Stereoselective synthesis of [5-[4,4,4,4′,4′,4′- hexafluoro-N-(2-hydroxyethoxy)-D-valine]]- and 5-[4,4,4,4′,4′, 4′-hexafluoro-N-(2-hydroxyethoxy)-L-valine]cyclosporin A

Article abstract of DOI:10.1002/hlca.201000175

Addition of various amines to the 3,3-bis(trifluoromethyl)acrylamides 10a and 10b gave the tripeptides 11a - 11f, mostly as mixtures of epimers (Scheme 3). The crystalline tripeptide 11f₂ was found to be the N-terminal (2-hydroxyethoxy)-substituted (R,S,S)-ester HOCH₂CH ₂O-D-Val(F₆)-MeLeu-Ala-O^tBu by X-ray crystallography. The C-terminal-protected tripeptide 11f₂ was condensed with the N-terminus octapeptide 2b to the depsipeptide 12a which was thermally rearranged to the undecapeptide 13a (Scheme 4). The condensation of the epimeric tripeptide 11f₁ with the octapeptide 2b gave the undecapeptide 13b directly. The undecapeptides 13a and 13b were fully deprotected and cyclized to the [5-[4,4,4,4′,4′,4′-hexafluoro- N-(2-hydroxyethoxy)-D-valine]]- and [5-[4,4,4,4′,4′,4′- hexafluoro-N-(2-hydroxyethoxy)-L-valine]]cyclosporins 14a and 14b, respectively (Scheme 5). Rate differences observed for the thermal rearrangements of 12a to 13a and of 12b to 13b are discussed.

Full text of DOI:10.1002/hlca.201000175

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Stereoselective Synthesis of [5-[4,4,4,4’,4’,4’-Hexafluoro-N-
(2-hydroxyethoxy)-d-valine]]- and [5-[4,4,4,4’,4’,4’-Hexafluoro-N-
(2-hydroxyethoxy)-l-valine]cyclosporin A1)
by Marcel K. Eberle and Reinhart Keese*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern
(e-mail: reinhart.keese@ioc.unibe.ch)
Addition of various amines to the 3,3-bis(trifluoromethyl)acrylamides 10a and 10b gave the
tripeptides 11a – 11f, mostly as mixtures of epimers (Scheme 3). The crystalline tripeptide 11f2 was found
to be the N-terminal (2-hydroxyethoxy)-substituted (R,S,S)-ester HOCH2CH2O-d-Val(F6)-MeLeu-Ala-
OtBu by X-ray crystallography. The C-terminal-protected tripeptide 11f2 was condensed with the N-
terminus octapeptide 2b to the depsipeptide 12a which was thermally rearranged to the undecapeptide
13a (Scheme 4). The condensation of the epimeric tripeptide 11f1 with the octapeptide 2b gave the
undecapeptide 13b directly. The undecapeptides 13a and 13b were fully deprotected and cyclized to the
[5-[4,4,4,4’,4’,4’-hexafluoro-N-(2-hydroxyethoxy)-d-valine]]- and [5-[4,4,4,4’,4’,4’-hexafluoro-N-(2-hy-
droxyethoxy)-l-valine]]cyclosporins 14a and 14b, respectively (Scheme 5). Rate differences observed
for the thermal rearrangements of 12a to 13a and of 12b to 13b are discussed.
Introduction. – Cyclosporin A (1a), the active ingredient of both Sandimmuneꢀ and
Neoraleꢀ, is a powerful immunosuppressant embodying eleven amino acids in a cyclic
array (for a comprehensive review, see [1]). Seven of the amino acids are methylated at
the N-atom, one of these being N-methyl-l-valine (amino acid 11 ¼ AA11). One l-
valine (AA5) is found among the four other amino acids that form H-bridges to give
cyclosporin A a distinct structure (Fig. 1).
Fluorine atoms, especially as trifluoromethyl (CF3) groups [2][3], have been
introduced into many positions of bioactive molecules, e.g., angiotensin [3b,g]. These
groups usually resist metabolic changes leading to the prospect of prolonging the
bioavailability of such modified molecules. Due to profound changes in their polarities
such polyfluorinated products may have enhanced lipophilicities and show novel
chemical and physiological properties.
To the best of our knowledge, F-atoms have been introduced into cyclosporine A
(1a) in two cases only. The 1h-fluorocyclosporin A (1f) was obtained by Eberle from 1a
via 1b (the known intermediate for the preparation of the main metabolite (1c) of
cyclosporine A [4]) and by the sequence 1b ! 1d ! 1e ! 1f [5]. The preparation of [8-
(b-fluoro-d-alanine)]cyclosporin (1g) was reported by Patchett et al. [6].
We described the synthesis of (ꢀ)-(4,4,4,4’,4’,4’-hexafluoro-d-valine (d-Val(F6)) in
high enantiomer excess by means of the addition of (þ)-(R)-1-phenylethylamine
(¼(þ)-(aR)-a-methylbenzenemethanamine) to the a-position of benzyl b,b-bis(tri-
1
)
This work was performed from 1998 to 2002.
ꢁ 2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

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Fig. 1. Cyclosporin (1a) and some derivatives
fluoromethyl)acrylate (¼ benzyl 4,4,4-trifluoro-3-(trifluoromethyl)but-2-enoate) as
key step [7]. Similarly, (þ)-4,4,4,4’,4’,4’-hexafluoro-l-valine (Val(F6)) was prepared
in high enantiomer excess (see below). The ready access to chiral hexafluorovaline is
based on the observation by Knunjants et al. [3a] that b,b-bis(trifluoromethyl)acrylate
reacts with NH3 in the a-position of the C¼C bond. Hexafluoroacetone was used as the
starting material for our preparation of (S)-5,5,5,5’,5’,5’-hexafluoroleucine [8].
The amino acids with geminal CF3 groups stimulated us to replace l-valine (AA5)
in cylcosporin A (1a) by hexafluoro-l- and hexafluoro-d-valine. The feasibility of this
plan is based on the observation [9] that the amide bonds of cyclosporin A can be
cleaved selectively on the side of the N-atom of l-valine (between AA4 and AA5) or
d-alanine (between AA7 and AA8) via thioamides and their corresponding thio-
amidates (pseudopeptide bonds) [9]. In the case of monothioamides, this leads to 4,5-
secocyclosporin and 7,8-secocyclosporin, respectively. In the case of the bis-thioamide
[4Y5, CSꢀNH; 7Y8, CSꢀNH]cyclosporin, a concomitant cleavage of the corresponding
bis-thioamidate leads to fragmentation of the cyclosporin producing the octapeptide 2a
and the tripeptide S-benzyl ester N-Boc-Val-MeLeu-Ala-SCH2Ph (3; Scheme 1).
While N-methyl-l-leucine (AA6) is located in the center of the tripeptide fragment,
l-valine (AA5) is strategically positioned at the N-terminus of the tripeptide 3
(Scheme 1). Thus, the problem of replacing l-valine (AA5) in cyclosporin A is reduced
to the preparation of the tripeptide Val(F6)-MeLeu-Ala as building block. [Val(F6)5]-
cyclosporin would subsequently be prepared by two condensation steps between this
tripeptide and the octapeptide 2b, readily available from 2a [9]. The protecting groups
to be used during the synthesis depend on the specific strategy used for the cyclization
of the linear undecapeptide to the [Val(F6)5]cyclosporin. When the cyclization is
carried out between AA4 and AA5 (4/5 variant), then a benzyl ester of the tripeptide
may be tolerated. However, when the cyclization is carried out between AA7 and AA8
(7/8 variant), then a tert-butyl ester of the tripeptide would be preferred.
The conventional ꢃlinearꢄ approach to the synthesis of the desired tripeptide would
require the condensation of activated N-protected hexafluoro-l-valine with an ester

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Scheme 1. Cleavage Reaction of Cyclosporin A (1a)
MeLeu-Ala-OR. In view of the rather high sensitivity of hexafluoro-d-valine benzyl
ester towards Na2CO3, leading to rapid racemization [7], the use of activated esters of
enantiomerically pure Val(F6) was considered to give the tripeptide as a mixture of
epimers. We, therefore, favored the preparation of the desired tripeptide from the N-
terminal b,b-bis(trifluoromethyl)acrylamide of an ester Leu-Ala-OR1 via addition of
NH3 or amines to the a-position of the C¼C bond of the acrylamide moiety. This
sequence is shorter by a few steps than the conventional linear approach. Furthermore,
it was our hope that the chiral centers in MeLeu-Ala would enhance the chiral
induction in the addition step. We considered such a selectivity to be more likely than
the formation of a 1:1 mixture of diastereoisomers observed in the reaction of (1R)-1-
phenylethylamine with benzyl b,b-bis(trifluoromethyl)acrylate [7].
Results and Discussion. – (þ)-4,4,4,4’,4’,4’-Hexafluoro-l-valine (Val(F6)). First, we
prepared the hitherto unreported (þ)-4,4,4,4’,4’,4’-hexafluoro-l-valine (7b) via sepa-
ration of the diastereoisomers obtained from the addition of (þ)-(1R)-1-phenylethyl-
amine (5) to benzyl b,b-bis(trifluoromethyl)acrylate (4) [7] (Scheme 2). Treatment of
this 1:1 mixture of diastereoisomeric adducts with TsOH in Et2O led to 6a and 6b and
subsequently to the rapid precipitation of the (R,R)-adduct 6a. Hydrogenolysis of the
corresponding HCl salt readily gave (ꢀ)-hexafluoro-d-valine 7a in 38% yield and 98%
ee. The (S,R)-diastereoisomer 6b used for the preparation of (þ)-hexafluoro-l-valine
7b was obtained pure by repeated precipitation of 6a in the presence of an excess of
TsOH in Et2O.

