Immunomodulators
09861680 ยท 2018-01-09
Assignee
Inventors
- Claudio Mapelli (Langhorne, PA)
- Martin Patrick Allen (Flemington, NJ)
- Paul Michael Scola (Glastonbury, CT)
Cpc classification
C07K7/54
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K38/12
HUMAN NECESSITIES
C07K7/54
CHEMISTRY; METALLURGY
Abstract
The present disclosure provides novel macrocyclic peptides which inhibit the PD-1/PD-L1 and PD-L1/CD80 protein/protein interaction, and thus are useful for the amelioration of various diseases, including cancer and infectious diseases.
Claims
1. A compound of formula (I) ##STR00052## or a pharmaceutically acceptable salt thereof, wherein: A is ##STR00053## wherein: denotes the point of attachment to the carbonyl group and
denotes the point of attachment to the nitrogen atom; z is 0; w is 1; R.sup.14 and R.sup.15 are hydrogen; and R.sup.z is-C(O)NHR.sup.16; wherein R.sup.16 is CHR.sup.17C(O)NH.sub.2; wherein R.sup.17 is selected from hydrogen and CH.sub.2OH; R.sup.a, R.sup.c, R.sup.e, R.sup.f, R.sup.h, R.sup.j, R.sup.m, and R.sup.n are hydrogen; R.sup.1 is benzyl optionally substituted with hydroxy; R.sup.2 is hydrogen or methyl; or, R.sup.b and R.sup.2, together with the atoms to which they are attached, can form a ring as described below; R.sup.3 is CH.sub.2C(O)NH.sub.2; R.sup.4 is hydrogen, or, R.sup.4 and R.sup.d, together with the atoms to which they are attached, can form a ring as described below; R.sup.5 is (CH.sub.2)imidazolyl; R.sup.6 is (CH.sub.2).sub.2CO.sub.2H; R.sup.7 is hydrogen or R.sup.7 and R.sup.g, together with the atoms to which they are attached, can form a ring as described below; R.sup.8 is (CH.sub.2)indolyl; R.sup.9 is (CH.sub.2).sub.2NH.sub.2; R.sup.10 is selected from (CH.sub.2)indolyl and (CH.sub.2)benzothienyl, each optionally substituted with CH.sub.2CO.sub.2H; R.sup.11 is selected from hydrogen, methyl, and butyl; R.sup.12 is selected from methyl and butyl; R.sup.13 is isobutyl; R.sup.b is selected from C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkyl, carboxyC.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl, hydroxyC.sub.1-C.sub.6alkyl, (NR.sup.aR.sup.b)C.sub.1-C.sub.6alkyl wherein R.sup.a and R.sup.b are independently selected from a hydrogen and C.sub.1-C.sub.6alkyl, and phenylC.sub.1-C.sub.6alkyl wherein the phenyl is optionally substituted with one, two, three, four or five groups independently selected from C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6alkyl, cyano, halo, and nitro; or, R.sup.b and R.sup.2, together with the atoms to which they are attached, form a ring a selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole, wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy; R.sup.d is selected from hydrogen, C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkyl, carboxyC.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl, hydroxyC.sub.1-C.sub.6alkyl, (NR.sup.aR.sup.b)C.sub.1-C.sub.6alkyl wherein R.sup.a and R.sup.b are independently a selected from hydrogen and C.sub.1-C.sub.6alkyl, and phenylC.sub.1-C.sub.6alkyl wherein the phenyl is optionally substituted with one, two, three, four or five groups independently selected from C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6alkyl, cyano, halo, and nitro; or, R.sup.d and R.sup.4, together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and a tetrahydrothiazole, wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, hydroxy, and phenyl; R.sup.g is selected from hydrogen, C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkyl, carboxyC.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl, hydroxyC.sub.1-C.sub.6alkyl, (NR.sup.aR.sup.b)C.sub.1-C.sub.6alkyl wherein R.sup.a and R.sup.b are independently a selected from hydrogen and C.sub.1-C.sub.6alkyl, and phenylC.sub.1-C.sub.6alkyl wherein the phenyl is optionally substituted with one, two, three, four or five groups independently selected from C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6alkyl, cyano, halo, and nitro; or R.sup.g and R.sup.7, together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and a tetrahydrothiazole, wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy; R.sup.k is selected from C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkyl, carboxyC.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl, hydroxyC.sub.1-C.sub.6alkyl, (NR.sup.aR.sup.b)C.sub.1-C.sub.6alkyl wherein R.sup.a and R.sup.b are independently selected from hydrogen and C.sub.1-C.sub.6alkyl; and phenylC.sub.1-C.sub.6alkyl; and R.sup.1 is C.sub.1-C.sub.2alkyl; provided that at least one of R.sup.b, R.sup.d, R.sup.g, R.sup.k, and R.sup.l is selected from C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkyl, carboxyC.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl, hydroxyC.sub.1-C.sub.6alkyl, (NR.sup.aR.sup.b)C.sub.1-C.sub.6alkyl wherein a R.sup.a and R.sup.b are independently selected from hydrogen and C.sub.1-C.sub.6alkyl, and phenylC.sub.1-C.sub.6alkyl wherein the phenyl is optionally substituted with one, two, three, four or five groups independently selected from C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6alkyl, cyano, halo, and nitro.
2. A compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.17 is hydrogen; and R.sup.2 is hydrogen or methyl.
3. A compound selected from: ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
Description
EXPERIMENTAL PROCEDURES
(1) The abbreviations used in the present application, including particularly in the illustrative schemes and examples which follow, are well-known to those skilled in the art. Some of the abbreviations used are as follows: DMF for N,N-dimethylformamide; HATU for O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HCTU for O-(6-C.sub.1-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; TFA for trifluoroacetic acid; DBU for 1,8-diazobicyclo[5.4.0]undec-7-ene; DIAD for diisopropyl azodicarboxylate; TIS for triisopropylsilane; DMSO for dimethylsulfoxide; MeCN or ACN for acetonitrile; DCM for 1,1-dichloromethane; DIEA or DIPEA for diisopropylethylamine; Fmoc for 9-fluorenylmethyloxycarbonyl; NMM for N-methylmorpholine; DMAP for 4-N,N-dimethylaminopyridine; NMP for N-methylpyrrolidone; Ac for acetyl; and Et for ethyl.
(2) Analytical Data:
(3) Mass Spectrometry: ESI-MS(+) signifies electrospray ionization mass spectrometry performed in positive ion mode; ESI-MS() signifies electrospray ionization mass spectrometry performed in negative ion mode; ESI-HRMS(+) signifies high-resolution electrospray ionization mass spectrometry performed in positive ion mode; ESI-HRMS() signifies high-resolution electrospray ionization mass spectrometry performed in negative ion mode. The detected masses are reported following the m/z unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions.
(4) Analysis LCMS Condition A:
(5) Column: Waters BEH C18, 2.150 mm, 1.7-m particles; Mobile Phase A: water with 0.05% TFA; Mobile Phase B: Acetonitrile with 0.05% TFA; Temperature: 50 C.; Gradient: 2% B to 98% B over 2 min., then a 0.5 min. hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.
(6) Analysis LCMS Condition C:
(7) Column: Waters BEH C18, 2.150 mm, 1.7-m particles; Mobile Phase A: water with 0.2% Formic Acid and 0.01% TFA; Mobile Phase B: Acetonitrile with 0.2% Formic acid an 0.01% TFA; Temperature: 50 C.; Gradient: 2% B to 80% B over 2 min., 80% B to 98% B over 0.1 minute then a 0.5 min. hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.
(8) Analysis LCMS Condition D:
(9) Column: Waters BEH C18, 2.150 mm, 1.7-m particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 70 C.; Gradient: 0-100% B over 3 min., then a 2.0-minute hold at 100% B; Flow: 0.75 mL/min; Detection: UV at 220 nm.
(10) Analysis LCMS Condition E:
(11) Column: Waters BEH C18, 2.150 mm, 1.7-m particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; temperature: 50 C. or 70 C.; Gradient: 0-100% B over 3 min., then a 0.75 or 2.0-minute hold at 100% B; Flow: 0.75 or 1.11 mL/min; Detection: UV at 220 nm.
(12) Analysis LCMS Condition F:
(13) Column: Waters XBridge C18, 2.150 mm; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 35 C.; Gradient: 0-100% B over 4 min., then a 1-minute hold at 100% B; Flow: 4 mL/min; Detection: UV at 220 nm.
(14) Analysis LCMS Condition G:
(15) Column: Waters BEH C18, 2.050 mm, 1.7-m particles; Mobile Phase A: 5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10 mM ammonium acetate; Temperature: 50 C.; Gradient: 0-100% B over 3 min., then a 0.5-minute hold at 100% B; Flow: 0.5 mL/min; Detection: UV at 220 nm.
