Chemical and Pharmaceutical Bulletin
Online ISSN : 1347-5223
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Practical Synthesis of Spermine, Thermospermine and Norspermine
Yuka KariyaYuta AsanumaMakoto InaiTomohiro AsakawaKyoko Ohashi-ItoHiroo FukudaMasahiro EgiToshiyuki Kan
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2016 Volume 64 Issue 9 Pages 1403-1407

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Abstract

Polyamines, such as spermine (1), thermospermine (2) and norspermine (3), are widely distributed in nature, and have multiple biological activities. In addition, many of their conjugates have potential for pharmacological use. Here, we present a solid-phase synthesis using our nitrobenzenesulfonyl (Ns) strategy, which can provide 1, 2 and 3 on a gram scale. This approach should be suitable for facile construction of a diverse library of polyamines.

Polyamines, especially of the spermine family (such as 1, 2 and 3) are ubiquitous in microorganisms, plants, and animals, and have multiple biological activities.14) They interact with nucleic acids and modulate DNA synthesis and cell proliferation.57) Due to their biological significance, practical and versatile synthetic methods are needed, but the lability of these compounds to oxidation, as well as their highly polar nature, makes handling difficult. We have shown that the nitrobenzenesulfonyl (Ns) moiety is available as both a protecting and activating group of amino group for flexible and practical synthesis of these compounds.8,9) More recently, some of the present authors reported that thermospermine (2) plays a key role in regulation of cell division and differentiation in plant meristems.10) Inspired by this interesting biological activity, we wished to synthesize thermospermine (2) and related compounds on a large scale for biological testing and mechanistic investigations. Here, we describe practical gram-scale syntheses of 1, 2 and 3 by means of a solid-phase method employing our Ns strategy (Fig. 1).

Fig. 1. Structures of Spermine (1), Thermospermine (2) and Norspermine (3)

As shown in Chart 1, thermospermine (2) was synthesized by orthogonal extension of bromide 4 and alcohol 7. After conversion of 4 and 7 to Ns amide, protection of sulfonamide with a tert-butoxycarbonyl (Boc) group and alcohol with a tert-butyldimethylsilyl (TBS) group afforded 6 and 8, respectively. Upon treatment of 6 and 8 with Cs2CO3 in the presence of tetrabutylammonium iodide, the desired coupling reaction proceeded smoothly to give 9. Selective deprotection of TBS ether was carried out by treatment with 10-camphorsulfonic acid (CSA) in methanol. Although Mitsunobu reaction of alcohol 10 and Ns-amide proceeded smoothly, we converted 10 to iodide 11 in a two-step sequence for ease of purification. Furthermore, iodide 11 opens up the possibility of further alkylation with sulfonamides such as 8 and 12 to extend the polyamine chain. In order to synthesize thermospermine (2), alkylation of sulfonamide 12 was conducted with iodide 11, which was readily prepared from diaminopropane by using our protocol.1113) After removal of the Boc group of 13, primary amine 14 was attached to our resin 15, which is readily obtained from Merrifield resin.1416) The alkoxytrityl chloride resin 15 offers significant advantages in terms of high reactivity, because the reactive site is well separated from the bulky polystyrene backbone. After deprotection of the Ns group was accomplished by treatment with 2-mercaptoethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), the product was cleaved from the resin under acidic conditions. Upon removal of the solvent, 2 was obtained in high purity. All spectroscopic data (1H- and 13C-NMR spectra and MS) of synthetic 2 were identical with those reported in the literature.17) This synthetic protocol was applicable on a gram scale and we were able to synthesize 1 g of 2 in a pure form without the need for HPLC purification. Synthetic thermospermine (2) showed the expected activity for regulation of cell division and differentiation in meristems.10)

Chart 1. Synthesis of Thermospermine (2) via an Orthogonal Extension Strategy

Reagents and conditions: (a) 5, Na2CO3, CH2Cl2/H2O, (b) Boc2O, Et3N, DMAP, CH2Cl2, (c) TBSCl, imidazole, DMF, (d) Cs2CO3, TBAI, MeCN, 60°C, (e) CSA, MeOH, (f) MsCl, Et3N, CH2Cl2, (g) NaI, butanone, 60°C, (h) Cs2CO3, MeCN, 60°C, (i) SOCl2, MeOH, (j) 15, N,N-diisopropylethylamine, DMAP, DMF, (k) 2-mercaptoethanol, DBU, DMF, (l) 3% TFA in CH2Cl2.

