First total synthesis of versicotide A, B and C

Article abstract of DOI:10.1039/d0ra09635k

The syntheses of versicotides A-C, natural products containing anthranilic acid and NMe-Ala, were achieved by solid phase peptide synthesis on 2-chlorotrityl resin followed by solution phase macrocyclization. Using an oxyma additive, the difficult coupling reactions to the deactivated aromatic amine of o-aminobenzoic acid, were performed in high yield, avoiding anthranilic rearrangements or side reactions. This journal is

Full text of DOI:10.1039/d0ra09635k

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First total synthesis of versicotide A, B and C†
ab
a
a
*
Laura Posada,
Danilo Davyt
and Gloria Serra
Cite this: RSC Adv., 2020, 10, 43653
The syntheses of versicotides A–C, natural products containing anthranilic acid and NMe-Ala, were
achieved by solid phase peptide synthesis on 2-chlorotrityl resin followed by solution phase
macrocyclization. Using an oxyma additive, the difficult coupling reactions to the deactivated aromatic
amine of o-aminobenzoic acid, were performed in high yield, avoiding anthranilic rearrangements or
side reactions.
Received 12th November 2020
Accepted 24th November 2020
DOI: 10.1039/d0ra09635k
rsc.li/rsc-advances
Particularly, marine fungi are subjected to many factors that
contribute to gene modications and to adaptive processes
1. Introduction
producing unique secondary metabolites. Aspergillus genus is
an important source of bioactive secondary metabolites with
high structural diversity.10 Versicotides are natural products
produced by fungi of the Aspergillus family. Versicotides A (1)
and B (2), Fig. 1, were rst isolated by Li and co-workers from
Aspergillus versicolor ZLN-60 extracts of the Yellow Sea sedi-
ment.11 Later, the same group employed the OSMAC (one strain
many compounds) approach on this fungus, resulting in the
isolation and characterization of a new compound with struc-
tural similarities named versicotide C (3),12 the rst natural
cyclic hexapeptide containing two anthranilic acids (Anth).
Further on, three new cyclopeptides were isolated from Asper-
gillus versicolor LZD-14-1 namely versicotides D–F,13 along with
the previously known versicotides A and B.
The presence of two rigid b-amino acids, Anth units, and two
N-methyl-alanine (NMe-Ala) in 1–3 add further constraints to
these cyclopeptides. In the case of the reported isolation of
versicotide C (3), the authors stated that, in DMSO-d6, it pres-
ents one trans amide bond and one cis amide bond on its
structure.12
In the past years, a signicant number of macrocyclic peptides,
with very interesting biological activities, have been described
in the literature.1 They are known to be promising candidates in
the search for new drugs as they show enhanced metabolic
stability in comparison with their corresponding linear
peptides, and could bind to receptors with high selectivity.2
Moreover, chemoinformatic exploratory analyses of synthetic
peptide combinatorial data sets have suggested that N-methyl-
ation and cyclization shis their position toward the chemical
space dened by sets of FDA approved drugs. Therefore, this
modications could result in compounds with biological
activity and suitable physicochemical properties.3 In addition, it
was demonstrated that multiple backbone N-methylation of
cyclic peptides remarkably improves their cell permeability and
therefore can be utilized in the design of new orally available
drugs.4 Recent studies suggest that because of their conforma-
tional exibility, cyclopeptides behave as molecular chameleons
as they adjust their conformations and polar surface areas
according with the properties of surrounding environment.5 On
the other hand, constrained structures are expected to present
a minor loss of conformational entropy upon binding, resulting
in higher target affinity.6 Thus, many groups investigate the
addition of further constraints to cyclopeptides in order to
obtain a potential bioactive conformer.7
Regarding their biological activity, 1 and 2 showed no cyto-
toxicity against murine leukemia P388, human hepatoma cell
line BEL-7402 and human leukemia cell HL-60; versicotides A–C
showed no activity against cancer cell lines A-549, HeLa and
SMMC-7721. In addition, versicotides D–F exerted lipid
lowering effects through the regulation of cholesterol efflux to
HDL in RAW264.7 macrophages, while 1–3 do not exhibit this
effect.
Natural products are a continuous source of inspiration for
the discovery and development of new drug candidates.8 Those
proceeding from marine environments, have been attracting
special interest for their unexplored structural diversity.9
a
´
´
´
´
´
Quımica Farmaceutica, Departamento de Quımica Organica, Facultad de Quımica,
´
Universidad de la Republica, General Flores 2124, CC1157, Montevideo, Uruguay.
E-mail: gserra@fq.edu.uy
b
´
´
Graduate Program in Chemistry, Facultad de Quımica, Universidad de la Republica,
Uruguay
† Electronic supplementary information (ESI) available. CCDC 2023773. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/d0ra09635k
Fig. 1 Versicotides A–C and analogue 4.
