Overcoming Chemical Challenges in the Solid-Phase Synthesis of High-Purity GnRH Antagonist Degarelix. Part 1.

Article abstract of DOI:10.1021/acs.oprd.9b00430

The highly potent, long-acting, gonadotropin-releasing hormone antagonist Degarelix is known to be very efficient for prostate cancer treatment. The synthesis of decapeptide Degarelix is complicated because of the presence in its sequence of several unnatural α-amino acids, which are prone to rearrangements and side reactions. In particular, the rearrangement of the dihydroorotic (Hor) moiety with following hydantoin formation in the presence of bases represents one of the major problems. In this study, we describe a novel chemical strategy to overcome this obstacle by the use of the corresponding p-nitrophenylalanine derivative, which is reduced on the solid support to p-aminophenylalanine and acylated with dihydroorotic acid at the end of the solid-phase synthesis. Thus, the contact of Hor with the bases required for Fmoc deprotection is completely avoided. This approach provides a superior purity of Degarelix when the synthesis is carried out in the industrial scale as well.

Full text of DOI:10.1021/acs.oprd.9b00430

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Full Paper
Overcoming chemical challenges in the solid-phase
synthesis of high-purity GnRH antagonist Degarelix
Ivan Guryanov, Andrea Orlandin, Angelo Viola, Barbara Biondi, Denis
Badocco, Fernando Formaggio, Antonio Ricci, and Walter Cabri
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00430 • Publication Date (Web): 12 Nov 2019
Downloaded from pubs.acs.org on November 12, 2019
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Overcoming chemical challenges in the solid-phase synthesis of high-purity
GnRH antagonist Degarelix.
Ivan Guryanov,*,‡,§,ǁ Andrea Orlandin, Angelo Viola, Barbara Biondi, Denis Badocco, Fernando
‡
‡
§
§
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*,‡
‡
Formaggio, Antonio Ricci, and Walter Cabri
‡Fresenius Kabi iPSUM Srl, via San Leonardo 23, Villadose (RO), 45010 Italy
§ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, Padova, via Marzolo 1,
3
5131 Italy
ǁSt. Petersburg State University, Institute of Chemistry, St. Petersburg, Peterhof, Universitetskij pr.
2
6, 198504 Russia
Keywords: degarelix, dihydroorotic acid, Fmoc deprotection, peptide, reduction
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ABSTRACT
The highly potent, long-acting, gonadotropin-releasing hormone antagonist Degarelix is known to
be very efficient for prostate cancer treatment. The synthesis of the decapeptide Degarelix is
complicated because of the presence in its sequence of several unnatural α-amino acids, which are
prone to rearrangements and side reactions. In particular, the rearrangement of the dihydroorotic
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(Hor) moiety with following hydantoin formation in the presence of the bases represents one of the
major problems. In this study we describe a novel chemical strategy to overcome this obstacle by the
use of the corresponding p-nitrophenylalanine derivative, which is reduced on the solid support to p-
aminophenylalanine and acylated with dihydroorotic acid at the end of the solid-phase synthesis.
Thus, the contact of Hor with the bases required for Fmoc deprotection is completely avoided. This
approach provides a superior purity of Degarelix when the synthesis is carried out in industrial scale
as well.
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INTRODUCTION
Cancer is one of the major public health problems worldwide. According to the World Health
Organization, globally, nearly 1 in 6 deaths is due to cancer that is responsible for an estimated 9.6
1
million deaths in 2018. Among other types of malignancies, prostate cancer accounts about 20% of
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new diagnoses in men and it is the third most common cause of cancer death in US. The treatment
of prostate cancer depends on the stage of the disease and includes radical prostatectomy, external
beam radiotherapy or brachytherapy in organ-confined case and androgen-deprivation therapy
ADT) at the metastatic stage.3
(
The principal mechanism of ADT is the prevention of androgen receptor signalling, which is
involved in the growth of the cancer cells. Generally, it can be achieved by (1) direct blocking of
androgen receptors or (2) suppression of circulating testosterone through the action of the
gonadotropin-releasing hormone (GnRH) agonists (via negative feedback loop) or GnRH
antagonists. This class of molecules, blocking the GnRH receptors in the pituitary, prevents the
production of the luteinizing hormone (LH) and downstream production of testosterone.4,5
According to the latter strategy, several GnRH agonists, such as Leuprolide, Goserelin, Triptorelin,
and two antagonists (Abarelix and Degarelix) were developed for the prostate cancer treatment.6-8 The
noticeable allergic effects of Abarelix led to its withdrawal from the US market. On the contrary,
Degarelix was shown to have a significantly lower risk of histamine release than Abarelix and to induce
almost immediate and long-lasting decrease of serum testosterone concentration. After the approval by
FDA in 2008, it became widely used in patients with advanced prostate cancer.9
Degarelix, formerly known as FE200486, is a synthetic, linear decapeptide chemically
described as N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-
alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-dioxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-
[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl-D-
alaninamide (Figure 1).
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O
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NH2
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Figure 1. Chemical structure of Degarelix.
This peptide molecule contains several unnatural α-amino acids, five of which are D-amino
acids. It is currently marketed as the acetate salt with the brand name Firmagon (EU and US) or
Gonax (Japan). After reconstitution with water and subcutaneous injection, it forms a gel-like depot,
which provides sustained release of the drug and ensures its prolonged action.
The high proteolytic stability of Degarelix and the resistance to the fast glomerular clearance
are due to the unique combination in its sequence of the hydrophilic and hydrophobic amino acids.
