Abstract Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 46 Synthesis and Biological Evaluation of Two New Analogues of Gonadotropin Releasing Hormone (GnRH)D-alanine 8 and D-alanine 10 Kawkab Y. Saour *.1 * Departement of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract So far synthesis of Gonadotropin Releasing Hormone (GnRH) analogues reported in the literature has clarified some aspects of structural activity of the naturally released GnRH. As a part of continuing efforts for further understanding of this relationship, the present investigation was undertaken which involved synthesis and biological evaluation of two GnRH analogues, firstly, by replacement of the amino acid L-Argenine in the 8 th position at the backbone structure of the natural hormone by the amino acid D-Alanine; and secondly, by replacement of the amino acid L-Glycine in the 10 th position by D-Alanine also at the backbone structure of the nature hormone, to obtain the following analogues respectively: PGlu-His-Trp-Ser-Tyr-Gly-Leu-DAla-Pro-Gly-NH2 (Analogue I: D-Alanine 8 GnRH), PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-DAla-NH2 (Analogue II: D-Alanine 10 GnRH), which were synthesized by applying conventional solution method. Peptides were purified by several recrystallization using appropriate solvent and proved to be homogenous. Conformity of the synthetic procedure was achieved by applying different physico-chemical analyses including Melting Point (mp), Thin Layer Chromatography (Tlc.), Infrared Spectroscopy (IR), Elemental Analysis (CHN), Amino Acid Analysis (AAA), and Nuclear Magnetic Resonance (H 1 NMR).Preliminary biological activity of the two analogues was determined by testing their effects of parenteral administration on ascorbic acid depletion from the ovary of pseudopregnant mice and compared with that of natural GnRH hormone. Analogue II showed significant ascorbic acid depletion as compared to the native hormone while the percentage in ascorbic acid depletion after administration of analogue I were not significant as compared to the native hormone. Key Words: Gonadotropine releasing hormone, Peptide synthesis, Biological activity of GnRH الخالصة نمذ كشفج يًبثالث هىسيىٌ Gonadotropin Releasing Hormone (GnRH) انًُشىسة فٍ األدبُبث انعبنًُت ُُت انجزَئُت نههىسيىٌ انطبُعُت، وكجزء يٍ يسبعُُب بهزا االحجبِ وانشايُت انً االسخزادة بعضب يٍ جىاَب انعاللت نهفعبنُت انحُىَت وانب -يٍ انخفهى األعًك واألشًم نهذِ انعاللت فمذ حى حُبول هزِ انذساست وانخٍ حشًم ححضُش يًبثهٍُ نههىسيىٌ انطبُعٍ ورنك عٍ طشَك: L-Arginine بذالً يٍ انحبيط األيٍُُ احالل انحبيط األيٍُُ -D-Alanine 1 فٍ انًىلع 8 L-Glycine ٍُُفٍ انًىلع 10بذال يٍ انحبض األي D-Alanine احالل انحبيط األيٍُُ -2 -عهً انخىانٍ وكًب َهٍ: pGlu His Trp Ser Tyr Gly Leu Dala Pro Gly-NH2 (Analogue I: D-Alanine 8 GnRH) pGlu His Trp Ser Tyr Gly Leu LArg Pro DAla-NH2 (Analogue II: D-Alanine 10 GnRH) وانخٍ خهمج ببسخخذاو طشق انًحهىل انًعهىدة وحى حُمُت انًًبثالث انًحضشة عٍ طشَك اعبدة انبهىسة ببسخخذاو يزَببث يالئًت. أيب انخٍ انسخخذيج بهذف انخىصم نهخىاص انًًُزة نهًًبثالث انًحضشة فمذ كبَج كشويبحىكشافُب انطبمت انخمُُبث انفُزَبوَت وانكًُُبوَت انشلُمت، لُبس دسجت االَصبس، يطُبف األشعت ححج انحًشاء، االسخذاسة انبصشَت ححهُم انعُبصش ويٍ ثى ححهُم انحىايط األيُُُت انببَىنىجٍ انبذائٍ حُث اسخُذ عهً حمذَش حأثُش انهىسيىٌ انًصُع عهً يعذل ويطُبف انشٍَُ انُىوٌ انًغُبطُسٍ. حى اجشاء انخمُُى اَخفبض حشكُز حبيط االسكىسبُك