SYNTHESIS OF A-AMANITIN AND ITS DERIVATIVES
20220298204 · 2022-09-22
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Inventors
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C07C229/30
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C07C229/22
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C07C233/49
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C07C227/20
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C07C229/22
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C07K7/64
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C07C271/22
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C07K7/56
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International classification
Abstract
The present invention relates to the chemical synthesis of α-amanitin and its derivatives. The present invention also relates to intermediate products of the α-amanitin synthesis.
Claims
1. A method for preparation of a compound of formula (Iox) ##STR00107## wherein a) a compound of formula (IIox) ##STR00108## wherein X and Y are H, or Y is OH and X is OR.sup.PGP wherein R.sup.PGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, or X and Y are selected from F, Cl, Br, and I, particularly X and Y are H, or Y is OH and X is OR.sup.PGP Z and W are H, or Z is OH and W is OR.sup.PGOH, wherein R.sup.PGOH is a protecting group for hydroxyl-groups, particularly a hydroxyl-protecting group cleavable with fluoride ions, is reacted with a peptide bond forming reagent, particularly with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid, more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (a1), and for Y being OH and/or Z being OH, the compound is reacted with a deprotection agent removing R.sup.PGP and/or R.sup.PGOH, or wherein b) the compound of formula (II) ##STR00109## wherein X, Y, Z and W have the same meanings as defined above, is reacted with a peptide bond forming reagent, particularly with HATU, in a reaction step (a2), yielding a compound of formula (I) ##STR00110## wherein the sulfur atom is subsequently oxidized, particularly i. using manganese ions, more particularly the compound is reacted with a compound of formula (XXII) ##STR00111## and with Mn(OTf).sub.2 and H.sub.2O.sub.2, ii. using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide; or iii. using iodine and oxygen; in a reaction step (b2); and for Y being OH and/or Z being OH, the compound is reacted with a deprotection agent removing R.sup.PGP and/or R.sup.PGOH, particularly for R.sup.PGP with reductive conditions and for R.sup.PGOH with fluoride ions, to yield the compound characterized by (Iox).
2. A method for preparation of a compound of formula (I) ##STR00112## wherein a compound of formula (II) ##STR00113## wherein X and Y are H, or Y is OH and X is OR.sup.PGP wherein R.sup.PGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, or X and Y are selected from F, Cl, Br, and I, particularly X and Y are H or Y is OH and X is OR.sup.PGP Z and W are H, or Z is OH and W is OR.sup.PGOH, wherein R.sup.PGOH is a protecting group for hydroxyl-groups, particularly a hydroxyl-protecting group cleavable with fluoride ions, is reacted with a peptide bond forming reagent, particularly with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid, more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU in a reaction step (a), and for Y being OH and/or Z being OH, the compound is reacted with a deprotection agent removing R.sup.PGP and/or R.sup.PGOH in a reaction step (b) to yield the compound characterized by (I).
3. The method according to claim 1 or 2, wherein a compound of formula (III) ##STR00114## and a compound of formula (IV) or (IVox) ##STR00115## wherein R.sup.NHB is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, or an amino protecting group cleavable with hydrogenolysis, particularly Cbz, most particularly R.sup.NHB is Fmoc, W and X have the same meaning as outlined in claim 1, wherein the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and preactivated (IV) or preactivated (IVox) and (III) are reacted with a peptide bond forming reagent, particularly with HATU, or the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and the carboxyl-group of compound (III) is preactivated, particularly with an O-PFP-ester, O-PCP-ester, or OSu-ester, and preactivated (IV) or preactivated (IVox) and preactivated (III) are reacted, in a reaction step (c), and the compound is reacted with a deprotection agent removing R.sup.NHB in a reaction step (d), particularly with a base if R.sup.NHB is Fmoc, or with hydrogenolysis if R.sup.NHB is Cbz, to yield the compound characterized by (II) or (IIox).
4. The method according to claim 3, wherein a compound of formula (IV) ##STR00116## wherein R.sup.COOX is a carboxyl-protecting group, particularly tButyl, R.sup.NHX is an amino-protecting group, particularly Teoc or Fmoc, more particularly Teoc, X has the same meaning as outlined in claim 1, wherein the sulfur atom is subsequently oxidized, particularly i. using manganese ions, more particularly the compound is reacted with a compound of formula (XXII) ##STR00117## and with Mn(OTD.sub.2 and H.sub.2O.sub.2, ii. using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide; or iii. using iodine and oxygen; in a reaction step (d2), and the compound is reacted with a deprotection agent removing R.sup.COOX, particularly with a strong acid, more particularly with TFA, and with a deprotection agent removing R.sup.NHX, particularly in case of R.sup.NHX being Teoc with a strong acid, more particularly with TFA, or in case of R.sup.NHX being Fmoc with alkaline conditions, to yield the compound characterized by (IVox).
5. The method according to claim 4, wherein a compound of formula (V) ##STR00118## wherein R.sup.NHF is an amino protecting group, particularly an amino protecting group cleavable with fluoride ions or strong acids, more particularly Teoc, or an amino protecting group cleavable with alkaline conditions, more particularly Fmoc, R.sup.COOA is a carboxyl-protecting group, particularly a carboxyl-protecting group cleavable under strongly acidic conditions, more particularly tert-butyl, X has the same meaning as outlined in claim 1, is reacted with a peptide bond forming reagent, particularly with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid, more particularly with T3P, HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (e), and the compound is reacted with a deprotection agent removing R.sup.NHF and R.sup.COOA in a reaction step (f), particularly with TFA, to yield the compound characterized by (IV).
6. The method according to claim 5, wherein a compound of formula (VI) ##STR00119## and a compound of formula (VII) ##STR00120## wherein R.sup.NHA is an amino protecting group, particularly an amino protecting group cleavable under acidic conditions, more particularly Boc, R.sup.COOA, R.sup.NHF and X have the same meaning as outlined in claims 1 and 5, wherein compound (VI) is preactivated with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, followed by a reaction with the silylated compound (VII), or is preactivated as in OSu-ester, followed by a reaction with the compound (VII) in a reaction step (g), and the compound is reacted with a deprotection agent removing R.sup.NHA in a reaction step (h), particularly with acidic conditions, to yield the compound characterized by (V).
7. The method according to claim 6, wherein a compound of formula (VIII) ##STR00121## and a compound of formula (IX) ##STR00122## wherein R.sup.COOZ is a carboxyl-protecting group, particularly a carboxyl-protecting group cleavable with Zn, more particularly Tce, or R.sup.COOZ is H, R.sup.COOA, R.sup.NHF, R.sup.NHA and X have the same meaning as outlined in claims 1, 5, and 6, are reacted in a reaction step (i), and if R.sup.COOZ is a carboxyl-protecting group, the compound is reacted with a deprotection agent removing R.sup.COOZ in a reaction step (j), particularly with Zn, to yield the compound characterized by (VI).
8. The method according to claim 3, wherein a compound of formula (X) ##STR00123## and a compound of formula (XI) ##STR00124## and a compound of formula (XII) ##STR00125## wherein R.sup.Pep is an active ester, particularly O-pentafluorophenol or OSu-ester, R.sup.NHB is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, are reacted with solid phase peptide synthesis in a reaction step (k), to yield the compound characterized by (III).
9. A method for preparation of a compound of formula (XIII), (XIIIC), (XIIIN), or (XIIICN) ##STR00126## wherein a compound of formula (XIV) ##STR00127## wherein R.sup.COOS is a carboxyl-protecting group, particularly tert-butyl, R.sup.NHZ is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, or an amino protecting group cleavable under reductive conditions, more particularly trifluoroacetyl; is reacted with Osmium(IV)-oxide in a reaction step (I), particularly in CHCl.sub.3/H.sub.2O, and optionally, the compound is reacted with a deprotection agent removing R.sup.NHR and/or R.sup.COOS in a reaction step (m), particularly with silylating agents for R.sup.COOS and reductive conditions or alkaline conditions for R.sup.NHR, more particularly with TMSOTf and lutidine for R.sup.COOS and/or sodium borohydride or alkaline conditions for R.sup.NHZ to yield the compound characterized by (XIII), (XIIIC), (XIIIN), or (XIIICN).
10. A method for preparation of a compound of formula (XV) ##STR00128## wherein a compound of formula (XVI) ##STR00129## and a compound of formula (XVII) or (XVIIs) ##STR00130## wherein R.sup.NHR an amino protecting group cleavable under reductive conditions, more particularly trifluoroacetyl, R.sup.COOA is a carboxyl-protecting group, particularly a carboxyl-protecting group cleavable under strongly acidic conditions, more particularly tert-butyl, are reacted with [(p-cymene)RuCl.sub.2].sub.2 in a reaction step (n) yielding the compound (XXIII) or (XXIV) ##STR00131## a) the compound (XXVI) is reacted with a deprotection agent removing R.sup.COOA in a reaction step (o), and is reacted with acylase in a reaction step (p), or b) the compound (XXIII) is reacted with a deprotection agent removing R.sup.COOA in a reaction step (o), and is reacted with a deprotection agent removing R.sup.NHR in a reaction step (q), particularly with reductive conditions, to yield the compound characterized by (XV).
11. A method for preparation of a compound of formula (XVIII) ##STR00132## wherein a compound of formula (XIX) ##STR00133## and a compound of formula (XX) ##STR00134## wherein R.sup.PGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, are reacted with Ni.sup.2+ in a reaction step (r) to yield the compound characterized by (XVIII).
12. A method for preparation of a compound of formula (Iox), wherein a compound of formula (I) ##STR00135## wherein the sulfur atom is oxidized, i. using manganese ions, more particularly the compound is reacted with a compound of formula (XXII) ##STR00136## and with Mn(OTf).sub.2 and H.sub.2O.sub.2, ii. using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide; or iii. using iodine and oxygen; yielding the compound (Iox).
13. A method for preparation of a compound of formula (XXIII) or (XXIIIox) ##STR00137## wherein a compound of formula (IV) or (IVox), respectively, ##STR00138## and a compound of formula (X) ##STR00139## wherein X, W, and R.sup.NHB have the same meanings as defined in claims 1 and 3, wherein the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and preactivated (IV) or preactivated (IVox) and (X) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, more particularly with COMU, in a reaction step (s) to yield the compound (XXIII) or (XXIIIox), respectively.
