Development of a practical process for the opening of macrocyclic Cyclosporin a and amino acid deletion

Article abstract of DOI:10.1021/op5003038

A practical and robust process for the derivatization of Cyclosporin A was demonstrated. The processes rely on the opening of Cyclosporin A and removal of amino acid fragments via Edman degradation, with the isolation of crystalline tetrafluoroboric salts of the corresponding acyclic polypeptides.

Full text of DOI:10.1021/op5003038

Communication
pubs.acs.org/OPRD
Development of a Practical Process for the Opening of Macrocyclic
Cyclosporin A and Amino Acid Deletion
†
‡
†
†
†
Bernard Riss, Arnaud Grandeury, Thorsten Gut, Manuela Seeger-Weibel, Christian Zuercher,
†
,†
Jinjing Li, and Fabrice Gallou*
†
‡
Chemical and Analytical Development and Pharmaceutical and Analytical Development, Novartis Pharma AG, CH-4002 Basel,
Switzerland
7
peptide. But the most widely encountered approach for
ABSTRACT: A practical and robust process for the
derivatization of Cyclosporin A was demonstrated. The
processes rely on the opening of Cyclosporin A and
removal of amino acid fragments via Edman degradation,
with the isolation of crystalline tetrafluoroboric salts of the
corresponding acyclic polypeptides.
derivatization still remains the selective opening of the
macrocycle at the most readily accessible positions, namely,
either the amino acid residues 1 and 11, or between 3 and 4,
using the Cyclosporin A nomenclature, followed by the removal
of the undesired amino acid residue(s) traditionally done via
8
Edman degradation. Such an approach was disclosed in the
9
mid 1980s and allows for robust and scalable protocols but
suffers from a severe limitation, namely, the high demand in
purification via costly methods, such as preparative chromatog-
raphy. In this paper, we would like to describe our efforts to
solve such process issues which led us to the development of a
streamlined and cost-effective process to advanced acyclic
ince its discovery by isolation from fungus Tolypocladium
1
S
inflatum (Beauveria nivea), Cyclosporin A has created
tremendous fascination and received enormous interest both
from the academic community and from the industry. The
most significant application was made in transplantation with
Cyclosporin A sold under the trade name Sandimmun that still
constitutes one of the most important immunosuppressant to
10
polypeptide derivatives of Cyclosporin A.
BACKGROUND
2
■
date. Continued efforts throughout the industry have led to a
The classical opening of Cyclosporin A between amino acid
residue 3 (sarcosin) and 4 (methyl leucine) starts with masking
of the free hydroxyl, so as to avoid isomerization ((1) in
equilibrium with (1-Iso), see Figure 2 and Scheme 1) between
the amide and ester forms. Not doing so leads to an equilibrium
plethora of other derivatives in various indications such as an
3
antiviral agent, multiple sclerosis. Solid-phase synthesis has
4
been one of the strategies used in discovery mostly. More
traditional solution phase approaches have relied on the
derivatization of the more readily accessible handle 1 (amino
acid residue 1 based on Cyclosporin A nomenclature as
depicted in Figure 1 below), easily derivatized via either the
11
between the amide and the corresponding ester form that
reduces the efficiency of the subsequent steps and further
complicates the process. The steric hindrance around the
hydroxyl group does not allow for many practical and
economically viable options to mask this functionality. The
acetate is therefore traditionally chosen and incorporated for
industrial applications via a well-established process, which
leads the desired monoacetylated product in high yields (>90%
isolated yield) by direct crystallization and in purity typically
around 95%. Care should nevertheless be taken of the
temperature of the reaction so as not to overacetylate the
polypeptide. The chemistry and process on this step were
performing sufficiently well to leave this part of the process
untouched.
The macrocyclic peptide is then classically opened using a
Meerwein salt, triethyl or trimethyloxonium tetrafluoroborate,
subsequently subjected to methanolysis and acid hydrolysis.
Although of limited selectivity, this step is remarkable from a
complexity standpoint. Indeed, despite an excess of alkylating
agent required to allow for sufficient reactivity, a remarkable
selectivity for such a functionalized system is observed. The
selectivity is presumably due to both steric and conformational
reasons, due to the reduced steric hindrance at the sarcosine
Figure 1. Structure of Cyclosporin A (1) and its nomenclature.
