Synthesis and characterization of a linker for primary amines used in the solid phase organic synthesis of spermidine

Abstracts

A linker resin for the synthesis of functionalized spermidine in good yield is described, along with its characterization by infrared (IR), 13C solid-state nuclear magnetic resonance with cross polarization and magic angle spinning (13C CPMAS NMR) and ¹H high resolution magic angle spinning nuclear magnetic resonance (¹H HRMAS NMR. This linker has been regenerated after cleavage of spermidine and re-used without loss of efficiency.

polyamines; 13C CPMAS NMR; HRMAS NMR; solid phase organic synthesis; spermidine

Neste trabalho é descrita a preparação de uma resina modificada, empregada como linker para a preparação de espermidina funcionalizada, em bom rendimento, assim como sua caracterização por infravermelho (IV), ressonância magnética nuclear de 13C no estado sólido com polarização cruzada e rotação no ângulo mágico (RMN-CPMAS de 13C) e ressonância magnética nuclear de ¹H de alta resolução com rotação no ângulo mágico (RMN-MAS de ¹H de alta resolução). O linker foi regenerado após clivagem da espermidina e reutilizado sem perda de eficiência.

ARTICLE

Synthesis and characterization of a linker for primary amines used in the solid phase organic synthesis of spermidine

Emerson T. da Silva^I; Rosane A. S. San GilI, ^*; Stefano Caldarelli^II; Fabio Ziarelli^III; Edson L. S. Lima^I

^IInstituto de Química, Universidade Federal do Rio de Janeiro, CP 068556 (Ed.CT), 21945-970 Rio de Janeiro-RJ, Brazil

^IIAix-Marseille Université ISm2 UMR 6263 Campus de Saint Jérôme, 13397 Cedex 20, Marseille, France

^IIIAix-Marseille Université Spectropôle - Fédération de Sciences Chimiques de Marseille, FR 1739 Campus de Saint Jérôme, 13397 Cedex 20, Marseille, France

ABSTRACT

A linker resin for the synthesis of functionalized spermidine in good yield is described, along with its characterization by infrared (IR), ¹³C solid-state nuclear magnetic resonance with cross polarization and magic angle spinning (¹³C CPMAS NMR) and ¹H high resolution magic angle spinning nuclear magnetic resonance (¹H HRMAS NMR. This linker has been regenerated after cleavage of spermidine and re-used without loss of efficiency.

Keywords: polyamines, ¹³C CPMAS NMR, HRMAS NMR, solid phase organic synthesis, spermidine

RESUMO

Neste trabalho é descrita a preparação de uma resina modificada, empregada como linker para a preparação de espermidina funcionalizada, em bom rendimento, assim como sua caracterização por infravermelho (IV), ressonância magnética nuclear de ¹³C no estado sólido com polarização cruzada e rotação no ângulo mágico (RMN-CPMAS de ¹³C) e ressonância magnética nuclear de ¹H de alta resolução com rotação no ângulo mágico (RMN-MAS de ¹H de alta resolução). O linker foi regenerado após clivagem da espermidina e reutilizado sem perda de eficiência.

Introduction

Spermidine is a naturally occurring polyamine, which like other amines as spermine and putrescine, is a growth regulator, besides possessing other unknown biological functions.¹ In the last years a great deal of attention has been given to naturally occurring polyamines, especially because of their involvement in the regulation of cellular functions such as cell proliferation and differentiation. Antiangiogenic, anticancer, and neurotoxins are some biological activities associated with polyamines and derivatives.^2-4 The inhibition of enzymes involved in the metabolism of parasitic protozoa has been recognized as a promising strategy for the chemotherapy of tropical diseases.^5,6 Trypanothione, structurally characterized as N1,N8-bis(glutathionyl)spermidine, is a polyamine derivative used by trypanosomatids as a defense against reactive oxygen species during their infective cycle. The metabolism of trypanothione is a potential target for drug development against trypanosomiases and leishmanioses.^7,8 As part of a research program aiming at the synthesis of polyamine derivatives, we became interested in the preparation of selectively protected spermidines.⁹ There are however many synthetic problems associated with polyamine chemistry, such as the amine differentiation and the ability to handle these exceptionally polar compounds. To overcome these obstacles we decided to explore polyamine spermidine synthesis on a solid support with the use of a linker derived from dimethyl-barbituric acid.

Solid phase organic synthesis, SPOS, present established advantages over organic reactions in solution,^10-15 notably the possibility of using large excess of reagents and a simplification of the isolation procedures with no need of purifications in each step are among the most relevant ones.

Various examples exist of the solid phase synthesis of polyamine conjugates for different purposes.^16-19 A previous paper²⁰ described the synthesis of an orthogonally protected spermidine, using the Merrifield resin.²¹ In the present paper a linker resin prepared from dimethyl-barbituric acid and the glutaric anhydride was successfully used in the solid-phase synthesis of a selectively protected spermidine.

