pubs.acs.

org/OPRD Article

Regioisomer Formation with Agmatine Guanidino Group, Its

Implications for Agmatine Peptide Cyclization, and Application of
Bis-Boc-Agmatine
Yi Yang* and Lena Hansen
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ABSTRACT: Agmatine peptides play a significant role in the therapeutic design. Imparting an agmatine moiety to a peptide
molecule through amine amidination with electrophilic guanylating agents could be challenging given the lability of the various
nucleophilic groups on peptides and the harsh reaction conditions. Agmatine sulfate could function as an agmatine synthon, but its
sparse solubility in common organic solvents limits its application for peptide syntheses. Neutralization of agmatine sulfate could
effectively enhance its solubility in organic solvents. Nevertheless, the deprotonated guanidino group poses a competition reaction
against the agmatine 4-amino group in the envisioned amidation reaction. The formed regioisomeric impurity bears an unpaired
amino group that could be undesirably entangled in the following reaction steps, further complicating and jeopardizing the agmatine
peptide synthesis. Bis-Boc-agmatine, a guanidino-protected species, could be readily fused to peptide molecules exclusively through
its amino group without forming regioisomeric impurities. A successful synthesis of an agmatine-bearing cyclic peptide FE 201836
has been accomplished using bis-Boc-agmatine as the building block in this study. Moreover, the strategy of managing the detected
isomeric impurity in the synthetic process has been advocated through isolating the isomeric intermediate, tracing its further
transformation in the following synthetic steps, and detecting the presence of the ultimate isomeric impurity in the final product.
KEYWORDS: agmatine peptide regioisomer, agmatine sulfate, freebase agmatine, Bis-Boc-agmatine, guanidino reactivity,
guanidino protection, peptide cyclization side reaction, intermediate isomer fate study

■ INTRODUCTION
Agmatine, also known as 1-(4-aminobutyl)guanidine, is a
time. Moreover, the substrate amino group for the amidination
modification should be selectively liberated in the presence of
compound derived from arginine decarboxylation by arginine other protected nucleophilic functional groups, potentially
decarboxylase (ADC). It has wide therapeutic applications, further restricting the application of the guanylating strategy
including diabetes mellitus, neurotrauma and neurodegener- for peptide manufacturing concerning the cost-effectiveness
ative diseases, opioid addiction, mood disorders, cognitive and product quality.
disorders, and cancer.1 Agmatine moiety is also found in a Desmopressin analogue FE 201836,14 a cyclic thioether
variety of natural compounds such as E-642 or peptide drugs peptide that functions as a vasopressin-2 receptor agonist and
like blenoxane3 and is frequently employed as an arginine aims at nocturia, bears an agmatine residue at its C-terminus
surrogate at the peptide C-terminus to explore receptor−ligand (Scheme 1).15 It is noted that FE 201836 has a Thi
interactions.4
In consideration of the significant role of agmatine in (thienylalanine) residue and a thioether moiety that are highly
therapeutic design, a variety of agmatine construction strategies sensitive to electrophile modification such as oxidation and
have been developed. Amidination of amine is one of the most iodination even under mild conditions.16 The construction of
prominent guanidine synthetic methodologies to build the target agmatine moiety through the amidination of the
agmatine structure. Direct or indirect electrophilic guanylating incorporated 1,4-butanediamine precursor by guanylating
agents including carbodiimide,5 O-methylisourea,6 thiourea,7 agents under harsh conditions might affect the integrity of
isothiourea,8 amino(imino)methanesulfonic acid,9 chlorofor- the Thi and/or thioether moieties. Given these restrictions, an
mamidine,10 1H-pyrazole-1-carboxamidine,11 N-triflylguani- alternative mild synthetic strategy should be pursued in the
dine,12 and cyanamide13 have been traditionally employed to context of agmatine assembly.
transform amines to the corresponding protected or non-
protected guanidine compounds.
Despite the accomplishments of the synthetic strategy of the Received: April 5, 2022
guanylating agent, the production of agmatine-bearing
peptides, particularly those with large volumes in industries,
remains restricted by the conventional processes due to the
corresponding amidination conditions that normally request a
metal-based catalyst, heating, oxidant, and/or long reaction