5
hydroxyl or the olefin moieties. Seebach in his seminal paper
on the development of chemistry on the Cyclosporin A core
exemplified the strong conformational bias of the macrocyclic
structure and took advantage of this feature to selectively
6
alkylate the sarcosine amino acid residue 3. More recently, in
collaboration with Beller, we demonstrated the direct
functionalization of the macrocyclic polypeptide core via the
selective reduction of the Abu fragment of the macrocyclic
Received: September 19, 2014
©
XXXX American Chemical Society
A
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Organic Process Research & Development
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Figure 2. Equilibrium amide (1)−ester (1-Iso).
Scheme 1. Original synthesis from Cyclosporin A 1−4
Scheme 2. Opening of the macrocylic structure from 2 to 3
B
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Table 1. Qualitative overview of salt screening for 3
HCl
32 solvents
TFA
H SO4
H PO4
pTsOH
HPF6
HBF4
2
3
screening
6 solvents
6 solvents
ethers
+
6 solvents
6 solvents
30 solvents
ethers, esters
+
44 solvents
ethers, esters
+++
positive hits
crystallinity
ketones, ethers, esters
++
−
+
−
−
−
−
Table 2. Solvent screening for 3
residue and the transannular intramolecular interaction within
tetrafluoroborate that differs from lot to lot with the finely
dispersed powder generates variability and complexity for the
process.
This first opened-form intermediate (dodecapeptide) 3 can
be used as such as a platform for derivatization, or amino acids
can be deleted sequentially, which is classically done via Edman
degradation. The major hurdle is the requirement for a labor-
intensive purification of the degradation product. The default
method of choice is a purification via column chromatography
of the trifluoroacetate salt, generated from the previous step.
1
2
the macrocycle. It is worth mentioning that the limited
solubility of the Meerwein salt adds a number of constraints to
the process. For instance, the reversible nature of the
transformation (hydrolysis of the intermediate imino-ether
back to the amide) creates some complications in the analytical
control strategy, the purification by column chromatography
generates a bottleneck in the overall process, and a high
number of operations can lead to decomposition if pH is not
carefully controlled during the solvent switch especially.
Associated with the use of trimethyloxonium tetrafluoroborate
also comes the requirement for a high solubilizing non-
coordinating solvent, typically a chlorinated solvent such as
dichloromethane. Besides, the quality of the trimethyloxonium
RESULTS
■
With these challenges identified, we aimed at identifying a more
robust process more specifically for the ring opening and
C
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Organic Process Research & Development
Communication
Scheme 3. Edman degradation process (from 3 to 4)
Table 3. Qualitative overview of salt screening for 4
HCl
32 solvents
TFA
H SO4
H PO4
pTsOH
HPF6
HBF4
2
3
screening
6 solvents
6 solvents
ethers
+
6 solvents
6 solvents
30 solvents
ethers, esters
+
46 solvents
ethers, esters
+++
positive hits
crystallinity
ketones, ethers, esters
++
−
+
−
−
−
−
Edman degradation steps (steps from intermediate 2 to 4,
Scheme 2), which could provide practically sufficiently pure
form of the intermediates in a minimum number of operations.
Our hope was to find new crystalline intermediates along the
sequence. Our strategy consisted in testing various protecting
groups for the free hydroxyl functionality on the amino acid
residue 1 (silyl ethers, polyethers, benzyl groups, esters) and
evaluate their crystallinity along the sequence from inter-
mediate 2 to 4. Very rapidly, we faced the known challenge of
reaching out to the hydroxyl group in a practical and
of hydantoin indeed has to be removed, along with a significant
amount of carry-over impurities resulting from the nature of the
original fermentation product. Purification by column chroma-
tography was the method of choice prior to our work that
allowed for obtention of material of acceptable quality of the
resulting product 4 (purity >90%). We were again aiming for a
chromatography-free process and were fortunate to identify
rapidly an alternative to remove the hydantoin side-product. A
selective liquid−liquid extraction, using the solvent already
present in the process, indeed allowed us to wash away the
hydantoin in a first operation. By adjusting the amount of water
at completion of the reaction, the Edman degradation product
indeed remained in the aqueous phase at this low pH (<1),
while the reagent and the hydantoin go almost completely into
the toluene phase. A second extraction results in the full
removal of hydantoin and product 4 can be extracted with 2-
methyl-tetrahydrofuran after further dilution with water.
