Synthesis and in vitro Toxicity of New Dodecaborate-Containing Amino Acids
Irina N. Slepukhina, Detlef Gabel
Department of Chemistry, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany
gabel@chemie.uni-bremen.de
Abstract: The new unnatural, boron-containing amino acids were synthesized. They were evaluated by in vitro toxicity tests using V 79 Chinese hamster cells.
Introduction
Although the concept of boron neutron capture therapy (BNCT) was proposed by Locher some 75 years ago1, the successful application of BNCT to the treatment of cancer still presents an issue in medical research. One of the conditions for this therapy to be successful depends on the ability to transport sufficient concentrations of boron to the tumor tissue and preferably localize the boron into the cell. The three most important parameters for compound development have essentially not changed since early days of BNCT: (1) achieving tumor concentrations in the range of 20-35 µg 10B/g; (2) a tumor:normal tissue differential greater than 1 and preferably 3-5;
(3) sufficiently low toxicity so that the dose administered would be well tolerated first in animals and subsequently in patients.
Various carrier molecules have been used to deliver boron to the tumor cells. These include carbohydrates2-4, amino acids5-8, nucleosides9,10, porphyrins11, antibodies12,13, and liposomes14. While these carrier molecules are under development, mercapto-undecahydro-closo-dodecaborat (BSH) and 4-dihydroxyborylphenylalanine (BPA) are being used clinically for the treatment of brain tumors and malignant melanoma in human patients15-17. The interest in the development of boron-containing amino acids and is certainly in part due to the fact that BPA is one of the two clinically used BNCT agents.
Water-soluble functionalized derivatives of the dodecahydro-closo-dodecaborate anion [B12H12]2- are promising candidates for BNCT for cancer. Such boron containing structures allow to obtain molecules that carry a large number of boron atoms per molecule. The traditional approach for the functionalization of the [B12H12]2- anion consists of the introduction of a reactive centre such as an amino-18,19, mercapto-20,21, or hydroxy-18,22,23 group into the boron cage, followed by attachment of the side chain containing a functional group.
Here, we describe the synthesis of unnatural, boron-containing amino acids.
Acetamidodiethylmalonate is a very convenient precursor for the synthesis of amino acids. After hydrolysis it gives amino acids as a racematic mixture. Synthesis of amino acids from hydrolysis of hydantoins seems for us an interesting possibility for the synthesis of α-alkyl amino acids.
Such kind of amino acids already have pharmaceutical application24.
Following synthesis of water-soluble amino acids, we cannot neglect their biological test as a BNCT agent. Toxicity tests, by using V 79 Chinese hamster cells, were carried out to give the first necessary information on toxicity, required for future application of these compounds.
For the task of synthesis amino acids the S-cyanoethyl derivative of BSH (CE-BSH)22 and the oxygen analogue of BSH [HO-B12H11]2-(BOH) were chosen.
Results and discussion
It is known that educts such as CE-BSH and BOH easily undergo alkylation reactions18,21. We have developed two convenient methods for the preparation of boronated amino acids containing the B12H11 cage.
The first pathway (Scheme 1) includes alkylation of the nucleophilic cyanoethyl-derivative of the BSH cluster. Selective removal of the remaining cyanoethyl group yields 4 and 5. In the case of acetamidodiethylmalonate derivative 4, hydrolysis with 6M NaOH was carried out under reflux for 3 days. Since the tetramethylammonium salt of amino acid 6 dissolves in water, we isolated it as tetraphenylphosphonium salt, which is not water-soluble.
Alkylation of 1 with chloroacetone gives the sulfonium salt 3. Such kind of ketone can easily form hydantoins in a Bucherer-Berg reaction25. We could obtain 7 from 3 and from 5. In the first case, the cyanoethyl group was removed during the reaction (Scheme 1). The general procedure is to warm the ketone derivatives 3 and 5 with 2 moles of sodium cyanide and 5 moles of ammonium carbonate in 50 per cent ethanol for 4 hours. The preparation of the hydantoin from the sulfonium salt 3 to 7 directly gives only 30% of yield, whereas the overall yield of the second way (from 3 via 5 to 7) is 60-65%. Probably, the basicity of the bases as NaCN and (NH4)2CO3
is not enough for elimination of acrylonitrile. Prolonged alkaline hydrolysis gives amino acid 8 with a quaternary carbon atom.