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Recently, as part of a general program to obtain anti-
plasmodial candidates,14 our group reported the synthesis of
versicotide D and analogues and their evaluation as antima-
larials against the chloroquine sensitive strain P. falciparum
3D7, where versicotide D showed EC50 ¼ 1.5 mM.15
There are few examples in the literature related to the
synthesis of cyclopeptide natural products containing anthra-
nilic acid,16 and they are all broadly in line to the fact that amide
bond formation involving the amine of Anth is a challenge not
only because of the deactivation of the aromatic amine via
resonance, but also due to rearrangements in which the Anth
motif can be involved.17 Frequently, long, complicated, multi-
stage and low yielding routes have been used to achieve the
synthesis of natural products containing this motif. An example
is the total synthesis of deoxo-solomonamide B in which Anth
motif was masked with an indole group that is cleaved by ozo-
nolysis in the last steps of the synthetic route.18 Therefore,
a simpler methodology to obtain compounds with Anth in good
yield is a chemical challenge. In addition, as many cyclo-
peptides containing Anth, present diverse bioactivities such as
increasing blood pressure,16b lipid lowering effect,12 anti-pro-
liferative,16a,16d anti-inammatory,18 insecticide,16e the interest
on this issue is enhanced. Here, we report the total synthesis of
versicotides A–C and analogue 4. The structures of the obtained
compounds were conrmed by MS, NMR and, in the case of
versicotide A (1), by X-ray analysis. A deep analysis of NMR data
(1H, 13C, COSY, HSQC and HMBC) was done in order to deter-
mine the adopted conformations in the used solvents, along
with temperature variation studies.
Scheme 1 Solution synthesis of cyclopentapeptide analogue 4.
Attempts to apply this strategy for the synthesis of versico-
tide A were not successful. The coupling reaction between
BocNMeAla-Anth (8) and Ala-Anth(OMe) using EDCI and Cl-
HOBt rendered the corresponding tetrapeptide intermediate
in very low yield along with the benzoxazinone 9, Scheme 2.
According with previous results,21 this heterocycle is derived
from the cyclodehydration reaction of 8.
2.2. SPPS synthesis of linear precursors and solution phase
cyclization
2. Results and discussion
2.1. Solution phase approach
In view of the limited achievements of the solution phase
synthetic methodology, we decided to explore the synthesis of
the linear precursors of versicotides A–C on solid phase and
cyclize them on solution phase. The linear sequence of each one
was chosen taking into account the characteristics of Anth
explained before. Based on this, Anth amine was not chosen as
the N-terminal in any case. Furthermore, we realized in early
steps of this work that using Anth as the C terminal on solid
phase peptide synthesis (SPPS) was not a good choice either, as
it leads to very low resin loadings and it could give a great steric
hindrance for the rst coupling to occur. Because of this, the
linear sequences chosen were: NHMeAla-Anth-NMeAla-Anth-
Ala-OH for 1, NHMe-Ala-Anth-Ala-Anth-NMeAla-OH for 2 and
NMeAla-Anth-Ala-NMeAla-Anth-Ala for 3.
First, we decided to prepare the cyclo[Ala-Anth-Ala-Anth-Ala] (4),
analogue of 1 and 2, and to investigate the solution phase
synthesis of the peptide precursor containing only Ala and
Anth. The in situ acyl chloride formation using triphosgene19
made it possible to couple Ala to the amine of methyl anthra-
nilate in 51% yield, Scheme 1. No racemization was observed by
1H-NMR analysis. However, advanced intermediates could not
be prepared using triphosgene. In an attempt to circumvent
this, we tried to apply the methodology described by Xin and
Burguess,20 which employed a combination of EDCI and HOBt.
As HOBt and HOAt present restrictions for their overseas
shipment, we decided to substitute HOBt by Cl-HOBt.
Dipeptide 5 and tetrapeptide 6 were obtained in 42% and
10% yield, respectively. No racemization during the coupling
reaction steps was detected according with the NMR spectra of
6. However, in the case of the synthesis of 6 many by-products,
as fragments of the desired compound, were observed. Two of
the detected by-products were methyl anthranilate and the tri-
peptide NH2-Anth-Ala-Anth-OMe, derived from fragmentation
of the labile Anth amide bond according with Hamada et al.
results.17b The preparation of pentapeptide 7 from 6 resulted in
a high yielding process (92%). Finally, deprotection and cycli-
zation in diluted conditions (2 mM) rendered analogue 4 in 35%
yield.
Our rst efforts to obtain the linear precursor of versicotide
A, 10, by SPPS on 2-chlorotrityl chloride resin (2-CTC), were not
successful. The use of HBTU, HATU, or DIC/Cl-HOBt for the
Scheme 2 Synthesis of benzoxazinone 9 from BocNMeAla-Anth.