فٍ يببَط انفئشاٌ كبربت انحًم. نمذ أدي حمٍ انًًبثهٍُ انًحضشٍَ انً حصىل اَخفبض يعُىٌ فٍ يعذل حشكُز حبيط األسكىسبُك فٍ انًببَط ( فٍ يعذل حشكُز حبيط األسكىسبُك بًُُب أظهش انًًبثم األول اَخفبضIIنهًًبثم ) َسبت نهفعبنُت انببَىَىنىجُت انمُبسُت نههىسيىٌ انطبُعٍ. Introduction The hypothalamus releases Gonadotropin Releasing Hormone (GnRH), a decapeptide that stimulates the anterior pituitary to secrete Leutinzing Hormone (LH), and Follicular Stimulating Hormone (FSH) respectively. This peptide controls and regulates both male and female reproduction system. GnRH is a modified decapeptide: PyroGlu-His-Trp-Ser- Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Figure1). The pyroglutamte at the N terminus and the C- terminal amide distinguish this peptide from unmodified decapeptides (1-3) . 1 Corresponding author E-mail : dr.ksaour@yahoo.com Received : 9/2/2009 Accepted : 23/6/2009 mailto:dr.ksaour@yahoo.com Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 47 Figure1: Basic structure of Gn-RH hormone The gonadotropins have a close functional relationship to estrogen, progesterone, and testosterone. They are called gonadotropins because of their action on the gonads. They control ovulation, spermatogenesis, and development of sex organs, and they maintain pregnancy. Included in this group GnRH, LH, FSH, Chorionic gonadotropin (CG; hCG is human gonadotropin), a glycoprotein produced by the placenta; its pharmacological actions are essentially the same as those of LH (4-9) . GnRH interacts with high-affinity receptors on the gonadotropes in the anterior pituitary, leading to the biosynthesis and release of the gonadotropins LH and FSH. The pulse- timing and concentration levels of GnRH are critical for the maintenance of gonadal steroidogenesis and for normal reproductive function (10-12) .Because of its simple structure there has been an enormous amount of interest in development of analogues as medicinal agents. Over 3000 GnRH analogues have been synthesized and studied in an attempt to evaluate their structure activity relationship (3) .The structure activity data reviewed in the previous works provide that Arg 8 was identified as being critical for high affinity binding to mammalian receptors alsoa number of early studies results showed that D-Arg, Gln, Leu, Ornithin, diaminobuteryl substitution for Arg 8 result in substantial decrease in activity while Lys retained most of the activity, it has been shown also that replacement of GlyNH2 at position10 by Ala resulted in mild reduction in activity, therefore we attempted to replace D-Ala at position8 and position10 respectively in the back bone structure of the native hormone in the hope of gaining more information of the structure activity relationship of the GnRH hormone (23). This paper reports the synthesis and preliminary biological evaluation of D-Ala 8 and D-Ala 10 GnRH analogues using conventional solution method by stepwise elongation manner. The products obtained could provide enough materials for further chemical and physical characterization and for biological evaluation and for future work, especially for broad biological testing of the hormone, which is indicated because of its possibly far-reaching in clinical medicine (13) . Materials and Methods All the chemicals used in this study were analar grade purchased from Sigma Company. Due to the presence of numerous