The hydrophobic residues allow non-covalent binding to the cell membranes and hydrophobic
carrier proteins, such as serum albumin, and protection against proteolytic degradation (“whole
body depot effect”). On the contrary, the presence of the hydrophilic residues, in particular, 4-
[(aminocarbonyl)amino]-phenylalanine [Aph(Cbm)] and 4-[(dihydroorotyl)amino]-phenylalanine
[
Aph(Hor)], with their strong ionic and H-bonding interactions, enhances water solubility of
Degarelix and reduces its tendency to form gels with respect to other potent GnHR antagonists (such
as Acyline).10 However, these substitutions still permit self-association with the formation of
amyloid” type fibrils, which dissolve over a long period (amyloid t1/2 = 15 days).10,11 For these
“
reasons, Degarelix shows a prolonged bioavailability and duration of action if compared to other
derivatives of the same therapeutic class.
The presence of a number of the amino acids with particular properties makes the preparation
of Degarelix rather challenging. Aph residues can be introduced into the sequence of Degarelix both
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as side-chain modified Aph(Cbm) or Aph(Hor), or as aniline precursors with orthogonal protecting
groups. The initial scheme of Degarelix synthesis, developed by the Ferring Research Institute, was
based on the tert-butyloxycarbonyl (Boc) solid-phase peptide synthesis (SPPS) with the stepwise
elongation of the peptide sequence and the final cleavage of the peptide from the resin by the
treatment with HF in the presence of anisole as a scavenger.12,13 All of the amino acids were coupled
as ready-to-use derivatives, with the exceptions of Aph(Cbm) and Aph(Hor), which were generated
from the corresponding anilines by Fmoc deprotection followed by the reaction with tert-butyl
isocyanate and Hor, respectively. Though this method allowed obtaining Degarelix in high yields, it
was not suitable for the industrial production of the peptide because of the involvement of hazardous
and environmentally unfriendly reagents. Therefore, various alternative synthetic schemes were
proposed for Degarelix preparation, mostly based on the Fmoc/tBu (tert-butyl) strategy, which
allows milder reaction conditions.14-26 In this case, both solid- and liquid-phase approaches, or
combination thereof, could be applied. Moreover, the high potency of Degarelix inspired research of
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the peptide analogs with enhanced activity by substitution of some amino acids and/or side-chain
modifications with various success.27-29
One of the major challenges in the preparation of Degarelix as an active pharmaceutical
ingredient (API) is the high sensitivity of the Hor moiety to the presence of the bases. In this case, a
rapid rearrangement of its six-membered ring (with hydrolysis to the N-carbamoylaspartyl
intermediate) to the five-membered ring hydantoin takes place (Scheme 1).30-33
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O
O
HN
-
OH
H
NH2
H
OH /H2O
N
N
O
N
O
N
H
H
O
O
DIHYDROOROTIC ACID
DERIVATIVE
-H2O
O
N
NH2
O
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H
-
O
OH /H O
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O
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N
ASPARTIMIDE
DERIVATIVE
N
H
O
H
OH
HN
O
HN
-
H2O
O
NH
NH2
O
HYDANTOIN
DERIVATIVE
Scheme 1. Rearrangement of the Hor residue in the presence of bases.
Due to the high similarity of the chemical structure of the hydantoin to Hor, the identification
of the hydantoin impurity in Degarelix samples is very difficult (e.g., HPLC retention time of the
hydantoin impurity is very close to that of Degarelix in almost all of the chromatographic
conditions). For the same reason, this impurity noticeably complicates the HPLC purification of the
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final peptide to get the purity necessary for the release of Degarelix API in the market. This
unfortunate situation has to be taken into account for the preparation of Degarelix using the Fmoc-
protecting group and the subsequent repetitive deprotection cycles with the organic bases. For
1
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example, in the recent patent of Zhang et al. it was shown that the treatment of Degarelix with a
2% solution of 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in DMF, which is often used in SPPS for
Fmoc deprotection, resulted in the formation of 1.8% hydantoin. The amount of the impurity
increased up to 7%, when 5% of water was added. These results suggest that the use of high-purity
reagents is required when the peptide is synthesized by the standard SPPS technique.
The development of methods for the preparation of the crude peptide Degarelix without the
hydantoin impurity will significantly lower the cost of production in a large scale with high yields.
From this point of view, the formation of the Aph derivatives from the corresponding Phe(p-NO2)
residue in SPPS could be a promising approach for introduction of the Aph(Hor) and/or Aph(Cbm)
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residues into the Degarelix sequence, in particular for the large-scale production of the peptide. In
this case, the introduction of the Hor moiety can be carried out at the very last step of the synthesis,
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thus avoiding its contact with the bases. The reduction methods of the aromatic nitro group often
involve activated metal catalysis or transition metal catalyzed hydrogenation, where other functional
groups can be affected.36,37 Consequently, many of these methods are not applicable in SPPS.
Sodium dithionite and stannous chloride showed to be promising alternatives to the catalytic
hydrogenation.38,39 They can function as reducing agents for nitro derivatives on solid supports and
allow a complete conversion to the corresponding anilines. However, in the case of sodium
dithionite, a phase-transfer catalyst, such as tetrabutylammonium hydrogen sulfate (TBAHS), and a
water/organic solvent mixture are needed to ensure the transfer of the inorganic salt to the organic
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phase, which contains molecule to be reduced attached to the solid support. On the contrary, the
reaction with stannous chloride can be carried out with high yields in a homogeneous way in an
organic solvent in the presence of DIPEA or DBU.39,41
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In our effort to discover innovative syntheses of high-purity Degarelix, here we describe a
novel approach, where the rearrangement of the Hor moiety of Degarelix is suppressed or absent
(Scheme 2). It comprises the introduction of the Hor moiety at the very last step of the synthesis,
when the treatment with the bases for Fmoc-deprotection is not required any more. This approach
can increase the purity of the crude peptide, thus facilitating its purification and increasing its final
yield, which is particularly important for the industrial production. Moreover, it can facilitate the
preparation of other active pharmaceutical ingredients characterized by the Hor moiety as well.