Major efforts were then devoted to finding a crystalline form,
free base or salt. The free base did not result in any lead, but
several salts were shown to be promising (Table 3). Of
particular interest again was the tetrafluoroborate salt. The
latter offered the advantage of being readily available and
potentially already present as the acidic reagent triggering the
Edman degradation. An extensive solvent screening demon-
strated again that ethers and esters resulted in highly crystalline
product 4 in good purity (Table 4). However, the isolated 4
seemed to lose crystallinity upon drying and retained a
significant amount of solvent even under extensive and various
drying conditions. Further optimization and solvent harmo-
nization led again to the use of 2-methyl-tetrahydrofuran and
TBME as the solvent and antisolvent, respectively. Anhydrous
conditions were again required and critical to generate material
of high crystallinity. Eventually, the optimized process resulted
in an isolable product 4 for the first time reported that would
become amorphous upon drying. It therefore rendered
subsequent operations difficult (mediocre filtration, poor
drying, presence of residual solvent, typically 2−8%) but
would still allow for efficient upgrade in quality of this
intermediate.
13
economically sound process. While most compounds could
form, based on our preliminary experience, a high-yielding
process could hardly be envisioned for anything but the acetate.
We therefore focused on the acetate protected Cyclosporin A
8
(
2) which was opened under reported conditions and focused
our efforts on finding a crystalline form of its resulting products
and 4, free base or salt. An extensive screening was done first
3
on intermediate 3 (see overview in Table 1). The free base did
not result in any lead. Several salts were shown to be promising.
Of particular interest was the tetrafluoroborate salt, which could
be generated straight from the reaction conditions thanks to the
use of the Meerwein salt in the ring opening. A large
preliminary solvent screen showed that ethers and esters
resulted in highly crystalline ring-opening product (dodecapep-
tide) in good purity (Table 2). Further optimization and
solvent harmonization then led to the use of 2-methyl-
tetrahydrofuran (2-MeTHF) and tert-butylmethyl ether
(
TBME) as the solvent and antisolvent of choice, respectively.
It is worth pointing out that anhydrous conditions are here
required to generate material of high crystallinity. The
crystallized product displayed good physical properties, fast
filtration, and easy drying, which provided a handle for reliable
14
isolation and purification.
Edman Degradation Step. In the next stage that consists
in the deletion of the leucine amino acid residue 4 via an
Edman degradation (Scheme 3), the chemical transformation
was carried out under standard conditions as reported in early
8
protocols using phenyl isothiocyanate to activate the terminal
basic amine and trigger the formation of hydantoin. The major
challenges associated with this process rely in the isolation and
purification of the resulting product. A stoichiometric amount
The ultimate process consisted in charging the undecapep-
tide 3 in toluene and treating it with a solution of sodium
D
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Organic Process Research & Development
Communication
Table 4. Solvent screening for 4
Scheme 4. Edman degradation process (from 4 to 5)
E
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Organic Process Research & Development
Communication
carbonate in order to liberate the base. After phase split,
phenylisothiocyanate was added giving raise to the thiourea
intermediate, which is treated with a solution of fluoroboric
acid in methanol to trigger the hydantoin formation and the
degradation. The later is removed by selective extraction from
toluene after dilution of the reaction mixture with water, then
the product was extracted with methyl-THF, dried by
azeotropic distillation, crystallized from 2-methyl-tetrahydrofur-
as a precursor to the Meerwein reagent in the macrocyclic
peptide opening.
A future area of improvement indicated by this analysis will
be the water consumption. Water is indeed still a major element
on our mass balance and the current area of efforts.
CONCLUSION
■
15
In conclusion, we have identified three new crystalline forms of
acyclic derivatives of Cyclosporin A and seco-Cyclosporin A
and developed scalable processes for their formation. These
processes have allowed for more rapid and practical entry into
Cyclosporin derivatives. These processes were scaled up to
multitons scale and led to significant improvement of the
environmental footprint. This is to our knowledge the first
example of crystalline intermediates of such complex
polypeptides, which paves the way for easier entry into new
derivatives.
an/tert-butylmethyl ether, and is isolated by filtration.
The terminal amino acid deletion process can be repeated via
the same process, and the subsequent Edman degradation
product be recrystallized as an HBF salt in an analogous way as
4
reported above (Scheme 4).
