NOVEL COMPOUNDS (IMMUNORHELINS)

Abstract

The present invention provides immune stimulating peptides (immunorhelins) capable of stimulating GnRH receptors when dosed to human patients or cells. These immunorhelins have utility in treating viral diseases and cancer.

Claims

1. A compound of formula (I) ##STR00517## or a pharmaceutically acceptable salt thereof, and wherein ##STR00518## wherein R.sub.5=Me, Et, CH.sub.2CF.sub.3, iPr, nPr, nBu, iBu, sBu, tBu, cyclopropyl CH.sub.2CONH.sub.2 or NHCONH.sub.2 and with the proviso that the invention does not include the following compounds: TABLE-US-00012 Pro- viso R1 R2 R3 R4 R5 P1  CH.sub.2CONH.sub.2 P2  CH.sub.2CONH.sub.2 P3  CH.sub.2CONH.sub.2 P4  CH.sub.2CONH.sub.2 P5  CH.sub.2CONH.sub.2 P6  CH.sub.2CONH.sub.2 P7  Et P8  Et P9  CH.sub.2CONH.sub.2 P10 CH.sub.2CONH.sub.2 P11 CH.sub.2CONH.sub.2 P12 Et P13 Et P14 Et P15 NHCONH.sub.2 P16 NHCONH.sub.2 P17 Et P18 NHCONH.sub.2 P19 Et P20 NHCONH.sub.2 P21 Et

2. A compound according to claim 1, wherein at least one of R.sub.1, R.sub.3 and R.sub.4 are selected from type II according to the list below, and those of R.sub.1, R.sub.3 and R.sub.4 which are not selected from type II are selected from Type I according to the list below: TABLE-US-00013 Group Type I Type II R.sub.1 R.sub.3 R.sub.4

3. A compound according to claim 2, wherein at least two or three of R.sub.1, R.sub.3 and R.sub.4 are selected from type II according to the list below: TABLE-US-00014 Group Type I Type II R.sub.1 R.sub.3 R.sub.4

4. A compound according to claim 2, wherein one of R.sub.1, R.sub.3 and R.sub.4 is selected from Type I according to the list below and two of R.sub.1, R.sub.3 and R.sub.4 are selected from type II according to the list below: TABLE-US-00015 Group Type I Type II R.sub.1 R.sub.3 R.sub.4

5. A compound according to any of claims 1 to 4, wherein R.sub.5=Et or CH.sub.2CONH.sub.2.

6. A compound according to any of claims 1 to 4 wherein R.sub.5=Me, iPr, nPr, nBu, iBu, sBu or tBu.

7. A compound according to any one of claims 1 to 6, wherein the compound of formula (I) is selected from: TABLE-US-00016 Com- pound no. R1 R2 R3 R4 R5 1 CH.sub.2CONH.sub.2 2 CH.sub.2CONH.sub.2 3 CH.sub.2CONH.sub.2 4 CH.sub.2CONH.sub.2 5 CH.sub.2CONH.sub.2 6 Et 7 CH.sub.2CONH.sub.2 8 Et 9 Et 10 Et 11 Et 12 Et 13 Et 14 CH.sub.2CONH.sub.2 15 Et 16 CH.sub.2CONH.sub.2 17 Et 18 Et 19 Et 20 Et 21 Et 22 Et 23 CH.sub.2CONH.sub.2 24 CH.sub.2CONH.sub.2 25 CH.sub.2CONH.sub.2 26 Et 27 CH.sub.2CONH.sub.2 28 CH.sub.2CONH.sub.2 29 Et 30 CH.sub.2CONH.sub.2 31 Et 32 Et 33 CH.sub.2CONH.sub.2 34 Et 35 Et 36 Et 37 CH.sub.2CONH.sub.2 38 CH.sub.2CONH.sub.2 39 CH.sub.2CONH.sub.2 40 CH.sub.2CONH.sub.2 41 CH.sub.2CONH.sub.2 42 Et 43 CH.sub.2CONH.sub.2 44 Et 45 Et 46 CH.sub.2CONH.sub.2 47 Et 48 Et 49 Et 50 Et 51 CH.sub.2CONH.sub.2 52 CH.sub.2CONH.sub.2 53 CH.sub.2CONH.sub.2 54 CH.sub.2CONH.sub.2 55 CH.sub.2CONH.sub.2 56 Et 57 CH.sub.2CONH.sub.2 58 CH.sub.2CONH.sub.2 59 Et 60 Et 61 Et 62 Et 63 Et 64 CH.sub.2CONH.sub.2 65 CH.sub.2CONH.sub.2 66 CH.sub.2CONH.sub.2 67 Et 68 CH.sub.2CONH.sub.2

