Syntheses of crowned polyamines using isolable succinimidyl esters of N -tritylated linear amino acids and peptides

Partial and total syntheses of polyamines incorporating crown ether moieties have been effected using N-tritylated linear amino acids, like β -alanine ( β Ala) and γ -aminobutyric acid ( γ Aba), to introduce the N–3-C and N–4-C polyamine structural units, respectively. The partial syntheses involve the acylation of commercially available crown bearing amino function(s) or aza-oxa crown ethers with the isolable succinimidyl esters of Trt-β Ala-OH or Trt-β Ala-γ Aba-OH, followed by LiAlH 4 -mediated reduction of the resulting amides, whereas in the total syntheses the crown and the aza-oxa crown ether moieties are built-up from commercially available starting materials like polyethylene glycols, epichlorohydrin and dibenzylamine.


Introduction
Linear polyamines (PAs), like spermidine (SPD) and spermine (SPM) and their conjugates (PACs) with other natural products are widely distributed in the living organisms and are associated with interesting biological functions.For this reason, they have attracted considerable interest, and a variety of PA analogues and PACs have been already synthesized in order to determine structure-activity relationship and to identify possible leads for the development of PA-based pharmaceuticals. 1 On the other hand, the cyclic oligomers of ethylene oxide, collectively known as crown ethers (CEs), and their analogues and derivatives are widely used for complexing and separating metal or organic cations, as well as in the areas of phase transfer catalysis, host-guest and supramolecular chemistry. 2Different types of CEs have been synthesized in order to improve their properties and find suitable applications.In addition, a variety of pharmaceutically interesting compounds have been equipped with crown moieties in order to modify their physiological action. 3Accordingly, we decided to examine the possibility of introducing CE moieties into PA molecules and study the complexing and biological properties of this new family of organic compounds, which we collectively coin as crowned polyamines (CRPAs).We now wish to report a general method that we have recently developed which facilitates access to CRPAs;.CEs is commercially available 4 but we have developed alternative methods of preparation.For the introduction of CE moieties into various polyamine skeletons as the purpose of this work, we have chosen the commercially available aza-oxa CEs 1 and 2 (Figure 1), the lariat aminomethyl CE 3 and the diaminodibenzo-CE 4, a mixture of the isomers 4a and 4b.Furthermore, we used simple commercially available building blocks, like polyethylene glycols, epichlorihydrin, and dibenzylamine, in order to build-up various CE skeletons to the appropriate size and content of N atoms, and the 'isolable′ active esters 5 and 6, readily available from the corresponding linear amino acids β-alanine (βAla) and γ-aminobutyric acid (γAba), 5 as N-3-C and N-3-C-N-4-C synthons for the assembly of various PA skeletons.Scheme 1. Syntheses of succinimidyl active esters and of linear polyamines (the numbers on the structures serve for the presentation of the NMR data).(i) Me 3 SiCl, CH 2 Cl 2 /MeCN (5:1), reflux, 30 min.(ii) Et 3 N/TrtCl, 0 ºC, 1 h then 25 ºC, 3 h.(iii) MeOH, 97% (7) or 79% (8).(iv) HOSu/DCC, THF/DMF (3:1), 0 ºC, 1 h then 25 o C, 12 h, 85% (5) or 88% (6).(v) Me 3 SiCl, CH 2 Cl 2 , 25 ºC, 10 min.(vi) 5/Et 3 N, 0 ºC, 45 min then 25 ºC, 15 min.(vii) PUT or DAO/Et 3 N, DMF, 25 ºC, 12-24 h, 90% (9a) or 78% (9b) or 82% (11).(viii) LiAlH 4 , THF, reflux, 2-4 d; 68% (10a) or 61% (10b).(ix) Boc 2 O/Et 3 N/cat.DMAP, CHCl 3 , 0 ºC, 30 min then 25 ºC, 12 h, 30%.(x) TFA/CH 2 Cl 2 (1:1), 25 ºC, 30 min, 75%.
O-Silylation of βΑla, followed by N-tritylation and desilylation with MeOH, 7 provided Trt-βAla 7 in 97% yield.SAE 5 was then obtained in 85% yield by the condensation of 7 with of N-hydroxysuccunimide (HOSu) in the presence of N,N′-dicyclohexylcarbodiimide (DCC).5a On the other hand, O-silylation of γAba, followed by N-acylation with SAE 5 and desilylation with MeOH gave the N-tritylated dipeptide 8 in 79% yield.From this compound, SAE 6 was easily obtained in 88% yield upon routine activation also with HOSu in the presence of DCC.5b The applicability of these compounds to build any type of tetra-or hexa-amines of the 3-n-3 or the 3-4-n-4-3 types was readily exemplified by the facile preparation of the tetra-amine derivatives 10a and 10b (n = 4 and 8, respectively), and the hexa-amine 14 (n = 4).Thus, condensation of SAE 5 with 1,4-diaminobutane (putrescine, PUT) gave the expected bisamide 9a in 90% yield.This was then reduced with LiAlH 4 to produce N 1 ,N 12 -Trt 2 -SPM 10a in 68% yield.5a Later, the selective direct ditritylation of SPM to produce 10a was reported. 8On the other hand, bisacylation of 1,8-diaminooctane (DAO), followed by LiAlH 4 reduction of the bisamide 9b produced the N 1 ,N 16 -ditritylated tetra-amine 10b in 61% yield. 4 Similarly, bisacylation of PUT with SAE 6 produced the tetra-amide 11 in 82% yield.LiAlH 4 reduction of all four amide functions took place unexceptionally and the thus obtained crude PA derivative 12 was per-tertbutoxycarbonylated to facilitate purification by flash column chromatography (FCC).The resulting pure, fully protected, hexa-amine 13 (30% overall yield) was deprotected with a 50% solution of trifluoroacetic acid (TFA) in CH 2 Cl 2 to produce the 3-4-4-4-3 hexa-amine 14 as the hexatrifluoroacetate salt, in 75% yield. 4 In this general methodology, diversion may readily arise from the linear or even branched (e.g.chiral) amino acids or peptides, and the linear or branched or cyclic α,ω-diaminoalkanes used to furnish the N-C n -N central core of the polyamine.Finally, TFA detritylation gave the tetra(trifluoroacetate) salt of CE 23 as in 90% yield. 4 This compound may be considered as a crowned oxa-SPM analogue.When SAE 6 was used to bisacylate diaza-oxa-18-CE-6 2, the expected tetra-amide 24 was obtained in 92% yield.LiAlH 4 reduction of this compound gave a 64% yield of CE derivative 25, from which the hexa(trifluoroacetate) salt of CRPA 26 was obtained in 90% yield. 4 This compound may be considered as a dioxa-3-4-8-4-3 PA analogue.It should be noted that the corresponding dioxa-3-8-3 PA analogue has been obtained by bisalkylation of CE 2 with acrylonitrile and subsequent catalytic hydrogenation over Raney Ni. 10

