Synthesis of diketopiperazines containing prolinyl unit - cyclo(L-prolinyl-L-leu cine), cyclo(L-prolinyl-L-isoleucine) and cyclo(L-tryptophyl-L-proline)

Diketopiperazines cyclo(L-prolinyl-L-isoleucine) 4a, cyclo(L-prolinyi-L-Ieucinc) 4b and cyclo(L-tryptophyl-L-proline) 6 were prepared from their respective suitably protected amino acid derivatives by standard peptide chemistry. Cyclo(L-(4-hydroxyprolinyi)-L-phenylalanine) 3, 4a and cyclo(L-prolinyl-L-tyrosine) 5 ~ere tested for their antibacterial activity.

2,5-Diketopiperazines, fonned by cyclization of dipeptides, are interesting compounds because of their important biological properties I-J. Diketopipcrazines have been isolated from plants mainly marine sponges 4 , microorganisms as well as from higher animals 5 . 2,5-Diketopiperazines are also obtained as by-products during the process of acidic or enzymatic digestion ofproteinous stutTusing mammalian or bacterial protease 6 . Suzuki eta/. 7 synthesized a number of analogs cyclo(Tyr-Arg) 1 and have studied their analgesic properties in mice. Many of these diketopiperazines contain proline and hydroxyproline and most of them exhibit important biological properties 7 . Recently, the diketopiperazines, cyclo[L-( 4-hydroxyproline )-L-leucine ], cyclol L-( 4-hydroxyprolinyl)-o-leu- 2 3 Chart l ~~ cine], 2a and 2b and cyclo(L-( 4-hydroxyprolinyl)-L-phenylalanine]8 3 have been isolated from Palythoa sp., a marine bactcrium 3 , and arc shown to stimulate plant growth. Herein we report the preparation of dikctopipcrazincs 4a, 4b and 6 and the biological activity of 3, 4a and 5 9 (Chart I).

