Synthesis of 28.
strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, parainfluenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, Reovirus-1, Sindbis, Reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain IIIB, and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on inhibition of virusinduced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa), or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque-forming units (PFU) (VZV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%. Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cytopathicity in CEM cell cultures was measured in microtiter 96-well plates containing 3 105 CEM cells/mL infected with 100 CCID50 of HIV per milliliter and containing appropriate dilutions of the test compounds. After 4–5 days of incubation at 37°C in a CO2-controlled humidified atmosphere, CEM giant (syncytium) cell formation was examined microscopically. The EC50 (50% effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50%.
Cytostatic activity assays All assays were performed in 96-well microtiter plates. To each well (5–7.5) 104 tumor cells and a given amount of the test compound were added. The cells were allowed to proliferate ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37°C in a humidified CO2-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The 50% inhibitory concentration (IC50) was defined as the concentration of the compound that inhibited cell proliferation by 50%. Data are derived from two to three independent experiments (the errors were calculated according to a formula given in the Supporting Information).
Results and discussion All three tested libraries of compounds exert no significant antiviral activity in cell culture at subtoxic concentrations (see Supporting Information Tables S1–S4). Antiproliferative activity was tested against three cancer cell lines (Table 1). Generally, compounds exhibit moderate activity in micromolar concentration range and show no selectivity to a specific cancer cell line. From the compounds in library 1, the highest activity was observed for 4 and 8, containing the longest alkyl linker between the adamantyl and the phthalimide moiety (CC50 range between 7.2 and 12 mg/mL). Also, antiproliferative activity can be achieved by incorporation of an amino substituent at the phthalimide 3-position, but only for the 1-adamantyl derivative 10 but not the adamantyl derivative 12. Contrary to our previous report [6] a change of the substitution on the adamantane, 1- versus 2-, virtually does not affect the antiproliferative activity. However, replacement of the adamantane with a homoadamantane skeleton (5 vs. 16) enhanced the cytostatic effect. A minor additional activity enhancement was also observed by substitution of the adamantane skeleton at the position 3 with a cyclohexanone ring (compound 20), whereas other groups (COOH, COOCH3, and CH2CH2CN) had no effect. All compounds in library 2 exhibited similar modest www.archpharm.com
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Table 1. Antiproliferative activities against L1210, CEM, and HeLa tumor cells. IC50 (mmol/L)a) Comp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Thalidomide a) b)
L1210
CEM
HeLa
>300 200 100 >300 30 7 30 ! >300b) 220 10 >300 31 0 >300 29 3 >300 >300 >300 60 ! 300b) 138 3 16 10 300 80 >300 >300 50 30 280 80 170 0 113 3 210 10 270 3 128 7 >300 >300 >200 >77
>300 70 10 >300 34 6 7 ! 300b) 190 10 300 37 6 >300 30 3 >300 >300 >300 150 120 120 30 30 10 210 160 >300 >300 100 0 110 60 190 10 103 3 280 100 300 50 100 60 >300 >300 >200 >42
>300 90 20 300 22 1 300 230 30 300 26 3 >300 33 3 200 100 300 >300 30 ! 300b) 150 60 80 30 120 80 320 50 >300 70 10 180 130 180 0 64 6 290 40 220 50 90 10 >300 >300 >200 >77
Fifty percent inhibitory concentration. No dose response between these concentrations.