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Scheme 2. General scheme of the Degarelix synthesis via Phe(p-NO ) reduction on the solid
2
support.
RESULTS AND DISCUSSION
In order to find suitable conditions for the reduction of the nitro group on the solid support,
two approaches were used: sodium dithionite- and stannous chloride-mediated reductions. In the
first case, the phase-transfer reagent (TBAHS) was introduced into the reaction mixture in order to
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afford the transfer of the salt to the organic phase. The efficiency of the reduction of the Phe(p-
NO ) residue was initially investigated using model pentapeptide with the sequences comprising the
2
C-terminal segment of Degarelix (Table 1).
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Table 1. Reduction of the Phe(p-NO ) residue under various conditions
2
Nitro group
reduction, %
Fmoc
deprotection, %
Entry
Chemical structure
Conditions
0.5 mL of DMF + 0.5 mL of
stock solution**, 2 h
1
2
0
-
Fmoc-D-Phe(p-NO
Lys(Boc, iPr)-Pro-D-Ala-NH-
2
)-Leu-
0.5 mL of DCM + 0.5 mL of
stock solution, 2 h
0
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100
12
-
resin*
2.5 mL of SnCl
in DMF) with 1.5 eq of DIPEA
70 μL), 12 h
2 2
·2H O (1.5 M
3
100
(
*200 mg of peptide resin, loading 0.65 mmol/g.
2 3
**Stock solution: Na 1050 mg (5 mmol), K CO 970 mg (7 mmol), TBAHS 170 mg (0.5 mmol) in 5 mL of water.
2
S
2
O
4
The reduction of the nitro group with sodium dithionite in DCM-water mixture occurred with
high yield in 2 h (Table 1, Entry 2). Nevertheless, a partial Fmoc deprotection was seen due to the
presence of the potassium carbonate in the mixture and the alkaline environment. Interestingly, the
starting material was completely recovered in the case of DMF-water (Table 1, Entry 1) and no
traces of the corresponding Aph were found. On the contrary, during the reduction of the nitro group
with stannous chloride, no Fmoc cleavage was observed and the reaction was completed in 12 h
(Table 1, Entry 3 and Figure 2). The appearance of p-aminophenylalanine in the amino acid
sequence caused the increase of hydrophilicity and the decrease of the retention time of the peptide.
This last approach demonstrated the feasibility of the designed reduction strategy of the Phe(p-NO2)
on the solid phase.
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Figure 2. HPLC chromatograms of Fmoc-D-Phe(p-NO )-Leu-Lys(iPr)-Pro-D-Ala-NH (A), and
2
2
Fmoc-D-Aph-Leu-Lys(iPr)-Pro-D-Ala-NH (B) (for details, see Experimental Part; analytical
2
method 1).
According to this, we started evaluating also the construction of the Aph(Cbm) residue in the
peptide already attached to the solid support (Scheme 2, step 3). In the synthesis proposed by the
Ferring Research Institute, the Cbm functionality was introduced by the reaction of the Aph side-
chain free amino group with tert-butyl isocyanate for 12 h with formation of the corresponding tert-
butyl protected urea derivative.12 Nevertheless, upon completion of the synthesis of Degarelix we
found that the complete removal of the tert-butyl group from the Aph(Cbm) residue required the
treatment of the peptide with TFA over a long time, when we observed a partial degradation of the
peptide (data not shown). Therefore, we applied another approach, where potassium cyanate is used
in the presence of acetic acid to liberate in situ cyanic acid, which can react with the free amino
4
3
group with formation of the Cbm moiety. The use of the solution of potassium cyanate in diluted
acetic acid gave unsatisfactory results since, after treatment of the peptide overnight, only about
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10% of the Cbm peptide derivative was found in the reaction mixture (Figure S1, A). By performing
the same reaction in glacial acetic acid, the conversion was complete but, along with the expected
product, a compound with m/z Δmass -1 was found (Figure S1, B). The hypothesis is that the
interaction of acetic acid and cyanic acid turned into the formation of the mixed anhydride, leading
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to acetylation of the amino group. Indeed, the replacement of acetic acid with a 20% solution of
phosphoric acid to afford cyanic acid resulted in the formation of the desired peptide in good HPLC
purity (Figure S1, C), demonstrating by that the correctness of the hypothesized side reaction and
the possibility to build up the Aph(Cbm) amino acid via the Phe(p-NO ) intermediate.
2
Lastly, in order to evaluate the possibility of the construction of the Aph(Hor) moiety during
the SPPS protocol, two SPPS syntheses of Degarelix were performed: (1) the reduction of the nitro
group followed by the Hor moiety incorporation at the step of the hexapeptide and (2) the reduction
and Hor insertion at the end of the synthesis of the peptide chain (Figure 3).
Figure 3. HPLC runs of crude Degarelix, prepared with the nitro group reduction and incorporation
of the Hor moiety at the level of the hexapeptide (3 g of Rink amide resin, A) and at the end of
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solid-phase synthesis (500 g of Rink amide resin, B) (for details, see Experimental Part; analytical
method 2).
