Bioorganic & Medicinal Chemistry 22 (2014) 3906–3912
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis, reactivity and biological activity of N(4)-boronated derivatives of 20 -deoxycytidine Joanna Nizioł a, Zbigniew Zielin´ski b, Andrzej Les´ c,d, Magdalena Da˛browska b, Wojciech Rode b, Tomasz Ruman a,⇑ a
´ ców Warszawy Ave., 35-959 Rzeszów, Poland Rzeszów University of Technology, Faculty of Chemistry, Bioorganic Chemistry Laboratory, 6 Powstan Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland University of Warsaw, Faculty of Chemistry, Quantum Chemistry Laboratory, 1 Pasteur Street, Warsaw 02-093, Poland d Pharmaceutical Research Institute, Rydygier Street 8, Warsaw 01-793, Poland b c
a r t i c l e
i n f o
Article history: Received 30 April 2014 Revised 2 June 2014 Accepted 6 June 2014 Available online 16 June 2014 Keywords: Boron nucleoside Boron nucleotide NMR Solvolysis In vivo phosphorylation
a b s t r a c t By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp3 hybridization. The latter allowed successful modification of deoxycytidine in the reaction with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane of the deoxynucleoside amino group. For new compounds, detailed NMR, LDI HRMS (Laser Desorption/Ionization High-Resolution Mass Spectrometry) analyses along with in vivo phosphorylation studies, toxicity assays and DFT modelling are presented. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Boron analogues and derivatives of biologically active compounds are gaining increased attention in the last years.1–4 Being often isosteric and isoelectronic to carbon-based, natural compounds they show unique properties, for example a capability to form tetrahedral sp3 hybridized boron ate-complexes. The latter results from coordination of a biomolecule electron pair to the boron atom. The ate-complexes formation results in effective inhibition of the target enzyme.5–7 Additionally, boron-containing compounds may be useful in boron-neutron capture therapy (BNCT).8–11 The field of boron derivatives/analogues of nucleosides and nucleotides has been recently reviewed.12,13 Beside several sugar-substituted boron derivatives of nucleosides/nucleotides, only a few nucleobase-substituted derivatives are known. The latter category includes cyanoborane adducts, containing the N-BH2CN moiety, of 20 -deoxyinosine, 20 -deoxyguanosine, 20 -deoxyadenosine and 20 -deoxycytidine,14 and borane complexes with CN or COOMe groups at boron atom.15 An important achievement in the boron nucleoside field was synthesis of 5-dihydroxyboryl (C(5)-B(OH)2) derivatives of uracil and uridine by Schinazi et al.16 So far, the only known boron nucleoside analogue is the benzoborauracil ⇑ Corresponding author. E-mail address: [email protected] (T. Ruman). http://dx.doi.org/10.1016/j.bmc.2014.06.014 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
nucleoside 4-[5-O-(tert-butyldimethylsilyl)-2,3-O-disopropylidene-D-ribofuranosyl]-1-hydroxy-2-methyl-1H-2,4,1-benzodiazaborin-3-one presented by Zhuo et al.17 By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents and/or thymidylate synthase (EC 2.1.1.45) inhibitors, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp3 hybridization. 2. Results and discussion The search for stable boron nucleosides led us to the conclusion that boron should be attached to nucleoside via a single CAB bond, where C is an aliphatic carbon atom of sp3 hybridization. In boron chemistry bonds of this type are known to be the most stable, being resistant to deboronation.25 The synthetic route applied in the present work (Fig. 1) applies commercially available 2-(iodomethyl)4,4,5,5-tetramethyl-1,3,2-dioxaborolane (PinBCH2I) undergoing reaction with amino group of deoxycytidine (dCyd). The described synthetic path shows a considerable potential due to high yield, relatively mild conditions and high purity of the crude product. This approach appears the most effective for synthesis of boroncontaining nucleosides and should be useful for boronation of other nucleosides containing amino group(s).
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J. Nizioł et al. / Bioorg. Med. Chem. 22 (2014) 3906–3912 NH2
A O
N
O
HN B
N
N O
PinBCH2I K2CO 3, DMSO
N
O
HO
O O
HO HO
HO
dC
1 O
B
O
HN
O
N
HO
N+ DO O
N D + D 2O - D2O
HO
B-
+
O O
D
N DO O
B N
O
B-
D
O
N
N D
O
O
DO
N
O
DO DO
DO
1 1. D2O Figure 1. Synthesis of 1 (A) and NAB coordinated (1)/solvated (1D2O) species equilibrium in deuterium oxide (B).