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coupling involving the amine of Anth led to incomplete or no we notice this throws false negative results as an incomplete
coupling at all. Doi and co-workers described the SPPS of Anth coupling was observed by HPLC-MS analysis of a small sample
containing peptides on the 2-CTC resin by generating an acyl cleaved from the resin. Nevertheless, with oxyma/DIC, we
chloride of the corresponding amino acid with triphosgene and conrmed optimal coupling aer 60 min of reaction time by
adding it to the resin without previous purication.22 During HPLC and 1H-NMR analysis. For the solution phase cyclization,
attempts to apply this methodology to obtain the desired HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
peptide, it was possible to prepare the tripeptide FmocNMeAla- b]pyridinium 3-oxid hexauorophosphate was employed as
Anth-Ala, but subsequent coupling reactions produced cleavage coupling reagent and dilution of the linear peptide was taken to
of the growing peptide from the resin.
5 mM concentration. Aer purication, 1 was obtained in 49%
Albericio et al. described the impressive coupling efficiency yield and no dimer formation was observed.
of oxyma (ethylcianoacetate oxime), in combination with DIC,23
Chromatographically pure versicotide A (1) showed three
surpassing HOAt coupling agent in more demanding peptide different conformers in the NMR spectra using DMSO-d6 as
models. Taking into account their results, we decided to select it solvent. To discard epimerization, 1H-NMR studies using
as an excellent option to overcome those difficulties. Conse- temperature gradient were performed. The spectra showed
quently, the use of oxyma for the coupling involving the amine variations of NMe and a methine peak integrations arising from
of Anth allowed us to prepare the dipeptide FmocNMeAla-Anth- conformers in slow exchange (see ESI†). The amide of NMe-
OH by SPPS, Scheme 3, in excellent yield. Moreover, the amino acids can present cis or trans conguration that
synthesis of the open precursor of versicotide A (10) was ach- depends, in many cases, on the used solvent. Taking into
ieved in 90% yield, based on the determination of the resin account that the N-methyl resonance of the amides in trans
loading. Completion of each coupling step was veried by conguration are shied downeld relative to the cis because of
Kaiser or chloranil test, for primary or secondary amines, the shielding effect of the carbonyl group,24 we observed that the
respectively. These colorimetric assays were not useful for major conformer, presents the two N-methyl amide bonds in cis
monitoring couplings to anthranilic acid amine group because conguration, Scheme 3. The chemical shis of this conformer
match with the reported ones for the isolated natural product.
The other two conformers would be in (cis, trans) and (trans,
trans) conguration (see ESI†). X-ray crystallographic structure
of versicotide A, Fig. 2, obtained from a crystal grown in
methanol, also agrees with previously described data for the
natural product, and presents both NMe-amides in cis
conguration.
Following the same procedure, the linear precursor of ver-
sicotide B (11), Scheme 3, was successfully synthesized in 87%
global yield (based on the determination of the resin loading).
However, cyclization using HATU in diluted conditions (5 mM),
led to a small amount of 2 (18% yield). The macrolactamization
reaction was repeated using EDCI/oxyma and 2 was puried by
ash chromatography to obtain 54% yield. The product pre-
sented a single spot by TLC in different mobile phases and two
peaks by HPLC. The analysis of both peaks by FIA-LC-ESI-MS
showed the signal of m/z 480.3 corresponding to [M + H]+ ion
1
of versicotide B (2). The H-NMR of each sample in DMSO-d6
showed a greater number of signals than expected. Finally,
a thermal gradient of the 1H-NMR in DMSO-d6 showed that
some signals corresponding to NMe and a methine change
Scheme 3 SPPS and solution macrocyclization of versicotides A (1)
and B (2).
Fig. 2 X-ray crystallographic structure of 1.25
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1
ꢀ
their proportion at higher temperatures. This fact allowed us to by a H-NMR experiment of 3 at 80 C which showed that the
discard epimerization and to conclude that both samples peaks assignable to the (cis, cis) conformer at 25 ꢀC, disappeared
contain a mixture of conformers of 2; in one of them both NMe- at this temperature (see ESI†).
