Accepted Manuscript Title: Synthesis of 19-norcalcitriol analogs with elongated side chain Authors: Pawel Brzeminski, Adrian Fabisiak, Katarzyna Sektas, Klaudia Berkowska, Ewa Marcinkowska, Rafal R. Sicinski PII: DOI: Reference:
S0960-0760(17)30222-4 http://dx.doi.org/10.1016/j.jsbmb.2017.08.008 SBMB 5008
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
8-5-2017 10-8-2017 13-8-2017
Please cite this article as: Pawel Brzeminski, Adrian Fabisiak, Katarzyna Sektas, Klaudia Berkowska, Ewa Marcinkowska, Rafal R.Sicinski, Synthesis of 19-norcalcitriol analogs with elongated side chain, Journal of Steroid Biochemistry and Molecular Biologyhttp://dx.doi.org/10.1016/j.jsbmb.2017.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis of 19-norcalcitriol analogs with elongated side chain
Pawel Brzeminskia, Adrian Fabisiaka, Katarzyna Sektasa, Klaudia Berkowskab, Ewa Marcinkowskab, Rafal
R. Sicinskia,* a
Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
b
Department of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland
*
To whom correspondence should be addressed: Prof. Rafal R. Sicinski Department of Chemistry University of Warsaw Pasteura 1 02-093 Warsaw, Poland Telephone: (+48) 22-55-26-252 Email: [email protected]
Graphical abstract
Highlights
Starting from the natural compounds synthesis of the building blocks was achieved
19-Norcalcitriol analog with elongated side chain obtained by Wittig-Horner coupling
The side chain of the synthesized analog can protrude from the VDR binding pocket
Abstract Pronounced biological potency of 19-norvitamin D compounds as well as interesting biological action of the vitamin D analogs possessing elongated side chains encouraged us to expand the scope of our structureactivity studies to encompass such modifications of the 1α,25-(OH)2D3 (calcitriol) molecule. The aim of our studies was the synthesis of calcitriol analog, designed on the basis of results of molecular modeling and docking experiments, and characterized by a presence of a long, nitrogen-containing substituent attached to carbon 26, and an exomethylene moiety transferred from C-10 to C-2. The convergent synthesis of such 19norcalcitriol compound, described in this communication, consisted of the preparation and combining four building blocks. The crucial point of the synthesis, coupling of the known A-ring phosphine oxide and the synthesized Grundmann ketone analog, was achieved using Wittig-Horner protocol. It provided the protected analog of 2-methylene-1α,25-dihydroxy-19-norvitamin D3 which was further transformed into the target compound.
Keywords: Secosteroids; 19-Norcalcitriol; Vitamin D receptor; Wittig-Horner reaction
1. Introduction Since the discovery of the most active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1, calcitriol; Chart 1), representing its hormonal form, responsible for all known genomic and non-genomic actions [1,2], numerous analogs of this compound have been synthesized and biologically tested [3,4]. The main reason of a broad interest in such structure-activity studies was the search for compounds with separated calcemic and antiproliferative activities. In 1998, an interesting structural modification of calcitriol molecule was described involving a formal “shift” of its A-ring exomethylene substituent that resulted in 19norcalcitriols (2 and 3) with enhanced calcemic activity [5,6]. The biological responses of 1α,25dihydroxyvitamin D3 are mediated by the nuclear vitamin D receptor (VDR), a ligand-dependent transcription regulator [7]. Therefore, effective binding of the vitamin D ligand to this protein is of crucial importance. The crystal structures of the binary hVDR-1 complex and ternary tridecapeptide-rVDR-3 complex were solved in 2000 and 2004 by Moras [8] and DeLuca [9] group, respectively. Knowledge of the structure of the receptor’s ligand binding domain significantly facilitated the docking experiments performed with the synthetic vitamin D analogs. Continuing our structure-activity studies in the vitamin D area, we turned our attention to compounds possessing an elongated substituent at C-17. Analysis of the literature data reveals that such calcitriol analogs can display various biological actions depending on their side chain structure (length, rigidity, bulkiness, etc.). Thus, some of them with long and bulky side chains (4) are VDR superagonists [10], whereas some analogs containing an alkoxycarbonyl group (5) [11] or bulky adamantyl substituent (6) [12] at the side-chain terminus, have antagonistic activity. In this communication, we describe the synthesis of 2-methylene-1,25-dihydroxy-19-norvitamin D3 analog 7, substituted at C-26 with 7-aminoheptyl moiety functionalized at nitrogen atom; the stereogenic center at C-25 has S-configuration. The results of molecular modeling and docking experiments demonstrate
that after docking to the VDR, the side chain of such compound will protrude from the binding pocket. The synthesis of this target molecule required coupling of four building blocks. The 19-norvitamin D skeleton was formed by Wittig-Horner coupling of the known A-ring phosphine oxide 8 [5,13] with an “upper” C/D-ring fragment, namely, Grundmann ketone type 9.
