Cyclopeptide alkaloids: Stereochemistry and synthesis of the precursors of discarines C and D and myrianthine A

Article abstract of DOI:10.1021/np400313w

The stereochemistry of discarines C (1) and D (2) and myrianthine A (3), three cyclopeptide alkaloids isolated from Discaria febrifuga, was determined by a combination of NMR studies of 1-3, enantioselective gas chromatography, and comparison of NMR data

Full text of DOI:10.1021/np400313w

Article
pubs.acs.org/jnp
Cyclopeptide Alkaloids: Stereochemistry and Synthesis of the
Precursors of Discarines C and D and Myrianthine A
Marco A. Mostardeiro,†,⊥ Vinicius Ilha,‡,⊥ Janice Dahmer,‡ Miguel S. B. Caro,§ Ionara I. Dalcol,‡
Ubiratan F. da Silva,‡ and Ademir F. Morel*,‡
†Departamento de Química, Universidade Federal de Sergipe, CEP 49100-000. Rod. Marechal Randon, s/n Jardin Rosa Elze, Sao
̃
Cristovao, SE, Brazil
́
̃
‡Departamento de Química, Universidade Federal de Santa Maria, CEP 97105-900, Camobi, Santa Maria, RS, Brazil
§Departamento de Química, Universidade Federal de Santa Catarina, CEP 88040-970, Florianop
olis, SC, Brazil
́
S
* Supporting Information
ABSTRACT: The stereochemistry of discarines C (1) and D (2) and
myrianthine A (3), three cyclopeptide alkaloids isolated from Discaria
febrifuga, was determined by a combination of NMR studies of 1−3,
enantioselective gas chromatography, and comparison of NMR data with
those of synthetic tripeptides. For the synthesis of peptides, the
nonproteinogenic amino acid 3-phenylserine was also obtained in its
four diastereoisomeric forms (L and D threo, obtained by recrystallization
of the diastereoisomeric tripeptide, and L and D erythro, obtained by a
Mitsunobu reaction with the threo-tripeptides). The general synthetic
strategy described in this paper allows the tripeptide to be obtained with
the free N-terminal extremity protected or dimethylated. This strategy
also allows the synthesis of the corresponding peptide with an
imidazolidinone ring.
yclopeptide alkaloids are polyamide bases composed of
13-, 14-, or 15-membered cyclic ethers, found mainly in
presence of this unit in the structures of this type of alkaloid
may be responsible for or contribute to their activity.6 Because
3-phenylserine has two asymmetric carbons, there is the
possibility of four diastereoisomeric forms [(D/L) erythro and
(D/L) threo].
C
plants of the Rhamnaceae family.1 Their structures comprise a
p-hydroxystyrylamine unit derived from tyrosine, a peptide
fragment formed by an α-amino acid, a β-hydroxyl amino acid
(3-hydroxyproline, 3-hydroxyleucine, or 3-phenylserine), and
an N-mono- or dimethylated terminal amino acid.
Taking into account that the biological activity may be
related to the stereochemistry of the amino acid, it is important
to clearly determine the configuration of the chiral centers of
the units that form the natural product. As the stereochemistry
of these alkaloids was undetermined, we proposed to determine
the absolute configuration of their proteinogenic amino acids,
leucine, isoleucine, and phenylalanine, and of their N-
dimethylated amino acid (N,N′-dimethylleucine, common in
the three alkaloids) by enantioselective gas chromatography.7
Because the amino acid 3-phenylserine degrades under the
conditions of acid hydrolysis of the alkaloids, its configuration
Discaria americana Gill & Hook (Rhamnaceae), also known
as “quina, quina-do-campo, quina-do-Brasil”,2 is a small shrub
found in southern Brazil. It is used in folk medicine to treat
diseases such as diabetes, skin diseases, and stomach ailments,
as a heart tonic, and as an agent for the treatment of fevers.3
The isolation and characterization of various 14-membered-ring
cyclopeptide alkaloids from plants of the genus Discaria have
been described in former publications.4 From Discaria
americana we have isolated, among other alkaloids, the
discarines A, B, C, and D and myrianthine A, which were
previously tested for their antimicrobial activity against Gram-
negative and Gram-positive bacteria and fungi.3
For the present study, we selected the alkaloid discarines C
(1) and D (2) and myrianthine A (3), because they present in
their structures the same β-hydroxy amino acid (3-phenyl-
serine) and the same dimethylated terminal amino acid (N,N′-
dimethylleucine), differing only by the α-amino acid of the
macrocycle (leucine, phenylalanine, isoleucine, respectively).