Again, compound 5 can be isolated and purified as a
1
6
crystalline salt. It is interesting to note that crystallinity is
practically worse and worse as the acyclic polypeptide is getting
shorter, which is easily experienced by the fast collapse of the
crystalline form onto amorphous ones as observed by XRPD.
GREEN CHEMISTRY METRICS
EXPERIMENTAL SECTION
■
■
The process mass intensity (PMI) is a well-accepted metric of
greenness currently recommended by the ACS Green Chemical
P r e p a r a t i o n
o f
2 :
S
R e f
4 .
(3S,6S,9S,12S,15R,18S,21S,24S,27S,30S)-Methyl-27-
((1R,2R,E)-1-acetoxy-2-methylhex-4-en-1-yl)-30-ethyl-
3 , 9 , 1 8 , 2 1 - t e t r a i s o b u t y l - 6 , 2 4 - d i i s o p r o p y l -
8 , 1 2 , 1 5 , 1 7 , 2 0 , 2 3 , 2 6 , 3 2 - o c t a m e t h y l -
4 , 7 , 1 0 , 1 3 , 1 6 , 1 9 , 2 2 , 2 5 , 2 8 , 3 1 - d e c a o x o -
2,5,8,11,14,17,20,23,26,29,32-undecaazatetratriacontan-34-
oateHydrogen(tetrafluoridoborate) (1:1) 3. Acetyl-Cyclo-
sporin A (ca. 184 kg tel quel, ca. 148 mol) was reacted with
trimethyloxonium tetrafluoroborate (58.8 kg, ca. 398 mol, 2.5
equiv) at 20−25 °C in dichloromethane (330 L). After 20 h,
acetonitrile (360 L) and water (1200 L) were added to perform
the hydrolysis. After 3 h, at 20−25 °C, the phases were
separatedm and the organic phase was washed with 400 kg of
water in order to increase the pH to 2−3. Then, the reaction
mixture was concentrated and dried by azeotropic distillation at
jacket temperature 45 °C, 150 mbar with 2-methyl-tetrahy-
drofuran (solvent exchange acetonitrile, dichloromethane/2-
methyl-tetrahydrofuran). The desired product was then crystal-
lized from 2-methyl-tetrahydrofuran (1600 L) and tert-
butylmethyl ether (700 L) to provide a white crystalline
17
Pharmaceutical Roundtable. It is defined as the quantity of
raw material input in kilograms per quantity of bulk API in
kilograms. We wanted to assess the evolution of mass
utilization related to the implementation of the two newly
found crystalline compounds 3 and 4, using PMI as the
1
8,19
representative metrics. (see Table 5 below).
Table 5
solvent aqueous
process
old
step
PMI
PMI
PMI
comments
overall
step 1
step 2
step 3
284.2
21.7
233.8
6.4
28.5
10.9
5.1
pyridine process
61.7
52.1
175.3
202.2
12.5
isolation after column
chromatography
new
overall
step 1
step 2
step 3
184.0
8.7
54.5
4.3
124.8
2.5
toluene process
107.3
44.8
16.4
35.8
94.7
27.6
crystallization HBF salt
4
crystallization HBF salt
4
1
powder (117.5 kg, after drying, ca. 86 mol). H NMR (400
MHz, DMSO): 0.69 (d, J = 6.6 Hz, 3 H); 0.71 (d, J = 6.5 Hz, 3
H); 0.8, (m, 6 H); 0.82−0.89 (m, 24 H); 0.90 (d, J = 6.6 Hz, 3
H); 0.93 (d, J = 6.6 Hz, 3 H); 1.16 (m, 6 H); 1.23−1.50 (m, 4
H); 1.52 (m, 1 H); 1.32−1.73 (m, 8 H); 1.59 (d, J = 6.0 Hz, 3
H); 1.65 (m, 2 H); 1.65−2.13 (m, 2 H); 1.93 (s, 3 H); 2.03 (m,
1 H); 2.19 (m, 1 H); 2.45 (s, 3 H); 2.72 (s, 3 H); 2.84 (s, 3 H);
2.86 (s, 3 H); 2.99 (s, 3 H); 3.02 (s, 3 H); 3.06 (s, 3 H); 3.62−
3.68 (m, 3 H); 3.78−3.87 (m, 1 H); 4.23 (m, 1 H); 4.53 (d, J =
18.6 Hz, 1 H); 4.10−4.26, (m, 1 H); 4.60 (m, 1 H); 4.62 (m, 1
H); 4.66 (m, 1 H); 5.02 (m, 1 H); 5.13 (dd, J = 11.3 Hz, 4.7
Hz, 1 H); 5.26 (m, 1 H); 5.29 (m, 1 H); 5.32 (m, 1 H); 5.36
(m, 1 H); 5.39 (m, 2 H); 7.72 (d, J = 7.3 Hz, 1 H); 8.14 (d, J =
7.3 Hz, 1 H); 8.21−8.35 (m,, 1 H); 8.85 (br s, 1 H); 8.96 (d, J
= 8.4 Hz, 1 H). HR-MS (EI): m/z: calcd for C H N O [M
While the benefits were clearly visible to us in our production
facilities having experienced both processes on scale, we wanted
to measure more precisely the process improvements from a
mass intensity standpoint and better understand future areas of
improvement. Overall, the manufacturing process greatly
improved thanks to our newly discovered solid state forms.