2. Materials and methods The target 26-substituted analog of 2-methylene-19-norcalcitriol 7 was synthesized at the Department of Chemistry, University of Warsaw, according to the synthetic route presented in Schemes 1, 2 and 3. All the prepared compounds exhibited spectroscopic and analytical data consistent with their structures. Full details of their synthesis will be reported elsewhere.
3. Results and discussion 3.1. Chemical synthesis of the 1α,25S-dihydroxy-2-methylene-19-norvitamin D3 compound 7 3.1.1. Chemical synthesis of the 4-(triethylsilyl)oxy-4-methylhept-6-enenitrile (15) The preparation of the nitrile 15 (Scheme 1), required for the construction of the steroidal side chain, started from the known compound 10, prepared from geraniol according to the literature procedure [14]. Ozonolysis of 10 followed by ozonide reduction provided the mono-protected triol 11, which gave olefin 12 by following the Grieco elimination protocol. After deprotection, its primary hydroxyl was converted into the nitrile group and the tertiary hydroxyl in the formed 14 was silylated to yield the desired fragment 15. 3.1.2. Chemical synthesis of the building block 9 Substitution of the known bicyclic tosylate 16 (Scheme 2), prepared from the vitamin D2 [15,16], with an anion of the nitrile 15, followed by reduction with potassium, furnished diether 17. Deprotection of both hydroxyl groups yielded the diol 18 which was subjected to the cross-methatesis reaction with the TBS-
protected hept-6-en-1-ol. The obtained mixture of the unsaturated, mono-protected triols 19 was then hydrogenated to the saturated compound 20. Oxidation of its secondary hydroxyl group led to the desired building block 9. 3.1.3. Coupling of the building blocks 8 and 9 leading to the target compound 7 The Wittig-Horner coupling of the bicyclic ketone 9 with an anion of the phosphine oxide 8, prepared by us from the quinic acid [5], gave the expected protected 2-methylene-19-norvitamin 21 (Scheme 3). Cleavage of the silyl ethers followed by selective tosylation of the primary hydroxyl in the tetraol 22 gave the tosylate 23. The latter was converted into the iodide 24 which upon the treatment with an excess of 2-(2’aminoethyl)pyridine afforded the target compound 7 [17]. 3.2. Molecular modeling 3.2.1. Conformational search The calculation of optimized geometries and steric energies were carried out using the algorithm from the MM+ HyperChem (release 8.0) software package (Hypercube Inc.). The procedure used for generation of the respective side-chain conformers of the vitamin D analog 7 was analogous to that described by us previously and involved the Conformational Search module [18]. The calculated global minimum conformers were next energy-minimized using PCModel (release 9.0) program (Serena Software). 3.2.2. Docking procedure The energy-minimized conformer of the vitamin D compound 7, calculated by HyperChem and PCModel as described above, was used as the ligand in the docking simulations performed with the Molegro Virtual Docker (release 4.0) software (CLC bio, QIAGEN). Compound 7 was docked into the ligand binding pocket of the hVDR (PDB Code: 1DB1) [8] and the calculated complexes were analyzed, taking into account the energy criterion, the number of hydrogen bonds and parallel orientation of the ligand’s intercyclic conjugated 5,7-diene fragment with respect to the tryptophan molecule (Trp 286) [19]. The docking
experiments revealed that the vitamin 7 anchors the binding pocket similarly as the vitamin D hormone 1 [8], creating hydrogen bonds with the same residues (Figure 1). 3.3. Measurement of binding to the vitamin D receptor 3.3.1. VDR binding assay Binding affinity of the analog to VDR was evaluated using a PolarScreenTM Vitamin D Receptor Competitor Assay Kit under manufacturer’s conditions (Invitrogen, Carlsbad, CA). 