NC S
-NC S
2-2Me4N+ R-X acetonitrile
R-X: Br(CH2)4C(COOEt)2(NHCOCH3) ClCH2COCH3
1 2: R=-(CH2)4C(COOEt)2(NHCOCH3) 3: R=-CH2COCH3
Me4NOH Me4N+
S
2-2Me4N+
4: R=-(CH2)4C(COOEt)2(NHCOCH3) 5: R=-CH2COCH3
NaCN, (NH4)2CO3
S (CH2)4 COOH NH2
R R
1) NaOH, HCl 2) Ph4PBr
2-2Ph4P+
S
2-2Me4N+ HN NH
O
O in case of 3 in case of 5
1) NaOH, HCl 2) Ph4PBr S
2-2Ph4P+ COOH NH2
6
7 8
in case of 4
Scheme 32. Synthesis of sulfur-containing amino acids (Pathway 1).
For the preparation of oxygen derivatives we used an analoguos synthetic way (Scheme 2).
The essential difference between the schemes lies in the boron-carry nucleophilic educts.
Alkylation of the hydroxyl group attached to the boron cluster 9 could be achieved in acetone using K2CO3 as a base. Similar to Peymann18 we showed that alkylation of BOH afforded monoalkylated derivatives.
2-2) CsF, methanol
R-X: Br(CH2)4C(COOEt)2(NHCOCH3) ClCH2COCH3
O
2-2Cs+
NaCN, (NH4)2CO3
O (CH2)4 COOH NH2 R
1) NaOH, HCl 3) Ph4PBr
2-2Ph4P+
O
2-2Cs+ HN NH
O
O
1) NaOH, HCl 2) Ph4PBr
O
2-2Ph4P+ COOH NH2 12
13 14
OH
2But4N+
1) K2CO3
acetone R-X
9 10: R=-(CH2)4C(COOEt)2(NHCOCH3) 11: R=-CH2COCH3
in case of 10
in case of 11
Scheme 33. Synthesis of oxygen-containing amino acids (Pathway 2).
The solubility of the newly synthesized amino acids depends of the cation. The tetraphenylphosphonium cation makes the amino acids soluble in methanol and acetonitrile, the tetrabutylammonium cation – in dichloromethane and water; tetramethylammonium salts of the amino acids prepared here dissolve in water. It is interesting to notice that others derivatives of B12H122- with tetrabutylammonium or tetramethylammonium cation are usually not soluble in water.
An analogous acid 12 was synthesized by Bregadze and co-workers26 from the oxonium derivative [B12H11O(CH2)4]- through ring-opening nucleophilic attack of the malonate carbanion of the THF derivative of B12H122-.
The in vitro toxicity test was carried out with two amino acids having different number of carbons and different heteroatom between cluster and side chain. The amino acids 8 and 12 were converted to water-soluble Cs+ salts 8a and 12a. For amino acid 8a LD50 was 5.7 mM and for 12a LD50 was 5.0 mM. According to this results, we can say that addition of amino acid group to the, e.g. BSH cage, do not change toxicity value (LD50 for the sodium salt of BSH, determined by the same assay, was 5.5 mM)28. Toxicity of p-boronophenylalanine (BPA), which was done B 16 human melanoma cells, was 8.55 mM29.
concentration (M)
1e-4 1e-3 1e-2
survival (%)
20 40 60 80 100 120
Figure 1. The percentage of cell survival as a function of the concentration of (S-(2-amino-2-carboxyl)propyl)-thio-undecahydro-closo-dodecaborate(2-) bis-cesium salt 8a.
concentration (M)
1e-5 1e-4 1e-3 1e-2 1e-1 1e+0
survival (%)
0 20 40 60 80 100 120 140
Figure 2. The percentage of cell survival as a function of the concentration of (S-(5-amino-5-carboxyl)penthyl)-oxy-undecahydro-closo-dodecaborate(2-) bis-cesium salt 12a.
Experimental Section
General Methods. All reagents were obtained from Aldrich Chemical Co., Acros Chemicals and Roth Chemicals and used without further purification. Acetonitrile was dried over P2O5 and then distilled.
The NMR measurements (11B, 1H) were carried out on a Bruker DPX 200 spectrometer. IR (cm-1) spectra were determined as KBr disc on a Biorad FTS 155 spectrometer. HPLC was carried out on a RP-18 LiChroCart 125x4 mm column30 with 57% MeOH/43% H2O 10 mM tetrabutylammonium hydrogen sulfate pH=7.0 as mobile phase. Detection was at 220 nm.