This indeed allowed to bypass a couple of mass-intensive and
time-consuming column chromatographies. Combined with
solvent recycling made feasible after streamlining of the solvent
strategy, it resulted in the improvement of our environmental
footprint by ca. 40%, as illustrated by reductions in overall PMI
from 284.2 to 184.0. Overall solvent consumption was reduced
by 70%, and aqueous waste reduced by 54%. One element that
is not included is the ability of using much lower grade
Cyclosporin A as a starting material, which further reinforces
our improvement in mass utilization as our starting material
itself did not require intensive purification. In addition, usage of
chlorinated solvents was reduced by greater than 90% and
promising results indicate it could be fully replaced when using
the more readily soluble dimethoxycarbenium tetrafluoroborate
65
118 11 14
+
+ H ]: 1275.87815, found: 1275.75512.
Methyl-(5S,8S,11S,14S,17S,20R,23S,26S,29S)-8-[(1R,2R,4E)-
1-(acetyloxy)-2-methylhex-4-en-1-yl]-29-amino-5-ethyl-
3,9,12,15,18,20,23,27,30-nonamethyl-14,17,26-tris(2-methyl-
propyl)-4,7,10,13,16,19,22,25,28-nonaoxo-11-(propan-2-yl)-
3,6,9,12,15,18,21,24,27-nonaazahentriacontan-1-oate
Hydrogen(tetrafluoridoborate) (1:1) 4. The previously pre-
F
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Organic Process Research & Development
Communication
pared salt (160 kg, ca. 120 mol) was charged in a suitable
reactor, suspended in toluene (420 L) and submitted to react at
ca. 30% by weight, and tert-butylmethyl ether (about 20 mL)
was added. Seeding with wet material induced crystallization of
the desired product, which was isolated by filtration and dried
2
0−25 °C with sodium carbonate (23 kg, ca. 217 mol), and
1
water (250 L). The resulting mixture was stirred at 20−25 °C
for 30 min, and the phases were separated. Phenylisothiocya-
nate (19 kg, ca. 140 mol) was added within 1 h at 20−25 °C,
and the resulting mixture was stirred until completion as
detected by HPLC. Then Methanol (236 kg) was added,
followed by fluoroboric acid 50% in water (31 kg, ca. 176 mol).
The mixture was stirred for an additional 1 h at 20−25 °C and
controlled by an IPC. Then, water (240 L) was added, and after
phase split the upper (toluene) phase was removed. The lower
layers were extracted (twice) with more toluene (210 kg each).
Then 2-methyl-tetrahydrofuran (400 kg) and water (800 kg)
were added. The aqueous phase was removed and back-
extracted with 2-methyl-tetrahydrofuran (270 kg). Then tert-
butylmethyl ether (120 kg) and water (160 kg) were added to
the combined organic phases. After phase split, the remaining
organic phase was concentrated and dried by azeotropic
distillation at 45 °C, 300 mbar with 2-methyl-tetrahydrofuran.