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Scheme 2. Synthesis of Hexafluorovalines 7a and 7b
a) MeOH, 08 ! r.t. b) TsOH, Et2O. c) NaHCO3. d) HCl, Et2O. e) H2, Pd/C.
The oily (S,R)-diastereoisomer 6b then led to the solid HCl salt 6d, from which (þ)-
hexafluoro-l-valine 7b was obtained in 29% yield and 98% ee. The stereoselective
transformations of the readily available enantiomeric hexafluorovalines 7a and 7b as
well as of the diastereoisomerically pure intermediates 6a and 6b are to be explored
further. Obviously, (þ)-hexafluoro-l-valine 7b could be obtained more directly by
starting with the addition of (ꢀ)-(1S)-1-phenylethylamine to 4 leading to the
enantiomers of 6a and 6b. In this case, the TsOH addition salt of the enantiomer of
6b should precipitate providing access to 7b in fewer steps.
As mentioned above, the replacement of Me by CF3 groups as, e.g., in valine, should
lead to a noticeable change in the chemical properties. Indeed, the lipophilicity of the
hexafluorovaline anion, determined across the H2O/nitrobenzene interface, is higher
than that of the valine anion by ca. 8 kJ/mol [10][11].
Tripeptides. In an exploratory approach, the known dipeptide 8a [12] was treated
with 3,3-bis(trifluoromethyl)acryloyl chloride (¼4,4,4-trifluoro-3-(trifluoromethyl)-
but-2-enoyl chloride; 9) [3a] to give the N-acylated dipeptide 10a as a low-melting
yellow solid (Scheme 3). The mass spectrum (MS) showed the expected molecular-ion
1
peak at m/z 496. The H-NMR spectrum of 10a revealed the presence of a s at d 7.12
assigned to the olefinic H-atom. The MeN group gave rise to a s at d 2.90. Two m at d
4.55 and 5.05 were assigned to the two HꢀC(a) of the dipeptide.
Treatment of 10a with an EtOH solution of anhydrous NH3 resulted in the
formation of the tripeptide 11a which was isolated as a single diastereoisomer in 37%
yield (Scheme 3). The MS of 11a showed the expected molecular-ion peak at m/z 513.
Signals due to three H-atoms were detected between d 4.15 and 5.15. These were
assigned to the three HꢀC(a) supporting the formation of a tripeptide via addition of
NH3 to the a-position of the acryloyl moiety of 10a. The m between d 3.9 and 4.05 was
assigned to the H-atom of the (CF3)2CH group. The MeN group of MeLeu was
observed as a s at d 3.05. Furthermore, in the 13C-NMR spectrum, three ds were

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Scheme 3. Synthesis of the Tripeptides 11a – 11f2 from the Dipeptides 8a or 8c
observed between d 49 and 55. These were assigned to the three N-substituted C(a)-
atoms adding further support to the formation of the tripeptide 11a. These results are in
agreement with and represent the first example of an anti-Michael addition of NH3 to a
b,b-bis(trifluoromethyl)acrylamide moiety as in 10a (see also [7]). The formation of a
second diastereoisomer was not observed.
t
Reaction of the crude tripeptide 11a with di(tert-butyl) dicarbonate [O(CO2 Bu)2]
gave the expected N-Boc-substituted product 11b in 24% yield from 10a as a crystalline
product. This is supported by its MS, which showed a signal for [M þ H]þ at m/z 614. In
the 1H-NMR spectrum, the signals in the region between d 4.5 and 5.35 were assigned
to the three HꢀC(a) of the tripeptide 11b. The H-atom of the (CF3)2CH group
appeared as a m between d 3.8 and 4.0. The tBu and the MeN groups gave rise to ss at d
1.41 and 3.30, respectively. Again the formation of a second diastereoisomer was not
observed.
Next, the addition of NH2OH to 10a was investigated (Scheme 3). Two epimers
were formed in a ratio of 1:6 and were isolated in pure form by column
chromatography (silica gel). The less polar isomer 11c1 was obtained as an oil, while
the more polar major isomer 11c2 was isolated in crystalline form. The MS of both
isomers showed peaks for [M þ H]þ at m/z 530 with slight differences in their
fragmentation patterns. In the 1H-NMR spectrum of the less polar 11c1, the signals at d
3.5, 3.7, and 5.10 are assigned to three HꢀC(a). For the more polar crystalline isomer
11c2, 1H-NMR signals for two HꢀC(a) were observed between d 4.45 and 4.60 and that
of the third HꢀC(a) at d 5.35. The configuration of the newly generated chiral centers
of the isomers was not determined.
The addition of amines to the a-position of b,b-bis(trifluoromethyl)acrylic acid
rather than to the b-position was first observed by Knunjants et al. [13]. Subsequently,
Eremeev et al. explored the enantioselective addition of achiral amines to b,b-
bis(trifluoromethyl)acrylamides containing a chiral amide [14]. In all cases, an a-

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addition to the b,b-bis(trifluoromethyl)acryloyl moiety with moderate deꢄs of 20 – 40%
was observed. As mentioned above, the addition of (1R)-1-phenylethylamine to benzyl
b,b-bis(trifluoromethyl)acrylate to the a-position led to a 1:1 mixture of diaster-
eoisomers, from which hexafluoro-d- and hexafluoro-l-valine were obtained.
This unique addition of nucleophiles to the a- rather than to the b-position of the
acrylate is due to the presence of the two CF3 groups in the b-position. Based on
detailed computational results for the gas phase and a solvent with a formal dielectric
constant of 7.0, this regioselectivity (anti-Michael addition) is interpreted in terms of a
kinetically controlled addition of nucleophiles like cyanide in the a-position leading to
an anion in the b-position, stabilized by the two adjacent CF3 groups [15][16]. In
addition, the partial positive charge and the coefficient of the LUMO are larger in the
a-position of the b,b-bis(trifluoromethyl)acryloyl moiety. Whether the addition of the
nucleophilic N-atom of the amine is synchronized with the protonation of the incipient
anion (in MeOH as solvent) is an open question. We surmised that the depsipeptides
10a and 10b, in which the b,b-bis(trifluoromethyl)acryloyl moiety is attached to the
dipeptide MeLeu-Val, would lead to a rather high diastereoselectivity in the a-addition
of a primary amine. Apart from the addition of NH3, all amines investigated gave the
two diastereoisomers in a rather low ratio (11c 1:6, 11d 3 :4, 11e 1:1, and 11f 2 :3),
similar to those described by Ermeleev et al. mentioned above. In the case of 11f, the
two epimers were separated, and their configuration was established (see below).
Attempted Condensation of the Ester Val(F6)-MeLeu-Ala-OCH2Ph 11a with the
Octapeptide 2b. With the tripeptide 11a at hand, the formation of the amide bond
between the octapeptide acid 2b and the NH2 group of Val(F6) in 11a was explored.
Treatment of 2b and 11a with N-ethyl-N’-[3-(dimethylamino)propyl]carbodiimide
(EDC), N,N-dimethylpyridin-4-amine (DMAP), and a catalytic amount of 1-hydroxy-
7-aza-1H-benzotriazole (HOAt) over extended periods of time, up to 7 days, gave only
trace amounts of the desired undecapeptide. This is in contrast with the observed
acylation of the tripeptide 11a with (Boc)2O to give the N-Boc-substituted tripeptide
11b and also with the ready N-acylation of (ꢀ)-hexafluoro-d-valine 7a with Mosherꢀs
acid chlorides, described earlier [7]. It may be argued that hexafluorovaline is about a
thousand times weaker as a base than valine [7]. Furthermore, Val(F6) might be
sterically more hindered than Val itself, being one of the more hindered amino acids. In
comparison to a Me group, the van-der-Waals radius of a CF3 group is 35% larger, and
the van-der-Waals volume is ca. 2.5 times as large [17]. Due to these considerations we
did not try to optimize the peptide-forming conditions but decided to explore an
alternative route. We surmised that an intramolecular peptide-forming reaction might
be successful, where the intermolecular peptide-bond formation had failed. As an
alternative reaction sequence, we visualized a ꢃlasso techniqueꢄ involving a-additions to
b,b-bis(trifluoromethyl)acrylamide derivatives 10a and 10b of (w-hydroxyalkyl)amines
with the functional groups being separated by two or three C-atoms. O-Acylation of the
terminal OH group of the tripeptide with MeLeu in the N-protected octapeptide 2b
followed by an intramolecular O to N migration of the acyl group should generate the
desired amide bond. Finally, ring closure would involve the intramolecular formation of
the peptide bond between the least hindered alanines AA7 and AA8 producing [5-
[hexafluoro-N-(oxyalkyl)-l-valine]]cyclosporins. Alternatively, 3-aminopropan-1-ol as
the auxiliary may be replaced by the isosteric 2-(aminooxy)ethanol leaving the option

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for a reductive cleavage of the NꢀO bond either at the secocyclosporin or cyclosporin
t
level. It should be noted that both termini of a 7,8-secocyclosporin would tolerate Bu
protecting groups, one as an ester and the other one as an N-Boc group. Both may be
removed simultaneously prior to cyclization.
More Tripeptides as tert-Butyl Esters. For reasons mentioned above, we focused our
attention on the preparation of tripeptides protected at the C-terminus (acid side) as
tert-butyl esters. Thus, addition reactions of 2-aminoethanol, 3-aminopropan-1-ol, and
2-(aminooxy)ethanol with the b,b-bis(trifluoromethyl)acrylamide moiety of the
dipeptide tert-butyl ester 10b were investigated. The hitherto unknown dipeptide 8b
was prepared from commercial l-alanine tert-butyl ester hydrochloride and N-
[(benzyloxy)carbonyl]-protected N-methyl-l-leucine [18] under standard peptide-
forming conditions. Analytical data supported the structure assigned to 8b. Removal of
the (benzyloxy)carbonyl group via hydrogenation over Pd/C gave 8c, which was treated
immediately with 9 to give the N-acylated dipeptide 10b in 98% yield. The MS of the
product showed the expected [M þ H]þ peak at m/z 463. The fragment at m/z 407 may
be due to the loss of 2-methylprop-1-ene. Both 1H- and 13C-NMR spectra are
compatible with the assigned structure.
The N-alkylated tripetides 11d and 11e were prepared from 10b via the addition of
2-aminoethanol and 3-aminopropanol, respectively. In both cases, mixtures of
diastereoisomers were isolated, from which samples of the pure isomers were secured
in both instances. The combined yield for the 2-aminoethanol addition products was
42% with an estimated ratio of 3 :4 for the two diastereoisomers 11d1 and 11d2. The less
polar minor product 11d1 was isolated as a liquid. According to its high-resolution MS,
11d1 has a molecular-ion peak at m/z 524 ([M þ H]þ). In the 1H-NMR spectrum, sharp
t
ss at d 1.45 and 2.99 were assigned to the Bu and the MeN group, respectively. Sharp
signals corresponding to these groups were detected in the 13C-NMR spectrum at d 81.9
and 30.8, respectively. Three C-atoms assigned to the HꢀC(a) groups were observed
between d 56 and 48. The CH2N and the CH2O C-atoms gave rise to signals at d 49.4
and 61.3, respectively. The more polar major isomer 11d2 was isolated as a crystalline
1
material also showing the [M þ H]þ peak at m/z 524. According to its H-NMR
spectrum, it may be concluded that 11d2 is present as a single conformer with a s at d
2.98 assigned to the MeN group. Other analytical data corresponded to those observed
for the isomer 11e1.
The addition of the homologous 3-aminopropanol to 10b resulted in the formation
of the two epimers 11e1 and 11e2 in a 1:1 ratio obtained as liquids in a combined yield of
35%. In both cases, the MS showed the presence of the [M þ H]þ peak at m/z 538. In
1
addition, both isomers were fully characterized by H- and 13C-NMR and IR spectra.
Finally, the reaction between 2-(aminooxy)ethanol [19] and 10b led to a mixture of
the two epimeric tripeptides 11f1 and 11f2 in an estimated ratio of 2 :3. These were only
partially separated by column chromatography in a combined yield of 92%. In the MS
of the two isomers, the oily 11f1 and the more polar, crystalline 11f2 showed the
expected [M þ H]þ peak at m/z 540.36 and 540.42, respectively. The less polar, liquid
tripeptide 11f1, although pure according to TLC and HPLC, seems to be present in
1
CDCl3 solution in more than one conformation as determined by H-NMR spectro-
scopy. The signals at d 3.02 and 2.79 were assigned to the MeN group of this tripeptide
and appeared in a ratio of 2 :1. When the spectrum of 11f1 was measured in CD3OD