ESI MS measurements were performed on a Bruker Esquire-LC ion trap mass spectrometer.
Samples were dissolved in solvents as indicated in the description of each compound, at concentrations of about 10-5 to 10-6 mol/L and injected into the mass spectrometer via a syringe pump at a flow rate of 2 µL/min. Spectra were recorded in the positive and negative ion mode
for 1 minute and averaged to correct for signal fluctuations. Intensity of peaks is given in percentage of the most intense peak.
The m/z values given correspond to the maxima of the isotopic distributions. In the analytical description of the compounds, peaks containing boron are indicated by “*”. As the boron-containing compounds have a broad isotopic distribution and therefore allow the assignment of the charge of the compound, z is given in parentheses after each m/z value.
The cell toxicity experiments were done using WST assay which is the colorimetric quantification of cell proliferation and cell viability, based on the reduction of the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) to formazan by a mitochondrial succinate-tetrazolium-reductase system in viable cells31. A decrease in the number of viable cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample leading to a decrease in the amount of formazan dye formed.
F10 medium containing 10% newborn calf serum NCS was used for growing V 79 Chinese hamster cells.
S-(2-cyanoethyl)-S-(5-bis(ethoxycarbonyl)-5-acetamidopentyl)-sulfonio-undecahydro-closo-dodecaborate(-) tetramethylammonium salt (2). S-(2-cyanoethyl)-thio-undecahydro-closo-dodecaborate(2-) bis-tetrametylammonium salt 1 (0.39 g, 1.04 mmol) was suspended in 15 ml of dry acetonitrile at room temperature. A solution of 0.55 g (1.56 mmol) 4-bromobutylacetamidodiethylmalonate32 was added. The reaction mixture was refluxed during 12 hours. After cooling, the precipitate of tetramethylammonium bromide was removed by filtration. Acetonitrile was evaporated and the resulting yellow oil was crystallized from diethylether. The resulting compound was used without further purification. Yield: 93% (0.55 g), mp 70-72°C, HPLC 7.26 min, 1H-NMR (CD3CN, ppm): 7.73 (1H, s, -NH-), 3.54 (4H, q, -C(-COOCH2CH3)2), 2.85-2.20 (18H, m, -SCH2CH2CN, -SCH2CH2CN, -S-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), N+(CH3)4), 1.5 (2H, t, -S-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 1.33 (3H, s, -NHCOCH3), 1.15 (4H, m, -S-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 0.57 (6H, t, -C(-COOCH2CH3)2(NHCOMe)), 2.3-(-0.7) (broad, B12H11), IR (cm-1): 3397(m) (-NH-), 2983(w), 2502(s) (B-H), 2361(w), 1736(m) (C=Oester), 1669(m) (C=Oamide), 1488(m), 1372(w), 1287(m), 1211(m), 1044(w), 949(w), 720(w), 525(w), MS: (pos. ESI, CH3CN) 74.0 (cat+, 50%), 647.6* (A-+2cat+, 100%), (neg. ESI, CH3CN) 141.0* (B12H11-, 10%), 499.3* (A-, 100%), 11B-NMR (CD3CN, ppm): -15.28 (broad, 12B).
S-(2-cyanoethyl)-S-(2-oxopropyl)-sulfonio-undecahydro-closo-dodecaborate(-)
tetramethylammonium salt (3). The tetramethylammonium salt of 1 (2.55 g, 6.79 mmol) was dissolved in 70 ml of dry acetonitrile at room temperature. Chloroacetone (0.94 g, 10.2 mmol) was added. The reaction mixture was refluxed during 12 hours (HPLC control). After cooling,
acetonitrile was evaporated under reduced pressure. The resulting green oil was crystallized from diethylether. Recrystallization from acetonitrile gave 2.31 g of white crystals of compound 3.