(16) Analysis LCMS Condition H:
(17) Column: Waters BEH C18, 2.050 mm, 1.7-m particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50 C.; Gradient: 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
(18) Analysis LCMS Condition I:
(19) Column: Waters BEH C18, 2.050 mm, 1.7-m particles; Mobile Phase A: 5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10 mM ammonium acetate; Temperature: 50 C.; Gradient: 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 0.5 mL/min; Detection: UV at 220 nm.
(20) Analysis LCMS Condition J:
(21) Waters CSH C18, 2.150 mm, 1.7-m particles; Mobile Phase A: 5:95 acetonitrile:water with trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with trifluoroacetic acid; Temperature: 70 C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 2.0-minute hold at 100% B; Flow: 0.75 mL/min; Detection: UV at 220 nm.
(22) General Procedures:
(23) Symphony Method A:
(24) All manipulations were performed under automation on a Symphony peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a Symphony polypropylene vessel fitted with a bottom frit. The vessel connects to the Symphony peptide synthesizer through both the bottom and the top of the tube. All solvents, DMF, DCM, amino acids and reagents are added through the bottom of the vessel and pass up through the frit to contact the resin. All solutions are removed through the bottom of the vessel. Periodic agitation describes a brief pulse of N.sub.2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 15 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution was used within 5 days of preparation. DMF=dimethylformamide; DCM=dichloromethane; THF=tetrahydrofuran; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; NMIVI=n-Methyl morpholine; DIPEA=diisopropylethylamine; DMAP=N,N-dimethylpyridin-4-amine; Rink resin=4-(2,4-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-aminomethyl resin; Sieber resin=Fmoc-amino-xanthen-3-yloxy, where 3-yloxy describes the position and type of connectivity to the polystyrene resin. The resins used are Merrifield polymer (polystyrene) with either a Rink or a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.35 mmol/g or 0.71 mmol/g loading, respectively. Other common acid sensitive resins can also be used in the synthesis such as functionalized Chlorotrityl Resin. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH (Dab=2,4-diaminobutyric acid); Fmoc-Dap(Boc)-OH (Dap=2,3-diaminopropionic acid); Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-t-Hyp(tBu)-OH (t-Hyp=trans-4-hydroxyproline); Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH (Sar=Sarcosine or [N-Me]Gly); Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.
(25) The procedures of Symphony Method A describe an experiment performed on a 0.050-0.100 mmol scale, where the scale is determined by the resin substitution. This scale corresponds to approximately 143-286 mg or 70-140 mg of the Rink or Sieber-Merrifield resins, respectively, described above. All procedures can be scaled beyond the 0.050-0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as Swelling procedure. Coupling of amino acids to a primary amine N-terminus used the Standard-coupling procedure described below. Coupling of amino acids to a secondary amine N-terminus used the Secondary-amine coupling procedure described below.
(26) Swelling Procedure:
(27) To a Symphony polypropylene reaction vessel was added Merrifield Rink or Sieber resin (70 mg, 0.050 mmol or 140 mg, 0.100 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit.
(28) Standard-Coupling Procedure:
(29) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The procedure was repeated one more time. The resin was washed 6 times as follows: for each wash, DMF (2.5-5.0 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25-2.5 mL, 5 eq), then HATU (0.2M in DMF, 1.25-2.5 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25-2.5 mL, 10 eq). The mixture was periodically agitated for one hour, then the reaction solution was drained through the frit. The resin was washed with DMF (2.5-5.0 mL) five times, stirring it for 30 seconds each time. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:5:85 v/v/v, 2.5-5.0 mL). The mixture was periodically agitated for 20 min., then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (2.5-5.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(30) Secondary Amine-Coupling Procedure:
(31) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The procedure was repeated one more time. The resin was washed 6 times as follows: for each wash, DMF (2.5-5.0 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5-5.0 mL, 10 eq), then HATU (0.2M in DMF, 2.5-5.0 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5-5.0 mL, 20 eq). The mixture was periodically agitated for two hours, then the reaction solution was drained through the frit. The resin was washed with DMF (2.5-5.0 mL) five times, stirring it for 30 seconds each time. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:5:85 v/v/v, 2.5-5.0 mL). The mixture was periodically agitated for 20 min., then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (2.5-5.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(32) Secondary Amine-Coupling without Fmoc Deprotection Procedure:
(33) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5-5.0 mL, 10 eq), then HATU (0.2M in DMF, 2.5-5.0 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5-5.0 mL, 20 eq). The mixture was periodically agitated for six hours, then the reaction solution was drained through the frit. The resin was washed with DMF (2.5-5.0 mL) five times, stirring it for 30 seconds each time. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:5:85 v/v/v, 2.5-5.0 mL). The mixture was periodically agitated for 20 min., then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (2.5-5.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(34) Chloroacetic Anhydride Capping Procedure:
(35) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed once with DMF (2.5-5.0 mL). To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5-5.0 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added NMM (0.8M in DMF, 3.0 mL, 48 eq) followed by Chloroacetic anhydride (0.4M in DMF, 3.0 mL, 24 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was washed once with DMF (4.0 mL). To the reaction vessel was added NMM (0.8M in DMF, 3.0 mL, 48 eq) followed by Chloroacetic anhydride (0.4M in DMF, 3.0 mL, 24 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was washed six times with DMF (3.0 mL) with periodically agitation of the mixture for 30 seconds before the solution was drained through the frit. The resin was then washed five times with DCM (3.0 mL) with periodic agitation of the resulting mixture for 30 seconds before the solution was drained through the frit. The resulting resin was then dried with a stream of Nitrogen for 10 mins.
(36) Symphony Method B:
(37) All manipulations were performed under automation on a Symphony peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a Symphony 20 mL polypropylene tube fitted with a bottom frit. The tube connects to the Symphony peptide synthesizer through both the bottom and the top of the tube. All Solvents, DMF, DCM, amino acids and reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. Periodic agitation describes a brief pulse of N2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 15 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution was used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; NMM=N-methyl morpholine; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where 3-yloxy describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Other common acid sensitive resins can also be used in the synthesis such as Rink or functionalized Chloro trityl Resin. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-t-Hyp(tBu)-OH (t-Hyp=trans-4-hydroxyproline; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.
(38) The procedures of Symphony Method B describes an experiment performed on a 0.10 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.10 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as Swelling procedure. Coupling of amino acids to a primary amine N-terminus used the Standard-coupling procedure described below. Coupling of amino acids to a secondary amine N-terminus used the Secondary amine-coupling procedure B, Custom amino acids are coupled via a manual blank addition of the amino acid Custom amino acids-coupling procedure described below, and Chloroacetyl Anhydride is added to the final position of the sequence using the Final capping procedure described below.
(39) Swelling Procedure:
(40) To a Symphony polypropylene reaction vessel was added Merrifield Sieber resin (140 mg, 0.100 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit.
(41) Standard-Coupling Procedure:
(42) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq), then HATU (0.2M in DMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac.sub.2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(43) Secondary Amine-Coupling Procedure:
(44) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq), then HATU (0.2M in DMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 60 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq.), then HATU (0.2M in DMF, 2.5 mL, 10 eq.), and finally NMM (0.8M in DMF, 2.5 mL, 20 eq.). The mixture was periodically agitated for 60 minutes, then the reaction solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac.sub.2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(45) Custom Amino Acids-Coupling Procedure:
(46) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq), then HATU (0.2M in DMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for six hours, then the reaction solution was drained through the frit. The resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac.sub.2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(47) Final Capping Procedure:
(48) The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed once with DMF (2.5-5.0 mL). To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added NMM (0.8M in DMF, 2.5 mL, 20 eq) followed by the addition of the Chloroacetic anhydride (0.4M in DMF, 2.5 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added NMM (0.8M in DMF, 2.5 mL, 10 eq) followed by the addition of the Chloroacetic anhydride (0.4M in DMF, 2.5 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac.sub.2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DCM (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was then dried with a stream of Nitrogen for 10 mins.
(49) Manual Procedures
(50) Bromoacetic Acid Coupling:
(51) All manipulations were performed manually at room temperature, unless noted otherwise. The resin was washed three times as follows: the resins from four 0.100 mmol scale Symphony syntheses used to prepare Resin Intermediates A and B were combined into a 50 mL fritted glass reactor equipped with a three-way stopcock and washed with DMF (10 mL) three times with agitation by N.sub.2 bubbling from the bottom of the reaction vessel. To the reaction vessel was added piperidine:DMF (20:80 v/v, 10 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The procedure was repeated once more. The resin was washed six times with DMF (10 mL). To the reaction vessel was added a solution of bromoacetic acid (10.0 eq) in DMF (5.0 mL) and DIC (10.3 eq). The mixture was agitated by N.sub.2 bubbling for one hour, then the reaction solution was drained through the frit. The resin was washed with DMF (5 mL) five times. The resulting resin was suspended in DCM/DMF (3:2) and divided, by volume, into seven equal parts (0.057 mmol), which were transferred into 25 mL fritted syringes. Each resin aliquot was used directly in the next on-resin N-alkylation step.