© XXXX American Chemical Society https://doi.org/10.1021/acs.oprd.2c00098

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Scheme 1. Chemical Structure of FE 201836 potential for various diseases.17 It is also frequently applied as a
nutrition supplement.18 Such a cost-effective starting material
could potentially be employed as the agmatine surrogate for
preparing the FE 201836 peptide.
In the FE 201836 synthetic process, precursor linear peptide
Intermediate A is first assembled on 2-chlorotrityl chloride
(CTC) resin, cleaved by 10% (v/v) HFIP (hexafluoroisopro-
panol)/DCM (dichloromethane), and isolated through pre-
cipitation of the concentrated peptide solution in MTBE
(methyl tert-butyl ether). Agmatine sulfate is supposed to be
conjugated to Intermediate A at the subsequent step to
construct the protected linear FE 201836. However, agmatine
sulfate is sparsely soluble in common organic solvents and
could not be directly coupled to the complementary peptide in
solution at a decent concentration. Therefore, agmatine sulfate
is first neutralized by 2 equiv NaOH in aqueous solution and
isolated through lyophilization; 1.92 equiv of thus obtained

■
freebase agmatine are solubilized in DMF and coupled with 1
RESULTS AND DISCUSSION equiv Intermediate A by 1.05 equiv PyBOP (benzotriazol-1-
FE 201836 Synthesis through Agmatine Sulfate yloxytripyrrolidinophosphonium hexafluorophosphate) and 2.5
Neutralization Strategy and the Formation of Regioiso- equiv DIPEA (diisopropylethylamine) to assemble the
meric and Dimeric Impurities. Agmatine sulfate (Scheme protected linear FE 201836. The latter is subjected to
2) is a commercially available compound with wide therapeutic deprotection treatment by TFA (trifluoroacetic acid)/TIS
(triisopropylsilane)/H2O mixture to derive the linear FE
Scheme 2. Chemical Structure of Agmatine Sulfate 201836. The complementary amino and carboxylate group
from the linear FE 201836 is spliced with 1.3 equiv HBTU and
6 equiv DIEPA in DMF through a pseudo-high-dilution
strategy19 to construct the target cyclic product FE 201836
while minimizing the undesired intermolecular reaction and
the resultant dimeric impurity. The process of FE 201836
synthesis is illustrated in Scheme 3.

Scheme 3. FE 201836 Synthesis through Freebase Agmatine Coupling (a) solid-phase peptide synthesis; (b) 10% HFIP/DCM;
(c) freebase agmatine/PyBOP/DIPEA/DMF; (d) TFA/TIS/H2O; (e) HBTU/DIEPA/DMF)

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Scheme 4. Freebase Agmatine-Induced Regioisomeric and Dimeric Impurity Formation

Scheme 5. FE 201836 Isomer A/B Formation Accounted to the Protected Linear FE 201836 Regioisomer (1)

The subject freebase agmatine coupling reaction was It is generally believed that the nucleophilicity of the
conducted in DMF at room temperature and was quantita- guanidino group is by and large restrained under acidic to weak
tively completed within 2 h. The isolated protected linear FE basic conditions. The potentials of the nucleophilicity of the
201836 product was analyzed by RP-HPLC, and two major guanidino group are significantly attenuated through the
impurities with abundances of 9.5 and 10.9% were detected (It protonation under prevalent pH conditions of the organic
is of note that all impurity quantities in this study are derived reactions. Nevertheless, guanidine compounds are categorized
from the peak area percentage of the corresponding RP-HPLC as organic superbase20 and possess excellent nucleophilicities
without being adjusted by the UV extinction coefficient). LC/ that could bestow them the role of nucleophilic catalysts in
MS analysis revealed that they might correspond to isomer 1 organic synthesis.21 In the subject reaction, the guanidino
and agmatine-crosslinked protected linear FE 201836 dimer 2, group from the agmatine is neutralized in advance of its
respectively (Supporting Information). Subsequently, the coupling to the peptide molecule, awakening its nucleophil-
protected linear FE 201836 crude product was treated with a icity. The deprotonated guanidino group thus competes with
concentrated TFA solution to remove the protecting groups. the 4-amino group on agmatine toward the activated peptide
The concomitantly resultant linear FE 201836 regioisomer carboxylate group to form a prolyl guanidine regioisomeric
impurity 3 and agmatine-crosslinked linear FE 201836 dimer impurity 1. This process, in principle, resembles the lactam
were purified and analyzed by NMR (Supporting Informa- formation that occurs in the process of Fmoc-Arg-OH
tion). It turned out that the subject regioisomeric impurities activation between the guanidino side chain and backbone
and dimeric impurities were initiated with the function carboxylate group.22 Noted that there is an unpaired free
between the carboxylate group from Intermediate A and the amino group on the regioisomeric impurity 1 that could
guanidino group on agmatine. accommodate another Intermediate A peptide molecule in the
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Scheme 6. Formation of Dimeric Impurities between Two Linear FE 201836 Regioisomer (3), and between One Linear FE
201836 Regioisomer (3) and One Linear FE 201836 (4)