14. A method for preparation of a compound of formula (XXVI) or (XXVIox) ##STR00140## wherein a compound of formula (XXVIII) or (XXVIIIox), respectively, ##STR00141## and a compound of formula (XXV) ##STR00142## wherein X, W, and R.sup.NHB have the same meanings as defined in claims 1 and 3, R.sup.NHB2 is an amino-protecting group, particularly an amino-protecting group cleavable under acidic conditions, more particularly Boc; R.sup.COOY is a carboxyl-protecting group, particularly fluorenylmethyl or benzyl, more particularly fluorenylmethyl; wherein (IV) or (IVox) and (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (t) to yield the compound (XXVI) or (XXVIox), respectively.
15. A method for preparation of a compound of formula (XXVII) or (XXVIIox) ##STR00143## wherein a) a compound of formula (IV) or (IVox), ##STR00144## and a compound of formula (X) ##STR00145## wherein X, W, and R.sup.NHB have the same meanings as defined in claims 1 and 3, wherein the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and preactivated (IV) or preactivated (IVox) and (X) are reacted with a peptide bond forming reagent, particularly with COMU, in a reaction step (s) to yield the compound (XXIII) or (XXIIIox), respectively, and subsequently compound (XXIII) or (XXIIIox) and compound (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU in a reaction step (u) to yield the compound (XXVII) or (XXVIIox), respectively; or b) a compound (XXIII) or (XXIIIox) and a compound (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU in a reaction step (u) to yield the compound (XXVII) or (XXVIIox), respectively; or c) a compound of formula (XXVIII) or (XXVIIIox), respectively, ##STR00146## and a compound of formula (XXIX) ##STR00147## wherein X, W, and R.sup.NHB have the same meanings as defined in claims 1 and 3, R.sup.NHB2 is an amino-protecting group, particularly an amino-protecting group cleavable under acidic conditions, more particularly Boc; wherein R.sup.COOY is a carboxyl-protecting group, particularly fluorenylmethyl or benzyl, more particularly fluorenylmethyl; wherein (XXVIII) or (XXVIIIox) and (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (t) to yield the compound (XXVI) or (XXVIox), respectively and subsequently compound (XXVI) or (XXVIox) and compound (X) are reacted with a peptide bond forming reagent, particularly with HATU, in a reaction step (v) to yield the compound (XXVII) or (XXVIIox), respectively or d) a compound (XXVI) or (XXVIox) and a compound (X) are reacted with a peptide bond forming reagent, particularly with HATU, in a reaction step (v) to yield the compound (XXVII) or (XXVIIox), respectively.
16. A compound of the general formula (I) ##STR00148## wherein Y is H and Z is H, Y is H and Z is OH, Y is OH and Z is H Y is F, Cl, I or Br, and Z is OH, or Y is F, Cl, I or Br, and Z is H, particularly Y and Z are independently selected from OH and H; or a compound of the general formula (II) ##STR00149## wherein X is H and W is H, X is OH and W is OH, X is H and W is OH, X is OH and W is H, X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H, particularly X and W are independently selected from OH and H; or a compound of the general formula (IIox) ##STR00150## wherein X is H and W is H, X is OH and W is OH, X is H and W is OH, X is OH and W is H, X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H, particularly X and W are independently selected from OH and H; or a compound of the general formula (IVox) ##STR00151## wherein X is H or OH, or X is F, Cl, I or Br particularly X is H or OH; or a compound of the general formula (XXVIII) ##STR00152## wherein X is H and W is H, X is OH and W is OH, X is H and W is OH, X is OH and W is H, X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H, particularly X and W are independently selected from OH and H; or a compound of the general formula (XXVIIIox) ##STR00153## wherein X is H and W is H, X is OH and W is OH, X is H and W is OH, X is OH and W is H, X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H, particularly X and W are independently selected from OH and H; or a compound of the general formula (XXVI) ##STR00154## wherein X is H and W is H, X is H and W is OH, X is OH and W is H, X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H, particularly X is H and W is H, or X is H and W is OH, or X is OH and W is H; or a compound of the general formula (XXVIox) ##STR00155## wherein X is H and W is H, X is OH and W is OH, X is H and W is OH, X is OH and W is H, X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H, particularly X and W are independently selected from OH and H.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0286]
[0287]
[0288]
[0289]
[0290]
[0291]
[0292]
[0293]
[0294]
[0295]
[0296]
[0297]
[0298]
[0299]
EXAMPLES
Example 1
Strategy for the Synthesis of (2S,3R,4R)-4,5-dihydroxyisoleucine Derivatives
[0300] 9 Step Synthesis Via Ruthenium-Catalyzed Allylic Alkylation
[0301] The (2S,3R,4R)-4,5-dihydroxyisoleucine derivative 13 was synthesized in 9 steps (
[0302] Separation of the desired
[0303] Asymmetric Synthesis Strategies Via Regioselective Ruthenium-Catalyzed Allylic Alkylation and Possible Shortcuts
[0304] In order to achieve a higher overall yield of the synthesis route described in the previous section a chiral alkylating reagent during the allylic alkylation reaction was used (
[0305] The overall synthesis strategy for the enantiomerically pure Fmoc-protected (2S,3R,4R)-4,5-dihydroxyisoleucine derivative (
[0306] The diastereomeric ratio (dr) was calculated to be 86:14 towards the (2R,3S)-diastereomer and 99:1 towards the (2S,3R)-configured diastereomer. The former was separated conveniently by the following acylase reaction which led to the formation of a enantiomerically pure didehydroisoleucine 7 with a dr of 99:1. Because of the enantiomeric purity the asymmetric dihydroxylation also resulted in a higher yield as there were only two diastereomers that needed separation afterwards instead of four. The overall yield starting from glycine tert-butyl ester (1) after 9 steps was calculated to be 17-21%.
[0307] The high enantiomeric excess of 5 also made it possible to take two shortcuts resulting in a higher yield (
[0308] One shortcut was the direct Tfa-deprotection of didehydroisoleucine 5 followed by Fmoc-protection. This way, the tBu-cleavage and reattachment was omitted resulting in the formation of the dihydroxylation substrate within two steps instead of four. The enantiomeric excess of 13 following shortcut 1 was calculated by chiral HPLC and resulted to be 95%. The overall yield was calculated to be 24-29%.
[0309] A second shortcut was the direct Sharpless dihydroxylation of the allylic alkylation product 5 followed by the protection of the side chain with the TBS protecting groups. The diastereomers were separated by column chromatography on silica gel after cleavage of the Tfa protecting group using LiOH and Fmoc-protection of the C-terminus (11). The enantiomeric excess of 13 following shortcut 2 was calculated by chiral HPLC and resulted to be 70%. Both shortcuts enable the synthesis of Fmoc-4,5-dihydroxyisoleucine in 7 steps instead of 9 and provided higher overall yields.
Example 2
Strategy for the Synthesis of (S)-6-hydroxytryptophan Derivatives
[0310] The (S)-6-hydroxytryptophan derivative 33 was synthesized in four steps, starting with an alkylation of the commercially available 6-benzoxyindol (29) using
3-(6-Benzoxy-1H-indol-3-yl)-2-acetylaminopropionic acid (30)
[0311] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm)=1.79 (s, 3H) 2.92 (dd, J=14.68, 8.66 Hz, 1H) 3.09 (dd, J=14.68, 4.89 Hz, 1H) 4.42 (td, J=8.22, 5.14 Hz, 1H) 5.09 (s, 2H) 6.72 (d, J=6.27 Hz, 1H) 6.89 (d, J=2.01 Hz, 1H) 6.98 (d, J=2.01 Hz, 1H) 7.27-7.33 (m, 1H) 7.35-7.41 (m, 3H) 7.42-7.48 (m, 2H) 8.11 (d, J=7.78 Hz, 1H) 10.64 (s, 1H).
[0312] .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ (ppm)=173.59, 169.20, 154.45, 137.68, 136.67, 128.39, 127.62, 127.48, 122.27, 121.85, 118.78, 110.00, 109.21, 95.95, 69.49, 52.98, 27.22, 22.42.
[0313] HRMS (ESI): m/z calc. für C.sub.20H.sub.20N.sub.2O.sub.4 (M+H).sup.+353.1496, found 353.1487.
(S)-3-amino-3-(6-(benzoxy)-1H-indol-3-yl)propanoic acid—Schiff Base Complex (32)
[0314] 1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=1.27-1.39 (m, 1H) 1.61-1.74 (m, 2H) 1.76-1.88 (m, 1H) 2.00-2.13 (m, 1H) 2.71-3.05 (m, 4H) 3.23 (dd, J=14.65, 3.97 Hz, 1H) 4.01 (d, J=12.51 Hz, 1H) 4.20 (t, J=4.65 Hz, 1H) 5.00 (s, 2H) 6.55 (d, J=2.44 Hz, 1H) 6.62-6.74 (m, 2H) 6.79 (s, 1H) 6.88 (d, J=1.83 Hz, 1H) 7.03 (dd, J=9.31, 2.44 Hz, 1H) 7.12 (d, J=8.70 Hz, 1H) 7.16-7.33 (m, 7H) 7.34-7.57 (m, 5H) 8.09 (d, J=9.31 Hz, 3H) 8.38 (br. s., 1H) 8.80 (d, J=1.68 Hz, 1H).
[0315] 13C NMR (100 MHz, CDCl3): δ (ppm)=179.92, 156.24, 141.28, 137.77, 137.47, 135.19, 134.01, 133.58, 133.51, 132.64, 131.17, 130.41, 130.04, 129.64, 129.32, 128.84, 128.13, 127.73, 125.83, 124.16, 123.53, 123.36, 120.68, 110.92, 110.18, 96.86, 71.78, 70.98, 63.47, 58.66, 30.95, 22.83.
[0316] HRMS (ESI): m/z calc. C.sub.43H.sub.35Cl.sub.3N.sub.4NiO.sub.4 (M+H).sup.+835.1150, found 835.1159.
[0317] The dr was determined by chiral HPLC with a CHIRALPAK AD-H column (hexane/isopropanol=55/45, λ=280 nm, 0.8 mL/min). t.sub.R (major diastereomer)=13.29 min, >99:1 dr, t.sub.R(minor diastereomer)=17.28 min.
(S)-6-benzoxytryptophan (33):
[0318] HRMS (ESI): m/z calc. C.sub.18H.sub.18N.sub.2O.sub.3(M+H).sup.+ 311.1390, found 311.1391.