2.1. NMR studies The product of condensation of dCyd and PinBCH2I, N(4)-[B(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidine (1), shown in Figure 1, is believed to form an additional 5-membered ring in which 3-nitrogen atom donates lone pair of electrons to the boron atom, as it has been shown in other similar structures.22,23 The modification reaction did not occur in N(3) position as similar reaction did not occur fir dU or dT nucleosides. The 1H and 11B NMR spectra of 1 are shown in Figure 2 D-1 and B-1, respectively. The 11B NMR chemical shift of 1 in deuterium oxide was in 7–8 ppm range (Fig. 2, B-1), strongly suggesting a relatively high electron density on boron atom that most probably is negatively charged. There is no evidence of boron trigonal (sp2) species existence in water solution, as apparent with the absence of 11B NMR chemical shifts are in the 18–40 ppm range.21–25 Mentioned structure have negatively charged boron moiety due to OD ion attached to boron due to the solvolysis process. The process of water/heavy water ionization is most probably a concerted reaction in which hydrogen-bonded water dissociation and formation of new (NAH/NAD and BAO) bonds proceed in one fast step. Similar structures known from literature proved to behave in a similar way in protic solvents.22,23 The pinacol moiety methyl groups in 1D2O are in the form of two resonances of 6H+6H pattern as shown in 1H NMR spectrum. The latter pattern is due to lack of up/down symmetry of tetrahedral boron surroundings, considering hypothetical O,O,B plane based on PinB moiety. It should be noted that theoretical 1 structure containing coordinative N(3)AB bond should present 3H+3H+3H+3H pinacol methyl resonance pattern due to lack of up/down and left/right symmetry. It is reasonable to assume that in water solution, solvated compound (1D2O) may be represented by two resonance structures, characterized by either C(4)AN single bond (amino structure) or C(4)@N double bond (imino structure). The estimation of amino/imino equilibrium made with the aid of DFT calculations suggest that the imino structure is strongly dominating (vide infra; chapter 3.3). Above mentioned solvolysis process produces negatively charged boron with OD ion and deuterium counter-cation attached to N(3) nitrogen in the amino form and to N(4) in the imino structure (1D2O in Fig. 1B).
In order to estimate coordination equilibrium and stability of 1D2O, NMR-controlled experiments were conducted. The problem of boron coordination equilibrium is of great importance because all boron atom-containing biologically active compounds studied proved to form coordinate or even covalent bonds between boron and their molecular targets. Compound 1D2O may be considered relatively stable as there was no noticeable change in 1H NMR spectrum following a few days incubation in deuterium oxide. Literature contain examples of synthesis a-aminoboronic acids and their esters, but unfortunately most of them undergo a spontaneous B-to-N migration.24–26 The stability of 1D2O is certainly linked with (i) steric hindrance of proximal nucleoside and (ii) dominating imino resonance structure. The latter electronic effect may suggest that nitrogen atom is of relatively low basicity for B-N migration to occur. The NMR results of an experiment monitoring coordination of an aromatic base, pyridine, are shown in Figure 2A–D, spectra no. 4. There are small changes in NMR spectra observed, but most importantly, pinacol methyl resonances show their starting 6H+6H pattern. There are almost no 11B resonance chemical shift differences, even with a 22-fold molar excess of py (calcd for 1). It may be concluded that either pyridine does not take part in ionization or its influence on ionization by attaching D+ is not fast enough, compared to ionization by the N(3)-B system. NMR experiment with the use of acetic acid gave unexpected results. The pinacol methyl moiety resonance of 1D2O ca. 10 min after addition of 2.8 mol equiv of AcOH was found in the form of a single 12H peak (Fig. 2C-2). The 11B NMR resonance was found to be at 5.1 ppm suggesting higher electron density at boron atom, compared to the starting form. Moreover, an additional resonance of low-intensity at 19.8 ppm appeared in position typical for boric acid.27,28 The single 1H NMR pinacol methyl resonance pattern and also chemical shift of discussed resonance (1.15 ppm) suggested the presence of free pinacol alcohol which was a product of acidcatalyzed hydrolysis. Similar process in which proximal nitrogen facilitate hydrolysis was described by Santos group.29 The proposed equilibria of the processes are shown in Figure 3A. Upon addition of acetic acid, the starting 1D2O was transformed into its hydrolyzed and solvated form 2D2O, with 11B NMR resonance chemical shift in a very good agreement with similar systems
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J. Nizioł et al. / Bioorg. Med. Chem. 22 (2014) 3906–3912
4
A
B
4
C
7.7
3
3
19.8
2
2
19.8
1
1
4
5.2
3
5.1
2
7.7
1
D 4
3
2
1
H(1’)
H(6)
H(5)
H(4’)
H(3’)
H(5’)
BCH2
H(2’)
Me
Figure 2. NMR spectra of 1 in D2O (D-1) and also 1 titrated with acetic acid (D-2), N-acetylcysteine methyl ester (D-3) and pyridine (D-4). Inserts at the top of figure contain magnified 1H NMR spectra fragments with H(6) resonances (A 1–4), pinacol moiety methyl resonances (C 1–4) and 11B NMR spectra (B 1–4) for 1 and above mentioned titration experiments.