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the signals were observed at d 3.05 and 2.85 in a ratio of 10 :1. In the 13C-NMR
spectrum of the tripeptide 11f1, a t detected at d 61.0 was assigned to the CH2OH C-
atom based on a comparison with the spectra of the pure isomers of 11d1, 11d2, 11e1, and
1
11e2. The solid tripeptide 11f2 showed a first-order H-NMR spectrum in CDCl3
indicating the presence of a single conformation on the NMR time scale. Interestingly,
1
other esters of the tripeptides 11a – 11e described above all show H-NMR spectra in
CDCl3 compatible with the presence of a single conformer for each tripeptide. The
13C-NMR signal observed at d 60.4 for the isomer 11f2 was assigned to the CH2OH C-
atom.
Most differences between the 13C-NMR chemical shifts of the corresponding pairs
of C-atoms for the diastereoisomers 11d1/11d2, 11e1/11e2, and 11f1/11f2 are small. Yet
significant shift differences of 0.55, 0.50, and 0.45 ppm, respectively, were observed for
the signals of the CH2 groups of the N-methyl-l-leucine moiety (MeLeu). In all three
cases, the CH2 group associated with the less polar compound, i.e., 11d1, 11e1, and 11f1,
appeared at higher field. Based on these observed shift differences, the less polar
isomers 11d1 and 11e1 were assigned the all-(S)-configuration. In retrospect, the
observed chemical shift for the corresponding CH2 at d 36.5 for 11a, obtained via the
addition of NH3 to 10a, might suggest the (R)-configuration for the newly generated
chiral center in 11a. On the other hand, the observed chemical shift for the
corresponding CH2 groups at d 35.93 for 11c2, obtained via the addition of NH2OH
to 10a, would suggest the (S)-configuration for the newly generated chiral center in
11c2.
X-Ray Analysis of Tripeptide 11f2. As mentioned above, the more polar epimeric
tripeptide 11f2 was obtained as a crystalline product. A suitable crystal was grown from
Et2O/hexane and was subjected to a single-crystal X-ray-analysis [20]. The config-
uration was determined to be (R,S,S) for tripeptide 11f2. Surprisingly, and contrary to
observations in solution (see NMR spectra), this diastereoisomer exists as a mixture of
two different conformations in the solid state. As a consequence of the X-ray structure
determination, the liquid, less polar tripeptide 11g1 was assigned the (S,S,S)
configuration.
Undecapeptides (Secocyclosporins). The depsipeptide 12a was prepared from the
N-Boc-protected octapeptide 2b and the (R,S,S)-tripeptide 11f2 (Scheme 4). This
reaction was allowed to run for 10 – 14 days at room temperature under conventional
peptide-forming conditions. The extended reaction times were due to the fact that the
tripeptide 11f2 and the condensation product 12a could not be distinguished on TLC
(Scheme 4). Thus, the presence of unreacted tripeptide had to be avoided. Chroma-
tography of the crude product mixture gave a compound whose ESI-MS showed the
expected [M þ H]þ peak at m/z 1545.21 for the condensation product 12a. An
interesting fragment peak was observed at m/z 351.25. Such fragments were
conspicuously absent in either of the MS of 11f1 and 11f2. In the 13C-NMR spectrum
of 12a, the C-atoms of the hydroxyethoxy group were detected at d 72.2 and 76.0, while
the corresponding signals for the tripeptides 11f1 and 11f2 appeared at d 61.0 and 75.0,
and d 60.5 and 75.4 ppm, respectively.
The doubly protected depsipeptide 12a was heated to reflux in toluene during 60 h
in which most of the starting material gave rise to a new, more polar product. According
to spectral evidence, the depsipeptide had thermally rearranged to the protected

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Scheme 4. Condensation Reactions of 11f1 and 11f2 with 2b and Formation of the Rearranged Products
13a and 13b
undecapeptide 13a, a 7,8-secocyclosporin encompassing the modified amino acid N-2-
hydroxyethoxy)-4,4,4,4’,4’,4’-l-hexafluoro-d-valine as AA5. The ESI-MS of this novel
undecapeptide 13a (calc. mass 1543.93) is distinguishable from the depsipeptide 12a by
its fragmentation pattern. The new product 13a shows a peak at m/z 1544.74 for [M þ
H]þ. A peak found at m/z 1527.11 is most likely due to the loss of H2O from AA1. A
peak at m/z 1485.12 may be explained with the loss of OCH2CH2O from the molecular
ion. Such a fragmentation is possible only for the peptide 13a and not for the
depsipeptide 12a. It was especially gratifying to observe this fragment on the way to [d-
Val(F6)5]cyclosporin, raising hope for a reductive cleavage of the incorporated
auxiliary 2-(aminooxy)ethanol. Each of the three aforementioned peaks in the ESI-MS

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of 13a showed three satellite peaks of [M þ 22]þ. This is due to to the replacement of a
proton by Naþ. The absence of a peak at m/z 562 in the MS of 13a further supports the
notion of a fragment at m/z 539 and not the presence of an impurity in either 12a or 13a.
In the 13C-NMR spectrum of 13a, the signals assigned to the C-atoms of the 2-
hydroxyethoxy group were shifted upfield and were observed at d 65.0 and 62.7,
respectively.
The condensation between the N-Boc-octapeptide 2b and the liquid diastereoiso-
meric (S,S,S)-tripeptide 11f1 was carried out under similar conditions as described for
the reaction of 11f2 and 2b. When a toluene solution of a sample, assumed to be the
depsipeptide 12b, was heated to reflux, no change was observed on TLC leading to the
conclusion that the condensation product was the undecapeptide 13b. The ESI-MS of
13b showed the [M þ H]þ peak at m/z 1545.09 and a fragment peak at m/z 1527.08 due
to the loss of H2O, most likely from AA1. Both aforementioned peaks in the ESI-MS of
13b showed the satellite peaks of [M þ 22]þ corresponding to the Naþ adducts. In the
13C-NMR spectrum of 13b, the signals assigned to the C-atoms of the 2-(hydroxy-
ethoxy) group were observed at d 72.2 and 62.8, respectively. This led to the conclusion
that the rearrangement to 13b had occurred under the conditions of the condensation
of the tripeptide 11f1 with the octapeptide 2b.
Rearrangements. The difference between the depsipeptides 12a and 12b in their
behavior under thermal conditions deserves some comment. Compound 12a had to be
heated to reflux in toluene for several days to achieve the conversion to the more polar
peptide 13a. The corresponding rearrangement from the depsipeptide 12b to the
undecapeptide 13b apparently took place at room temperature. This may be explained
considering the transition states A and B (Fig. 2) leading to the diastereoisomeric 3-
oxy-tetrahydro-1,4,2-dioxazines, assumed intermediates for the O-to-N shift of the acyl
groups. The two negatively polarized groups, the developing O-anion and the
electronegative (CF3)2CH group, achieve maximal separation if the two groups can
be placed on opposite sides of the tetrahydro-1,4,2-dioxazine ring and assuming an
antiperiplanar conformation in the developing six-membered heterocyclic ring. For the
case of the O-to-N acyl shift involving the hexafluoro-l-valine moiety (see A), fewer
1,3-interactions are found than in the case of the hexafluoro-d-valine moiety (see B),
making the transition state in the former case more favorable.
5-[Hexafluoro-N-(hydroxyethoxy)valine]]cyclosporins. The linear undecapeptide
13a was deprotected concomitantly at both termini by treatment with CF3COOH and
Fig. 2. Structural models A and B for the transition states of the rearrangements 13a ! 14a and 13b ! 14b,
respectively

Helvetica Chimica Acta – Vol. 93 (2010)
1593
subjected to ring closure under standard peptide-forming conditions but at high
dilution. Following column chromatography (silica gel), the cyclic compound 14a was
obtained (Scheme 5). Its ESI-MS showed the expected peaks for [M þ H]þ at m/z
1370.85 and for [M þ Na]þ at m/z 1392.97. In the 13C-NMR spectrum of 14a, the signals
assigned to the C-atoms of the 2-hydroxyethoxy substituent of AA5 were observed at d
71.2 and 62.7, respectively.
Scheme 5. Formation of the Derivatives of Cyclosporine 14a and 14b from 13a and 13b
Likewise, deprotection of the termini (N-Boc and tert-butyl ester) of 13b with
CF3COOH followed by the treatment with EDC gave the hexafluoro derivative 14b of
cyclosporin A. The ESI-MS showed the expected peak for [M þ H]þ at m/z 1370.81 and
a peak at m/z 1374.86 ([M þ Na ꢀ H2O]þ). In the 13C-NMR spectrum of 14b, the
signals assigned to the C-atoms of the 2-hydroxyethoxy substituent of AA5 were
observed at d 71.8 and 62.5, respectively.
1
The H-NMR spectra of the novel cyclosporins 14a and 14b did not show the
resolution usually associated with cyclosporins. This may be due to the fact that the
amide H-atom of the hexafluorovaline residue (AA5) is replaced by a side chain (2-
hydroxyethoxy) precluding the formation of a H-bridge to the C¼O group of AA2
(Fig. 1).
Concluding Remarks. – The 4,4,4,4’,4’,4’-hexafluoro-N-(2-hydroxyethoxy)-d- or -l-
valine was incorporated stereoselectively into cyclosporin A replacing the l-valine-
residue (AA5) of the natural product 1a. This was accomplished via the preparation of
the tripeptides HOCH2CH2O-Val(F6)-MeLeu-Ala-OtBu (11f1, (S,S,S) and HOCH2-
CH2O-d-Val(F6)-MeLeu-Ala-OtBu 11f2, (R,S,S)) as building blocks. These epimeric
compounds were prepared via the addition of 2-(aminooxy)ethanol to the acrylamide
10b. The diastereoisomers 11f1 and 11f2 were condensed with the octapeptide 2b. The
depsipeptide 12a was obtained from 11f2 ((R,S,S)) and rearranged at 1108 to the
undecapeptide 13a. The condensation of octapeptide 2b with the tripeptide 11f1