complicated amino acids, some difficulties for the synthesis of the decapeptide should be expected. We therefore applied intermediates, which supposedly could be purified easily. Pyro Glutamic acid has been prepared according to Budavari (14) . C-terminal protecting esters of amino acids also prepared according to Huber and Brenner (15) . They are Histidine, Serine, Tyrosine, Leucine, Proline, Glycine, and D- Alanine ethyl esters by dissolving 2 gm (1 mol.) of the amino acid in 15 ml. ethanol. Then the mixture was cooled to -10 o C, thionyl chloride was added gradually with stirring. The mixture was left for one hour at this temperature, then left over night at 40 o C. The mixture was refluxed in water bath at 55-60 o C for three hours. Purification was done under vacuum using ether to obtain the precipitate C O H N CH C CH2 O N NH H N CH C CH2 O HN H N CH C CH2 O OH H N CH C CH2 O OH H N CH C H O H N CH C CH2 O CH CH3 CH3 H N CH C CH2 O CH2 CH2 NH C NH2 NH N C O H N CH C H NH2 O Fig. basic structure of Gn - RH hormone O 1 2 3 4 5 6 7 8 9 10 PGlu His Trp Ser Tyr Gly Leu Arg Pro Gly -NH2 Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 48 N-terminal protection Benzyloxy carbonyl amino acids (Z) were prepared according to Zervas and Bergmann and Baily (16) . They are Z Tryptophan, ZD-Alanine, Z-Glycine, and Z- Arginine. Conventional solution method was applied as a coupling method between the protected amino acid for peptide bond formation using Dicyclohexyl carbodiimide DCC in the presence of 1-hydroxy benzotriazole HBT and N-methyl morpholine (25) NMM. Deprotection of C- terminal protecting groups (saponification) was performed using 1.5 equivalent of sodium hydroxide (NaOH) solution (1 N). Table 1 showed the physico-chemical properties of these esters. Deprotection of the N-terminal protecting groups was performed using hydrobromic acid (HBr) in glacial acetic acid (equimolar). Table 2 showed the physico- chemical properties of these Z protected groups (16) . The intermediates and segment peptides had been purified by repeating recrystalization several times (4 times) using different solvents like diethyl ether, petroleum ether (40-60 o ), ethyl acetate, absolute ethanol, distilled water (D.W.), chloroform. Ascending thin layer chromatography was run on Kieslgel Gf 254 type (60) Merck, for checking the purity of the prepared compounds as well as monitoring the reaction process. Spots were revealed by reactivity with iodine vapour or irradiation with U.V. light or by ninhydrine (15) spraying reagent 2% in absolute ethanol chromatograms were eluted by the following systems:- Acetic acid 30% Methanol Chloroform A 20 45 60 Ammonia Methanol Chloroform B 20 45 60 Water Acetic acid Butanol C 45 10 40 Table 1: Physico-chemical properties of the prepared amino acid esters Name of amino acid ester RfA MP(C o ) νcm-1 1. Histidine methyl ester 0.42 205-207 3180 for primary amine, 1750 for (C=O) of ester. 2. Serine methyl ester 0.46 163-165 3400 for primary amine, 1750 for (C=O) of ester,1260 for (C-O)of ester. 3. Leucine methyl ester 0.62 190-192 As in (2.) 4. Proline methyl ester 0.49 150-153 As in (2.) 5. Glycine methyl ester 0.58 69-72 As in (2.) 