(52) N-Gly-Alkylation On-Resin Method A:
(53) All manipulations were performed manually at room temperature, unless noted otherwise. To the resin from the bromoacetylation step (0.057 mmol) was added a solution of the required amine (10 eq.) and DIEA (11 eq.) in DMF (mL) and the resulting mixture was stirred for one hour. The resin was washed five times with DMF (10 mL). The procedure was repeated once more. When the hydrochloride salt of the amine was used, an additional 10 eq. of DIEA was used. Reaction progress was monitored by TFA micro-cleavage of small resin samples. Upon reaction completion, the N-alkylated resin was washed five times with DMF (10 mL) and placed back into a Symphony reaction vessel for completion of sequence assembly on the Symphony peptide synthesizer.
(54) N-Gly-Alkylation On-Resin Method B:
(55) All manipulations were performed manually at room temperature, unless noted otherwise. To the resin from the bromoacetylation step (0.057 mmol) was added a solution of the required amine (10 eq.) and DIEA (11 eq.) in DMF (mL) and the resulting mixture was stirred for one hour. The resin was washed five times with DMF (10 mL). The procedure was repeated once more. When the hydrochloride salt of the amine was used, an additional 10 eq. of DIEA was used. The resin was then treated with a solution of amine (10-20 eq.) and DMAP (21 eq.) for one to sixteen hours. Reaction progress was monitored by TFA micro-cleavage of small resin samples. Upon reaction completion, the N-alkylated resin was washed five times with DMF (10 mL) and placed back into a Symphony reaction vessel for completion of sequence assembly on the Symphony peptide synthesizer.
(56) N-Gly-Alkylation On-Resin Method C:
(57) All manipulations were performed manually at room temperature, unless noted otherwise. This procedure was used when the required amine was ethylamine. To the resin from the bromoacetylation step (0.057 mmol) was added a solution of the ethylamine hydrochloride (10 eq.) and DIEA (21 eq.) in DMF (mL) and the resulting mixture was stirred for one hour. The resin was washed five times with DMF (10 mL). The procedure was repeated once more. The resin was then treated with a 2 M solution of ethylamine in THF (10 mL) for sixteen hours. Reaction progress was monitored by TFA micro-cleavage of small resin samples. Upon reaction completion, the N-alkylated resin was washed five times with DMF (10 mL) and placed back into a Symphony reaction vessel for completion of sequence assembly on the Symphony peptide synthesizer.
(58) N-Gly-Alkylation On-Resin Method D
(59) All manipulations were performed manually at room temperature, unless noted otherwise. To the bromoacetylatedresin (0.100 mmol) was added a solution of the required amine (10 eq., 1.0 mmol) and DBU (5 eq., 0.5 mmol) in DMF (3 mL) and the resulting mixture was stirred for three hours. The resin was washed once with DMF (5 mL). The procedure was repeated once more, but the reaction was allowed to proceed for 16 hrs. The resin was washed four times with DMF (4 mL) and DCM (4 mL), and was then placed back into a Symphony reaction vessel for completion of sequence assembly on the Symphony peptide synthesizer.
(60) Alkylation Method A:
(61) A solution of the alcohol corresponding to the alkylating group (0.046 g, 1.000 mmol), triphenylphosphine (0.131 g, 0.500 mmol), and DIAD (0.097 mL, 0.500 mmol) in 3 mL of THF was added to nosylated resin (0.186 g, 0.100 mmol), and the reaction mixture was stirred for 16 hours at room temperature. The resin was washed three times with THF (5 mL) Tetrahydrofuran, and the above procedure was repeated 1-3 times. Reaction progress was monitored by TFA micro-cleavage of small resin samples treated with a solution of 50 L of TIS in 1 mL of TFA for 1.5 hours.
(62) Alkylation Method B:
(63) The nosylated resin (0.100 mmol) was washed three times with N-methylpyrrolidone (NMP) (3 mL). A solution of NMP (3 mL), Alkyl Bromide (20 eq, 2.000 mmol) and DBU (20 eq, 0.301 mL, 2.000 mmol) was added to the resin, and the reaction mixture was stirred for 16 hours at room temperature. The resin was washed with NMP (3 mL) and the above procedure was repeated once more. Reaction progress was monitored by TFA micro-cleavage of small resin samples treated with a solution of 50 L of TIS in 1 mL of TFA for 1.5 hours.
(64) Nosylate Formation:
(65) A solution of collidine (10 eq.) in DCM (2 mL) was added to the resin, followed by a solution of Nos-Cl (8 eq.) in DCM (1 mL). The reaction mixture was stirred for 16 hours at room temperature. The resin was washed three times with DCM (4 mL) and three times with DMF (4 mL). The alternating DCM and DMF washes were repeated three times, followed by one final set of four DCM washes (4 mL).
(66) Nosylate Removal:
(67) The resin (0.100 mmol) was swelled using three washes with DMF (3 mL) and three washes with NMP (3 mL). A solution of NMP (3 mL), DBU (0.075 mL, 0.500 mmol) and 2-mercaptoethanol (0.071 mL, 1.000 mmol) was added to the resin and the reaction mixture was stirred for 5 minutes at room temperature. After filtering and washing with NMP (3 mL), the resin was re-treated with a solution of NMP (3 mL), DBU (0.075 mL, 0.500 mmol) and 2-mercaptoethanol (0.071 mL, 1.000 mmol) for 5 minutes at room temperature. The resin was washed three times with NMP (3 mL), four times with DMF (4 mL) and four times with DCM (4 mL), and was placed back into a Symphony reaction vessel for completion of sequence assembly on the Symphony peptide synthesizer.
(68) Chloroacetyl Chloride Coupling Procedure:
(69) To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 5 minutes and then the solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added 3.5 mL of a solution of DIPEA (40 eq), and chloroacetyl chloride (20 eq) in DMF. The mixture was periodically agitated for three hours, then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (5.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, CH.sub.2C.sub.12 (5.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. The resin was then dried under high vacuum.
(70) CEM Method A:
(71) All manipulations were performed under automation on a CEM Liberty microwave peptide synthesizer (CEM Corporation). All procedures unless noted were performed in a 30 or 125 mL polypropylene tube fitted with a bottom frit to a CEM Discovery microwave unit. The tube connects to the CEM Liberty synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top and bottom of the tube, which washes down the sides of the tube equally. All solutions are removed through the bottom of the tube except while transferring resin from the top. Periodic bubbling describes a brief bubbling of N2 gas through the bottom frit. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution was used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where 3-yloxy describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH
(72) The procedures of CEM Method A describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as Resin-swelling procedure. Coupling of amino acids to a primary amine N-terminus used the Single-coupling procedure described below. Coupling of amino acids to a secondary amine N-terminus used the Secondary amine-coupling procedure described below. Coupling of chloroacetyl group to the N-terminus of the peptide is described by the Chloroacetyl chloride coupling procedure or Chloroacetic acid coupling procedure detailed above.
(73) Resin-Swelling Procedure:
(74) To 50 mL polypropylene conical tube was added Merrifield: Sieber resin (140 mg, 0.100 mmol). Then DMF (7 mL) was added to the tube followed by DCM (7 mL). The resin was then transferred to the reaction vessel from top of the vessel. The procedure is repeated additionally two times. DMF (7 mL) was added followed by DCM (7 mL). The resin was allowed to swell with N2 bubbling from the bottom of the reaction vessel for 15 minutes before the solvent was drained through the frit.
(75) Standard Coupling Procedure:
(76) To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N2 bubbling for 5 minutes at 75 C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50 C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65 C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.
(77) Double-Couple Coupling Procedure:
(78) To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N2 bubbling for 5 minutes at 75 C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50 C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N2 bubbling for 5 minutes at 75 C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50 C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65 C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.
(79) Custom Amino Acids-Coupling Procedure:
(80) To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid solution (1.25 mL to 5 mL, 2.5 eq to 10 eq) containing HATU (2.5 eq to 10 eq), and finally DIPEA (2M in NMP, 0.5 mL to 1 mL, 20 eq). The mixture was mixed by N2 bubbling for 5 minutes to 2 hours at 25 C. to 75 C., then the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65 C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.
(81) Chloroacetyl Chloride Coupling Procedure:
(82) To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added 3.0 mL of a solution of DIPEA (4.0 mmol, 0.699 mL, 40 eq), and chloroacetyl chloride (2.0 mmol, 0.160 mL, 20 eq) in DMF. The mixture was periodically agitated for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2C.sub.12 (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.