The comparison of the HPLC runs reported in Figure 3 shows a higher overall purity of the
Degarelix obtained via the second approach with respect to the one resulting from the coupling of
the dihydroorotic acid before elongation of the Degarelix sequence (87% vs 75%) with the amount
of hydantoin impurity less than 0.15%. This behavior is related to the sensitivity of the Hor moiety
to the SPPS conditions, that enhance the Hor-to-hydantoin isomerism, and to the acetylation of the
Hor moiety, when it is present during N-terminal capping. This evidence confirms the validity of the
process as a robust manufacturing strategy for the large-scale production of the peptide, especially
with insertion of the Hor at the end of the Degarelix synthesis.
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An important aspect to be carefully evaluated while using tin(II) chloride for the industrial
applications is regulatory requirements. Accordingly to the regulatory agencies new guidance, tin(II)
chloride, albeit considered safer respect to other toxic metals, has to be absent or present at low ppm
45
level in the final API. For this reason, the Degarelix API and some its intermediates were analyzed
by the inductively coupled plasma mass spectrometry (ICP-MS) to evaluate the amount of residual
tin salts. The results showed that tin content is only 0.7% (7000 ppm) in the crude Degarelix and it is
below the limit of quantification (0.31 ppb) for the peptide API after HPLC purification (Table 2).
Thus, tin(II) chloride excess used at the step of nitro group reduction can be successfully eliminated
without the use of scavengers or special purification steps (Table 2).
Table 2. ICP-MS analysis results of Degarelix and its intermediates
Sample
)-Leu-Lys(Boc,iPr)-Pro-D-Ala-Resin
Sn content, % (w/w)
<LOQ
Fmoc-Rink amide resin
Fmoc-D-Phe(p-NO
2
<LOQ
Crude Degarelix
Degarelix after HPLC purification
0.700±0.018
<LOQ
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Finally, the identity of Degarelix obtained by the approach reported herein was confirmed by
1H and homo- and heteronuclear NMR analysis by complete assignment of the NMR peaks (Tables
S1, S2, Figures S2-8, the data are shown for the API produced with the process comprising the Hor
attachment at the last step of the synthesis).
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CONCLUSIONS
In summary, this work describes the novel approach for the solid-phase synthesis of the GnRH
antagonist Degarelix, which helps to prevent the rearrangement of the Hor moiety in the presence of
the organic bases. It comprises the reduction of the nitro group-containing precursor of the peptide
and subsequent incorporation of the Hor moiety in the very last step of the synthesis, avoiding by
that the contact of Hor with the standard chemicals used in the SPPS. The here described optimized
process was successfully scaled-up to 500 g of the starting resin, providing Degarelix API with a
very high HPLC purity. This approach can be used for the preparation not only of Degarelix, but
also of other peptides of potential pharmaceutical interest containing the Hor moiety in their amino
acid sequence.
EXPERIMENTAL PART
Materials and Methods
Iris Biotech: N,N-dimetylformamide (DMF), dichloromethane (DCM), N,N-diisopropylethylamine
(DIPEA), trifluoroacetic acid (TFA), piperidine, Fmoc-protected Rink amide resin; Sigma Aldrich:
acetonitrile (MeCN) for mass spectrometry (MS) (>99,9%), TFA for MS (>99,9%), methyl tert-
butyl ether (MTBE), triisopropylsilane (TIS), acetic anhydride, tetrabutylammonium hydrogen
sulfate (TBAHS); Carbosynth: Ethyl (hydroxyimino)cyanoacetate (Oxyme Pure), N,N′-
diisopropylcarbodiimide (DIC); GL Biochem: Fmoc-Nal-OH, Fmoc-Cpa-OH, Fmoc-Pal-OH, Fmoc-
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Ser(tBu)-OH, Fmoc-Phe(p-NO )-OH, Fmoc-D-Phe(p-NO )-OH, Hor, Fmoc-D-Aph(Cbm)-OH,
2
2
Fmoc-Leu-OH, Fmoc-Lys(Boc, iPr)-OH, Fmoc-Pro-OH, Fmoc-D-Ala-OH, Fmoc-Lys(Boc)-OH.
HPLC and UPLC chromatographies: HPLC and UPLC analyses were performed on Agilent
Technologies 1200 and Agilent Technologies 1290 instruments, respectively, using C18 Zorbax
Eclipse Plus column (4.6×50 mm, 1.8 μm) for HPLC analyses and Extended C18 Agilent column
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(
4.6×50 mm, 5 μm) for UPLC analyses. Analytical method 1: eluent A, TFA/H O 0.1% v/v; eluent
2
B: TFA/MeCN 0.07% v/v; detection at 224 or 215 nm; gradient mode: 0 min – 25% of eluent B, 20
min – 45% of eluent B. Analytical method 2: gradient mode: 0 min – 25% of eluent B, 30 min –
4
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5% of eluent B. The preparative purification was carried out using Phenomenex Luna C18 250 ×
1.2 mm column and the eluent A, TFA/H O 0.1% v/v; eluent B: water/MeCN 10/90 (v/v) with
2
0.07% TFA. The following gradient elution was applied: 0 min – 17% of eluent B, 5 min – 15% of
eluent B, 15 min – 35% of eluent B, 45 min – 50 of eluent B, 47 min – 50 of eluent B.
Mass spectrometry analyses: mass spectra were acquired on a Mariner API-ToF workstation
(PerSeptive Biosystems, Framingham, MA) and in the LC-MS mode on Agilent 6530 mass accuracy
Q-ToF, operating in the positive mode.