Results and Discussion

Dimethyl-barbituric acid was transformed into 1,3-dimethyl-5-glutaryl-barbituric acid 1 by reaction with glutaric anhydride in THF at 81% of yield.²² This intermediate was linked to the polymeric resin using standard solid phase conditions by use of the diisopropylcarbodiimide (DIC) and dimethylamine pyridine (DMAP) to produce the linker resin 2 (Scheme 1).²³ The aminomethyl resin was prepared from Merrifield resin, purchased from Aldrich, as previously described,²⁴ and Fmoc loading assay²⁵ showed a loading of 0.64 mmol g^-1.

Most compounds in Scheme 1 can be characterized by standard liquid-state methods, while for functionalized resins, as 2, adapted analytical techniques must be used to follow the reaction in the solid phase. Typically infrared spectroscopy^26-29 and solid state NMR techniques^{10, 30-32} are proper to the task. Figure 1 illustrates the solid-state ¹³C cross polarization magic angle spinning (CP/MAS) spectra³³ of compounds 1 and 2. The signal corresponding to COOH (at 182.5 ppm) is not present in the spectra of resin 2. Conversely, the formation of the N-C=O bond caused the broadening of the signal at δ 170. 2D TOCSY (total correlation spectroscopy) HR-MAS NMR³³ also confirmed the amide linkage (Figure 2).

The resin 2 was then used in the synthesis of spermidine as demonstrated in Scheme 2. In order to initiate the preparation the 2-nitrossulfonamide was anchored on the resin 2 by reaction in THF under reflux.³⁴ The resulting resin 3 then reacted with 4-bromobutylphthalimide in acetonitrile in the presence of Cs₂CO₃ as base, generating the resin 4.³⁵ Protected spermidine 5³⁶ was cleaved from resin 4 after treatment with hydrate hydrazine at room temperature. Spermidine 5 was previously prepared by our group in solution system utilizing Fukuyama's sulfonamide.³⁷

Resin 2 was then reconstituted (Scheme 3) by treatment of resin 4, after cleavage of spermidine, with isopropyl nitrite in presence of H₂O/AcOH in THF at room temperature overnight (Scheme 3).

Conclusion

In summary this communication reports a resin linker for the synthesis of functionalized spermidine. The resin was successfully prepared and characterized using solid state spectroscopic methods, infrared and NMR techniques in phase solid proving the efficiency of this method. This linker was regenerated after cleavage of spermidine and re-used without loss of efficiency. It can be used similarly to other primary amines.

Acknowledgements

The authors would like to thank CAPES-COFECUB for support (grant No. 436/03), FAPERJ for schollarship (E. T. da Silva) and CAPES for a visiting professor grant (grant No. 014/2008, S. Caldarelli).

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22. Compound 1 (5-(4-carboxybutyryl)-1,3-dimethyl-2,4,6-pyrimidinetrione): to a solution of dimethylbarbituric acid (500 mg, 3.21 mmol) in dry THF (15 mL) was added glutaric anhydride (366 mg, 3.21 mmol) and a few crystals of DMAP. The mixture was stirred at reflux during 8 h under nitrogen, after which the solvent was removd in the rotovap. The residue was purified by column chromatography on silica gel (230-400 mesh, AcOEt/hexane 50%) to afford the desired product as a pale white solid, mp 84-86 °C. Rf = 0.58 (AcOEt/hexane 50%); -¹H NMR (CDCl₃, 200 MHz): δ 17.59 (1H, s, OH), 3.30 (3H, s, N-CH₃), 3.25 (3H, s, N-CH₃), 3.16 (2H, t, Csp²-CH₂, J 7.5 Hz); 2.42 (2H, t, -CH₂-CO₂H, J 7.5 Hz) 2.0 (2H, qui, -CH₂-CH₂-CH₂ -); ¹³C NMR (CDCl₃, 50 MHz): δ 20.44, 28.06, 28.21, 96.02, 150.57, 161.28, 169.60; IR ν_max/cm^-1: 3432, 3007, 2962, 1724, 1705, 1669, 1552, 1489, 1264, 1169, 1017, 800, 754, 424 (KBr). Elemental anal. calc. for C₁₁H₁₄N₂O₆ : C, 48.89; H, 5.22; N, 10.37; found: C, 48.86; H, 5.16; N, 10.67%.

23. Resin 2: aminomethyl resin (1 g; load 0.64 mmol g^-1) was swelled with DMF (15 mL), then were added 457 mg (1.8 mmol; 2.8 equiv.) of compound 1, a few crystals of DMAP and DIC (1.8 mol; 2.8 equiv.) and the mixture gently agitated overnight when ninhydrin test indicated the total consumption of free amino group. The resin was filtered and washed with DMF (3 × 15 mL), THF (3 × 15 mL), EtOH (3 × 15 mL), CH₂Cl₂ (3 ×15 mL). Then the resin was dried under vacuum. IR ν_max/cm^-1: 3412, 3326, 3058, 3023, 2916, 2848, 1723, 1668, 1557, 1490, 1450, 1353, 1026, 1269, 754, 535 (KBr). ¹H HRMAS NMR TOCSY correlation peaks of the (XC=OCH₂CH₂CH₂COH=) spin system: 3.2 (CH₂COH=); 2.3 (-CH₂-CO₂H); 2.05 ppm (-CH₂-CH₂-CH₂ -).