A
D
+
N
N
O
B-
DO O
D
N D
+AcOH - (Me 2COH) 2
O
-AcOH +(Me 2COH) 2
+
OAc
B-
D
N DO OD
N
N DO OD
N H
+D 2O -AcOH
O
-D2O +AcOH
D
+
N
N
N
B-
O
R
2.D2O
1.D2O
B
N D
R
R
OD
B-
+
O
DO O
D
+
N
N D
R'SH - (Me 2COH) 2
O
-R'SH +(Me 2COH) 2
N R
R
1.D2O
SR'
D
+
N
DO OD N H
+D 2O -R'SH
O
-D2O +R'SH
B-
OD
DO OD N D
N
O
R
2. D2O
SH
R'SH =
B-
COOMe NHAc
Figure 3. Acetic acid- and thiol (R0 SH)-catalyzed hydrolysis of 1D2O pinacol fragment. R = 2-deoxyribose moiety.
containing negatively charged tetrahedral C-B(OH)3 moiety.22,23,31 These results was rather surprising as pinacol-protected boronic esters belong to the most stable forms of protection of boronic acids. However, the hydrolysis of pinacol moiety into pinacol alcohol was few times mentioned in literature.21,30,32,33
The study of interactions of cysteine side chain with boron atom was of great interest because of possible S-B coordination of potential inhibitor 1 inside the active centre of thymidylate synthase, the enzyme being an important molecular target in anticancer, antivirus, antifungal and antiprotozoal chemotherapy.34–37
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J. Nizioł et al. / Bioorg. Med. Chem. 22 (2014) 3906–3912
The N,C-protected cysteine N-acetylcysteine methyl ester was chosen as a model of thymidylate synthase active centre catalytic Cys (e.g., Cys198 in human thymidylate synthase). The resulting 1H and 11B NMR spectra are shown in Figure 2, panels D-3 and B-3, respectively. Based on the NMR data, addition of protected cysteine resulted in hydrolysis of pinacol moiety, yielding products very similar to those resulting from acetic acid-catalyzed hydrolysis (Fig. 3B). As in the acetic acid hydrolysis experiment, an additional peak of boric acid (19.8 ppm) in 11B NMR spectrum was also found. The observed cysteine side chain hydrolytic activity results apparently from a relatively high SH group acidity, reflected by the pKa value of ca. 6.0–6.5, but also from a very high nucleophilicity of thiolate anion.38 Of note are potential very significant consequences of the latter hydrolytic activity. In particular, the hydrolytic cleavage of pinacol catalyzed by amino acid side chain, results in formation of boronic acid derivative characterized by a considerably lower steric hindrance caused by boron atom surroundings, thus probably facilitating formation of a protein–boronic acid ate-complex. Moreover, pinacol moiety may be considered a hydrophobic anchor, removable by the target enzyme that greatly enhances cell wall transport of the nucleoside derivative.
extracted with 60% aqueous methanol at 20 °C as previously described.39 Supernatant diluted 10-times with ethanol was analyzed with the use of high-resolution laser desorption/ionization mass spectrometry on 109AgNPET plate.40 Spectra obtained from two independent MS experiments are shown in Figure 4. The detailed MS data is also shown in Table 1. The results obtained in reflectron-mode positive ionization LDI MS experiments demonstrate the presence in the extract of 1 and its mono-, di- and triphosphates. Of note is that all of the ion formulas shown in Table 1 are having Dm/z values lower than 10 ppm, with majority of them (7 out of 9) lower than 5 ppm. Thus, at this level of mass determination accuracy, the results leave no doubt that 1 enters the cells and undergoes a series of phosphorylations up to the triphosphate level, although the results allow no quantitative assessment of either the compound’s cellular uptake or phosphorylation. Nevertheless, intracellular nucleotide formation may positively influence uptake by tumor cells and cause retention of 1,39 pointing to the need of further studies of both phenomena, as well as of potential incorporation of 1 to DNA. Integration with DNA or location of the boron compound in close proximity to the nucleus is extremely desirable for BNCT, as the required cellular boron concentration has been assessed 2 to 5-fold lower with the boron compound located in the nuclear or perinuclear region. It should be pointed out that the above results provide a further example of our 109AgNPET method’s high potential in analysis of biological material.40
2.2. Biological activity studies In order to test cytotoxic activity of 1, the compound was used to inhibit human colorectal adenocarcinoma C85 cell growth and found to cause a rather weak inhibition, reflected by the EC50 value of 6.2 ± 2.3 mM (a mean result ± % difference between the mean and each of the two results). The latter appears to be in accord with the requirement for a boron delivery agent to be used in BNCT to exhibit low toxicity.7 Further studies are needed to verify whether the toxicity is low also with normal cells. Potential uptake and phosphorylation of 1 by cultured tumor cells was also tested. C85 cells exposed to the 1 for 48 h were
A
571
572
573
574
E
Intens. [a.u.]
732.5
x105 5
B
574.0754
575
576
577
m/z
733.5
734.0
734.5
735.0
591.4
735.5
m/z
In order to estimate electronic structure of substituted amino group and its surrounding in 1D2O and 2D2O, DFT calculations were performed. The essential informations from DFT calculations are shown in Table 2. It is clear, that C(4)AN(4) bond lengths of 1D2O and 2D2O are very similar being approx. 1.3 Å. The typical
591.6
591.8
592.0
592.2
592.4
737.0
737.5
738.0
738.5
696.0261
652.0637
592.6
592.8
593.0
m/z
651.0
651.5
652.0
652.5
G
737.9758
736.5
D
C
592.1070
F
733.9800
733.0
591.2
2.3. DFT calculations
653.0
653.5
694.0
m/z
694.5
695.0
695.5
696.0
696.5
697.0
697.5
698.0
m/z
H
590.1066
809.9145
739.0
739.5
740.0 m/z
586
587
588
589
590
591
592
593
594
595
596 m/z
808.0
808.5
809.0
I
J
809.5
810.0
810.5
811.0
811.5
812.0 m/z
696.0118
4 3 692
694
696
698
700
702
m/z
2 1 0
100
200
300
400
500
600
700
Figure 4. An overlay of two (violet and black lines) independent LDI MS spectra of 1-exposed tumor cell extract analyzed on magnified fragments of both spectra.
