Recent developments in guanylating agents

Guanidines are important molecules with a wide range of interesting properties. In this overview we summarize recent advances in the development of guanylating reagents which we define as compounds forming a guanidine structure by a chemical transformation. We cover important classes of guanylating agents developed in the last two decades and representative examples are reported


Introduction
Guanidines possess great biochemical and pharmaceutical importance.Guanidine itself is a strong base (pKa of conjugated acid is 12.5) as are substituted guanidines.1a Guanidine was first prepared by Strecker in 1861 by oxidizing guanine.The biological role, chemical and biochemical properties of natural and synthetic guanidine derivatives have been outlined.1b The natural amino acid, L-arginine 1, is often found at active (or catalytic) sites in proteins and enzymes; it is critical for the normal function of living organisms.Many natural guanidines have been isolated and tested for biological activity.The isolation, structural identification and synthesis of naturally occurring guanidines have been reviewed by  The isolation and synthesis of guanidine metabolites from Ptilocaulis spiculifer has also been summarized.3e Isolation of guanidine compounds as metabolites provides leads for the prevention of metabolic disorders and helps the prognosis of cancer, cardiovascular diseases, diabetes etc. 3f Recent examples of synthetic, biologically-active guanidines include antimicrobial activity, 4a,b thrombin inhibitors, 5 Na + /H + exchanger (NHE) inhibitors, 6a,b transport for the delivery of anti-cancer agents, 7a,b anti-influenza agents.7d Figure 1 shows a few examples of superpotent sweeteners with the guanidine core structure; 8a which have attracted a great deal of recent interest.8b  Guanidines are also known as useful basic catalysts.1a,9 Several reports describe the synthesis of chiral guanidines and their use in asymmetric synthesis.10a-c An excellent review of preparative methods for guanidines was prepared by Anslyn et al., 11a and others have emphasized solid phase synthesis.11b,c Their wide importance has prompted the investigation of new approaches to guanidine derivatives.In this overview we summarize recent advances in the development of guanylating reagents (i.e.compounds which form the guanidine structure by a chemical transformation) for guanidine synthesis.Functionalizations of pre-existing guanidine cores, e.g., by alkylation, arylation or acylation are not covered.We do not attempt to catalog exhaustively the enormous range of synthetic guanidine derivatives, but limit our review to reports describing the discovery of guanylating reagents, and investigations of reaction conditions (time, temperature, catalysis etc.) undertaken to improve the yields of guanidines.Reports of the preparation of guanidine compounds by previously developed methods (such as the synthesis of guanidine natural products and oligomers) are also outside the scope of this report.