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and one or more pharmaceutically acceptable excipients.

9. A compound according to any one of claims 1 to 7 for use in medicine.

10. A compound according to any one of claims 1 to 7 for use in the treatment of viral infections or cancer.

11. A compound selected from P1-P21 as defined in claim 1 for use in the treatment of viral infections or cancer.

12. A method for treating or preventing a disease caused by a viral infection comprising administering to a human or animal subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 7.

13. A method for treating or preventing a disease caused by a viral infection comprising administering to a human or animal subject in need thereof a therapeutically effective amount of a compound selected from P1-P21 as defined in claim 1.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0102] FIG. 1: Expression of MHC class I after stimulation of T cells with increasing concentrations of GnRH II. PBMCs from a healthy donor was stimulated with GnRH II and IL-2 for 72 hours. Data points represent mean fluorescent intensity of MCH class I expression on CD4.sup.+ T cells (blue triangles) or CD8+ T cells (black squares) measured with flow cytometry.

[0103] FIGS. 2A-2B: Expression of MHC class I after stimulation of T cells with increasing concentrations of GnRH I analogue (red) and GnRH II. (black). PBMCs from a healthy donor was stimulated with GnRH I analogue or with GnRH II and IL-2 for 72 hours. Data points represent mean fluorescent intensity of MCH class I expression on CD4+ T cells (FIG. 2A) or CD8+ T cells (FIG. 2B) measured with flow cytometry.

[0104] FIG. 3: Expression of MHC class I after stimulation of CD4+CD14+ monocytes with increasing concentrations of GnRH I analogue (red) and GnRH II (black). CD14+ monocytes PBMCs from a healthy donor was stimulated with GnRH I analogue or with GnRH II and IL-2 for 72 hours. Data points represent mean fluorescent intensity of MCH class I expression on CD4+CD14+ monocytes measured with flow cytometry.

[0105] FIG. 4: GnRH receptor expression in human T cells analysed with quantitative real-time PCR. The bars represent ratios of GnRHR I or GnRHR II mRNA normalized to RNA polymerase II expression in sorted naive T cells (white bars) or memory T cells (gray bars). MCF-7 breast cancer cell line (black bar) was used as a positive control.

EXPERIMENTAL

[0106] General Biology Methods

[0107] The preferential effect of the compounds of the invention on GnRH receptors may be tested using one or more of the methods described below:

[0108] 1. Expression of GnRH Receptors on T Cells

[0109] Human naive and memory T cells were labeled with fluorescent surface marker antibodies CD45RA, CD45RO and CD4 and sorted with flow cytometry. Total RNA was extracted with Rnaeasy kit (Qiagen) and reversed transcribed with iScript select cDNA synthesis kit (Biorad). The template cDNA was amplified with SYBR Green (Applied Biosystem) and run on CFX96 PCR (Biorad). Ratios of Type I GnRH Receptor and Type II GnRH Receptor mRNA were normalized to RNA polymerase 11 expression in sorted naive T cells or memory T cells. The MCF-7 breast cancer cell line was used as a positive control.