Total syntheses of polyamines incorporating crown ether moieties
Having established the viability of this method we went on to synthesize more complex CRPAs.Taking into consideration the fact that the CEs so far used are commercially available compounds but very expensive for large scale applications, we decided to develop an alternative method for their preparation.For example, we envisaged that aminomethyl CE 3 could be readily produced using as a key-reaction the nucleophilic displacement of the Cl atom of epichlorohydrin (27) by a synthetically suitable amine.We have used two amines in the past, namely TrtNH 2 11 and Bn 2 NH, 5b to introduce the amino function into the γ-carboxy group of kainic acid, and at position 8 of SPD, respectively.It should be noted that aminomethyl CEs have been obtained so far either from the corresponding hydroxymethyl CEs through phthalimidation, followed by hydrazinolysis 12 or displacement of the Cl atom of chloromethylated glycol or oligoethylene glycols by ammonia or primary amines followed by CE formation by reaction with the appropriate oligoethylene glycol ditosylates or dichlorides. 13n addition, it had been reported that the reaction of hindered amines with 27 leads to either 3azetidinols in fair yields with primary amines and to a variety of products, some of which coming from EtOH used as the reaction solvent, with secondary amines. 14To avoid these solvent-induced side-reactions we performed our exploratory experiments in 27 as the reaction medium.Much cleaner and reproducible results we obtained with Bn 2 NH in place of TrtNH 2 , and therefore, we used Bn 2 NH for the preparation of aminomethyl CEs.
In fact, reaction of Bn 2 NH with 27 in the presence of i Pr 2 NEt as HCl scavenger, at 110 ºC for 2 h produced a mixture of two compounds which were readily separated in a small scale experiment by FCC and identified as the chlorohydrin 28 and the epoxide 29 (Scheme 4).Although this reaction mixture would have been used as such for the following transformation, namely the reaction with an oligoethylene glycol (OEG) in the presence of NaH, much better results were obtained when this mixture (ratio 28:29 = 3:2; or 2:3) by NMR, was first cleanly converted to the epoxide 29 (67% overall yield based on Bn 2 NH) by treatment with NaH in THF at ambient temperature.NaH-catalyzed nucleophilic ring-opening of epoxides by OEGs have been used for the production of higher homologues of OEGs, suitable for CEs preparation. 15hus, treatment of epoxide 29 with diethylene glycol (DEG) in the presence of a catalytic quantity of NaH at refluxing THF for 2 days produced the anticipated triethylene glycol (TEG) derivative 30 in 75% yield.Preparation of the desired aminomethyl CE derivative 31 required as the final step the condensation of diol 30 with triethylene glycol ditosylate (TGT).Although several OEG ditosylates are commercially available, we used for the large scale preparation of DGT the method prescribed in ref. 2d, p. 84, an adaptation of the method by Ouchi et al, 16 TGT and tetraethylene glycol ditosylate (TEGT; for its application cf. the following discussion).This method involves treatment of OEGs with tosyl chloride in the presence of NaOH in a mixture of THF/H 2 O (2:1) as the solvent.On the other hand, an improved method has been recently published for the preparation of CEs exploiting the significant enhancement of the alkylation efficiency of alkoxides by ditosylates by using an unwashed 60% NaH dispersion in anhydrous DMSO. 17 Indeed, using this method, the anticipated compound 31 was obtained in 57% yield.From this compound, the amino function was unmasked by catalytic hydrogenolysis at 3 atm and ambient temperature, using Pearlman′s catalyst [Pd(OH) 2 on C] to remove both benzyl groups.5b,6b The aminomethyl CE 3 (obtained in 96% yield) was acylated without any further purification by SAE 5 to introduce the first N-3-C-N unit of SPM into CE.FCC purification afforded amide 32a in 93% yield.This compound was detritylated with a solution of TFA in CH 2 Cl 2 , and the amino function was acylated with SAE 6 in the presence of Et 3 N in order to introduce the remaining 4-C-N-3-C-N of SPM into CE.The expected trisamide 33 was obtained in 75% overall yield.Reduction of this amide with LiAlH 4, followed by complete benzyloxycarbonylation with benzyloxycarbonyl chloride (ZCl) produced the fully protected crowned SPM (CRSPM) 34 in 60% overall yield. 