Scheme 7. Formation of Amidinated FE 201836 Isomers

reaction, as to generate the agmatine-crosslinked dimeric detectability, and purge ratio of the ultimate isomer impurities
impurity 2. This process is elucidated in Scheme 4. if any from the final product. To this end, linear FE 201836
The regioisomer/dimer impurity formation mechanism has regioisomer 3 was isolated by the chromatographic purification
been further corroborated by the simulation reaction in which and separately subjected to the following cyclization. The
saturated NaOH/DMF solution (pH 9−10) is employed as derived product was analyzed by LC/MS. Both Isomers A and
the solvent for the topic agmatine coupling reaction. Under B displayed in Scheme 5 have been detected with 60.8 and
such strong alkaline conditions, regioisomer and dimer 5.1%, without knowing the exact correlation. As both FE
impurity contents have been increased to 12.4 and 13.6%, 201836 Isomers A and B have one unpaired amino group in
respectively. This observation sustains the claim that the their structures, they could be amidinated by the excess HBTU
neutralization of agmatine sulfate by 2 equiv NaOH induces in the cyclization reaction mixture and converted to the
the undesired acylation of the guanidino group and the corresponding guanidine counterparts as delineated in Scheme
subsequent dimeric impurity formation. 7.23 Both of the amidinated Isomers A and B have been
Fate Study of the Linear FE 201836 Regioisomeric detected in the crude product, and one of them is
Impurity. It is to note that the formation of the protected predominantly formed in this reaction up to a 23.0% content.
linear FE 201836 regioisomer 1 would interfere with the Also, a dimer is detected in the reaction mixture, which should
subsequent cyclization reaction after the TFA treatment since be attributed to one of the six possible isomeric dimers
the resultant linear FE 201836 regioisomer 3 bears one induced by the linear FE 201836 isomer (Scheme 6). Since
carboxylic group and two amino groups, which could there is an unpaired amino group from each of these six
theoretically forge two regioisomeric impurities (Isomer A isomeric dimers, they remain susceptible to the amidination by
and B) in the final product of FE 201836 (Scheme 5). HBTU. Indeed, 2 amidinated isomeric dimers have been
Additionally, linear FE 201836 regioisomer 3 could also be detected in the crude product.
entangled in intermolecular functions at the cyclization step, Given the above experiment and analyses, it could be
either with another molecule 3 or with an ordinary linear FE concluded that the isolated linear FE 201836 regioisomer
201836 molecule 4, giving rise to the formation of six and four could be subjected to various transformations, including
dimeric impurities, respectively (Scheme 6). cyclization, dimerization, and amidination. The dimerization
It is always imperative to explore the fate of the between two linear FE 201836 regioisomers is actually
regioisomeric impurities detected from the intermediates in beneficial for the target peptide synthesis since two
their transformation at the subsequent synthetic steps, regioisomeric impurities are consumed to form another
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Scheme 8. Bis-Boc-Agmatine-Directed FE 201836 Synthesis (a) Bis-Boc-agmatine/PyBOP/DIPEA/DMF; (b) TFA/TIS/H2O;