Thioureas
Thioureas are common reagents for the synthesis of guanidines.Usually the conversion of a thiourea into a guanidine requires initial activation.However, in many cases the characterization, isolation or even definition of active intermediates is not described.
Scheme 1 presents the conversion of thioureas 2 into guanidines 4 in a suitable solvent (THF, acetonitrile, or chloroform) containing copper sulphate -silica gel in the presence of tertiary amines. 12The formation of the intermediate carbodiimide 3 (non-isolable under these conditions) proceeds quickly and the overall reaction time is short (Table 1).The presence of a tertiary amine, e.g., triethylamine, accelerates the desulfurization.The procedure allows the preparation a very wide range of di-, tri-and tetra-substituted guanidines.Electron withdrawing substituents in the thiourea fragment also accelerate the reaction.Many recent synthetic approaches utilize protected thioureas, containing electron withdrawing protecting groups which can be removed by standard methods.Thus, di-Boc protected thiourea 5 is converted into guanidines 7 by amines in the presence of 1-methyl-2-chloropyridinium iodide 13 6 (Mukaiyama's reagent, Scheme 2, Table 2) as follows: (i) primary and unhindered secondary amines are guanylated in high (>80%) yield using a slight excess of reagent in anhydrous DMF (entries 1-4); (ii) for hindered or unreactive amines, methylene chloride provides a substantial increase in yield (entries 7 and 8) over reactions run in DMF (entries 5 and 7).The effect of solvent on yield probably results from the instability of the carbodiimide intermediates.When nucleophilic attack by an amine is slow, competitive decomposition of the carbodiimide occurs.In methylene chloride, the reactions are heterogeneous owing to sparing solubility of the di-Boc protected thiourea; it is believed that this results in slower production of the di-Boc carbodiimide and consequently, more efficient consumption of the thioureum by less reactive amines.The guanylation of resin-bound amines under these conditions was also examined.Ethoxycarbonyl substituted thioureas 8 are prepared using the corresponding isothiocyanate (Scheme 3).Guanidines 9 are prepared by displacement of sulfur in the presence of ethyl-3aminopropyl carbodiimide hydrochloride (EDCI) 14 in 48 h without the formation of any major side products.The guanidines obtained via EDCI coupling (Table 3) were easily purified by flash chromatography.Deprotection can be achieved by the treatment with Me 3 SiBr.
Scheme 3 Amines can be converted into guanidines 11 by the reaction di-Boc thiourea 5 15 in the presence of mercuric chloride or copper(II) chloride in the presence of triethylamine (Scheme 4).The influence of the desulphurizing agent is shown in Table 4.
Scheme 4 N-Boc-N'-alkyl or aryl thioureas 12 reacted smoothly with an aryl or alkyl amine in the presence of HgCl 2 16a (Scheme 5).The N-boc-N',N"-disubstituted guanidine products 13 are easily deprotected, as shown in Scheme 5, providing an efficient route to N,N'-disubstituted guanidines14, which compares favorably with other methods (Table 5).For an adaptation to solid phase synthesis see ref 16 b .Carbamoyl isothiocyanates 15 possess several advantages for the preparation of thioureas 17 (Scheme 6, Table 6): These reagents provide a protecting group throughout the synthesis, facilitating purification, and they show increased reactivity over alkyl isothiocyanates, forming thioureas even with hindered amines.A second amine can be coupled to the carbamoyl thiourea 16 using EDCI, forming 1,3-disubstituted and 1,1,3-trisubstituted guanidines 17 through either a stepwise or one-pot synthesis.To gauge the steric and electronic limitations of this procedure, amines of varying reactivity (A-G) were investigated for their ability to form protected thiourea and guanidine; yields for reactions of ethoxycarbonyl isothiocyanate are shown in Table 6.This procedure is general for other carbamoyl isothiocyanates (ethyl carbamate, benzyl carbamate (Cbz), 2,2,2-trichloro-1,1-dimethylethyl carbamate, fluorenylmethyl carbamate (Fmoc), and phenyl carbamate) which allows synthetic flexibility in the deprotection.N-Hydroxyguanidines 19 were prepared from 1-benzyloxy-3-Cbz-thiourea 18 18 (Scheme 7, Table 7).The Cbz strategy was chosen because cleavage of the Cbz and benzyl groups could be accomplished simultaneously.The solvent of choice was DMF; mercuric chloride provided efficient desulphurization.
Scheme 7 The desulphurization of a thiourea to a carbodiimide and subsequent reaction with an amine was applied to solid phase synthesis. 19The thiourea 20 was treated with an amine (5 equiv.) at 50°C in CHCl 3 in the presence of DIC (diisopropylcarbodiimide) (5 equiv.)and DIEA (5 equiv.) to give the resin bound guanidine 21.The disubstituted guanidine 22 was then cleaved under mild Rink resin cleavage conditions (25% TFA/CH 2 Cl 2 at room temperature) (Scheme 8, Table 8).The approach of Scheme 9 combines the advantages of traditional solution phase chemistry with the application of polymeric reagents. 20The desired compounds are obtained in a high throughput manner without additional purification, and in satisfactory purity.No base is required.The use of TFA-cleavable Pbf-group protection/activation is an advantageous alternative for the synthesis of guanidines 21a (Scheme 10).Primary or secondary amines, including tertbutylamine and diisopropylamine, in the presence of Mukaiyama's reagent, give guanidines 27 in high yields at room temperature and in 12-18 h.The high efficiency of Mukaiyama's reagent in promoting the guanidinylating reaction contrasts with its role in sulfamoylthiourea based systems.The transformation also succeeds with EDCI in place of Mukaiyama's reagent.No heavy metal salt or excessive heating was needed.