Example 3
Synthesis of α-Amanitin and Amaninamide
[0319] Synthesis of the Peptide Building Blocks
[0320] The thioether building units 40 and 41 were readily established by treatment of a fully protected L-cystine derivative (35) with sulfuryl chloride. Cleavage of the disulfide afforded the highly reactive sulfenyl chloride monomer 36, which in the following step is susceptible for an electrophilic aromatic substitution (S.sub.EAr) either solely N-terminally protected or fully protected 6-hydroxytryptophan and tryptophan derivative 38 and 39. The use of the TCE-protecting group at the C-terminus helped to suppress the formation of undesired side-products with residual sulfuryl chloride from the sulfenyl chloride formation, but was not imperative for the reaction to take place (
[0321] The tripeptide building block H-Gly-Ile-Gly-OH (45) was synthesized in solution phase by first synthesizing a N-terminally Cbz- and C-terminally Bn-protected tripeptide, followed by simultaneous Cbz- and Bn-deprotection by hydrogenolysis using H.sub.2 and Pd/C as catalyst. (
[0322] The Fmoc-Asn-Hyp-DHIL(TBS).sub.2-OH tripeptide 48 was synthesized on solid phase using the CTC-resin. The use of Fmoc-Asn-OPfp (47) during the coupling of asparagine with no protecting group at the side chain suppressed the formation of the dehydration product (a tripeptide containing Fmoc-p-cyanoalanine) to the extent of only 10% (
[0323] A C-terminally 9-Fluorenylmethyl ester-protected dipeptide building block 66 was synthesized by esterification of trans-N-(Boc)-4-hydroxy-L-proline (63) with 9-fluorenylmethanol affording fully protected 4-hydroxy-L-proline 64. Boc-deprotection under acidic conditions, followed by coupling with Boc-Asn-OH using EDC and HOBt and repeated Boc-deprotection under acidic conditions led to the formation of dipeptide building block 66 with no formation of the dehydration side product present.
[0324] Assembly of the Peptide Building Blocks Towards (S)-Deoxy-(O)-Benzyl α-amanitin and (S)-Deoxy Amaninamide.
[0325] First, monocyclic thioethers 55 and 56 were synthesized in order to obtain the bicyclic structures of (S)-Deoxy (O)-benzyl-α-amanitin and (S)-Deoxy amaninamide (
[0326] A second pathway leading to the formation of (S)-Deoxy-(O)-benzyl-α-amanitin and (S)-Deoxy amaninamide was the direct coupling of DHIL-derivative 13 to the fully deprotected monocyclic pentapeptides 55 and 56 by using a silylating agent for the N-terminus of the pentapeptides and an active ester forming agent for the carboxylic function of the DHIIe derivative, analogous to the method described above. Monocyclic hexapeptides 67 and 68 could then be coupled to the C-terminally Fm-protected dipeptide building block 66 leading to the formation monocyclic octapeptides 69 and 70. The final cyclization was then performed after simultaneous Fmoc and Fm-cleavage and subsequent TBS-deprotection from the DHIL residue.
[0327] Sulfide Oxidation Affording α-Amanitin and Amaninamide
[0328] The asymmetric oxidation of the tryptathionine moiety leading to (O)-Benzyl-α-amanitin and amaninamide (62) was achieved by using a manganese complex as a catalyst with a porphyrine inspired chiral ligand, following a protocol reported by Gao et al (D. Wen, L. Jun, S. Gao, Org. Lett. 2013, 15, 22, 5658-61). Hydrogenolysis with H.sub.2 and Pd/C of (O)-Benzyl-α-amanitin in THF afforded the natural product α-amanitin (61) after 30 min of reaction time.
[0329] Materials and Methods
tert-Butyl (2,2,2-trifluoroacetyl)glycinate (2)
[0330] ##STR00054##
[0331] To a solution of glycine tert-butyl ester hydrochloride (10.0 g, 137 mmol, 1.00 eq) in MeOH (150 ml) triethylamine (17.4 ml, 125 mmol, 2.10 eq) was added dropwise. After stirring for 5 min ethyl trifluoroacetate (16.4 ml, 137 mmol, 2.3 eq) was added and the mixture was stirred for 16 h at room temperature during which time a clear solution formed. Then, the reaction mixture was concentrated under reduced pressure and the resulting residue acidified with 2 N HCl before being extracted with EtOAc (3×100 ml). The organic layers were combined, then washed with sat. NaHCO.sub.3 (2×100 ml), distilled H.sub.2O (2×100 ml) and brine (2×100 ml) and dried over MgSO.sub.4. The solvent was removed in vacuo to give the product (2) as a yellow oil (13.5 g, quant.).
[0332] .sup.1H NMR (CDCl.sub.3-d.sup.1, 400 MHz): δ=1.50 (s, 9H), 4.02 (d, J=5.02 Hz, 2H), 6.89 ppm (br s, 1H).
[0333] .sup.13C NMR (CDCl.sub.3-d.sup.1, 100 MHz): δ=27.91, 41.94, 83.52, 115.60 (q, J=287.28 Hz), 157.06 (q, J=39.60 Hz), 167.28 ppm.
[0334] HRMS (ESI): m/z calculated: C.sub.8H.sub.12F.sub.3NO.sub.3(M−H).sup.− 226.0686, found 226.0693.
But-3-en-2-yl tert-butyl carbonate (4):
[0335] ##STR00055##
[0336] But-3-en-2-ol (1.50 g, 20.8 mmol, 1.00 eq) was added slowly to a solution of NaH (1.50 g, 62.4 mmol, 3.00 eq) in dry THF (40 ml) at 0° C. Then Boc.sub.2O (5.9 g, 27 mmol, 1.3 eq) was added in portions over 10 min under vigorous stirring at this temperature. The reaction mixture was allowed to warm to room temperature overnight and then diluted with Et.sub.2O after 16 h of vigorous stirring. Excess sodium hydride was quenched by the slow addition of water. The resulting mixture was then extracted with diethyl ether (3×50 ml). The combined organic extracts were washed with brine (1×50 ml), dried over MgSO.sub.4 and concentrated under reduced pressure.
[0337] After purification by column chromatography on silica gel (hexane/EtOAc, 10:1) the product (4) was obtained as a colourless liquid (3.6 g, 20.3 mmol, quant.).
[0338] .sup.1H NMR (CDCl.sub.3-d.sup.1, 400 MHz): δ=1.36 (d, J=6.53 Hz, 3H), 1.49 (s, 9H), 5.13-5.17 (m, 2H), 5.25-5.31 (m, 1H), 5.83-5.91 ppm (m, 1H).
[0339] .sup.13C NMR (CDCl.sub.3-d.sup.1, 100 MHz): δ=19.72, 27.48, 73.72, 81.62, 115.78, 137.78, 137.17, 152.53 ppm.
[0340] HRMS (ESI): m/z calculated: C.sub.9H.sub.16O.sub.3(M+H).sup.+ 173.1172, found 173.1171.
tert-Butyl 3-methyl-2-(2,2,2-trifluoroacetamido)pent-4-enoate (Trifluoroacetyl 4,5-didehydroisoleucine tert-butyl ester) (5)
[0341] ##STR00056##
[0342] LHMDS (1 M in THF, 11 mmol, 2.5 eq) was added slowly to a solution of trifluoroacetyl glycine tert-butylester (2, 1.0 g, 4.4 mmol, 1.5 eq) in dry THF (12 ml) at −78° C. After stirring for 10 min a solution of dried ZnCl.sub.2 (720 g, 5.30 mmol, 1.20 eq) in dry THF (6 ml) was added at this temperature and stirring was continured for another 30 min at −78° C. Meanwhile a solution was prepared from [(p-cymene)RuCl.sub.2].sub.2 (37 mg, 0.06 mmol, 0.02 eq) and triphenylphosphine (32 mg, 0.12 mmol, 0.04 eq) in dry THF (6 ml) and stirred for 10 min at room temperature. Then, the allylic carbonate (4) (517 mg, 3.00 mmol, 1.00 eq) was added and the resulting solution was added to the chelated enolate at −78° C. The reaction mixture was allowed to warm to room temperature overnight. After diluting with EtO.sub.2 (100 ml) the reaction mixture was hydrolyzed by addition of 1 M KHSO.sub.4 until the precipitate was fully dissolved in the organic layer. Then, the layers were separated, the aqueous layer was extracted with diethyl ether (3×50 ml) and the combined organic layers were washed with brine (100 ml) and dried over MgSO.sub.4. The solvent was removed in vacuo and the crude product was purified by column chromatography on silica gel (hexane/EtOAc, 10:1), which afforded the product (5) as a colourless oil (740 mg, 88%).
[0343] .sup.1H NMR (CDCl.sub.3-d.sup.1, 400 MHz): δ=1.10 (d, J=7.03 Hz, 3H), 1.49 (s, 9H), 2.82-2.89 (m, 1H), 4.48-4.54 (m, 1H), 5.07-5.20 (m, 2H), 5.70 (s, 1H), 6.70 ppm (d, J=7.53 Hz, 1H).
[0344] .sup.13C NMR (CDCl.sub.3-d.sup.1, 100 MHz): δ=15.75, 27.97, 40.21, 56.71, 83.35, 117.15 (q, J=273.2 Hz), 117.46, 136.78, 157.03 (q, J=39.60 Hz), 168.89 ppm.
3-methyl-2-(2,2,2-trifluoroacetamido)pent-4-enoic acid (Trifluoroacetyl 4,5-didehydroisoleucine) (6)
[0345] ##STR00057##
[0346] Fully protected didehydroisoleucine (5) (1.0 g, 3.6 mmol, 1.0 eq) was dissolved in a solution of 95% TFA in DCM. After stirring for 2 h at room temperature the solvent was evaporated in vacuo to afford the trifluoroacetylated didehydroisoleucine (6) (800 mg, quant.) as a white solid.
[0347] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=6.63 (br, 1H), 5.66-5.77 (m, 1H) 5.19-5.27 (m, 2H), 4.66 (q, J=4.2 Hz, 1H), 2.90-2.99 (m, 1H), 1.15 (d, J=7.04 Hz, 3H)
[0348] .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=175.37, 157.67, 136.37, 118.37, 117.00, 56.43, 39.56, 16.32
[0349] HRMS (ESI): m/z calc for C.sub.8H.sub.9F.sub.3NO.sub.3 (M−H).sup.− 224.0529, found 224.0536.