1α,25-(OH)2D3 and the vitamin D compound 7 were evaluated within the concentration range 10-13–10-5 M. The tested compounds were incubated for 4 hours at RT in order to reach equilibrium. The polarized fluorescence of every plate was measured three times using Envision multiplate reader (PerkinElmer, Waltham, MA) and mean fluorescence polarization was calculated from these measurements. The whole assay was repeated three times. IC50 values were calculated in GraphPad Prism (GraphPad Software, San Diego CA) using the average of values obtained. 3.3.2. Affinity of the analog 7 to VDR The affinity of the vitamin D compound 7 to VDR was assessed using a fluorescence polarizationbased competition assay. In this assay recombinant human VDR is added to a fluorescent VDR ligand to form a complex, resulting in a high fluorescence polarization value. Then the tested compounds are added to the complex into 386-well plates. The tested compounds displace the fluorescent ligand from the complex, resulting in lower polarization value. Dose-response curves were plotted and IC50 values were calculated from these dose-response curves. The binding affinity of the tested analog was compared to 1α,25-(OH)2D3, for which the RBA was determined as 100% and is presented in Table 1. 3.4. Conclusion In this communication, a convergent synthesis of the new 1α,25S-dihydroxy-2-methylene-19norvitamin D3 analog 7 has been presented with a significantly modified side-chain bearing terminal fragment
capable of chelation of transition metal ions. During this synthesis, three chiral building blocks were used, obtained from the natural compounds (geraniol, vitamin D2 and quinic acid), and combined for the assembly of the target vitamin. Such strategy can be obviously applied for the synthons required for the synthesis of other vitamin D compounds modified in the different parts of their molecules. The results of our docking experiments of the final compound 7 seemed to support our expectations since the side chain of this new analog protruded from the VDR binding pocket. Therefore, its terminal moiety could be used for formation of the complexes with the metal cations. However, the results of the VDR binding assay indicate that further side-chain modification is necessary because the binding affinity of the synthesized analog 7 is decreased by three orders of magnitude in comparison with 1α,25-(OH)2D3.
Acknowledgements This study was supported by grant OPUS 8, 2014/15/B/ST5/02129 from the National Science Center, Poland.
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S. Yamada, Identification of a highly potent vitamin D receptor antagonist: (25S)-26-adamantyl-25-hydroxy2-methylene-22,23-didehydro-19,27-dinor-20-epi-vitamin D3 (ADMI3), Archives of Biochemistry and Biophysics 460 (2007) 240–253. [13]
D.R. Laplace, M. Van Overschelde, P.J. Clercq, A. Verstuyf, J.M. Winne, Synthesis of 2-ethyl-19-nor
analogs of 1α,25-dihydroxyvitamin D3, European Journal of Organic Chemistry 4 (2013) 728–735. [14]
D. Kakati, N.C. Barua, Total synthesis and assignment of the absolute stereochemistry of xanthangelol
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H.F. DeLuca, A. Flores, P. Grzywacz, L.A. Plum, M. Clagett-Dame, (20S)-2-Methylene-19-nor-22-
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7: UV (in EtOH) max 244.5, 253.0 (ε 44500), 262.0 nm; 1H NMR (500 MHz, CDCl3) δ 0.544 (3H, s,
18-H3), 0.933 (3H, d, J = 6.3 Hz, 21-H3), 1.155 (3H, s, 27-H3), 2.28 (1H, dd, J = 13.5, 8.4 Hz, 10α-H), 2.33 (1H, dd, J = 13.4, 6.0 Hz, 4β-H), 2.56 (1H, dd, J = 13.4, 3.2 Hz, 4α-H), 2.81 (1H, d, J = 12 Hz, 9β-H), 2.86
(1H, dd, J = 13.5, 4.8 Hz, 10β-H), ca. 3.0 (3H, m, NHCH2CH2), 3.33 (4H, m, pyr-CH2CH2), 4.47 (2H, m, 1βand 3α-H), 5.08 and 5.11 (1H and 1H, each s, =CH2), 5.88 and 6.35 (1H and 1H, each d, J = 11.1 Hz, 7- and 6-H), 7.22 (2H, m, Hpyr-5 and Hpyr-3), 7.68 (1H, dt, J = 1.8, 7.7 Hz, Hpyr-4), 8.43 (1H, br d, J = 4.6 Hz, Hpyr6); HRMS (ESI) exact mass calcd for C41H67N2O3 (M+ + H) 635.5152, measured 635.5156. [18]
R.R. Sicinski, H.F. DeLuca, Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro
analogues of 1α,25-dihydroxyvitamin D3, Bioorganic and Medicinal Chemistry 7 (1999) 2877–2889. [19]
W. Sicinska, W.M. Westler, H.F. DeLuca, NMR assignments of tryptophan residue in apo and holo
LBD-rVDR, Proteins 61 (2005) 461–467.