Scheme 10
Solid phase strategy for the preparation of guanidines 30 has also applied triphenylphosphine dichloride as a desulphurizing agent.21b The immobilised thiourea 28 was treated with triphenylphosphine dichloride freshly prepared from triphenylphosphine with hexachloroethane in THF.The use of base proved to be detrimental.

Scheme 11
The preparation of N,N'-disubstituted acylguanidines from primary amides 31, isothiocyanates 32 and amines 21b has utilized three alternative one pot procedures: HgCl 2 , EDCI and Mukaiyama's reagent; each showed comparable yields of guanidines.

Isothioureas
As well as thioureas, isothioureas, particularly S-methylisothioureas, are well developed as guanylating agents due to their easy preparation and availability.
Guanidines have been successfully prepared from N-arylsulfonyl S-methylisothioureas. 22 The Mtr-reagent 35 (Mtr is 4-methoxy-2,3,6-trimethylphenylsulfonyl) reacted with piperidine or aniline in the presence of triethylamine and Hg(ClO 4 ) 2 in refluxing THF (or toluene) to produce mono Mtr-protected guanidines 36 in moderate to good yields (Schemes 13, Table 10).Higher yields were obtained when the reactions were carried out in refluxing THF in the presence of triethylamine and mercuric perchlorate.

36
Scheme 13  Hg(ClO 4 ) 2 (1.1 eq.), Et 3 N (2 eq.), THF, ∆ Reagents 35 and 37 (Schemes 13,14) were evaluated with several substrates to determine optimal conditions (Tables 10, 11).The reagents were reacted with Boc-Lys-OMe.HC1 and Bocp-aminophenylalanine-OMe.The protected lysine ester (Table 11, Entries 1-3) has a primary aliphatic amine side chain but appears to react with 35 and 37 less readily and in lower yield, than the other substrates.The Boc-p-aminophenylalanine-OMe substrate reacts efficiently with both reagents in high yield to produce the target guanylated amino acids (Entry 4).These results indicate that arylsulfonyl isothioureas react with the less nucleophilic electron deficient amines more readily than the more basic primary aliphatic amines.The relatively easy activation of thioureas 39 as thiazetidines 40 provides tri-and tetrasubstituted guanidines 41 23 (Scheme 15, Table 12) in good to excellent yields.
Scheme 15 Reactions of S-methylisothioureas 42 24 with various cyclic amines in refluxing tert-butyl alcohol gave almost a hundred salts of guanidine (Table 13, See Supplemental Materials) in fair to good yields (Scheme 16), accommodating phenyl substituents ranging from strongly electron withdrawing to strongly electron donating, as well as those with bulky substituents in the orthoposition.Either the isothiourea or the amine should be in the form of a soluble salt to achieve the satisfactory reaction rates.

Scheme 16
Mild and efficient promotion by mercuric chloride converts di-Cbz-isothioureas 44 into protected guanidines 45 25 (Scheme 17, Table 14).Free and Cbz-protected guanidinoacids 47 were prepared similarly (Scheme 18, Table 15) from 44 and 46 by in situ carboxyl protection with trimethylsilyl chloride at the first stage of the reaction. 26  Guanidinoureas 49 and 50 were obtained by condensation of N-Cbz-ureido-N`-Cbz-Smethylisothiourea 48 27 with amines in the presence of triethyl amine in DMF at 20 o C (Scheme 19.Table 16).In the reaction with butylamine, a triazinedione by-product 52 formed, but all the other cases gave exclusively guanidine compounds.A library of acylguanidinoureas has been reported.28a Isothioureas 53 were carbamoylated by a resin bound reagent, then acylated.Susequent aminolysis of the thiomethyl group in the presence of mercuric chloride led to 54 which were cleaved to give the guanidine library (Scheme 20, Table 17).
Scheme 20 Another solid support approach for acyl guanidine synthesis utilizes resin bound Smethylthioureas 55. 28b Displacements of the methylthio group with ammonia, primary or secondary amines and aniline all proceeded cleanly at room temperature to give 56 which we than cleaved to 57.