800 109
900
m/z
AgNPET (bottom, J). Inserts A–I presents
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J. Nizioł et al. / Bioorg. Med. Chem. 22 (2014) 3906–3912
Table 1 HRMS data for tumor cell extract analyzed on Compound 1-Monophosphate 1-Diphosphate 1-Triphosphate
1
109
AgNPET
Ion formula +
[C16H25BN3O9PNaK2+EtOH] [C16H27BN3O9P109Ag+H2O]+ [C16H27BN3O12P2Na2+H2O]+ [C16H25BN3O12P2NaK3+MeOH]+ [C16H27BN3O15P3K2109Ag+H2O]+ [C16H28BN3O15P3Na2]+ [C16H28BN3O15P3109AgNa]+ [C16H26BN3O15P3Na4]+ [C16H25BN3O15P3Na4K]+
m/zcalcd
m/zexp
Dm/z1
Dm/z (ppm)
592.1015 574.0734 590.1067 696.0081 809.9178 652.0625 737.9775 696.0264 733.9822
592.1070 574.0754 590.1066 696.0118 809.9145 652.0637 737.9758 696.0252 733.9800
0.0055 0.0020 0.0001 0.0037 0.0033 0.0012 0.0017 0.0012 0.0022
9.3 3.5 0.2 5.3 4.1 1.8 2.3 1.7 3.0
Dm/z = m/zcalcd m/zexp; m/zcalcd m/zcalcd; m/zexp m/z found (experimental).
4. Experimental section
Table 2 Calculated CAN bond lengths of 1D2O and 2D2O Compound
Bond
Length (Å)
1D2O
C(4)AN(4)
1.299 1.473
2D2O
N(4)ACH2 N(3)AC(4) N(3)AC(2) N(1)A(C10 )
1.385 1.390 1.373
C(4)AN(4) N(4)ACH2 N(3)AC(4) N(3)AC(2) N(1)AC(10 )
1.300 1.469 1.387 1.390 1.375
CAN single bond length of aliphatic systems is in 1.4–1.5 Å range, which suggest that a substantially shorter C(4)AN(4) bond of 1D2O and 2D2O should be considered rather double bond. It is reasonable to state that this unusual electronic configuration is connected with protonation of nucleobase ring and also water ionization. Thus, in both 1D2O and 2D2O structures, free of interactions with other molecules, the C(4)-imino structure appears energetically favored. The latter is of interest, considering potential interaction of the corresponding nucleotide with thymidylate synthase. Namely the enzyme’s active center discriminates between the substrate, dUMP, and dCMP by the mechanism involving an active center asparagines residue hydrogen-bonding to dUMP C(4)@O and non-dissociated N(3)AH groups41–43, and in dUMP analogues the C(4)@O group may be imitated by the C(4)@NA group.44,45 The latter group is found in the C(4)-imino structure of 2D2O that in solution apparently exists in equilibrium with the amino structure (C(4)ANA). 3. Conclusions The synthetic route applied to synthesize N(4)-[B-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidine (1) shows a considerable potential due to high yield, relatively mild conditions and high purity of the crude product. NMR investigation suggested the new compound to be in zwitterionic form in heavy water (1D2O), having OD anion attached to boron atom and D+ to the proximal nucleobase-N(3) nitrogen. Hydrolysis of the pinacol moiety was observed under very mild conditions upon addition of small amounts of acetic acid or protected cysteine. The product of hydrolysis (2D2O) shows much lesser steric hindrance around boron atom, the latter causing an important influence on boron coordination properties. Potential uptake and phosphorylation of 1 by cultured tumor cells was also tested. The results obtained in LDI MS experiments showed the presence of in vivo-synthesized mono-, di- and triphosphates of 1.