[0110] Primer sequences:

TABLE-US-00006 Type I GnRH Receptor fwd 5′-tgc ctc ttc atc atc cct ct-3′ rev 5′-gca aat gca acc gtc att tt-3′ Type II GnRH Receptor fwd 5′-act gtt caa tgg ctg gct gt-3′ rev 5′-gcc ccc aga agt ttc ctt ac-3′

[0111] I. GnRH I vs GnRH II assay

[0112] Compounds were tested on cells made to express Type I or Type II GnRH Receptors by transfection. The cells were exposed to labelled GnRH compound, washed and then assessed by measuring the label on the cells. The label was either measured directly (radioactive isotope label or fluorescent label) or indirectly (biotin labelled peptide).

[0113] Signalling induced by the GnRH compounds was measured in the cell lines expressing Type I GnRH and Type 11 GnRH Receptors respectively. GnRH compounds were investigated for their respective affinity to GnRH I and GnRH II receptors using competition assays. Calcium flux was measured using cells labelled with Fluo-4-Direct either using a flow cytometer or by live cell imaging microscopy, in order to evaluate their potency establishing ED50 values. Signalling was also studied by western blotting using antibodies to p-ERK or p-JNK.

[0114] To assess the effects of cellular activation on the production of LH and FSH and compare it with stimulation of immune related functions, the effects of the compounds were studied on pituitary cells and immune cells expressing either Type 11 GnRH or Type I GnRH Receptors.

[0115] II. Immune Stimulation Assay

[0116] The potency of compounds in inducing activation of immune cells can be assessed using an assay such as the following:

[0117] Human peripheral blood mononuclear cells (PBMCs) were purified from healthy donors with Ficoll-Hypaque density centrifugation. Cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 μg/mL ampicillin, 100 μg/mL streptomycin and IL-2 (100 U/mL) for 72 hours in 37° C., 5% CO.sub.2 Cells were stimulated with increasing concentrations of GnRH II or GnRH I analogues and analysed for expression of cell specific surface activation markers CD25, CD69 and MHC class I with monoclonal antibodies from BD Pharmingen using flow cytometry.

[0118] Solubility and Stability Data (Examples 2 and 3)

[0119] Each compound (0.2 mg) is added to PBS (pH 7.4, 0.2 mg/ml) and sonicated for 10 mins then shaken for 20 mins. A T=0 h sample (80 μl) was taken for LC/MS analysis. The solutions were then incubated (37° C. with agitation) in a Techne Roller-Blot Hybridiser HB-3D. Further samples were taken at T=4, 24, 96 h for LC/MS analysis.

[0120] LC/MS analysis was conducted on an Agilent HP1100 HPLC system fitted with a diode array detector (DAD), Waters ZQ single quadrupole MS and a Waters X select CSH C18, 2.1 mm×50 mm, 3.5 μm column. LC/MS data was collected for compound identity. LC/UV area under curve (AUC) data was collected for each compound at 280 nm at each of the four timepoints (T=0, 4, 24, 96 h). The trend in this data was interpreted to give each compound a solubility rating of 1 (good) to 5 (poor) and a stability rating (T %).

[0121] Calcium Assay in CHO-K1 Cells (Genscript)