The β-hydroxy amino acids are important representatives of the
subclass of nonproteinogenic amino acids that are present in
many biologically active natural products.5 Therefore, the
1
was proposed using H and 13C NMR data.
In 1997 Han et al. reported that frangufoline, a sedative 14-
membered cyclopeptide alkaloid, was converted, via an
enzymatic process in rodents, to the tripeptide M1, [(S)-
(N,N′-dimethylphenylalanyl)-(2S,3S)-3-[(formylphenoxy)-
leucyl]-(S)-leucine. On the basis of these results, Han et al.
suggested that frangufoline, once absorbed, rapidly undergoes
metabolic conversion to M1, which would be the pharmaco-
Received: April 15, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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logically active metabolite.8 On the basis of these results, and to
determine the absolute configuration of these alkaloids, a series
of peptide fragments, linear precursors of alkaloids 1−3, was
synthesized in all four diastereoisomeric forms of the
nonproteinogenic amino acid 3-phenylserine.
The tripeptide precursors prepared in this study were
evaluated for antimicrobial activity by direct bioautography
assays. Configuration−activity relationships of the β-hydroxy
amino acids in the peptides were also examined.
For the L-erythro series, the signal of the α-carbon (C-4)
appears at ca. δ 55.0, whereas for the D-erythro it appears at ca. δ
53.0. Important information is also observed for the β-carbon
(C-3): in the L-erythro series the signal appears at δ 81.5,
whereas for the D-erythro configuration it appears at ca. δ 87.0.
Therefore, the carbon chemical shifts of C-3 and C-4 provide
information for determining the absolute configuration of the
β-OH-amino acid unit in the erythro form; however little
information was found for the threo series.11
RESULTS AND DISCUSSION
■
We began this study by determining the stereochemistry of the
cyclopeptide alkaloids, of which the α-amino acids of the
macrocycle are leucine in compound 1, phenylalanine in
compound 2, and isoleucine in compound 3. N,N′-
Dimethylleucine is the terminal unit common in all three
alkaloids. Its absolute configuration was determined by chiral
phase gas chromatography (CPGC) using 2,6-Me-3-Pe-β-CD
as the stationary phase.7 With this purpose, the alkaloid
discarines C and D and myrianthine A were hydrolyzed and
their amino acids derivatized and analyzed through the CPGC.
In discarine C, leucine has the L(S) configuration, in discarine
D phenylalanine has the L(S) configuration, and in myrianthine
A the isoleucine is in the L(S) form. The common N,N′-
dimethylleucine has the same L(S) configuration.
On the basis of analysis of the 13C NMR data, it was possible
to establish the configuration 3S, 4S for the β-OH-amino acid
unit in alkaloids 1−3, because C-4 presents a chemical shift
around δ 56.0, while C-3 is at approximately δ 82.0. For the D-
erythro series (3R, 4R), these signals should appear at δ 53.0
and 87.0, respectively.9
After determining the configuration of the alkaloids, we
initiated the synthesis of their fragment precursors. The
synthetic route shown in Scheme 1 used the classical
techniques for peptide synthesis and allowed the synthesis of
the tripeptide precursors of these alkaloids in order to aid in the
elucidation of the stereochemistry of the alkaloids.
The synthesis began with dipeptide 4 obtained in agreement
with the literature.12 The dipetide Z-L-Leu-D,L-threo-Phe-
(βOH)-OH (4) was coupled to the amino acids leucine,
isoleucine, and phenylalanine, thereby obtaining the respective
tripeptides (Z-L-Leu-D,L-threo-Phe(βOH)-L-Leu-OMe, Z-L-Leu-
D,L-threo-Phe(βOH)-L-Phe-OMe, and Z-L-Leu-D,L-threo-Phe-
(βOH)-L-Ile-OMe), yielding pure diastereoisomeric forms
through successive recrystallization with ethyl ether/petroleum
ether (40−60 °C). Following the synthesis, the protecting
group benzyloxycarbonyl was removed through catalytic
hydrogenation (Pd/C 10%) and bubbling of hydrogen,
providing the peptides with free amino function, now ready
to be N-dialkylated through a reductive methylation.