Next, we turned our attention to the synthesis of spermine 1 and norspermine 3. Considering the symmetrical structures of 1 and 3, we expected that these polyamines could be synthesized via a simultaneous two-dimensional extension strategy, as illustrated in Chart 2. Although we have reported the synthesis of several protected spermine derivatives in the solution phase, as well as in the solid phase,14) the present method should be more convenient. Upon treatment of 2.2 eq of sulfonamide 17 with dibromobutane 18a or dibromopropane 18b in the presence of Cs2CO3 and a catalytic amount of n-tetrabutylammonium iodide, the desired double alkylation reaction proceeded readily to provide 19a and b, respectively. This one-step construction is superior to the stepwise-elongation of protected spermine and norspermine from diaminopropane. After removal of the Boc group, attachment of 20a or b to 15 was performed by treatment with a catalytic amount of N,N-dimethyl-4-aminopyridine (DMAP) and diisopropylethylamine. Subsequently, similar treatment to that used for the preparation of thermospermine (2) provided the natural products 1 and 3.

Chart 2. Synthesis of Spermine (1) and Norspermine (3) via a Simultaneous Extension Strategy

Reagents and conditions: (a) Cs2CO3, TBAI, MeCN, 60°C, (b) SOCl2, MeOH, (c) 15, N,N-diisopropylethylamine, DMAP, DMF, (d) 2-mercaptoethanol, DBU, DMF, (e) 3% TFA in CH2Cl2.

In conclusion, we have developed a practical, large-scale synthesis of polyamines via efficient extension reaction on a solid support based on the Ns strategy. This method does not require tedious purification of polyamines, which can easily be prepared on a gram scale. The method should be applicable to various types of polyamines, including long-chain polyamines (LCPAs). This application is currently being investigated in our laboratories, and the results will be reported in due course.

Experimental

General Comments

Nuclear magnetic resonance [1H-NMR (500 MHz), 13C-NMR (125 MHz)] spectra were determined on JEOL ECA-500 instrument. Chemical shifts for 1H-NMR were reported in parts per million downfields from tetramethylsilane (δ) as the internal standard and coupling constants were in hertz (Hz). The following abbreviations are used for spin multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad. Chemical shifts for 13C-NMR were reported in ppm relative to the centerline of a triplet at 77.0 ppm for deuteriochloroform. High-resolution (HR)-MS were obtained on a BRUKER DALTONICS micrOTOF electrospray ionization (ESI). IR spectra were recorded on a SHIMADZU IRPrestige-21. Analytical TLC was performed on Merck precoated analytical plates, 0.25 mm thick, silica gel 60 F254. Preparative TLC separations were made on 7×20 cm plates prepared with a 0.25 mm layer of Merck silica gel 60 F254. Compounds were eluted from the adsorbent with 10% MeOH in chloroform. Column chromatography separations were performed on KANTO CHEMICAL Silica Gel 60 (spherical) 40–50 µm, Silica Gel 60 (spherical) 63–210 µm or Silica Gel 60 N (spherical, neutral) 63–210 µm. Reagents and solvents were commercial grades and were used as supplied with the following exceptions. 1) Dichloromethane, tetrahydrofuran and toluene: dried over molecular sieves 4A. 2) MeOH and acetonitrile: dried over molecular sieves 3A. All reactions sensitive to oxygen and/or moisture were conducted under an argon atmosphere.