amides are in trans conguration, Scheme 3, and in the other
conformer, one amide is in cis and the other in trans congu-
ration, (see ESI†) according with the literature.24
3. Experimental section
3.1. General procedure. Solid phase peptide synthesis
In a similar way, the synthesis of the linear precursor of
versicotide
C (12), Scheme 4, was successfully achieved
2-Chlorotrityl chloride resin (2-CTC) (100–300 mesh, 1.20 mmol
gꢁ1) was added to a syringe peptide synthesis vessel. The resin
was swelled in CH2Cl2 (3 ꢂ 5 min). A solution of rst protected
amino acid Fmoc-AA-OH (1 eq. for 0.8 mmol gꢁ1 loading) and
DIPEA (3 eq.) in CH2Cl2 was added and the resin was shaken 10
minutes. Then, an extra 7.0 eq. of DIPEA were added and shaking
was continued for 50 min. MeOH (0.8 mLgꢁ1 of resin) was added
to the previous mixture in order to cap unreacted functional
groups on the resin, and shaken for 10 min. Aer ltering, the
resin was washed with CH2Cl2 (ꢂ3), MeOH (ꢂ3), CH2Cl2 (ꢂ3),
DMF (ꢂ3). The resin was washed with DMF (ꢂ3) and Fmoc
protecting group was removed by treating the resin with piperi-
dine–DMF solution (1 : 4) for 1, 5 and 5 minutes successively. In
exceptional cases deprotection step was accomplish by a single
treatment with piperidine–DMF solution for 5 minutes, in order
to prevent side reactions. Then, the resin was washed with DMF
(ꢂ3), CH2Cl2 (ꢂ3) and DMF (ꢂ3). A solution of Fmoc-AA-OH or
Fmoc-Anth-OH (3 eq.) and DIPEA (6 eq.) in DMF was added to the
resin, followed by a solution of HBTU, for coupling to primary
amines, or HATU (2.9 eq.) in DMF, in case of coupling to an N-
methylated amino acid. The mixture was stirred for 60 min. Aer
the coupling was completed, the resin was washed with DMF
(ꢂ3) and CH2Cl2 (ꢂ3). Completion of the coupling was moni-
tored by colorimetric assays; Kaiser test in case of primary amines
and Chloranil test for secondary amines. Coupling procedure was
repeated in case of positive results. For the coupling of subse-
quent Fmoc-AA to anthranilic acid, a solution of Fmoc-AA-OH (5
eq.), Oxyma Pure (5 eq.), and DIC (5 eq.) was added to the vessel.
The mixture was stirred for 60 min. Then, the resin was washed
with DMF (ꢂ3) and CH2Cl2 (ꢂ3). Deprotection and coupling
cycles were repeated with the appropriate amino acids or Anth to
provide the desired compound. The peptide was cleaved from the
resin by treatment with 1% TFA in CH2Cl2 for 2–3 minutes at
room temperature followed by ltration and collection of the
ltrate in MeOH. The treatment was repeated three times and
then the resin washed with CH2Cl2 (ꢂ5) and MeOH (ꢂ3). Solvents
were removed in vacuo to obtain the crude peptide. LC-MS was
used to identify the product.
employing Oxyma + DIC in key steps, leading to 98% overall
yield (based on the determination of the resin loading).
Then, macrocyclization in solution phase, using HATU in
DCM (5 mM), rendered versicotide C (3) in 49% yield aer
purication and no dimer formation was observed. NMR
spectra of synthetic versicotide C in DMSO-d6, match those of
the isolated natural product, even though the authors informed
a single conformer with one NMe amide bond in cis and the
other one in trans conguration.12 A deep analysis of NMR
spectra, with special regard to signal integrations, lead us to
conclude that two conformers of 3, in a 1 : 0.2 ratio, are present
in both acetone-d6 and DMSO-d6. The conformers, which
present symmetrical structure, were easily distinguished by the
NMR signals of the two N-Me groups. The two corresponding N-
methyl amide bonds of one conformer are in trans congura-
tion, Scheme 4, and of the other conformer, in cis conguration.
The presence of these two conformers were further conrmed
3.2. Solution phase synthesis
3.2.1. General procedures for coupling reaction
3.2.1.1. Method I. EDC$HCl (1.5 eq.), Cl-HOBt (1.5 eq.) and
DIPEA (2.0 eq.) were added to a solution of Boc protected amino
acid (Boc-AA-OH, 1.0 eq.) in DCM at 0 ꢀC under N2 atmosphere.
N-Terminus deprotected linear peptide or amino acid ester
(NH2-AA-COOEt) was added and the reaction mixture was stir-
red at 0 ꢀC for 10 min and then at room temperature, overnight.
DCM was removed under vacuum and AcOEt was added. The
organic phase was washed with 0.1 M HCl aqueous solution
Scheme 4 SPPS and solution macrocyclization of versicotide C (3)
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(30 mL ꢂ 2), brine (10 mL), saturated NaHCO3 solution (30 mL for C25H32N5O6 ([M + H]+) 498.2, found 498.4. Macrocyclization
ꢂ 2) and brine (10 mL), dried over MgSO4 and ltered. The reaction was performed following the general procedure
solvent was removed under vacuum to give the crude material. method I (dilution 5 mM, 3 days), starting from the tri-
The crude material was puried by ash chromatography.