(83) Global Deprotection Method A:
(84) All manipulations were performed manually at room temperature unless noted otherwise. The procedure of Global Deprotection Method A describes an experiment performed on a 0.057-0.100 mmol scale, where the scale is determined by the amount of Rink or Sieber linker bound to the resin. The procedure can be scaled beyond 0.057-0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A deprotection solution was prepared using trifluoroacetic acid:triisopropylsilane:dithiothreitol (95:2.5:2.5 v:v:w) or trifluoroacetic acid:triisopropylsilane:dithiothreitol (96.5:2.5:1.0 v:v:w). The solution was pre-cooled in ice prior to adding it to the resin. To the resin in a 25 mL fritted syringe was added the deprotection solution (2.5-4.0 mL). The mixture was mixed in a shaker for 60 min. The solution was filtered through the frit into cold diethyl ether (30 mL). The precipitated solid was centrifuged for 3 min. The supernatant solution was decanted and the solid was re-suspended in diethyl ether (15 mL). This procedure was repeated two more times. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.
(85) Cyclization Method A:
(86) All manipulations were performed manually unless noted otherwise. The procedure of Cyclization Method A describes an experiment performed on a 0.057-0.100 mmol scale. The crude peptide solid was dissolved in a solution of acetonitrile:aqueous 0.1M ammonium bicarbonate buffer (1:3 or 1:2, v:v; 30-40 mL), and the pH of the solution was carefully adjusted to 8.5-9.0 using aqueous NaOH (1.0 M). The solution was then mixed using a shaker for 12 to 18 hours. The reaction solution was concentrated and the residue was then dissolved in acetonitrile:water. This solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.
Preparation of Intermediate Resins for Examples 3003-3050
(87) Preparation of Intermediate Resin A:
(88) ##STR00005##
(89) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(90) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(91) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(92) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(93) Fmoc-Ala-OH Symphony Method B: Standard-coupling procedure was followed. The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
Preparation of Intermediate Resin B:
(94) ##STR00006##
(95) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(96) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(97) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(98) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(99) Fmoc-Nle-OH Symphony Method B: Standard-coupling procedure was followed. The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
Preparation of Intermediate Resin C:
(100) ##STR00007##
(101) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(102) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(103) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(104) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(105) Fmoc-nMethyl-Nle-OH Symphony Method B: Standard-coupling procedure was followed;
(106) Fmoc-Nle-OH Symphony Method B: Secondary amine-coupling procedure was followed.
(107) The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
(108) Preparation of Intermediate Resin D:
(109) ##STR00008##
(110) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(111) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(112) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(113) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(114) Fmoc-nMethyl-Nle-OH Symphony Method B: Standard-coupling procedure was followed;
(115) Fmoc-Ala-OH Symphony Method B: Secondary amine-coupling procedure was followed.
(116) The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
(117) Preparation of Intermediate Resin E:
(118) ##STR00009##
(119) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(120) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(121) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(122) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(123) Fmoc-nMethyl-Nle-OH Symphony Method B: Standard-coupling procedure was followed;
(124) Fmoc-nMethyl-Nle-OH Symphony Method B: Secondary amine-coupling procedure, was followed;
(125) Fmoc-Trp(Boc)-OH Symphony Method B: Secondary amine-coupling procedure, was followed;
(126) Fmoc-Dab(Boc)-OH Symphony Method B: Standard-coupling procedure was followed;
(127) Fmoc-Trp(Boc)-OH Symphony Method B: Standard-coupling procedure was followed;
(128) Fmoc-Hyp(tBu)-OH Symphony Method B: Standard-coupling procedure was followed;
(129) Fmoc-Glu(tBu)-OH Symphony Method B: Secondary amine-coupling procedure was followed;
(130) Fmoc-His(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(131) Fmoc-Pro-OH Symphony Method B: Standard-coupling procedure was followed;
(132) Fmoc-Asn(Trt)-OH Symphony Method B: Secondary amine-coupling procedure was followed;
(133) Fmoc-Ala-OH Symphony Method B: Standard-coupling procedure was followed. The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
Preparation of Intermediate Resin F:
(134) ##STR00010##
(135) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(136) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(137) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(138) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(139) Fmoc-nMethyl-Nle-OH Symphony Method B: Standard-coupling procedure was followed.
(140) The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), and was then transferred to a BioRad tube, and treated with the following solution: Bromoacetic acid (10 eq., 1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reaction mixture was stirred for 3 hours. After filtration and a DMF wash (3 ML), the resin was re-treated with the following solution: Bromoacetic acid (10 eq., 1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reaction mixture was stirred for 16 hours. After filtration, the resin was washed five times with DMF (3.0 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
(141) Preparation of Intermediate Resin G:
(142) ##STR00011##
(143) To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol) and the vessel was placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(144) Fmoc-Gly-OH Symphony Method B: Resin-swelling procedure was followed;
(145) Fmoc-Cys(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(146) Fmoc-Leu-OH Symphony Method B: Standard-coupling procedure was followed;
(147) Fmoc-nMethyl-Nle-OH Symphony Method B: Standard-coupling procedure was followed;
(148) Fmoc-nMethyl-Nle-OH Symphony Method B: Secondary amine-coupling procedure was followed;
(149) Fmoc-Trp(Boc)-OH Symphony Method B: Secondary amine-coupling procedure was followed;
(150) Fmoc-Dab(Boc)-OH Symphony Method B: Standard-coupling procedure was followed;
(151) Fmoc-Trp(Boc)-OH Symphony Method B: Standard-coupling procedure was followed;
(152) Fmoc-Hyp(tBu)-OH Symphony Method B: Standard-coupling procedure was followed;
(153) Fmoc-Glu(tBu)-OH Symphony Method B: Secondary amine-coupling procedure was followed;
(154) Fmoc-His(Trt)-OH Symphony Method B: Standard-coupling procedure was followed;
(155) Fmoc-Pro-OH Symphony Method B: Standard-coupling procedure was followed;
(156) Fmoc-Asn(Trt)-OH Symphony Method B: Secondary amine-coupling procedure was followed.
(157) The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 mins with periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodic Nitrogen stirring was continued for 5 mins. The resulting resin was washed six times with DMF (2.5 mL), and was then transferred to a BioRad tube, and treated with the following solution: Bromoacetic acid (10 eq., 1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reaction mixture was stirred for 3 hours. After filtration and a DMF wash (3 ML), the resin was re-treated with the following solution: Bromoacetic acid (10 eq., 1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reaction mixture was stirred for 16 hours. After filtration, the resin was washed five times with DMF (3.0 mL), then five times with DCM (2.5 mL), and was used as an intermediate for the N-alkylation procedures.
(158) Preparation of Intermediate Resin H.
(159) ##STR00012##
(160) To four Symphony reaction vessels was added Rink resin (mg, 0.100 mmol) and the vessels were placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(161) Symphony Method A: Resin-swelling procedure was followed;
(162) Symphony Method A: Standard coupling procedure was followed with Fmoc-Gly-OH;
(163) Symphony Method A: Standard coupling procedure was followed with Fmoc-Cys(Trt)-OH;
(164) Symphony Method A: Standard coupling procedure was followed with Fmoc-Leu-OH;
(165) Symphony Method A: Secondary-amine Coupling procedure was followed with Fmoc-[N-Me]Nle-OH;
(166) Symphony Method A: Secondary-amine Coupling procedure was followed with Fmoc-[N-Me]Nle-OH
(167) Symphony Method A: Secondary-amine Coupling procedure was followed with Fmoc-Trp(Boc)-OH;
(168) Symphony Method A: Standard coupling procedure was followed with Fmoc-Dab(Boc)-OH;
(169) Symphony Method A: Standard coupling procedure was followed with Fmoc-Trp(Boc)-OH.
(170) The resulting resins were combined into a 50 mL fritted glass reactor and washed with DMF as described in the above bromoacetic acid coupling procedure.
(171) Preparation of Intermediate Resin I.
(172) ##STR00013##
(173) To four Symphony reaction vessels was added Rink resin (mg, 0.100 mmol) and the vessels were placed on the Symphony peptide synthesizer. The following procedures were then performed sequentially:
(174) Symphony Method A: Resin-swelling procedure was followed;
(175) Symphony Method A: Standard coupling procedure was followed with Fmoc-Gly-OH;
(176) Symphony Method A: Standard coupling procedure was followed with Fmoc-Cys(Trt)-OH;
(177) Symphony Method A: Standard coupling procedure was followed with Fmoc-Leu-OH;
(178) Symphony Method A: Secondary-amine coupling procedure was followed with Fmoc-[N-Me]Nle-OH;
(179) Symphony Method A: Secondary-amine coupling procedure was followed with Fmoc-[N-Me]Nle-OH
(180) Symphony Method A: Secondary-amine coupling procedure was followed with Fmoc-Trp(Boc)-OH;
(181) Symphony Method A: Standard coupling procedure was followed with Fmoc-Dab(Boc)-OH;
(182) Symphony Method A: Standard coupling procedure was followed with Fmoc-Trp(Boc)-OH.