Yield: 93%, mp 105-107°C, HPLC 4.58 min, 1H-NMR (CD3CN, ppm): 4.32 (2H, d, -S-CH2COCH3), 3.44 (12H, s, N+(CH3)4), 2.35-3.10 (4H, m, -S-CH2CH2CN), 2.35 (3H, s, -S-CH2COCH3), 2.10-(-0.90) (broad, B12H11), IR (cm-1): 3429(m), 3020(w), 2966(w), 2921(w), 2504(s) (B-H), 2249(w) (C≡N), 1723(m) (C=Oketon), 1486(s), 1414(w), 1360(w), 1287(w), 1155(w), 1045(m), 949(s), 822(w), 720(w), 558(w), MS: (pos. ESI, CH3CN) 74.0 (cat+, 100%), 432* (A-+2cat+, 20%), (neg. ESI, CH3CN) 141.0* (B12H11-, 10%), 284.2* (A-, 100%), 11B-NMR (CD3CN, ppm): -9.50 (1B, B1), -12.73, -14.43 (11B, B2-12).
S-(5-bis(ethoxycarbonyl)-5-acetamidopentyl)-thio-undecahydro-closo-dodecaborate(2-) bis-tetramethylammonium salt (4). The tetramethylammonium salt of 2 (0.5 g, 0.87 mmol) was dissolved in 50 ml of hot acetone. Tetramethylammonium hydroxide as a 25% solution in methanol was added in equimolar amounts. The resulting compound precipitated, was filtered off, and recrystallized from hot water:methanol 1:1. Yield: 78% (0.4 g), mp 163-165°C, HPLC 8.75 min, 1H-NMR (CD3CN, ppm): 7.10 (1H, s, -NH-), 4.16 (4H, q, -C(-COOCH2CH3)2(NHCOMe)), 3.09 (24H, s, N+(CH3)4), 2.32 (2H, t,
-S-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 2.10 (2H, t,
-S-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 2.08 (3H, s, -NHCOCH3), 1.50 (4H, m, -S-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 1.20 (6H, q, -C(-COOCH2CH3)2), 2.30-(-0.70) (broad, B12H11), IR (cm-1): 3431(m) (-NH-), 3234(w) (-NH-), 3028(w), 2481(s) (B-H), 1738(s) (C=Oester), 1638(m) (C=Oamide), 1489(w), 1291(w),1218(w), 1186(w),951(w), 843(w), 720(w), 669(w), 618(w), 525(w), MS: (pos. ESI, CH3CN) 74.0 (cat+, 100%), 667* (A2-+3cat+, 60%), (neg. ESI, CH3CN) 141.0* (B12H11-, 10%), 185.6* ((A2- -COOC2H5)/2, 25%), 162.6* (A2- -(COOC2H5)(OC2H5), 100%), 222.6* (A2-/2, 100%), 11B-NMR (CD3CN, ppm): -15.04 (broad, 12B).
S-(2-oxopropyl)-thio-undecahydro-closo-dodecaborate(2-) bis-tetramethylammonium salt (5). The bis-tetramethylammonium salt of 3 (2.3 g, 6.43 mmol) was dissolved in 180 ml of hot acetone. Tetramethylammonium hydroxide as a 25% solution in methanol was added in equimolar amounts. The resulting compound precipitated, was filtered off, and washed with diethylether. Yield: 72% (1.75 g), mp 238-240°C, HPLC 4.94 min, 1H-NMR (CD3CN, ppm):
3.01 (24H, s, N+(CH3)4), 2.23 (3H, s, -S-CH2COCH3), 2.08 (2H, s, -S-CH2COCH3), 2.30-(-0.30) (broad, B12H11), IR (cm-1): 3438(m), 3026(w), 2484(s) (B-H), 1702(m) (C=Oketon), 1487(m), 1353(w), 1287(w), 1235(w), 1050(w), 949(m), 844(w), 722(w), 667(w), MS: (pos. ESI, CH3CN) 74.2 (cat+, 100%); 452.6* (A2-+3cat+, 20%); (neg. ESI, CH3CN) 87.1* (B12H11SH2-/2, 10%),
115.0* (A2-/2, 100%), 141.0* (B12H11-, 20%), 11B-NMR (CD3CN, ppm): 0.66 (1B, B1), 9.88, -12.17 (11B, B2-6).
S-(5-amino-5-carboxylpentyl)-thio-undecahydro-closo-dodecaborate(2-) bis-tetraphenylphosphonium salt (6). The tetramethylammonium salt 4 (2.25 g, 3.8 mmol) was suspended in 50 ml of 6M NaOH. During heating the salt dissolved. Hydrolysis was carried out for 3 days (HPLC control). The cold reaction mixture was neutralized with 6M HCl to pH 7.
Ph4PBr (3.1 g, 7.6 mmol) in 50 ml of water was added. The precipitated amino acid 6 was filtered off and washed with water. Recrystallization from methanol gave 1.8 g of compound 6.