uoroacetate salt of linear peptide NHMe-Ala-Anth-Ala-NMe-
3.2.1.2. Method II. Bis(trichloromethyl)carbonate (0.33 eq.) Ala-Anth-Ala-OH (200 mg, 0.33 mmol), using HATU as
was added to a solution of Boc-Ala-OH (1 eq.) in dry THF under coupling agent. Further purication by ash chromatography
N2 atmosphere at 0 ꢀC. 2,4,6-Colidine (2.6 eq.) were added to the using AcOEt as mobile phase, rendered the desired macrocycle
solution and a white suspension was formed. The reaction in 50% yield (78 mg, 0.16 mmol). HPLC analysis employing
mixture was stirred for 5 minutes and the suspension was H2O : MeCN and 0.1% formic acid in a 90 : 10 to 0 : 100 linear
added to a solution of N-terminus deprotected linear peptide or gradient showed two peaks of retention times 7.9 and 8.1 min in
amino acid ester (NH2-AA-COOEt) (1 eq.) in dry THF, followed 1 : 1.4 ratio. Thermal analysis by NMR, proved them to be
by DIPEA (1 eq.). The reaction mixture was stirred overnight and conformers. 85% total purity.
concentrated under vacuum. 30 mL of AcOEt were added and
3.3.2.2. Cyclo-[NMeAla-Anth-NMeAla-Anth-Ala] (1). White
the organic phase was washed with HCL 5% (3 ꢂ 5 mL), brine (3 solid (Y ¼ 50%). Rf ¼ 0.32 (AcOEt). [a]2D5 ¼ ꢁ215.7 (c 0.275,
1
ꢂ 5 mL) and NaHCO3 (3 ꢂ 5 mL), dried with Na2SO4, ltered CHCl3). H NMR (400 MHz, DMSO-d6) Three conformers were
and concentrated. The crude material was puried by ash present: conformer A : conformer B : conformer
C
in
chromatography.
1 : 0.4 : 0.1 ratio. d 1.30–1.37 (m, 3Ha, 6Hb, 6Hc), 1.41 (d, J ¼
3.2.2. Ester hydrolysis. 1 eq. of the ester was dissolved in 6.8 Hz, 3Ha, 3Hb), 1.53 (d, J ¼ 6.7 Hz, 3Ha), 1.58 (d, J ¼ 6.9 Hz,
THF and 3 eq. of de LiOH dissolved in water. The reaction was 3Hc), 2.70 (s, 3Ha), 2.75 (s, 3Hb), 2.77 (s, 3Hb), 2.84 (s, 3Ha), 2.87
stirred for two hours at room temperature. THF was removed (s, 3Hc), 3.23 (s, 3Hc), 4.68–4.80 (m, 1Ha, 2Hc), 4.81–4.92 (m,
under vacuum. HCl 5% was added to the remaining solution to 1Ha), 5.00–5.08 (m, 1Hb), 5.20 (q, J ¼ 6.8 Hz, 1Ha), 5.26–5.34 (m,
a pH of 3. The solution was extracted with AcOEt. The organic 1Hc), 5.35–5.44 (m, 2Hb, 1Hc), 7.14–7.20 (m, 1Ha, 1Hb), 7.22–
layers were dried with Na2SO4, ltered, and the solvent removed 7.28 (m, 1Ha, 2Hc), 7.40–7.58 (m, 3Ha, 1Hb, 3Hc), 7.77 (d, J ¼
under vacuum.
8.2 Hz, 1Hb), 7.86 (dd, J ¼ 8.0, 0.9 Hz, 1Hb), 7.98–8.09 (m, 2Ha,
3.2.3. Boc deprotection procedure. A threefold excess of 1Hc), 8.48 (dd, J ¼ 8.3, 0.7 Hz, 1Hc), 8.54 (dd, J ¼ 8.4, 0.4 Hz,
a solution of 1.8 M HCl in dioxane was added to the Boc- 1Hb), 8.60 (dd, J ¼ 8.4, 0.7 Hz, 1Ha), 8.73 (s, 1Hc), 9.04 (s, 1Hb),
aminoacid. The solution was stirred at room temperature for 9.27 (d, J ¼ 3.7 Hz, 1Hb), 9.40 (d, J ¼ 4.2 Hz, 1Ha), 9.51 (s, 1Ha),
an hour and the solvent removed under vacuum. The obtained 11.43 (s, 1Hb), 12.22 (s, 1Ha), 12.67 (s, 1Hc). 13C NMR (100 MHz,
hydrochloride derivative was used in the next step without DMSO-d6) d 13.5 (Cb), 15.0 (Ca), 15.3 (Ca), 15.5 (Cb), 16.6 (Ca),
further purication.