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Helvetica Chimica Acta – Vol. 93 (2010)
((S,S,S)) produced the undecapeptide 13b at room temperature. The peptides 13a and
13b were cyclized to the derivatives 14a and 14b of cyclosporin A, respectively.
The route via an intramolecular condensation was necessitated following our
observation that an intermolecular condensation of 2b with the secondary-amino
group of the tripeptide 11a did not produce the undecapeptide in reasonable yield. On
the other hand, intramolecular amide-bond formations were successful employing a
lasso technique via the depsipeptides 12a and 12b to produce the substituted
secocyclosporins 13a and 13b, respectively. Successful intramolecular transformations
where corresponding intermolecular reactions had failed, have been described
previously in the literature [21]. As an example of the ꢃlasso techniqueꢄ thioamides
were used in the total synthesis of vitamin B12 to form enamines with the extrusion of
the S-atom [22].
This work was financially supported by the Swiss National Science Foundation (Project No. 20-
43565.95). We thank Dr. L. Tenud (Lonza Ltd., Visp, Switzerland) for a generous gift of fluorinated
chemicals. We are grateful to Prof. F. Scholz, University of Greifswald, Germany, for the measurement of
the the lipophilicity of 4,4,4,4’,4’,4’-hexafluoro-l-valine.
Experimental Part
General. All reactions were performed under Ar or N2. Chemicals were purchased from commercial
suppliers and used without further purification. N-Ethyl-N’-[3-(dimethylamino)propyl]carbodiimide
(EDC), N,N-dimethylpyridin-4-amine (DMAP), 1-hydroxy-1H-benzotriazole (HOBt), 1-hydroxy-1H-
7-azabenzotriazole (HOAt), di(tert-butyl) dicarbonate ((Boc)2O). Commercial solvents (except quality
grade) were distilled prior to their use. TLC: silica-gel plates SIL G/UV254 (SiO2; Macherey & Nagel).
Column chromatography (CC): SiO2 columns. NMR Spectra: Bruker AC-300 (300 (1H) and 75 MHz
(13C)) and Bruker DRX-400 (376 (19F)); in CDCl3; d in ppm rel. to Me4Si (d ¼ 0) for 1H to CDCl3 (d ¼
77.0) for 13C, and to CCl3F (external probe, d ¼ 0.0) for 19F-NMR, J in Hz; assignments tentative. EI- and
ESI-MS (pos. mode): Micromass-Autospec and Micromass-Platform spectrometer, resp.; in m/z (rel. %).
N-[(tert-Butoxy)carbonyl]-d-alanyl-N-methyl-l-leucyl-N-methyl-l-leucyl-N-methyl-l-valyl-(2S,3R,
4R,6E)-3-hydroxy-4-methyl-2-(methylamino)oct-6-enoyl-(2S)-2-aminobutanoyl-N-methylglycyl-N-
methyl-l-leucine (2b). The side-chain O-acetyl-substituted N-Boc-octapeptide-SCH2Ph (prepared from
1b) [9] (6.0 g, 5.13 mmol) in DMF (60 ml) was stirred at r.t. overnight in the presence of 2n NaOH
(40 ml, 80 mmol). Then, the mixture was neutralized with AcOH and concentrated under aspirator
vacuum at 60 – 708. The residue was dissolved in Et2O and washed with 1n NaOH (5 ꢁ 50 ml) and then
with brine (2ꢁ). This was repeated four times. The combined org. phase was dried (MgSO4) and
concentrated: 2b (2.3 g, 44%). ESI-MS: 1044.88 (35, [M þ Na]þ), 1026.23 (48, [M þ Na ꢀ H2O]þ),
1022.75 (34, Mþ), 1004.75 (38, [M ꢀ H2O]þ).
Benzyl (2S)-4,4,4-Trifluoro-2-{[(1R)-1-phenylethyl]amino}-3-(trifluoromethyl)butanoate 4-Methyl-
benzenesulfonate (¼4,4,4,4’,4’,4’-Hexafluoro-N-[(1R)-1-phenylethyl]-l-valine Phenylmethyl Ester 4-
Methylbenzenesulfonate (1:1); 6b). A soln. of benzyl 4,4,4-trifluoro-3-(trifluoromethyl)but-2-enoate
(4; 17.0 g, 57 mmol) in MeOH (50 ml) was cooled in an ice bath and treated with commercial (þ)-(1R)-1-
phenylethylamine (5); (6.9 g, 57 mmol) and kept at r.t. overnight. Then, TsOH · H2O (10.8 g, 57 mmol)
was added. The soln. was concentrated to ca. 10 ml, and Et2O was added to precipitate a total of 13.5 g
(22.8 mmol) of 6a ((R,R)). The Et2O soln. containing 6b ((S,R)) was treated with sat. NaHCO3 soln.,
dried (Na2SO4), and evaporated: 13.1 g of 6b as an oil.
Benzyl (2S)-4,4,4-Trifluoro-2-{[(1R)-1-phenylethyl]amino}-3-(trifluoromethyl)butanoate Hydro-
chloride (¼4,4,4,4’,4’,4’-Hexafluoro-N-[(1R)-1-phenylethyl]-l-valine Phenylmethyl Ester Hydrochloride
(1:1); 6d). A soln. of 6b (13.1 g, 30 mmol) in Et2O was treated with an ice-cold soln. of HCl in Et2O
(100 ml) and the mixture concentrated. Fresh Et2O (10 ml) was added and the HCl salt 6d crystallized.
After 2 h, the solid was filtered off to give 7.7 g (59%) of 6d, containing 3% of 6c (NMR).

Helvetica Chimica Acta – Vol. 93 (2010)
1595
Crystallizations from CH2Cl2/Et2O and CH2Cl2/hexane gave a total of 3.98 g (30.9%) of pure 6d. M.p.
1
133 – 1358. [a]D ¼ þ13.20 (c ¼ 1.09, CHCl3). H-NMR: 1.98 (d, J ¼ 7, Me); 4.22 (s, 1 H); 4.50 – 4.65 (m,
1 H); 4.80 – 5.0 (dd, CH2); 5.45 – 5.65 (m, 1 H); 7.15 – 7.25 (m, 2 H); 7.30 – 7.47 (m, 6 H); 7.53 – 7.7 (m,
2 H); 8.0 – 9.5 (br., 1 H). 13C-NMR: 164.9 (s); 134.7 (s); 133.0 (s); 130.0 (d); 129.3 (d); 129.1 (d); 129.0
(d); 128.9 (d); 128.6 (d); 69.7 (t); 62.4 (d); 54.5 (d); 48.2 (m, J ¼ 29.0 Hz); 19.37 (q). 19F-NMR: ꢀ 62.93;
ꢀ 64.06. EI-MS: 419 (1), 418 (2), 404 (8), 328 (9), 284 (31), 268 (5), 120 (45), 105 (100), 91 (67), 77 (25).
HR-MS: 419.13165 (Mþ, C20H19F6NOþ2 ; calc. 419.131999).
(þ)-4,4,4,4’,4’,4’-Hexafluoro-l-valine Hydrochloride (7b · HCl). A soln. of 6d (4.0 g, 8.8 mmol) in
MeOH (50 ml) was hydrogenated overnight in the presence of 5% Pd/C (1.5 g). The mixture was filtered
through Celite and concentrated, and Et2O was added (10 ml). The product 7b · HCl solidified and was
filtered to give 0.67 g of crystalline material. The residue of the filtrate was dissolved in H2O and
evaporated. This procedure was repeated and yielded an additional 1.38 g of 7b · HCl: total yield 2.05 g
(94%). M.p. 180 – 1828. [a]D ¼ þ11.4 (c ¼ 1.67, H2O). 1H-NMR (CD3OD): 4.22 (br. s, 1 H); 4.63
(m, 1 H). ESI-MS: 225 (0.2), 208 (0.1), 80 (100), 160 (8), 140 (6), 113 (18), 112 (10), 111 (5), 69
(30).
The N-acylation of 7b · HCl with (ꢀ)-(S)-Mosherꢄs acid chloride was performed as described for the
acylation of 7a · HCl with (þ)-(R)-Mosherꢄs acid chloride [8].
[(Benzyloxy)carbonyl]-N-methyl-l-leucine-l-alanine tert-Butyl Ester (8b). A soln. of commercial l-
alanine tert-butyl ester hydrochloride (4.1 g, 22.7 mmol), EDC (5.0 g, 26.2 mmol), Z-MeLeu [14] (6.3 g,
22.6 mmol), and Et3N (10 ml, 100 mmol) in CH2Cl2 (200 ml) was kept at r.t. for 2 h. The soln. was
concentrated, and AcOEt (200 ml) was added. The mixture was washed with 0.5n HCl soln. (100 ml) and
sat. Na2CO3 soln., dried (MgSO4), and concentrated to give the crude product (9.58 g). This was filtered
through SiO2 with AcOEt/hexane 1:4 to give 7.56 g of pure 8b (82.4%). TLC (AcOEt/hexane 1:3):
single spot, Rf 0.62. [a]D ¼ ꢀ50.6 (c ¼ 1.45, CHCl3). IR (film): 3332 (NH), 1736 (ester C¼O), 1670
(amide C¼O, urethane). 1H-NMR: 0.75 – 0.95 (m, 2 Me); 1.26 (d, J ¼ 7, Me); 1.42 (s, tBu); 1.40 – 1.50 (br.
m, CH); 1.62 – 1.70 (m, CH2); 2.81 (s, MeN); 4.25 – 4.41 (m, 1 HꢀC(a)); 4.55 – 4.80 (m, 1 HꢀC(a)); 5.16
(s, CH2O); 6.20 – 6.55 (br., NH); 7.25 – 7.41 (m, Ph). 13C-NMR: 171.5, 170.2, 156.9 (3 C¼O); 136.3 (arom.
C); 128.2, 127.7, 127.4 (5 arom. CH); 81.4 (Me3C); 67.2 (CH2O); 56.5 (CH(a)); 48.3 (MeN); 36.4 (CH2);
29.4 (CH(a)); 27.6 (Me3C); 24.4 (CH); 22.9, 21.5, 17.9 (3 Me). EI-MS: 406 (0.2, Mþ), 350 (0.6, [M ꢀ 56]þ,
loss of CH2¼CMe2), 333 (0.21, [M ꢀ 73)]þ, loss of C4H9O), 262 (0.25, [M ꢀ 144]þ, loss of Ala-OtBu), 234
(26, [M ꢀ 172]þ, loss of O¼C-Ala-OtBu), 190 (49, [234 – 44], loss of CO2), 91 (100, C6H5CHþ2 ). HR-MS:
407.2541 ([M þ H]þ, C22H35N2Oþ5 ; calc. 407.2546).
N-Methyl-l-leucine-(S)-alanine tert-Butyl Ester (8c). A soln. of 8b (7.3 g, 18 mmol) in MeOH
(150 ml) was hydrogenated under normal pressure in the presence of 10% Pd/C (1 g) until the H2 uptake
stopped (ca. 325 ml, 15 mmol). The soln. was filtered through Celite and concentrated to give crude 8c
(4.10 g, 84%) which was used immediately for the next step. TLC (AcOEt/hexane 1:3): no starting 8b
left.
N-Methyl-N-[4,4,4-trifluoro-1-oxo-3-(trifluoromethyl)but-2-en-1-yl]-l-leucine-l-alanine Benzyl Es-
ter (10a). A soln. of the dipeptide 8a [9] (300 mg, 1 mmol) and Et3N (2 g, 20 mmol) in CH2Cl2 (20 ml) was
treated with 4,4,4-trifluoro-3-(trifluoromethyl)but-2-enoyl chloride (9; 450 mg, 2 mmol) [3a]. After
90 min at r.t., Et2O (100 ml) was added, the org. phase washed with H2O, dried (Na2SO4), and
concentrated, and the residue subjected to CC (SiO2, AcOEt/hexane 1:3): 10a (480 mg, 96%). [a]D ¼
ꢀ52.8 (c ¼ 2.38, CHCl3). 1H-NMR: 0.92 (d, J ¼ 6.5, Me); 0.96 (d, J ¼ 6.5, Me); 1.45 (d, J ¼ 7.5, Me);
1.25 – 1.55 (m, CH, CH2); 2.90 (s, MeN); 4.50 – 4.65 (m, 1 HꢀC(a)); 5.05 – 5.10 (m, 1 HꢀC(a)); 5.11 –
5.21 (dd, CH2O); 6.40 – 6.52 (d, NH); 7.12 (s, C¼CH); 7.30 – 7.45 (m, Ph). MS: 496 (1, Mþ), 477 (1.5,
[M ꢀ F]þ), 440 (5, [M ꢀ 56]þ, loss of C4H8), 420 (5, [440 ꢀ HF]þ), 405 (1, [M ꢀ benzyl]þ, 318 (5, [M ꢀ
Ala-OCH2Ph]þ), 290 (11, [318 ꢀ CO]þ), 178 ([Ala-OCH2Ph]þ), 91 (32, C7H7þ ), 58 (100, C4H1þ0 ).
N-Methyl-N-[4,4,4-trifluoro-1-oxo-3-(trifluoromethyl)but-2-en-1-yl]-l-leucine-l-alanine tert-Butyl
Ester (10b). A soln. of 8c (5.5 g, 20 mmol) and Et3N (6 ml, 60 mmol) in CH2Cl2 (100 ml) was cooled
in an ice/salt bath. A soln. of 4,4,4-trifluoro-3-(trifluoromethyl)but-2-enoyl chloride [3a] (9; 6.0 g,
26.5 mmol) in CH2Cl2 (60 ml) was added dropwise. After the addition was complete, the mixture was
stirred at r.t. for 1 h, then a sat. K2CO3 soln. was added and the mixture extracted with CH2Cl2 and dried
(MgSO4). The solvent was evaporated and the crude product (11.5 g) filtered through SiO2 first with