[0122] Test article sample solutions were dissolved in HBSS buffer (with 20 mM HEPES buffer, pH 7.4) to form the 5× working solution. FLIPR® Calcium 4 assay kit from Molecular devices (R8141) was used as required. In brief, CHO-K1 cells expressing GnRHR (CHO-K1/GnRHR/Ga15, Genscript accession NM_000406) were respectively cultured in the 10-cm dishes and maintained at 37° C./5% CO2. CHO-K1 cells expressing GNRHR were respectively seeded in a 384-well black-wall, clear-bottom plate at a density of 15,000 cells per well in 20 μl of growth medium about 18 hours prior to the day of experiment and maintained at 37° C./5% CO2. Then, 20 μl of dye loading solution was added into the wells and the plates were subsequently placed into a 37° C. incubator for 60 minutes, followed by a 15 minutes' incubation at room temperature. At last, 10 μl of compounds or control agonist were added into respective wells of the assay plate during reading in FLIPR. The plate containing 5× compound and control agonist solution was placed in FLIPR. Solutions were added into the cell plate automatically at the 20 seconds and the fluorescence signal was monitored for an additional 100 seconds (21 sec to 120 sec.). Data were recorded by ScreenWorks (version 3.1) as FMD files with FLIPR and stored on the GenScript computer network for off-line analysis. Data acquisition and analyses was performed using ScreenWorks (version 3.1) program and exported to Excel. The average value of the first 20 seconds' reading was calculated as the baseline and the relative fluorescent units (ΔRFU) intensity values were calculated by subtracting the average value of baseline from the maximum fluorescent units (21s to 120s).

[0123] The % Stimulation was calculated with the following equation:

% Stimulation=(ΔRFUCompound−ΔRFUBackground)/(ΔRFUAgonist control−ΔRFUBackground)×100

[0124] Dose response curves were fitted with four-parameter-logistic-equation by the software GraphPad Prism 6.

[0125] Equation: four parameter logistic equation.

Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log EC50−X)×Hillslope))

[0126] X is the logarithm of concentration. Y is the response.

[0127] Materials

[0128] Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources.

[0129] General Synthesis Method

[0130] Method A

##STR00374##

[0131] Peptides were prepared using standard Fmoc solid-phase synthesis as per the diagram above. Protected amino acids (Fmoc and tBu or Trt if necessary) were used, and synthesis was performed on 2-chlorotrityl polystyrene resin. Reactions are carried out in the order A, B, C followed by multiple iterations of B and C to build up the desired peptide. When the final amino acid (pyroglutamate—note, reaction C is used to do this, though the amino acid is not Fmoc protected) have been added the final two reactions—D and E—take place in that order to generate a compound of the invention.

[0132] Reaction A: The resin was suspended in dichloromethane (10-20 volume equivalents compared to the resin) and stirred at room temperature. Fmoc protected amino acid (2 equivalents) was added to 1 equivalent of resin in the presence of diisopropylethylamine (6 equivalents). The reaction was stirred for 0.5 to 1 hour at room temperature. The resin was collected by filtration and washed 6 times with DMF and then used directly in the next step.

[0133] Reaction B: The Fmoc protecting group was removed by the treatment of piperidine (20%) in dimethylformamide (5-10 volume equivalents compared to the resin) at room temperature. The reaction was stirred for up to 1 hour and the resin collected b filtration and then the resin was washed 6 times with DMF and used directly in the next step.

[0134] Reaction C: Fmoc-protected amino acid (4 equivalents) was dissolved in DMF and DIPEA (2 equivalents) added. After stirring at room temperature for one minute these were added to the resin supported amino acid (1 equivalent) from Reaction B was treated with HBTU (1 equivalent) added. The reaction was stirred for up to one hour and before the resin was collected by filtration and washed 6 times with DMF and used directly in the next step. The next step was either reaction B or reaction D depending on the target sequence.

[0135] Reaction D: The protected peptide was cleaved from the resin by treatment with 3-5% trifluoroacetic acid in dichloromethane. The resin was removed by filtration and the peptide accrued by precipitation with ice cold diethyl ether and collection by centrifugation. The solid was washed in further diethyl ether and then dried under vacuum before being used in the next step.

[0136] Reaction E: The C-terminal amide was formed by dissolving the peptide from Reaction D (1 equivalent) in DMF, monoalkylamine (20-50 equivalents) and HBTU (2-3 equivalents) were added and the reaction stirred at room temperature for up to 3 hours.

[0137] The reaction was diluted with water and the crude peptide was then purified as detailed below.