4.1. Materials and methods 1 H NMR spectra were obtained with the Bruker Avance II spectrometer, operating in the quadrature mode at 500 MHz. All 11B spectra were performed using 5 mm pure quartz NMR tube. The residual peaks of deuterated solvents were used as internal standards. Reagents and deuterated solvents of the highest commercially available grade were purchased from Aldrich. DMSO was dried by vacuum distillation over anhydrous magnesium sulfate. All procedures, including preparation of samples for the NMR measurements, were carried out under nitrogen. All reagents, with the exception of boranes, were dried by triple aseotropic distillation from deuterated chloroform. High-resolution COSY spectra were prepared using 4096 4096 measurement points. 11B NMR spectra were referenced to external BF3OEt2 sample (0.0 ppm). All 1H NMR experiments were made with HDO-suppression method applied. LDI time-of-flight (TOF) mass spectrometry experiments were performed using a Bruker Autoflex Speed reflectron time-of-flight mass spectrometer, equipped with a SmartBeam II laser (352 nm). The laser impulse energy was approximately 60– 120 lJ, the laser repetition rate was 1000 Hz, and the deflection value was set on m/z
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis, reactivity and biological activity of N(4)-boronated derivatives of 20 -deoxycytidine Joanna Nizioł a, Zbigniew Zielin´ski b, Andrzej Les´ c,d, Magdalena Da˛browska b, Wojciech Rode b, Tomasz Ruman a,⇑ a
´ ców Warszawy Ave., 35-959 Rzeszów, Poland Rzeszów University of Technology, Faculty of Chemistry, Bioorganic Chemistry Laboratory, 6 Powstan Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland University of Warsaw, Faculty of Chemistry, Quantum Chemistry Laboratory, 1 Pasteur Street, Warsaw 02-093, Poland d Pharmaceutical Research Institute, Rydygier Street 8, Warsaw 01-793, Poland b c
a r t i c l e
i n f o
Article history: Received 30 April 2014 Revised 2 June 2014 Accepted 6 June 2014 Available online 16 June 2014 Keywords: Boron nucleoside Boron nucleotide NMR Solvolysis In vivo phosphorylation
a b s t r a c t By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp3 hybridization. The latter allowed successful modification of deoxycytidine in the reaction with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane of the deoxynucleoside amino group. For new compounds, detailed NMR, LDI HRMS (Laser Desorption/Ionization High-Resolution Mass Spectrometry) analyses along with in vivo phosphorylation studies, toxicity assays and DFT modelling are presented. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Boron analogues and derivatives of biologically active compounds are gaining increased attention in the last years.1–4 Being often isosteric and isoelectronic to carbon-based, natural compounds they show unique properties, for example a capability to form tetrahedral sp3 hybridized boron ate-complexes. The latter results from coordination of a biomolecule electron pair to the boron atom. The ate-complexes formation results in effective inhibition of the target enzyme.5–7 Additionally, boron-containing compounds may be useful in boron-neutron capture therapy (BNCT).8–11 The field of boron derivatives/analogues of nucleosides and nucleotides has been recently reviewed.12,13 Beside several sugar-substituted boron derivatives of nucleosides/nucleotides, only a few nucleobase-substituted derivatives are known. The latter category includes cyanoborane adducts, containing the N-BH2CN moiety, of 20 -deoxyinosine, 20 -deoxyguanosine, 20 -deoxyadenosine and 20 -deoxycytidine,14 and borane complexes with CN or COOMe groups at boron atom.15 An important achievement in the boron nucleoside field was synthesis of 5-dihydroxyboryl (C(5)-B(OH)2) derivatives of uracil and uridine by Schinazi et al.16 So far, the only known boron nucleoside analogue is the benzoborauracil ⇑ Corresponding author. E-mail address: [email protected] (T. Ruman). http://dx.doi.org/10.1016/j.bmc.2014.06.014 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
nucleoside 4-[5-O-(tert-butyldimethylsilyl)-2,3-O-disopropylidene-D-ribofuranosyl]-1-hydroxy-2-methyl-1H-2,4,1-benzodiazaborin-3-one presented by Zhuo et al.17 By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents and/or thymidylate synthase (EC 2.1.1.45) inhibitors, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp3 hybridization. 2. Results and discussion The search for stable boron nucleosides led us to the conclusion that boron should be attached to nucleoside via a single CAB bond, where C is an aliphatic carbon atom of sp3 hybridization. In boron chemistry bonds of this type are known to be the most stable, being resistant to deboronation.25 The synthetic route applied in the present work (Fig. 1) applies commercially available 2-(iodomethyl)4,4,5,5-tetramethyl-1,3,2-dioxaborolane (PinBCH2I) undergoing reaction with amino group of deoxycytidine (dCyd). The described synthetic path shows a considerable potential due to high yield, relatively mild conditions and high purity of the crude product. This approach appears the most effective for synthesis of boroncontaining nucleosides and should be useful for boronation of other nucleosides containing amino group(s).
3907
J. Nizioł et al. / Bioorg. Med. Chem. 22 (2014) 3906–3912 NH2
A O
N
O
HN B
N
N O
PinBCH2I K2CO 3, DMSO
N
O
HO
O O
HO HO
HO
dC
1 O
B
O
HN
O
N
HO
N+ DO O
N D + D 2O - D2O
HO
B-
+
O O
D
N DO O
B N
O
B-
D
O
N
N D
O
O
DO
N
O
DO DO
DO
1 1. D2O Figure 1. Synthesis of 1 (A) and NAB coordinated (1)/solvated (1D2O) species equilibrium in deuterium oxide (B).