9
1
According to the literature, in the H NMR spectra of the
alkaloid frangulanine, the erythro form of the β-hydroxy amino
acid shows a J3,4 of ca. 8.0 Hz (ϕ of 0−20° or 150−180°), and
the fairly rigid geometry of frangulanine permits only the latter
geometry, i.e., erythro configuration. Moreover, the threo 3-
hydroxyleucine shows a J3,4 of ca. 2.0 Hz (ϕ of ca. 60° or 120°).
The fairly rigid structures of this macrocycle permit only the
geometry with angles of ca. 60° in the threo configuration of the
alkaloids (Figure 1).10 Moreover, this information provides
only the relative configuration (erythro/threo) of this unit, but
not the absolute configuration (L or D erythro/threo β-OH-
amino acid). However, the 13C NMR data provide information
on the absolute configuration of this unit.9,11
Thus, the reaction of the amines with formaldehyde under
hydrogenation, catalyzed by Pd/C (10%) and hydrogen
pressure, afforded six desired tripeptides as methyl esters 11−
13. Surprisingly, during reductive methylation, byproducts
containing the imidazolidinone ring (14−16) were observed.
These products are also possible synthetic intermediates of
alkaloids such as sativanine-B13 and nummuraline-G.14
Reductive methylation using sodium cyanoborohydride gave
the same mixture of products, with a small increase in the
proportion of byproduct.12
The formation of these byproducts is related to a second step
of the reaction mechanism. After the insertion of the first
methyl group, by formation of an imine followed by reduction,
insertion of the second imine group occurs. This intermediate
3
Figure 1. The Karplus relationship for JH3−H4 coupling constants in
alkaloids 1−3.
B
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a
Scheme 1. Synthetic Route to Tripeptide Fragments 5−16
a
Reagents and conditions: (a) DCC, HOBt, NMM, THF, methyl ester of L-amino acid, 0 °C to rt; (b) 10% Pd/C (catalytic), H2 by bubbling,
MeOH, 4 h, rt, 92%; (c) CH2O, 10% Pd/C, H2 42.6 psi of pressure, MeOH, overnight, rt.
a
Scheme 2. Synthetic Route to Tripeptide Fragments 17a,b−22a,b
a
Reagents and conditions: (a) PPh3, 4-hydroxybenzaldehyde, DEAD, THF, 12 h, rt (Note: numbering of alkaloids 1−3 is applied, cf. schemes).
undergoes intramolecular nucleophilic attack by part of a pair of
free electrons of the −NH adjacent to this unit, forming the
cyclized product before it undergoes catalytic reduction.
Our next step following the proposed approach was the
inversion of the configuration in the threo-3-phenylserine unit
present in the tripeptides. Since the alkaloid is in the erythro
configuration, this was possible using Mitsunobu’s reaction15
(Scheme 2), which resulted in the configuration erythro-3-
phenylserine. Thus, the tripeptides 11−16 were reacted with 4-
hydroxybenzaldehyde, triphenylphosphine (PPh3), and diethyl
azodicarboxylate (DEAD) at room temperature, efficiently
producing compounds 17a,b−22a,b (a = L erythro/threo 3-
phenylserine, and b = D erythro/threo 3-phenylserine), which
contain the new linear alkyl−aryl ether bond, also present in
the alkaloids.