Bromide 6

To a solution of 4 (10.0 g, 45.7 mmol) in CH2Cl2 (100 mL) and water (100 mL) were added Na2CO3 (9.68 g, 91.4 mmol) and NsCl (222 g, 43.5 mmol) at 0°C. The mixture was stirred at room temperature for 1 h. Then the mixture was quenched with 10% citric acid and extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was used for the following reaction without further purification. To a solution of crude material in CH2Cl2 (300 mL) were added Et3N (8.35 mL, 65.3 mmol), DMAP (532 mg, 4.35 mmol), and Boc2O (11.4 g, 52.2 mmol) at 0°C. The mixture was stirred at room temperature for 1 h. Then the mixture was quenched with saturated aqueous NH4Cl solution and extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was crystallized from EtOAc and n-hexane to afford 6 (18.4 g, quant. for the 2 steps from 4) as a light yellow solid. FT-IR (film) cm−1: 719, 743, 773, 853, 1123, 1150, 1175, 1256, 1288, 1339, 1366, 1541, 1724, 2980, 3102. 1H-NMR (CDCl3) δ: 1.38 (s, 9H), 2.32 (dt, J=6.8, 7.1 Hz, 2H), 3.46 (t, J=6.8 Hz, 2H), 3.92 (t, J=7.1 Hz, 2H), 7.71–7.79 (m, 3H), 8.28–8.34 (m, 1H) 13C-NMR (CDCl3) δ: 27.8, 28.2, 29.6, 33.1, 46.7, 85.4, 124.3, 131.8, 133.2, 133.3, 134.3, 147.5, 150.2. HR-MS (ESI-TOF) m/z: 445.0057 (Calcd for C14H19BrN2O6SNa+ [M+Na]+: 445.0039).

Ns Amide 8

To a solution of 7 (1.00 g, 13.3 mmol) in CH2Cl2 (26.5 mL) and water (26.5 mL) were added Na2CO3 (1.41 g, 13.3 mmol) and NsCl (2.79 g, 12.6 mmol) at 0°C. The mixture was stirred at room temperature for 3 h. Then the mixture was quenched with 10% citric acid and extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was used for the following reaction without further purification. To a solution of crude material in N,N-dimethylformamide (DMF) (1.26 mL) were added imidazole (2.57 g, 37.8 mmol) and TBSCl (2.85 g, 18.9 mmol) at 0°C under an argon atmosphere. The mixture was stirred at room temperature for 3 h. Then the mixture was quenched with saturated aqueous NH4Cl solution and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane–EtOAc=4 : 1) to afford 8 (4.68 g, 96% for the 2 steps from 7) as a yellow oil. FT-IR (film) cm−1: 780, 837, 854, 1094, 1169, 1345, 1360, 1420, 1536, 1560, 2858, 2929, 3349. 1H-NMR (CDCl3) δ: 0.04 (s, 6H), 0.86 (s, 9H), 1.73 (tt, J=5.7, 6.2 Hz, 2H), 3.23 (q, J=6.2 Hz, 2H), 3.68 (t, J=5.7 Hz, 2H), 5.68 (t, J=5.4 Hz, 1H), 7.70–7.74 (m, 2H), 7.81–7.86 (m, 1H), 8.10–8.14 (m, 1H). 13C-NMR (CDCl3) δ: −5.5, 18.2, 25.8, 31.8, 41.8, 61.0, 125.1, 131.0, 132.6, 133.4, 133.6, 148.1. HR-MS (ESI-TOF) m/z: 375.1406 (Calcd for C15H27N2O5SSi+ [M+H]+: 375.1404).

TBS-Protected 2-Mer 9

To a solution of 6 (30.0 g, 70.9 mmol), 8 (19.0 g, 50.6 mmol) and tetrabutylammonium iodide (TBAI) (3.70 g, 10.1 mmol) in MeCN (253 mL) was added Cs2CO3 (49.5 g, 152 mmol) at 0°C under an argon atmosphere. The mixture was stirred at 60°C for 2 h. Then the mixture was quenched with saturated aqueous NH4Cl solution and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane–EtOAc=1 : 2) to afford 9 (29.6 g, 82%) as a light yellow amorphous solid. FT-IR (film) cm−1: 657, 777, 837, 851, 961, 984, 1103, 1123, 1142, 1175, 1258, 1292, 1356, 1377, 1441, 1462, 1539, 1551, 1734, 2858, 2885, 2932, 2995, 3100. 1H-NMR (CDCl3) δ: 0.01 (s, 6H), 1.33 (s, 9H), 0.85 (s, 9H), 1.76 (dt, J=5.7, 7.4 Hz, 2H), 2.01 (quint, J=7.4 Hz, 2H), 3.38–3.46 (m, 4H), 3.60 (t, J=5.7 Hz, 2H), 3.72 (t, J=7.4 Hz, 2H), 7.58–7.62 (m, 1H), 7.63–7.78 (m, 5H), 7.98–8.04 (m, 1H), 8.23 (d, J=7.4 Hz, 1H). 13C-NMR (CDCl3) δ: −5.4, 18.3, 25.9, 27.8, 31.4, 44.7, 45.0, 45.4, 60.3, 85.4, 124.2, 124.5, 130.9, 131.8, 131.9, 133.1, 133.6, 134.5, 147.6, 148.0, 150.2. HR-MS (ESI-TOF) m/z: 739.2085 (Calcd for C29H44N4O11S2SiNa+ [M+Na]+: 739.2109).