Then, after volume adjustment to 290 L, tert-butylmethyl ether
under vacuum (yield 15.0 g, 13.2 mmol). H NMR (400 MHz,
DMSO): 0.66−0.75 (m, 7 H); 0.76−0.90 (m, 27 H); 1.04 (s 6
H); 1.18 (br d, J = 6.6 Hz, 3 H); 1.26 (br d, J = 6.6 Hz, 3H);
1.31−1.45 (m, 5 H); 1.45−1.58 (m, 7 H); 1.60 (br s, 4 H);
1.62−1.77 (m, 3 H); 1.90−1.97 (m, 3 H); 2.08−2.23 (m, 2 H);
2.46 (s, 3 H); 2.49−2.51 (m, 1 H); 2.76 (s, 2 H); 2.83−2.86
(m, 2 H); 2.99−3.10 (m, 5 H); 3.59−3.65 (m, 2 H); 3.72−3.80
(m, 2 H); 3.88 (br d, J = 17.2 Hz, 1 H); 4.25 (br d, J = 17.2 Hz,
1 H); 4.47−4.56 (m, 1 H); 4.58−4.69 (m, 2 H); 5.03 (br d, J =
10.6 Hz, 1 H); 5.24−5.40 (m, 6 H); 8.11 (d, J = 6.9 Hz, 1 H);
8.12−8.21 (m, 1 H); 8.28 (d, J = 6.9 Hz, 1 H); 8.69 (d, J = 8.1
+
Hz, 1 H). HR-MS (EI): m/z: calcd for C53
H
96
N
O
9
12 [M + H ]:
1050.71002, found: 1050.71704.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +41 61 324 9911; fax: +41 61 324 8536. E-mail address:
fabrice.gallou@novartis.com.
■
(
1280 kg) was added, and the solution was seeded at 35−40 °C
Present Address
J.L.: Chemical and Analytical Development, Changshu, China.
with a suspension of freshly prepared crystals of compound 4
(
(
300 g) in 2-methyl-tetrahydrofuran/tert-butylmethyl ether
3:1, 2 L). After a slow cooling to 17 °C within 12 h, the
Notes
The authors declare no competing financial interest.
desired product was isolated by centrifugation and dried at 50
C under vacuum to yield a white powder (ca. 136.0 kg, ca. 94
°
1
ACKNOWLEDGMENTS
mol, after drying). H NMR (400 MHz, DMSO): 0.69 (d, J =
.2 Hz, 3 H); 0.73 (d, J = 7.0 Hz, 3 H); 0.81 (t, J = 7.3 Hz, 7.3
Hz, 3 H); 0.82 (m, 3 H); 0.85 (m, 9 H); 0.88 (m, 6 H); 0.91
d, J = 7.0 Hz, 3 H); 0.93 (d, J = 6.6 Hz, 3 H); 0.99 (d, J = 7.0
■
The authors would like to thank Yaohui Ji, Shangjun Teng,
Lukas Hueber, Matthias Kastner, Lukas Oberer, Alexandre
Luneau, and Thomas Lochman for their participation on the
project.
6
(
Hz, 3 H); 1.1, (d, J = 6.6 Hz, 6 H); 1.30−1.55 (m, 9 H); 1.60
(
(
d, J = 5.5 Hz, 3 H); 1.56−1.72 (m, 4 H); 1.93 (s, 3 H); 2.09-
REFERENCES
m, 3 H); 2.74 (s, 3 H); 2.84, (s, 3 H); 3.06 (s, 3 H); 2.87 (s, 3
■
H); 2.94 (s, 3 H); 3.02 (s, 3 H); 3.06 (s, 3 H); 3.68 (s, 3 H);
(1) Dreyfuss, M. H.; Hofmann, H. H.; Kobel, H.; Pache, W.;
Tscherter, H. Eur. J. Appl. Microbiol. 1976, 3, 125−133.
2) Since its introduction on the market in 1993 for the prevention of
3
2
.88 (d, J = 17.2 Hz, 1 H); 4.10 (d, J = 18.7 Hz, 1 H); 4.24 (m,
H); 4.39−4.62, (m, 1 H); 4.66 (m, 1 H); 5.02 (m, 1 H); 5.08
(
organ rejection, Cyclosporin A, sold under the trade name
Sandimmun, has been one of the most prescribed immuno-
suppressant and has seen its medical uses expanded to rheumatoid
arthritis and psoriasis and as an ophthalmic emulsion for the treatment
of dry eyes.