(2S,3S)-2-amino-3-methylpent-4-enoic acid (4,5-Didehydroisoleucine) (7)
[0350] ##STR00058##
[0351] To a solution of the racemic trifluoroacetylated didehydroisoleucine 6 (700 mg, 3.10 mmol, 1.00 eq) in Sørensen phosphate buffer (15 ml, pH 7.5) 4 M KOH (777 μl, 3.10 mmol, 1.00 eq) and acylase I from Aspergillus melleus (300 mg) was added. After 6 h at 36° C. the reaction mixture was filtered through a Amicon Ultra centrifugal filter unit (cut off 10 kDa). The resulting the amino acid buffer mixture was then submitted to the subsequent Fmoc protection without any further purification.
[0352] HRMS (ESI): m/z calc for C.sub.6H.sub.11NO.sub.2 (M+H).sup.+ 130.0863, found 130.0858.
Fmoc-4,5-didehydroisoleucine (8)
[0353] ##STR00059##
[0354] To a stirring solution of 4,5-didehydroisoleucine 8 in Sørensen phosphate buffer (15 ml) was added a solution of Fmoc-OSu (823 mg, 2.44 mmol, 1.05 eq) in acetone (10 ml). After stirring for 16 h the reaction mixture was diluted with 50 ml water, acidified to pH 2 with 1 N HCl and extracted with ethyl acetate (3×30 ml). The combined organic layers were washed with brine, dried over MgSO.sub.4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (1% MeOH/DCM) to obtain the Fmoc didehydroisoleucine 8 as a white solid (810 mg, 75%, 2 steps).
[0355] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ (ppm)=8.49 (br, 1H), 7.68 (d, J=7.5 Hz, 2H), 7.44-7.54 (m, 2H), 7.32 (t, J=7.02 Hz, 2H), 7.23 (t, J=7.02 Hz, 2H), 5.45-5.73 (m, 1H), 5.18-5.32 (m, 1H), 5.01-5.17 (m, 1H), 4.30-4.37 (m, 2H), 4.12-4.19 (m, 1H), 2.39-2.54 (m, 1H), 2.44-2.52 (m, 1H), 1.60 (d, J=6.0 Hz, 2H), 1.05 (d, J=7.0 Hz, 1H)
[0356] .sup.13C-NMR (100 MHz, DMSO-d.sub.6): δ (ppm)=177.01, 156.04, 143.58, 141.45, 137.20, 130.67, 127.93, 127.02, 125.20, 124.13, 120.18, 117.60, 67.46, 58.04, 53.33, 47.25, 39.65, 35.09
[0357] HRMS (ESI): m/z calc for C.sub.21 H.sub.21NO.sub.4 (M+H).sup.+ 374.1363, found 374.1360.
Fmoc-4,5-didehydroisoleucine tert-butyl ester (9)
[0358] ##STR00060##
[0359] BF.sub.3*OEt.sub.2 (1.69 ml, 13.7 mmol, 6.0 eq) was added to a stirring solution of Fmoc-protected 4,5-didehydroisoleucine (8) (800 mg, 2.28 mmol, 1.00 eq) in 10 ml .sup.tBuOAc. The reaction mixture was stirred at rt for 5 min, then cooled to 0° C. and neutralized with saturated NaHCO.sub.3. The reaction mixture was then extracted with EtOAc (3*50 ml), washed with 1 M HCl (3×20 ml) and brine (2×20 ml) and dried over MgSO.sub.4. After evaporation of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (hexane/EtOAc, 10:1) to obtain the fully protected 4,5-didehydroisoleucine (9) as a colourless oil (650 mg, 70%).
[0360] .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm)=1.07-1.14 (m, 3H), 1.49 (s, 9H), 2.74-2.85 (m, 1H), 4.21-4.34 (m, 2H), 4.39 (m, J=6.90, 6.90 Hz, 2H), 5.08-5.18 (m, 2H), 5.24 (s, 1H), 5.68-5.79 (m 1H), 7.33 (t, J=7.53 Hz, 2H), 7.41 (t, J=7.53 Hz, 2H), 7.61 (d, J=7.53 Hz, 2H), 7.78 (d, J=7.53 Hz, 2H)
[0361] .sup.13C NMR (100 MHz, CDCl.sub.3): δ (ppm)=15.66, 27.75, 40.07, 46.86, 58.02, 66.69, 81.93, 116.32, 119.64, 124.81, 126.72, 127.36, 137.45, 140.97, 143.56, 155.92, 170.27
[0362] HRMS (ESI): m/z calc for C.sub.26H.sub.29NO.sub.4 (M+H).sup.+ 430.1989, found 430.1982.
Fmoc-4,5-dihydroxyisoleucine tert-butyl ester (10)
[0363] ##STR00061##
[0364] N-methylmorpholine-N-oxide (287 mg, 2.45 mmol, 1.30 eq) was added to a stirring solution of 9 and potassium osmate dihydrate (45.2 mg, 122 μmol, 0.05 eq) in 15 ml of a 4:1 mixture of CHCl.sub.3 and water and stirred for 20 min at rt. Then, Fmoc-protected 4,5-dihydroxyisoleucine tent-butyl ester (10) (1.00 g, 2.45 mmol, 1.00 eq) was added to the biphasic mixture. The resulting mixture was stirred at rt for 16 h and diluted with 100 ml DCM. Afterwards, saturated sodium metabisulfite solution (20 ml) was added and extracted with DCM (3×10 ml) after being stirred for 30 min. the combined organic phases were dried over NaSO.sub.4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (0.6% MeOH/DCM to 1.0% MeOH/DCM gradient) to obtain the 4,5-dihydroxyisoleucine derivative 10 as white crystals (439 mg, 40%).
[0365] .sup.1H NMR (CDCl.sub.3-d.sup.1, 400 MHz): δ=1.00 (d, J=7.03 Hz, 3H), 1.50 (s, 9H), 1.94-2.04 (m, 3H), 3.57 (dd, J=10.79, 3.01 Hz, 1H), 3.72 (t, J=9.50 Hz, 1H), 3.76-3.82 (m, 1H), 4.18-4.27 (m, 2H), 4.37-4.47 (m, 2H), 5.90 (d, J=8.28 Hz, 1H), 7.33 (t, J=7.03 Hz, 2H), 7.41 (t, J=7.28 Hz, 2H), 7.61 (d, J=7.28 Hz, 2H), 7.77 ppm (d, J=7.53 Hz, 2H)
[0366] .sup.13C NMR (CDCl.sub.3-d.sup.1, 100 MHz): δ=10.41, 27.99, 38.32, 47.14, 57.85, 64.71, 67.18, 71.70, 82.74, 119.96, 125.02, 127.06, 127.71, 141.27, 143.63, 156.82 ppm
[0367] HRMS (ESI): m/z calc for C.sub.25 H.sub.31NO.sub.6 (M+H).sup.+ 442.2224, found 442.2222.
[0368] tert-butyl(2S,3R,4R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,5-bis((tert-butyldimethylsilyl)oxy)-3-methylpentanoate (11)
##STR00062##
[0369] Under a nitrogen atmosphere tert-butyldimethylsilyl chloride (546 mg, 3.62 mmol, 8.0 eq) was added to a stirring solution of Fmoc-4,5-dihydroxyisoleucine tert-butyl ester (10, 200 mg, 452 μmol, 1.00 eq) in 4 ml of a 1:1 mixture of dry DMF and pyridine. Then, DMAP (8.3 mg, 68 μmol, 0.15 eq) was added and the resulting mixture was stirred for 24 h at rt. Afterwards, the reaction mixture was diluted with 50 ml EtOAc and washed with 1 M HCl (3×20 ml) and brine (2×20 ml), dried over MgSO.sub.4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 19:1) to obtain the fully protected 4,5-dihydroxyisoleucine (9) as a colourless oil (271 mg, 90%).
[0370] .sup.1H NMR (CDCl.sub.3-d.sup.1, 500 MHz): δ=0.06-0.09 (m, 6H), 0.11 (s, 3H), 0.17 (s, 3H), 0.90-0.95 (m, 18H), 1.00 (d, J=7.02 Hz, 3H), 1.49 (s, 9H), 2.35-2.44 (m, 1H), 3.44 (dd, J=9.69, 8.32 Hz, 1H), 3.52-3.57 (m, 1H), 3.82-3.86 (m, 1H), 4.19-4.26 (m, 2H), 4.39 (d, J=7.02 Hz, 2H), 5.99 (d, J=8.24 Hz, 1H), 7.30 (t, J=7.78 Hz, 2H), 7.40 (t, J=7.40 Hz, 2H), 7.63 (dd, J=9.77, 7.78 Hz, 2H), 7.76 (d, J=7.63 Hz, 2H)
[0371] .sup.13C NMR (CDCl.sub.3-d.sup.1, 126 MHz): −5.50, −5.38, −4.64, −4.09, 10.40, 18.05, 18.26, 25.87, 25.90, 28.04, 35.51, 47.27, 59.12, 64.18, 66.80, 73.74, 81.38, 119.88, 125.20, 126.98, 127.55, 141.28, 144.02, 144.17, 156.46, 171.22
[0372] HRMS (ESI): m/z calculated: C.sub.37H.sub.59NO.sub.6Si.sub.2(M+H).sup.+ 670.3953, found 670.3945.
(2S,3R,4R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,5-bis((tert-butyldimethylsilyl)oxy)-3-methylpentanoic acid (13)
[0373] ##STR00063##
[0374] A solution of fully protected 4,5-dihydroxyisoleucine 11 (100 mg, 149 μmol, 1.0 eq) was dissolved in 2 ml dry DCM, treated with 2,6 lutidine (173 μl, 1.49 mmol, 10.0 eq) and cooled to 0° C. Then, TMSOTf (135 μl, 746 μmol, 5.0 eq) was added and the reaction mixture was allowed to warm to room temperature overnight. The solution was diluted with Et.sub.2O (10 ml) followed by the addition of Sørensen phosphate buffer (pH=7; 5 ml). Afterwards, the pH of the mixture was adjusted to 2 by the dropwise addition of NaHSO.sub.4-solution (10%). The phases were separated and the aqueous phase was extracted with Et.sub.2O (3×10 ml). The combined organic phases were washed with brine, dried over NaSO.sub.4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (1% MeOH/DCM) to furnish the final product (13) as a colourless oil (90.5 mg, quant.).