ICP-MS analysis: inductively coupled plasma mass spectrometry analysis (ICP-MS) for the
determination of the Sn content was carried out with an Agilent Technologies 7700x ICP-MS
(Agilent Technologies International Japan, Ltd., Tokyo, Japan). ICP-MS instrument was equipped
with an octopole collision cell operating in kinetic energy discrimination mode used for the removal
of polyatomic interferences and argon-based interferences. The optimal performance was obtained
4
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by using the collision cell in He mode for Au and He-He mode for Sn. The ICP-MS was tuned
daily using a tuning solution containing 1 μg/L 140Ce, Li, Tl, and Y (Agilent Technologies,
7
205
89
UK). A 100 μg/L solution (Aristar, BDH, UK) containing 115In, prepared in HNO 3.5% was used as
3
internal standard through addition to the sample solution via a T-junction.
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Multi-element calibration standard CCS-5 “Inorganic ventures” containing B, Ge, Hf, Mo, Nb,
P, Re, S, Sb, Si, Sn, Ta, Ti, W, Zr (100 μg/mL) in aqueous HF 1.0 % (v/v) and HNO 7.14 % (v/v)
3
was used for Sn determination. Multi-element calibration solutions were prepared by gravimetric
serial dilution at five different concentrations from 250 ng/L to 100 μg/L using 3.5% HNO solution
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in MilliQ water. The parameters of the calibration lines were obtained by using the Theil-Sen non-
parametric regression technique.47
In a typical experiment about 2 mg of the sample were accurately weighed and digested using
microwaves in 5 g of 69% solution of nitric acid accordingly to the following procedure: ramp
temperature from RT to 220 °C in 5 min, then 220 °C for 5 min, pressure 400 PSI, power 300 W and
stirring mode “medium”. Then the solutions were diluted with the same solvent used for calibrations
and analyzed.
NMR analysis: all NMR experiments were carried out at 298 K in DMSO-d solution (peptide
6
concentration 1.2 mM) using a Bruker AVANCE DRX-400 spectrometer and the TOPSPIN
software package. The homonuclear spectra were acquired by collecting 512 experiments consisting
of 64–80 scans and 2 K data points. The spin systems of coded amino acid residues were identified
using standard DQF-COSY spectra. In the latter case, the spin-lock pulse sequence was 70 ms long.
ROESY experiments were utilized for sequence-specific assignment. At the beginning of the
experiments the build-up curve of the volumes of NOE cross-peaks as a function of the mixing time
(
50–500 ms) was obtained in order to avoid the problem of spin diffusion. The mixing time of the
ROESY experiment used for interproton distance determination was 150 ms (i.e., in the linear part
of the NOE build-up curve). To fully assign all the resonances also heteronuclear experiments,
HMQC and HMBC were acquired.
Peptide synthesis
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Small scale synthesis. Solid-phase syntheses of the peptides and intermediates in a small scale were
carried out using Biotage Syrowave (automated syntheses). In a typical procedure 0.2 g of the Rink
amide resin with the loading of 0.65 mmol/g were used. The resin was swollen in 2 mL of DMF for
30 min, Fmoc deprotected with 20% piperidine in DMF (two cycles for 5 and 20 min, respectively),
and washed 4 times with 2 mL of DMF. The solid-phase synthesis was carried out using 3 eq of
protected amino acids that were preactivated during 3 min with 3 eq of Oxyma Pure and 3 eq of
DIC, and subsequentry coupled in 1 h. The unreacted peptide chains were capped with acetic
anhydride (10 eq) in the presence of DIPEA (10 eq). The cycles of coupling and deprotection were
continued to obtain the desired peptide sequences. At the end of the synthesis, the peptide was
cleaved from the resin with 3 mL of the mixture TFA/TIS/water (95:2.5:2.5, v/v) and precipitated in
10 mL of MTBE. The product was filtered off and dried.
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Synthesis of Degarelix with nitro-group reduction during SPPS. The synthesis was carried out using
Biotage MultiSynTech semi-automated synthesizer and 3 g of Rink amide resin (loading 0.65
mmol/g). The resin was swollen in 18 mL of DMF for 30 min, Fmoc deprotected with 20%
piperidine in DMF (two cycles for 5 and 20 min) and washed 4 times with 18 mL of DMF. The
solid-phase synthesis was carried out using 3 eq of protected amino acids that were preactivated
during 3 min with 3 eq of Oxyma Pure and 3 eq of DIC, and subsequentry coupled in 1 h. The
cycles of coupling and deprotection were continued to obtain Fmoc-Phe(pNO )-D-Aph(Cbm)-Leu-
2
Lys(iPr, Boc)-Pro-D-Ala-Rink amide resin. Then the resin was treated with a solution of tin(II)
chloride dihydrate (4.4 g, 17.7 mmol) in 14.5 ml of DMF in the presence of DIPEA (0.40 mL, 2.3
mmol). The reaction was carried out overnight under nitrogen bubbling and the resin was washed
with DMF (2×18 mL), a solution of 1 eq of DIPEA in 18 mL of DMF and DMF (2×18 mL). The
obtained Fmoc-Aph-D-Aph(Cbm)-Leu-Lys(iPr, Boc)-Pro-D-Ala-Rink amide resin was then treated
with a solution of Hor-OH (1.5 eq), diisopropylcarbodiimide (1.5 eq) and HOBt (1.5 eq) in 18 mL of
DMF. After 1.5 h the solvent was filtered and the resin was washed with DMF (2×18 mL) to have
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Fmoc-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr, Boc)-Pro-D-Ala-Rink amide resin. The synthesis was
then continued to get H-D-Nal-D-Cpa-D-3-Pal-Ser(tBu)-Aph-D-Aph(Cbm)-Leu-Lys(iPr, Boc)-Pro-
D-Ala-Rink resin. The unreacted peptide chains and Nal residue were acetylated with acetic
anhydride (10 eq) in the presence of DIPEA (10 eq). At the end of the synthesis, the peptide was
cleaved from the resin with 20 mL of the mixture TFA/TIS/water (95:2.5:2.5, v/v) and precipitated
in 100 mL of MTBE. The product was filtered off, washed several times with MTBE and dried.