The screen for antibacterial activity of all tripeptides
synthesized was evaluated by means of direct bioautography
using a TLC bioassay.16 This method was selected because it
was the same method used previously by us for evaluation of
antimicrobial activity of 1−3.4m
The synthesized intermediates were more active against the
test microorganisms (<50 μg, considered active) than the
natural alkaloids 1−3 (>50 μg, considered inactive). Unfortu-
nately it was not possible to make a correlation between the
erythro/threo stereochemistry and antibacterial activity because
the synthesized erythro derivatives are structurally different from
the threo derivatives. However, it was possible to compare the
structure−activity relationship between the two threo isomers
(D and L) and between the two erythro isomers (D and L). For
example, the derivative H2N-L-Leu-L-threo-Phe(βOH)-L-Phe-
OMe (9a) was more active (3.1 μg) than the derivative H2N-L-
Leu-D-threo-Phe(βOH)-L-Phe-OMe (9b), which was consid-
ered inactive (>50 μg) against the bacterium Staphylococcus
epidermidis. In contrast, 9a was less active (12.5 μg) than 9b
(1.5 μg) against Escherichia coli. Comparing the activities of
compounds with an imidazolidinone ring, 15a and 15b, we
observed that 15b, with D-threo configuration, was more active
against S. epidermidis (6.2 μg), E. coli (3.1 μg), Klebsiella
Confirmation of the inversion of configuration at C-3 of the
1
3-phenylserine was obtained through H and 13C NMR data
allied to chiral phase gas chromatography. Thus, the H-3 that
had a coupling constant of about 2.0 Hz (threo form) moved to
6.0 Hz (erythro form), and the C-3 went from δ 71.0 to δ 79.0.
This confirmed the assignment of the amino acid 3-phenyl-
serine in the form of the erythro alkaloid discarines C and D and
myrianthine A, made previously from NMR data.
C
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been previously reported.12 It is described again in Supporting
Information.
pneumoniae (6.2 μg), and Salmonella setubal (12.5 μg) than its
diastereomer 15a, with the β-OH-amino acid in the L-threo
form (50 μg, >50 μg, 50 μg, 25 μg, respectively). From the
intermediates with the β-OH-amino acid in the erythro form,
only compounds 18a and 19a, with the L-erythro configuration,
showed activity against any of the test bacteria, while the D-
erythro diastereomers were inactive.
The activity of these peptides against bacterial species
suggests that these compounds may have an important role in
the biological activity of the corresponding alkaloids. In
conclusion the absolute configuration of discarines C (1) and
D (2) and myrianthine A (3) was determined by
enantioselective gas chromatography (CPGC) and NMR
spectroscopy data. Alkaloids 1−3 have the same absolute
configuration: (3S,4S) (L-erythro), (7S), and (23S).
Several peptide precursors of alkaloids 1−3 were synthesized
in the different diastereoisomeric forms of the nonproteino-
genic amino acid 3-phenylserine and used as models in NMR
spectroscopy. The synthetic peptides were tested against a
collection of Gram-positive and Gram-negative bacteria, and
the results showed that the synthesized tripeptides were more
active (<50 μg) than the corresponding alkaloids (>100 μg).
In addition, we observed that, in compounds 18a and 19a,
the L-erythro form of the β-OH-amino acid inserted in the
peptide was more active than the D-erythro form, whereas most
of the peptides synthesized in the threo form of its β-OH-amino
acid were active for some kind of bacteria, the D-threo form
being the most active. However, the results were not sufficient
to determine a structure−activity relationship against the tested
microorganisms. Therefore, further investigations will be made
in order to propose a mechanism of action and structure−
activity correlations.
General Procedure for the Synthesis of Tripeptides 11−16.
A solution of individual diastereomeric tripeptide, 8−10 (6.0 mmol
each), in MeOH (10 mL) and a 10% palladium-on-charcoal catalyst
(0.6 mmol) was prepared in a reaction bottle, and formaldehyde (12.0
mmol) was added. The reaction bottle was placed in a Parr-type
hydrogenator at 42.6 psi of H2. The reaction was monitored by
pressure decrease of H2 in the container and by TLC (CHCl3/MeOH,
99:1). Catalyst was removed in a column packed with diatomaceous
earth (Celite). The crude products (ca. 3.0 g) were separated by
chromatography on a column of silica gel (230−400 mesh), eluting
with CHCl3/MeOH (99:1).
(S)-Methyl-2-((2S,3R)-2-((S)-2-(dimethylamino)-4-methylpenta-
namido)-3-hydroxy-3-phenylpropanamido)-4-methylpentanoate
1
(11a): yield 66%; yellowish, viscous mass; H NMR (CDCl3) δ 7.41
(1H, d, J = 7.8 Hz), 7.37 (2H, m), 7.29 (2H, m), 7.23 (1H, m), 5.40
(1H, d, J = 2.3 Hz, H-3), 4.66 (1H, dd, J = 7.8, 2.3 Hz, H-4), 4.35 (1H,
m, H-7), 3.75 (3H, s), 2.78 (1H, dd, J = 8.7, 5.2 Hz, H-23), 2.04 (6H,
s), 1.60 (1H, m), 1.59 (3H, m), 1.34 (1H, m), 1.23 (2H, m), 0.90 (3H,
d, J = 6.1 Hz), 0.89 (3H, d, J = 6.1 Hz), 0.80 (3H, d, J = 6.5 Hz), 0.75
(3H, d, J = 6.5 Hz); 13C NMR (CDCl3) δ 174.9, 172.6, 171.6, 139.2,
128.3, 127.5, 125.6, 71.3 (CH, C-3), 67.4 (CH, C-23), 56.8 (CH, C-
4), 52.2, 50.8 (CH, C-7), 42.0, 40.9, 36.6, 25.6, 24.7, 23.3, 22.7, 21.7,
21.6; ESIMS m/z 450 (M + H)+.