(c) HBTU/DIEPA/DMF)

impurity that should be readily removed from the product by Global deprotection of 5 with a solution consisting of 60
chromatographic purification. However, the linear FE 201836 equiv TFA, 1.5 equiv TIS, and 6 equiv H2O gives linear FE
regioisomer could also be dimerized with a molecule of linear 201836 after 2 h at room temperature with 91.0% purity and
FE 201836 to form a mixed dimer, as illustrated in Scheme 6. quantitative uncorrected yield. Linear FE 201836 is then
Such side reactions consume a linear FE 201836 molecule and cyclized through a pseudo-high dilution strategy to obtain the
reduce the reaction yield. final product FE 201836 with 87.8% purity. Less than 1.5%
The most prominent conclusion drawn from the fate study dimeric impurity is generated in this process. The crude
of linear FE 201836 regioisomer is that the FE 201836 Isomer product is analyzed by LC/MS (Supporting Information). No
A/B, as well as regioisomeric and dimeric impurities, once cyclic isomer, amidinated dimer, agmatine-crosslinked dimer,
formed, could be detected with high detectability and and dimer isomer has been detected, verifying bis-Boc-
unambiguously distinguished from the product FE 201836 agmatine’s efficacy in suppressing the freebase agmatine-
with the applied RP-HPLC method. Hence, from the induced side reactions from the counterpart process. The
perspective of the FMEA (failure mode effect analysis) purity of the crude product has been significantly increased
concept, the integrity of product quality will be sustained by from 70.9% (freebase agmatine process) to 87.8% (bis-Boc-
such a high detectability since their removal by the purification agmatine process), and the complexity of the downstream
process could be unambiguously followed and assessed. processing is pronouncedly alleviated. The utilization of bis-
FE 201836 Synthesis through the Bis-Boc-Agmatine Boc-agmatine in place of freebase agmatine has thus
Strategy. Given the regioisomer formation induced by the prominently optimized the overall performance of the
manufacturing process.

■
freebase agmatine and its implications in the cyclization
reaction, as well as the low yield and purity (71.0%) of the
crude product, an alternative agmatine surrogate should be CONCLUSIONS
employed for the FE 201836 synthesis. The introduction of In this study, side reactions related to the freebase agmatine’s
Boc-protecting groups on guanidino moiety can drastically application in peptide synthesis have been detected and
enhance the solubility of parent agmatine molecules in organic elucidated. Deprotonated guanidino group from the freebase
solvents, which could thus preclude the unnecessary guanidino agmatine poses a competition reaction against its 4-amino
neutralization. Actually, Fmoc-Arg(Boc)2-OH has already been group in the target amidation reaction, giving rise to the
widely employed as a building block for peptide synthesis in formation of an acyl guanidine regioisomeric impurity. The
the case of challenging Arg(Pbf) deprotection. Similarly, bis- unpaired amino group on the formed isomer could be
Boc-agmatine24 is deemed a potentially viable building block entangled in the subsequent reactions and further complicate
for FE 201836 preparation, bypassing the step of agmatine the impurity profile of the product. Bis-Boc-agmatine, an
sulfate neutralization and the formation of various regioiso- agmatine building block protected at the guanidino group,
meric and dimeric impurities that significantly reduce the could be readily prepared and utilized in the agmatine peptide
production yield. The process of bis-Boc-agmatine-directed FE synthesis. No isomeric impurities and resultant dimeric
201836 synthesis is depicted in Scheme 8. impurities are formed from the bis-Boc-agmatine process.
It turns out that 1.05 equiv bis-Boc-agmatine is sufficient to The bis-Boc-protecting groups on the guanidino functionality
drive the condensation reaction to completion within 30 min can be readily and quantitatively removed by the TFA
at ambient temperature by 1.05 equiv PyBOP and 2.5 equiv treatment. Significant increases in product purity and
DIPEA in DMF. No isomeric or dimeric impurities are production yield are accomplished by virtue of the application
generated in this process. It is to note that the stoichiometry of of bis-Boc-agmatine in FE 201836 manufacturing. This study
bis-Boc-agmatine should be rigorously controlled since the also highlights the necessity and the methodology to
excess bis-Boc-agmatine could be carried over to the investigate the fate of the isomeric impurity detected from
cyclization step as agmatine, which would interfere with the the intermediate product, which is paramount for securing the
integrity of the subject API products.