Yield: 47%, mp 73-75°C, HPLC 3.98 min, 1H-NMR (CD3CN, ppm): 7.84-7.68 (40H, m, ((C6H5)4P+)2), 3.01 (1H, t, -S-CH2CH2CH2CH2CH(COOH)(NH2)), 2.21 (2H, t, -S-CH2CH2CH2CH2CH(COOH)(NH2)), 1.65-1.32 (6H, m, -S-CH2CH2CH2CH2CH(COOH)(NH2)), 2.30-(-0.70) (broad, B12H11), IR (cm-1): 3430(m), 3060-2849(w) (-NH3+), 2479(s) (B-H), 1636(m) (br, NH3+, COO-), 1585(m) (CHarom), 1437(s) ((C6H5)4P+)2), 1317(w), 1189(w) (CCN), 1108(s) ((C6H5)4P+)2), 1044(w), 996(w), 836(w), 757(w), 722(s) ((C6H5)4P+)2), 689(m), 529(s) ((C6H5)4P+)2), MS: (pos. ESI, CH3CN) 339.0 (cat+, 100%), (neg. ESI, CH3CN) 141.0* (B12H11-, 40%), 642.3* (A2-+cat+, 100%), 561* (A2--81, 30%), 11B-NMR (CD3CN, ppm): -7.18 (1B, B1), -16.31 (11B, B2-12).
5-(Methylene-thio-undecahydro-closo-dodecaboratyl)-5-methylhydantoin (2-) bis-tetramethylammonium salt (7). A mixture of 5 (2.13 g, 5.6 mmol), 50% aqueous ethanol (45 ml), sodium cyanide (0.54 g, 10.12 mmol) and ammonium carbonate (2.85 g, 29.8 mmol) were placed in an autoclave and kept at 60°C for 4 hour. The mixture was acidified with 6M HCl, and filtered through a pad of activated carbon. Solvents were removed by evaporation at room temperature, and the solid material which remained was recrystallized from water. Yield: 57%
(1.75 g), mp>250°C, HPLC 5.52 min, 1H-NMR (CD3CN, ppm): 8.19 (1H, s, -CONHCOhydant), 6.11 (1H, s, -CNHCOhydant), 3.09 (24H, s, N+(CH3)4), 2.75 (2H, m, -S-CH2-Cq-), 1.32 (3H, s, -Cq -CH3), 2.00-(-0.80) (broad, B12H11), IR (cm-1): 3362(m), 3181(m) (-NH-), 3030(w) (-NH-), 2925(w), 2488(s) (B-H), 1773(m) (COhydant), 1718(s) (COhydant), 1630(w), 1487(s), 1416(m), 1285(w), 1168(w), 1050(m), 949(m), 840(w), 733(w), 718(w), 646(w), 588(w), MS: (pos. ESI, CH3CN) 74.1 (cat+, 100%), (neg. ESI, CH3CN), 87.1* (B12H11SH2-/2, 10%), 141.0* (B12H11-, 5%), 150.1* (A2-/2, 100%), 374.2* (A2-+cat+, 70%), 11B-NMR (CD3CN, ppm): 0.66 (1B, B1), -9.88, -12.17 (11B, B2-12).
5-(Methylene-thio-undecahydro-closo-dodecaboratyl)-5-methylhydantoin (2-) bis-tetramethylammonium salt (7). A mixture of 3 (0.3 g, 0.84 mmol), 50% aqueous ethanol (5 ml), sodium cyanide (0.08 g, 1.68 mmol) and ammonium carbonate (0.43 g, 4.48 mmol) were placed in an autoclave and kept at 60°C for 4 hour. The mixture was acidified with 6M HCl, and
filtered through a pad of activated carbon. Solvents were removed by evaporation at room temperature, and the solid material which remained was recrystallized from water. Yield: 30%
(0.11 g).
S-(2-amino-2-carboxypropyl)-thio-undecahydro-closo-dodecaborate(2-) bis-tetraphenylphosphonium salt (8). Compound 7 (1.7 g, 3.87 mmol) was suspended in 30 ml of 6M NaOH. During heating the salt dissolved. Hydrolysis was carried out for 24 hours (HPLC control). After cooling, 6M HCl was added until pH 7. Ph4PBr (3.25 g, 7.74 mmol) in 50 ml of water was added. The precipitated amino acid was filtered off and washed with water.