4 Complete benzyloxycarbonylation serves two purposes.It facilitates purification by FCC of the crude product produced by LiAlH 4 reduction of the amide functions and allows selective removal of the Trt group in the presence of the Z groups by mild acidolysis for further primary amino function modifications.For example, detritylation of CRSPM derivative 34 followed by guanidylation of the free primary amino group with the commercially available reagent 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea (BZMTU) produced the fully protected guanidylated CRSPM derivative 36 in 70% yield.Complete deprotection is then readily effected by catalytic hydrogenolysis to produce the final product, the guanidylated CRSPM 37 in 70% yield.Natural and synthetic guanidylated polyamines 1a and other types of organic compounds incorporating the guanidinium function 18 show very interesting biological properties, due to the fact that this function interacts strongly through hydrogen bonds and electrostatic interactions with other functional groups present in enzymes or receptors.
We then decided to synthesize diaza-oxa CEs incorporating PA elements in their skeleton, such as the CRPAs 23, 43, 48 and 51 (Scheme 5).We envisaged that CEs of this type might be readily available from a common precursor, i.e. the ditritylated oxo-SPM analogue 41, by alkylation with various OEG diiodides (OEGIs).We based our reasoning on a well-established methodology (the Dale reaction) to produce aza-oxa CEs through the alkylation of primary or secondary diamines by OEGIs in the presence of anhydrous Na 2 CO 3 . 19OEGIs are readily available from the corresponding ditosylates OEGTs through the classical displacement with NaI in acetone (see experimental section) whereas oxa-SPM derivative would be assembled from the diamine 39 and SAE 5.Although several OEG α,ω-diamines (OEGAs) are commercially available, we obtained the required OEGA 39 from DGT through the classical two-steps sequence involving displacement with potassium phthalimide, followed by hydrazinolysis.For the purpose of this work, we isolated the bisphthalylhydrazide salt of OEGA 39 and used it as such for the coupling with SAE 5 in the presence of Et 3 N.The expected bisamide 40 was obtained in 77% yield.LiAlH 4 reduction of bisamide 40 proceeded unexceptionally to give the key-intermediate 41 in 85% yield.Bisalkylation of this intermediate with TGI in dry MeCN in the presence of anhydrous Na 2 CO 3 produced ditritylated CRPA 22 in very good yield (73%), whereas its bisalkylation by TEGI in the presence of anhydrous K 2 CO 3 gave access to the next higher CE homologue 42 in 66% yield.These CEs incorporate a symmetric oxa-3-5-3 tetraamine skeleton and can be considered as oxa-SPM analogues.Detritylation of these compounds provided the corresponding CRPAs 23 and 43, which upon coupling with the SAE 6, also in the presence of Et 3 N, gave the tetra-amides 44 and 45 in 68 and 55% yield, respectively.These tetra-amides were subsequently reduced with LiAlH 4 at refluxing THF to produce the corresponding crude ditritylated CRPAs 46 and 49.For purification by FCC, these compounds were per-tert-butoxycarbonylated with bis(tert-butyl)dicarbonate (Boc 2 O) in the presence of a catalytic amount of 4-(dimethylamino)pyridine (DMAP) to give the fully protected CRPAs 47 and 50 in 35% and 26% overall yields, respectively, based on the starting tetra-amides.Finally, TFA-mediated detritylation of these intermediates gave the desired CRPAs 48 and 51 in 88% and 83% yield, respectively.These CEs incorporate a symmetric oxa-3-4-3-5-3-4-3 octa-amine skeleton.