Figure captions Chart 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), its analogs and building blocks used in the synthesis. Figure 1. View of the three-dimensional structure of ligand binding cavity of the human VDR with the docked vitamin D analog 7 (Molegro Virtual Docker). The six amino acids (Tyr 143, Ser 237, Arg 274, Ser 278, His 305 and His 397) forming the hydrogen bonds with the ligand are depicted. The intercyclic diene fragment of the ligand is parallel to the tryptophan rings (Trp 286).
Figure 1. View of the three-dimensional structure of ligand binding cavity of the human VDR with the docked vitamin D analog 7 (Molegro Virtual Docker). The six amino acids (Tyr 143, Ser 237, Arg 274, Ser 278, His 305 and His 397) forming the hydrogen bonds with the ligand are depicted. The intercyclic diene fragment of the ligand is parallel to the tryptophan rings (Trp 286).
.
Scheme 1. (a) 1. O3, CH2Cl2, -78 oC; 2. NaBH4, CH2Cl2, 80%; (b) 1. 2-nitrophenylselenocyanate, Bu3P, THF, rt; 2. H2O2, pyridine, CH2Cl2, 0 oC, 74%; (c) TBAF, THF, rt, 95%; (d) p-TsCl, pyridine, 0 oC; (e) NaCN, DMSO, 80 oC, 68% (2 steps); (f) TfOTES, 2,6lutidine, CH2Cl2, -78 oC, 60%.
Scheme 2. (a) 15, LDA, THF, -78 oC; (b) K, HMPA, t-BuOH, THF, 0 oC; 64% (2 steps); (c) TBAF, THF, rt, 87%; (d) CH2=CH(CH2)5OTBS, 2nd Generation Grubbs’ catalyst, CH2Cl2, 40 oC, 59%, mixture of isomers; (e) 10% Pd/C, H2, AcOEt, rt, 84%; (f) Dess-Martin periodinane, CH2Cl2, rt, 89%.
Scheme 3. (a) 8, PhLi, THF, -78 °C, 71%; (b) TBAF, THF, rt; 93%; (c) p-TsCl, DMAP, CH2Cl2, 0 oC; (d) NaI, acetone, 80 oC, 71% (2 steps); (e) 2-(2’-aminoethyl)pyridine, MeOH, reflux, 56%.
Chart 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), its analogs and building blocks used in the synthesis.