2.1. NMR studies The product of condensation of dCyd and PinBCH2I, N(4)-[B(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidine (1), shown in Figure 1, is believed to form an additional 5-membered ring in which 3-nitrogen atom donates lone pair of electrons to the boron atom, as it has been shown in other similar structures.22,23 The modification reaction did not occur in N(3) position as similar reaction did not occur fir dU or dT nucleosides. The 1H and 11B NMR spectra of 1 are shown in Figure 2 D-1 and B-1, respectively. The 11B NMR chemical shift of 1 in deuterium oxide was in 7–8 ppm range (Fig. 2, B-1), strongly suggesting a relatively high electron density on boron atom that most probably is negatively charged. There is no evidence of boron trigonal (sp2) species existence in water solution, as apparent with the absence of 11B NMR chemical shifts are in the 18–40 ppm range.21–25 Mentioned structure have negatively charged boron moiety due to OD ion attached to boron due to the solvolysis process. The process of water/heavy water ionization is most probably a concerted reaction in which hydrogen-bonded water dissociation and formation of new (NAH/NAD and BAO) bonds proceed in one fast step. Similar structures known from literature proved to behave in a similar way in protic solvents.22,23 The pinacol moiety methyl groups in 1D2O are in the form of two resonances of 6H+6H pattern as shown in 1H NMR spectrum. The latter pattern is due to lack of up/down symmetry of tetrahedral boron surroundings, considering hypothetical O,O,B plane based on PinB moiety. It should be noted that theoretical 1 structure containing coordinative N(3)AB bond should present 3H+3H+3H+3H pinacol methyl resonance pattern due to lack of up/down and left/right symmetry. It is reasonable to assume that in water solution, solvated compound (1D2O) may be represented by two resonance structures, characterized by either C(4)AN single bond (amino structure) or C(4)@N double bond (imino structure). The estimation of amino/imino equilibrium made with the aid of DFT calculations suggest that the imino structure is strongly dominating (vide infra; chapter 3.3). Above mentioned solvolysis process produces negatively charged boron with OD ion and deuterium counter-cation attached to N(3) nitrogen in the amino form and to N(4) in the imino structure (1D2O in Fig. 1B).
In order to estimate coordination equilibrium and stability of 1D2O, NMR-controlled experiments were conducted. The problem of boron coordination equilibrium is of great importance because all boron atom-containing biologically active compounds studied proved to form coordinate or even covalent bonds between boron and their molecular targets. Compound 1D2O may be considered relatively stable as there was no noticeable change in 1H NMR spectrum following a few days incubation in deuterium oxide. Literature contain examples of synthesis a-aminoboronic acids and their esters, but unfortunately most of them undergo a spontaneous B-to-N migration.24–26 The stability of 1D2O is certainly linked with (i) steric hindrance of proximal nucleoside and (ii) dominating imino resonance structure. The latter electronic effect may suggest that nitrogen atom is of relatively low basicity for B-N migration to occur. The NMR results of an experiment monitoring coordination of an aromatic base, pyridine, are shown in Figure 2A–D, spectra no. 4. There are small changes in NMR spectra observed, but most importantly, pinacol methyl resonances show their starting 6H+6H pattern. There are almost no 11B resonance chemical shift differences, even with a 22-fold molar excess of py (calcd for 1). It may be concluded that either pyridine does not take part in ionization or its influence on ionization by attaching D+ is not fast enough, compared to ionization by the N(3)-B system. NMR experiment with the use of acetic acid gave unexpected results. The pinacol methyl moiety resonance of 1D2O ca. 10 min after addition of 2.8 mol equiv of AcOH was found in the form of a single 12H peak (Fig. 2C-2). The 11B NMR resonance was found to be at 5.1 ppm suggesting higher electron density at boron atom, compared to the starting form. Moreover, an additional resonance of low-intensity at 19.8 ppm appeared in position typical for boric acid.27,28 The single 1H NMR pinacol methyl resonance pattern and also chemical shift of discussed resonance (1.15 ppm) suggested the presence of free pinacol alcohol which was a product of acidcatalyzed hydrolysis. Similar process in which proximal nitrogen facilitate hydrolysis was described by Santos group.29 The proposed equilibria of the processes are shown in Figure 3A. Upon addition of acetic acid, the starting 1D2O was transformed into its hydrolyzed and solvated form 2D2O, with 11B NMR resonance chemical shift in a very good agreement with similar systems
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4
A
B
4
C
7.7
3
3
19.8
2
2
19.8
1
1
4
5.2
3
5.1
2
7.7
1
D 4
3
2
1
H(1’)
H(6)
H(5)
H(4’)
H(3’)
H(5’)
BCH2
H(2’)
Me
Figure 2. NMR spectra of 1 in D2O (D-1) and also 1 titrated with acetic acid (D-2), N-acetylcysteine methyl ester (D-3) and pyridine (D-4). Inserts at the top of figure contain magnified 1H NMR spectra fragments with H(6) resonances (A 1–4), pinacol moiety methyl resonances (C 1–4) and 11B NMR spectra (B 1–4) for 1 and above mentioned titration experiments.