[0375] 1H NMR (CDCl.sub.3-d.sup.1, 500 MHz): δ=0.02-0.14 (m, 12H), 1.04 (d, J=7.02 Hz, 3H), 2.36-2.47 (m, 1H), 3.55 (t, J=8.24 Hz, 1H), 3.58-3.64 (m, 1H), 3.79-3.88 (m, 1H), 4.24 (t, J=6.79 Hz, 1H), 4.37-4.49 (m, 3H), 6.10 (d, J=7.32 Hz, 1H), 7.31 (t, J=7.48 Hz, 2H), 7.40 (t, J=7.32 Hz, 2H), 7.61 (t, J=7.63 Hz, 2H), 7.76 (d, J=7.48 Hz, 2H)
[0376] .sup.13C NMR (CDCl.sub.3-d.sup.1, 126 MHz): −5.48, −5.41, −4.74, −4.07, 11.07, 18.00, 18.25, 25.79, 25.87, 37.01, 47.23, 57.77, 64.68, 67.00, 73.97, 119.95, 125.05, 125.12, 127.03, 127.64, 141.33, 143.86, 156.43
[0377] HRMS (ESI): m/z calculated: C.sub.33H.sub.51NO.sub.6Si.sub.2 (M+H).sup.+ 614.3328, found 614.3324.
((2S,3S)-3-methyloxiran-2-yl)methyl 4-methylbenzenesulfonate ((S,S)-19)
[0378] ##STR00064##
[0379] A flame dried flask was charged with 10 g of crushed activated 3 Å molecular sieves and flushed with nitrogen for several minutes. Then, DCM (200 ml) was added and the flask was cooled to −20° C. (+)-Diisopropyl tartrate (DIPT) (1.75 g, 7.49 mmol, 0.06 eq), crotyl alcohol (9.00 g, 125 mmol, 1.00 eq) and Ti(OiPr).sub.4 (1.77 g, 6.24 mmol, 0.05 eq) were added sequentially at this temperature. The resulting mixture was stirred for 15 min at −20° C., then a solution of tert-butyl hydroperoxide (TBHP, 5 M in DCM) (50.0 ml, 150 mmol, 2.00 eq) was added dropwise. The reaction mixture was stirred for 2 h at this temperature. Careful quenching of the excess TBHP was carried out by careful addition of trimethyl phosphite (22.0 ml, 187 mmol, 1.50 eq) at −20° C. after which trimethyl amine (26.1 ml, 187 mmol, 1.50 eq), DMAP (1.83 g, 15.0 mmol, 0.12 eq) and a solution of p-toluenesulfonyl chloride (23.8 g, 125 mmol, 1.00 eq) in DCM (100 ml) was added sequentially. The temperature was raised to −10° C. and the reaction mixture stirred for 36 h. Afterwards the mixture was filtered through a pad of Celite and washed with DCM. The filtrate was then washed with 10% tartaric acid (2×100 ml), saturated NaHCO.sub.3 (2×100 ml) and brine (2×100 ml). The organic phase was dried over MgSO.sub.4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 2:1) and recrystallized (Et.sub.2O/hexane) in order to afford the tosylate (S,S-19))) as white needles (20.2 g, 67%).
[0380] .sup.1H NMR (CDCl.sub.3-d.sup.1, 400 MHz): δ=1.30 (d, J=5.27 Hz, 3H), 2.46 (s, 3H), 2.87-2.94 (m, 1H), 2.92 (s, 1H), 3.98 (dd, J=11.42, 5.90 Hz, 1H), 4.18 (dd, J=11.42, 3.89 Hz, 1H), 7.36 (d, J=8.28 Hz, 2H), 7.80 ppm (d, J=8.53 Hz, 2H).
[0381] .sup.13C NMR (CDCl.sub.3-d.sup.1, 100 MHz): δ=16.95, 21.64, 52.77, 55.45, 70.03, 127.94, 129.89, 132.70, 145.06 ppm.
[0382] HRMS (ESI): m/z calculated: C.sub.11H.sub.14O.sub.4S (M+H).sup.+ 243.0686, found 243.0678.
(S)-But-3-en-2-ol ((S)-3)
[0383] ##STR00065##
[0384] A solution of tosylate (S,S)-19 in dry THF (20 ml) was added to a suspension of Zinc-Copper-couple (6 g) and dry NaI (22.3 g, 149 mmol, 3.00 eq) in dry THF (150ml). The resulting suspension was stirred for 2 h at 70° C., cooled to room temperature and filtered through a pad of silica. Afterwards the THF-butenol mixture was distilled under reduced pressure (200 mbar, 100° C.) while the collecting flask was cooled to −78° C. (dry ice). The THF-butenol solution was then submitted to the next step without further purification.
(S)-But-3-en-2-yl tert-butyl carbonate ((S)-4)
[0385] ##STR00066##
[0386] The butenol-THF solution of cooled to 0° C. and NaH (5.82 g, 145 mmol, 3.00 eq) was carefully added under a nitrogen atmosphere in small portions. Then Boc.sub.2O (11.7 g, 53.4 mmol, 1.1 eq) was added in portion-wise over 10 min under vigorous stirring at this temperature. The reaction mixture was allowed to warm to room temperature overnight and then diluted with Et.sub.2O after 16 h of vigorous stirring. Excess sodium hydride was quenched by the slow addition of water. The resulting mixture was then extracted with diethyl ether (3×50 ml). The combined organic extracts were washed with brine (1×50 ml), dried over MgSO.sub.4 and concentrated under reduced pressure. After purification by column chromatography on silica gel (hexane/EtOAc, 10:1) the product (S)-4 was obtained as a colourless liquid (6.25 g, 75% two steps).
3-(6-Benzoxy-1H-indol-3-yl)-2-acetylaminopropionic acid (30)
[0387] ##STR00067##
[0388] A suspension of
(S)-3-amino-3-(6-(benzoxy)-1H-indol-3-yl)propanoic acid—Schiff Base Complex (32)
[0389] ##STR00068##
[0390] A suspension of 30 (1.03 g, 3.32 mmol) in 40% NaOH (H.sub.2O/MeOHv/v=1:1, 14 mL/mmol) was stirred at 110° C. for 4 h, neutralized with conc. HCl to pH=7 and the solvent was removed in vacuo. The crude product was dissolved in MeOH (20 mL/mmol) and Ni(OAc).sub.2*6 H.sub.2O (966 mg, 3.32 mmol) and (S)-28 (1.78 g, 3.65 mmol) were added followed by K.sub.2CO.sub.3 (2.09 g, 15.1 mmol). The resulting mixture was refluxed for 16 h and the precipitate was filtered of and washed with DCM. The solvent of the filtrate was removed in vacuo and the crude product was suspended in DCM, washed with H.sub.2O and dried with Na.sub.2SO.sub.4. After removal of the solvent in vacuo the crude product was purified with DCM/MeOH (v/v=40:1) to give the product 32 as an orange solid (2.44 g, 88%).
(S)-6-benzoxyttyptophan (33)
[0391] ##STR00069##
[0392] To a solution of 32 (180 mg, 0.51 mmol) in MeOH was added 6 M HCl (15 mL) The solution was stirred for 45 min at 70° C. and conc. NH.sub.4OH-solution was added until pH=7 was reached. The aqueous layer was washed with EtOAc (2×15 mL), and the pH was adjusted to 10. The precipitate was centrifuged and washed two times with water (pH=10), to give (S)-6-benzoxytryptophan as a white solid.
(S)-3-(6-(benzyloxy)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propanoic acid (33a)
[0393] ##STR00070##
[0394] Triethylamine (446 μL g, 3.22 mmol, 2.00 eq.) and TeocOSu (626 mg, 2.42 mmol, 1.50 eq.) was added successively to a solution of L-6-benzoxytryptophan (33, 0.5 g, 1.61 mmol, 1.00 eq.) in DMF (20 mL). The reaction mixture was stirred at r.t. for 2 h, then concentrated under reduced pressure. The aqueous layer was carefully acidified to pH=4 by dropwise addition of 1
[0395] HRMS (ESI): m/z calc. for C.sub.17H.sub.24N.sub.2O.sub.4Si (M+H).sup.+ 455.1997, found 455.1999.
((2-(trimethylsilyl)ethoxy)carbonyl)-
[0396] ##STR00071##
[0397] Triethylamine (11.3 mL g, 80.9 mmol, 1.5 eq.) and TeocOSu (18.2 g, 70.0 mmol, 1.30 eq.) was added successively to a solution of
[0398] HRMS (ESI): m/z calc. for C.sub.17H.sub.24N.sub.2O.sub.4Si (M+H).sup.+ 349.1578, found 349.1582.
2,2,2-trichloroethyl ((2-(trimethylsilyl)ethoxy)carbonyl)-L-tryptophanate (38)
[0399] ##STR00072##
[0400] To a solution of N-Teoc protected
[0401] HRMS (ESI): m/z calc. for C.sub.19H.sub.25Cl.sub.3N.sub.2O.sub.4Si (M+H).sup.+ 479.0722, found 479.0721.
2,2,2-trichloroethyl (S)-3-(6-(benzyloxy)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)-amino)propanoate (39)
[0402] ##STR00073##
[0403] To a solution of compound 33a (1.0 g, 2.2 mmol, 1.0 eq.) in DCM (8.8 mL) at 0° C. was added DMAP (40.3 mg, 0.329 mmol, 0.15 eq.) and EDC*HCl (548 mg, 2.86 mmol, 1.30 eq.) successively. After stirring at 0° C. for 10 min 2,2,2-trichloroethanol (0.42 mL, 4.4 mmol, 2.00 eq.) was added and the solution was stirred for 2 h at r.t. The reaction mixture was diluted with DCM (50 mL), washed with 0.5 M HCl (2×25 mL), sat. NaHCO.sub.3 solution (25 mL) and brine (25 mL). After drying over Na.sub.2SO.sub.4 and removal of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (0.5% MeOH/DCM) to afford compound 39 as a yellow oil (1.1 g, 85%).
[0404] HRMS (ESI): m/z calc for C.sub.26H.sub.31Cl.sub.3N.sub.2O.sub.5Si (M+H).sup.+ 585.1141, found 585.1139.
[0405] Synthesis of (N-Boc).sub.2-cystine-(OtBu).sub.2 (35)
##STR00074##
[0406] A solution of
[0407] HRMS (ESI): m/z calc for C.sub.24H.sub.44N.sub.2O.sub.8S.sub.2 (M+H).sup.+ 553.2612, found 553.2615.
tert-butyl S-(6-(benzyloxy)-3-((S)-3-oxo-3-(2,2,2-trichloroethoxy)-2-(((2-(trimethylsilyl)ethoxy) carbonyl)amino)propyl)-1H-indol-2-yl)-N-(tert-butoxycarbonyl)-
[0408] ##STR00075##
[0409] To a solution of (N-Boc).sub.2-
[0410] HRMS (ESI): m/z calc. for C.sub.38H.sub.52Cl.sub.3N.sub.3O.sub.9SSi (M+H).sup.+ 860.2332, found 860.2323.
tert-butyl N-(tert-butoxycarbonyl)-S-(3-((S)-3-oxo-3-(2,2,2-trichloroethoxy)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propyl)-1H-indol-2-yl)-L-cysteinate (41)
[0411] ##STR00076##
[0412] To a solution of (N-Boc).sub.2-
[0413] HRMS (ESI): m/z calc. for C.sub.3H.sub.46Cl.sub.3N.sub.3O.sub.8SSi (M+H).sup.+ 754.1913, found 754.1917.
(S)-3-(6-(benzyloxy)-2-(((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)thio)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propanoic acid (49)
[0414] ##STR00077##
[0415] A solution of tryptathionine derivative 39 (1.63 mmol, 1.00 eq.) in DMF (8.4 mL) was treated with CH.sub.3COOH (0.8 mL) and zinc (3.51 g, 53.6 mmol, 33.0 eq.) for 2 h at 45° C. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (50 mL) and washed with 10% KHSO.sub.4 solution (2×25 mL) and brine (2×25 mL). After drying over Na.sub.2SO.sub.4 and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (AcN/H.sub.2O 50% to 100% gradient) to give compound 49 as a yellow solid (840 mg, 83% over 2 steps).
[0416] HRMS (ESI): m/z calc. for C.sub.36H.sub.51N.sub.3O.sub.9SSi (M+H).sup.+ 730.3183, found 730.3188.
(S)-3-(2-(((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)thio)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propanoic acid (50)
[0417] ##STR00078##
[0418] A solution of tryptathionine derivative 38 (9.15 mmol, 1.00 eq.) in DMF (40 mL) was treated with CH.sub.3COOH (4 mL) and zinc (20.0 g, 302 mmol, 33.0 eq.) for 2 h at 45° C. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (200 mL) and washed with 10% KHSO.sub.4 solution (2×50 mL) and brine (2×50 mL). After drying over Na.sub.2SO.sub.4 and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (AcN/H.sub.2O 50% to 100% gradient) to afford compound 50 as a yellow oil (5.0 g, 88%. over 2 steps).
[0419] HRMS (ESI): m/z calc. for C.sub.31H.sub.46Cl.sub.3N.sub.3O.sub.8SSi (M+H).sup.+ 624.2769, found 624.2775.
((benzyloxy)carbonyl)glycyl-L-isoleucine (44)
[0420] ##STR00079##
[0421] To a solution of Cbz-glycine (42, 10.0 g, 32.7 mmol, 1.00 eq.) in acetone (100 mL) was added a suspension of
[0422] HRMS (ESI): m/z calc. for C.sub.16H.sub.22N.sub.2O.sub.5 (M+H).sup.+ 323.1601, found 323.1606.
[0423] Glycyl-L-isoleucylglycine (45)
##STR00080##
[0424] Dipeptide 44 (10.1 g, 31.3 mmol, 1.00 eq.) and benzyl glycinate (8.21 g, 40.7 mmol, 1.30 eq.)
[0425] were dissolved in dry DMF (125 mL). Then, COMU (17.4 g, 40.7 mmol, 1.30 eq.) and DIPEA (12.6 mL, 72.1 mmol, 3.00 eq.) were added at 0° C. The reaction mixture was allowed to warm to r.t. overnight and diluted with EtOAc (300 mL) afterwards. After washing with a solution of 10% KHSO.sub.4-solution (2×100 mL) the fully protected tripeptide precipitated in the organic phase. The organic phase was cooled to 4° C. for 4 h in order for the peptide to precipitate, then the precipitate was filtered and washed with cold EtOAc. The precipitate was redissolved in a 1:1 mixture of water and THF (260 mL). Pd/C (1 g) was added to the solution after degassing with N.sub.2 for 30 min. Then, the reaction mixture was degassed with hydrogen for 1 h. After vigorous stirring at room temperature under 1.0 atm of hydrogen overnight, the catalyst was filtered through a pad of Celite. Afterwards, the mixture was concentrated under reduced pressure to obtain the product 45 as a white solid (5.71 g, 74%)
[0426] HRMS (ESI): m/z calc. for C.sub.10H.sub.19N.sub.3O.sub.4 (M+H).sup.+ 246.1448, found 246.1440.
[0427] Synthesis of Pentapeptide 51:
##STR00081##
[0428] A solution of thioether building block 49 (111 mg, 0.14 mmol, 1.00 eq.) in AcN (0.7 mL) was treated with collidine (37 μL, 0.27 mmol, 2.0 eq) and N,N′-disuccinimidyl carbonate (39 mg, 0.15 mmol, 1.1 eq.) and stirred for 1 h at r.t. A solution of tripeptide 45 (44 mg, 0.18 mmol, 1.3 eq) in a 1:4 mixture of AcN/H.sub.2O (1 mL) was added and the reaction mixture was stirred for 2 h at r.t. Afterwards, the mixture was diluted with EtOAc (20 mL), 10% KHSO.sub.4-solution (20 mL) was added and the aqueous layer was extracted with EtOAc (2×20 mL). The organic layer was washed with brine (2×20 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure which afforded pentapeptide 51 as a yellow solid (115 mg, 90%).
[0429] HRMS (ESI): m/z calc. for C.sub.46H.sub.68N.sub.6O.sub.12SSi (M+H).sup.+ 957.4458, found 957.4457.
[0430] Synthesis of Pentapeptide 52:
##STR00082##
[0431] A solution of tryptathionine building block 50 (2.0 g, 2.5 mmol, 1.0 eq.) in AcN (10 mL) was treated with collidine (659 μL, 4.95 mmol, 2.00 eq) and N,N′-disuccinimidyl carbonate (697 mg, 2.72 mmol, 1.10 eq.) and stirred for 1 h at r.t. A solution of tripeptide 45 (790 mg, 3.22 mmol, 1.30 eq.) in a 1:4 mixture of AcN/H.sub.2O (18 mL) was added and the reaction mixture was stirred for 2 h at r.t. Afterwards, the mixture was diluted with EtOAc (100 mL), 10% KHSO.sub.4-solution (20 mL) was added and the aqueous layer was extracted with EtOAc (2×50 mL). The organic layer was washed with brine (2×50 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure which afforded pentapeptide 52 as a yellow solid (2.15 g, 93%).
[0432] HRMS (ESI): m/z calc. for (M+H).sup.+ C.sub.36H.sub.51Cl.sub.3N.sub.6O.sub.11S 851.4039, found 851.4058.
[0433] Fully Protected Cyclic Pentapeptide 53:
##STR00083##
[0434] Pentapeptide 51 (151 mg, 0.180 mmol, 1.00 eq.) was dissolved in 1 mL of a solution of p-toluenesulfonic acid in THF (1.8
[0435] HRMS (ESI): m/z calc. for C.sub.41H.sub.58N.sub.6O.sub.9SSi (M+H).sup.+ 839.3828, found 839.3839.
[0436] Fully Protected Cyclic Pentapeptide (54):
##STR00084##
[0437] Pentapeptide 52 (700 mg, 0.822 mmol, 1.00 eq.) was dissolved in 10 mL of 2 M HCl in dioxane and stirred for 40 min at r.t. Then, the reaction mixture diluted with 40 mL of dioxane and the solvent was evaporated under reduced pressure. The precipitate was dissolved in 8 mL DMF and diluted with 82 mL DCM. Afterwards, DIPEA (279 μL, 1.64 mmol, 2.00 eq.) and T3P (50% in EtOAc, 977 μL, 1.64 mmol, 2.00 eq.) were added. After the solution was stirred for 5 h at r.t., ⅓ of the the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHSO.sub.4-solution (20 mL), sat. NaHCO.sub.3-solution (20 mL), water (20 mL) and brine (20 mL). The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN/H.sub.2O 50% to 100% gradient) to afford cyclic pentapeptide 54 as a yellow solid (420 mg, 72%).
[0438] HRMS (ESI): m/z calc. for C.sub.34H.sub.52N.sub.6O.sub.8SSi (M+H).sup.+ 733.3409, found 733.3409.
[0439] Fully Deprotected Monocyclic Pentapeptide 55:
##STR00085##
[0440] Monocyclic Pentapeptide 53 (125 mg, 0.17 mmol, 1.00 eq.) was stirred in TFA/DCM/TIPS (8:1.5:0.5) for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/H.sub.2O 20% to 100%) to afford the fully deprotected monocyclic pentapeptide 55 as a white powder (100 mg, quant.).
[0441] HRMS (ESI): m/z calc. for C.sub.31H.sub.38N.sub.6O.sub.7S (M+H).sup.+ 639.2595, found 639.2590.
[0442] Fully Deprotected Monocyclic Pentapeptide 56:
##STR00086##
[0443] Monocyclic Pentapeptide 54 (250 mg, 0.34 mmol, 1.00 eq.) was stirred in TFA/DCM/TIPS (8:1.5:0.5) for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/H.sub.2O 10% to 30%) to afford the fully deprotected monocyclic pentapeptide 56 as a white powder (200 mg, quant.).
[0444] HRMS (ESI): m/z calc. for C.sub.24H.sub.32N.sub.6O.sub.6S (M+H).sup.+ 533.2177, found 533.2188.
[0445] Tripeptide (48):
##STR00087##
[0446] HRMS (ESI): m/z calc. for C.sub.42H.sub.64N.sub.4O.sub.10Si.sub.2 (M+H).sup.+ 841.4233, found 841.4253.
[0447] Monocyclic Octapeptide 57:
##STR00088##
[0448] A solution of fully deprotected monocyclic pentapeptide 55 (50.0 mg, 0.078 mmol, 1.00 eq.) and MSA (13.8 μL, 0.86 mmol, 1.1 eq.) in DMA (1.5 mL) was stirred for 2 h at 50° C. Separately, a solution of Fmoc-Asn-Hyp-DHIIe(TBS).sub.2-OH (98.8 mg, 0.12 mmol, 1.50 eq.), COMU (36.9 mg, 0.086 mmol, 1.10 eq.) and DIPEA (15.0 μL, 0.083 mmol, 1.10 eq.) in DMA (0.4 mL) was stirred for 30 min at 0° C. The silylated monocyclic peptide was then added to the activated tripeptide and stirred for 1 h at 0° C. then at 35° C. for 3 h. Et.sub.2NH (82.0 μL, 0.078 mmol, 10.0 eq.) was added and stirred for 1 h at r.t. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column, gradient A) to afford octapeptide 57 as a white solid (65.5 mg, 68%).
[0449] HRMS (ESI): m/z calc. for C.sub.58H.sub.90N.sub.10O.sub.14SSi.sub.2 (M+H).sup.+ 1239.5970, found 1239.5980.
[0450] Monocyclic Octapeptide 58:
##STR00089##
[0451] A solution of fully deprotected monocyclic pentapeptide 56 (40.0 mg, 0.075 mmol, 1.00 eq.) and MSA (12 μL, 0.075 mmol, 1.00 eq.) in DMA (1.5 mL) was stirred for 2 h at 50° C. Similtaneously, a solution of Fmoc-Asn-Hyp-DHIIe(TBS).sub.2-OH (95 mg, 0.11 mmol, 1.50 eq.), COMU (35.0 mg, 0.083 mmol, 1.10 eq.) and DIPEA (14.0 μL, 0.083 mmol, 1.10 eq.) in DMA (0.4 mL) was stirred for 30 min at 0° C. The silylated monocyclic peptide was then added to the activated tripeptide and stirred for 1 h at 0° C. then at 35° C. for 3 h. Et.sub.2NH (77 μL, 0.75 mmol, 10 eq.) was added and stirred for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column, gradient A) to afford octapeptide 58 as a white powder (70 mg, 68%).
[0452] HRMS (ESI): m/z calc. for C.sub.51H.sub.84N.sub.10O.sub.13SSi.sub.2 (M+H).sup.+ 1133.5551, found 1133.5549.
[0453] Monocyclic Octapeptide 59:
##STR00090##
[0454] A solution of TBAF in THF (1
[0455] HRMS (ESI): m/z calc. for C.sub.46H.sub.62N.sub.10O.sub.14S (M+H).sup.+ 1011.4240, found 1001.4247.
[0456] Monocyclic Octapeptide 60:
##STR00091##
[0457] A solution of TBAF in THF (1
[0458] HRMS (ESI): m/z calc. for C.sub.39H.sub.56N.sub.10O.sub.13S (M+H).sup.+ 905.3822, found 905.38113.
[0459] 2-((9H-fluoren-9-yl)methyl) 1-(tert-butyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (64):
##STR00092##
[0460] A solution of N-Boc-protected (2S,4R)-4-hydroxyproline 63 (5.0 mg, 22 mmol, 1.0 eq.) in DMF (20 mL) was added dropwise to a solution of 9-Fluorenemethanol (8.5 mg, 43 mmol, 2.0 eq.), EDC*HCl (8.3 g, 43 mmol, 2.0 eq.) and DMAP (396 mg, 3.24 mmol, 0.150 eq.) in DCM (220 mL). The reaction mixture was stirred at r.t. for 2 h. Then, 10% KHSO.sub.4 solution (50 mL) was added. The organic phase was washed with brine (50 mL) and dried over NaSO.sub.4. Afterwards, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate=1:1) to afford compound 64 as a white solid (5.0 g, 56%).
[0461] (9H-fluoren-9-yl)methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate (64a):
##STR00093##
[0462] 64 (5.0 g, 22 mmol, 1.0 eq.) was treated with 4 M HCl in Dioxane (30 mL) at r.t. for 1 h. Afterwards, the solvent was evaporated under reduced pressure to afford the product (64a) as a white solid (3.7 g, quant.).
[0463] HRMS (ESI): m/z calc. for C.sub.19H.sub.19NO.sub.3 (M+H).sup.+ 310.1438, found 310.1426.
(9H-fluoren-9-yl)methyl (2S,4R)-1-((tert-butoxycarbonyl)-L-asparaginyl)-4-hydroxypyrrolidine-2-carboxylate (65)
[0464] ##STR00094##
[0465] N-Boc-protected asparagine (1.7 g, 7.3 mmol, 1.5 eq.), 64a (1.5 g, 4.8 mmol, 1.0 eq.), EDC*HCl (1.4 g, 7.3 mmol, 1.5 eq.) and HOBt*H.sub.2O (1.5 g, 9.7 mmol, 2.0 eq.) were dissolved in DMF (72 mL) and stirred at r.t. for 16 h. The reaction mixture was diluted with EtOAc (200 mL). Then, 10% KHSO.sub.4 solution (50 mL) was added. The organic phase was washed with 10% KHSO.sub.4 solution (50 mL) and brine (2×50 mL). After drying over NaSO.sub.4 and removal of the solvent under reduced pressure the crude product was purified by C18 reversed phase chromatography (AcN/H2O 20% to 70%) to afford dipeptide 65 as a white powder (1.6 g, 78%).
[0466] HRMS (ESI): m/z calc. for C.sub.28H.sub.33N.sub.3O.sub.7(M+H).sup.+ 524.2391, found 524.2396.
(9H-fluoren-9-yl)methyl (2S,4R)-1-(L-asparaginyl)-4-hydroxypyrrolidine-2-carboxylate (66)
[0467] ##STR00095##
[0468] 65 (1.0 g, 1.9 mmol, 1.0 eq.) was treated with 4 M HCl in Dioxane (30 mL) at r.t. for 1 h. Afterwards, the solvent was evaporated under reduced pressure to afford the product (66) as a white solid (870 mg, quant.).
[0469] HRMS (ESI): m/z calc. for C.sub.23H.sub.25N.sub.3O.sub.5(M+H).sup.+ 424.1866, found 424.1858.
[0470] Monocyclic Hexapeptide 67:
##STR00096##
[0471] A solution of fully deprotected monocyclic pentapeptide 55 (42 mg, 0.66 mmol, 1.00 eq.) and MSA (11.6 μL, 0.723 mmol, 1.10 eq.) in DMA (2 mL) was stirred for 2 h at 50° C. Simultaneously, a solution of Fmoc-DHIIe(TBS).sub.2-OH (13, 52 mg, 0.85 mmol, 1.30 eq.), COMU (36 mg, 0.85 mmol, 1.30 eq.) and DIPEA (15 μL, 0.85 mmol, 1.30 eq.) in DMA (0.4 mL) was stirred for 30 min at 0° C. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for 1 h at 0° C. then at r.t. overnight. Afterwards, the mixture was diluted with EtOAc (50 mL) and washed with 10% KHSO.sub.4 solution (3×5 mL). The organic phase was washed with brine (2×20 mL), dried over NaSO.sub.4 and evaporated under reduced pressure. The crude product of 67 was used in the next step without any further purification.
[0472] HRMS (ESI): m/z calc. for C.sub.64H.sub.87N.sub.7O.sub.12SSi.sub.2 (M+H).sup.+ 1234.5745, found 1234.5745.
[0473] Monocyclic Hexapeptide 68:
##STR00097##
[0474] A solution of fully deprotected monocyclic pentapeptide 56 (100 mg, 0.188 mmol, 1.00 eq.) and MSA (33.2 μL, 0.207 mmol, 1.10 eq.) in DMA (4 mL) was stirred for 2 h at 50° C. Simultaneously, a solution of Fmoc-DHIIe(TBS).sub.2-OH (13, 149 mg, 0.244 mmol, 1.30 eq.), COMU (104 mg, 0.244 mmol, 1.30 eq.) and DIPEA (42.5 μL, 0.244 mmol, 1.30 eq.) in DMA (1.25 mL) was stirred for 30 min at 0° C. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for 1 h at 0° C. then at r.t. overnight. Afterwards, the mixture was diluted with EtOAc (100 mL) and washed with 10% KHSO.sub.4 solution (3×10 mL). The organic phase was washed with brine (2×25 mL), dried over NaSO.sub.4 and evaporated under reduced pressure. The crude product of 68 was used in the next step without any further purification.
[0475] HRMS (ESI): m/z calc. for C.sub.57H.sub.81N.sub.7O.sub.11SSi.sub.2 (M+H).sup.+ 1128.5326, found 1128.5316.
[0476] Monocyclic Octapeptide 69:
##STR00098##
[0477] Crude monocyclic hexapeptide 67 (0.66 mmol, 1.0 eq.), dipeptide 66 (42 mg, 0.10 mmol, 1.50 eq.) were dissolved in DMF (1.5 mL). Then, DIPEA (17.3 mL, 0.10 mmol, 1.50 eq.) and HATU (38 mg, 0.10 mmol, 1.5 eq) were added at 0° C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified by C18 reversed phase chromatography (AcN/H.sub.2O 60% to 100%) to fully protected octapeptide 69 as a white solid (60 mg, 55% over two steps).
[0478] HRMS (ESI): m/z calc. for C.sub.87H.sub.110N.sub.10O.sub.16SSi.sub.2 (M+H).sup.+ 1639.7433, found 1639.7404.
[0479] Monocyclic Octapeptide 70:
##STR00099##
[0480] Crude monocyclic hexapeptide 68 (0.188 mmol, 1.00 eq.), dipeptide 68 (119 mg, 0.282 mmol, 1.50 eq.) were dissolved in DMF (3 mL). Then, DIPEA (49.1 mL, 0.282 mmol, 1.50 eq.) and HATU (108 mg, 0.282 mmol, 1.50 eq) were added at 0° C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified by C18 reversed phase chromatography (AcN/H2O 60% to 100%) to fully protected octapeptide 70 as a white solid (170 mg, 59% over two steps).
[0481] HRMS (ESI): m/z calc. for C.sub.80H.sub.104N.sub.10O.sub.15SSi.sub.2 (M+H).sup.+ 1533.7015, found 1533.7004.