Scheme 21
Commercially available di-Boc-S-methylisothiourea 58 reacted with amines in the presence of mercuric chloride at 0-20 o C affording, after simple work up, the guanylated products 59 in goods yields 29  Guanidinoglycosides 61 were prepared by the intramolecular cyclization of β-amino N-Fmoc-protected acyl isothioureas 60 (Scheme 23).A catalytic amount of DBU (0.7% equiv) is optimal for converting these isothioureas into guanidinoglycosides (35-66%) within an hour.

Scheme 23
S-Linked isothioureas 64 were formed via bis-electrophilic chlorothioformimines 63, as key intermediates. 31Dithiocarbamates were prepared from amines, carbon disulfide and benzyl chloride.Benzyl chloride was chosen for this study because it mimics the Merrifield resin.The dithiocarbamates 62 are quantitatively converted into the corresponding chlorothioformimines 63 by treatment at 60 °C with phosgene in toluene for 12 h.The first amine converts chlorothioformimines 63 into the isothiourea 64 without double addition.The second substitution to give 65 was effected at 100 °C, with an excess of a third amine, optimally in toluene.The reported reaction sequence is well adapted for SPS since it allows, in a four-step process, the addition of three primary amines under reaction conditions compatible with a wide variety of functional groups (Scheme 24).Moreover since the reaction conditions are suitable for automation and high-throughput synthesis, it appears possible to prepare large libraries of guanidines by this traceless linker strategy.

Scheme 24
S-Linked isothioureas 66 as a masked guanidine scaffold allow the parallel synthesis of mono and dialkylated guanidines in high yield and purity 32 (Scheme 25, Table 19).This procedure allows a high level of diversity using parallel array or combinatorial synthesis.The initial Mitsunobu step allows the use of either primary or secondary alcohols to generate 67 with the first point of diversity.Subsequent treatment of the resin bound N-alkyl isothioureas 67 with ammonia or primary amines liberates traceless guanidines 68 with a second point of diversity.Another combinatorial synthesis of guanidines utilizes resin bound thiourea 70, which after conversion into the isothiourea 71 by treatment with iodomethane, reacted with primary or secondary amines to give polymer supported guanidines 72.Cleavage provided access to a variety of guanidines 73 33 (Scheme 26, Table 20).Guanidinoacetic acids were prepared as outlined in Scheme 30 on solid support via treatment of anchored amines with intermediate solution-generated carbodiimides 85. 37 This particular protocol is extremely practical.Conversion of thioureas 84 to carbodiimides 85 on treatment with Mukaiyama's reagent (2-chloro-1-methypyridinium iodide) is almost instantaneous at room temperature though brief sonication was desirable to accelerate solubilization of the reagent.The carbodiimides are very nonpolar and are generally isolated by extraction into hexanes and filtration through a short silica plug using the same solvent.Reaction times for addition of the carbodiimides 85 to Wang-supported glycine or alanine 86 varied, but the transformation was conveniently monitored via the ninhydrin test.

Scheme 30
Another approach via carbodiimides involves their preparation on solid support followed by the reaction with amines to furnish resin bound guanidines 38 (Scheme 31).The sequence commenced with coupling of the p-bromomethyl benzoic acid to a primary amine of a Rinkextended macrocrown.The p-bromomethylbenzamide obtained underwent nucleophilic displacement with azide to afford the α-azido-p-toluamide 88.Treatment with triphenylphosphine and phenyl isothiocyanate provided the carbodiimide 89, presumably via an in situ Staudinger reaction, to generate the intermediate iminophosphorane and subsequent aza-Wittig coupling with the isothiocyanate.Reaction of the carbodiimide with N-phenylpiperazine yielded a polymer-bound guanidine 90, which was cleaved with TFA/H 2 0 (95:5) to afford 91.

Scheme 31
Cyanamides serve as suitable starting materials for the preparation of guanidines: a recent example describes the formation of 92 in situ and immediate reaction with excess of amine 39 to yield 93 (Scheme 32, Table 24).
For Entry 1, R-Ar = For R and R A recent investigation showed that the presence of electron withdrawing groups in the pyrazole ring and/or on one or both of the carboximidamide nitrogens can be advantageous for the yield of guanidines and allow milder reaction conditions.Thus, di-Boc-4-nitropyrazole-1carboximidamide 96 has been used for the preparation of protected guanidines 97 41 (Scheme 34).A 4-nitro group in the pyrazole ring facilitates the reaction and gives higher yields (Table 26) Scheme 34 The use of N-Boc-N'-tosylpyrazole-1-carboximidamide 100 is very efficient for the preparation of guanidines 101 and for coupling to peptides 42 (Scheme 34).This reagent was also used for coupling with peptides in solution and on solid supports.The reactions of a variety of amines with di-Boc-pyrazole-1-carboximidamide 102 have also been examined 43 (Scheme 35, Table 27).In adidition to the guanylation of simple amines, several amino acids were converted into guanidino acids.Amino acids have a low solubility in many organic solvents, but reaction can be achieved in water, or aqueous acetonitrile to give satisfactory results.Pyrazole-1-carboximidamide connected to a solid support by an oxycarbonyl linker 104 can be acylated to 105 and thus becomes a potent reagent for the generation of guanidines 106 on solid supports 44 (Scheme 36) by utilizing electronically diverse acylating agents and amines.Guanidine derivatives from nitroanilines were obtained in good yields for a range of acylated derivatives of pyrazole-1-carboximidamide.
Marianoff and coworkers described a practical two-step procedure based on thioureas 117 oxidized with H 2 O 2 . 49A high yield of pure sulfonic acid 118 was obtained rapidly in a short reaction time when the reaction was run as a slurry in water.The rate of reaction was dependent on the concentration of catalyst (Na 2 MoO 4 ) employed.In general, the sulfonic acid derivatives are stable at room temperature and are the preferred intermediates.The oxidation products were isolated by filtration and air-dried for use in the displacement reaction (Scheme 40, Table 31).
The second step of the sequence, displacement of the oxidized sulfur with amine nucleophiles, to give 119 was carried out under mild conditions (Scheme 40).Yields of the displacement reactions are reported in Table 32.Similar investigation on the use of aminoiminomethansulfonic acids 121 has been described for the conversion of amino acids into a guanidino acids 122 50 (Scheme 41, Table 33).This method is advantageous for the preparation of di-and tri-substituted guanidine acids due to their synthesis from isothiouronium compounds.In a recent, one-pot procedure, quaternary ammonium permanganate in the presence of amine showed advantages over other oxidizing agents for thioureas 120 52 (Scheme 43, Table 33).Modification of benzotriazole-1-carboxamidine by introducing electron withdrawing groups, Boc on both nitrogens of the amidine moiety and nitro or chloro group in benzotriazole to give 131, enhanced the ability of the benzotriazole moiety as a leaving group 54 (Scheme 45).Di(benzotriazolyl)carboximidamide 132 has been developed as a new guanylating agent, for the synthesis of tri-136 and tetra-substituted guanidines 135. 55The sequential condensation of two amines with di(benzotriazolyl)carboximidamide is insensitive to electronic and steric effects allowing the use of a wide variety of amines and guanidines as free bases.The products were obtained in high yields under neutral and mild conditions using an easy purification protocol (Scheme 46, Tables 37, 38).Analogous to the di(benzotriazolyl)carboximidamide reagent 132, di(imidazol-1yl)carboximidamide 138 was later synthesized by treatment of cyanogens bromide with imidazole. 56Reagent 138 can also be converted into substituted guanidines 141 by sequential displacement of imidazole moieties with amines.Further, through its N-cyano derivative 139 it provides an access to substituted cyanoguanidines 143 (Scheme 47, Table 38).Benzotriazolylcarboximidoyl chlorides 145 (stable, odorless and convenient to handle) also allow the preparation of unsymmetrical guanidines 57 (Scheme 48).

Conclusions
We have attempted to summarize recent advances in guanidine synthesis from the point of view of guanylating agents.

Table 1 .
One pot desulfurization of thioureas and reaction with an amine by the procedure of Scheme 1

Table 3 .
One pot preparation of guanidines by EDCI desulfurization and reaction of with amines (of Scheme 3)

Table 4 .
Preparation of guanidines in the presence of copper or mercuric salts (seeScheme 4)

Table 8 .
Conversion of thioureas to guanidines on solid phase (of Scheme 8)

Table 12 .
Synthesis of polysubstituted guanidines via thiazetidine derivatives 40 (ofScheme 15) Simple crystallization after work up provided pure materials.The utility of generating a protected guanidine was established further by converting Gly-Gly to α-(bisbenzyloxycarbonyl)guanidinoacetylglycine.

Table 18 .
(Scheme 22).The results, summarized in Table18, illustrate the broad application of the reagent.Since the synthesis is equally successful with aliphatic and aromatic amines.Sterically hindered amines react well as do anilines with electron donating groups.Electron deficient anilines react slowly to afford the guanylated product in acceptable yields.Preparation of guanidines 59 1 NH 2

Table 21 .
35nthesis of cyanoguanidines 76 in the presence of 1,4-diazabicyclo[2.2.2]octane (see Readily available dithiobiuret 77 and amines in refluxing isopropanol35provide a convenient method for the preparation of mono N-substituted guanylthioureas 79 in moderate to very good yields (Scheme 28, Table22).

Table 22 .
36nthesis of guanylthioutreas from dithiobiuret 78 by method of Scheme 28 Aryliminophosphoranes 80 were converted into N 1 ,N 2 ,N 3 -triarylguanidines 83 in good yields by reaction with isocyanates 81 followed by treatment of the intermediate N1,N 2diarylcarbodiimides 82 with aromatic amines in the presence of TBAF.36The presence of one equiv of TBAF is essential and the reaction takes place at room temperature within 10 min to give the desired guanidine (Scheme 29).As the commercially available THF solution of TBAF containes ca. 5 wt% water, it was pretreated with anhydrous MgSO 4 .

Table 24 .
Synthesis of diarylguanidines 93 by method of Scheme 32Pyrazole-1-carboximidamides 94 are now frequently used for the preparation of guanidines 95.The reaction of 2,4-dimethylpyrazole-1-carboximidamide with amines was considered in 1953 as abnormal because the initial desire was to substitute an NH 2 group in analogy to ureas and related compounds 40a (Scheme 33).The guanidines prepared are listed in Table25.For related work see references 40 b,c .

Table 30 .
Conversion of secondary amines into guanidines by di-protected triflylguanidine 115

Table 33 .
Synthesis of guanidines 122 via oxidized thioureas 121Continued optimization of the protocol for guanidine preparation led to use of the oxidized product without isolation 51 (Scheme 42).Oxidizing agents are compared in Table32and the oxidation rate for aliphatic derivatives was improved by addition of a catalytic amount of triethyl amine.The guanidines 124 were obtained by treatment with amines in water with short reaction times, excellent conversion, and simple isolation.However, trisubstituted thioureas resisted the oxidation.

Table 35 .
Synthesis of guanidines from oxidation of thioureas by quaternary ammonium permanganate (Scheme 43)

Table 36 .
53F/PhCH 2 N + (C 2 H 5 ) 3 MnO 4 -carboxamidinium tosylate 12953prepared from benzotriazole 128 with cyanamide 127 in refluxing 1,4-dioxane in the presence of p-TsOH, is an efficient general reagent for the synthesis of mono and disubstituted guanidines 130. Reactions are conveniently carried out using equimolar amine in (i) DMF-diisopropylethylamine at room temperature, (ii) acetonitrile or (iii) the absence of solvent.Product isolation is facile as the precipitated guanidine can be filtered from the ether soluble benzotriazole by-poduct when DMF is used.The product precipitates during the reaction, while in the absence of solvent product can be isolated chromatographically.Under mild conditions, benzotriazole-1-carboxamidinium tosylate gives guanidines in moderate to good yields and offers advantages over previous procedures (Scheme 44, Table36).Synthesis of guanidines from benzotriazole-1-carboxamidinium tosylate