A
D
+
N
N
O
B-
DO O
D
N D
+AcOH - (Me 2COH) 2
O
-AcOH +(Me 2COH) 2
+
OAc
B-
D
N DO OD
N
N DO OD
N H
+D 2O -AcOH
O
-D2O +AcOH
D
+
N
N
N
B-
O
R
2.D2O
1.D2O
B
N D
R
R
OD
B-
+
O
DO O
D
+
N
N D
R'SH - (Me 2COH) 2
O
-R'SH +(Me 2COH) 2
N R
R
1.D2O
SR'
D
+
N
DO OD N H
+D 2O -R'SH
O
-D2O +R'SH
B-
OD
DO OD N D
N
O
R
2. D2O
SH
R'SH =
B-
COOMe NHAc
Figure 3. Acetic acid- and thiol (R0 SH)-catalyzed hydrolysis of 1D2O pinacol fragment. R = 2-deoxyribose moiety.
containing negatively charged tetrahedral C-B(OH)3 moiety.22,23,31 These results was rather surprising as pinacol-protected boronic esters belong to the most stable forms of protection of boronic acids. However, the hydrolysis of pinacol moiety into pinacol alcohol was few times mentioned in literature.21,30,32,33
The study of interactions of cysteine side chain with boron atom was of great interest because of possible S-B coordination of potential inhibitor 1 inside the active centre of thymidylate synthase, the enzyme being an important molecular target in anticancer, antivirus, antifungal and antiprotozoal chemotherapy.34–37
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The N,C-protected cysteine N-acetylcysteine methyl ester was chosen as a model of thymidylate synthase active centre catalytic Cys (e.g., Cys198 in human thymidylate synthase). The resulting 1H and 11B NMR spectra are shown in Figure 2, panels D-3 and B-3, respectively. Based on the NMR data, addition of protected cysteine resulted in hydrolysis of pinacol moiety, yielding products very similar to those resulting from acetic acid-catalyzed hydrolysis (Fig. 3B). As in the acetic acid hydrolysis experiment, an additional peak of boric acid (19.8 ppm) in 11B NMR spectrum was also found. The observed cysteine side chain hydrolytic activity results apparently from a relatively high SH group acidity, reflected by the pKa value of ca. 6.0–6.5, but also from a very high nucleophilicity of thiolate anion.38 Of note are potential very significant consequences of the latter hydrolytic activity. In particular, the hydrolytic cleavage of pinacol catalyzed by amino acid side chain, results in formation of boronic acid derivative characterized by a considerably lower steric hindrance caused by boron atom surroundings, thus probably facilitating formation of a protein–boronic acid ate-complex. Moreover, pinacol moiety may be considered a hydrophobic anchor, removable by the target enzyme that greatly enhances cell wall transport of the nucleoside derivative.
extracted with 60% aqueous methanol at 20 °C as previously described.39 Supernatant diluted 10-times with ethanol was analyzed with the use of high-resolution laser desorption/ionization mass spectrometry on 109AgNPET plate.40 Spectra obtained from two independent MS experiments are shown in Figure 4. The detailed MS data is also shown in Table 1. The results obtained in reflectron-mode positive ionization LDI MS experiments demonstrate the presence in the extract of 1 and its mono-, di- and triphosphates. Of note is that all of the ion formulas shown in Table 1 are having Dm/z values lower than 10 ppm, with majority of them (7 out of 9) lower than 5 ppm. Thus, at this level of mass determination accuracy, the results leave no doubt that 1 enters the cells and undergoes a series of phosphorylations up to the triphosphate level, although the results allow no quantitative assessment of either the compound’s cellular uptake or phosphorylation. Nevertheless, intracellular nucleotide formation may positively influence uptake by tumor cells and cause retention of 1,39 pointing to the need of further studies of both phenomena, as well as of potential incorporation of 1 to DNA. Integration with DNA or location of the boron compound in close proximity to the nucleus is extremely desirable for BNCT, as the required cellular boron concentration has been assessed 2 to 5-fold lower with the boron compound located in the nuclear or perinuclear region. It should be pointed out that the above results provide a further example of our 109AgNPET method’s high potential in analysis of biological material.40
2.2. Biological activity studies In order to test cytotoxic activity of 1, the compound was used to inhibit human colorectal adenocarcinoma C85 cell growth and found to cause a rather weak inhibition, reflected by the EC50 value of 6.2 ± 2.3 mM (a mean result ± % difference between the mean and each of the two results). The latter appears to be in accord with the requirement for a boron delivery agent to be used in BNCT to exhibit low toxicity.7 Further studies are needed to verify whether the toxicity is low also with normal cells. Potential uptake and phosphorylation of 1 by cultured tumor cells was also tested. C85 cells exposed to the 1 for 48 h were
A
571
572
573
574
E
Intens. [a.u.]
732.5
x105 5
B
574.0754
575
576
577
m/z
733.5
734.0
734.5
735.0
591.4
735.5
m/z
In order to estimate electronic structure of substituted amino group and its surrounding in 1D2O and 2D2O, DFT calculations were performed. The essential informations from DFT calculations are shown in Table 2. It is clear, that C(4)AN(4) bond lengths of 1D2O and 2D2O are very similar being approx. 1.3 Å. The typical
591.6
591.8
592.0
592.2
592.4
737.0
737.5
738.0
738.5
696.0261
652.0637
592.6
592.8
593.0
m/z
651.0
651.5
652.0
652.5
G
737.9758
736.5
D
C
592.1070
F
733.9800
733.0
591.2
2.3. DFT calculations
653.0
653.5
694.0
m/z
694.5
695.0
695.5
696.0
696.5
697.0
697.5
698.0
m/z
H
590.1066
809.9145
739.0
739.5
740.0 m/z
586
587
588
589
590
591
592
593
594
595
596 m/z
808.0
808.5
809.0
I
J
809.5
810.0
810.5
811.0
811.5
812.0 m/z
696.0118
4 3 692
694
696
698
700
702
m/z
2 1 0
100
200
300
400
500
600
700
Figure 4. An overlay of two (violet and black lines) independent LDI MS spectra of 1-exposed tumor cell extract analyzed on magnified fragments of both spectra.