[0138] Method B

##STR00375## ##STR00376##

[0139] Peptides were prepared using standard Fmoc solid-phase synthesis as per the diagram above. Protected amino acids (Fmoc and tBu or Trt if necessary) were used, and synthesis was performed on Ramage resin. Reactions are carried out in the order A, B. C followed by multiple iterations of B and C to build up the desired peptide. When the final amino acid (pyroglutamate—note, reaction C is used to do this, though the amino acid is not Fmoc protected) have been added the final two reactions—D and E—take place in that order to generate a compound of the invention.

[0140] Reaction F: Fmoc Ramage resin is suspended in DMF (5-10 volume equivalents compared to resin) containing 20% piperidine. The reaction was stirred for up to 1 hour at room temperature and the resin collected by filtration and washed 6 times with DMF and used directly in the next reaction.

[0141] Reaction G: Fmoc-protected amino acid (5 equivalents) was dissolved in DMF and DIPEA (2 equivalents) added. After stirring at room temperature for one minute these were added to the resin supported amino acid (1 equivalent) from Reaction F was treated with HBTU (1 equivalent) added. The reaction was stirred for up to one hour and before the resin was collected by filtration and washed 6 times with DMF and used directly in the next step.

[0142] Reaction H: The Fmoc protecting group was removed by the treatment of piperidine (20%) in dimethylformamide (5-10 volume equivalents compared to the resin) at room temperature. The reaction was stirred for up to 1 hour and the resin collected b filtration and then the resin was washed 6 times with DMF and used directly in the next step.

[0143] Reaction I: Fmoc-protected amino acid (4 equivalents) was dissolved in DMF and DIPEA (2 equivalents) added. After stirring at room temperature for one minute these were added to the resin supported amino acid (1 equivalent) from Reaction H was treated with HBTU (1 equivalent) added. The reaction was stirred for up to one hour and before the resin was collected by filtration and washed 6 times with DMF and used directly in the next step. The next step was either reaction H or reaction J depending on the target sequence.

[0144] Reaction J: The peptide was cleaved from the resin by treatment with 90% trifluoroacetic acid with 2.5% water, 2.5% triisopropylsilane and 5% dichloromethane. The resin was removed by filtration and the peptide accrued by precipitation with ice cold diethyl ether and collection by centrifugation. The crude peptide was then purified as detailed below.

[0145] Purification

[0146] The crude peptides were individually dissolved in acetonitrile/H.sub.2O (1:1, v/v) and purified by preparative HPLC with a C18 column using a water (0.1% TFA)-acetonitrile (0.1% TFA) gradient. The final purity of the peptides was confirmed by analytical HPLC. Peptide was lyophilized before storage at −20° C.

[0147] Compound Analysis—Identity and Purity

[0148] Analysis Method A

[0149] For analysis, the compounds were dissolved in methanol:water (9:1, 0.1 mg/ml) and a 150 μl portion was placed in an HPLC microvial and centrifuged at 14000 rpm for 3 minutes. The sample was then examined by high performance liquid chromatography with diode array (HPLC-DAD) and mass spectrometry (HPLC-MS) detection. HPLC-DAD-MS was performed using an Agilent 1100 HPLC system comprising of quaternary pump, auto sampler, column oven and diode array detector coupled to a Waters ZQ single quadrupole mass spectrometer. The same reverse-phase Waters Xselect CSH C18, 2.1 mm×50 mm, 3.5 μm particle size column was used for all compounds and was fitted with a Waters VanGuard CSH C18, 2.1 mm×5 mm, 3.5 μm particle size guard column and Waters Acquity, 0.2 μm in-line column filter. The column was used at a flow rate of 1 ml/min maintained at a temperature of 60° C. The solvents used were 0.17% formic acid in 95% acetonitrile, 5% water (solvent B) and 10 mM ammonium formate, 0.2% formic acid in water (solvent A), with a gradient as follows: 5% solvent B from 0 to 0.2 min, 5 to 50% solvent B from 0.2 to 9.3 min, 50 to 95% solvent B from 9.3 to 9.5 min, 95% solvent B from 9.5 to 11 min, 95 to 5% solvent B from 11 to 11.05 min and re-equilibration with 5% solvent B from 11.05 to 11.5 min. Nitrogen was used as auxiliary and sheath gas. Source voltage was set at 3400 V, cone voltage set at 31 V with a gas flow of 50 L/hour, drying gas flow rate at 550 L/hour and drying gas temperature at 350° C.