cytostatic activity, so that no SAR could be established. On the contrary, the amino acid derivatives from library 3 exhibit no antiproliferative activity at all. Thus, the presence of a heterocyclic ring (phthalimide or other aza-heterocycle) is essential for the antiproliferative effect. It was shown that the activity of thalidomide, a clinically used anticancer drug with the phthalimide pharmacophore, is related to the modulation of tumor necrosis factor TNF-a, especially in multiple myeloma. Namely, TNF-a is a multifunctional cytokine playing a key role in both apoptosis and cell survival, as well as in inflammation and immunity [17]. TNF-a also shows the capacity to form a voltagedependent cationic channel in lipid bilayers [11, 18]. Thalidomide was proposed to elicit its anti-HIV by accelerating the decay of the TNF-a mRNA [19]. Consequently, the antiproliferative activity of the compounds presented herein may be perhaps related to TNF-a inhibition. This concept is to be further tested, and if shown to be correct, it is anticipated to have impact in further development of anticancer agents. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Conclusion Three libraries of adamantane derivatives were synthesized and screened for antiviral and antiproliferative activity. Whereas none of the compounds exhibited pronounced activity against virus infection, antiproliferative activity was found against L1210, CEM, and HeLa for 4, 8, and 10.
Experimental General 1
H and 13C NMR spectra were recorded on a Bruker spectrometer at 300 or 600 MHz, respectively. All NMR spectra were measured in CDCl3 or DMSO-d6 using tetramethylsilane as a reference. Melting points were obtained using an Original Kofler Mikroheitztisch apparatus (Reichert, Wien) and were uncorrected. IR spectra were recorded on an ABB Bomem M-102 spectrophotometer. Elemental analyses were carried out on a Perkin-Elmer 2400 Series II CHNS Analyzer at the Ruđer Boškovic Institute. Silica gel (Merck 0.05–0.2 mm) was used for chromatographic purifications. Solvents were purified by distillation. The chemicals for synthesis were obtained from usual commercial sources.
3-(N-Phthalimido)adamantane-1-carboxylic acid methyl ester (14) In a round bottom flask (50 mL) equipped with a stopper, phthalic anhydride (889 mg, 6 mmol) was melted. To the melt, ester 30 (1.14 g, 3 mmol) was added as a CH2Cl2 solution in several portions. After the addition was completed, the reaction mixture was stirred over 10 min with the stopper, after which stopper was removed and stirring continued for another 10 min to remove water. To the cooled reaction mixture, CH2Cl2 (100 mL) was added, and the solution washed with 10% acetic acid (3 30 mL) and 10% solution of NaHCO3 (3 30 mL). Washed organic layer was dried over anhydrous MgSO4, filtered, and the solvent was removed on a rotary evaporator. The crude reaction mixture was purified by column chromatography on SiO2 using CH2Cl2 as an eluent. Colorless crystals (832 mg, 45%); m.p. 98–99°C; IR (KBr) nmax/cm1 3343, 2957, 2925, 2890, 2852, 1767, 1724, 1463, 1436, 1371, 1346, 1320, 1079, 876, 714, 642; 1H NMR (DMSO-d6, 600 MHz) d/ppm 7.75 (m, 2H), 7.67 (m, 2H), 3.67 (s, 3H, OCH3), 2.64 (s, 2H), 2.57 (dd, 2H, J ¼ 12.2 Hz, J ¼ 1.3 Hz), 2.45 (d, 2H, J ¼ 11.7 Hz), 2.29 (br s, 2H), 1.96 (dd, 2H, J ¼ 12.6 Hz, J ¼ 1.3 Hz), 1.87 (d, 2H, J ¼ 12.3 Hz), 1.78 (ddd, 1H, J ¼ 12.6 Hz, J ¼ 1.9 Hz, J ¼ 1.6 Hz), 1.67 (ddd, 1H, J ¼ 12.6 Hz, J ¼ 1.9 Hz, J ¼ 1.6 Hz); 13C NMR (DMSO-d6, 150 MHz) d/ppm 176.7 (s), 169.5 (s), 133.6 (d, 2C), 131.8 (s, 2C), 122.5 (d, 2C), 60.0 (s), 51.7 (q), 42.7 (s), 40.8 (t), 39.2 (t, 2C), 37.6 (t, 2C), 35.1 (t), 28.2 (d, 2-C); Elemental analysis calcd. for C20H21NO4 (Mr 339.38) C 70.78, H 6.24, N 4.13%; found: C 70.78, H 6.30, N 4.12%. www.archpharm.com