Yield: 1.53 g (48%). The identity of the intermediates obtained was confirmed by ESI MS analysis
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after small scale cleavage or complete cleavage for the final product:
+
= 866.48, [M+H]+
H-Phe(pNO )-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH : [M+H]
=
exp
2
2
calc
8
66.51
+
= 835.51, [M+H]+ = 835.46
H-Aph-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH : [M+H]
2
calc
exp
+
= 976.53, [M+H]+exp = 976.47
calc
H-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH : [M+H]
2
Degarelix: [M+H]+ = 1631.75, [M+H]+ = 1631.73
calc
exp
Synthesis of Degarelix with nitro-group reduction at the end of SPPS. The synthesis was carried out
using CS-Bio 936 system and 500 g of Rink amide resin (loading 0.65 mmol/g). The resin was
swollen in 3 L of DMF for 30 min, Fmoc deprotected with 20% piperidine in DMF (two cycles for 5
and 20 min) and washed 4 times with 3 L of DMF. The solid-phase synthesis was carried out using
3
eq of protected amino acids that were preactivated during 3 min with 3 eq of Oxyma Pure and 3 eq
of DIC, and subsequentry coupled in 1 h. The cycles of coupling and deprotection were continued to
obtain Ac-D-Nal-D-Cpa-D-3-Pal-Ser(tBu)-Phe(pNO )-D-Aph(Cbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-
2
Rink resin, which was treated with a solution of tin(II) chloride dihydrate (733 g, 3.24 mol) in 2.5 L
of DMF in the presence of DIPEA (66 mL, 0.37 mol). The reaction was carried out overnight under
nitrogen bubbling. The Ac-D-Nal-D-Cpa-D-3-Pal-Ser(tBu)-Aph-D-Aph(Cbm)-Leu-Lys(iPr,Boc)-
Pro-D-Ala-Rink resin was then washed with DMF (2×3 L) than with 1 eq of DIPEA in 3 L of DMF
and washed with DMF (2x3 L). A solution of Hor-OH (1.5 eq), diisopropylcarbodiimide (1.5 eq)
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and HOBt (1.5 eq) in 3 L of DMF was added to the resin and stirred for 1.5 h. The solvent was
filtered off and the resin was washed with DMF (2×3 L), DCM (2×3 L) and dried. At the end of the
synthesis the peptide was cleaved from the resin with 7.5 L of the mixture TFA/TIS/water
(95:2.5:2.5 v/v) and precipitated in 22 L of MTBE. The product was filtered off and dried. Yield:
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91 g (55%). A part of the crude peptide was purified using preparative HPLC for further ICP-MS
and NMR analysis. The identity of the peptides obtained was confirmed by ESI MS after small scale
cleavage or complete cleavage for the final product:
[M+H]+calc
Ac-Nal-Cpa-Pal-Ser-Phe(p-NO )-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH :
=
2
2
1
521.70, [M+H]+ = 1521.72
exp
Ac-Nal-Cpa-Pal-Ser-Aph-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2: [M+H]+
=
1491.73,
calc
[M+H]+ = 1491.74
exp
Degarelix: [M+H]+calc = 1631.75, [M+H]+exp = 1631.73
Reaction of the peptide resin with potassium cyanate. The Fmoc-D-Aph-Leu-Lys(iPr, Boc)-Pro-D-
Ala-Rink amide resin was treated with a solution of KOCN (130 mg, 1.6 mmol, 10 eq) in 2.5 mL of
1
0% acetic acid, glacial acetic acid or a solution of 20% phosphoric acid in DMF. The reaction was
carried out overnight and the peptide-resin was washed with DMF (3×2 mL). A sample of the
peptide was cleaved using the mixture 1% water/TFA for HPLC analysis. The identity of the peptide
obtained was confirmed by ESI MS analysis after small scale cleavage:
+
= 896.50, [M+H]+ = 896.52
exp
Fmoc-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH : [M+H]
2
calc
ASSOCIATED CONTENT
Supporting information
HPLC profiles of the reaction of the pentapeptide with potassium cyanate solution and NMR spectra
1
13
of Degarelix with the tables containing H and C chemical shift values of Degarelix molecule.
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REFERENCES
(1) World Health Organization, http://www.who.int/mediacentre/factsheets/fs297/en/.
(
2) Siegel, R. L.; Miller K. D.; Jemal, A. Cancer Statistics, 2017. CA Cancer J. Clin. 2017, 67, 7-
30.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(3) Doehn, C.; Sommerauer, M.; Jocham, D. Degarelix and its Therapeutic Potential in the
Treatment of Prostate Cancer. Clin. Interv. Aging 2009, 4, 215-223.
(4) Clinton, T. N.; Woldu, S. L.; Raj, G. V. Degarelix Versus Luteinizing Hormone-Releasing
Hormone Agonists for the Treatment of Prostate cancer. Expert Opin. Pharmacother. 2017,
1
8, 825-832.