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33. Solid state ¹³C CPMAS spectra were acquired on a Bruker DRX300 spectrometer (7.05 T), operating at 75.4 MHz, using a 4 mm Bruker CPMAS probehead and 4 mm ZrO₂ rotor with Kel-F stoppers, spinning at 5 and 7 kHz. Acquis. parameters: P₁ 5 μs, contact time (optimized) of 3 ms, recycle time 2 s, number of scans 1000. Hexamethyl benzene was used as external reference (CH₃ at 17.3 ppm). ¹H HRMAS NMR spectra were obtained on a Bruker DSX400 instrument, equipped with a 4 mm dual ¹H/¹³C high resolution MAS probehead, using 1 to 2.5 mg of sample swollen in 12 to 50 μL CD₂Cl₂, pulse delay 10 s, 256 scans and spinning rate of 4000 Hz. 2D-TOCSY spectra were recorded with a mixing time of 85 ms, 16 transients and 3500 Hz of rotation frequency. Chemical shifts (δ) for ¹H MAS NMR are reported in ppm relative to residual signal of CD₂Cl₂.

34. Resin 3: Resin 2 (500 mg; load: 0.55 mmol g^-1) was swelled with dry THF (10 mL) and submitted to reaction with 2-nitrosulfonamidopropylamine (616 mg; 10 equiv.) under reflux for 20 h. After this time the resin was filtered and washed with THF (3 × 10 mL), isopropanol (3 × 10 mL), MeOH (3 × 10 mL), CH₂Cl₂ (3 × 10 mL). The resin was dried under vacuum. IR ν_max/cm^-1: 3415, 3024, 2921, 1706, 1672, 1643, 1590, 1492, 1451, 1348, 1166, 758, 700, 539 (KBr).

35. Resin 4: To a suspension of resin 3 (500 mg; load 0.49 mmol g^-1) swelled with dry DMF was added 798 mg (2.45 mmol; 10 equiv.) of cesium carbonate and 346 mg (1.22 mmol; 5 equiv.) of N-(4-bromobutyl)phthalimide. Ther reaction was gently stirred at 80 °C for 26 h. Then, the resin was filtered and washed with DMF (3 × 10 mL), isopropanol (3 × 10 mL), MeOH (3 × 10 mL), H₂O (3 × 10 mL), MeOH (3 × 10 mL), CH₂Cl₂ (3 × 10 mL). The resin was dried under vacuum. IR ν_max/cm^-1: 3409, 3024, 292, 1704, 1640, 1584, 1492, 1451, 1372, 1272, 1166, 758, 700, 541(KBr).

36. Compound 5: Resin 4 (500 mg; load 0.45 mmol g^-1) was swelled with dry THF (10 mL) and submitted to reaction with 5 equiv. of hydrazine hidrate at room temperature for 30 min. After the resin was filtered and washed with THF (3 × 5 mL) and CH₂Cl₂ (3 × 5 mL). The solvent was removed at reduced pressure and the residue obtained was poured into water and extracted with ethyl acetate (3 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated, furnishing an oil that after purification by flash chromatography (SiO₂, 230-400 mesh, MeOH/Et₃N 50%) yield 80% of spermidine 5 as a colorless oil. Rf= 0.65 (MeOH/Et₃N 50%); IR ν_max/cm^-1: 3450, 2930, 2854, 1711, 1593, 1543, 1477, 1161 (film); ¹H NMR (200 MHz, CDCl₃): δ 1.47-1.85 (m, 6H, CH₂), 2.41 (m, 4H, -CH₂-NNs-CH₂-); 2.79 (sl, 2H, NH₂); 3.58 (t, 2H, J 6.91 Hz, -CH₂NPht); 7.55-8.10 (m, 8H, ArH); ¹³C NMR (50 MHz, CDCl₃): δ 23.48, 26.49, 37.85, 39.69, 58.52, 52.04, 52.99, 123.38, 124.30, 131.10, 131.95, 132.24, 133.10, 133.90, 134.08, 148.20, 168.50.

37. da Silva, E. T. ; Fona, F. S.;Lima, E. L. S.; J. Braz. Chem. Soc. 2004, 15, 433.

Submitted: May 17, 2010

Published online: August 12, 2010

Publication Dates

• Publication in this collection
  26 Jan 2011
• Date of issue
  Jan 2011

History

• Received
  17 May 2010
• Accepted
  12 Aug 2010