[0150] Compound Analysis—Solubility and Stability in Solution

[0151] Analysis Method B

[0152] For solubility and stability analysis, the compounds were dissolved (0.2 mg/ml) in phosphate buffer solution (PBS, 10 mM, pH 7.4) and shaken at room temperature for 20 minutes. A T=0 hour sample was taken (80 μl) and centrifuged at 14000 rpm for 3 minutes then analysed by Analysis method A as above. The bulk samples were placed in a Techne Roller-Blot HB-3D Rolling Hybridiser at 37° C. and only removed when a sample (80 μl) was taken at time points T=4, 24 and 96 hours. The samples were centrifuged at 14000 rpm for 3 mins then analysed by HPLC-DAD-MS as above. The UV area under curve at 280 nm was recorded at each time point.

EXAMPLES

Example 1—Compound Synthesis

[0153] Compounds of the invention were made according to the methods set out in the General Synthesis Method.

TABLE-US-00007 Syn- Com- thesis pound Meth- no. R1 R2 R3 R4 R5 od 6 [00377] [00378] [00379] [00380] Et A 8 [00381] [00382] [00383] [00384] Et A 9 [00385] [00386] [00387] [00388] Et A 10 [00389] [00390] [00391] [00392] Et A 11 [00393] [00394] [00395] [00396] Et A 12 [00397] [00398] [00399] [00400] Et A 13 [00401] [00402] [00403] [00404] Et A 14 [00405] [00406] [00407] [00408] CH.sub.2CONH.sub.2 B 16 [00409] [00410] [00411] [00412] CH.sub.2CONH.sub.2 B 20 [00413] [00414] [00415] [00416] CH.sub.2CONH.sub.2 B 23 [00417] [00418] [00419] [00420] CH.sub.2CONH.sub.2 B 24 [00421] [00422] [00423] [00424] CH.sub.2CONH.sub.2 B 25 [00425] [00426] [00427] [00428] CH.sub.2CONH.sub.2 B 27 [00429] [00430] [00431] [00432] CH.sub.2CONH.sub.2 B 28 [00433] [00434] [00435] [00436] CH.sub.2CONH.sub.2 B 30 [00437] [00438] [00439] [00440] CH.sub.2CONH.sub.2 B 33 [00441] [00442] [00443] [00444] CH.sub.2CONH.sub.2 B 42 [00445] [00446] [00447] [00448] Et A 44 [00449] [00450] [00451] [00452] Et A 45 [00453] [00454] [00455] [00456] Et A 47 [00457] [00458] [00459] [00460] Et A 48 [00461] [00462] [00463] [00464] Et A 49 [00465] [00466] [00467] [00468] Et A 50 [00469] [00470] [00471] [00472] Et A 56 [00473] [00474] [00475] [00476] Et A 59 [00477] [00478] [00479] [00480] Et A 60 [00481] [00482] [00483] [00484] Et A 61 [00485] [00486] [00487] [00488] Et A 62 [00489] [00490] [00491] [00492] Et A 63 [00493] [00494] [00495] [00496] Et A 64 [00497] [00498] [00499] [00500] CH.sub.2CONH.sub.2 65 [00501] [00502] [00503] [00504] CH.sub.2CONH.sub.2 66 [00505] [00506] [00507] [00508] CH.sub.2CONH.sub.2 67 [00509] [00510] [00511] [00512] Et 68 [00513] [00514] [00515] [00516] CH.sub.2CONH.sub.2