(
5) Friedlander, T. W.; Ryan, C. Novel Hormonal Approaches in Prostate Cancer. Curr. Oncol.
Rep. 2009, 11, 227-234.
(
6) Hoda, M. R.; Kramer, M. W.; Merseburger, A. S.; Cronauer, M. V. Androgen Deprivation
Therapy with Leuprolide Acetate for Treatment of Advanced Prostate Cancer. Expert Opin.
Pharmacother. 2017, 18, 105-113.
(7) Merseburger, A. S.; Hupe, M. S. An Update on Triptorelin: Current Thinking on Androgen
Deprivation Therapy for Prostate Cancer. Adv. Ther. 2016, 33, 1072-1093.
(8) Mongiat-Artus, P.; Teillac, P. Abarelix: the First Gonadotrophin-Releasing Hormone
antagonist for the treatment of prostate cancer. Expert Opin. Pharmacother. 2004, 5, 2171-
2
179.
(9) Steinberg, M. Degarelix: a Gonadotropin-Releasing Hormone Antagonist for the Management
of Prostate Cancer. Clin. Therapeutics 2009, 31, 2312-2331.
(
10) Nestor, J. J. The Medicinal Chemistry of Peptides. Curr. Med. Chem. 2009, 16, 4399-4418.
11) Zhou, N.; Gao, X.; Lv, Y.; Cheng, J.; Zhou W.; Liu, K. Self-Assembled Nanostructures of
Long-Acting GnRH Analogs Modified at Position 7. J. Pept. Sci. 2014, 20, 868-875.
(
2
0
ACS Paragon Plus Environment

Page 21 of 24
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
(12) Sample, G.; Jiang, G. GnRH Antagonists Being Modified in Positions 5 and 6. WO 98/46634
A1, October 22, 1998.
(
13) Jiang, G.; Stalewski, J.; Galyean, R.; Dykert, J.; Schteingart, C.; Broqua, P.; Aebi, A.; Aubert,
M. L.; Semple, G.; Robson, P.; Akinsanya, K.; Haigh, R.; Riviere, P.; Trojnar, J.; Junien J. L.;
Rivier, J. E. GnRH Antagonists:ꢀ a New Generation of Long Acting Analogues
Incorporating p-Ureido-Phenylalanines at Positions 5 and 6. J. Med. Chem. 2001, 44, 453-467.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(14) Zhang, H.; Fomsgaarg, J.; Staerkaer, G. Method for the Manufacture of Degarelix.
WO2010121835 A1, October 28, 2010.
(15) Tovi, A.;ꢁEidelman, C. Process for Production of Degarelix. WO2011066386 A1, June 03,
2
011.
(16) Youjin, C.; Fei, L.; Shengkun, X.; Jian, L.; Yaping, M.; Jiancheng, Y. Solid-Phase Synthetic
Process for Degarelix. CN102329373 A, January 25, 2012.
(
17) Kalita, D.; Layek, M.; Rao, A. V. D.; Potula, V. A.; Gajare, V; Balakumaran, K.; Nilsson, A.;
Jiang, G. Process for the Manufacture of Degarelix and its Intermediates. WO2012055903 A1,
May 03, 2012.
(
18) Rasmussen, J. H.; Fomsgaard, J.; Hansen, S.; Rasmussen, P. H.; Wachs W. O. Process for the
Manufacture of Degarelix and its Intermediates. WO2012055905 A1, May 03, 2012.
19) Schwach, G.; Nilsson, A.; Gottschalk, B.; Tine, E.; Rasmussen, J. H.; Moernstam, B.; Tsirk,
A.; Annby U.; Fomsgaard, J. Manufacture of Degarelix. WO2013178788 A2, December 05,
(
2
013.
(20) Li B.; Yuan, Q.; Chen, M.; Fang, S.; Dong, X.; Li, N. Method for Synthesizing Degarelix.
CN102952174 A, March 06, 2013.
(21) Yun, X.; Wu, S.; Yuan, J,; Zhang, W.; Chen, C. Synthesis of Degarelix by Solid-Phase
Segment Method. CN103351428 A, October 16, 2013.
2
1
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Organic Process Research & Development
Page 22 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
2
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3
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3
3
3
3
3
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(22) Dong, S.; Cao, S.; Li, Z.; Ge, X. Solid-Phase Synthesis Method of Degarelix. CN103992392
A, August 20, 2014.
(
23) Li, X.; Dong, H.; Guo, D.; Zeng, D.; Wen, Y. Method for Synthesizing Degarelix.
CN104177478 A, December 03, 2014.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
24) Zhongyong W.; Xiang, L.; Qi, X. Preparation Method for Degarelix. CN105085634 A,
November 25, 2015.
(25) Guo, D.; Zeng, D.; Tong, G.; Wen, Y. Method for Synthesizing Degarelix. US2017260247,
CN105524143, September 14, 2017.
(26) Xu, F.; Gu, H.; Wang, X.; Zhou, J.; Sun, M. Synthetic Method of Degarelix. CN106589071 A,
April 26, 2017.
(27) Samant, M. P.; Gulyas, J.; Hong, D. J.; Croston, G.; Rivier C.; Rivier, J. Iterative Approach to
the Discovery of Novel Degarelix Analogues:ꢀ Substitutions at Positions 3, 7, and 8. Part II. J.
Med. Chem. 2005, 48, 4851-4860.