Conclusions
Linear and crowned polyamines of variable length or ring-sizes and number of nitrogen functions in their skeleton can be readily assembled using the isolable succinimidyl active esters of the N-tritylated amino acid βAla and peptide βAla-γAba to couple to α,ω-diamino-alkanes and -dibenzocrown ethers, diaza-oxa crown ethers and aminomethyl crown ethers, followed by LiAlH 4 -mediated reduction of the thus obtained polyamides.The required amino components were either commercially available or were readily synthesized in our laboratory employing, also commercially available, building blocks like oligoethylene glycols, epichlorohydrin and dibenzylamine and the alkylation of either oligoethylene alkoxides by oligoethylene tosylates or oligoethylene diamines by oligoethylene diiodides as key-reactions for the assembly of the crown or diaza-oxa crown ether skeleta, respectively.Further applications of this general approach to the synthesis of other types of crowned polyamines and conjugates and the evaluation of the metal-complexing and biological properties of the novel crowned polyamines described in this work are currently under investigation.

Experimental Section
General Procedures.Melting points were determined with a Buchi SMP-20 apparatus and are uncorrected.IR spectra were recorded for KBr pellets on a Perkin Elmer 16PC FT-IR spectrophotometer. 1 H-NMR spectra were obtained at 400.13 MHz and 13 C-NMR spectra at 100.62 MHz on a Bruker DPX spectrometer.CDCl 3 and tetramethylsilane (TMS) were used as the solvent and internal standard, respectively, unless otherwise stated.Chemical shifts are reported in δ units, parts per million () downfield from TMS.The assignments of the 1 H spectra are based on chemical shift arguments, analysis of coupling patterns and signal intensities whereas the 13 C spectra were assigned taking into consideration chemical shift arguments.Data for the aromatic region (trityl group) are omitted for 1 H and 13 C NMR spectra of the N-tritylated compounds for the sake of brevity.The values observed for compound 7 are typical: 1 H NMR: δ 7.417 (6H, d, J = 8.0 Hz, o-H), 7.268 (6H, t, J = 7.6 Hz, m-H), 7.191 (3H, t, J = 7.2 Hz, p-H). 13C NMR: δ 146.321 (ipso-C), 128.957 (o-C), 128.460 (m-C), 126.630 (p-C).
Electron-impact ionization mass spectra (EI-MS) were obrtained on a Fisons VG 7070E mass spectrometer with electron beam energy of 70 eV.Fast atom bombardment (FAB) mass spectra were recorded on a Fisons VG ZAB 2F operating at an accelerating potential of 8 kV and neutral xenon beam of 9 eV and using m-nitrobenzyl alcohol (NBA) as the matrix.Liquid secondary ion ionization (LSI) mass spectra in the positive mode were obtained on a Fisons VG ZAB-T four-sector instrument.The compounds were ionized using a caesium ion gun operated at an acceleration voltage of 30 kV.NBA was also used as the matrix solution which was carefully mixed with a 1 µL of sample solution in CHCl 3 on the tip of the probe prior to analysis.Electron-spray ionization (ESI) mass spectra were recorded on a Micromass-Platform LC spectrometer using MeOH or MeCN as solvents.The ESI high-resolution MS (HR-MS) experiments were carried out in a hybrid QqTOF mass spectrometer equipped with an ion spray ionization source.Compounds were dissolved in a solution containing 0.1% acetic acid in methanol/water 50/50 and analyzed by direct infusion (5 L/min) at the optimum ion spray voltage of 4800V.The nitrogen gas flow was set at 30 psi, whereas the orifice, the focusing ring and the skimmer voltages were kept at 30, 50 and 25V, respectively.MS/MS experiments were performed in the collision cell q on the isotopically pure ( 12 C) peak of the selected precursor ions by keeping the Q1 at 20 or 30V and unit resolution, and scanning the TOF analyser.All the acquisitions were averaged over 50 scans at 8000 TOF resolution with a standard deviation of 0.01 cps.Microanalyses were performed on a Carlo Erba EA 1108 CHNS elemental analyzer in the Center of Instrumental Analysis of the University of Patras.