17.5 (Cb), 28.7 (Cb), 29.4 (Ca), 29.8 (Ca), 38.7 (Cb), 45.5 (Cb), 46.7
(Ca), 55.7 (Cb), 56.2 (Ca), 59.2 (Ca), 116.9 (Ca), 119.4 (Cb), 119.5
(Ca), 119.7 (Cb), 122.9 (Ca), 123.0 (Cb), 124.4 (Ca), 124.8 (Cb),
125.4 (Ca), 125.5 (Ca), 126.2 (Cb), 126.6 (Cb), 127.5 (Ca), 129.4
3.3. Macrocyclization in solution phase
3.3.1. Method I. Macrocyclization reaction of the corre- (Cb), 129.6 (Ca), 130.6 (Cb), 130.7 (Ca), 133.1 (Cb), 133.8 (Ca),
sponding linear peptide was performed in diluted conditions 136.0 (Cb), 136.2 (Cb), 136.7 (Ca), 139.2 (Cb), 140.3 (Ca), 167.9
(1–5 mM) using HBTU or HATU (1.5 eq.), DIPEA (3 eq.), 4-DMAP (Ca), 168.5 (Ca), 168.8 (2Cb), 169.3 (Cb), 169.5 (Ca), 169.6 (Ca),
(catalytic) in dried CH2Cl2 at room temperature during 3 days. 169.9 (Cb), 170.1 (Cb), 173.0 (Ca), 173.1 (Cb). ESI-MS m/z calc. for
The reaction mixture was washed with HCl 5% and then with
C
25H30N5O6 ([M + H]+) 480.2, found 480.3.
saturated aqueous NaHCO3, dried over MgSO4, ltered and
3.3.2.3. Cyclo-[NHMe-Ala-Anth-Ala-Anth-NMeAla] (versicotide
concentrated in vacuo. The crude was puried by ash chro- B) (2). The triuoroacetate salt of NHMe-Ala-Anth-Ala-Anth-
matography to obtain the pure macrocycle. NMeAla-OH was obtained as a white solid following the
3.3.2. Method II. The triuoroacetate salt of the corre- general SPPS procedure. The global yield based on the deter-
sponding linear peptide was dissolved in dried CH2Cl2 and mination of the resin loading, was 87% (350 mg, 0.57 mmol).
diluted to a concentration of 1–5 mM. DIPEA (1 eq.) was added ESI-MS m/z calc. for C25H32N5O6 ([M + H]+) 498.2, found 498.4.
to enable dissolution. EDCI (1.2 eq.) and oxyma (1.2 eq.) were Macrocyclization reaction was performed following the general
added at 0 ꢀC and the reaction mixture was stirred for 10 procedure method I or II (dilution 5 mM, 3 days), starting from
minutes. Then, the reaction mixture is allowed to reach room the triuoroacetate salt of linear peptide NHMe-Ala-Anth-Ala-
temperature and stirred for 3 days. The reaction mixture was NMe-Ala-Anth-Ala-OH (200 mg, 0.33 mmol). Cyclization
washed with HCl 5% and then with saturated aqueous NaHCO3, method I, using HATU as coupling agent, rendered the desired
dried over MgSO4, ltered and concentrated in vacuo. The crude macrocycle in 18% yield (28 mg, 0.06 mmol) aer purication
was puried by ash chromatography to obtain the pure by ash chromatography using AcOEt as mobile phase. HPLC
macrocycle.
analysis employing H2O : MeCN and 0.1% formic acid in
3.3.2.1. Cyclo-[NHMeAla-Anth-NMeAla-Anth-Ala] (versicotide a 90 : 10 to 0 : 100 linear gradient showed two peaks tr ¼ 7.5 and
A) (1). The triuoroacetate salt of NHMeAla-Anth-NMeAla-Anth- 7.8 min. Cyclization method II, using Oxyma and EDCI as
Ala-OH was obtained as a white solid following the general SPPS coupling reagents rendered the desired macrocycle in 54% yield
procedure. The global yield, based on the determination of the (65 mg, 0.13 mmol) aer purication by ash chromatography
resin loading, was 90% (352 mg, 0.58 mmol). ESI-MS m/z calc. using AcOEt : EP (3 : 2) as mobile phase. HPLC analysis
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employing H2O : MeCN and 0.1% formic acid in a 90 : 10 to 128.6(2C), 133.2(2C), 141.6(2C), 168.3(2C), 169.8(2C), 172.3(2C).
0 : 100 linear gradient in 15 min showed two peaks tr ¼ 7.5 and (cis, cis) Conformer: d 17.1(2C), 18.1(2C), 28.4(2C), 46.1(2C),
7.8 min in a 1.4 : 1 ratio. Collection of the peaks at the end of 56.4(2C), 119.8(2C), 120.1(2C), 128.1(2C), 133.3(2C), 141.6(2C),
the column and LC-MS analysis revealed both peaks have the 142.9(2C), 169.7(2C), 170.7(2C), 171.1(2C). ESI-MS m/z calc. for
same mass as the natural product versicotide B. Thermal
analysis by NMR proved them to be conformers. 91% total
purity.
C
28H35N6O6 ([M + H]+) 551.3, found 551.3.