800 109
900
m/z
AgNPET (bottom, J). Inserts A–I presents
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Table 1 HRMS data for tumor cell extract analyzed on Compound 1-Monophosphate 1-Diphosphate 1-Triphosphate
1
109
AgNPET
Ion formula +
[C16H25BN3O9PNaK2+EtOH] [C16H27BN3O9P109Ag+H2O]+ [C16H27BN3O12P2Na2+H2O]+ [C16H25BN3O12P2NaK3+MeOH]+ [C16H27BN3O15P3K2109Ag+H2O]+ [C16H28BN3O15P3Na2]+ [C16H28BN3O15P3109AgNa]+ [C16H26BN3O15P3Na4]+ [C16H25BN3O15P3Na4K]+
m/zcalcd
m/zexp
Dm/z1
Dm/z (ppm)
592.1015 574.0734 590.1067 696.0081 809.9178 652.0625 737.9775 696.0264 733.9822
592.1070 574.0754 590.1066 696.0118 809.9145 652.0637 737.9758 696.0252 733.9800
0.0055 0.0020 0.0001 0.0037 0.0033 0.0012 0.0017 0.0012 0.0022
9.3 3.5 0.2 5.3 4.1 1.8 2.3 1.7 3.0
Dm/z = m/zcalcd m/zexp; m/zcalcd m/zcalcd; m/zexp m/z found (experimental).
4. Experimental section
Table 2 Calculated CAN bond lengths of 1D2O and 2D2O Compound
Bond
Length (Å)
1D2O
C(4)AN(4)
1.299 1.473
2D2O
N(4)ACH2 N(3)AC(4) N(3)AC(2) N(1)A(C10 )
1.385 1.390 1.373
C(4)AN(4) N(4)ACH2 N(3)AC(4) N(3)AC(2) N(1)AC(10 )
1.300 1.469 1.387 1.390 1.375
CAN single bond length of aliphatic systems is in 1.4–1.5 Å range, which suggest that a substantially shorter C(4)AN(4) bond of 1D2O and 2D2O should be considered rather double bond. It is reasonable to state that this unusual electronic configuration is connected with protonation of nucleobase ring and also water ionization. Thus, in both 1D2O and 2D2O structures, free of interactions with other molecules, the C(4)-imino structure appears energetically favored. The latter is of interest, considering potential interaction of the corresponding nucleotide with thymidylate synthase. Namely the enzyme’s active center discriminates between the substrate, dUMP, and dCMP by the mechanism involving an active center asparagines residue hydrogen-bonding to dUMP C(4)@O and non-dissociated N(3)AH groups41–43, and in dUMP analogues the C(4)@O group may be imitated by the C(4)@NA group.44,45 The latter group is found in the C(4)-imino structure of 2D2O that in solution apparently exists in equilibrium with the amino structure (C(4)ANA). 3. Conclusions The synthetic route applied to synthesize N(4)-[B-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidine (1) shows a considerable potential due to high yield, relatively mild conditions and high purity of the crude product. NMR investigation suggested the new compound to be in zwitterionic form in heavy water (1D2O), having OD anion attached to boron atom and D+ to the proximal nucleobase-N(3) nitrogen. Hydrolysis of the pinacol moiety was observed under very mild conditions upon addition of small amounts of acetic acid or protected cysteine. The product of hydrolysis (2D2O) shows much lesser steric hindrance around boron atom, the latter causing an important influence on boron coordination properties. Potential uptake and phosphorylation of 1 by cultured tumor cells was also tested. The results obtained in LDI MS experiments showed the presence of in vivo-synthesized mono-, di- and triphosphates of 1.
4.1. Materials and methods 1 H NMR spectra were obtained with the Bruker Avance II spectrometer, operating in the quadrature mode at 500 MHz. All 11B spectra were performed using 5 mm pure quartz NMR tube. The residual peaks of deuterated solvents were used as internal standards. Reagents and deuterated solvents of the highest commercially available grade were purchased from Aldrich. DMSO was dried by vacuum distillation over anhydrous magnesium sulfate. All procedures, including preparation of samples for the NMR measurements, were carried out under nitrogen. All reagents, with the exception of boranes, were dried by triple aseotropic distillation from deuterated chloroform. High-resolution COSY spectra were prepared using 4096 4096 measurement points. 11B NMR spectra were referenced to external BF3OEt2 sample (0.0 ppm). All 1H NMR experiments were made with HDO-suppression method applied. LDI time-of-flight (TOF) mass spectrometry experiments were performed using a Bruker Autoflex Speed reflectron time-of-flight mass spectrometer, equipped with a SmartBeam II laser (352 nm). The laser impulse energy was approximately 60– 120 lJ, the laser repetition rate was 1000 Hz, and the deflection value was set on m/z