Recrystallization from methanol gives 1.8 g of compound 8. Yield: 49%, mp 132-134°C, HPLC 3.97 min, 1H-NMR (CD3CN, ppm): 7.84-7.68 (40H, ((C6H5)4P+)2), 2.85 (2H, m, -S-CH2-C-), 1.38 (-C-CH3), 2.5-(-0.2) (broad, B12H11), IR (cm-1): 3430(m), 3055(w) (NH3+), 2488(s) (B-H), 1613 (br, NH3+, COO-), 1483(w), 1437(m) ((C6H5)4P+)2), 1109(s) ((C6H5)4P+)2), 995(w), 756(w), 722(m) ((C6H5)4P+)2), 690(m), 529(s) ((C6H5)4P+)2), MS: (pos. ESI, CH3CN) 339.0 (cat+, 100%), (neg. ESI, CH3CN) 87.1* (B12H11SH2-/2, 80%), 141.0* (B12H11-, 20%), 614.0* (A2-+cat+, 100%), 630.1* (A2--H++OH-, 40%), 646.1* (A2--2H++2OH-, 15%), 662.1* (A2--3H++3OH-, 10%), 678.1* (A2--4H++4OH-, 5%), 694.1* (A2--5H++5OH-, 2%), 11B-NMR (CD3CN, ppm): -0.66 (1B, B1), -9.88, -12.17 (11B, B2-12).
S-(2-amino-2-carboxylpropyl)-thio-undecahydro-closo-dodecaborate(2-) bis-ceasium salt (8a). 1.8 g (1.9 mmol) of 8 was dissolved in 10 ml of methanol. CsF 0.6 g (4.1 mmol) was dissolved in 3 ml of methanol and added to the stirred solution of 8. Precipitated compound 8a was filtered off and washed with 5 ml of methanol. Recrystallization from water gave 0.9 g of 8a. Yield: 91.8%, MS: (pos. ESI, H2O) 133.0 (cat+, 100%), (neg. ESI, H2O) 87.1* (B12H11SH 2-/2, 100%), 141.0* (B12H11-, 30%), 408.6* (A2-+cat+, 20%).
O-(5-bis(ethoxycarbonyl)-5-acetamidopentyl)-oxy-undecahydro-closo-dodecaborate(2-) bis-cesium salt (10). 1.24 g (3.5 mmol) of 4-bromobutylacetamidodiethylmalonate and 1.77 g (12.8 mmol) of K2CO3 were added to solution of 2.06 g (3.2 mmol) of hydroxyl-undecahydro-closo-dodecaborate(2-) bis-tetrabutylammonium salt 9 in 50 ml of acetone. The reaction mixture was refluxed during 3 days, then cooled to room temperature and filtered off. The compound 10 was isolated as cesium salt by addition of 0.97 g (6.4 mmol) CsF in methanol to the filtrate followed by the recrystallization of the precipitate from water. Yield: 53%, (1.17 g) mp>250°C, HPLC 3.19 min, 1H-NMR (D2O, ppm): 4.14 (4H, q, -C(-COOCH2CH3)2(NHCOMe)), 3.36 (2H, t, -O-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 2.05 (2H, m, -O-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 1.95 (3H, s, NHCOCH3), 1.41 (4H, m, -O-CH2CH2CH2CH2C(NHCOMe)(COOEt)2), 1.15 (6H, t, -C(-COOCH2CH3)2), 2.4-(-0.6) (broad, B12H11), IR (cm-1): 3567(m), 3431(m) (NH), 2983(w), 2492(s) (B-H), 1734(m) (C=Oester),
1653(m) (C=Oamide), 1373(w), 1203(w), 1170(w), 1155(w), 1094(w), 1033(m), 908(w), 720(w), 668(w), MS: (pos. ESI, H2O) 132.7 (cat+, 100%), 827.8* (A2-+3cat+, 60%), (neg. ESI, H2O) 79.2* (B12H11OH2-/2, 10%), 141.0* (B12H11-, 20%), 214.8* (A2-/2, 25%), 562.6* (A2-+cat+, 100%), 11B-NMR (CD3CN, ppm): -0.78 (1B, B1), -9.29 (5B, B2-6), -10.32 (5B, B7-11), -12.40 (1B, B12).
O-(2-oxopropyl)-oxy-undecahydro-closo-dodecaborate(2-) bis-cesium salt (11).