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N1,N2-{bis[3-(Methyloxocarbonyl)adamantan-1-yl]}phthalamide (29) Colorless crystals, 240 mg (5.4%); m.p. 215–216°C; IR (KBr) nmax/cm1 3283, 2912, 2857, 1729, 1648, 1546, 1534, 1454, 1434, 1366, 1244, 1102; 1H NMR (CDCl3, 600 MHz) d/ppm 7.74 (m, 2H), 7.43 (m, 2H), 6.41 (s, 2H, NH), 3.66 (s, 6H, OCH3), 2.30 (s, 4H), 2.25 (br s, 4H), 2.15 (d, 4H, J ¼ 11.4 Hz), 2.01 (d, 4H, J ¼ 11.4 Hz), 1.90 (d, 4H, J ¼ 12.0 Hz), 1.86 (d, 4H, J ¼ 12.0 Hz), 1.71 (d, 2H, J ¼ 12.5 Hz), 1.67 (d, 2H, J ¼ 12.5 Hz); 13C NMR (CDCl3, 75 MHz) d/ppm 176.7 (s, 2C), 168.4 (s, 2C), 134.9 (s, 2C), 129.9 (d, 2C), 128.2 (d, 2C), 51.8 (s, 2C), 51.6 (q, 2C), 42.5 (s, 2C), 42.0 (t, 2C), 40.5 (t, 4C), 37.7 (t, 4C), 35.2 (t, 2C), 28.95 (d, 4C).
N-(3-Carboxyadamantan-1-yl)-2-carbamoylbenzoic acid (28) A round bottom flask (50 mL) was charged with ester 14 (500 mg, 1.47 mmol), CH3OH (10 mL) and an aqueous solution of Na2CO3 (20 mL, 10%). The mixture was heated at reflux temperature for 2 days. The cooled reaction mixture was washed with CH2Cl2 (3 25 mL) and basic aqueous layer acidified by use of 1 M HCl until pH 2 was reached. The resulting suspension was extracted with EtOAc (3 45 mL) and the combined extracts were dried over anhydrous MgSO4. After filtration and removal of the solvent, the pure product was obtained (277 mg, 58%) in the form of colorless crystals. Colorless crystals, m.p. 295–297°C; IR (KBr) nmax/cm1 3366, 3032, 2917, 1689, 1675, 1532, 1310, 1287; 1H NMR (DMSO-d6, 600 MHz) d/ppm 12.40 (br s, 2H, COOH), 7.79 (s, 1H, NH), 7.74 (dd, 1H, J ¼ 1.0 Hz, J ¼ 7.7 Hz), 7.53 (ddd, 1H, J ¼ 1.3 Hz, J ¼ 7.5 Hz, J ¼ 7.7 Hz), 7.45 (ddd, 1H, J ¼ 1.3 Hz, J ¼ 7.5 Hz, J ¼ 7.7 Hz), 7.34 (dd, 1H, J ¼ 1.0 Hz, J ¼ 7.7 Hz), 2.14 (s, 2H), 2.12 (br s, 2H), 2.03 (d, 2H, J ¼ 11.7 Hz), 1.91 (d, 2H, J ¼ 11.7 Hz), 1.76–1.71 (m, 4H), 1.60–1.57 (m, 2H); 13C NMR (DMSO, 150 MHz) d/ppm 177.8 (s), 168.2 (s), 167.8 (s), 139.6 (s), 131.1 (d), 129.0 (d), 128.6 (d), 127.7 (d), 51.6 (s), 41.8 (t), 41.5 (s), 40.0 (t, 2C), 37.7 (t, 2C), 35.2 (t), 28.6 (d); Elemental analysis calcd. for C19H21NO5 (Mr 343.14) C 66.46; H 6.16; N 4.08%; found: C 65,92; H 6,39; N 4,03%. These materials are based on work financed by the Foundation for Science of the Republic of Croatia (HRZZ grant no. 02.05/25), the Ministry of Science Education and Sports of the Republic of Croatia (grant no. 098-0982933-2911). We thank Leentje Persoons, Frieda De Meyer, Lies Van den Heurck, Steven Carmans, Anita Camps, Lizette van Berckelaer, and Leen Ingels for excellent technical assistance for the biological assays. The research of JB was supported by grants of the KU Leuven (GOA 10/14). The authors have declared no conflict of interest.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 334–340
Full Paper Antiproliferative and Antiviral Activity of Three Libraries of Adamantane Derivatives 1, Margareta Sohora1, Nikola Cindro1, Kata Mlinaric -Majerski1, Erik De Clercq2*, and Nikola Basaric 2 Jan Balzarini * 1 2
Department of Organic Chemistry and Biochemistry, Ruđer Boškovic Institute, Zagreb, Croatia Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium
Three libraries of adamantane derivatives were synthesized and evaluated for antiviral and antiproliferative activities against a broad variety of DNA and RNA viruses. Whereas none of the compounds exhibit antiviral activity at subtoxic concentrations, antiproliferative activity was found against murine leukemia cells (L1210), human T-lymphocyte cells (CEM), and cervix carcinoma cells (HeLa) for 4, 8, and 10. Keywords: Adamantanes / Antiproliferative activity / Antiviral activity / Phthalimides / Polycyclic compounds / Unnatural amino acids Received: September 11, 2013; Revised: November 15, 2013; Accepted: November 15, 2013 DOI 10.1002/ardp.201300345
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Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Introduction A number of different antiviral drugs exist nowadays in the market. However, combating viral diseases and finding appropriate drugs with minimal side effects remain a challenge in chemistry, pharmacy, and medicine. A major problem in antiviral therapy is the rapid mutations of viruses leading to development of drug resistance. Amantadine (1-aminoadamantane hydrochloride) and rimantadine [1-(2aminoethyl)adamantane hydrochloride] are widely used as anti-influenza agents that block M2 protein ion channel, which prevents fusion of the virus with the host-cell membrane and release of viral RNA into the cytoplasm [1]. The same compounds have been shown to exhibit inhibitory activity – although rather modest – against some other viruses such as HIV [2], hepatitis [3], and herpes simplex virus [4]. However, inappropriate use of these drugs in many countries contributed to an increasing number of resistant influenza viruses [5]. In the search of new drugs and novel mechanisms of action based on yet unknown intracellular targets, it is important to screen new libraries of molecules and develop Correspondence: Dr. Nikola Basari c, Department of Organic Chemistry and Biochemistry, Ruđer Boškovi c Institute, Bijeni cka Cesta 54, 10 000 Zagreb, Croatia. E-mail: [email protected] Fax: þ385 1 4680 195
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new structure–activity relationships (SAR). Herein, we report an investigation of the antiviral activity in vitro of three libraries of adamantane derivatives. In addition, the same libraries of compounds were also tested for antiproliferative activity. Recently, we have reported an investigation of antiproliferative activity of a series of adamantylphthalimides, where all investigated compounds exhibited higher activity than the clinically used drug thalidomide [6].
Materials and methods Structure design The first library of compounds (Fig. 1) is composed of adamantylphthalimides since adamantylphthalimides were previously reported to be endowed with anti-HIV activity [7]. Structural design of the phthalimide compounds is in connection with a pharmacophore phenytoin (PHT) that exhibits anticonvulsant activity. This membrane-reactive drug has been reported to inhibit HIV binding to CD4 positive lymphocytes [8], and reduced the CD4 receptor availability for ligand interaction [9]. It was also proposed that
*Additional correspondence: Prof. Erik De Clercq, E-mail: [email protected]; Prof. Jan Balzarini, E-mail: [email protected]
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Figure 1. Library 1: adamantylphthalimides.