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Helvetica Chimica Acta – Vol. 93 (2010)
AcOEt/hexane 1:9 then with AcOEt/hexane 1:4: pure 10b (9.13 g, 98%). Amber waxy substance. TLC
(AcOEt/hexane 1:2): single spot, Rf 0.5. [a]D ¼ ꢀ83.0 (c ¼ 1.675, CHCl3). IR (film): 3319 (NH), 1731
(ester C¼O), 1632 – 1681 (amide C¼O). 1H-NMR: 0.87 (d, J ¼ 6.7, 3 H); 0.91 (d, J ¼ 6.6, 3 H); 1.28 (d,
t
J ¼ 7.0, 3 Me); 1.41 (s, Bu); 1.35 – 1.50 (m, CH); 1.61 – 1.70 (m, CH2); 2.86 (s, MeN); 4.28 – 4.35 (m,
1 HꢀC(a)); 5.10 – 5.30 (m, 1 HꢀC(a)); 6.50 (d, J ¼ 7.35, NH); 7.08 (s, ¼CH). 13C-NMR: 171.5, 168.5,
163.3 (3 C¼O); 135.9 (¼CH); 120.2 (q, J ¼ 274, CF3CH); 119.9 (q, J ¼ 274, CF3CH); 81.9 (Me3C); 53.9
(CH(a)); 48.7 (MeN); 36.1 (CH2); 31.2 (CH(a)); 27.8 (Me3C); 24.6 (CH); 22.9 (Me); 21.8 (Me); 18.0
(Me). 19F-NMR: ꢀ 62.20; ꢀ 64.23. EI-MS: 463.2 (20, [M þ H]þ), 407.1 (60, [M þ H ꢀ 56]þ, loss of
CH2¼CMe2), 318.0 (90, [M ꢀ Ala-OtBu]þ), 290.0 (100, [318 ꢀ F þ H]þ), 190.9 (6, C5HF6Oþ), 162.9 (4,
C4HF6þ ), 144 (5, [M ꢀ 318]þ). HR-MS: 463.2041 ([M þ H]þ, C19H29F6N2Oþ4 ; calc. 463.2032).
4,4,4,4’,4’,4’-Hexafluorovalyl-N-methyl-l-leucyl-l-alanine Benzyl Ester (11a). A soln. of 10a (1 g,
2 mmol) in EtOH (10 ml) was added to liq. NH3 (20 ml). The mixture was allowed to warm to r.t. The
solvent was evaporated, and the crude product was subjected to CC (SiO2, AcOEt/hexane 1:3, then
AcOEt/hexane 1:2 (!10a (374 mg)), then AcOEt/hexane 1:1): 11a (243 mg, 37% based on trans-
formed 10b) as a single diastereoisomer (tentatively (R,S,S)). 1H-NMR: 0.85 (d, J ¼ 7, 3 H); 0.92 (d, J ¼ 7,
3 H); 0.95 – 1.00 (m, CH); 1.38 (d, J ¼ 8, Me); 1.65 – 1.90 (m, NH2, CH2); 3.05 (s, MeN); 3.85 – 4.02 (m,
CF3CH); 4.15 – 4.22 (d, 1 HꢀC(a)); 4.50 – 4.65 (m, 1 HꢀC(a)); 5.10 – 5.15 (d, 1 HꢀC(a)); 5.16 – 5.20
(dd, CH2O); 6.55 – 6.68 (d, NH); 7.31 – 7.42 (m, Ph). 13C-NMR: 172.7, 172.0, 170.5 (3 C¼O); 129.0 (CH);
121.3 (CH); 67.7 (CH2O); 55.5, 49.4, 48.7 (3 CH(a)); 36.5 (CH2); 30.9 (MeN); 25.2 (CH); 23.7, 21.8, 18.7
(3 Me). EI-MS: 513.3 (0.5, Mþ), 457.2 (1.5, [M ꢀ CH2¼CMe2]þ), 440 (0.5, [457 ꢀ NH3]þ), 335.2 (5.5,
[M ꢀ Bn ꢀ Ala]þ), 307.1 (60, [335 ꢀ C¼O]þ), 278.1, [335 ꢀ C4H9]þ), 249.1 (42, C7H7F6N2Oþ), 180.0
([249 ꢀ CF3]þ), 100.1 (100, C4H8N2Oþ), 91 (50, C7Hþ7 ).
N-[(tert-Butyl)carbonyl]-4,4,4,4’,4’,4’-hexafluorovalyl-N-methyl-l-leucyl-l-alanine Benzyl Ester
(11b). As described for 11a, with 10a (340 mg, 0.68 mmol) in THF (10 ml). The solvent was evaporated.
Fresh THF (30 ml) was added together with (Boc)2O (1.0 g, 4.6 mmol). The mixture was kept at r.t.
overnight. The solvent was evaporated and the residue subjected to CC (SiO2, AcOEt/hexane 1:4): 11b
(100 mg, 24%; tentatively (R,S,S)). Crystals. 1H-NMR: 0.86 (d, J ¼ 6, 3 H); 0.94 (d, J ¼ 6, 3 H); 1.37 (d,
J ¼ 7, Me); 1.41 (s, tBu); 1.29 – 1.50 (m, 1 H); 1.60 – 1.78 (m, 2 H); 3.30 (s, MeN); 3.81 – 3.98 (m, CF3CH);
4.49 – 4.62 (m, 1 HꢀC(a)); 5.02 – 5.11 (m, 1 HꢀC(a)); 5.15 – 5.20 (dd, CH2O); 5.25 – 5.35 (m,
1 HꢀC(a)); 6.48 – 6.58 m, NH); 7.30 – 7.40 (m, Ph). EI-MS: 614.2 (15, [M þ H]þ), 558.1 (14, [614 ꢀ
56]þ), 435.1 (18, [M ꢀ Bn ꢀ Ala]þ), 379.0 (100, [435 ꢀ 56]þ), 335.0 (10, [379 ꢀ CO2]þ), 307.1 (11, [N ꢀ
Boc ꢀ Val(F6) ꢀ ketene]þ).
4,4,4,4’,4’,4’-Hexafluoro-N-hydroxyvalyl-N-methyl-l-leucyl-l-alanine Benzyl Ester (11c1/11c2). A
soln. of 10a (270 mg, 0.54 mmol) in CH2Cl2 (5 ml) was mixed with a soln. of NH2OH in EtOH (1 ml,
prepared from NH2OH · H2O (1 g) and AcONa (1 g)) and was kept at r.t. for 2 h. The mixture was diluted
with AcOEt, washed with H2O, dried (Na2SO4), and concentrated. The residue (300 mg) was subjected
to CC (SiO2, AcOEt/hexane 1:2): less polar, pure 11c1 (20 mg) as an oil and pure 11c2 (120 mg) as
crystals.
Diasteroisomer 11c1 (tentatively (R,S,S)): TLC: Rf 0.35 (AcOEt/hexane 1:2). 1H-NMR: 0.90 (d, J ¼
6, 3 H); 0.94 (d, J ¼ 6, 3 H); 1.34 (d, J ¼ 7, 3 H); 1.60 – 1.80 (m, CH, CH2); 3.06 (s, MeN); 3.65 – 3.80 (m,
CF3CH); 4.14 – 4.25 (m, NH); 4.38 – 4.8 (m, 2 HꢀC(a)); 5.05 – 5.21 (m, 1 HꢀC(a)); 5.14 – 5.20 (dd,
CH2O); 5.65 – 5.80 (br., OH); 6.45 – 6.60 (d, NH); 7.30 – 7.40 (m, Ph). EI-MS: 530.1 (22, [M þ H]þ), 351.1
(100, [M ꢀ Ala-OCH2Ph]þ), 323.1 (19, [351 ꢀ CO]þ).
Diastereoisomer 11c2 (tentatively (S,S,S)): TLC: Rf 0.24 (AcOEt/hexane 1:2). M.p. 98 – 1028.
1
[a]D ¼ ꢀ57.8 (c ¼ 1.45, CHCl3). H-NMR: 0.85 – 0.92 (d, 3 H); 0.94 – 0.99 (d, 3 H); 1.30 – 1.35 (d, Me);
1.18 – 1.45 (m, CH); 1.60 – 1.75 (m, 1 H); 1.78 – 1.95 (m, CH2); 3.0 (s, MeN); 3.70 – 3.85 (m, CF3CH);
4.45 – 4.60 (m, 2 HꢀC(a)); 5.15 (s, CH2O); 5.30 – 5.40 (m, 1 HꢀC(a)); 5.81 – 5.91 (d, 1 H); 6.30 – 6.42 (m,
1 H, NHOH); 6.90 – 7.05 (d, CONH); 7.20 – 7.40 (m, Ph). 13C-NMR: 17.8, 21.0, 23.3 (3 Me); 24.4 (CH);
30.6 (MeN); 35.93 (CH2); 48.0 (m, CF3CH); 48.4 (CH(a)); 55.7 (CH(a)); 56.2 (CH(a)); 67.2 (CH2);
122.7 (q, J ¼ 280, CF3); 128.1 (CH); 128.4 (CH); 128.6 (CH); 135.3 (s); 169.7, 171.6, 172.5 (3 C¼O). 19F-
NMR (CDCl3): ꢀ 0.013; ꢀ 0.091. EI-MS: 530.2 (14, [M þ H]þ), 393.1 (5, [M ꢀ BnOCOH]þ), 351.1 (100,
[M ꢀ Bn ꢀ Ala]þ), 323.1 (32, [351 ꢀ CO]þ), 305.1 (13, [MeLeu ꢀ Val( F 6)Bn]þ), 196 (11,
(CF3)2CHNHOHþ).