(S)-Methyl-2-((2R,3S)-2-((S)-2-(dimethylamino)-4-methylpenta-
namido)-3-hydroxy-3-phenylpropanamido)-4-methylpentanoate
(11b): yield 70%; yellow, viscous mass; 1H NMR (CDCl3) δ 7.75 (1H,
d, J = 7.8 Hz), 7.39 (2H, m), 7.31 (2H, m), 7.27 (1H, m), 7.23 (1H, d,
J = 7.8 Hz), 5.41 (1H, d, J = 2.2 Hz, H-3), 4.63 (1H, dd, J = 7.8, 2.2
Hz, H-4), 4.52 (1H, m, H-7), 3.74 (3H, s), 2.82 (1H, dd, J = 8.7, 4.9
Hz, H-23), 2.05 (6H, s), 1.61(2H, m), 1.59 (1H, m), 1.27 (1H, m),
1.24 (2H, m), 0.90 (3H, d, J = 5.8 Hz), 0.89 (3H, d, J = 5.8 Hz), 0.81
(3H, d, J = 6.5 Hz), 0.75 (3H, d, J = 6.5 Hz); 13C NMR (CDCl3) δ
174.8, 172.7, 171.7, 139.2, 128.3, 127.5, 125.6, 71.3 (CH, C-3), 67.3
(CH, C-23), 56.8 (CH, C-4), 52.3, 50.9 (CH, C-7), 41.9, 40.9, 36.6,
25.6, 24.7, 23.3, 22.7, 21.7, 21.6; ESIMS m/z 450 (M + H)+.
(S)-Methyl-2-((2S,3R)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-hydroxy-3-phenylpropanamido)-3-phenyl-
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
determined with a MQAPF-301 apparatus and are uncorrected. H
■
1
propanoate (12a): yield 73%; white solid; mp 99−101 °C; H NMR
1
(CDCl3) δ 7.67 (1H, d, J = 8.0 Hz), 7.38 (1H, d, J = 8.3 Hz), 7.35
(1H, d, J = 7.2 Hz), 7.28 (1H, m), 7.21 (1H, m), 7.25 (1H, m), 7.19
(1H, m), 7.11 (1H, d, J = 6.8 Hz), 5.38 (1H, d, J = 2.0 Hz, H-3), 4.80
(1H, dt, J = 7.6, 7.2, 5.6 Hz, H-7), 4.65 (1H, dd, J = 8.0, 2.0 Hz, H-4),
3.72 (3H, s), 3.13 (1H, dd, J = 14.0, 7.2 Hz), 3.02 (1H, dd, J = 14.0,
5.6 Hz), 2.72 (1H, dd, J = 8.4, 5.2 Hz, H-23), 1.97 (6H, s), 1.43/1.35
(2H, m), 1.20 (1H, m), 0.80 (3H, d, J = 6.5 Hz), 0.76 (3H, d, J = 6.5
Hz); 13CNMR (CDCl3) δ 174.6, 171.3, 171.0, 139.3, 135.7, 129.0,
128.3, 128.1, 127.4, 126.9, 125.5, 71.4 (CH, C-3), 66.9 (CH, C-7),
57.1 (CH, C-4), 53.4 (CH, C-7), 52.2, 41.8, 37.6, 36.3, 25.5, 23.2,
21.8; ESIMS m/z 484 (M + H)+.
and 13C NMR spectra were recorded at 400.1/100.6 MHz, and 2D
experiments were acquired with the standard pulse program on a
Bruker DPX-400 spectrometer using CDCl3 as solvent and TMS as
internal standard. Optical rotations were taken on a Perkin−Elmer 341
digital polarimeter. Low-resolution ESIMS were recorded on an
Agilent LC/MS/MS model 6460. Thin-layer chromatography was
performed on precoated TLC plates (Merck, silica gel 60 F-254) using
CHCl3/MeOH (99:1). The spots were detected using one or more of
the following methods: UV (254 nm), ninhydrin (0.1% in EtOH), o-
toluidine, and the modified Dragendorff reagent. All reagents and
amino acids used, except 3-phenylserine (obtained according to Shaw
et al.17), possess the S-configuration (L-configuration) and are
commercially available (Aldrich/Fluka and Merck). Note: The
numbering adopted to identify stereogenic centers (1H and 13C) of
synthetic peptides in experimental NMR data follows the same
numbering given to the skeleton of alkaloids 1−3. This numbering is
different from that used in the IUPAC nomenclature.