PHT suppresses the influx of Ca2þ ions that occurs shortly after HIV infection [10]. Later, it was discovered that some of the HIV proteins may organize as sodium channel entities in planar lipid bilayers [11]. Screening indicated that the 4-aminophthalimide pharmacophore and N-(l-adamantyl) substitutions are required for their anti-HIV properties [7]. The structures in library 1 are 1- or 2-adamantyl derivatives ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wherein adamantane and phthalimide are connected or separated by an alkyl spacer of different length (compounds 1–8). Furthermore, structures were modified by substitution of the phthalimide by an amino or a nitro group (compounds 9–12), or by additional substitution of the adamantane moiety (compounds 13–15, 19, and 20), and a change of the polycyclic skeleton (compounds 16–18). www.archpharm.com
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The third library of compounds (Fig. 3) is composed of adamantane amino acids. Anticancer activity of cage amino acids and the corresponding peptides has recently been reported [13]. Moreover, it has been demonstrated that dipeptides with incorporated cage amino acids exhibit HIV protease inhibition [14].
Synthesis
Figure 2. Library 2: adamantyl aza-heterocyclic derivatives.
The second library (Fig. 2) is composed of different azaheterocyclic derivatives of adamantane. The design of compounds is based on reports of antiviral activity of a series of polycyclic amines and aza-heterocycles [12]. It was found that the size of the heterocyclic ring influences the activity against influenza A virus, with a higher activity with five- than with four- and three-membered rings [12]. On the other hand, substitution of the heterocylic nitrogen increases the anti-HIV activity [12]. Compounds in library 2 are derivatives of azepine (21–23) or pyrrolidine (24–26), that are also characterized by a different connectivity to the adamantane skeleton.
Compounds in library 1 were prepared according to the described procedures [6, 15]. Adamantylphthalimides 1–8 were prepared in a condensation reaction of phthalic anhydride and the corresponding 1- or 2-aminoalkyladamantane [15a]. Aminophthalimide derivatives 10 and 12 were obtained from the nitro derivatives 9 and 11, respectively, by reduction with Zn. Homo-, 16, and protoadamantanes, exo-17 and endo-18, were made by applying a Mitzunobu protocol, from phthalimide and the corresponding polycyclic alcohols [15b]. Compounds 19 and 20 were obtained in a photochemical reaction from carboxylic acid 13 in the presence of acrylonitrile or cyclohexenone, respectively [15c], whereas 15 was obtained as one of the products in the photochemical reaction of 2 [16]. Compounds in library 2 were obtained in photochemical reactions from the compounds in library 1 (21 from 6, 22 from 1, 23 from 3, 24 from 2, and 25 and 26 from 5) [16]. Amino acid 27, from library 3 was obtained by a base-hydrolysis of 22 [16a]. Amino acid derivatives 14, and 29 were synthesized from ester 30 in the condensation reaction with phthalic anhydride (Scheme 1). Base hydrolysis of ester 14 furnished diacid 28 (Scheme 2). Phthalimido acid 13 was also obtained in the condensation reaction of phthalic anhydride and 3-aminoadamantane-1-carboxylic acid [15c]. All chiral compounds were synthesized as racemates.
Antiviral activity assays The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK VZV strain 07–1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV)
Figure 3. Library 3: adamantane amino acid derivatives.
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Scheme 1. Synthesis of 29.
Scheme 2. Synthesis of 28.
strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, parainfluenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, Reovirus-1, Sindbis, Reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain IIIB, and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on inhibition of virusinduced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa), or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque-forming units (PFU) (VZV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%. Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cytopathicity in CEM cell cultures was measured in microtiter 96-well plates containing 3 105 CEM cells/mL infected with 100 CCID50 of HIV per milliliter and containing appropriate dilutions of the test compounds. After 4–5 days of incubation at 37°C in a CO2-controlled humidified atmosphere, CEM giant (syncytium) cell formation was examined microscopically. The EC50 (50% effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50%.