N-Trityl-β-alanine (7).
To a magnetically stirred suspension of βAla (17.82 g, 0.2 mol) in anhydrous CH 2 Cl 2 (250 mL) and MeCN (50 mL) was added Me 3 SiCl (27.9 mL, 0.22 mol), and the reaction mixture was refluxed with exclusion of moisture for 30 min.To the ice-cooled (0 ºC) resulting solution anhydrous Et 3 N (61 mL, 0.44 mol) was added dropwise followed by the addition of TrtCl (58.5 g, 0.21 mol) in three equal portions within 15 min.Stirring was continued at 0 ºC for 1 h, and at ambient temperature for 3 h.MeOH (20 mL) was then introduced into the reaction mixture and the solvents were evaporated to dryness.To the resulting residue, aqueous NaOH solution (1N, 700 mL) was added and then extracted twice with Et 2 O.The aqueous phase was then ice-cooled and brought to pH 5 by the dropwise addition of glacial AcOH.A 5% aqueous citric acid solution was then added to further acidify the reaction mixture until no more precipitate was formed.The precipitate was filtered and washed on the filter twice with H 2 O and then with ice-cold Et 2 O and finally dried under vacuo at 40 ºC overnight to give pure product 7 (64.3g. 97%), mp 166-69 ºC; R f (G) 0.33.FT-IR: 3455-2500 (CO-OH), 3298 (NH-Trt), 1708 (CO-OH), 1594 (Ph C=C) cm -1 .EI-MS (m/z): 331 (M), 243 (Trt), 165 (Trt-PhH). 1

ISSN 1424-6376
Page 97 © ARKAT USA, Inc However, in a small scale experiment (2 mL from each of Bn2NH and iPr2NEt and 3 mL of 27), the resulting mixture was separated by FCC using solvent system A as eluant to give pure alcohol 28 and epoxide 29, both as oils.