Helvetica Chimica Acta – Vol. 93 (2010)
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4,4,4,4’,4’,4’-Hexafluoro-N-(2-hydroxyethyl)-l- and 4,4,4,4’,4’.4’-Hexafluoro-N-(2-hydroxyethyl)-d-
valyl-N-methyl-l-leucine-l-alanine tert-Butyl Ester (11d1 and 11d2, resp.). To a soln. of 10b (924 mg,
2 mmol) in EtOH (50 ml) was added 2-aminoethanol (490 mg, 8 mmol) and kept at r.t. overnight. The
solvent was evaporated, the residue dissolved in AcOEt, the soln. washed with H2O dried (Na2SO4), and
concentrated, and the residue (920 mg) separated by CC (SiO2, hexane/AcOEt 2 :1): less polar 11d1
((S,S,S); 120 mg) as an oil, a mixture of both isomers (150 mg), and more polar 11d2 ((R,S,S); 160 mg) as
crystals. Combined yield 42%.
(S,S,S)-Isomer 11d1: TLC (AcOEt/hexane 1:1): single spot, Rf 0.36. [a]D ¼ ꢀ72.1 (c ¼ 1.56, CHCl3).
IR (film): 3460, 3342, 1735, 1625 – 1675. 1H-NMR: 0.86 (d, J ¼ 6.62, 3 H); 0.92 (d, J ¼ 6.62, 3 H); 1.24 (d,
J ¼ 7.35, Me); 1.41 (s, tBu); 1.33 – 1.46 (m, CH); 1.58 – 1.76 (m, CH2); 2.49 – 2.59 (m, 1 H of CH2N); 2.75 –
2.85 (m, 1 H of CH2N); 1.90 – 2.60 (br., NH, OH); 2.95 (s, MeN); 3.55 – 3.67 (m, CH2O, CF 3CH); 4.05 (d,
J ¼ 8.45, 1 HꢀC(a)); 4.25 – 4.38 (m, 1 HꢀC(a)); 4.99 – 5.08 (m, 1 HꢀC(a)); 6.53 (d, J ¼ 7.35, O¼CNH).
13C-NMR: 171.7, 171.7, 168.9 (3 C¼O); 123.0 (q, J ¼ 281, CF3); 81.9 (Me3C); 61.4 (CH2O); 55.6 (CH(a));
54.0 (CH(a)); 50.2 (m, J ¼ 26.2, CF3CH); 49.5 (CH2N); 48.6 (CH(a)); 36.1 (CH2); 30.9 (MeN); 27.9
(Me3C); 24.9 (CH); 23.1 (Me); 21.8 (Me); 17.9 (Me). 19F-NMR (CDCl3): ꢀ 61.74; ꢀ 64.74. EI-MS: 524
(100, [M þ H]þ), 468 (30, [M ꢀ 56]þ, loss of CH2¼CMe2), 379 (28, [M ꢀ 144]þ, loss of Ala-OtBu), 351 (1,
[M ꢀ 172]þ, loss of O¼C-Ala-OtBu), 243 (1, [M ꢀ 280]þ, loss of HOCH2CH2-Val(F6)-NHMe, 224 (53,
(CF3)2CHCHNHCH2CH2OH]þ), 206 (12, [224 ꢀ F þ H]þ), 144 (4, [Ala-OtBu]þ), 128 (3,
[CH2¼CHCOOtBu]þ). HR-MS: 524.256160 ([M þ H]þ, C21H36F6N3Oþ5 ; calc. 524.255916).
(R,S,S)-Isomer 11d2: M.p. 86 – 878. TLC (AcOEt/hexane 1:1): single spot, Rf 0.27. [a]D ¼ ꢀ52.5 (c ¼
1
1.57, CHCl3). IR (film): 3450, 3332, 1734, 1630 – 1685. H-NMR: 0.82 (d, J ¼ 6.3, 3 H); 0.89 (d, J ¼ 6.6,
t
3 H); 1.28 (d, J ¼ 7.3, Me); 1.40 (s, Bu); 1.31 – 1.48 (m, CH); 1.61 – 1.69 (m, CH2); 2.00 – 2.60 (br., NH,
OH); 2.66 – 2.83 (m, CH2N); 2.95 (s, MeN); 3.40 – 3.65 (m, CF3CH, CH2O); 4.15 (d, J ¼ 6.6, 1 HꢀC(a));
4.36 – 4.40 (m, 1 HꢀC(a)); 5.10 – 5.20 (m, 1 HꢀC(a)); 6.66 (d, J ¼ 7.36, NH). 13C-NMR: 172.3, 171.8,
169.8 (3 C¼O); 123.1 (q, J ¼ 280, CF3); 82.5 (Me3C); 61.9 (CH2O); 55.3 (CH(a)); 54.2 (CH(a)); 50.1 (m,
J ¼ 26.2, CF3CH); 49.9 (CH2N); 48.8 (CH(a)); 36.6 (CH2); 30.5 (MeN); 27.9 (Me3C); 24.7 (CH); 23.2
(Me); 21.3 (Me); 18.4 (Me). 19F-NMR (CDCl3): ꢀ 61.88; ꢀ 64.91. EI-MS: 524 (100, [M þ H]þ), 468 (35,
[M ꢀ 56]þ, loss of CH2¼CMe2), 379 (33, [M ꢀ 144]þ, loss of Ala-OtBu), 351 (4, [M ꢀ 172]þ, loss of O¼C-
Ala-OtBu), 243 (4, [M ꢀ 280]þ, loss of HOCH2CH-Val(F6)-NHMe); 224 (51, (CF3)2CHCHNH-
CH2CH2OHþ), 206 (14, [224 ꢀ F þ H]þ), 144 (7, [Ala-OtBu]þ), 128 (38, CH2¼CHCOOtBuþ). HR-MS:
524.2543 ([M þ H]þ, C21H36F6N3Oþ5 ; calc. 524.2559).
4,4,4,4’,4’,4’-Hexafluoro-N-(3-hydroxypropyl)-l- and 4,4,4,4’,4’,4’-Hexafluoro-N-(3-hydroxypropyl)-
d-valyl-N-methyl-l-leucyl-l-alanine tert-Butyl Ester (11e1 and 11e2, resp.). As described for 11d1/11d2,
with 10b (700 mg, 1.5 mmol), EtOH (30 ml), and 3-aminopropan-1-ol (700 mg, 9 mmol). The crude
product (1.3 g) was separated by CC (SiO2, AcOEt/hexane 1:2): less polar 11e1 ((S,S,S); 97 mg), a
mixture of both isomers (98 mg), and more polar 11e2 ((R,S,S); 85 mg) as oils. Combined yield 35%.
(S,S,S)-Isomer 11e1: TLC (AcOEt/hexane 1:1): single spot, Rf 0.30. [a]D ¼ ꢀ79.2 (c ¼ 1.07, CHCl3).
IR (film): 3334, 1735, 1639 – 1670, 1522. 1H-NMR: 0.84 (d, J ¼ 6.3, 3 H); 0.92 (d, J ¼ 6.6, 3 H); 1.22 (d, J ¼
t
7.0, Me); 1.40 (s, Bu); 1.36 – 1.45 (m, CH); 1.59 – 1.69 (m, 2 CH2); 2.29 – 2.59 (br., OH); 2.44 – 2.55 (m,
1 H of CH2N); 2.74 – 2.86 (m, 1 H of CH2N); 2.94 (s, MeN); 3.51 – 3.66 (m, CF3CH); 3.69 (t, J ¼ 5.9,
CH2O); 3.99 (d, J ¼ 8.1, 1 HꢀC(a)); 4.24 – 4.40 (m, 1 HꢀC(a)); 5.45 – 5.55 (m, 1 HꢀC(a)); 6.52 (d, J ¼
7.4, O¼CNH). 13C-NMR (CDC3): 171.6, 168.8 (2 C¼O); 81.9 (Me3C); 62.1 (CH2O); 54.9 (CH(a)); 54.2
(CH(a)); 50.1 (m, J ¼ 26, CF3CH); 48.6 (CH(a)); 46.4 (CH2N); 36.1 (CH2); 31.9 (CH2); 30.5 (MeN);
27.9 (Me3C); 24.9 (CH); 23.1 (Me); 21.8 (Me); 17.9 (Me). 19F-NMR (CDCl3): ꢀ 61.89; ꢀ 64.77. ESI-MS:
560.2538 ([M þ Na]þ, C22H37F6N3NaOþ5 ; calc. 560.2535), 538.2223 (100, Mþ), 482.1693 (95, [M ꢀ
C4H8]þ), 393.1564 (41, [M ꢀ Ala-OtBu]þ), 156.1052 (17, C8H14NO2þ, MeLeu fragment), 128.1088 (62,
C7H14NOþ, MeLeu fragment).
(R,S,S)-Isomer 11e2: TLC (AcOEt/hexane 1:1): single spot, Rf 0.18. [a]D ¼ ꢀ58.1 (c ¼ 1.115,
1
CHCl3). IR (film): 3331, 1732, 1632 – 1670, 1537. H-NMR: 0.82 (d, J ¼ 6.3, Me); 0.89 (d, J ¼ 6.6, Me);
1.27 (d, J ¼ 7.0, Me); 1.40 (s, tBu); 1.35 – 1.40 (m, CH); 1.54 – 1.72 (m, 2 CH2); 2.30 – 2.67 (br., OH); 2.46 –
2.62 (m, 1 H of CH2N); 2.77 – 2.88 (m, 1 H of CH2N); 2.99 (s, MeN); 3.47 – 3.62 (m, CF3CH); 3.67 (t, J ¼
5.5, CH2O); 4.04 (d, J ¼ 7.7, 1 H ꢀC(a)); 4.27 – 4.38 (m, 1 HꢀC(a)); 5.08 – 5.60 (m, 1 HꢀC(a)); 6.62 (d,
J ¼ 7.0, NH). 13C-NMR: 171.8, 171.6, 169.8 (3 C¼O); 122.9 (q, J ¼ 281, CF3); 82.2 (Me3C); 61.9 (CH2O);