■
target amidation cyclization with its amino group (Supporting
Information). The protected intermediate 5 is isolated through
the precipitation in a 20% iPrOH (isopropanol) aqueous EXPERIMENTAL SECTION
solution while maximizing the removal of the excess bis-Boc- Materials. 2-Chlorotrityl chloride resin (100−200 mesh,
agmatine. The intermediate 5 is obtained with 92.8% purity loading: 1.60 mmol/g) was purchased from Chemical and
and an uncorrected yield of 96.7%. Biopharmaceutical Laboratories of Patras SA. Fmoc-Pro-OH
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(purity by HPLC ≥98.5%), Fmoc-Asn(Trt)-OH (purity by MTBE was added to the suspension over 0−5 min. The
HPLC ≥99.0%), and Fmoc-Val-OH (purity by HPLC precipitated peptide was filtered. The filter cake was washed
≥98.5%) were procured from Flamma. Fmoc-Cys- four times with 1.0 mL of MTBE. The product was dried
((CH3)2COOtBu)-OH (purity by HPLC ≥98.5%), Fmoc- under vacuum at 30 °C for a minimum of 3 h.
Thi-OH (purity by HPLC ≥98.5%), and Boc-chlorophenyla- Cyclization of the Isolated Linear FE 201836
nine (purity by HPLC ≥98.5%) were obtained from Flamma Regioisomer Obtained from the Freebase Agmatine
Honkai Pharmaceutical Co., Ltd. Agmatine sulfate (assay Strategy. The cyclization reactor is blanketed with nitrogen.
≥97%), Oxyma (assay ≥97%), HOBt.H2O (wetted with not Linear FE 201836 regioisomer (0.073 g, 0.061 mmol) was
less than 14 wt % water, 98% purity), DIC (assay ≥99%), added in a flask. DMF (0.365 mL) was charged to peptide, and
piperidine (99%), PyBOP (purity by HPLC ≥99.0%), DIPEA the mixture was stirred until full dissolution. The peptide
(≥99%), and triisopropylsilane (98%) were procured from solution was transferred into Syringe 1. HBTU (0.030 g, 0.08
Sigma-Aldrich. Bis-Boc-Agmatine (purity by HPLC ≥97%) mmol) was added to another flask. DMF (0.219 mL) was
was purchased from Shanghai ACI Biotech Co., Ltd. TFA charged into the HBTU, and the mixture was stirred until full
(peptide grade) was obtained from Iris-Biotech. DMF (purity dissolution. The HBTU solution was transferred into Syringe
by GC ≥99.5%) was procured from Merck. Isopropanol (grade 2. DMF (0.511 mL) was added into the cyclization reactor.
for liquid chromatography) was purchased from VWR. NaOH The peptide solution and HBTU solution were added in
(98.5%) was procured from Thermo Fisher. Syringe 1 and Syringe 2, respectively, simultaneously and
Agmatine Sulfate Neutralization and Coupling of continuously over 50−60 min into the cyclization reactor. The
Freebase Agmatine to Intermediate A. Agmatine sulfate mixture was stirred in cyclization reaction through all of the
(10 g, 43.8 mmol) was added into a flask and fully dissolved in processes. DIPEA (0.047 mL, 0.267 mmol) was added
84 mL of H2O. NaOH (3.52 g, 88.0 mmol) was dissolved in 40 manually batchwise into the cyclization reactor over the
mL of H2O in another flask. The NaOH solution was same period of the peptide and HBTU addition (10−12
transferred into the agmatine sulfate solution, and the mixture additions of equal size with the first addition at time 0.) Record
was stirred for at least 5 min. The solution was lyophilized to the pH of the reaction solution after each DIPEA addition. If
isolate the freebase agmatine (Na2SO4 salt) product. the pH is below 8, add extra DIPEA to increase the pH above
The reactor is blanketed with nitrogen. Freebase agmatine 8. After adding the above solutions and DIPEA, the reaction
(1.67 g, 6.14 mmol) was added to the reactor. DMF (64 mL) mixture was stirred for another 10 min and the reaction
was charged into the reactor, and the mixture was stirred. The conversion was analyzed by HPLC. If the reaction is complete,
freebase agmatine will not be entirely dissolved. Intermediate A 9.85 mL of cold (2−8 °C) 1% AcOH aq. solution was added to
peptide (4 g, 3.20 mmol) was added in a flask to which 1.75 g the peptide solution and the combined solution was stored