Iodide 2-Mer 11

To a solution of 9 (5.18 g, 7.01 mmol) in MeOH (24.1 mL) was added CSA (838 mg, 3.61 mmol) at 0°C under an argon atmosphere. The reaction mixture was stirred at room temperature for 1 h. Then the mixture was quenched with Et3N, and concentrated under reduced pressure. The residue was poured into saturated aqueous NH4Cl solution, and the aqueous layer was extracted thoroughly with CH2Cl2. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. To a solution of the crude of alcohol 10 in CH2Cl2 (24.5 mL) were added Et3N (3.10 mL, 22.1 mmol) and MsCl (1.79 mL, 22.1 mmol) at 0°C under an argon atmosphere. The reaction mixture was stirred at room temperature for 10 min. Then the mixture was quenched with saturated aqueous NH4Cl solution, and it was extracted thoroughly with CH2Cl2. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude material of mesylate was used for the following reaction without further purification. To a solution of the crude of mesylate in 2-butanone (25.4 mL) was added NaI (3.42 g, 22.8 mmol) at 0°C under an argon atmosphere. The reaction mixture was stirred at 60°C for 1 h. Then the mixture was quenched with saturated aqueous Na2S2O3 solution, and it was extracted thoroughly with CH2Cl2. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane–EtOAc=1 : 1) to afford 11 (4.02 g, 78% for the 3 steps from 9) as a light yellow solid. FT-IR (film) cm−1: 842, 852, 974, 1103, 1123, 1157, 1260, 1292, 1356, 1369, 1438, 1543, 1734, 2937, 2980, 3099. 1H-NMR (CDCl3) δ: 1.37 (s, 9H), 2.02 (quint, J=7.4 Hz, 2H), 2.13 (dt, J=7.4, 6.8 Hz, 2H), 3.17 (t, J=6.8 Hz, 2H), 3.39–3.47 (m, 4H), 3.73 (t, J=7.4 Hz, 2H), 7.62–7.67 (m, 1H), 7.68–7.78 (m, 5H), 8.06–8.11 (m, 1H), 8.26–8.33 (m, 1H). 13C-NMR (CDCl3) δ: 1.5, 27.8, 28.6, 32.0, 45.1, 45.3, 47.9, 85.5, 124.3, 124.4, 130.1, 131.8, 131.9, 132.8, 133.2, 133.7, 134.3, 147.5, 147.9, 150.1. HR-MS (ESI-TOF) m/z: 735.0262 (Calcd for C23H29IN4O10S2Na+ [M+Na]+: 735.0262).