(
(
m, 1 H); 5.26 (m, 2 H); 5.32 (m, 1 H); 5.37 (m, 1 H); 5.39
m, 2 H); 7.84 (d, J = 7.3 Hz, 1 H); 7.98 (br s, 3 H); 8.07 (d, J
=
7.3 Hz, 1 H); 8.13 (d, J = 7.3 Hz, 1 H). HR-MS (EI): m/z:
+
calcd for C H N O [M + H ]: 1149.77843, found:
58
105 10 13
1
149.80164.
3S,6S,9R,12S,15S,18S,21S,24S)-Methyl 21-((1R,2R,E)-1-
acetoxy-2-methylhex-4-en-1-yl)-24-ethyl-3,12,15-triisobutyl-
(
3) Faulds, D.; Goa, K. L.; Benfiled, P. Drug 1993, 45, 953−995 and
(
references therein.
(4) (a) Wenger, R. M. Pharm. Rev. 1989, 41, 243−247. (b) Angell, Y.
M.; Thomas, T. L.; Flenkle, G. R.; Ricj, D. H. J. Am. Chem. Soc. 1995,
1
4
8-isopropyl-6, 9, 11,14, 17, 20, 26-heptamethyl-
,7,10,13,16,19,22,25-octaoxo-2,5,8,11,14,17,20,23,26-non-
1
17, 7279−7280. (c) Raman, P.; Stokes, S.; Angell, Y. M.; Flentke, G.
R.; Rich, D. H. J. Org. Chem. 1998, 63, 5734−5735.
5) (a) Rich, D. H.; Sun, C.-Q.; Guilliaume, D.; Dunlap, B.; Evans, D.
aazaoctacosan-28-oateHydrogen(tetrafluoridoborate)
(
(
1:1) 5. Compound 4 (20.0 g, 16.2 mmol) was added portion-
A.; Weber, A. E. J. Med. Chem. 1989, 32, 1982−1987. (b) Eberle, M.
K.; Nuninger, F. J. Org. Chem. 1992, 57, 2689−2691. (c) Park, S. B.;
Meier, G. P. Tetrahedron Lett. 1989, 30, 4215−4218. (d) Smulik, J. A.;
Diver, S. T.; Pan, F.; Liu, J. O. Org. Lett. 2002, 4, 2051−2054.
wise to a mixture of water (30 mL), followed by toluene (60
mL) and sodium carbonate (2.8 g, 26.4 mmol). After stirring
for 6 h at 15−20 °C, the phases were split, and
phenylisothiocyanate(2.6 g) was added dropwise to the
remaining organic phase. After 30 min stirring at 20 °C,
methanol (40 mL) was added, followed by aqueous fluoroboric
acid 48% (4.2 g, 23.0 mmol). After 10 h stirring at 20 °C, water
(e) Lazarova, T.; Chen, J. S.; Hamann, B.; Kang, J. M.; Homuth-
Trombino, D.; Han, F.; Hoffmann, E.; McClure, C.; Eckstein, J.; Or, Y.
S. J. Med. Chem. 2003, 46, 674−676. (f) Debiopharm SA,
WO0001715. (g) Debiopharm SA, WO0046239. (g) Traber, R.;
Hofmann, H.; Kobel, H. J. Antibiot. 1989, 42, 591−597. (h) Lawen, A.;
Traber, R.; Geyl, D.; Zocher, R.; Kleinkauf, H. J. Antibiot. 1989, 42,
(
20 mL) were added, and the upper organic phase removed.
Toluene (40 mL) was added to extract more of the hydantoin
byproduct. The washing was repeated twice to remove
hydantoin. Then methyl-tetrahydrofuran (100 mL) was
added. The aqueous phase was removed, and the organic
phase washed with water (20 mL) in order to increase the pH
of the solution. Then, the volume was reduced to decreased the
water content to below 0.5%, the concentration was adjusted to
1
283−1289. (i) Loor, F.; Tiberghien, F.; Wenandy, T.; Didier, A.;
Traber, R. J. Med. Chem. 2002, 45, 4598−4612.
6) (a) Seebach, D.; Beck, A. K.; Bossler, H. G.; Gerber, C.; Ko, S. Y.;
(
Murtiashaw, C. W.; Naef, R.; Shoda, S.; Thaler, A.; Krieger, M.;
Wenger, R. Helv. Chim. Acta 1993, 76, 1564−1590. (b) Papageorgiou,
C.; Kallen, J.; France, J.; French, R. Bioorg. Med. Chem. 1997, 5, 187−
192.