(3.36 mmol) of PyBOP was also charged. DMF (64 mL) was overnight at 2−8 °C. The mixture was filtered through a 2.4
added to the flask, and the mixture was stirred until complete μm filter. The storage container was rinsed with 0.46 mL of
dissolution, and 1.41 mL (8.00 mmol) of DIPEA is H2O, the rinsing solution was passed through the same filter,
subsequently charged. The solution was stirred for 5 min and the filtrate was collected in a flask. The derived peptide
and transferred to the reactor with freebase agmatine/DMF solution was analyzed by RP-HPLC and LC/MS.
suspension to start the coupling reaction. The mixture was Cyclization of the Linear FE 201836 Obtained from
stirred for 30 min, and the conversion was controlled with the the Freebase Agmatine. A representative linear FE 201836
developed RP-HPLC method. The reaction was continued starting material containing both the target linear FE 201836
until less than 2% of Intermediate A is detected (maximum and its regioisomer was subjected to the cyclization process
reaction time 2 h). Cold water (2−8 °C) (256 mL) was added identical to that of the aforementioned cyclization reaction of
within 50−60 min to the reaction solution to precipitate the the isolated linear FE 201836 regioisomer.
protected linear FE 201836 product. The suspension was kept Conjugation of Intermediate A with Bis-Boc-agma-
under stirring, and another 256 mL of 2−8 °C cold water was tine. The reactor is blanketed with nitrogen. Intermediate A
added within 5 min into the reaction vessel. The product was (20 g, 16.0 mmol) was fully dissolved in 160 mL of DMF. The
filtered, and the filter cake was washed two times in 32 mL of mixture was stirred until the full dissolution, and the
water at room temperature and again two times with 32 mL of Intermediate A solution was transferred into the reactor.
MTBE (methyl tert-butyl ether) at room temperature. The PyBOP (8.73 g, 16.8 mmol) was added to the reactor. The
filter cake was dried under vacuum at a temperature below 40 mixture was stirred until the full dissolution. DIPEA (7.03 mL,
°C until <1% weight change in 12 h is accomplished. A 40.0 mmol) was added into the reactor, and the mixture was
protected linear FE 201836 product (4.0 g, 2.93 mmol, 91.6% stirred at ambient temperature for 5 min. Bis-Boc-agmatine
yield) was obtained. (5.55 g, 16.8 mmol) was dissolved in 40 mL of DMF, and the
Global Deprotection of the Protected Linear FE bis-Boc-agmatine solution was added into the reactor to
201836 Derived from the Freebase Agmatine Strategy. initiate the coupling reaction. The reaction mixture was stirred
The reactor is blanketed with nitrogen. TFA global at room temperature for 30 min, and the conversion rate was
deprotection cocktail [composition: 7.04 mL (91.8 mmol) of controlled by RP-HPLC. The reaction was stopped when less
TFA, 0.14 mL (0.72 mmol) of TIS, and 0.14 mL (0.72 mmol) than 2% of Intermediate A is left in the reaction mixture
of H2O] (7.32 mL) was prepared and transferred into the (maximum reaction time 2 h). Cold (2−8 °C) 20%
reactor and cooled to 10−15 °C. Protected linear FE 201836 isopropanol aqueous solution (1000 mL) was added to the
(0.916 g, 0.67 mmol) was added to the TFA cocktail in the reaction mixture within 50−60 min to precipitate the bis-Boc-
reactor. The peptide TFA solution was stirred at room protected linear FE 201836 product. The suspension was
temperature for 2 h. The cooled MTBE (≤−20 °C) (7.33 mL) stirred at 2−8 °C for another 30 min after adding the
was continuously added into the peptide solution over 40−50 isopropanol solution. The peptide product was filtered, and the
min to precipitate the product. Another 7.33 mL of cold filter cake was washed sequentially two times with 40 mL of
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■
pubs.acs.org/OPRD Article