(183) Symphony Method A: Standard coupling procedure was followed with Fmoc-t-Hyp(tBu)-OH;
(184) Symphony Method A: Secondary-amine Coupling procedure was followed with Fmoc-Glu(OtBu)-OH;
(185) Symphony Method A: Standard coupling procedure was followed with Fmoc-His(Trt)-OH.
(186) The resulting resins were combined into a 50 mL fritted glass reactor and washed with DMF as described in the above bromoacetic acid coupling procedure.
Preparation of Example 3003
(187) ##STR00014##
(188) Example 3003 was prepared following the general synthetic sequence described below, starting from Intermediate Resin H. Intermediate Resin A (0.400 mmol) was placed into a 50 mL fritted glass reactor. The following procedures were then performed sequentially:
(189) Bromoacetic Acid Coupling procedure was followed;
(190) N-Gly-Alkylation On-resin Method C procedure was followed;
(191) Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure was followed with Fmoc-Glu(OtBu)-OH;
(192) Symphony Method A: Standard coupling procedure was followed with Fmoc-His(Trt)-OH;
(193) Symphony Method A: Standard coupling procedure was followed with Fmoc-Pro-OH;
(194) Symphony Method A: Secondary-amine coupling procedure was followed with Fmoc-Asn(Trt)-OH;
(195) Symphony Method A: Standard coupling procedure was followed with Fmoc-[N-Me]Ala-OH;
(196) Symphony Method A: Secondary-amine coupling procedure was followed with Fmoc-Tyr(tBu)-OH;
(197) Chloroacetic Anhydride capping procedure was followed;
(198) Global Deprotection Method A was followed;
(199) Cyclization Method A was followed.
(200) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 6.3 mg, and its estimated purity by LCMS analysis was 99% using Analysis LCMS condition D.
(201) Analysis LCMS condition D: Retention time=1.88 min; ESI-MS(+) m/z 935.1 (M+2H).
(202) ESI-HRMS(+) m/z:
(203) Calculated: 934.4692 (M+2H).
(204) Found: 934.4674 (M+2H).
Preparation of Example 3004
(205) ##STR00015##
(206) Example 3004 was prepared following the general synthetic sequence described for the preparation of Example 3003, starting from Intermediate Resin H, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(207) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 6.6 mg, and its estimated purity by LCMS analysis was 95% using Analysis LCMS condition D.
(208) Analysis LCMS condition D: Retention time=1.87 min; ESI-MS(+) m/z 961.9 (M+2H).
(209) ESI-HRMS(+) m/z:
(210) Calculated: 965.4771 (M+2H).
(211) Found: 965.4754 (M+2H).
Preparation of Example 3005
(212) ##STR00016##
(213) Example 3005 was prepared following the general synthetic sequence described for the preparation of Example 3003, starting from Intermediate Resin H, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method B procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(214) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 3.9 mg, and its estimated purity by LCMS analysis was 100% using Analysis LCMS condition D.
(215) Analysis LCMS condition D: Retention time=1.72 min; ESI-MS(+) m/z 950.4 (M+2H).
(216) ESI-HRMS(+) m/z:
(217) Calculated: 949.4563 (M+2H).
(218) Found: 949.4547 (M+2H).
Preparation of Example 3006
(219) ##STR00017##
(220) Example 3006 was prepared following the general synthetic sequence described for the preparation of Example 3003, starting from Intermediate Resin H, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(221) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 13.8 mg, and its estimated purity by LCMS analysis was 100% using Analysis LCMS conditions D and E.
(222) Analysis LCMS condition D: Retention time=1.90 min; ESI-MS(+) m/z 950.5 (M+2H).
(223) Analysis LCMS condition E: Retention time=1.58 min; ESI-MS(+) m/z 950.1
(224) ESI-HRMS(+) m/z:
(225) Calculated: 949.4745 (M+2H).
(226) Found: 949.4725 (M+2H).
Preparation of Example 3007
(227) ##STR00018##
(228) Example 3007 was prepared following the general synthetic sequence described for the preparation of Example 3003, starting from Intermediate Resin H, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method B procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(229) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 3.9 mg, and its estimated purity by LCMS analysis was 100% using Analysis LCMS condition D.
(230) Analysis LCMS condition D: Retention time=1.96 min; ESI-MS(+) m/z 969.3 (M+2H).
(231) ESI-HRMS(+) m/z:
(232) Calculated: 968.4629 (M+2H).
(233) Found: 968.4617 (M+2H).
Preparation of Example 3008
(234) ##STR00019##
(235) Example 3008 was prepared following the general synthetic sequence described below, starting from Intermediate Resin I. Intermediate Resin B (0.400 mmol) was placed into a 50 mL fritted glass reactor. The following procedures were then performed sequentially:
(236) Bromoacetic Acid Coupling procedure was followed;
(237) N-Gly-Alkylation On-resin Method A procedure was followed;
(238) Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure was followed with Fmoc-Asn(Trt)-OH;
(239) Symphony Method A: Standard coupling procedure was followed with Fmoc-[N-Me]Ala-OH;
(240) Symphony Method A: Secondary-amine coupling procedure was followed with Fmoc-Tyr(tBu)-OH;
(241) Chloroacetic Anhydride capping procedure was followed;
(242) Global Deprotection Method A was followed;
(243) Cyclization Method A was followed.
(244) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 10.6 mg, and its estimated purity by LCMS analysis was 99% using Analysis LCMS condition D.
(245) Analysis LCMS condition D: Retention time=1.87 min; ESI-MS(+) m/z 974.3 (M+2H).
(246) ESI-HRMS(+) m/z:
(247) Calculated: 973.4745 (M+2H).
(248) Found: 973.4729 (M+2H).
Preparation of Example 3009
(249) ##STR00020##
(250) Example 3009 was prepared following the general synthetic sequence described for the preparation of Example 3008, starting from Intermediate Resin I, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(251) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 35 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 9.1 mg, and its estimated purity by LCMS analysis was 100% using Analysis LCMS condition E.
(252) Analysis LCMS condition E: Retention time=1.56 min; ESI-MS(+) m/z 958.0 (M+2H).
(253) ESI-HRMS(+) m/z:
(254) Calculated: 957.4720 (M+2H).
(255) Found: 957.4693 (M+2H).
Preparation of Example 3010
(256) ##STR00021##
(257) Example 3010 was prepared following the general synthetic sequence described for the preparation of Example 3008, starting from Intermediate Resin I, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method B procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(258) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 4.7 mg, and its estimated purity by LCMS analysis was 97% using Analysis LCMS condition D.
(259) Analysis LCMS condition D: Retention time=1.83 min; ESI-MS(+) m/z 977.7 (M+2H).
(260) ESI-HRMS(+) m/z:
(261) Calculated: 976.4604 (M+2H).
(262) Found: 976.4592 (M+2H).
Preparation of Example 3011
(263) ##STR00022##
(264) Example 3011 was prepared following the general synthetic sequence described for the preparation of Example 3003, starting from Intermediate Resin H, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(265) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 10.6 mg, and its estimated purity by LCMS analysis was 89% using Analysis LCMS conditions D and E.
(266) Analysis LCMS condition D: Retention time=1.85 min; ESI-MS(+) m/z 942.9 (M+2H).
(267) Analysis LCMS condition E: Retention time=1.47 min; ESI-MS(+) m/z 942.6 (M+2H).
(268) ESI-HRMS(+) m/z:
(269) Calculated: 941.9723 (M+2H).
(270) Found: 941.9747 (M+2H).
Preparation of Example 3012
(271) ##STR00023##
(272) Example 3012 was prepared following the general synthetic sequence described for the preparation of Example 3003, starting from Intermediate Resin H, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(273) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 3.4 mg, and its estimated purity by LCMS analysis was 95% using Analysis LCMS conditions D and E.
(274) Analysis LCMS condition D: Retention time=1.95 min; ESI-MS(+) m/z (M+2H), not detected.
(275) Analysis LCMS condition E: Retention time=1.60 min; ESI-MS(+) m/z (M+2H), not detected.
(276) ESI-HRMS(+) m/z:
(277) Calculated: 942.4667 (M+2H).
(278) Found: 942.4659 (M+2H).
Preparation of Example 3013
(279) ##STR00024##
(280) Example 3013 was prepared following the general synthetic sequence described for the preparation of Example 3008, starting from Intermediate Resin I, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method C procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(281) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 40 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 5.8 mg, and its estimated purity by LCMS analysis was 97% using Analysis LCMS conditions D and E.
(282) Analysis LCMS condition D: Retention time=1.80 min; ESI-MS(+) m/z 942.9 (M+2H);
(283) Analysis LCMS condition D: Retention time=1.56 min; ESI-MS(+) m/z 943.0 (M+2H).
(284) ESI-HRMS(+) m/z:
(285) Calculated: 942.4667 (M+2H).
(286) Found: 942.4656 (M+2H).