Protected Thermospermine 13

To a solution of iodide 11 (4.70 g, 6.60 mmol) and Ns amide 12 (2.00 g, 5.37 mmol) in MeCN (15.7 mL) was added Cs2CO3 (4.60 g, 14.1 mmol) at 0°C under an argon atmosphere. The mixture was stirred at 60°C for 2 h. Then the mixture was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane–EtOAc=1 : 1) to afford 13 (5.14 g, quant.) as a light yellow amorphous solid. FT-IR (film) cm−1: 837, 851, 961, 984, 1103, 1123, 1142, 1175, 1258, 1292, 1356, 1377, 1441, 1462, 1539, 1551, 1734, 2858, 2885, 2932, 2955, 3099. 1H-NMR (CDCl3) δ: 1.32 (s, 9H), 1.42 (s, 9H), 1.43–1.49 (m, 2H), 1.56 (quint, J=6.8 Hz, 2H), 1.86 (m, 2H), 1.96 (quint, J=7.4 Hz, 2H), 3.08 (q, J=6.2 Hz, 2H), 3.27–3.33 (m, 6H), 3.36 (t, J=7.4 Hz, 2H), 3.69 (t, J=7.4 Hz, 2H), 4.64 (br s, 1H), 7.58–7.65 (m, 2H), 7.65–7.78 (m, 7H), 7.97–8.04 (m, 2H), 8.23–8.30 (m, 1H). 13C-NMR (CDCl3) δ: 25.4, 27.0, 27.8, 28.4, 28.6, 31.9, 44.7, 45.0, 45.3, 47.3, 85.5, 124.16, 124.24, 124.4, 130.1, 131.8, 131.9, 132.8, 133.2, 133.7, 134.3, 147.5, 147.9, 150.1, 156.9. HR-MS (ESI-TOF) m/z: 980.2435 (Calcd for C38H51N7O16S3Na+ [M+Na]+: 980.2447).

Ns-Protected Thermospermine on Resin 16

To a solution of 13 (5.14 g, 5.37 mmol) in MeOH (33.0 mL) was added SOCl2 (4.09 mL, 56.4 mmol) at 0°C under an argon atmosphere. The mixture was stirred at room temperature for 2 h. Then the reaction mixture was evaporated to give the crude of 14. To a suspension of the freshly prepared resin 15 (27.2 mmol),16) the crude material of 14 and DMAP (3.33 g, 27.2 mmol) in DMF (215 mL) was added N,N-diisopropylethylamine (DIPEA) (14.2 mL, 81.7 mmol). The mixture was slowly stirred for 24 h at room temperature. Then the mixture was washed with DMF, H2O, 10% MeOH in CH2Cl2, CH2Cl2 and dried in vacuo for 5 h. For analytical purpose, a small amount of the resin 16 was treated with 3% trifluoroacetic acid (TFA) in CH2Cl2, the suspension was filtered and the resin was washed with 10% MeOH in CH2Cl2. The filtrate was concentrated in vacuo to afford primary amine 14 as a brown oil. HR-MS (ESI-TOF) m/z: 758.1603 (Calcd for C28H36N7O16S3+ [M+H]+: 758.1579).

Thermospermine (2)

To a suspension of the resin 16 in N,N-dimethylformamide (DMF) (180 mL) were added DBU (23.1 mL, 164 mmol) and 2-mercaptoethanol (11.5 mL, 164 mmol) at 0°C under an argon atmosphere. The mixture was slowly stirred at room temperature for 24 h. Then the mixture was washed with CH2Cl2, MeOH, dimethyl sulfoxide (DMSO), CH2Cl2, MeCN and dried in vacuo for 10 h. The resin was treated with 3% TFA in CH2Cl2, the suspension was filtered and the resin was washed with 10% MeOH in CH2Cl2. The combined washings were evaporated and dried in vacuo to afford a pale yellow amorphous solid of 2 (1.1 g, quant. for the 4 steps from 13). FT-IR (film) cm−1: 1136, 1163, 1198, 1670. 1H-NMR (CD3OD) δ: 1.71–1.83 (m, 4H) 2.02–2.18 (m, 4H), 2.98 (t, J=7.4 Hz, 2H), 3.04–3.10 (m, 4H), 3.11–3.19 (m, 6H). 13C-NMR (CD3OD) δ: 24.1, 24.2, 25.3, 25.5, 37.8, 40.0, 45.8, 46.0, 48.2. HR-MS (ESI-TOF) m/z: 225.2045 (Calcd for C10H26N4Na+ [M+Na]+: 225.2050).