Chloroacetone (0.25 g, 2.7 mmol) and 0.7g (5 mmol) of K2CO3 were added to solution of 0.8 g (1.25 mmol) of bis-tetrabutylammonium salt 9 in 30 ml of acetone. The reaction mixture was refluxed during 3 days, then cooled to room temperature and filtered. 11 was isolated as cesium salt by addition of 0.38 g (2.5 mmol) of CsF in methanol to the filtrate followed by the recrystallization of the precipitate from water. Yield: 68.4% (0.41 g), mp>250°C, HPLC 4.23 min, 1H-NMR (D2O, ppm): 4.30 (2H, s, -O-CH2COCH3), 2.13 (3H, s, -O-CH2COCH3), 2.3-(-0.4) (broad, B12H11), IR (cm-1): 3564(w), 3436(m), 2480(s) (B-H), 1731(m) (C=Oketon), 1411(w), 1385(w), 1352(w), 1199(w), 1153(m), 1019(m), 897(w), 723(w), 685(w), 612(w), MS: (pos.
ESI, H2O) 132.9 (cat+, 100%), (neg. ESI, H2O), 79.3* (B12H11OH2-/2, 10%), 107.2* (A2-/2, 100%), 141.0* (B12H11-, 10%), 347.2* (A2-+Cs+, 10%), 11B-NMR (D2O, ppm): 6.58 (1B, B1), -17.24 (10B, B2-7), -21.79 (1B, B12).
O-(5-amino-5-carboxypentyl)-oxy-undecahydro-closo-dodecaborate(2-) bis-tetraphenylphosphonium salt (12). Bis-cesium salt of 10 (0.53 g, 0.9 mmol) was suspended in 10 ml of 6M NaOH. During heating the salt dissolved. Hydrolysis was carried out for 2 days (HPLC control). The cold reaction mixture was neutralized. Ph4PBr 0.79 g (1.8 mmol) in 20 ml of water was added. The precipitated amino acid was filtered and washed with water.
Recrystallization from methanol gave 0.46 g of compound 12. Yield: 53%, mp 230-232°C, HPLC 3.52 min, 1H-NMR (DMSO, ppm): 7.91-7.66 (40H, m, ((C6H5)4P+)2), 3.30 (1H, t, -O-CH2CH2CH2CH2CH(COOH)(NH2), 3.00 (2H, m, -O-CH2CH2CH2CH2CH(COOH)(NH2)), 1.78-1.30 (6H, m, -O-CH2CH2CH2CH2CH(COOH)(NH2)), 2.3-(-0.7) (broad, B12H11), IR (cm-1):
3434(s), 3055-2849(w) (-NH3+), 2467(s) (B-H), 1634 (w, br, NH3+, COO-), 1585 (w) (CHarom), 1484(m) ((C6H5)4P+)2), 1336(w),1108(m) ((C6H5)4P+)2), 1047(w), 1021(w), 996(w), 899(w), 756(w), 722(m) ((C6H5)4P+)2), 689(m), 668(w), 527(m) ((C6H5)4P+)2), MS: (pos. ESI, CH3CN) 339.0 (cat+, 100%), (neg. ESI, CH3CN) 79.3* (B12H11OH2-/2, 60%), 141.0* (B12H11-, 50%), 626.8* (A2-+cat+, 100%), 11B-NMR (DMSO, ppm): -5.77 (1B, B1), -14.92 (5B, B2-6), -16.93 (5B, B7-11), -18.91 (1B, B12).
O-(5-amino-5-carboxylpentyl)-oxy-undecahydro-closo-dodecaborate(2-) bis-cesium salt (12a). 0.46 g (0.5 mmol) of 12 was dissolved in 5 ml of methanol. CsF 0.16 g (1.03 mmol) was dissolved in 3 ml of methanol and added to stirring solution of 12. The precipitated compound
12a was filtered off and washed with 5 ml of methanol. Recrystallization from water gave 0.25 g of 12a. Yield: 92.5%, MS: (pos. ESI, H2O) 133.0 (cat+, 100%), 556.9* (A2-+2cat++H+, 5%), 686.0* (A2-+3cat+, 5%), (neg. ESI, H2O) 87.1* (B12H11SH2-/2, 100%), 141.0* (B12H11-, 30%), 408.6* (A2-+cat+, 20%).