TABLE-US-00008 Retention Time m/z Compound Salt (Analysis (Analysis number form method A) method A) 6 TFA 4.15 1213.8 8 TFA 5.5 1312.7 9 TFA 6.64 1246.8 10 TFA 4.27 1286.9 11 TFA 5.35 1220.9 12 TFA 6.75 1319.7 13 TFA 5.53 1293.7 14 TFA 6.13 1318.7 16 TFA 4.93 1292.8 20 TFA 4.02 1358.9 23 TFA 4.79 1296.7 24 TFA 5.84 1395.7 25 TFA 7.19 1329.6 27 TFA 4.91 1369.6 28 TFA 5.92 1303.8 30 TFA 7.20 1402.7 33 TFA 6.00 1376.4 42 TFA 3.90 1183.8 44 TFA 5.23 1282.8 45 TFA 6.35 1216.8 47 TFA 4.00 1256.8 48 TFA 5.12 1190.8 49 TFA 6.56 1290.0 50 TFA 5.30 1263.7 56 TFA 3.42 1297.7 59 TFA 5.22 1330.8 60 TFA 3.81 1370.6 61 TFA 4.36 1304.7 62 TFA 5.60 1403.5 63 TFA 4.69 1377.7 64 TFA 6.19 1391.7 65 TFA 5.09 1366.0 66 TFA 4.92 1384.7 67 TFA 4.47 1396.9 68 TFA 3.83 1285.8

Example 2—Solubility Analysis

[0154] The solubility of compounds of the invention was tested as described in the general methods. Solubility was then graded according to a rating between 1 to 5, where 1 is most soluble and 5 is least soluble.

TABLE-US-00009 Compound number Solubility grading Buserelin acetate 1 Triptorelin acetate 2 Naferelin acetate 2 Histrelin acetate 4 Leuprorelin acetate 2 Buserelin TFA 2 Triptorelin TFA 2 Naferelin TFA 1 Histrelin TFA 2 Leuprorelin TFA 1 6 1 8 2 9 1 10 1 11 1 12 4 13 3 14 4 16 2 20 3 23 1 24 5 25 4 33 2 30 1 28 2 27 1 56 1 67 1 59 4 63 5 62 5 61 3 60 2 42 1 44 2 45 2 50 1 49 4 48 2 47 1 68 1 66 5 14 4 65 4 64 5 16 2 20 3

Example 3—Stability Analysis

[0155] The stability of compounds of the invention in aqueous media (PBS ph 7.4) was tested as described in the general methods. Stability was then graded according to a rating where t½>96 minutes was shown as + and stability less than this was shown as -.

TABLE-US-00010 Compound number Stability grading 6 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 16 + 20 + 23 + 24 − 25 − 33 − 30 − 28 + 27 + 56 + 67 − 59 − 63 − 62 − 61 + 60 + 42 + 44 + 45 + 50 + 49 + 48 + 47 + 68 + 66 + 14 + 65 + 64 − 16 + 20 +

Example 4—GnRH R Stimulation

[0156] The ability of compounds of the invention to stimulate GnRHR was assessed by using a calcium assay with CHO-K1 cells (Genscript), see the general methods for details. Activity was then recorded as percentage stimulation at 1 μM.

TABLE-US-00011 Compound number GNRHR Stimulation at 1 μM Buserelin 94 Leuprorelin 107 (n = 2) Goserelin 99 Gonadorelin 102 Nafarelin 100 (n = 2)  6 88  8 86  9 78 10 81 11 76 12 80 13 87 14 78 16 76 20 75 23 106 24 87 25 82 33 67 30 12 28 95 27 108 56 96 67 117 59 85 63 77 62 77 61 112 60 102 42 102 44 111 45 105 50 105 49 77 48 118 47 119 68 86 66 83 14 78 65 72 64 50 16 76 20 75

REFERENCES

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[0168] All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.