Preparation of Example 3014
(287) ##STR00025##
(288) Example 3014 was prepared following the general synthetic sequence described for the preparation of Example 3008, starting from Intermediate Resin I, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(289) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 40 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 10.2 mg, and its estimated purity by LCMS analysis was 90% using Analysis LCMS condition D.
(290) Analysis LCMS condition D: Retention time=1.75 min; ESI-MS(+) m/z 950.5 (M+2H).
(291) ESI-HRMS(+) m/z:
(292) Calculated: 949.9722 (M+2H).
(293) Found: 949.9704 (M+2H).
Preparation of Example 3015
(294) ##STR00026##
(295) Example 3015 was prepared following the general synthetic sequence described for the preparation of Example 3008, starting from Intermediate Resin I, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method B procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(296) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 40-80% B over 40 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 3.4 mg, and its estimated purity by LCMS analysis was 90% using Analysis LCMS condition D.
(297) Analysis LCMS condition D: Retention time=1.72 min; ESI-MS(+) m/z 958.2 (M+2H).
(298) ESI-HRMS(+) m/z:
(299) Calculated: 957.4538 (M+2H).
(300) Found: 957.4521 (M+2H).
Preparation of Example 3016
(301) ##STR00027##
(302) Example 3016 was prepared following the general synthetic sequence described for the preparation of Example 3008, starting from Intermediate Resin I, using the following general procedures: Bromoacetic Acid Coupling procedure; N-Gly-Alkylation On-resin Method A procedure; Symphony Method A: Secondary-amine coupling without Fmoc deprotection procedure; Symphony Method A: Standard coupling procedure; Symphony Method A: Secondary-amine coupling procedure; Chloroacetic Anhydride capping procedure; Global Deprotection Method A; Cyclization Method A.
(303) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 35 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 4.0 mg, and its estimated purity by LCMS analysis was 98% using Analysis LCMS conditions D and E.
(304) Analysis LCMS condition D: Retention time=1.85 min; ESI-MS(+) m/z, (M+2H) not detected;
(305) Analysis LCMS condition E: Retention time=1.56 min; ESI-MS(+) m/z, (M+2H) not detected.
(306) ESI-HRMS(+) m/z:
(307) Calculated: 950.4642 (M+2H).
(308) Found: 950.4629 (M+2H).
Preparation of Example 3017
(309) ##STR00028##
(310) Example 3017 was prepared following the general synthetic sequence described below, starting from Intermediate Resin E. The following procedures were performed sequentially:
(311) Nosylate formation, Alkylation method B, Nosylate removal, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 5.1 mg, and its estimated purity by LCMS analysis was 100%.
(312) Analysis LCMS Condition H: retention time=1.71 min.; ESI-MS(+) m/z 956.2 (M+2H);
(313) Analysis LCMS Condition I: retention time=2.73 min.; ESI-MS(+) m/z 956.3 (M+2H).
Preparation of Example 3019
(314) ##STR00029##
(315) Example 3019 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from Intermediate Resin E. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(316) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 10.7 mg, and its estimated purity by LCMS analysis was 97%.
(317) Analysis LCMS Condition H: retention time=1.73 min.; ESI-MS(+) m/z 987.2 (M+2H).
(318) Analysis LCMS Condition I: retention time=2.81 min.; ESI-MS(+) m/z 987.2 (M+2H).
Preparation of Example 3020
(319) ##STR00030##
(320) Example 3020 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin A. The following procedures were performed sequentially: Nosylate formation, Alkylation method B, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(321) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 14.1 mg, and its estimated purity by LCMS analysis was 97%.
(322) Analysis LCMS Condition H: retention time=1.5 min.; ESI-MS(+) m/z 935.9 (M+2H).
(323) Analysis LCMS Condition J: retention time=1.26 min.; ESI-MS(+) m/z 935.0 (M+2H).
Preparation of Example 3021
(324) ##STR00031##
(325) Example 3021 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin B. The following procedures were performed sequentially: Nosylate formation, Alkylation method B, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(326) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 6.0 mg, and its estimated purity by LCMS analysis was 100%.
(327) Analysis LCMS Condition H: retention time=1.77 min.; ESI-MS(+) m/z 956.4 (M+2H).
(328) Analysis LCMS Condition J: retention time=1.47 min.; ESI-MS(+) m/z 956.3 (M+2H).
Preparation of Example 3022
(329) ##STR00032##
(330) Example 3022 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin D. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(331) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 10.7 mg, and its estimated purity by LCMS analysis was 100%.
(332) Analysis LCMS Condition H: retention time=1.26 min.; ESI-MS(+) m/z 942.8 (M+2H).
(333) Analysis LCMS Condition J: retention time=1.0 min.; ESI-MS(+) m/z 942.5 (M+2H).
Preparation of Example 3023
(334) ##STR00033##
(335) Example 3023 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin C. The following procedures were performed sequentially: Nosylate formation, Alkylation method B, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(336) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 6.9 mg, and its estimated purity by LCMS analysis was 98%.
(337) Analysis LCMS Condition H: retention time=1.67 min.; ESI-MS(+) m/z 955.9 (M+2H).
(338) Analysis LCMS Condition I: retention time=2.73 min.; ESI-MS(+) m/z 956.2 (M+2H).
Preparation of Example 3024
(339) ##STR00034##
(340) Example 3024 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin D. The following procedures were performed sequentially: Nosylate formation, Alkylation method B, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(341) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 9.2 mg, and its estimated purity by LCMS analysis was 100%.
(342) Analysis LCMS Condition H: retention time=1.59 min.; ESI-MS(+) m/z 935.0 (M+2H).
(343) Analysis LCMS Condition E: retention time=1.38 min.; ESI-MS(+) m/z 935.3 (M+2H).
Preparation of Example 3025
(344) ##STR00035##
(345) Example 3025 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin D. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(346) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 8.0 mg, and its estimated purity by LCMS analysis was 95%.
(347) Analysis LCMS Condition H: retention time=1.5 min.; ESI-MS(+) m/z 950.0 (M+2H).
(348) Analysis LCMS Condition J: retention time=1.37 min.; ESI-MS(+) m/z 949.6 (M+2H).
Preparation of Example 3026
(349) ##STR00036##
(350) Example 3026 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin E. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(351) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: Waters CSH C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 10.8 mg, and its estimated purity by LCMS analysis was 78%.
(352) Analysis LCMS Condition H: retention time=1.74 min.; ESI-MS(+) m/z 971.1 (M+2H).
(353) Analysis LCMS Condition J: retention time=1.37 min.; ESI-MS(+) m/z 971.1 (M+2H).
Preparation of Example 3028
(354) ##STR00037##
(355) Example 3028 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin D. The following procedures were performed sequentially: Nosylate formation, Alkylation method B, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(356) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.1 mg, and its estimated purity by LCMS analysis was 82%.
(357) Analysis LCMS Condition H: retention time=1.31 min.; ESI-MS(+) m/z 950.0 (M+2H).
(358) Analysis LCMS Condition J: retention time=1.16 min.; ESI-MS(+) m/z 950.0 (M+2H).
Preparation of Example 3029
(359) ##STR00038##
(360) Example 3029 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin D. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(361) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.7 mg, and its estimated purity by LCMS analysis was 78%.
(362) Analysis LCMS Condition H: retention time=1.97 min.; ESI-MS(+) m/z 967.7 (M+2H).
(363) Analysis LCMS Condition J: retention time=1.53 min.; ESI-MS() m/z 965.5 (M+2H).
Preparation of Example 3030
(364) ##STR00039##
(365) Example 3030 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin C. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(366) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 40 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.0 mg, and its estimated purity by LCMS analysis was 86%.
(367) Analysis LCMS Condition H: retention time=1.59 min.; ESI-MS(+) m/z 964.3 (M+2H).
(368) Analysis LCMS Condition J: retention time=1.27 min.; ESI-MS(+) m/z 964.3 (M+2H).
Preparation of Example 3032
(369) ##STR00040##
(370) Example 3032 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from intermediate resin E. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(371) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: Waters CSH C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 0-40% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.6 mg, and its estimated purity by LCMS analysis was 98%.
(372) Analysis LCMS Condition H: retention time=1.69 min.; ESI-MS(+) m/z 963.4 (M+2H).
(373) Analysis LCMS Condition J: retention time=1.14 min.; ESI-MS(+) m/z 964.2 (M+2H).
Preparation of Example 3033
(374) ##STR00041##
(375) Example 3033 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting from Intermediate resin D. The following procedures were performed sequentially: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(376) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 25-70% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 0.6 mg, and its estimated purity by LCMS analysis was 85%.
(377) Analysis LCMS Condition H: retention time=1.42 min.; ESI-MS(+) m/z 943.0 (M+2H).
(378) Analysis LCMS Condition J: retention time=1.13 min.; ESI-MS(+) m/z 1883.9 (M+H).