5-(Methylene-oxy-undecahydro-closo-dodecaboratyl)-5-methylhydantoin (2-) bis-cesium salt (13).A mixture of compound 11 (2.03 g, 4.2 mmol), 50% aqueous ethanol (45 ml), sodium cyanide (0.41 g, 8.37 mmol) and ammonium carbonate (2.1 g, 21.8 mmol) were placed in an autoclave and kept at 60°C for 4 hour. The mixture was acidified with 6M HCl and filtered through a pad of activated carbon. Solvents were removed by evaporation at room temperature, and the solid material which remained was recrystallized from water. Yield: 64% (1.14 g), mp>250°C, HPLC 3.96 min, 1H-NMR (D2O, ppm): 3.63 (2H, m, -O-CH2C), 1.19 (3H, s, CCH3), 2.6-(-0.4) (broad, B12H11), IR (cm-1): 3383(w), 3144(m) (-NH-), 3074(w), 2492(s) (B-H), 1765(m) (COhydant), 1723(s) (COhydant), 1630(w), 1407(s), 1315(w), 1260(w), 1168(m), 1075(w), 1021(w), 907(w), 726(w), 592(w), MS: (pos. ESI, H2O) 133.0* (cat+, 100%), 682.1* (A2-+3cat+, 5%), (neg. ESI, H2O) 79.3* (B12H11OH2-/2, 15%), 141.0* (B12H11-, 20%), 142.3* (A2-/2, 100%), 416.2* (A2-+cat+, 10%), 11B-NMR (DMSO, ppm): 6.69 (1B, B1), -16.83 (5B, B2-6), -17.86 (5B, B7-11), -22.49 (1B, B12).
O-(2-amino-2-carboxylpropyl)-oxy-undecahydro-closo-dodecaborate(2-) bis-tetraphenylphosphonium salt (14). Bis-cesium salt of 13 (1.1 g, 2.0 mmol) was suspended in 20 ml of 6M NaOH. During heating the salt dissolved. Hydrolysis was carried out for 24 hours (HPLC control). After cooling, 6M HCl was added to pH=7. Ph4PBr (1.68 g, 4.0 mmol) in 30 ml of water was added. Precipitated amino acid was filtered and washed with water.
Recrystallization from methanol gave 1.2 g of compound 14. Yeld: 64%, mp>250°C, HPLC 3.88 min, 1H-NMR (DMSO, ppm): 7.98-7.66 (40H, m, ((C6H5)4P+)2), 3.72-3.30 (2H, m, -O-CH2-C-), 1.14 (1H, s, -C-CH3), 2.30-(-0.50) (broad, B12H11), IR (cm-1): 3421(m), 3060(w) (NH3+), 2472(s) (B-H), 1618(m) (br, NH3+, COO-), 1585(m), 1483(m), 1436 ((C6H5)4P+)2), 1145(m), 1108(s) ((C6H5)4P+)2), 1049(w), 1017(w), 996(w), 759(w), 722(m) ((C6H5)4P+)2), 689(m), 527(s) ((C6H5)4P+)2), MS: (pos. ESI, CH3CN) 339.0 (cat+, 100%), (neg. ESI, CH3CN) 79.0*
(B12H11OH2-/2, 10%), 141.0* (B12H11-, 3%), 497.6* (B12H11OH2-+cat+, 5%), 553.6* (A2-+cat+ -COOH, 30%), 598.7* (A2-+cat+, 100%), 11B-NMR (DMSO, ppm): 6.96 (1B, B1), -16.57 (5B, B2-6), -17.92 (5B, B7-11), -22.61 (1B, B12).
Toxicity test
8000 V 79 Chinese hamster cells per well was cultivated in a 96-well-plate and incubated for 24 hours at 37°C and 5% CO2.Different concentrations of the substances were added: 0.78*10-4 -0.01 M of compound 8a and 0.78*10-4-0.1 M of compound 12a. Three and six wells, resp., were
used for each concentration. Each of the compounds was incubated with the cells for 18 hours at 37°C. At the end of the incubation time, the old medium was removed and 100 µl of fresh medium per well was added. Ten µl of diluted WST-1 reagent per well was added and incubated for 4 hours at 37°C. The absorbance was measured using Plate Reader MRX: Dynatech Laboratories. The wavelength for measurements was 630 nm.
Acknowledgment. This research was supported by stipend to I.S. from the Ernst A. C. Lange Foundation.
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