Cytostatic activity assays All assays were performed in 96-well microtiter plates. To each well (5–7.5) 104 tumor cells and a given amount of the test compound were added. The cells were allowed to proliferate ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37°C in a humidified CO2-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The 50% inhibitory concentration (IC50) was defined as the concentration of the compound that inhibited cell proliferation by 50%. Data are derived from two to three independent experiments (the errors were calculated according to a formula given in the Supporting Information).
Results and discussion All three tested libraries of compounds exert no significant antiviral activity in cell culture at subtoxic concentrations (see Supporting Information Tables S1–S4). Antiproliferative activity was tested against three cancer cell lines (Table 1). Generally, compounds exhibit moderate activity in micromolar concentration range and show no selectivity to a specific cancer cell line. From the compounds in library 1, the highest activity was observed for 4 and 8, containing the longest alkyl linker between the adamantyl and the phthalimide moiety (CC50 range between 7.2 and 12 mg/mL). Also, antiproliferative activity can be achieved by incorporation of an amino substituent at the phthalimide 3-position, but only for the 1-adamantyl derivative 10 but not the adamantyl derivative 12. Contrary to our previous report [6] a change of the substitution on the adamantane, 1- versus 2-, virtually does not affect the antiproliferative activity. However, replacement of the adamantane with a homoadamantane skeleton (5 vs. 16) enhanced the cytostatic effect. A minor additional activity enhancement was also observed by substitution of the adamantane skeleton at the position 3 with a cyclohexanone ring (compound 20), whereas other groups (COOH, COOCH3, and CH2CH2CN) had no effect. All compounds in library 2 exhibited similar modest www.archpharm.com
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Table 1. Antiproliferative activities against L1210, CEM, and HeLa tumor cells. IC50 (mmol/L)a) Comp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Thalidomide a) b)
L1210
CEM
HeLa
>300 200 100 >300 30 7 30 ! >300b) 220 10 >300 31 0 >300 29 3 >300 >300 >300 60 ! 300b) 138 3 16 10 300 80 >300 >300 50 30 280 80 170 0 113 3 210 10 270 3 128 7 >300 >300 >200 >77
>300 70 10 >300 34 6 7 ! 300b) 190 10 300 37 6 >300 30 3 >300 >300 >300 150 120 120 30 30 10 210 160 >300 >300 100 0 110 60 190 10 103 3 280 100 300 50 100 60 >300 >300 >200 >42
>300 90 20 300 22 1 300 230 30 300 26 3 >300 33 3 200 100 300 >300 30 ! 300b) 150 60 80 30 120 80 320 50 >300 70 10 180 130 180 0 64 6 290 40 220 50 90 10 >300 >300 >200 >77
Fifty percent inhibitory concentration. No dose response between these concentrations.
cytostatic activity, so that no SAR could be established. On the contrary, the amino acid derivatives from library 3 exhibit no antiproliferative activity at all. Thus, the presence of a heterocyclic ring (phthalimide or other aza-heterocycle) is essential for the antiproliferative effect. It was shown that the activity of thalidomide, a clinically used anticancer drug with the phthalimide pharmacophore, is related to the modulation of tumor necrosis factor TNF-a, especially in multiple myeloma. Namely, TNF-a is a multifunctional cytokine playing a key role in both apoptosis and cell survival, as well as in inflammation and immunity [17]. TNF-a also shows the capacity to form a voltagedependent cationic channel in lipid bilayers [11, 18]. Thalidomide was proposed to elicit its anti-HIV by accelerating the decay of the TNF-a mRNA [19]. Consequently, the antiproliferative activity of the compounds presented herein may be perhaps related to TNF-a inhibition. This concept is to be further tested, and if shown to be correct, it is anticipated to have impact in further development of anticancer agents. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Conclusion Three libraries of adamantane derivatives were synthesized and screened for antiviral and antiproliferative activity. Whereas none of the compounds exhibited pronounced activity against virus infection, antiproliferative activity was found against L1210, CEM, and HeLa for 4, 8, and 10.