3.3.2.7. Cyclo-[NH2Ala-Ala-Anth-Ala-Anth] (4). Compound 5
was prepared by solution phase synthesis, either by coupling
3.3.2.4. Cyclo-[NMeAla-Anth-Ala-Anth-NMeAla] (2). Yellow method I or II (yield 42% and 51% respectively). Half of
solid (Y ¼ 54%). ESI-MS m/z calc. for C25H30N5O6 ([M + H]+) dipeptide 5 was deprotected on the C terminal following the
480.2, found 480.6. 1H NMR (400 MHz, DMSO-d6) two general ester hydrolysis procedure. The other half was depro-
conformers A : B in 1 : 1.3 ratio. d 1.13 (d, J ¼ 6.6 Hz, 3Ha), 1.26 tected on the N terminal by Boc deprotection procedure. The
(d, J ¼ 6.7 Hz, 3Hb), 1.40–1.44 (m, 6Ha, 3Hb), 1.47 (d, J ¼ 6.5 Hz, resulting dipeptides were coupled following coupling method I
3Hb), 2.24 (s, 3Ha), 2.96 (s, 3Hb), 2.99 (s, 3Hb), 3.06 (s, 3Ha), 4.14– to obtain 6 in a 10% yield. 6 was subjected to Boc deprotection
4.25 (m, 1Ha), 4.52–4.68 (m, 1Hb), 4.70–4.87 (m, 1Ha), 5.08–5.17 conditions and then Boc-Ala-OH was coupled with coupling
(m, 1Hb), 5.17–5.26 (m, 1Ha), 5.30–5.41 (m, 1Hb), 7.12–7.22 (m, method I to obtain 7. 7 was deprotected rst on the C terminal
1Ha + 1Hb), 7.23–7.31 (m, 1Ha), 7.34 (d, J ¼ 6.7 Hz, 1Hb), 7.39– with ester hydrolysis procedure, and then at the N terminal with
7.58 (m, 2Ha, 2Hb), 7.60 (m, 2Ha), 7.81 (d, J ¼ 6.1 Hz, 1Hb), 8.07 Boc deprotection procedure. The resulting peptide was cyclized
(d, J ¼ 7.6 Hz, 1Hb), 8.16 (d, J ¼ 8.2 Hz, 1Ha), 8.44–8.5 (m, 2Ha,+ following macrocyclization method II to render cyclopeptide 4
2Hb), 9.07–9.20 (m, 2Ha), 9.24 (s, 1Hb), 9.36 (s, 1Hb), 10.58 (s, in 35% yield.
1Ha), 10.92 (s, 1Hb). 13C NMR (100 MHz, DMSO-d6) d 13.5 (Ca),
3.3.2.8. Cyclo-[Ala-Ala-Antr-Ala-Antr] (4). Yellow solid Rf ¼
1
13.8 (Cb), 14.8 (Ca), 16.7 (Ca), 17.1 (Cb), 17.5 (Cb), 28.7 (Ca), 30.3 0.41 (AcOEt : EP, 4 : 1). H-NMR (400 MHz, (CD3)2CO) d 1.27–
(Cb), 30.8 (Ca), 34.3 (Cb), 49.8 (Cb), 50.3 (Cb), 52.1 (Ca), 52.5 (Ca), 1.36 (m, 3H), 1.53–1.62 (m, 6H), 4.46–4.58 (m, 1H), 4.63–4.75
52.6 (Cb), 54.5 (Ca), 120.3, 120.6, 122.1, 123.1, 123.7, 124.3, (m, 1H), 4.80–4.95 (m, 1H), 7.08–7.19 (m, 2H), 7.72 (dd, J ¼ 7.6,
124.6, 125.6, 126.1, 127.3, 128.6, 129.2, 129.4 (Ca), 130.2, 130.6, 1.0 Hz, 1H), 7.48 (m, 2H), 7.85 (d, J ¼ 7.9 Hz, 1H), 7.98–8.06 (m,
132.1 (Ca), 132.9, 135.3, 136.2, 137.4 (Ca), 139.8, 141.6, 145.7, 1H), 8.10–8.19 (m, 1H), 8.20–8.26 (m, 1H), 8.35 (d, J ¼ 8.3 Hz,
168.4 (Cb), 168.6 (Ca), 169.6 (2Ca,b), 169.8 (Ca), 170.3 (Cb), 170.9 1H), 8.62 (d, J ¼ 8.7 Hz, 1H), 11.05 (s, 1H), 11.31 (s, 1H). 13C
(Cb), 171.3 (Ca), 171.9 (Ca), 172.3 (Cb).
NMR (100 MHz, (CD3)2CO) d (ppm) 15.5, 17.1, 17.4, 49.6, 50.4,
3.3.2.5. Cyclo-[NHMe-Ala-Anth-Ala-NMe-Ala-Anth-Ala] (versi- 51.2, 121.1, 121.2, 121.4, 121.5, 122.6, 122.8, 123.7, 125.3, 127.4,
cotide C) (3). The triuoroacetate salt of NHMe-Ala-Anth-Ala- 128.6, 131.4, 132.1, 168.3, 170.2, 171.2, 173.0, 173.1. MS m/z
NMe-Ala-Anth-Ala-OH was obtained as a white solid following calc. for C23H25N5O5 ([M + H]+) 452.19, found 452.2.