(S)-Methyl-2-((2R,3S)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-hydroxy-3-phenylpropanamido)-3-phenyl-
1
propanoate (12b): yield 70%; white solid; mp 97−98 °C; H NMR
(CDCl3) δ 7.52 (1H, d, J = 7.8 Hz), 7.32 (2H, m), 7.32 (2H, m), 7.30
(2H, m), 7.26 (1H, m), 7.25 (1H. overlap), 7.24 (1H, m), 7.14 (2H,
m), 5.38 (1H, d, J = 2.4 Hz, H-3), 4.78 (1H, dt, J = 7.1, 5.7 Hz, H-7),
4.61 (1H, dd, J = 7.8, 2.4 Hz, H-4), 3.70 (3H, s), 3.14 (1H, dd, J =
13.9, 7.1 Hz), 3.03 (1H, dd, J = 13.9, 5.7 Hz), 2.64 (1H, bs, H-23),
2.03 (6H, s), 1.37 (1H, m), 1.43/1.29 (2H, m), 0.79 (3H, d, J = 2.4
Hz), 0.77 (3H, d, J = 2.4 Hz); 13C NMR (CDCl3) δ 175.1, 171.5,
171.4, 139.3, 135.6, 129.2, 128.7, 128.3, 127.6, 127.2, 125.6, 71.6 (CH,
C-3), 67.1 (CH, C-23), 57.5 (CH, C-4), 53.6 (CH, C-7), 52.3, 42.1,
37.7, 37.1, 23.1, 22.0, 20.2; ESIMS m/z 484 (M + H)+.
Plant Material. The root bark of Discaria americana was collected
in Santana do Livramento (30°53′33″ S, 55°31′36″ N), Brazil, in
December 2007 and was authenticated by Prof. Renato Zachia
(Department of Botany, Federal University of Santa Maria, RS Brazil).
A specimen sample SMDB 2829 was deposited at the herbarium of the
Federal University of Santa Maria.
Extraction and Isolation. The alkaloids discarine C (1), discarine
D (2), and myrianthine A (3) were isolated from the roots of D.
americana, as described previously by Morel et al.4d,l,m Physical and
spectroscopic data for the natural alkaloids 1−3 are found as
Supporting Information.
General Procedure for Synthesis of Tripeptides 5−10. The
preparation and characterization of the tripeptides fragments 5−10 has
(2S,3S)-Methyl-2-((2S,3R)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-hydroxy-3-phenylpropanamido)-3-methyl-
pentanoate (13a): yield 69%; yellow, viscous mass; 1H NMR
(CDCl3) δ 7.71 (1H, d, J = 7.7 Hz), 7.49 (1H, d, J = 8.4 Hz), 7.38
(2H, m), 7.30 (1H, m), 7.23 (2H, m), 5.40 (1H, d, J = 2.2 Hz, H-3),
4.65 (1H, dd, J = 7.7, 2.2 Hz, H-4), 4.50 (1H, dd, J = 8.4, 5.16 Hz, H-
7), 3.75 (3H, s), 2.80 (1H, m, H-23), 2.04 (6H, s), 1.90 (1H, m), 1.39
D
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(1H, m), 1.26 (2H, m), 1.39/1.15 (2H, m), 0.89 (3H, t. J = 7.3 Hz),
0.88 (3H, d, J = 6.2 Hz), 0.80 (3H, d, J = 6.5 Hz), 0.75 (3H, d, J = 6.5
Hz); 13C NMR (CDCl3) δ 174.9, 171.6, 171.6, 139.2, 128.2, 127.5,
125.5, 71.3 (CH, C-3), 67.4 (CH, C-23), 57.0 (CH, C-4), 56.7 (CH,
C-7), 52.1, 42.0, 37.4, 36.5, 25.6, 24.9, 23.3, 21.7, 15.4, 11.4; ESIMS
m/z 450 (M + H)+.
