Immunorhelin compounds for intracellular infections

Abstract

The present invention provides immune stimulating peptides (immunorhelins) capable of activating GnRH receptors when administered to animal or human patients or cells. These immunorhelins have utility in treating intracellular bacterial, fungal, and protozoal infections.

Claims

1. A GnRH analog according to formula (I): ##STR00520## or a pharmaceutically acceptable salt thereof; wherein R.sub.1= ##STR00521## R.sub.2= ##STR00522## R.sub.3= ##STR00523## R.sub.4= ##STR00524## 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 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-00012 Group Type I Type II R.sub.1 R.sub.3 R.sub.4

2. The GnRH analog according to claim 1, wherein R.sub.5=Et or CH.sub.2CONH.sub.2.

3. The GnRH analog according to claim 1, wherein R.sub.5=Me, iPr, nPr, nBu, iBu, sBu, or tBu.

4. A pharmaceutical composition comprising a GnRH analog according to claim 1 and one or more pharmaceutically acceptable excipients.

5. The GnRH analog according to claim 1, wherein R.sub.1= ##STR00531##

6. The GnRH analog according to claim 5, wherein: R.sub.2= ##STR00532##

7. The GnRH analog according to claim 5, wherein: R.sub.1= ##STR00533## R.sub.3= ##STR00534## and R.sub.4= ##STR00535##

8. The GnRH analog according to claim 7, wherein the GnRH analog according to formula (I) is one of the following compounds: TABLE-US-00013 Compound no. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 28 CH.sub.2CONH.sub.2 35 Et 55 CH.sub.2CONH.sub.2 61 Et 41 CH.sub.2CONH.sub.2 or 48 Et or a pharmaceutically acceptable salt of any of these.

9. The GnRH analog according to claim 5, wherein: R.sub.1= ##STR00560## R.sub.3= ##STR00561## and R.sub.4= ##STR00562##

10. The GnRH analog according to claim 9, wherein the GnRH analog according to formula (I) is one of the following compounds: TABLE-US-00014 Compound no. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 27 CH.sub.2CONH.sub.2 34 Et 54 CH.sub.2CONH.sub.2 60 Et 40 CH.sub.2CONH.sub.2 or 47 Et or a pharmaceutically acceptable salt of any of these.

11. The GnRH analog according to claim 5, wherein R.sub.2= ##STR00587##

12. The GnRH analog according to claim 11, wherein the GnRH analog according to formula (I) is one of the following compounds: TABLE-US-00015 Compound no. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 5 CH.sub.2CONH.sub.2 11 Et 4 CH.sub.2CONH.sub.2 or 10 Et or a pharmaceutically acceptable salt of any of these.

13. The GnRH analog according to claim 1, wherein: R.sub.1= ##STR00604## R.sub.3= ##STR00605## and R.sub.4= ##STR00606##

14. The GnRH analog according to claim 13, wherein the GnRH analog according to formula (I) is one of the following compounds: TABLE-US-00016 Compound no. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 7 CH.sub.2CONH.sub.2 12 Et 30 CH.sub.2CONH.sub.2 36 Et 62 Et 43 CH.sub.2CONH.sub.2 or 49 Et or a pharmaceutically acceptable salt of any of these.

15. A GnRH analog or a pharmaceutically acceptable salt thereof, wherein the GnRH analog is one of the following compounds: 1: pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Arg-Pro-Gly-NH.sub.2, 2: pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Arg-Pro-Gly-NH.sub.2, 3: pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Tyr-Pro-Gly-NH.sub.2, 6: pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Arg-Pro-NHEt, 8: pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Arg-Pro-NHEt, 9: pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Tyr-Pro-NHEt, 23: pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-Gly-NH.sub.2, 24: pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-Gly-NH.sub.2, 25: pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-Gly-NH.sub.2, 26: pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Arg-Pro-NHEt, 29: pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-NHEt, 31: pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-NHEt, 32: pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-NHEt 33: pGlu-His-Trp-Ser-His-D-Nal-Trp-Tyr-Pro-Gly-NH.sub.2, 37: pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-Gly-NH.sub.2, 38: pGlu-His-Trp-Ser-Try-D-Leu-Trp-Arg-Pro-Gly-NH.sub.2, 39: pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-Gly-NH.sub.2, 42: pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-NHEt, 44: pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Arg-Pro-NHEt, 45: pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-NHEt, 50: pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-NHEt, 51: pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-Gly-NH.sub.2, 52: pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-NH.sub.2, 53: pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-Gly-NH.sub.2, 56: pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-NHEt, 57: pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-NH.sub.2, 58: pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-NH.sub.2, 59: pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-NHEt, 63: pGlu-His-Trp-Ser-His-D-Bhi-Trp-Tyr-Pro-NHEt, or 67: pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-NHEt.

Description

LEGENDS TO FIGURES

(1) 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.sup.+ T cells (black squares) measured with flow cytometry.