[0482] Monocyclic Octapeptide 71:
##STR00100##
[0483] Monocyclic octapeptide 69 (15 mg, 10.3 μmol, 1.00 eq.) was dissolved in DMF (0.1 mL). Et.sub.2NH (10.8 μL, 0.103 mmol, 10 eq.) was added and stirred for 2 h at r.t. The solvent was removed under reduced pressure and the precipitate was redissolved in THF (0.2 mL). Then, a solution of TBAF in THF (1
[0484] HRMS (ESI): m/z calc. for C.sub.46H.sub.62N.sub.10O.sub.14S (M+H).sup.+ 1011.4240, found 1001.4241.
[0485] Monocyclic Octapeptide 72:
##STR00101##
[0486] Monocyclic octapeptide 69 (20.0 mg, 14.8 μmol, 1.00 eq.) was dissolved in DMF (0.15 mL). Et.sub.2NH (15.5 μL, 0.148 mmol, 10 eq.) was added and stirred for 2 h at r.t. The solvent was removed under reduced pressure and the precipitate was redissolved in THF (0.3 mL). Then, a solution of TBAF in THF (1
[0487] HRMS (ESI): m/z calc. for C.sub.39H.sub.56N.sub.10O.sub.13S (M+H).sup.+ 905.3822, found 905.3810.
[0488] (S)-Deoxy-(O)-benzyl-α-amanitin (61a):
##STR00102##
[0489] Monocyclic octapeptide 59 or 71 (20.0 mg, 19.2 μmol, 1.00 eq.) was dissolved in DMF (19 mL). Then, DIPEA (6.71 μL, 38.5 μmol, 2.00 eq.) and HATU (4.98 mg, 38.5 μmol, 2.00 eq) were added at 0° C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column, gradient C) to afford (O)-Benzyl-α-amanitin (61-a, 13 mg, 68%) as a white powder.
[0490] HRMS (ESI): m/z calc. for C.sub.46H.sub.60N.sub.10O.sub.13S (M+H).sup.+ 993.4135, found 993.4145.
[0491] (S)-Deoxyamaninamide (72a):
##STR00103##
[0492] Monocyclic octapeptide 60 or 72 (20.0 mg, 21.4 μmol, 1.00 eq.) was dissolved in DMF (21 mL). Then, DIPEA (7.47 μL, 42.9 μmol, 2.00 eq.) and HATU (16.3 mg, 42.9 μmol, 2.00 eq) were added at 0° C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column, gradient B) to afford (S)-Deoxyamaninamide (72a, 14 mg, 74%) as a white powder.
[0493] HRMS (ESI): m/z calc. for C.sub.39H.sub.54N.sub.10O.sub.12S (M+H).sup.+ 887.3716, found 887.3718.
(O)-Benzyl-α-amanitin (61b):
[0494] ##STR00104##
[0495] The prophyrine derived ligand (22 μg, 0.45 μmol, 0.3 eq.) and MnOTf.sub.2 (16 μg, 0.45 μmol, 0.3 eq.) were dissolved in DCM (1 mL) and stirred for 3 h at r.t. Then Octapeptide 61a (1.5 mg, 1.5 μmol, 1.0 eq.) dissolved in DMF (500 μL), AcOH (0.21 μL, 3.8 μmol, 2.5 eq.) and H.sub.2O.sub.2 (0.11 μL, 4.5 μmol, 3.0 eq.) were added. The reaction mixture was cooled down to 0° C. and was stirred for 16 h at 0° C. The solvent was removed under reduced pressure and the crude product was used in the next step without further purification.
[0496] HRMS (ESI): m/z calc. for C.sub.46H.sub.60N.sub.10O.sub.14S (M+H).sup.+ 1009.4084 found 1009.4118.
[0497] Amaninamide (62):
##STR00105##
[0498] The prophyrine derived ligand (73 μg, 1.5 μmol, 0.3 eq.) and MnOTf.sub.2 (53 μg, 1.5 μmol, 0.3 eq.) were dissolved in DCM (1 mL) and stirred for 3 h at r.t. Then Octapeptide 72 (5 mg, 5 μmol, 1.0 eq.) dissolved in DMF (500 μL), AcOH (0.700 μL, 12.7 μmol, 2.5 eq.) and H.sub.2O.sub.2 (0.37 μL, 15.0 μmol, 3.0 eq.) were added. The reaction mixture was cooled down to 0° C. and was stirred for 16 h at 0° C. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column, gradient D) to afford amaninamide 62 as a white powder.
[0499] HRMS (ESI): m/z calc. for C.sub.39H.sub.54N.sub.10O.sub.13S (M+H).sup.+, found.
[0500] α-Amanitin (61):
##STR00106##
[0501] The crude product of 61b was dissolved in THF/H2O (2:1) and Pd/C (1 mg) was added. The reaction mixture was flushed with N.sub.2 for 10 min, then with H.sub.2 for 10 min and was stirred for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column) to afford α-Amanitin (61) as a white powder.
[0502] HRMS (ESI): m/z calc. for C.sub.39H.sub.54N.sub.10O.sub.14S (M+H).sup.+ 919.3614 found 919.3614.
[0503] Preparative HPLC Purification Gradients:
[0504] Gradient A: 0-25 min 40%-60% B, 25-35 min 100% B; 35-40 min 40% B 0.1% formic acid in water (Solvent A) and 0.1% formic acid in ACN (Solvent B).
[0505] Gradient B: 0-30 min 10%-30% B, 30-40 min 100% B; 40-50 min 10% B 0.1% formic acid in water (Solvent A) and 0.1% formic acid in ACN (Solvent B).
[0506] Gradient C: 0-60 min 5%-50% B, 60-65 min 100% B; 65-70 min 5% B 0.1% formic acid in water (Solvent A) and 0.1% formic acid in ACN (Solvent B).
[0507] Solid-Phase Peptide Synthesis
[0508] Automated or manual solid-phase peptide synthesis was performed in 50 μmol scale. Loading: To a 10 ml syringe reactor with frit and cap 1 g of tritylchloride polystyrene (TCP) resin (0.9 mmol/g) were added and 7 ml drydichloromethane (DCM). For resin loading with the first amino acids, the resin was pre-swollen for 10 min and the solvent was removed by evaporation in vacuum. A mixture of the amino acids (0.6 mmol) and 3 equivalents of N,N-diisopropylamine (DIPEA) dissolved in 5 ml dry DCM was added to the resin. The syringe was agitated for 30 min at room temperature. The solution was removed and the resin was washed (2×5 ml N,N-dimethylformamide(DMF), 2×5 ml DCM). Capping of non-reacted functional groups of the resin was performed with DCM, methanol and DIPEA 80:15:5 (2×10 ml, 10 min). After washing (5×5 ml DMF), Fmoc-removal was achieved with DMF/piperidine (4:1, 5 ml, 1×2 min, 1×20 min). After final washing (2×5 ml DMF, 1×5 ml methanol, 3×5 ml DCM), the resin was dried in vacuo. Coupling of Fmoc protected amino acids: To 200 mg of the resin (˜0.5 mmol/g), a 0.25M solution of the amino acid in DMF (2.5 eq relative to resin loading) was added. After addition of a 0.5M solution of DIPEA in DMF (2.5 eq) and a 0.25 M solution of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate TBTU in DMF (2.5 eq), the reaction solution was mixed for 15 min. A second coupling was performed for 15 min. Fmoc removal: DMF/piperidine (4:1, 2.5 ml) was added to the resin and mixed for 2.5 min. The procedure was repeated 4 times. The resin was washed with DMF (6×2.5 ml). After the final coupling cycle, the resin was washed with DCM (6×2 ml). Cleavage: After addition of the cleavage cocktail (DCM/HFIP 4:1, the syringe was shaken for 30 min. The solution was transferred to a flask and the solvent was removed in vacuo. Further instructions can be found in (Amblard M, Fehrentz J A, Martinez J, Subra G. Mol Biotechnol. 2006 July; 33(3):239-54).
[0509] Abbreviations
[0510] BF.sub.3*Et.sub.2O: boron trifluoride etherate
[0511] Bn: benzyl
[0512] Boc: tert-butyloxycarbonyl
[0513] BMIM-PF.sub.6: 1-butyl-3-methylimidazolium hexafluorophosphate
[0514] Cbz: benzyloxycarbonyl
[0515] COMBU: 4-{[1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)ylidenaminooxy](dimethylamino)methylen}morpholin-4-iumhexafluorophosphate
[0516] COMU: (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium-hexafluorophosphate
[0517] [(p-cymene) RuCl.sub.2].sub.2: (cymene)ruthenium dichloride dimer
[0518] DCM: dichloromethane
[0519] DHIL: dihydroxy-isoleucine
[0520] DIPEA: N,N-diisopropylethylamine
[0521] DMA: dimethylacetamide
[0522] DMF: dimethylformamide
[0523] (DHQD).sub.2PHAL: hydroquinidine 1,4-phthalazinediyl diether
[0524] Fm-9-Fluorenylmethyl
[0525] Fmoc: fluorenylmethyloxycarbonyl
[0526] Fmoc-OSu: 9-Fluorenylmethyl N-succinimidyl carbonate
[0527] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium
[0528] HBTU: 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate
[0529] HCTU: 2-(6-Chlor-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium-hexafluorophosphate
[0530] Hyp: trans-4-hydroxy-proline
[0531] LHMDS: lithium bis(trimethylsilyl)amide
[0532] MSA: N-Methyl-N-trimethylsilylacetamid
[0533] NMO: 4-methylmorpholine 4-oxide
[0534] PPh.sub.3: triphenylphosphine
[0535] PPO: Phthaloyl peroxide
[0536] T3P: 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide
[0537] TBAF: tetra-n-butylammonium fluoride
[0538] tBuOAc: tert-butyl-acetate
[0539] TBS: tert.-butyldimethylsilyl
[0540] TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate
[0541] Tce: trichloroethyl
[0542] Teoc: 2-(Trimethylsilyl)ethoxycarbonyl
[0543] TFA: trifluoroacetic acid
[0544] THF: tetrathydrofuran
[0545] TMSOTf: trimethylsilyl trifluoromethanesulfonate
[0546] TOMBU: N-{[1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)-ylidenaminooxy](dimethylamino)methylen}-N-methylmethanaminiumhexafluorophosphate