Protected Spermine 19a

To a solution of 17 (100 mg, 278 µmol) and TBAI (23 mg, 63 µmol) in MeCN (500 µL) were added Cs2CO3 (272 mg, 835 µmol) and 1,4-dibromobutane (18a) (15 µL, 126 µmol) at 0°C under an argon atmosphere. The mixture was stirred at 60°C for 2 h. Then the mixture was quenched with saturated aqueous NH4Cl solution and extracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane–EtOAc=1 : 2) to afford 19a (105 mg, quant.) as a light yellow amorphous solid. FT-IR (film) cm−1: 584, 737, 779, 853, 1061, 1126, 1163, 1252, 1269, 1344, 1367, 1439, 1458, 1516, 1545, 1701, 2934, 2976. 1H-NMR (CDCl3) δ: 1.43 (s, 18H), 1.47–1.53 (m, 4H), 1.70 (t, J=6.8 Hz, 4H), 3.05–3.15 (m, 4H), 3.23–3.33 (m, 8H), 4.83 (br s, 2H), 7.60–7.64 (m, 2H), 7.66–7.73 (m, 4H), 7.94–8.00 (m, 2H). 13C-NMR (CDCl3) δ: 25.0, 28.3, 28.6, 37.4, 45.1, 46.9, 77.3, 124.2, 130.4, 131.8, 133.0, 133.6, 147.9, 156.0. HR-MS (ESI-TOF) m/z: 795.2639 (Calcd for C32H48N6O12S2Na+ [M+Na]+: 795.2664).

Ns-Protected Spermine on Resin 21a

To a solution of 19a (105 mg, 278 µmol) in MeOH (1 mL) was added SOCl2 (404 µL, 5.56 mmol) at 0°C under an argon atmosphere. The mixture was stirred at room temperature for 2 h. Then the reaction mixture was evaporated to give the crude material of 20a. To a suspension of the freshly prepared resin (1.39 mmol) and the crude material of 20a in DMF (5 mL) were added DMAP (170 mg, 1.39 mmol) and DIPEA (726 µL, 4.17 mmol). The mixture was slowly stirred for 24 h at room temperature. Then the mixture was washed with DMF, CH2Cl2, MeOH, H2O, DMF, MeOH, CH2Cl2, MeCN, CH2Cl2 and dried in vacuo for 2 h. For analytical purpose, a small amount of the resin 21a was treated with 3% TFA in CH2Cl2, the suspension was filtered and the resin was washed with 10% MeOH in CH2Cl2. The filtrate was concentrated in vacuo to afford primary amine 20a as a brown oil. HR-MS (ESI-TOF) m/z: 572.1718 (Calcd for C22H32N6O8S2+ [M]+ 572.1718).

Spermine (1)

To a suspension of the resin 21a in DMF (5.00 mL) were added DBU (831 µL, 5.56 mmol) and 2-mercaptoethanol (391 µL, 5.56 mmol) at 0°C under an argon atmosphere. The mixture was slowly stirred at room temperature for 24 h. Then the mixture was washed with DMF, CH2Cl2, MeOH, DMSO, CH2Cl2, MeCN, CH2Cl2 and dried in vacuo for 2 h. The resin was treated with 3% TFA in CH2Cl2, the suspension was filtered and the resin was washed with 10% MeOH in CH2Cl2. The combined washings were evaporated and dried in vacuo to provide pale yellow amorphous solid of 1 (30 mg, 52% for the 4 steps from 19a) as the TFA salt. FT-IR (film) cm−1: 1177, 1120, 1670. 1H-NMR (CD3OD) δ: 1.78–1.84 (m, 4H), 2.08 (quint, J=7.9 Hz, 4H), 3.03–3.10 (m, 8H), 3.31 (t, J=7.9 Hz, 4H). 13C-NMR (CD3OD) δ: 24.2, 25.3, 37.8, 45.8, 48.2. HR-MS (ESI-TOF) m/z: 203.2236 (Calcd for C10H27N4+ [M+H]+ 203.2230).