G
dx.doi.org/10.1021/op5003038 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Communication
(
(
7) Submitted for publication.
8) Alisporivir was first claimed generically by Debiopharm in the
product case WO-00001715, which claims compositions comprising a
Cyclosporin A derivative. The synthesis of Alisporivir, a [d-
3
−
4−
MeAla] [EtVal] Cyclosporin A derivative, was initiated from the
starting linear peptide H-MeLeu-Val-MeLeu-Ala-d-Ala-MeLeu-
MeLeu-MeVal-MeBmt(OAc)-Abu-Sar-OMe. The peptide was pre-
pared in four steps, by acylation of Cyclosporin A, ring-opening, the
formation of a phenyl thiourea derivative and the treatment of the
thiourea with trifluoroacetic acid.
(
9) Debiopharm SA EP1793844, JP2008/514690, US2006/252675,
US2008/139463, WO2006/038088.
(
(
10) Novartis Pharma AG WO2013/167703.
11) Rueegger, A.; Kuhn, M.; Lichti, H.; Loosli, H.-R.; Huguenin, R.;
Quiquerez, C.; von Wartburg, A. Helv. Chim. Acta 1976, 59, 1075−
092.
12) Eberle, M. K.; Jutzi-eme, A. M.; Nuninger, F. J. Org. Chem. 1994,
9, 7249−7257.
13) (a) Eberle, M. K.; Nuninger, F. Tetrahedron Lett. 1994, 35,
477−6480. (b) Eberle, M. K.; Nuninger, F.; Weber, H. P. J. Org.
Chem. 1995, 60, 2610−2612.
14) Crystaline compound 3 displays limited stability and rapidly
becomes amorphous upon drying.
15) Crystaline compound 4 displays limited stability and becomes
amorphous almost immediately upon drying. Dry 4 is amorphous.
16) Compound 5 displays limited crystalinity. The main evidence
1
(
5
(
6
(
(
(
comes from the upgrade in the purity and rejection of the main
impurities.
(
17) ACS GCIPR: ACS GCIPR. American Chemical Society, Green
Chemistry Institute. Pharmaceutical Roundtable website http://www.
acs.org/gcipharmaroundtable, 2008. For more information about the
ACS Green Chemistry Institute Pharmaceutical Roundtable Solvent
Selection Guide, see: http://portal.acs.org/portal/PublicWebSite/
greenchemistry/industriainnovation/roundtable/index.htm (accessed
June 2014); green solvent selection guides. (a) Alfonsi, K.; Colberg,
J.; Dunn, P. J.; Fevig, T.; Jennings, S.; Johnson, T. A.; Kleine, H. P.;
Knight, C.; Nagy, M. A.; Perry, D. A.; Stefaniak, M. Green Chemistry
Tools to Influence a Medicinal Chemistry and Research Chemistry
Organisation. Green Chem. 2008, 10 (1), 31−36. (b) Henderson, R.
́ ́
K.; Jimenez-Gonzalez, C.; Constable, D. J. C.; Alston, S. R.; Inglis, G.
G. A.; Fisher, G.; Sherwood, J.; Binks, S. P.; Curzons, A. D. Expanding
GSK’s Solvent Selection Guide − Embedding Sustainability into
Solvent Selection Starting at Medicinal Chemistry. Green Chem. 2011,
1
3 (4), 854−862. (c) Prat, D.; Pardigon, O.; Flemming, H.-W.;
Letestu, S.; Ducandas, V.; Isnard, P.; Guntrum, E.; Senac, T.; Ruisseau,
S.; Cruciani, P.; Hosek, P. Sanofi’s Solvent Selection Guide: A Step
Toward More Sustainable Processes. Org. Process Res. Dev. 2013, 17
(
12), 1517−1525. (d) Prat, D.; Hayler, J.; Wells, A. Survey of solvent
selection guides. Green Chem. 2014, advanced paper.
18) Anastas, P. Green Chemistry: Theory and Practice; Oxford
University Press: Oxford, 1998.
19) Jimenez-Gonzalez, C.; Ponder, C. S.; Broxterman, Q. B.;
(
(
Manley, J. B. Org. Process Res. Dev. 2011, 15, 912−917 and references
therein..
H
dx.doi.org/10.1021/op5003038 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX