Sulfo-and Oxy-analogues of Arginine : Synthesis , Analysis and Preliminary Biological Screening

A novel methodology for the synthesis of sulfoand oxy-modified amino acid analogues of arginine (Arg) has been developed using both conventional and polymer assisted synthesis from ready available amino acid precursor. Introduction of guanidine group was made also by the MWA synthesis. The in vitro inhibitory effect of the amino acid analogues on the growth of murine erythroleukemia cells, clone F4N in culture was also studied. (doi: 10.5562/cca1780)


INTRODUCTION
As the building blocks of peptides and proteins, amino acids are vitally important biochemical species.A detailed knowledge of their chemical properties is essential to understanding the complex interplay between amino acid structure and biological function.
One of the most prominent members of coded amino acids is arginine (Arg), which has been intensively studied with the respect to biological properties and as a building block in peptide chemistry. 13][4] The guanidino group of Arg is the important detail of its biological specificity, a group with a high pK that is positively charged at all biologically relevant pH values.A highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is however, often associated with low selectively, excreted rapidly, or do not pass biological membranes resulting in a poor bioavailability.
A significant effort has therefore been focused on the design and preparation of arginine analogues which possess reduced basicity, resulting in increased stability towards enzymatic degradation. 57][8][9] Their effects on the growth of microorganisms, model plant systems, cultured tumor cells and their antitumor activity in vivo have been evaluated. 10,11][14][15] Consequently, these studies emphasize the need of milder and more practical conditions for preparation of these arginine analogues.
In this article we report optimization of the synthesis of previously described oxy-and sulfo-analogues of arginine. 8,9,16The optimization strategy included a polymer assisted synthesis (PAS) methodology, which combines advantages of traditional solution phase chemistry with application of polymeric agents.Moreover, the introduction of guanidine group was made also by the micro wave assisted (MWA) synthesis.This approach led to the desired compounds in a high throughput manner, in sufficient purity, without additional purification steps.
The in vitro inhibitory effect of the amino acid analogues on the growth of murine erythroleukemia cells, clone F4N in culture was also studied.

Z-Ser(OMes)-OBzl (2)
To the solution of Z-Ser-OBzl (1.2 g, 4 mmol) in DCM (10 ml) at -10 °C DIPEA (13.79 ml, 80 mmol) was added, and after that Mes-Cl (0.62 ml, 8 mmol) dropwise.The reaction mixture was stirred for 2 hours at room temperature.Product (2) was obtained without further purification after evaporation of the solution in vacuo (1.6 g ,70 %) and it was used for the next step.

Z-CH(CH 2 SO 3 H)-OBzl (3)
Method I: 2 (0.4 g, 1 mmol) was suspended in water (2 ml) and a solution of Na 2 SO 3 (0.6 g, 5 mmol) in water (5 ml) was added.The reaction mixture was stirred at room temperature for 24 hours.After acidification a new product (3) was obtained as white crystals from the reaction mixture.It was filtered out, washed with water and dried in vacuo (0.3 g, 75 % yield).
Method II.The same as in method I, but with the following conditions at microwave irradiation (300 W), 1 min to 40 °C, hold 5 min (0.35 g, 87 % yield).
Z-NCan-OBzl ( 9) 7 (0.4 g, 0.8 mmol) was dissolved in EtOH (4 ml) and N 2 H 4 •H 2 O (0.5 ml) was added.The mixture was stirred at room temperature for 24 hours and EtOH was evaporated in vacuo.The residue was recrystalized from mixture of EtOAc and petroleum ether to obtain 0.24 g (85 % yield) of 8. C 18 H 20 N 2 O 5 (344.37);R f (B) 0,74; R f (C) 0,61;  (10)  To the solution of 1H-pyrazol-1(N,N'-bis-Boc)-carboxamidine (0.55 g, 1.6 mmol) and DIPEA (0.7 ml, 4 mmol) in CH 3 CN (4 ml) at room temperature, 8 (0.57 g, 1.6 mmol) was added.The mixture was stirred for 24 hours at room temperature.The solvent was evaporated in vacuo and the residue was dissolved in EtOAc (20 ml) and was washed subsequently with 5 % HCl (2 ×10 ml), brine and was dried over Na 2 SO 4 .EtOAc was evaporated in vacuo and the resulting product was recrystalized from mixture of EtOAc and hexane to obtain 0.37 g (42 % yield).Z-NCav(Boc) 2 -OH (11) 9 (0.29 g, 0.6 mmol) was dissolved in a mixture of dioxane and water (volume ratio = 1 :1) and 1M solution of NaOH was added dropwise until the hydrolysis was complete.Dioxane was evaporated in vacuo and the aqueous solution was acidified to pH = 3 and extracted with EtOAc (3 ×10 ml).The combined organic layers were washed with brine to pH = 7 and dried over Na 2 SO 4 .EtOAc was evaporated under reduced pressure and the product was recrystalized from EtOH to obtain 0.18 g (76 % yield).

Z-NCav(Boc) 2 -OBzl
NCav ( 12) 10 (0.15 g, 0.3 mmol) was dissolved in DCM and mixture of HBr and CH 3 COOH (0.5 ml) was added.Deprotection was carried out for 1 hour at room temperature and the solvent was evaporated in vacuo.The solid residue was recrystalized from EtOEt to obtain 0.047 g (96 % yield).Boc-Ser-Wang-Resin (13)  In a round bottom flask the resin was suspended in 7 ml of mixture of CH 2 Cl 2 and DMF (volume ratio = 9 :1).In a separate flask, Boc-Ser (12) was dissolved in a minimum amount of DMF (4 ml).The same equivalence of HOBt was added.The mixture was stirred until the HOBt was dissolved.This solution was added to the resin.In a separate flask Et 3 N was dissolved in a minimum amount of DMF (1 ml).HBTU was added to the resin mixture then the DMAP solution was added.The mixture was shaken for 2 hours at room temperature.The resin was filtered and washed 3 times with DMF, then 3 times with DCM, and finally 3 times with methanol.In each wash, enough solvent was used to slurry the resin.After the final methanol wash, the resin was dried in vacuo to a constant weight.The substitution of the resin can be estimated from the weight gain of the resin.(14)  Method I. Boc-Ser-Resin (0.25 g, 0.18 mmol) was suspended in DMF (5 ml) and TPP (0.12 g, 0.45 mmol) and N-hydroxyphthalimide (0.06 g, 0.36 mmol) were added subsequently.DEAD (0.08 ml, 0.45 mmol) was added and the mixture was shaken for 2 hours at room temperature.The resin was filtered and washed 5 times with DMF.Method II.The same as in method I, but with the following conditions at microwave irradiation (300 W), (1 min to 40 °C, 4 ×30 s hold).

Boc-Ser(OPhth)-Resin
Boc-NCan-Resin (15) 14 (0.95 g, 2 mmol) and N 2 H 4 •H 2 O (2 ml) were suspended in EtOH (10 ml).The mixture was shaken for 2 hours at room temperature.The resin was filtered and washed 3 times with EtOH and 3 times with DMF.ture to 15.The mixture was shaken for 24 hours at room temperature.The resin was filtered and washed 3 times with CH 3 CN and 3 times with DMF.Method II.The same as in method I, but with the following conditions at microwave irradiation (300W), 1 min to 40 °C, hold 5 min.

NCav (11)
Cleavage of 11 from the resin was achieved with acidic hydrolysis with 5 ml of 95 % TFA for 30 min.The resin was filtered out and the solvent was evaporated in vacuo.NCav was obtained as a white solid (0.03 g, 53 % yield).

HPLC-characterization of Reaction Products 7 and 12
All samples were prepared in a concentration of 1 mg ml -1 and dissolved in the mobile phase.Mobile phases were degassed for 10 min by helium 5.0.

Determination of Purity of Crude Products 7 and 12
HPLC analysis of the products were performed on a column Beckman Ultrasphere TM RPC 18 (250 ×4.6 mm, 5 μm) with a mobile phase: mixture of 0.01 M K 2 HPO 4 (pH = 2.5) and acetonitrile (volume ratio = 80 : 20) over 30 min at a flow rate of 0.5 ml min -1 .Detection was performed at 230 nm.

Determination of Enantiomeric Purity of the Crude Products 7 and 12
Micro-HPLC analysis of the products were performed on a column packed with the macrocyclic antibiotic Ristocetin A immobilized on 3.5 m silica gel (100 ×1 mm) with a mobile phase: mixture of 0.1 % TEAA in water (pH = 6.9) and methanol (volume ratio = 80 : 20) at a flow rate 30 μl min -1 .Detection was performed at 208 nm.

Incubation with Drugs and In Vitro Cytotoxicity Test
Murine erythroleukemia cells, clone F4N 17 were cultured in Dulbecco's modified Eagle medium supplemented with 10 % calf serum, under a 5 % CO 2 atmosphere at 37 °C, and passed every day at a concentration of 5 ×10 5 cells ml -1 .
The inhibitors were dissolved immediately before use in DMSO to obtain stock solutions of different concentrations.Each of these solutions was used at 1 % concentration in the experiments with F4N cells.Control samples were incubated in the presence of 1 % DMSO.The final concentration of DMSO in the medium did not affect cell growth.
Exponentially growing cells (0.5 ×10 6 cells ml -1 ) were incubated in triplicate with increasing concentrations of the test complexes in 96-well microtiter plates.After 24, 48 and 72 h of drug treatment, the cells were counted hemocytometrically.The number of dead cells was determined by staining with trypan blue.The mean of triplicate determinations of three independent experiments was calculated.The 50 % inhibitory dose (IC50) was defined as drug concentration that reduces the number of living cells by 50 %.

RESULTS AND DISCUSSION
We have recently reported a general synthetic route to sulfo-and oxo-analogues of Arg. 8,9heme 1. Synthesis of NsArg and NCav.Reagents and conditions.
As outlined at the Scheme 1, sulfo-and oxoanalogues of Arg (Figure 1) were prepared for the first time starting from the same key compound Ser.The first steps of this route were common for obtaining the mesylated fully protected Ser (2) and after that we prepared NCav (12) and NsArg (7) analogues.
For the preparation of NsArg (7) we applied a new approach -synthesis of sulfonyl chloride (4) via sulfonic acid 18 (3) by conventional synthesis in solution or in conditions of microwave irradiation.In the second case the reaction time was shortened from 24 hours to 5 minutes.All other steps of this synthetic scheme have been previously described.
NCav (12) was synthesized by using two different approaches.The first was based on conventional methods in solution (Scheme 1), the second -on solid phase organic synthesis (Scheme 2).In some steps of both synthetic schemes we applied for first time microwave irradiation.In conditions of MWA, SPS on the first and second stages reaction was carried out at 40 degrees and an irradiation time of totally 2 minutes.Introduction of guanidine group was made also in the MWA synthesis at 40 degrees and irradiation time of 5 minutes.
The synthesis of the hydrazide derivatives of NsArg and NCav (Figure 1) has been described previously. 8,9Both of the hydrazides, NsArg-CONHNHC 6 H 5 (18) and NCav-CONHNHC 6 H 5 (19)  were prepared by a condensation reaction of the corresponding di-protected compound with the Boc-NHNHC 6 H 5 in the presence of DIPEA, using TBTU reagent.The condensation method we have applied afforded high purity products and column chromatography purification was not required.Moreover, it should be noted that under the mild conditions of the synthesis, undesirable side processes, including racemization, did not take place.Cleavage of the protecting Boc-group was achieved by ethyl acetate saturated by ethyl acetate saturated anhydrous HCI (mixture of 1.5 M HCl and EtOAC), or mixture of TFA and anisole (volume ratio = 9 : 1) in 95-98 % yields.
In conjunction with these synthesis routes, analytical methods are required to monitor the chemical and enantiomeric purity of the formed products.Using optimized conditions described above, excellent purity obtained of 7 (98 %) and 12 (96 %) in the crude HPLCchromatograms (Figures 2 and 3).For most applications the purity of these products will be sufficient.
To determine whether the MWA conditions resulted in racemization at the α-amino center, 7 and 12 were evaluated for enantiomeric purity.As several UAA were successfully analyzed by us using macrocyclic antibiotic phases, 19 it was decided to adapt this well established method for the chiral analysis of NCav and NsArg.
The resulting chromatograms did not show detect-  able diastereomeric contamination, indicating that the ee value of 12 is > 98.9 % (Figure 4).The elemental analysis and spectral data confirmed the structures of the sulfo-and oxo-analogues of Arg shown in Figure 1, and were in accordance with the data reported in our previous papers. 8,9

Cytotoxic Effect
The cytotoxicity of NCav, NCav-CONHNHC 6 H 5 , NsArg, NsArg-CONHNHC 6 H 5 in F4N cells was examined using cell-growth assay.The growth-inhibitory effects of 24, 48 and 72 h incubation of the cells with the drugs are expressed by IC50 values (Table 1).Clear differences in the cell growth were observed after 48 and 72 h of incubation.IC50 values of NCav were found to be 8 and 3.2 mmol dm -3 after 48 and 72 h incubation respectively, while those of NCav-CONH-NHC 6 H 5 were 2.5 and 1.3.The second compound proved to be 3 times more cytotoxic than NCav.Similar results were obtained for NsArg and its phenylhydrazide derivative NsArg-CONHNHC 6 H 5 .The concentrations of NsArg reducing the number of living cells by 50 % ranged from 4 to 2.4 mmol dm -3 for the different times of treatment, while those of NsArg-CONHNHC 6 H 5 ranged from 4.6 to 0.8 mmol dm -3 .These results con-    firm our previous findings, showing that CPH was more cytotoxic than Cav by almost an order of magnitude. 10sArg ant its phenylhydrazide derivative exhibit higher cytotoxicity compared to the respective NCav compounds.
In conclusion, the main goal of the chemical approach applied was a quick and efficient preparation of NCav and NsArg of high purity, in a quantity sufficient for biological testing.That was achieved, since the HPLC data of the amino acid analogues revealed 95 % average purity, based on the relative peak area with monitoring at 230 and 254 nm.In addition 1 % of the unwanted enantiomer was detected.All tested compounds showed cytotoxic activity against F4N cells.Phenylhydrazide derivatives proved to be more cytotoxic than their parent compounds in accordance with our previous results.

Scheme 2 .
Scheme 2. Synthesis of NCav on solid support.Reagents and conditions.

Table 1 .
Cytotoxicity of sulfo-and oxyguanidine arginine analogues in F4N cellsCompoundCell growth inhibition(a)   IC50 (b) / mmol dm -Exponentially growing cells were incubated in culture medium for 24, 48 and 72 h at 37 °C with varying amounts of the compounds and counted thereafter hemocytometrically.The number of dead cells was determined by trypane blue exclusion.(b)Drug concentration that reduces the number of living cells by 50 %.Values are means of triplicate determination in at least two independent experiments. (c) Results from our previous research.10Croat.Chem.Acta 84 (2011) 447.