6. D-Alanine methyl ester 0.66 172-174 As in (2.) Benzyloxy carbonyl N-protected amino acids (Z) was produced as precipitate except for Z- Argenine which was produced as oily residue using 0.2 mol. and 0.2 mol. of benzyl chloroformate, 4 volumes of 1-N sodium hydroxide. Table 2: Physico-chemical properties of the N- protected amino acids by benzyloxy carbonyl prepared (Z) in this work Name of Z- protected amino acid RfA MP(C o ) cm -1 1 . Z-tryptophan 0.84 114-121 3400 for secondary amine, 1500 secondary amide, 1680 for (C=O). 2 . Z-Glycine 0.82 124-129 3310 secondary amine, 1680 for (C=O). 3 . Z-Argenine 0.78 oily As above 4 . Z-D-Alanine 0.69 118-122 As above Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 49 Scheme I and scheme II showed the pattern followed to obtain analogue I and analogue II respectively. M.P., TLC., optical rotation, IR,CHN, and NMR were the analytical techniques utilized to the chemical evaluation of the different coupling procedures between amino acids and peptide intermediates and to confirm the success of the synthetic process for both analogues. All intermediates and peptides showed optical activity, and purity as revealed by TLC, and acceptable IR, CHN, H 1 NMR, and Amino acid analysis as well. Scheme I: Synthetic steps of Analogue I Scheme II: synthetic steps of Analogue II. Biological Activity of the prepared Analogues The potency of each analogue was estimated by comparing their effect in treated mice with 0.5,1.0,2.0 mcg/0.1 ml of buffered plasma albumin (BpA)/mouse, on ascorbic acid depletion as compared with standard GnRH preparation according to Bogdauove and Gay (17) modified procedure published in British Pharmacopoeia (1988).Female rats approximately 21 days old has been chosen of approximately equal weights with the range 12-13 gm, and then randomly distributed into four groups 5each.group1 control group2-4 test group. The mice were hormonally treated as follows all pseudo pregnant mice were subcutaneously injected with 50 units of Human Chorionic Gonadotrophin (HCG) at the first ,third & fifth day of the starting time of the experiment and after 6 days 5 mice in the groups 2-4 were injected with 0.5,1.0,2.0 mcg of D-alanine 8 GnRH/0.1 ml BpA subcutaneously and respectively while the mice in the first group were injected with BpA for comparison after 3 hours all mice were killed, their ovaries are removed ,weighed and placed immediqately in ice bath to avoid losses and dryness ,the ascorbic acid concentration was measured by homogenizing the 2 ovaries of each mice in 10 ml of metaphosphoric acid allowing the homogenate to stand for 30 minutes & centrifuge ,then 7 ml of the clear supernatant liquid was added to a freshly prepared mixture of 7 ml of acetic acid (pH=7) 3 ml distilled water and 2 ml of the dye 2,6 dichlorophenol indophenol standard solution ,30 seconds after the mixing ,the absorbance of the resulting solution was measured at the maximum at about 520 nm (17) .The result of the assay was calculated by standard statistical method using Complete Random Design (CRD) (18) .The biological activity of D-Alanine 10 analogues has been estimated by same manner.Table 3and 4 show the percent of depletion of ascorbic acid in the ovaries of the treated group. Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 50 Table 3: Average weight of pseudo pregnant mice ovaries, concentration of ascorbic acid, and percent of depletion of ascorbic acid in treated groups by analogue (II): Group number Concentration of D-alanine 8GnRH analogue mcg/mouse Average weight of mice * (gm) Average weight of ovaries/mg Concentration of ascorbic acid mcg/ovary % of depletion of ascorbic acid 1 control 12.11 117.03 65.49±0.156 2 0.5 12.34 117.38 61.109±0.195a 6.809 3 1.0 12.13 118.03 60.01±0.156b 8.37 4 2.0 12.17 117.52 59.18 ±0.372c 8.68 Table 4: Average weight of pseudo pregnant mice ovaries, concentration of ascorbic acid, and percent of depletion of ascorbic acid in the treated groups by analogue (I): Group number Concentration of D-alanine 8GnRH analogue mcg/mouse Average weight of mice (gm) Average weight of ovaries/mg Concentration of ascorbic acid mcg/ovary % of depletion of ascorbic acid 1 control 12.16 117.05 65.54 2 0.5 12.32 117.36 3.41 3 1.0 12.133 118.001 4.17 4 2.0 12.175 117.63 4.301 * Five mice in each group. a, b, and c indicate significant difference at percentage of error 0.01. Results and Discussion The results of physico-chemical evaluation of intermediate peptides were good indication of the success of the synthetic methodology using M.P, TLC, and IR as shown in Table 5. Elemental analysis(CHN), optical rotation, and sometimes AAA have been applied to add conformity to the success of the synthetic procedure: 1. For the dipeptide P Glu-His-OH: 25 ][ D  = -25.9 (C2 DMF) this indicates that this peptide is optically active. Elemental analysis (CHN); calculated: C 49.61, H 5.76 N 21.05, found: C 49.65 H 5.29 N 21.11. 2. For dipeptide Trp-Ser: 25 ][ D  = -46.4 (C2 DMF) this indicates the optical purity of the peptide, CHN analysis; calculated: C 59.97 H 5.73 N 14.94, found: C 60.06 H 5.77 N 15.53. ] 3. Tetrapeptide P Glu-His-Trp-Ser: 25 ][ D  = - 44 (C2 DMF), CHN analysis; calculated: C 55.64 H 5.42 N 18.18, found C 55.98 H 5.66 N 18.63. 4. Pentapeptide P Glu-His-Trp-Ser-Tyr: 25 ][ D  = -39.1 (C2 DMF), CHN analysis; calculated: C 58.1 H 5.45 N 15.85, found: C 58.78 H 5.66 N 16.23. 5. Peptide Z-Gly-Leu: 25 ][ D  = -40.6 (C1 DMF). CHN analysis; calculated C 51.3 H 8.61 N 14.96, found C 51.76 H 8.81 N 51.03. 6. For completion of Analogue I the following steps have been done Peptide D Ala-ProOCH3: 25 ][ D  = -14.0 (C1 DMF). CHN analysis; calculated: C 53.97 H 8.06 N 13.99, found: C 54.3 H 8.23 N 14.44. 7. Tripeptide D Ala-Pro-Gly-OCH3: 25 ][ D  = -12 (C1 DMF). Amino acid analysis was D-Ala 1.01, Pro 0.99, Gly 1.03. 8. Pentapeptide Z Gly-Leu-D Ala-Pro-Gly- OCH3: C 57.73 H 7.00 N 12.47 O 22.80, found: C 58.32 H 7.38 N 12.86 O 23.11. 9. Z Deprotected pentapeptide Gly-Leu-D Ala-Pro-Gly-OCH3: 25 ][ D  = -33 (C=2 DMF). CHN analysis; calculated: C 53.37 H 7.78 N 16.39 O 22.46, found: C 53.91 H 7.99 N 16.83 O 22.71. 10. Decapeptide PGlu-His-Trp-Ser-Tyr-Gly- Leu-DAla-Pro-Gly-OCH3: CHN analysis; calculated: C 57.22 H 6.26 N 16.38 O 20.15, found: C 57.49 H 6.57 N 14.01 O 20.65. 11. Aminolysis of the above decapeptide using ammonia and tri ethyl amine PGlu- His-Trp-Ser-Tyr-Gly-Leu-DAla-Pro-Gly- NH2 (Analogue I). 12. For Completion of Analogue II the following steps have been done: Dipeptide ZArg-Pro-OCH3: CHN analysis; calculated: C 57.25 H 6.97 N 16.70 O Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 51 19.09, found C 57.89 H 6.23 N 16.91 O 19.52. 13. Z Deprotected Arg-Pro-OCH3: CHN, calculated: C 50.49 H 8.13 N 24.55 O 16.83, found: C 51.01 H 8.43 N 25.03 O 17.22. 14. Tripeptide ZArg-Pro-DAla-OCH3: AAA Arg 0.89, Pro 0.93, D-Ala 1.09. 15. ZGly-Leu-Arg-Pro-DAla-OCH3: CHN analysis; calculated: C 56.33 H 7.33 N 16.99 O 19.38, found: C 57.01 H 7.45 N 17.23 O 19.58. 16. Z Deprotected ZGly-Leu-Arg-Pro-DAla- OCH3: CHN analysis; calculated: C 52.44 H 8.04 N 21.28 O 18.23, found: C 52.71 H 8.29 N 21.57 O 18.43. 17. Decapeptide Pglu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-DAla-OCH3: CHN analysis; calculated, C 56.50 H 6.49 N 18.51 O 18.50, found: C 61.01 H 6.84 N 18.91 O 18.79. 18. Aminolysis of the above decapeptide using ammonia and tri ethyl amine Pglu- His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-DAla- NH2. Table 5: Some physico-chemical properties of the synthesized intermediate peptides (Percent yield, Melting points, Rf values, and Characteristic IR spectra). Characteristic IR band (ν cm -1 ) Rf Value Melting Point Percent Yield Amino Acid Residues Peptide Sequence 3320 stretching vibration of secondary amine, 1630 stretching vibration of amide band. 0.73B 109-112 55% P Glu-His-OCH3 1-2 3320 stretching vibration of secondary amine disappearance of ester band at 1730, 1630 stretching vibration of amide band. 0.64A 214- 216 80% P Glu-His-OH 1-2 3220 stretching vibration of primary amine, 1660 stretching vibration of carbonyl of amide 1 band. 0.18A 128- 130 73% Trp-Ser-OCH3 3-4 3370 stretching vibration of secondary amine, 1640 stretching vibration of carbonyl of amide 1 band. 0.89B 198- 200 66% PGlu-His-Trp-Ser-OCH3 1-4 1590 c-o vibration of carboxyl group. 0.81A 172-177 62% PGlu-His-Trp-Ser-OH 1-4 3370 stretching vibration of secondary amine interfered with phenol stretching vibration at 3000, 1630 stretching vibration of carbonyl of amide 1 band. 6.78C 204- 206 64% PGlu-His-Trp-Ser- Tyr-OCH3 1-5 3600-3300 (broad) of phenol group of tyrosine, C=C-H stretching vibration of aromatic ring 3100-3030, 760 out plane bend of aromatic C C 0.78C 221-226 58% PGlu-His-Trp-Ser- Tyr-OH 1-5 3380 stretching vibration of secondary amine, 1640 stretching vibration of amide 1 band 0.48C 101- 103 81% Zgly-Leu-OCH3 6-7 2970-2920 and 2860-2830 stretching vibration of CH3 and CH2, disappearance of ester absorption. 0.53A 99-103 77% ZGly-Leu-OH 6-7 Completion of analogue I D- Ala 8 GnRH 1630 stretching vibration of carbonyl of amide 1 band. 0.68A 181- 186 64% ZD Ala- Pro-OCH3 8-9 3500 stretching vibration of carboxyl group interfered with secondary amine. 0.33C 162-165 60% ZD Ala-Pr-OH 8-9 1680 stretching vibration of carbonyl of amide 1. 0.55C 170- 174 72% ZD Ala-Pro-Gly-OCH3 8-10 0.86C 126- 128 59% D Ala-Pro-Gly-OCH3 8-10 1660 stretching vibration of carbonyl of amide 1 band, 1550 stretching vibration of secondary amine. 0.72B 152-156 67% Z Gly-Leu-D Ala-Pro-Gly- OCH3 6-10 1650-1620 C=O stretching vibration of amide band. 0.61B 143-146 55% Gly-Leu-D Ala-Pro-Gly- OCH3 6-10 1650-1680 N-H bend, 1460, 1380 C-H bend of CH3 and CH2, 1240 O-H bend. 0.89B 218-222 75% PGlu-His-Trp-Ser-Tyr-Gly- Leu-DAla-Pro-Gly-OCH3 1-10 (Aminolysis of the decapeptide (Analogue I) was performed using ammonia in tri ethyl amine) PGlu-His-Trp-Ser-Tyr-Gly-Leu-DAla-Pro-Gly-NH2. Yield 63%. 