Table 1. Affinities of the compounds to recombinant VDRa. . 1α,25-(OH)2D3 analog 7 IC50 (nM)
1.464
1345
RBAb
100
0.1
The VDR binding affinity is expressed as IC50 and percentage activity. bThe potency of 1α,25-(OH)2D3 is normalized to 100. RBA: relative binding affinity. a
S0960-0760(17)30222-4 http://dx.doi.org/10.1016/j.jsbmb.2017.08.008 SBMB 5008
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
8-5-2017 10-8-2017 13-8-2017
Please cite this article as: Pawel Brzeminski, Adrian Fabisiak, Katarzyna Sektas, Klaudia Berkowska, Ewa Marcinkowska, Rafal R.Sicinski, Synthesis of 19-norcalcitriol analogs with elongated side chain, Journal of Steroid Biochemistry and Molecular Biologyhttp://dx.doi.org/10.1016/j.jsbmb.2017.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis of 19-norcalcitriol analogs with elongated side chain
Pawel Brzeminskia, Adrian Fabisiaka, Katarzyna Sektasa, Klaudia Berkowskab, Ewa Marcinkowskab, Rafal
R. Sicinskia,* a
Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
b
Department of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland
*
To whom correspondence should be addressed: Prof. Rafal R. Sicinski Department of Chemistry University of Warsaw Pasteura 1 02-093 Warsaw, Poland Telephone: (+48) 22-55-26-252 Email: [email protected]
Graphical abstract
Highlights
Starting from the natural compounds synthesis of the building blocks was achieved
19-Norcalcitriol analog with elongated side chain obtained by Wittig-Horner coupling
The side chain of the synthesized analog can protrude from the VDR binding pocket
Abstract Pronounced biological potency of 19-norvitamin D compounds as well as interesting biological action of the vitamin D analogs possessing elongated side chains encouraged us to expand the scope of our structureactivity studies to encompass such modifications of the 1α,25-(OH)2D3 (calcitriol) molecule. The aim of our studies was the synthesis of calcitriol analog, designed on the basis of results of molecular modeling and docking experiments, and characterized by a presence of a long, nitrogen-containing substituent attached to carbon 26, and an exomethylene moiety transferred from C-10 to C-2. The convergent synthesis of such 19norcalcitriol compound, described in this communication, consisted of the preparation and combining four building blocks. The crucial point of the synthesis, coupling of the known A-ring phosphine oxide and the synthesized Grundmann ketone analog, was achieved using Wittig-Horner protocol. It provided the protected analog of 2-methylene-1α,25-dihydroxy-19-norvitamin D3 which was further transformed into the target compound.
Keywords: Secosteroids; 19-Norcalcitriol; Vitamin D receptor; Wittig-Horner reaction
1. Introduction Since the discovery of the most active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1, calcitriol; Chart 1), representing its hormonal form, responsible for all known genomic and non-genomic actions [1,2], numerous analogs of this compound have been synthesized and biologically tested [3,4]. The main reason of a broad interest in such structure-activity studies was the search for compounds with separated calcemic and antiproliferative activities. In 1998, an interesting structural modification of calcitriol molecule was described involving a formal “shift” of its A-ring exomethylene substituent that resulted in 19norcalcitriols (2 and 3) with enhanced calcemic activity [5,6]. The biological responses of 1α,25dihydroxyvitamin D3 are mediated by the nuclear vitamin D receptor (VDR), a ligand-dependent transcription regulator [7]. Therefore, effective binding of the vitamin D ligand to this protein is of crucial importance. The crystal structures of the binary hVDR-1 complex and ternary tridecapeptide-rVDR-3 complex were solved in 2000 and 2004 by Moras [8] and DeLuca [9] group, respectively. Knowledge of the structure of the receptor’s ligand binding domain significantly facilitated the docking experiments performed with the synthetic vitamin D analogs. Continuing our structure-activity studies in the vitamin D area, we turned our attention to compounds possessing an elongated substituent at C-17. Analysis of the literature data reveals that such calcitriol analogs can display various biological actions depending on their side chain structure (length, rigidity, bulkiness, etc.). Thus, some of them with long and bulky side chains (4) are VDR superagonists [10], whereas some analogs containing an alkoxycarbonyl group (5) [11] or bulky adamantyl substituent (6) [12] at the side-chain terminus, have antagonistic activity. In this communication, we describe the synthesis of 2-methylene-1,25-dihydroxy-19-norvitamin D3 analog 7, substituted at C-26 with 7-aminoheptyl moiety functionalized at nitrogen atom; the stereogenic center at C-25 has S-configuration. The results of molecular modeling and docking experiments demonstrate
that after docking to the VDR, the side chain of such compound will protrude from the binding pocket. The synthesis of this target molecule required coupling of four building blocks. The 19-norvitamin D skeleton was formed by Wittig-Horner coupling of the known A-ring phosphine oxide 8 [5,13] with an “upper” C/D-ring fragment, namely, Grundmann ketone type 9.
2. Materials and methods The target 26-substituted analog of 2-methylene-19-norcalcitriol 7 was synthesized at the Department of Chemistry, University of Warsaw, according to the synthetic route presented in Schemes 1, 2 and 3. All the prepared compounds exhibited spectroscopic and analytical data consistent with their structures. Full details of their synthesis will be reported elsewhere.