Protected Norspermine (19b)

To a solution of 17 (100 mg, 278 µmol) and TBAI (23 mg, 63 µmol) in MeCN (500 µL) were added Cs2CO3 (272 mg, 835 µmol) and 1,3-dibromopropane (18b) (12.8 µL, 126 µmol) at 0°C under an argon atmosphere. The mixture was stirred at 60°C for 2 h. Then the mixture was quenched with saturated aqueous NH4Cl solution and extracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane–EtOAc=1 : 1) to afford 19b (80.5 mg, 85%) as a light yellow amorphous solid. FT-IR (film): 565, 583, 739, 777, 853, 1163, 1346, 1368, 1512, 1543, 1690 cm−1; 1H-NMR (CDCl3) δ: 1.42 (s, 18H), 1.65–1.77 (m, 4H), 1.83 (t, J=7.2 Hz, 2H), 3.11 (q, J=6.3 Hz, 4H), 3.26 (t, J=7.4 Hz, 4H), 3.31 (t, J=7.2 Hz, 4H), 4.90 (br s, 2H), 7.59–7.64 (m, 2H), 7.67–7.77 (m, 4H), 7.94–7.99 (m, 2H). 13C-NMR (CDCl3) δ: 27.4, 28.4, 28.6, 37.4, 45.2, 45.6, 79.3, 124.2, 130.6, 131.9, 132.8, 133.7, 148.0, 156.0; HR-MS (ESI-TOF) m/z: 781.2532 (Calcd for C31H46N6O12S2Na+ [M+Na]+: 781.2507).

Ns-Protected Norspermine on Resin 21b

To a solution of 19b (8.10 g, 10.7 mmol) in MeOH (50 mL) was added SOCl2 (7.75 mL, 107 mmol) at 0°C under an argon atmosphere. The mixture was stirred at room temperature for 2 h. Then the reaction mixture was evaporated. The crude material of 20b was used for the following reaction without further purification. To a suspension of the freshly prepared resin (54.1 mmol) and the crude material of 20b in DMF (260 mL) were added DMAP (6.52 g, 53.4 mmol) and DIPEA (27.9 mL, 160 mmol). The mixture was slowly stirred for 24 h at room temperature. Then the mixture was washed with DMF, CH2Cl2, MeOH, H2O, DMF, MeOH, CH2Cl2, MeCN, CH2Cl2 and dried in vacuo for 10 h. For analytical purpose, a small amount of the resin the crude material of 21b was treated with 3% TFA in CH2Cl2, the suspension was filtered and the resin was washed with 10% MeOH in CH2Cl2. The filtrate was concentrated in vacuo to afford primary amine 20b as a brown oil. HR-MS (ESI-TOF) m/z: 559.1627 (Calcd for C21H31N6O8S2+ [M+H]+: 559.1639).

Norspermine (3)

To a suspension of the resin 21b in DMF (360 mL) were added DBU (31.9 mL, 213 mmol) and 2-mercaptoethanol (15.0 mL, 213 mmol) at 0°C under an argon atmosphere. The mixture was slowly stirred at room temperature for 24 h. Then the mixture was washed with DMF, CH2Cl2, MeOH, DMSO, CH2Cl2, MeCN, CH2Cl2 and dried in vacuo for 10 h. The resin was treated with 3% TFA in CH2Cl2, the suspension was filtered and the resin was washed with 10% MeOH in CH2Cl2. The combined washings were evaporated and dried in vacuo to provide a pale yellow amorphous solid of 3 (2.1 g, quant. for the 4 steps from 19b) as the TFA salt. FT-IR (film) cm−1: 1198, 1676, 2947. 1H-NMR (DMSO-d6) δ: 1.86–2.01 (m, 6H), 2.84–2.93 (m, 4H), 2.94–3.03 (m, 8H), 7.99 (br s, 6H), 8.99 (br s, 4H). 13C-NMR (DMSO-d6) δ: 22.5, 23.9, 36.3, 44.2, 115.6, 118.0, 158.8, 159.1. HR-MS (ESI-TOF) m/z: 189.2080 (Calcd for C9H25N4H+ (M+H)+: 189.2074).

Acknowledgments

This work was financially supported by MEXT/JSPS KAKENHI Grant Numbers 23390007, Grants-in-Aid for Scientific Research on Priority Areas 26102736 and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and Japan Agency for Medical Research and Development (AMED).

Conflict of Interest

The authors declare no conflict of interest.

References
 
© 2016 The Pharmaceutical Society of Japan
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