the general SPPS procedure. The global yield, based on the
See ESI† for HPLC chromatograms, MS and NMR spectra
determination of the resin loading, was 98% (240 mg, 0.35 data of the cyclopeptides.
mmol). ESI-MS m/z calc. for C28H37N6O7 ([M + H]+) 569.3, found
569.3. Macrocyclization reaction was performed following the
general procedure method I (dilution 5 mM, 3 days), starting
4. Conclusions
from the triuoroacetate salt of linear peptide NHMe-Ala-Anth- In conclusion, the syntheses of three natural products, versi-
Ala-NMe-Ala-Anth-Ala-OH (200 mg, 0.30 mmol), using HATU as cotides A–C, containing Anth, were accomplished in very good
coupling agent. Further purication by ash chromatography yields and purity aer searching for the most adequate meth-
using AcOEt as mobile phase, rendered the desired macrocycle odology and coupling reagents. The combination of oxyma and
in 49% yield (77 mg, 0.15 mmol). HPLC analysis employing DIC, provided a simple methodology to achieve difficult
H2O : MeCN and 0.1% formic acid in a 90 : 10 to 0 : 100 linear couplings involving the aromatic amine of Anth, in short times,
gradient showed 93% purity, tr ¼ 6.88 min.
to avoid possible rearrangements or side reactions, and with
(3). very good yields. This methodology could be applied to the
3.3.2.6. Cyclo-[NMe-Ala-Anth-Ala-NMe-Ala-Anth-Ala]
White solid (Y ¼ 49%). Rf ¼ 0.25 (AcOEt). [a]D25 ¼ ꢁ35.83 (c synthesis of interesting compounds containing this motif.
0.25, MeOH). 1H NMR (400 MHz, (CD3)2CO) two conformers
Macrocyclization of versicotides A and C was achieved with
were present: A (trans, trans)-conformer and B (cis, cis)- HATU and no epimerization was detected. For macro-
conformer in 1 : 0.2 ratio. (trans, trans) Conformer: d 1.43 (d, J ¼ cyclcization of versicotide B (2), Oxyma and EDCI provided
7.0 Hz, 3H), 1.50 (d, J ¼ 6.8 Hz, 3H), 3.53 (s, 3H), 3.93 (q, J ¼ higher yield. In the versicotides A–C NMR spectra, the presence
6.8 Hz, 1H), 5.26–5.36 (m, 1H), 6.91–6.99 (m, 1H), 7.34–7.41 (m, of well-dened peaks for each conformer shows that these
1H), 7.67 (d, J ¼ 7.2 Hz, 1H), 7.75 (dd, J ¼ 7.9, 1.4 Hz, 1H), 8.69 molecules are rigid enough for the conversion from one
(dd, J ¼ 8.4, 0.9 Hz, 1H), 11.88 (s, 1H). (cis, cis) Conformer: d 1.34 conformer into another to be slower than NMR time scale. A
(d, J ¼ 6.6 Hz, 1H), 1.67 (d, J ¼ 7.2 Hz, 1H), 2.73 (s, 3H), 4.69 (q, J deep analysis of versicotide C NMR spectra led us to conclude
¼ 7.2 Hz, 1H), 5.26–5.36 (m, 1H), 6.86–6.91 (m, 1H), 7.05–7.11 that a mixture of (cis, cis) and (trans, trans) symmetrical
(m, 1H), 7.88 (dd, J ¼ 8.1, 1.2 Hz, 1H), 8.07 (dd, J ¼ 8.4, 0.8 Hz, conformers is present in DMSO-d6 rather than a single (cis,
1H), 8.15 (d, J ¼ 9.5 Hz, 1H), 12.29 (s, 1H). 13C NMR (100 MHz, trans) conformer as previously reported for the natural product.
(CD3)2CO) (trans, trans) conformer:
d 12.7(2C), 18.1(2C), Biological evaluation against P. falciparum of compounds 1–4
38.4(2C), 46.7(2C), 63.1(2C), 119.5(2C), 119.9(2C), 122.6(2C), will be performed and reported in the due course.
43658 | RSC Adv., 2020, 10, 43653–43659
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RSC Advances
11 L. N. Zhou, H. Q. Gao, X. S. Cai, T. J. Zhu, Q. Q. Gu and
D. H. Li, Helv. Chim. Acta, 2011, 94, 1065.
Conflicts of interest
12 J. Peng, H. Gao, X. Zhang, S. Wang, C. Wu, Q. Gu, P. Guo,
T. Zhu and D. Li, J. Nat. Prod., 2014, 77, 2218.
There are no conicts to declare.
13 R. Chen, Z. Cheng, J. Huang, L. Dong, C. Wu, P. Guo and
W. Lin, RSC Adv., 2017, 7, 49235.
Acknowledgements
The authors acknowledge the collaboration of Leopoldo Sues-
cun and Alejandro Peixoto to obtain the versicotide A X-ray
crystallographic data. This work was supported by Grants
˜
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