1-10 Completion of analogue II D- Ala 10 GnRH 1675 stretching vibration of amide band. 0.68A 181- 183 79% Z Arg-Pro-OCH3 8-9 1660-1630 C=N stretching vibration of Arginine, disappearance of ester band. 0.55A 173-176 66% Z Arg-Pro-OH 8-9 3450 N-H stretching vibration of secondary amine, 1650 stretching vibration of carbonyl of amide 1, 1730 stretching vibration of carbonyl of ester group, 700 bending vibration of benzyl ring. 0.48A 169- 172 71% Z Arg-Pro-D Ala-OCH3 8-10 0.76B 116- 118 53% Arg- Pro- D Ala-OCH3 8-10 3500 N-H stretching vibration of terminal primary amine, 1630 stretching vibration of carbonyl of amide 1 band. 0.72A 147- 150 82% Z Gly- Leu- Arg- Pro- D Ala- OCH3 6-10 0.59A 140-146 60% Gly-Leu-Arg-Pro-D Ala- OCH3 6-10 0.87A oily 72% PGlu-His-Trp-Ser-Tyr-Gly- Leu-Leu-Arg-Pro-D Ala- OCH3 1-10 Aminolysis of the decapeptide (Analogue II) D Alanine 10 GnRH was performed using ammonia in tri ethyl amine. PGlu-His-Trp-Ser-Tyr-Gly-Leu-Leu-Arg-Pro-D Ala-NH2. Yield 68%. 1-10 Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 52 Chemical evaluation of analogue I (D- Ala 8 GnRH): Melting point ( o C) = 212- 214. Rf = 0.21A, 0.65B. C=1; -37 25 D ][ Optical Rotation in dimethyl formamide Elemental Analysis Amino acid composition of acid hydrolysates of the analogue was:- Tyr Ser Trp His Glu 1.03 0.98 1.1 0.99 1.01 Pro D-ala 2Gly 0.9 0.89 2.03 H 1 NMR resolution at 300 MHz in deteriuarated dimethyl sulfoxide using tetramethyl silane as a standard showed the following characteristic chemical shifts represented in ppm starting from Glycine amide ending with pyroglutamine as follows: NH2 7.21 prim(s). amide, NH 9.04 sec(s). amide, CH2 3.15;3.41 pyrrolidine (m), CH 4.40 pyrrolidine(m) , CH2 2.34;2.09 pyrrolidine(m) , CH2 2.02;1.92 pyrroline (m), NH 8.32 sec(s). amide, CH2 1.75 methylene(d) , CH 4.53 methine (m), NH 8.32 sec. (s) amide, H 4.09 methylene (d), CH2 4.09 methylene, OH 9.83 aromatic(s) C-OH, CH 6.95 1-benzene(s) , CH 6.68 1-benzene(s), CH 6.68 1-benzene(s) , CH 6.95 1-benzene(s) , NH 8.32 sec. amide(s) , CH2 4.16;3.91 methylene(d) , CH 4.62 methine(m) , CH 7.58 3-indole(s), NH 8.32 sec. amide(s), CH 7.66 imidazole(d) , NH 13.4 imidazole (d), CH 8.73 imidazole (d), NH 8.32 sec. amide(s) ,NH 7.79 pyrrolidin-2-one(s) , CH2 2.28;2.18 pyrrolidin-2-one(m) , CH2 2.46;2.21 pyrrolidin-2-one(m) . Chemical evaluation of analogue II D- Ala 10 GnRH Melting point ( o C) = 218- 220 Rf = 0.19A, 0.36B C=1; -28 25 D ][ Optical Rotation Elemental Analysis Amino Acid Analysis Tyr Ser Trp His Glu 1.09 1.11 0.89 0.98 1.08 D-ala Pro Arg Leu Gly 1.02 1.09 1.01 0.96 0.99 H 1 NMR resolution at 300 MHz in deteriuarated dimethyl sulfoxide using tetramethyl silane as a standard showed the characteristic chemical shifts represented in ppm starting from D-alanine amide and ending with pyroglutamine as follows:- NH2 7.21 prim. amide(s), CH3 1.48 methyl(d), CH 4.71 methine (m), NH 8.32 sec. amide (s) , CH2 3.51;3.41 pyrrolidine (m), CH 4.40 pyrrolidine(m) , CH2 2.34;2.09 pyrrolidine (m), CH2 2.02;1.92 pyrrolidine (m), NH2 6.63 amine, NH 2.0 amine(m), CH 4.53 methine(m) , NH 8.32 sec. amide(s) , CH2 1.75 methylene(d) , CH 4.53 methine(m) , NH 8.32 sec. amide (s), H 4.09 methylene, CH2 4.09 methylene, NH 9.04 sec. amide(s), OH 9.83 aromatic (s), (s) C-OH, CH 6.68 1- benzene, CH 6.68 1-benzene(s) , CH 6.95 1- benzene(s) , CH2 3.17;2.92 methylene(d) , CH 4.92 methine (m), OH 4.78 alcohol(s) , CH2 4.16;3.91 methylene (d), CH 4.62 methine (m), CH 7.34 3-indole (s),NH 10.85 3-indole(s) , CH 7.18 3-indole, CH 4.92 methine, NH 8.32 sec. amine(s), CH 7.66 imidazole (d), NH 13.4 imidazole (d),CH 8.73 imidazole (d), CH2 3.17; 2.92 methylene, CH 4.92 methine, NH 8.32 sec. amide(s) , NH 7.79 