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Helvetica Chimica Acta – Vol. 93 (2010)
55.2 (CH(a)); 54.1 (CH(a)); 50.1 (m, J ¼ 26.5, CF3CH); 48.7 (CH(a)); 46.3 (CH2N); 36.7 (CH2); 31.9
(CH2); 30.6 (MeN); 27.9 (Me3C); 24.7 (CH); 23.1 (Me); 21.4 (Me); 18.3 (Me). 19F-NMR (CDCl3):
ꢀ 62.03; ꢀ 64.96. ESI-MS: 560.2643 ([M þ Na]þ, C22H37F6N3NaOþ5 ; calc. 560.2535), 538.2339 (100, Mþ)
482.1798 (92, [M ꢀ C4H8]þ), 393.1633 (24, [M ꢀ Ala-OtBu]þ), 156.1066 (11, C8H14NOþ2 , MeLeu
fragment), 128.1095 (35, C7H14NOþ, MeLeu fragment).
4,4,4,4’,4’.4’-Hexafluoro-N-(2-hydroxyethoxy)-l-valyl- and 4,4,4,4’,4’,4’-Hexafluoro-N-(2-hydroxy-
ethoxy)-d-valyl-N-methyl-l-leucine-l-alanine tert-Butyl ester (11f1 and 11f2, resp.). As described for
11d1/11d2, with 10b (600 mg, 1.3 mmol), EtOH (20 ml) and 2-(aminooxy)ethanol [15] (300 mg,
3.9 mmol) at r.t. for 3 d. The crude product (858 mg) was separated by CC (SiO2, AcOEt/hexane
1:2): less polar 11f1 ((S,S,S); 141 mg, 20%) as an oil, a mixture of the diastereoisomers (284 mg, 41%),
and more polar 11f2 ((R,S,S); 217 mg, 31%) as crystals. Total yield 92%.
(S,S,S)-Isomer 11f1: TLC (AcOEt/hexane 1:1): single spot, Rf 0.37. [a]D ¼ ꢀ84.6 (c ¼ 1.095, CHCl3);
[a]D ¼ ꢀ87.7 (c ¼ 1.12, MeOH). IR (film): 3348, 1734, 1635 – 1670, 1517. 1H-NMR (CDCl3): 0.85 (d, J ¼
t
6.5, Me); 0.89 (d, J ¼ 6.5, Me); 1.23 (d, J ¼ 7.0, Me); 1.40 (s, Bu); 1.40 – 1.50 (m, CH); 1.55 – 1.78 (m,
CH2); 1.90 – 2.28 (m, NH); 2.45 – 2.65, 3.27 – 3.41 (br., OH); 2.78, 3.00 (2s, ratio 1:2, MeN); 3.52 – 3.98
(m, CHCF3, OCH2CH2O); 4.28 – 4.40 (m, 1 HꢀC(a)); 4.45 (dd, J ¼ 11.3, 9.5) and 4.64 (dd, J ¼ 11.3, 10.0,
1 HꢀC(a)); 4.83 (dd, J ¼ 10.5, 3.3) and 4.96 (dd, J ¼ 9.3, 6.0, 1 HꢀC(a)); 6.13 (d, J ¼ 11.5) and 6.14 (d,
J ¼ 11.5, NH); 6.48 (d, J ¼ 7.5) and 7.10 (d, J ¼ 8.0, NH). 1H-NMR (CD3OD): 0.85 (d, J ¼ 6.3, 3 H); 0.89
(d, J ¼ 6.3, 3 H); 1.23 (d, J ¼ 7.4, 3 H); 1.38 (s, tBuO); 1.43 – 1.75 (m, CH, CH2); 2.85, 3.05 (2s, ratio 1:10,
MeN); 3.50 – 3.75 (m, OCH2CH2O); 3.80 – 4.00 (m, CF3CH); 4.10 – 4.23 (dd, 1 HꢀC(a)); 4.55 (d, J ¼ 7.5,
1 HꢀC(a)); 5.05 – 5.15 (dd, 1 HꢀC(a)). 13C-NMR (CDCl3): 171.7 (C¼O); 171.1 (C¼O); 169.0 (C¼O);
122.7 (q, J ¼ 280, CF3); 122.5 (q, J ¼ 280, CF3); 81.9 (Me3CO); 75.0 (CH2ON); 61.0 (CH2OH); 59.7, 58.3
(CH(a)); 56.0, 55.0 (CH(a)); 48.5 (CH(a)); 48.3 (m, J ¼ 27, CF3CH); 36.2 (CH2); 31.3, 29.9 (MeN); 27.8
(Me3CO); 24.5 (CH); 23.0 (Me); 21.7 (Me); 17.9 (Me). 13C-NMR (CD3OD): 172.4 (C¼O); 172.2
(C¼O); 172.1 (C¼O); 124.5 (q, J ¼ 283, CF3); 124.3 (q, J ¼ 283, CF3); 82.6, 79.4 (Me3C); 76.1 (CH2ON);
60.7 (CH2OH); 56.5 (CH(a)); 56.4 (CH(a)); 50.1 (CH(a)); 49.2 (m, J ¼ 28, CF3CH); 38.3 (CH2); 31.8
(CH); 28.2 (Me3C); 25.7 (CH); 23.5 (Me); 22.3 (Me); 17.4 (Me). 19F-NMR (CD3OD): ꢀ 63.33 (minor);
ꢀ 63.44, ꢀ 65.34 (10%), ꢀ 65.61 (90%). ESI-MS (MeOH): 601.32 (20, [M þ HF þ NaF]þ), 562.37 (73,
[M þ Na]þ), 540.36 (17, [M þ H]þ), 484.23 (20, [M þ H ꢀ C4H8]þ), 395.22 (100, [M ꢀ 144]þ, loss of Ala-
OtBu), 367.31 (5, [M ꢀ 172]þ, loss of O¼C-Ala-OtBu).
(R,S,S)-Isomer 11f2: TLC (AcOEt/hexane 1:1): single spot, Rf 0.30. M.p. 97 – 988 (from Et2O/
1
hexane). [a]D ¼ ꢀ28.0 (c ¼ 1.095, CHCl3); [a]D ¼ ꢀ33.9 (c ¼ 1.04, MeOH). H-NMR: 0.76 (d, J ¼ 6.2,
t
Me); 0.84 (d, J ¼ 6.6, Me); 1.24 (d, J ¼ 7.0, Me); 1.35 (s, Bu); 1.52 – 1.72 (m, CH); 1.55 – 1.70 (m, 2 H);
2.98 (s, MeN); 3.20 – 3.55 (br., OH); 3.55 – 3.80 (m, OCH2CH2O, CHCF3); 4.25 – 4.38 (m, 1 HꢀC(a));
4.43 – 4.56 (m, 1 HꢀC(a)); 5.20 – 5.30 (m, 1 HꢀC(a)); 6.34 (d, J ¼ 11.7, NH); 6.95 (d, J ¼ 7.4, NH).
13C-NMR: 171.9 (C¼O); 171.8 (C¼O); 169.8 (C¼O); 122.67 (q, J ¼ 282, CF3); 122.5 (q, J ¼ 282, CF3);
81.9 (Me3CO); 75.4 (CH2ON); 60.5 (CH2OH); 55.3 (CH(a)); 54.7 (CH(a)); 48.6 (CH(a)); 48.2 (m, J ¼
27.5, CF3CH); 36.7 (CH2); 30.7 (MeN); 27.7 (Me3C); 24.3 (CH); 23.1 (Me); 21.0 (Me); 18.1 (Me). 19F-
NMR (CDCl3): ꢀ 62.68, ꢀ 65.19. ESI-MS (MeOH): 562.31 (70, [M þ Na]þ), 540.42 (40, [M þ H]þ),
484.35 (20, [M þ H ꢀ C4H8]þ), 395.22 (100, [M ꢀ 144]þ, loss of Ala-OtBu), 367.31 (10, [M ꢀ 172]þ, loss of
O¼C-Ala-OtBu). ESI-MS (neg. mode, MeCN/H2O/NEt3): 584.24 (25, [M þ CO2?]ꢀ), 558.38 (70, [M þ
F]ꢀ), 538.41 (15, [M ꢀ H]ꢀ), 498.35 (20, [M ꢀ H ꢀ 2 HF]ꢀ), 478.39 (35, [M ꢀ 1 H ꢀ 3 HF]ꢀ), 476.40 (95,
[478.39 ꢀ 2 H]ꢀ), 436.40 (55, [476.40 ꢀ 2 HF]ꢀ), 416.31 (100, [436.40 ꢀ HF]ꢀ), 359.32 (25,
C14H19F6N3Oꢀ).
The crystalline 11f2 was dissolved in Et2O (2 ml) and diluted with hexane (10 ml). The solvent was
allowed to slowly evaporate. Residual solvent was removed from the crystals which were subjected to X-
ray-analysis and found to be the (R,S,S)-isomer [20].
N-(tert-Butoxy)carbonyl]-d-alanyl-N-methyl-l-leucyl-N-methyl-l-leucyl-N-methyl-l-valyl-(2S,3R,
4R,6E)-3-hydroxy-4-methyl-2-(methylamino)oct-6-enoyl-(2S)-2-aminobutanoyl-N-methylglycyl-N-
methyl-l-leucyloxy-Y(OꢀCH2)-ethoxy-Y(OꢀNH)-4,4,4,4’,4’,4’-hexafluoro-d-valyl-N-methyl-l-leucyl-l-
alanine tert-Butyl Ester (12a). A mixture of N-Boc-octapeptide-OH 2b (2.30 g, 2.17 mmol), tripeptide
11f2 (650 mg, 1.18 mmol), EDC (600 mg, 3 mmol), DMAP (600 mg, 5 mmol), and a cat. amount of 1-
hydroxy-7-aza-1H-benzotriazole (HOAt) in CH2Cl2 (70 ml) was kept at r.t. for 14 d. The mixture was