Experimental General 1
H and 13C NMR spectra were recorded on a Bruker spectrometer at 300 or 600 MHz, respectively. All NMR spectra were measured in CDCl3 or DMSO-d6 using tetramethylsilane as a reference. Melting points were obtained using an Original Kofler Mikroheitztisch apparatus (Reichert, Wien) and were uncorrected. IR spectra were recorded on an ABB Bomem M-102 spectrophotometer. Elemental analyses were carried out on a Perkin-Elmer 2400 Series II CHNS Analyzer at the Ruđer Boškovic Institute. Silica gel (Merck 0.05–0.2 mm) was used for chromatographic purifications. Solvents were purified by distillation. The chemicals for synthesis were obtained from usual commercial sources.
3-(N-Phthalimido)adamantane-1-carboxylic acid methyl ester (14) In a round bottom flask (50 mL) equipped with a stopper, phthalic anhydride (889 mg, 6 mmol) was melted. To the melt, ester 30 (1.14 g, 3 mmol) was added as a CH2Cl2 solution in several portions. After the addition was completed, the reaction mixture was stirred over 10 min with the stopper, after which stopper was removed and stirring continued for another 10 min to remove water. To the cooled reaction mixture, CH2Cl2 (100 mL) was added, and the solution washed with 10% acetic acid (3 30 mL) and 10% solution of NaHCO3 (3 30 mL). Washed organic layer was dried over anhydrous MgSO4, filtered, and the solvent was removed on a rotary evaporator. The crude reaction mixture was purified by column chromatography on SiO2 using CH2Cl2 as an eluent. Colorless crystals (832 mg, 45%); m.p. 98–99°C; IR (KBr) nmax/cm1 3343, 2957, 2925, 2890, 2852, 1767, 1724, 1463, 1436, 1371, 1346, 1320, 1079, 876, 714, 642; 1H NMR (DMSO-d6, 600 MHz) d/ppm 7.75 (m, 2H), 7.67 (m, 2H), 3.67 (s, 3H, OCH3), 2.64 (s, 2H), 2.57 (dd, 2H, J ¼ 12.2 Hz, J ¼ 1.3 Hz), 2.45 (d, 2H, J ¼ 11.7 Hz), 2.29 (br s, 2H), 1.96 (dd, 2H, J ¼ 12.6 Hz, J ¼ 1.3 Hz), 1.87 (d, 2H, J ¼ 12.3 Hz), 1.78 (ddd, 1H, J ¼ 12.6 Hz, J ¼ 1.9 Hz, J ¼ 1.6 Hz), 1.67 (ddd, 1H, J ¼ 12.6 Hz, J ¼ 1.9 Hz, J ¼ 1.6 Hz); 13C NMR (DMSO-d6, 150 MHz) d/ppm 176.7 (s), 169.5 (s), 133.6 (d, 2C), 131.8 (s, 2C), 122.5 (d, 2C), 60.0 (s), 51.7 (q), 42.7 (s), 40.8 (t), 39.2 (t, 2C), 37.6 (t, 2C), 35.1 (t), 28.2 (d, 2-C); Elemental analysis calcd. for C20H21NO4 (Mr 339.38) C 70.78, H 6.24, N 4.13%; found: C 70.78, H 6.30, N 4.12%. www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2014, 347, 334–340
N1,N2-{bis[3-(Methyloxocarbonyl)adamantan-1-yl]}phthalamide (29) Colorless crystals, 240 mg (5.4%); m.p. 215–216°C; IR (KBr) nmax/cm1 3283, 2912, 2857, 1729, 1648, 1546, 1534, 1454, 1434, 1366, 1244, 1102; 1H NMR (CDCl3, 600 MHz) d/ppm 7.74 (m, 2H), 7.43 (m, 2H), 6.41 (s, 2H, NH), 3.66 (s, 6H, OCH3), 2.30 (s, 4H), 2.25 (br s, 4H), 2.15 (d, 4H, J ¼ 11.4 Hz), 2.01 (d, 4H, J ¼ 11.4 Hz), 1.90 (d, 4H, J ¼ 12.0 Hz), 1.86 (d, 4H, J ¼ 12.0 Hz), 1.71 (d, 2H, J ¼ 12.5 Hz), 1.67 (d, 2H, J ¼ 12.5 Hz); 13C NMR (CDCl3, 75 MHz) d/ppm 176.7 (s, 2C), 168.4 (s, 2C), 134.9 (s, 2C), 129.9 (d, 2C), 128.2 (d, 2C), 51.8 (s, 2C), 51.6 (q, 2C), 42.5 (s, 2C), 42.0 (t, 2C), 40.5 (t, 4C), 37.7 (t, 4C), 35.2 (t, 2C), 28.95 (d, 4C).