(2) FIG. 2: 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 analogoue or 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 (A) or CD8.sup.+ T cells (B) measured with flow cytometry.

(3) FIG. 3: Expression of MHC class I after stimulation of CD4.sup.+CD14.sup.+ monocytes with increasing concentrations of GnRH I analogue (red) and GnRH II (black). CD14.sup.+ 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.sup.+CD14.sup.+ monocytes measured with flow cytometry.

(4) 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.

(5) FIG. 5: Expected data: Number of mycobacteria in the lungs of infected mice are suppressed by compounds of the present invention in the presence or absence of testosterone. Colony Forming Units (CFU) are assessed by growing lysates of lung tissue from infected mice on bacterial plates and counting colonies.

EXPERIMENTAL

(6) General Biology Methods

(7) The preferential effect of the compounds of the invention on GnRH receptors may be tested using one or more of the methods described below:

(8) I. Expression of GnRH Receptors on T Cells

(9) 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 II expression in sorted naive T cells or memory T cells. The MCF-7 breast cancer cell line was used as a positive control.

(10) Primer Sequences:

(11) 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′

(12) I. GnRH I Vs GnRH II Assay

(13) 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).

(14) Signalling induced by the GnRH compounds was measured in the cell lines expressing Type I GnRH and Type II GnRH Receptors respectively. GnRH compounds were investigated for their respective affinity to type I GnRH and type II GnRH 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.

(15) 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 II GnRH or Type I GnRH Receptors.

(16) II. Expression of Cell Specific Surface Markers and MHC Class II and MHC Class I

(17) 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 ampicilin and 100 μg/mL streptomycin for 24-72 hours in 37° C., 5% CO.sub.2. Cells were stimulated with a compound according to the invention and analysed for expression of cell specific surface markers and MHC class II (monoclonal antibodies from BD Pharmingen) with flow cytomtery.

(18) To test a set of compounds according to the invention for their immunomodulatory properties in an in vitro assay and evaluate their ability to induce MHC class II expression on monocytes. First when a known GnRH analogue was used in a co-culture to stimulate monocytes. A small increase in MHC class expression from background of in MFI may be seen. In contrast, when a compound according to the invention is used we may detect a larger expression of cell surface expression of MHC class II. If this is the observation, we may have identified a compound with an effect on MHC class II expression, allowing increased turnover and presentation of MHC class II and class I peptides from the endosomal and lysosomal pathway. The findings will enhance the presentation of peptides derived from intracellular pathogens and promote CD4.sup.+ helper T as well as CD8+ T cells activation, expansion and induce sterilizing immunity.

(19) Materials

(20) Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources.

(21) Theoretical Example of the Compounds of the Invention on Intracellular Bacteria

(22) Material and Methods

(23) Male mice are infected with Mycobacterium tuberculosis by inhalation of an aerosol containing the bacteria. The infecting dose is between 100 and 1000 bacteria per mouse. The GnRHII or GnRHI related compound (alone or together with testosterone) or vehicle is administered by an appropriate route in an appropriate dose for 1-2 weeks following infection with the bacteria either. The mice are sacrificed and lungs removed and homogenized and plated on bacteria dishes containing medium that supports the growth of Mycobacterium tuberculosis. After 3-4 weeks incubation, in a heated cabinet at 37 degrees C., the amount of bacteria in the lungs of the mice are quantified by counting bacterial colonies on the plate.

(24) Expected Results

(25) Expected results of this experiments is that the amount of Mycobacterium tuberculosis bacteria in the lungs of mice are reduced in mice treated with GnRH compounds compared with control vehicle treated mice. We also expect GnRHII related compounds to be superior to GnRHI related compounds in this respect. We do not expect co-administration of testosterone will have any effect on the degree of effect of the GnRH compounds.

(26) Measurement of Castractive Effects and Compensation Thereof

(27) Castration induced by the compounds of the invention, as well as any compensation thereof can be determined by measurement of the circulating levels of the relevant sex hormones. How to carry out such measurements is known to the person skilled in the art.

(28) General Synthesis Method

(29) ##STR00377##

(30) 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.

(31) Reaction A:

(32) 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.

(33) Reaction B:

(34) 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.

(35) Reaction C:

(36) 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.

(37) Reaction D:

(38) 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.

(39) Reaction E:

(40) 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. The reaction was diluted with water and the crude peptide was then purified as detailed below.

(41) ##STR00378## ##STR00379##

(42) 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.

(43) Reaction F:

(44) 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.

(45) Reaction G:

(46) 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.

(47) Reaction H:

(48) 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.

(49) Reaction I:

(50) 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.

(51) Reaction J:

(52) 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.

(53) Purification

(54) 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.

(55) Compound Analysis—Identity and Purity

(56) Analysis Method A

(57) 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. HPLCDAD-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.

(58) Compound Analysis—Solubility and Stability in Solution

(59) Analysis Method B

(60) 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

(61) Compounds of the invention were made according to the methods set out in the General Synthesis Method.

(62) TABLE-US-00007 Com- pound Synthesis no. R1 R2 R3 R4 R5 Method  6 0 Et A  8 Et A  9 0 Et A 10 Et A 11 Et A 12 00 01 02 03 Et A 13 04 05 06 07 Et A 14 08 09 0 CH.sub.2CONH.sub.2 B 16 CH.sub.2CONH.sub.2 B 20 CH.sub.2CONH.sub.2 B 23 0 CH.sub.2CONH.sub.2 B 24 CH.sub.2CONH.sub.2 B 25 0 CH.sub.2CONH.sub.2 B 27 CH.sub.2CONH.sub.2 B 28 CH.sub.2CONH.sub.2 B 30 0 CH.sub.2CONH.sub.2 B 33 CH.sub.2CONH.sub.2 B 42 0 Et A 44 Et A 45 Et A 47 0 Et A 48 Et A 49 0 Et A 50 Et A 56 Et A 59 0 Et A 60 Et A 61 0 Et A 62 Et A 63 Et A 64 00 01 02 03 CH.sub.2CONH.sub.2 65 04 05 06 07 CH.sub.2CONH.sub.2 66 08 09 0 CH.sub.2CONH.sub.2 67 Et 68 CH.sub.2CONH.sub.2

(63) TABLE-US-00008 Compound Retention Time m/z number Salt form (Analysis method A) (Analysis 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

(64) 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.

(65) 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

(66) The stability of compounds of the invention in aqueous media (PBS ph7.4) was tested as described in the general methods. Stability was then graded according to a rating where t1/2>96 minutes was shown as + and stability less than this was shown as −.

(67) 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

(68) 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.

(69) 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

(70) Interferon-inducible effector mechanisms in cellautonomous immunity. MacMicking, J. D. Nat. Rev. Immunol. 12, 367-382 (2012). Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis. Noss, E. H. et al. J. Immunol. 167, 910-918 (2001). HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2. Lubben, N. B. et al. Mol Biol. Cell. 18 (3351-3365). Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation. Ramachandra, L., Noss, E., Boom, W. H. & Harding, C. V. J. Exp. Med. 194, 1421-1432 (2001). The ins and outs of MHC class II-mediated antigen processing and presentation. Paul A. Roche & Kazuyuki Furuta. Nature Reviews Immunology 15, 203-216 (2015) Secreted Toxoplasma gondii molecules interfere with expression of MHC-II in interferon gamma-activated macrophages. Leroux L-P, Dasanayake D, Rommerein L M, Fox B A, Bzik D J, Jardim A, Dzierszinski F S. International Journal for Parasitology 2015 45: 319-332 Protection form Direct Cerebral Cryptococcus Infection by Interferon gamma-dependent Activation of Microglial Cells. Zhou Q, Gault R A, Kozel T R, Murphy W J. The Journal of Immunology 2007: 178: 5753-5761. Mycobacterium tuberculosis EsxH inhibits ESCRT-dependent CD4+ T-cell activation Portal-Celhay C, Tufariello J A M, Srivastava S, Zahra A, Klevorn T, Grace P S, Mehra A, Park H S, Ernst J D, Jacobs Jr W R & Philips J A. Nature Microbiology 2, Article number: 16232 (2016) doi:10.1038/nmicrobiol.2016.232 Gonadotropin secretion and its control. Fink G, The physiology of reproduction 1998. Immunomodulatory actions of gonadal steroids may be mediated by gonadotropin-releasing hormone. Jacobson J D and Ansari M A, Endocrinology 2004; 145(1):330-6. Unusual morphologic features of uterine leiomyomas treated with gonadotropin-releasing hormoneagonists: massive lymphoid infiltration and vasculitis. McClean G and McCluggage W G, Int J Surg Pathol. 2003; 11(4):339-44. Massive lymphocytic infiltration of uterine leyomyomas associated with GnRH agonist treatment. Bardsley V et al., Histopathology 1998; 33(1):80-2. Chronic plasma cell endometritis in hysterectomy specimens of HIV-infected women: a retrospective analysis. Kerr-Layton J A et al., Infect Dis Obstet Gynecol. 1998; 6(4):186-90. Serum dihydrotestosterone and testosterone concentrations in human immunodeficiency virus-infected men with and without weight loss. Arver S et al., J Androl 1999; 20(5):611-8. Prevalence of endocrine dysfunction in HIV-infected men. Brockmeyer G et al., Horm Res 2000; 54(5-6):294-5. Gonadotropin-releasing hormone increases CD4-T-lymphocyte numbers in an animal model of immunodeficiency. Jacobson J D et al., J Allergy Clin Immunol. 1999; 104:653-8. A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q.12. Morgan et al., Endocrinology. 2003 February; 144(2):423-36 Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists. Eidne et al., J Olin Endocrinol Metab. 1987 March; 64(3):425-32

(71) All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.