pyrrolidin-2-one(s) , CH2 2.28;2.18 pyrrolidon-2-one (m), CH2 2.46;2.21 pyrrolidin-2-one (m). So, the synthetic approaches according to the step wise manner of this study were proved to be effective for the synthesis of homogeneous analogues as indicated from M.P., TLC., optical rotation, CHN, IR, and NMR. These analogues were preliminary estimated for GnRH activity, D- alanine 10 GnRH (Analogue II) was found to posses significant activity as shown in Table 3 while D-alanine 8 GnRH (Analogue I) showed lower activity as shown in table v. Chang et al (22,23) described the importance of basicaty of Arginine moiety at position (8) and the influence of it is multi structural function for biological activity there for injection of the analog D- alanine 10 GuRH in the mice lead to significant Depletion (1>0.01) in ascorbic acid concentration in the ovaries after 3hrs of the injection time as indicated in Table 3 the percent of decrease in ascorbic acid concentration was 6.089, 8.37, 8.68 % for the group. 2-4 respectively compared with the control. This is similar to that obtained by Gay and Bogudonve (17) who N H C 17.88 6.25 56.91 Calculated 17.91 6.55 56.56 Found N H C 19.91 6.49 56.21 Calculated 19.99 6.71 56.29 Found Iraqi J pharm Sci, Vol.18(2) 2009 Synthesis and biological evaluation of two new GNRH analogues 53 mentioned that the percent of decrease in ascorbic acid concentration in the ovary was 10% after 3hrs of the injection time 1.35 mcg of standard GnRH preparation in the mice. This is a clear indication of the presence of significant GnRH activity of the prepared D- alanine 10 GnRH in stimulating the release of leutenizing hormone (LH) which in its turn affect the yellow body cells in the ovaries of pseudo pregnant mice (pretreated with HCG) which lead to the consume of ascorbic acid content in these cell will be to stimulate consumed peroxidase enzyme. This will lead to the oxidation of ascorbic acid and in this oxidation state ascorbic acid can be utilized for the production of very important steroid (progesterone) which is released from the yellow body cells, other probable mechanism of LH function in decreasing ascorbic acid content in the ovary is by increasing the excretion of ascorbic acid, as the venous blood exuded from the ovary (19, 21) . D- alanine 10 GnRH ascorbic acid depletion >D- alanine 8 GnRH. The result of this preliminary biological study may indicate that Gly at COOH terminal is not essential for biological activity since its replacement by D-Ala which has a reverse stereochemistry showed no reduction in the potency of the native hormone and may be the most important finding from this study is that Arg at position 8 seems to be critical for high affinity binding to mammalian receptors because the substitution of Arg8 by D-Aala cause a marked reduction in the biological activity and this could be based from the suggestion that Arg8 of GnRG may interact with acidic moieties on the receptors (24) . Acknowledgement The author would like to acknowledge the College of Agriculture- Department of Animal Resources for their help in carrying the biological activity and supplying HCG hormone. References 1. Fink .G Gonadotropin secretion and its control.ln : Knobil E , Neill .J (eds) The Phsyiology of Reproduction. Raven press, New York, 1988. pp 1349-1377. 2. 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