125.6, 71.8 (CH, C-3), 69.3, 64.5 (CH, C-23), 59.2 (CH, C-4), 53.4
(CH, C-7), 52.4, 40.9, 38.9, 37.5, 24.8, 22.9, 22.4; ESIMS m/z 448 (M
+ H)+.
(S)-Methyl-2-((2S,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-ox-
oimidazolidin-1-yl)-3-phenylpropanamido)isomethylpentanoate
(16a): yield 28%; yellow, viscous mass; 1H NMR (CDCl3) δ 7.32 (2H,
d, J = 7.4 Hz), 7.25 (2H, t, J = 7.2 Hz), 7.18 (1H, d, J = 7.2 Hz), 6.87
(1H, d, J = 8.2 Hz), 5.40 (1H, d, J = 4.5 Hz, H-3), 4.65 (1H, d, J = 4.5
Hz, H-4). 4.43 (1H, dd, J = 8.2, 5.1 Hz, H-7), 4.55 (1H, d, J = 7.0 Hz),
3.78 (1H, d, J = 7.0 Hz), 3.63 (3H, s), 2.74 (1H, t, J = 6.4 Hz, H-23),
2.23 (3H, s), 1.81 (1H, m), 1.61 (1H, m), 1.21 (2H, m), 1.17/1.05
(2H, m), 0.82 (3H, t, J = 6.4 Hz), 0.81 (3H, d, J = 6.8 Hz), 0.76 (3H,
d, J = 6.6 Hz), 0.73 (3H, d, J = 6.6 Hz); 13C NMR (CDCl3) δ 175.2,
171.5, 168.9, 139.2, 128.6, 127.6, 125.8, 72.0 (CH, C-3), 65.3 (CH, C-
23), 59.9 (CH, C-4), 56.3 (CH, C-7), 52.1, 52.0, 41.6, 38.0, 37.3, 25.0,
24.5, 22.6, 22.2, 15.4, 11.3; ESIMS m/z 448 (M + H)+.
(2S,3S)-Methyl-2-((2R,3S)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-hydroxy-3-phenylpropanamido)-3-methyl-
1
pentanoate (13b): yield 66%; yellowish semisolid mass; H NMR
(CDCl3) δ 7.56 (1H, d, J = 7.6 Hz), 7.39 (2H, d, J = 7.4 Hz), 7.32
(2H, m), 7.28 (1H, d, overlap), 7.27 (1H, m), 5.45 (1H, d, J = 2.2 Hz,
H-3), 4.67 (1H, dd, J = 7.6, 2.2 Hz, H-4), 4.5 (1H, dd, J = 8.2, 4.8 Hz,
H-7), 3.71 (3H, s), 2.70 (1H, dd, J = 8.4, 5.1 Hz, H-23), 1.89 (1H, m),
1.48 (2H, m), 1.44 (1H, m), 1.45/1.20 (2H, m), 0.90 (3H, t, J = 7.3
Hz), 0.89 (3H, d, J = 6.9 Hz), 0.82 (3H, d, J = 8.1 Hz), 0.81 (3H, d, J
= 8.1 Hz); 13C NMR (CDCl3) δ 175.3, 171.8, 171.5, 139.4, 128.3,
127.5, 125.6, 71.6 (CH, C-3), 67.4 (CH, C-23), 57.8 (CH, C-4), 56.9
(CH, C-7), 52.1, 42.2, 37.5, 37.3, 25.5, 25.1, 23.2, 22.0, 15.6, 11.6;
ESIMS m/z 450 (M + H)+.