3. Results and discussion 3.1. Chemical synthesis of the 1α,25S-dihydroxy-2-methylene-19-norvitamin D3 compound 7 3.1.1. Chemical synthesis of the 4-(triethylsilyl)oxy-4-methylhept-6-enenitrile (15) The preparation of the nitrile 15 (Scheme 1), required for the construction of the steroidal side chain, started from the known compound 10, prepared from geraniol according to the literature procedure [14]. Ozonolysis of 10 followed by ozonide reduction provided the mono-protected triol 11, which gave olefin 12 by following the Grieco elimination protocol. After deprotection, its primary hydroxyl was converted into the nitrile group and the tertiary hydroxyl in the formed 14 was silylated to yield the desired fragment 15. 3.1.2. Chemical synthesis of the building block 9 Substitution of the known bicyclic tosylate 16 (Scheme 2), prepared from the vitamin D2 [15,16], with an anion of the nitrile 15, followed by reduction with potassium, furnished diether 17. Deprotection of both hydroxyl groups yielded the diol 18 which was subjected to the cross-methatesis reaction with the TBS-
protected hept-6-en-1-ol. The obtained mixture of the unsaturated, mono-protected triols 19 was then hydrogenated to the saturated compound 20. Oxidation of its secondary hydroxyl group led to the desired building block 9. 3.1.3. Coupling of the building blocks 8 and 9 leading to the target compound 7 The Wittig-Horner coupling of the bicyclic ketone 9 with an anion of the phosphine oxide 8, prepared by us from the quinic acid [5], gave the expected protected 2-methylene-19-norvitamin 21 (Scheme 3). Cleavage of the silyl ethers followed by selective tosylation of the primary hydroxyl in the tetraol 22 gave the tosylate 23. The latter was converted into the iodide 24 which upon the treatment with an excess of 2-(2’aminoethyl)pyridine afforded the target compound 7 [17]. 3.2. Molecular modeling 3.2.1. Conformational search The calculation of optimized geometries and steric energies were carried out using the algorithm from the MM+ HyperChem (release 8.0) software package (Hypercube Inc.). The procedure used for generation of the respective side-chain conformers of the vitamin D analog 7 was analogous to that described by us previously and involved the Conformational Search module [18]. The calculated global minimum conformers were next energy-minimized using PCModel (release 9.0) program (Serena Software). 3.2.2. Docking procedure The energy-minimized conformer of the vitamin D compound 7, calculated by HyperChem and PCModel as described above, was used as the ligand in the docking simulations performed with the Molegro Virtual Docker (release 4.0) software (CLC bio, QIAGEN). Compound 7 was docked into the ligand binding pocket of the hVDR (PDB Code: 1DB1) [8] and the calculated complexes were analyzed, taking into account the energy criterion, the number of hydrogen bonds and parallel orientation of the ligand’s intercyclic conjugated 5,7-diene fragment with respect to the tryptophan molecule (Trp 286) [19]. The docking
experiments revealed that the vitamin 7 anchors the binding pocket similarly as the vitamin D hormone 1 [8], creating hydrogen bonds with the same residues (Figure 1). 3.3. Measurement of binding to the vitamin D receptor 3.3.1. VDR binding assay Binding affinity of the analog to VDR was evaluated using a PolarScreenTM Vitamin D Receptor Competitor Assay Kit under manufacturer’s conditions (Invitrogen, Carlsbad, CA). 1α,25-(OH)2D3 and the vitamin D compound 7 were evaluated within the concentration range 10-13–10-5 M. The tested compounds were incubated for 4 hours at RT in order to reach equilibrium. The polarized fluorescence of every plate was measured three times using Envision multiplate reader (PerkinElmer, Waltham, MA) and mean fluorescence polarization was calculated from these measurements. The whole assay was repeated three times. IC50 values were calculated in GraphPad Prism (GraphPad Software, San Diego CA) using the average of values obtained. 