N-(3-Carboxyadamantan-1-yl)-2-carbamoylbenzoic acid (28) A round bottom flask (50 mL) was charged with ester 14 (500 mg, 1.47 mmol), CH3OH (10 mL) and an aqueous solution of Na2CO3 (20 mL, 10%). The mixture was heated at reflux temperature for 2 days. The cooled reaction mixture was washed with CH2Cl2 (3 25 mL) and basic aqueous layer acidified by use of 1 M HCl until pH 2 was reached. The resulting suspension was extracted with EtOAc (3 45 mL) and the combined extracts were dried over anhydrous MgSO4. After filtration and removal of the solvent, the pure product was obtained (277 mg, 58%) in the form of colorless crystals. Colorless crystals, m.p. 295–297°C; IR (KBr) nmax/cm1 3366, 3032, 2917, 1689, 1675, 1532, 1310, 1287; 1H NMR (DMSO-d6, 600 MHz) d/ppm 12.40 (br s, 2H, COOH), 7.79 (s, 1H, NH), 7.74 (dd, 1H, J ¼ 1.0 Hz, J ¼ 7.7 Hz), 7.53 (ddd, 1H, J ¼ 1.3 Hz, J ¼ 7.5 Hz, J ¼ 7.7 Hz), 7.45 (ddd, 1H, J ¼ 1.3 Hz, J ¼ 7.5 Hz, J ¼ 7.7 Hz), 7.34 (dd, 1H, J ¼ 1.0 Hz, J ¼ 7.7 Hz), 2.14 (s, 2H), 2.12 (br s, 2H), 2.03 (d, 2H, J ¼ 11.7 Hz), 1.91 (d, 2H, J ¼ 11.7 Hz), 1.76–1.71 (m, 4H), 1.60–1.57 (m, 2H); 13C NMR (DMSO, 150 MHz) d/ppm 177.8 (s), 168.2 (s), 167.8 (s), 139.6 (s), 131.1 (d), 129.0 (d), 128.6 (d), 127.7 (d), 51.6 (s), 41.8 (t), 41.5 (s), 40.0 (t, 2C), 37.7 (t, 2C), 35.2 (t), 28.6 (d); Elemental analysis calcd. for C19H21NO5 (Mr 343.14) C 66.46; H 6.16; N 4.08%; found: C 65,92; H 6,39; N 4,03%. These materials are based on work financed by the Foundation for Science of the Republic of Croatia (HRZZ grant no. 02.05/25), the Ministry of Science Education and Sports of the Republic of Croatia (grant no. 098-0982933-2911). We thank Leentje Persoons, Frieda De Meyer, Lies Van den Heurck, Steven Carmans, Anita Camps, Lizette van Berckelaer, and Leen Ingels for excellent technical assistance for the biological assays. The research of JB was supported by grants of the KU Leuven (GOA 10/14). The authors have declared no conflict of interest.
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