Preparation of Example 3037
(379) ##STR00042##
(380) Example 3037 was prepared following the general synthetic sequence described for the preparation of Example 3017, starting with Intermediate resin F. The following procedures were performed sequentially: N-Gly-Alkylation On-resin Method D, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(381) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 0-40% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 32.0 mg, and its estimated purity by LCMS analysis was 94%.
(382) Analysis LCMS Condition H: retention time=1.25 min.; ESI-MS(+) m/z 943.2 (M+2H);
(383) Analysis LCMS Condition J: retention time=1.14 min.; ESI-MS(+) m/z 943.2 (M+2H).
Preparation of Example 3038
(384) ##STR00043##
(385) Example 3038 was prepared following the general synthetic sequence described for the preparation of Example 3017, composed of the following general procedures, starting with intermediate resin F: N-Gly-Alkylation On-resin Method D, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(386) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 11.9 mg, and its estimated purity by LCMS analysis was 95%.
(387) Analysis LCMS Condition H: retention time=1.64 min.; ESI-MS(+) m/z 962.1 (M+2H);
(388) Analysis LCMS Condition J: retention time=1.33 min.; ESI-MS(+) m/z 962.0 (M+2H).
Preparation of Example 3041
(389) ##STR00044##
(390) Example 3041 was prepared following the general synthetic sequence described for the preparation of Example 3017, composed of the following general procedures, starting from intermediate resin D: Nosylate formation, Alkylation method A, Nosylate removal, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(391) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.8 mg, and its estimated purity by LCMS analysis was 81%.
(392) Analysis LCMS Condition H: retention time=1.68 min.; ESI-MS(+) m/z 970.0 (M+2H);
(393) Analysis LCMS Condition J: retention time=1.51 min.; ESI-MS(+) m/z 969.3 (M+2H).
Preparation of Example 3044
(394) ##STR00045##
(395) Example 3044 was prepared following the general synthetic sequence described for the preparation of Example 0001, composed of the following general procedures, starting with intermediate resin G: N-Gly-Alkylation On-resin Method D, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(396) The crude material was purified via preparative LC/MS with the following conditions: Column: waters CSH c-18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 19.4 mg, and its estimated purity by LCMS analysis was 96%.
(397) Analysis LCMS Condition H: retention time=1.55 min.; ESI-MS(+) m/z 964.1 (M+2H);
(398) Analysis LCMS Condition J: retention time=1.32 min.; ESI-MS(+) m/z 963.8 (M+2H).
Preparation of Example 3045
(399) ##STR00046##
(400) Example 3045 was prepared following the general synthetic sequence described for the preparation of Example 3017, composed of the following general procedures, starting with intermediate resin G: N-Gly-Alkylation On-resin Method D, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(401) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.5 mg, and its estimated purity by LCMS analysis was 94%.
(402) Analysis LCMS Condition H: retention time=1.60 min.; ESI-MS(+) m/z 957.1 (M+2H);
(403) Analysis LCMS Condition J: retention time=1.30 min.; ESI-MS(+) m/z 957.5 (M+2H).
Preparation of Example 3046
(404) ##STR00047##
(405) Example 3046 was prepared following the general synthetic sequence described for the preparation of Example 3017, composed of the following general procedures, starting with intermediate resin G: N-Gly-Alkylation On-resin Method D, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(406) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 7.2 mg, and its estimated purity by LCMS analysis was 100%.
(407) Analysis LCMS Condition H: retention time=1.71 min.; ESI-MS(+) m/z 982.9 (M+2H).
(408) Analysis LCMS Condition J: retention time=1.40 min.; ESI-MS(+) m/z 982.9 (M+2H).
Preparation of Example 3047
(409) ##STR00048##
(410) Example 3047 was prepared following the general synthetic sequence described for the preparation of Example 3017, composed of the following general procedures, starting with intermediate resin F: N-Gly-Alkylation On-resin Method D, Symphony Method B: Resin-swelling procedure, Symphony Method B: Standard-coupling procedure, Symphony Method B: Secondary amine-coupling procedure, Symphony Method B: Custom amino acids-coupling procedure, Symphony Method B: Final capping procedure, Global Deprotection Method A, and Cyclization Method A.
(411) The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: Waters CSH C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.0 mg, and its estimated purity by LCMS analysis was 97%.
(412) Analysis LCMS Condition H: retention time=1.44 min.; ESI-MS(+) m/z 936.1 (M+2H);
(413) Analysis LCMS Condition J: retention time=1.21 min.; ESI-MS(+) m/z 936.4 (M+2H).
(414) Experimental Procedures for Compounds 5001, 5002, 5003
(415) Analytical Data:
(416) Mass Spectrometry: ESI-MS(+) signifies electrospray ionization mass spectrometry performed in positive ion mode; ESI-MS() signifies electrospray ionization mass spectrometry performed in negative ion mode; ESI-HRMS(+) signifies high-resolution electrospray ionization mass spectrometry performed in positive ion mode; ESI-HRMS() signifies high-resolution electrospray ionization mass spectrometry performed in negative ion mode. The detected masses are reported following the m/z unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions.
(417) Analysis Condition A:
(418) Column: Waters BEH C18, 2.050 mm, 1.7-m particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50 C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 1 mL/min; Detection: UV at 220 nm.
(419) Analysis Condition B:
(420) Column: Waters BEH C18, 2.050 mm, 1.7-m particles; Mobile Phase A: 5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10 mM ammonium acetate; Temperature: 50 C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 0.5 mL/min; Detection: UV at 220 nm.
(421) Analysis Condition C:
(422) Column: Waters Aquity BEH C18 2.150 mm 1.7 m particles; Mobile Phase A: water with 0.05% TFA; Mobile Phase B: acetonitrile with 0.05% TFA; Temperature: 40 C.; Gradient: 0% B, 0-100% B over 1.5 minutes, then a 0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.
(423) General Procedures:
(424) Prelude Method A:
(425) All manipulations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a 10 mL polypropylene tube fitted with a bottom frit; where the scale of the reaction exceeded 0.100 mmol, a 40 mL polypropylene tube fitted with a bottom frit was used. The tube connects to a the Prelude peptide synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top of the tube, which washes down the sides of the tube equally. The remaining reagents are added through the bottom of the tube and pass up through the fit to contact the resin. All solutions are removed through the bottom of the tube. Periodic agitation describes a brief pulse of N2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 30 seconds. Chloroacetyl chloride solutions in DMF were used within 24 h of preparation. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solutions were used within 5 days of preparation. DMF=dimethylformamide; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine; Rink=(2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine, where 4-alkoxy describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Rink linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.
(426) Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.
(427) The procedures of Prelude Method A describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Rink linker bound to the resin. This scale corresponds to approximately 178 mg of the Rink-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as Resin-swelling procedure. Coupling of amino acids to a primary amine N-terminus used the Single-coupling procedure described below. Coupling of amino acids to a secondary amine N-terminus used the Double-coupling procedure described below. Coupling of chloroacetylchloride to the N-terminus of the peptide is described by the Chloroacetyl chloride coupling procedure detailed below.
(428) Resin-Swelling Procedure:
(429) To a 10 mL polypropylene solid-phase reaction vessel was added Merrifield: Rink resin (178 mg, 0.100 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (2.0 mL), upon which the mixture was periodically agitated for 10 minutes before the solvent was drained through the frit.
(430) Single-Coupling Procedure:
(431) To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF, 0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added acetic anhydride (2.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(432) Double-Coupling Procedure:
(433) To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF, 0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF, 0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added acetic anhydride (2.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.
(434) Chloroacetyl Chloride Coupling Procedure:
(435) To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added DIPEA (0.8M in DMF, 3.0 mL, 24 eq), then chloroacetyl chloride (0.8M in DMF, 1.65 mL, 13.2 eq). The mixture was periodically agitated for 30 minutes, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2C.sub.12 (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was placed under a N.sub.2 stream for 15 minutes.
(436) Symphony Method A:
(437) This collection of procedures is identical that of Prelude Method A except as noted. For all procedures a Symphony X peptide synthesizer (Protein Technologies) was used instead of a Prelude peptide synthesizer and all reagents were added through the top of the reaction vessel.
(438) Resin-Swelling Procedure:
(439) This procedure is identical to Prelude Method A: Resin-swelling procedure.
(440) Single-Coupling Procedure:
(441) This procedure is identical to Prelude Method A: Single-coupling procedure except that the concentration of DIPEA solution was 0.4M and 1.0 mL of this solution was delivered to the reaction.
(442) Double-Coupling Procedure:
(443) This procedure is identical to Prelude Method A: Double-coupling procedure except that the concentration of DIPEA solution was 0.4M and 1.0 mL of this solution was delivered to the reaction.
(444) Chloroacetyl Chloride Coupling Procedure:
(445) This procedure is identical to Prelude Method A: Chloroacetyl chloride coupling procedure.