cold water (2−8 °C) and two times with 80 mL of cold (2−8 ASSOCIATED CONTENT
°C) MTBE. The filter cake was dried under vacuum at a * Supporting Information
sı
temperature lower than 40 °C until <1% weight change is The Supporting Information is available free of charge at
accomplished in a 12 h period. Bis-Boc-protected linear FE https://pubs.acs.org/doi/10.1021/acs.oprd.2c00098.
201836 product (29.0 g) (92.8% purity) is obtained, Characterizations of the isomeric and dimeric impurity
corresponding to a 96.7% uncorrected yield. induced in the freebase agmatine coupling reaction by
Global Deprotection of Bis-Boc-Protected Linear FE LC/MS and NMR; investigation on the consequences of
201836. The reactor is blanketed with nitrogen. TFA global the freebase agmatine-induced regioisomeric; impurity
deprotection solution [consisting of 85.25 mL (1113.2 mmol) formation impurity profile of the FE 201836 crude
of TFA, 5.70 mL (27.8 mmol) of TIS, and 2.00 mL (111.3 product prepared from freebase agmatine strategy;
mmol) of H2O] (92.95 mL) was added into the reactor. The conjugation of Intermediate A with Bis-Boc-agmatine;
TFA solution was cooled to 10−15 °C. Bis-Boc-protected impurity profile of the linear FE 201836 obtained from
linear FE 201836 (29.01 g, 18.55 mmol) was charged into the Bis-Boc-agmatine strategy; and impurity profile of FE
TFA solution. The reaction mixture was stirred at room 201836 obtained from the Bis-Boc-agmatine strategy
temperature for 2 h. Cold MTBE (≤−20 °C) (185.90 mL) (PDF)
was added into the peptide solution continuously over 40−50
min to precipitate the product. Another 185.90 mL of cold
MTBE (≤−20 °C) was added to the peptide suspension over
■ AUTHOR INFORMATION
Corresponding Author
0−5 min. The precipitated peptide product was filtered, and Yi Yang − Chemical Development, Global Pharmaceutical
the filter cake was washed four times with MTBE (each time R&D, Ferring Pharmaceuticals A/S, Kastrup 2770,
with 23.24 mL) at room temperature. The product was dried Denmark; orcid.org/0000-0002-0003-8426;
under vacuum at 30 °C for minimum 3 h and until less than Phone: +45 28 78 73 74; Email: [email protected]
1% weight change in 3 h is accomplished. Linear FE 201836 Author
(bis-TFA salt) product (24.39 g, 20.45 mmol) is obtained, Lena Hansen − Chemical Development, Global
corresponding to a 110% uncorrected yield. Note that the Pharmaceutical R&D, Ferring Pharmaceuticals A/S,
derived linear FE 201836 crude product was quantified with an Kastrup 2770, Denmark
FE 201836 reference, and the assay was 15.5 mmol. Hence, the Complete contact information is available at:
overall corrected yield relating to peptide assembly, cleavage, https://pubs.acs.org/10.1021/acs.oprd.2c00098
bis-Boc-agmatine coupling, and global deprotection is 64.7%.
The linear FE 201839 product is obtained with a 91.0% purity, Notes
whereas no isomer or agmatine-crosslinked dimer is formed. The authors declare no competing financial interest.
Cyclization of Linear FE 201836 Derived from the Bis-
Boc-Agmatine Strategy. The linear FE 201836 material
derived from the bis-Boc-agmatine strategy was subject to the
■ ACKNOWLEDGMENTS
The authors thank Ileana Rodríguez León, Ph.D., and Jörgen
cyclization reaction through a pseudo-high-dilution strategy. Kjellgren Sjögren, Ph.D., from Ferring Pharmaceuticals A/S,
Linear FE 201836 (16.0 g, 13.42 mmol) was dissolved in 80 for the assistance with the LCMS analyses, Mai Schoenemann
mL of DMF. The peptide solution was transferred into feeding Jensen and Linda Nyboe, Ph.D., from Ferring Pharmaceuticals
vessel 1. HBTU (6.61 g, 17.44 mmol) was added to feeding A/S, for the purification of the linear FE 201836 isomer. The
vessel 2 and dissolved with 48 mL of DMF. DIPEA (14.02 mL, authors also thank Johan Evenäs, Ph.D., and Klara Jonasson,
80.49 mmol) was loaded in feeding vessel 3. DMF (112 mL) Ph.D., from Red Glead Discovery AB, for the NMR
was added together with ca. 200 μL of DIPEA into the spectroscopy study.
cyclization reactor. The peptide solution from feeding vessel 1,
HBTU solution from feeding vessel 2, and DIPEA from
feeding vessel 3 were added simultaneously and continuously
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