Results and discussion
The target molecules were individually synthesized using the plan depicted in Schemes I and 2 beginning with the appropriate amino acids. The amino group of the L-proline was first protected using bcnzylchlorofonnatc in alkaline medium. L-lsoleucinc and L-leucinc were converted into their corresponding ester hydrochloride by ref1uxing the amino acid in absolute ethanol in the presence of thionyl chloride. The ester hydrochlorides obtained were washed with K 2 C0 3 to obtain the pure ethyl esters 7a and 7b. The use of concentrated sulfuric acid as catalyst provided the desired esters in lower yields (45%).
Coupling of protected amino acids were accomplished with dicyclohexylcarbodiimidc (DCC) in dry DCM at room temperature to give dipeptides 8a and 8b.
Prior to cyclization of the dipeptides, the prolinylamino group was deprotected using transfer hydrogenation by refluxing the dipeptide in MeOH in the presence of Pd-C and cyclohexene afforded the N-deprotected products 9a and 9b. The peaks corresponding to the benzylic protons of the Z-group at 57.3 and 5.1 were absent in the 1 H NMR spectra of9a and 9b, confirming that the Z-group had been successfully removed.
The final step of the reaction involved the cyclization of the dipeptides 9a and 9b. This was first attempted using DMAP in refluxing toluene but the reaction was unsuccessful. Use of higher boiling solvents such as xylene did 766 using a catalytic amount of potassium carbonate finally yielded the cyclo(L-prolinyi-L-isoleucine) 4a. The same procedure yielded also the isomer 4b (Scheme 1).
For the diketopiperazine 6 the a-NH of tryptophan was protected as the BOC group using BOC anhydride in the presence of triethylamine (TEA). Coupling of the SOCprotected tryptophan I 0 with proline ethyl ester 11 using DCC and I, 1-hydroxybenzotriazole in the presence of TEA yielded the dipeptide 12.
Treatment of the dipeptide 12 with TFA gave the deprotected dipeptide 13. The absence of the intense singlet at o 1.1 ppm confirmed that BOC was effectively removed.
Antibacterial screening of all the compounds were carried out, using the Gram-positive bacterium Staphylococcus aureus, and the Gram-negative bacteria, Esherichia coli, Pseudomonas areuginosa and Salmonella typhimerium.
Blank tests showed that DMSO and water used in the preparation of the test solutions did not affect the growth of the microorganisms. Antibacterial activity of the different diketopiperazines 3, 4a and 5 was evaluated at concentration of1600 Jlg ml-1 . Cetyltrimethylammonium bromide (CTBA) was used as control. The diketopiperazine 3 containing a hydroxyl group was found active against P aeruginosa and S. aureus. The other dipeptides were found inactive. Hence, antibacterial activity of3 was further studied using different concentrations ( 1600, 800, 400, 200 and I 00 Jlg mJ-1 ) in DMSO and the zone of inhibition was measured. At a concentration of 1600 Jlg ml-1 , 3 inhibited the growth of P aeruginosa with an inhibition zone of 15.5 mm while it inhibited the growth of S. aureus with an inhibition zone of I 0.5 mm. CTBA inhibited the growth of both the bacteria with inhibition zone of20 mm. 3 inhibited the activity of S. aureus at a minimum concentration of 400 Jlg ml-1 (7.0 mm) while it was still active P aeruginosa at a concentration of I 00 Jlg ml-1 (9.5 mm). Experimental 1 Hand 13 C spectra (CDC1 3 ) were recorded on a Brucker Spectrospin instrument at 250 MHz with TMS as internal standard and IR spectra on a Mattson Genesis II series FTIR spectrophotometer. Chromatography refers to the 'flash column' technique over silica gel (70-230 mesh). TLC was carried out on plates pre-coated with silica and visualized by exposure to iodine vapour. C, H, N were analyzed on a LECO CHNS-932 analyzer. All chemicals were of A.R. grade except methanol, which was ofGPR grade. Methanol was distilled and kept over molecular sieves.
N-(Benzyloxycarbonyl}-L-proline : To a ice-cold (0°) solution of L-proline ( 1.5 g, 13.05 mmol) dissolved in 4 M NaOH (6.3 ml, 27.41 mmol, 2.1 eq), benzyl chlorofonnate (2.26 g, 15.66 mmol, 1.3 eq) was added dropwise over a period of 20 min with stirring while maintaining the temperature at 0°. The mixture was further stirred for 2 h with gradual wanning to room temperature while monitoring the progress of the reaction by TLC (methanol/glacial acetic acid, 19 : I, Rr 0.6). The reaction mixture was then diluted with water (I 0 ml) and extracted with diethyl ether (2 x I 0 ml). The resulting aqueous phase was acidified with HCl (I : 1). The liberated oil was extracted with ether (5 x 10 ml) and dried over anhyd. sodium sulfate. Evaporation of the solvent gave the product as a very pale yellow viscous liquid (2. Cyclo(L-prolinyl-L-isoleucine) 4a : To a solution of Ndeprotected peptide 9a (1.99 g, 7.80 mmol) in ethylene glycol (15 ml), potassium carbonate (I g, I eq) was added. The mixture was refluxed for 5 h, then diluted with water (15 ml) and extracted with ethyl acetate (5 x I 0 ml). The organic layer was dried (Na 2 S0 4 ) and evaporated to give a reddish-brown paste. The crude product was purified by column chromatography (EtOAc) to a brownish paste (0.716 g, 46%) in 30% overall yield starting from L-proline : hif 4.0 (I 1-1, m), 3.6 (I H, m), 3.5 (I 1-1, m) N-t-Butyloxycarbonyltryptophan 10 : To a stirred suspension oftryptophan (0.5 g, 2.5 mmol) in methanol, TEA (5 ml) and Boc 2 0 (0.53 g) were added. The reaction mixture was stirred for 24 h at room temperature. The reaction was monitored by TLC (EtOAc : Hex, I : I, Rr : 0.86). Methanol was then evaporated and a yellow· paste was obtained. A minimum amount of water was added to the reaction mixture and extracted with diethyl ether (2 x 25 ml), washed with brine (20 ml), dried (Na 2 S0 4 ) and on evaporation a yellow paste was obtained (0.60 g, 80%); "inax 3300 (NH), 1710, 1690 cm-1 (CO);~~ 8.1 (1H, s) L-Prolinyl ethyl ester 11 : To a stirred, ice-cold suspension of proline ( 1.7 g, 0.015 mol) in absolute ethanol (50 ml), thionyl chloride (2.73 g, 1.7 ml, 1.5 eq) was added dropwise and the mixture was refluxed for 3-4 h. The reaction mixture was then allowed to stand overnight and the ethanol was evaporated in vacuo to leave proline ester hydrochloride as a viscous colourless liquid, which was stirred with dry diethyl ether. The ether was removed by evaporation leaving a viscous colourless liquid (2.49 g, 90%); vmax 1730 cm-1 (CO); Jr 1 4.3 (I H, m), 4.0 (2H, q, J 7 Hz), 2.6 (2H, m), 1.9 (4H, t), 1.4 (3H, t, J 7 Hz). The resulting L-prolinyl ethyl ester hydrochloride (3.4 g) was dissolved in water and washed with a saturated solution of potassium carbonate. The mixture was then extracted with diethyl ether (5 x 15 ml). The organic layers were combined and evaporated to give a yellow paste (0.25 g, I 0%).