(446) Global Deprotection Method A:
(447) All manipulations were performed manually unless noted. The procedure of Global Deprotection Method A describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Rink linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A deprotection solution was prepared by combining in a 40 mL glass vial trifluoroacetic acid (22 mL), phenol (1.325 g), water (1.25 mL) and triisopropylsilane (0.5 mL). The resin was removed from the reaction vessel and transferred to a 4 mL glass vial. To the vial was added the deprotection solution (2.0 mL). The mixture was vigorously mixed in a shaker (1000 RPM for 1 minute, then 500 RPM for 1-2 h). The mixture was filtered through a 0.2 micron syringe filter and the solids were extracted with the deprotection solution (1.0 mL) or TFA (1.0 mL). To a 24 mL test tube charged with the combined filtrates was added Et.sub.2O (15 mL). The mixture was vigorously mixed upon which a significant amount of a white solid precipitated. The mixture was centrifuged for 5 minutes, then the solution was decanted away from the solids and discarded. The solids were suspended in Et.sub.2O (20 mL); then the mixture was centrifuged for 5 minutes; and the solution was decanted away from the solids and discarded. For a final time, the solids were suspended in Et.sub.2O (20 mL); the mixture was centrifuged for 5 minutes; and the solution was decanted away from the solids and discarded to afford the crude peptide as a white to off-white solid.
(448) Cyclization Method A:
(449) All manipulations were performed manually unless noted. The procedure of Cyclization Method B describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Rink linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. The crude peptide solids were dissolved in MeCN:aq. 0.1M NH.sub.4OAc (1:1) to a total volume of 18-22 mL, and the solution was carefully then adjusted to pH=8.5-9.0 using aq NaOH (1.0M). The solution was then allowed to stand without stirring for 6 days. The reaction solution was concentrated and the residue was then dissolved in DMSO:MeOH. This solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.
(450) General Synthetic Sequence A:
(451) General Synthetic Sequence A describes a general sequence of procedures that were used to afford the cyclic peptides described herein. For the purposes of this general procedure, the procedures of Symphony Method A are interchangeable with those of Prelude Method A. To a 10 mL polypropylene solid-phase reaction vessel was added Rink-Merrifield resin (178 mg, 0.100 mmol), and the reaction vessel was placed on the Prelude peptide synthesizer. Prelude Method A: Resin-swelling procedure was followed. Then a series of amino acids couplings was sequentially performed on the Prelude following Prelude Method A: Single-coupling procedure if the N-terminus of the resin-bound peptide was a primary amine or Prelude Method A: Double-coupling procedure if the N-terminus of the resin-bound peptide was a secondary amine. Prelude Method A: Chloroacetyl chloride coupling procedure was followed; then Global Deprotection Method A was followed; then Cyclization Method A was followed.
Preparation of Example 5001
(452) ##STR00049##
(453) Example 5001 was prepared following General Synthetic Sequence A. In the synthesis 2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(tert-butoxy)-2-oxoethyl)amino)acetic acid was used as indicated by the sequence. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 7.8 mg, and its estimated purity by LCMS analysis was 100%.
(454) Analysis condition A: Retention time=1.48 min; ESI-MS(+) m/z 956.8.1 (M2H)
(455) Analysis condition B: Retention time=2.82 min; ESI-MS(+) m/z 958.2 (M+2H).
Preparation of Example 5002
(456) ##STR00050##
(457) Example 5002 was prepared following General Synthetic Sequence A. In the synthesis 2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(tert-butoxy)-2-oxoethyl)amino)acetic acid was used as indicated by the sequence. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 55-95% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.2 mg, and its estimated purity by LCMS analysis was 81%.
(458) Analysis condition A: Retention time=1.51 min; ESI-MS(+) m/z 964.1 (M2H)
(459) Analysis condition B: Retention time=2.88 min; ESI-MS(+) m/z 966.5 (M+2H).
Preparation of Example 5003
(460) ##STR00051##
(461) Example 5003 was prepared following General Synthetic Sequence A. In the synthesis 2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(tert-butoxy)-2-oxoethyl)amino)acetic acid was used as indicated by the sequence. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19200 mm, 5-m particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammonium acetate; Gradient: 60-100% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge Phenyl, 19200 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-40% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.0 mg, and its estimated purity by LCMS analysis was 100%.
(462) Analysis condition A: Retention time=1.777 min; ESI-MS(+) m/z 972.40 (M+2H)
(463) Analysis condition B: Retention time=2.393 min; ESI-MS(+) m/z 968.25 (M2H)
(464) ESI-HRMS(+) m/z: Calculated: 971.9344 (M+2H). Found: 971.9311 (M+2H).
Methods for Testing the Ability of Macrocyclic Peptides to Compete for the Binding of Pd-1 to Pd-L1 Using Homogenous Time-Resolved Fluorescence (HTRF) Binding Assays
(465) The ability of the macrocyclic peptides of the present disclosure to bind to PD-L1 was investigated using a PD-1/PD-L1 Homogenous Time-Resolved Fluorescence (HTRF) binding assay.
(466) Methods
(467) Homogenous Time-Resolved Fluorescence (HTRF) Assays of Binding of Soluble PD-1 to Soluble PD-L1. Soluble PD-1 and soluble PD-L1 refers to proteins with carboxyl-end truncations that remove the transmembrane-spanning regions and are fused to heterologous sequences, specifically the Fc portion of the human immunoglobuling G sequence (Ig) or the hexahistidine epitope tag (His). All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 l of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 l of assay buffer and further incubation for 15 m. PD-L1 fusion proteins from either human, cynomologous macaques, mouse, or other species were used. HTRF detection was achieved using europium crypate-labeled anti-Ig monoclonal antibody (1 nM final) and allophycocyanin (APC) labeled anti-His monoclonal antibody (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 l was dispensed on top of binding reaction. The reaction was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 and 5 nM, respectively), CD80-His/PD-L1-Ig (100 and 10 nM, respectively) and CD80-His/CTLA4-Ig (10 and 5 nM, respectively). Binding/competition studies between biotinylated Compound No. 71 and human PD-L1-His were performed as follows. Macrocyclic peptide inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 minutes in 4 l of assay buffer followed by addition of biotinylated Compound No. 71 (0.5 nM final) in 1 l of assay buffer. Binding was allowed to equilibrate for 30 minutes followed by addition of europium crypated labeled Streptavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 l of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.
(468) Recombinant Proteins. Carboxyl-truncated human PD-1 (amino acids 25-167) with a C-terminal human Ig epitope tag [hPD-1 (25-167)-3S-IG] and human PD-L1 (amino acids 18-239) with a C-terminal His epitope tag [hPD-L1(19-239)-tobacco vein mottling virus protease cleavage site (TVMV)-His] were expressed in HEK293T cells and purified sequentially by recombinant Protein A affinity chromatography and size exclusion chromatography. Human PD-L2-His (Sino Biologicals), CD80-His (Sino Biologicals), CTLA4-Ig (RnD Systems) were all obtained through commercial sources.
(469) Sequence of Recombinant Human PD-1-Ig
(470) hPD1(25-167)-3S-IG
(471) TABLE-US-00001 (SEQIDNO:1) 1 LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSN 51 QTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVEARRNDSGTYLCG 101 AISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQGSPGGGG 151 GREPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISPTPEVTCVV 201 VDVSHEDPEVKFNWYVDGVEVHNAKTKPRESQYNSTYRVVSVLTVLHQDW 251 LNGKEYKCKVSNKALPAPIEKTISKAKGQPPEPQVYTLPPSRDELTKNQV 301 SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD 351 KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(472) Sequence of Recombinant Human PD-L1-TVMV-His (PD-L1-His)
(473) hPDL1(19-239)-TVMV-His
(474) TABLE-US-00002 (SEQIDNO:2) 1 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV 51 HGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY 101 GGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWT 151 SSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEE 201 NHTAELVIPELPLAHPPNERTGSSETVRFQGHHHHHH
(475) The results are shown in Table 1. As shown, the macrocyclic peptides of the present disclosure demonstrated potent inhibition of PD-1-Ig binding activity to PD-L1-TVMV-His (PD-L1-His). Ranges are as follows: A=0.10-10 M; B=0.01-0.099 M; C=0.005-0.0099 M.
(476) TABLE-US-00003 TABLE 1 Example Number HTRF IC50 (M) 3003 B 3004 B 3005 0.03 3006 B 3007 B 3008 0.20 3009 B 3010 B 3011 6.12E03 3012 B 3013 B 3014 B 3015 B 3016 B 3017 C 3019 B 3020 C 3021 C 3022 B 3023 B 3024 B 3025 B 3026 B 3028 A 3029 A 3030 B 3032 B 3033 A 3037 A 3038 2.36 3041 B 3044 B 3045 B 3046 B 3047 3048 A 3049 A 3050 A 5001 C 5002 C 5003 B
(477) It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.