(28) Samant, M. P.; Gulyas, J.; Hong, D. J.; Croston, G.; Rivier C.; Rivier, J. Synthesis, in Vivo
and in Vitro Biological Activity of Novel Azaline B Analogs. Bioorg. Med. Chem. 2005, 15,
2
894-2897.
(29) Samant, M. P.; Hong, D. J.; Croston, G.; Rivier C.; Rivier, J. Novel Gonadotropin-Releasing
Hormone Antagonists with Substitutions at Position 5. Biopolymers 2005, 80, 386-391.
(30) Meusel, M.; Gütschow, M. Recent Developments in Hydantoin Chemistry. A Review. Org.
Prep. Proced. Int. 2004, 36, 391-443.
(31) Kaneti, J.; Kirby, A. J.; Koedjikov A. H.; Pojarlieff, I. G. Thorpe–Ingold Effects in
Cyclizations to Five-Membered and Six-Membered Rings Containing Planar Segments. The
Rearrangement of N(1)-Alkyl-Substituted Dihydroorotic Acids to Hydantoinacetic Acids in
Base. Org. Biomol. Chem. 2004, 2, 1098-1103.
2
2
ACS Paragon Plus Environment

Page 23 of 24
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
(32) Koedjikov, A. H.; Blagoeva, I. B.; Pojarlieff I. G.; Stankevic, E. β-Ureido Acids and
Dihydrouracils. Part 15. Effect of Allylic Strain on Ring Opening of 1,6-Disubstituted
Dihydrouracils. J. Chem. Soc., Perkin Trans. II 1984, 1077-1081.
(33) Sander, E. G. The Alkaline Hydrolysis of the Dihydropyrimidines. J. Am. Chem. Soc. 1969,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
1, 3629-3634.
34) FDA Page on ANDAs for Certain Highly Purified Synthetic Peptide Drug Products that Refer
to Listed Drugs of rDNA Origin.
(
https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guida-
nces/UCM578365.pdf
(
35) Bellucci, M. C.; Sacchetti, A.; Volonterio, A. Multicomponent Approach to Libraries of
Substituted Dihydroorotic Acid Amides. ACS Comb. Sci. 2019, 21, 705-715.
36) Orlandi, M.; Brenna, D.; Harms, R.; Jost, S.; Tenaglia, M. Recent Developments in the
Reduction of Aromatic and Aliphatic Nitro Compounds to Amines. Org. Process Res. Dev.
2018, 22, 430-445.
(
(37) Downing, R. S.; Kunkeler, P. J.; van Bekkum, H. Catalytic Syntheses of Aromatic Amines.
Catal. Today 1997, 37, 121-136.
(38) Chandregowda, V.; Rao, G. V.; Reddy, G. C. Convergent Approach for Commercial Synthesis
of Gefitinib and Erlotinib. Org. Process Res. Dev. 2007, 11, 813-816.
(
39) Křupková, S.; Funk, P.; Soural M.; Hlaváč, J. 4‑Chloro-2-Fluoro-5-Nitrobenzoic Acid as a
Possible Building Block for Solid-Phase Synthesis of Various Heterocyclic Scaffolds. ACS
Comb. Sci. 2013, 15, 20-28.
(
40) Kaplánek R.; Krchňák, V. Fast and Effective Reduction of Nitroarenes by Sodium Dithionite
under PTC Conditions: Application in Solid-Phase Synthesis. Tetrahedron Lett. 2013, 54,
2600-2603.
2
3
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Organic Process Research & Development
Page 24 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(41) Neagoie C.; Krchňák, V. Piperazine Amide Linker for Cyclative Cleavage from Solid Support:
Traceless Synthesis of Dihydroquinoxalin-2-Ones. ACS Comb. Sci. 2012, 14, 399.
(
42) Guryanov, I.; Orlandin, A.; Biondi, B.; Formaggio, F.; Visentini, D. Process for the
Manufacture of Degarelix and its Intermediates. WO2017103275 A1, June 22, 2017.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(43) Davis, T. L. The Mechanism of Reactions in the Urea Series. Proc. Natl. Acad. Sci. U. S. A.
1
925, 11, 68-73.
(44) Blagbrough, I. S.; Mackenzie, N. E.; Ortiz, C.; Scott, A. I. The Condensation Reaction
Between Isocyanates and Carboxylic Acids. A Practical Synthesis of Substituted Amides and
Anilides. Tetrahedron Lett. 1986, 27, 1251.
(
45) ICH Q3D, Guideline for Elemental Impurities, International Council for Harmonization,
Harmonised Guideline Step 4, Dec 2014.
(
46) Badocco, D.; Lavagnini, I.; Mondin, A; Favaro, G.; Pastore, P. Definition of the Limit of
Quantification in the Presence of Instrumental and Non-Instrumental Errors. Comparison
Among Various Definitions Applied to the Calibration of Zinc by Inductively Coupled
Plasma–Mass Spectrometry. Spectrochim Acta B 2015, 114, 81-86.
(47) Lavagnini, I.; Badocco, D.; Pastore, P.; Magno, F. Theil-Sen Nonparametric Regression
Technique on Univariate Calibration, Inverse Regression and Detection Limits. Talanta 2011,
8
7, 180-188.
*
Correspondence to: Ivan Guryanov, Fresenius Kabi iPSUM Srl, via San Leonardo 23, Villadose
(RO), 45010, Italy. E-mail: ivan.guryanov1@gmail.com
Antonio Ricci, Fresenius Kabi iPSUM Srl, via San Leonardo 23, Villadose (RO), 45010, Italy. E-
mail: antonio.ricci@fresenius-kabi.com
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