2-Dibenzylaminomethyl-18-crown ether-6 (31).
To a solution of diol 30 (4.8 g, 13.4 mmol) in anhydrous DMSO (50 mL) was first added NaH (2.68 g of a 60% dispersion in oil, 67 mmol) and then TGT (6.15 g, 13.4 mmol).The resulting reaction mixture was stirred at ambient temperature for 2 d and then diluted with H 2 O (150 mL) and brine (50 mL).The resulting mixture was extracted three times with Et 2 O, the organic layers were combined, washed three times with brine and finally dried (anhydrous MgSO 4 ) and evaporated to dryness to leave an oily residue.This residue was subjected to FCC using solvent system G as eluant to give pure product 31 (3.62

2-Aminomethyl-18-crown ether-6 (3).
To a solution of crown ether 31 (2.2 g, 4.6 mmol) in MeOH (30 mL) was added of Pearlman′s catalyst (0.5 g), and the resulting mixture was subjected to catalytic hydrogenation at 3 atm pressure at ambient temperature for 3 d.Filtration of the catalyst and evaporation of the MeOH filtrate to dryness left the oily crown ether 3 (1.3 g, 96%), R f (G) 0.11.This product 3 was identical in all respects with a commercially available sample and was used without any further purification in the preparation of amide 32a.2-(5′-Tritylamino-3′-oxo-2′-azapentyl)-1,4,7,10,13,16-hexaoxacyclohexadecane (32a).To a solution of aminomethyl crown 3 (1.3 g, 4.43 mmol) and dry Et 3 N (0.62 mL, 4.43 mmol) in anhydrous DMF (5 mL) was added in three equal portions within 30 min SAE 5 (1.9 g, 4.43 mmol).The resulting solution was kept at ambient temperature overnight and was then diluted with CHCl 3 and washed sequentially with a 5% aqueous NaHCO 3 solution (twice) and with brine (once), dried (MgSO 4 ) and evaporated to dryness to leave an oily residue.Following FCC purification with solvent system J as eluant pure amide 32a (2.5 g, 93%) was obtained as a foam, R f (J) 0. This oil was dissolved in anhydrous DMF (3 mL) and treated at 0 ºC in turn with Et 3 N (0.5 mL, 3.8 mmol) and SAE 6 (0.98 g, 1.9 mmol) for 1 h and then at ambient temperature for 1 d.Identical work-up as for 32a and FCC purification using solvent system G for elution gave pure trisamide 33 (1.13 g, 79%) as a foam, R f (G) 0.26.FT-IR: 3284 (NH), 1652 (NHCO) cm -1 .A solution of trisamide 33 (0.7 g, 0.92 mmol) in anhydrous THF (8 mL) was added dropwise within 30 min to a magnetically stirred suspension of LiAlH 4 (0.31 g, 8.3 mmol) in refluxing THF (3 mL).The reaction mixture was stirred at this temperature for 4 d and was then worked-up as described for the preparation of 10a to give crude tetra-amine 34 (0.45 g, 0.63 mmol).This was dissolved in dry CHCl 3 (5 mL) and treated at 0 ºC in turn with anhydrous i Pr 2 NEt (0.43 mL, 2.5 mmol) and ZCl (0.29 mL, 2 mmol) for 30 min and at ambient temperature for overnight.The resulting solution was diluted with CHCl 3 and washed sequentially twice with a 5% aqueous NaHCO 3 solution and once with H 2 O, dried (MgSO 4 ) and evaporated to leave an oily residue.Upon FCC purification using solvent system F for elution pure product 34 (0.60 g, 60% based on trisamide 33) was obtained as an oil, R f (F) 0.36.HR-MS (m/z): Found 1,123.6032(M + +1), C 66 H 83 N 4 O 12 requires M + +1 = 1,123.6008.

Figure 1 .
Figure 1.Structures of compounds related to this work.

Scheme 2 .
Scheme 2. Partial syntheses of crown ethers incorporating polyamine elements (the numbers on the structures serve for the presentation of the NMR data).