3.3.2. Affinity of the analog 7 to VDR The affinity of the vitamin D compound 7 to VDR was assessed using a fluorescence polarizationbased competition assay. In this assay recombinant human VDR is added to a fluorescent VDR ligand to form a complex, resulting in a high fluorescence polarization value. Then the tested compounds are added to the complex into 386-well plates. The tested compounds displace the fluorescent ligand from the complex, resulting in lower polarization value. Dose-response curves were plotted and IC50 values were calculated from these dose-response curves. The binding affinity of the tested analog was compared to 1α,25-(OH)2D3, for which the RBA was determined as 100% and is presented in Table 1. 3.4. Conclusion In this communication, a convergent synthesis of the new 1α,25S-dihydroxy-2-methylene-19norvitamin D3 analog 7 has been presented with a significantly modified side-chain bearing terminal fragment
capable of chelation of transition metal ions. During this synthesis, three chiral building blocks were used, obtained from the natural compounds (geraniol, vitamin D2 and quinic acid), and combined for the assembly of the target vitamin. Such strategy can be obviously applied for the synthons required for the synthesis of other vitamin D compounds modified in the different parts of their molecules. The results of our docking experiments of the final compound 7 seemed to support our expectations since the side chain of this new analog protruded from the VDR binding pocket. Therefore, its terminal moiety could be used for formation of the complexes with the metal cations. However, the results of the VDR binding assay indicate that further side-chain modification is necessary because the binding affinity of the synthesized analog 7 is decreased by three orders of magnitude in comparison with 1α,25-(OH)2D3.
Acknowledgements This study was supported by grant OPUS 8, 2014/15/B/ST5/02129 from the National Science Center, Poland.
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Figure captions Chart 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), its analogs and building blocks used in the synthesis. Figure 1. View of the three-dimensional structure of ligand binding cavity of the human VDR with the docked vitamin D analog 7 (Molegro Virtual Docker). The six amino acids (Tyr 143, Ser 237, Arg 274, Ser 278, His 305 and His 397) forming the hydrogen bonds with the ligand are depicted. The intercyclic diene fragment of the ligand is parallel to the tryptophan rings (Trp 286).
Figure 1. View of the three-dimensional structure of ligand binding cavity of the human VDR with the docked vitamin D analog 7 (Molegro Virtual Docker). The six amino acids (Tyr 143, Ser 237, Arg 274, Ser 278, His 305 and His 397) forming the hydrogen bonds with the ligand are depicted. The intercyclic diene fragment of the ligand is parallel to the tryptophan rings (Trp 286).
.
Scheme 1. (a) 1. O3, CH2Cl2, -78 oC; 2. NaBH4, CH2Cl2, 80%; (b) 1. 2-nitrophenylselenocyanate, Bu3P, THF, rt; 2. H2O2, pyridine, CH2Cl2, 0 oC, 74%; (c) TBAF, THF, rt, 95%; (d) p-TsCl, pyridine, 0 oC; (e) NaCN, DMSO, 80 oC, 68% (2 steps); (f) TfOTES, 2,6lutidine, CH2Cl2, -78 oC, 60%.
Scheme 2. (a) 15, LDA, THF, -78 oC; (b) K, HMPA, t-BuOH, THF, 0 oC; 64% (2 steps); (c) TBAF, THF, rt, 87%; (d) CH2=CH(CH2)5OTBS, 2nd Generation Grubbs’ catalyst, CH2Cl2, 40 oC, 59%, mixture of isomers; (e) 10% Pd/C, H2, AcOEt, rt, 84%; (f) Dess-Martin periodinane, CH2Cl2, rt, 89%.
Scheme 3. (a) 8, PhLi, THF, -78 °C, 71%; (b) TBAF, THF, rt; 93%; (c) p-TsCl, DMAP, CH2Cl2, 0 oC; (d) NaI, acetone, 80 oC, 71% (2 steps); (e) 2-(2’-aminoethyl)pyridine, MeOH, reflux, 56%.
Chart 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), its analogs and building blocks used in the synthesis.
Table 1. Affinities of the compounds to recombinant VDRa. . 1α,25-(OH)2D3 analog 7 IC50 (nM)
1.464
1345
RBAb
100
0.1
The VDR binding affinity is expressed as IC50 and percentage activity. bThe potency of 1α,25-(OH)2D3 is normalized to 100. RBA: relative binding affinity. a