Helvetica Chimica Acta – Vol. 93 (2010)
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filtered through SiO2 (1 ꢁ 12 cm) with Cl2Cl2 and the filtrate concentrated: 12a 1.725 g, 95%). TLC
1
t
t
(AcOEt): 0.58. H-NMR: 0.63 – 1.05 (m, 38 H); 1.19 – 1.34 (m, 10 H); 1.42 (s, Bu); 1.45 (s, Bu); 1.20 –
1.53 (m, 6 H); 1.56 – 1.82 (m, 12 H); 1.82 – 2.11 (m, 2 H); 2.21 – 2.45 (m, 2 H); 2.70 – 3.30 (m, 21 H);
3.50 – 4.12 (m, 3 H); 4.18 – 4.69 (m, 5 H); 4.75 – 5.02 (m, 1 H); 5.04 – 5.62 (m, 7 H); 6.12 – 7.00 (m, 3 H).
13C-NMR: 15.6; 18.1; 18.2; 18.3; 18.4; 18.6; 19.8; 19.9; 21.6; 22.2; 22.8; 23.1; 23.6; 24.3; 24.9; 25.1; 27.2;
28.2; 28.6; 30.4; 30.5; 31.0; 31.1: 34.1; 36.4 (t); 36.6; 38.0; 38.4 (t); 40.2 (t); 43.6 (t); 46.9; 48.9; 51.5; 51.8;
54.8; 55.7; 62.8 (t); 62.8 (t); 69.3 (t); 72.2 (t); 128.8; 129.7; 137.2 (s); 155.2 (s); 168.3 (s); 168.8 (s); 170.8 (s);
171.5 (s); 171.7 (s); 173.1 (s); further signal 127.2. 19F-NMR: ꢀ 62.45; ꢀ 64.72. ESI-MS: 1567.23 (35,
[M þ Na]þ), 1549.17 (65, [M þ Na ꢀ H2O]þ), 1545.11 (15, [M þ H]þ), 1527.04 (20, [M þ H ꢀ H2O]þ),
562.21 (100), 539.27 (75).
Rearrangement of Depsipeptide (12a) to N2.8-[(tert-Butoxy)carbonyl]-[5-[4,4,4,4’,4’,4’-hexafluoro-N-
(2-hydroxyethoxy)-d-valine]]-7,8-secocyclosporin tert-Butyl Ester (¼ N-(tert-Butoxy)carbonyl]-d-alan-
yl-N-methyl-l-leucyl-N-methyl-l-leucyl-N-methyl-l-valyl-(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methyl-
amino)oct-6-enoyl-(2S)-2-aminobutanoyl-N-methylglycyl-N-methyl-l-leucyl-4,4,4,4’,4’,4’-hexafluoro-N-
(2-hydroxyethoxy)-d-valyl-N-methyl-l-leucyl-l-alanine tert-Butyl Ester; 13a). A soln. of the depsipep-
tide 12a (20 mg, 0.02 mmol) in toluene (10 ml) was heated to reflux for 60 h. The solvent was evaporated.
The more polar product was separated via prep. TLC (AcOEt). 1H-NMR: 0.50 – 1.00 (m, 38 H); 1.42 (2s,
18 H); 1.15 – 2.20 (m, 34 H); 2.25 – 2.41 (m, 2 H); 2.68 – 3.30 (m, 21 H); 3.55 – 3.90 (m, 2 H); 4.05 – 5.00
(m, 8 H); 5.05 – 5.63 (m, 8 H). 13C-NMR: 14.1; 15.2; 17.7; 17.8; 17.9; 18.0; 18.1; 18.2; 18.7; 18.9; 19.4; 19.5;
21.2; 21.7; 22.1; 22.3; 22.5; 22.8; 23.1; 23.9; 24.7; 24.8; 26.7; 27.0; 27.9; 28.2; 29.9; 30.0; 30.1; 30.4; 30.7; 36.7;
37.0 (t); 37.4; 37.9 (t); 39.8 (t); 46.5; 49.2 (t); 50.0; 51.1; 51.1; 51.4; 57.6; 62.6 (t); 63.4 (t); 64.2 (t); 65.0 (t);
77.2; 79.5; 127.6; 128.2; 155.2 (s); 170.8 (s); 171.6 (s); 172.0 (s); 173.1 (s); further signals at 127.1, 127.3, and
128.5. 19F-NMR (CDCl3): ꢀ 62.48; ꢀ 64.87. ESI-MS: 1566.92 (5, [M þ Na]þ), 1549.16 (10, [M þ Na ꢀ
H2O]þ), 1544.74 (8, [M þ H]þ), 1527.11 (8, [M þ H ꢀ H2O]þ), 1507.11 (C71H123F6N11NaO1þ5 ), 1485.12
(C71H124F6N11Oþ15, loss of OCH2CH2O), 1143.90 (94, C56H98N9NaOþ14 ), 1121.72 (88, C56H99N9O1þ4 ), 539.34
(100, C21H35F6N3Oþ6 ).
N
2.8-[(tert-Butoxy)carbonyl][5-[4,4,4,4’,4’,4’-hexafluoro-N-(2-hydroxyethyoxy)-l-valine]]-7,8-seco-
cyclosporin tert-Butyl Ester (¼ N-(tert-Butoxy)carbonyl]-d-alanyl-N-methyl-l-leucyl-N-methyl-l-leucyl-
N-methyl-l-valyl-(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)oct-6-enoyl-(2S)-2-aminobutano-
yl-N-methylglycyl-N-methyl-l-leucyl-4,4,4,4’,4’,4’-hexafluoro-N-(2-hydroxyethoxy)-d-valyl-N-methyl-l-
leucyl-l-alanine tert-Butyl Ester; 13b). A mixture of N-Boc-octapeptide-OH 2b (4.1 g, 4 mmol),
tripeptide 11f1 (1.0 g, 1.9 mmol, EDC · HCl (1.1 g, 5.7 mmol), DMAP (2.0 g, 16.4 mmol), and a cat.
amount of 1-hydroxy-7-aza-1H-benzotriazole (HOAt in CH2Cl2 (100 ml) was kept at r.t. for 14 d. The
soln. was filtered through SiO2 (1 ꢁ 15 cm): pure 13b (2.05 g, 72%). TLC (AcOEt): 0.59. 1H-NMR: 0.61 –
t
t
1.01 (m, 37 H); 1.39 (s, Bu); 1.42 (s, Bu); 1.15 ꢀ 1.62 (m, 19 H); 1.78 – 2.42 (m, 6 H); 2.67 – 3.26 (m,
21 H); 3.53 – 3.89 (m, 5 H); 3.92 – 4.10 (m, 1 H); 4.18 – 4.74 (m, 5 H); 4.83 – 4.99 (m, 1 H); 5.00 – 5.58 (m,
7 H); 6.07 – 6.97 (m, 3 H); 7.15 – 7.32 (m, 8 H). 13C-NMR: 15.6; 18.1; 18.2; 18.3; 18.4; 18.6; 19.1; 19.3; 19.8;
19.9; 20.3; 21.6; 22.5; 22.7; 23.1; 23.2; 23.5; 24.3; 24.8; 25.1; 27.2; 28.2; 28.6; 30.4; 30.5; 30.7; 31.0; 31.1; 34.1;
36.4 (t); 36.6; 38.0; 38.3 (t); 40.2 (t); 43.5 (t); 46.9; 48.9; 51.5; 51.8; 54.8; 55.7; 62.8 (t); 62.9 (t); 69.3 (t);
72.2 (t); 77.6; 78.3; 128.8; 129.7; 137.7 (s); 155.6 (s); 168.6 (s); 168.8 (s); 169.3 (s); 171.3 (s); 171.4 (s); 171.9
(s); 172.1 (s); 172.2 (s); 172.3 (s); 172.6 (s); 173.6 (s); further signal at 127.7. 19F-NMR(CDCl3): ꢀ 62.45;
ꢀ 64.72. ESI-MS: 1567.08 (25, [M þ Na]þ), 1549.01 (100, [M þ Na ꢀ H2O]þ), 1545.09 ([M þ H]þ),
1527.08 (16, [M ꢀ H2O]þ).
[5-[4,4,4,4’,4’,4’-Hexafluoro-N-(2-hydroxyethoxy)-d-valine]]cyclosporin (14a). The undecapeptide
13a (105 mg, 0.07 mmol) was stirred at r.t. for 2 h in CF3COOH (2 ml). The solvent was evaporated
under aspirator vacuum, and sat. Na2CO3 soln. (10 ml) was added. The mixture was extracted with
CH2Cl2 (2 ꢁ 25 ml) and the soln. dried (MgSO4). A cat. amount of HOAt, EDC · HCl (382 mg, 2 mmol),
and DMAP (366 mg, 3 mmol) were added to the soln. and stirred at r.t. for 5 d. The soln. was subjected to
CC (SiO2, AcOEt): 14a (8 mg). TLC (AcOEt): Rf 0.56. 1H-NMR: 0.60 – 1.00 (m, 33 H); 1.15 – 1.25 (m,
2 H); 1.35 – 2.15 (m, 26 H); 2.25 – 2.50 (m, 3 H); 2.52 – 3.33 (m, 20 H); 3.40 – 3.88 (m, 1 H); 4.20 – 4.40 (m,
5 H); 4.56 – 5.58 (m, 9 H); 6.55 – 7.70 (m, 5 H). 13C-NMR: 14.2; 15.2; 15.9; 17.8; 17.9; 19.8; 21.3; 21.4; 21.7;
21.9; 22.0; 22.8; 23.1; 25.0; 28.6; 29.2; 29.2; 29.6; 29.9; 30.0; 30.5; 35.6; 36.4; 36.6; 36.7; 37.0; 37.1; 37.2; 37.3;
37.4 (t); 37.7; 37.8; 39.9; 40.3 (t); 42.0; 43.0; 46.6; 49.4; 49.9 (t); 50.1; 50.2; 51.1; 51.8; 54.62; 57.0; 58.6; 59.0

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Helvetica Chimica Acta – Vol. 93 (2010)
(t); 62.6, 63.0 (t); 64.3, 64.7 (t); 65.0, 65.4 (t); 70.7, 71.8 (t); 77.6, 86.2, 87.6 (t); 128.1; 128.8; 159.8; 168.4;
171.1; 171.3; 171.4; 171.7; further signals at 126.7, 126.8, 127.2, 127.4, 128.9, 128.9, and 129.0. 19F-NMR
(CDCl3): ꢀ 76.45. ESI-MS: 1392.97 (5, [M þ Na]þ), 1370.85 (10, [M þ H]þ), 1352.59 (5, [M þ H ꢀ
H2O]þ), 1300.69 (8, [M þ H ꢀ CF3]þ), 1257.79 (20, [M þ H ꢀ CF3 ꢀ CH2CH2O]þ), 1239.85 (100, [M þ
H ꢀ CF3 ꢀ OCH2CH2OH]þ).
[5-[4,4,4,4’,4’,4’-Hexafluoro-N-(2-hydroxyethoxy)-l-valine]]cyclosporin (14b). As described for 14a,
with undecapeptide 13b (150 mg, 0.1 mmol), CF3COOH (0.5 ml), and CH2Cl2 (5 ml) at r.t. overnight.
Workup with sat. Na2CO3 soln. (10 ml) and CH2Cl2 (2 ꢁ 25 ml). Then with 1-hydroxy-7-aza-1H-
benzotriazole (HOAt), EDC · HCl (380 mg, 2 mmol), and DMAP (250 mg, 2 mmol) at r.t. for 4 d. Most
of the solvent was evaporated and the residue subjected to CC (SiO2, 4% MeOH/Et2O): 14b (9 mg).
TLC (AcOEt): Rf 0.4. 1H-NMR: 0.57 – 1.08 (m, 35 H); 1.09 – 1.35 (m, 10 H); 1.35 – 1.50 (m, 6 H); 1.51 –
1.85 (m, 16 H); 1.86 – 2.45 (m, 5 H); 2.65 – 3.29 (m, 6 MeN); 3.31 – 4.60 (m, 8 H); 4.65 – 5.57 (m, 8 H);
6.02 – 7.02 (m, 4 H). 13C-NMR: 18.2; 18.3; 18.4; 18.6; 18.7; 19.1; 19.3; 19.8; 19.9; 20.4; 21.5; 21.8; 22.1;
22.4; 23.6; 23.7; 23.9; 24.1; 24.3; 25.0; 25.0; 25.1; 25.3; 25.4; 25.5; 27.6; 30.2; 30.3; 30.4; 30.7; 30.8; 31.0;
31.1; 31.2; 31.6; 31.7; 32.0; 34.1; 35.7 (t); 37.6 (t); 38.0 (t); 45.7; 46.0; 49.2; 49.5; 49.9 (t); 50.1 (t); 50.6; 51.0;
52.7; 54.3; 55.5; 55.8; 56.8; 57.8; 62.9 (t); 72.2 (t); 77.6; 128.1; 128.5; 162.1; 168.1; 170.7; 170.8; 171.1; 171.6;
171.9; further signals at 125.9 and 127.8. 19F-NMR (CDCl3): ꢀ 62.31; ꢀ 64.79. ESI-MS: 1374.86 (10, [M þ
Na ꢀ H2O]þ), 1370.81 (45, [M þ H]þ), 1352.86 (55, [M þ H ꢀ H2O]þ), 688.02 (20, [M þ H þ Na ꢀ
H2O)2þ, 685.90 (100, [M þ 2 H]2þ), 677.02 (55, [M þ 2 H ꢀ H2O]2þ).
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Received May 3, 2010