(S)-Methyl-2-((2R,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-ox-
oimidazolidin-1-yl)-3-phenylpropanamido)isomethylpentanoate
1
(16b): yield 29%; yellowish, viscous mass; H NMR (CDCl3) δ 7.30
(S)-Methyl-2-((2S,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-ox-
oimidazolidin-1-yl)-3-phenylpropanamido)methylpentanoate
(14a): yield 32%; yellow, viscous mass; 1H NMR (CDCl3) δ 7.32 (1H,
d, J = 7.4 Hz), 7.25 (1H, t, J = 7.2 Hz), 7.18 (2H, d, J = 7.2 Hz), 6.87
(1H, d, J = 8.2 Hz), 5.40 (1H, d, J = 4.5 Hz, H-3), 4.65 (1H, d, J = 4.5
Hz, H-4), 4.55 (1H, d, J = 7.0 Hz), 4.43 (1H, dd, J = 8.2, 5.1 Hz, H-7),
3.78 (1H, d, J = 7.0 Hz), 3.63 (3H, s), 2.74 (1H, t, J = 6.4 Hz, H-23),
2.23 (3H, s), 1.81 (1H, m), 1.61 (1H, m), 1.21 (2H, m), 1.17/1.05
(2H, m), 0.82 (3H, t, J = 6.4 Hz), 0.81 (3H, d, J = 6.8 Hz), 0.76 (3H,
d, J = 6.6 Hz), 0.73 (3H, d, J = 6.6 Hz); 13C NMR (CDCl3) δ 175.1,
171.4, 168.8, 139.1, 128.5, 127.5, 125.7, 71.9 (CH, C-3), 65.3 (CH, C-
23), 59.8 (CH, C-4), 56.2 (CH, C-7), 52.0, 41.5, 37.9, 37.2, 24.9, 24.5,
22.6, 22.1, 15.3, 11.3; ESIMS m/z 448 (M + H)+.
(S)-Methyl-2-((2R,3S)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-ox-
oimidazolidin-1-yl)-3-phenylpropanamido)methylpentanoate
(14b): yield 27%; yellow, viscous mass; 1H NMR (CDCl3, 400.1 MHz)
δ 7.30 (1H, d, J = 7.4 Hz), 7.26 (1H, t, J = 7.4 Hz), 7.20 (2H, d, J = 7.0
Hz), 6.92 (1H, d, J = 8.0 Hz), 5.44 (1H, d, J = 4.7 Hz, H-3), 4.8 (1H,
d, J = 4.7 Hz, H-4), 4.45 (1H, d, J = 7.2 Hz), 4.45 (1H, dd, J = 8.0, 5.0
Hz, H-7), 3.80 (1H, d, J = 7.2 Hz), 3.68 (3H, s), 2.70 (1H, t, J = 6.0
Hz, H-23), 2.17 (3H, s), 1.80 (1H, m), 1.57 (1H, m), 1.20 (2H, m),
1.15/1.00 (2H, m), 0.80 (3H, t, J = 6.3 Hz), 0.78 (3H, d, J = 6.8 Hz),
0.76 (3H, d, J = 6.6 Hz), 0.73 (3H, d, J = 6.6 Hz); 13C NMR (CDCl3,
100.6 MHz) δ 174.10, 170.6, 168.9, 140.0, 129.5, 126.7, 125.9, 72.0
(CH, C-3), 64.3 (CH, C-23), 60.1 (CH, C-4), 57.2 (CH, C-7), 52.0,
51.4, 38.9, 37.4, 25.6, 23.5, 22.5, 21.6, 15.4, 11.6; ESIMS m/z 448 (M
+ H)+.
(2H, d, J = 7.4 Hz), 7.24 (2H, t, J = 7.2 Hz), 7.21 (1H, d, J = 7.2 Hz),
6.90 (1H, d, J = 8.0 Hz), 5.46 (1H, d, J = 4.2 Hz, H-3), 4.60 (1H, d, J =
4.2 Hz, H-4), 4.40 (1H, dd, J = 8.0, 5.2 Hz, H-7), 4.50 (1H, d, J = 7.0
Hz), 3.74 (1H, d, J = 7.0 Hz), 3.60 (3H, s), 2.70 (1H, t, J = 6.2 Hz, H-
23), 2.17 (3H, s), 1.80 (1H, m), 1.60 (1H, m), 1.23 (2H, m), 1.19/
1.05 (2H, m), 0.80 (3H, d, J = 6.2 Hz), 0.78 (3H, d, J = 6.8 Hz), 0.74
(3H, d, J = 6.4 Hz), 0.73 (3H, d, J = 6.4 Hz); 13C NMR (CDCl3) δ
174.9, 171.4, 167.1, 137.8, 129.2, 127.6, 125.6, 71.8 (CH, C-3), 65.2
(CH, C-23), 58.4 (CH, C-4), 56.8 (CH, C-7), 52.4, 51.9, 41.8, 38.1,
37.3, 24.9, 24.7, 22.9, 22.0, 15.4, 11.4; ESIMS m/z 448 (M + H)+.
General Procedure for the Synthesis of Tripeptides 17−22.
Tripeptides 12−16 (3.3 mmol) were added in a round-bottom flask
flamed under an argon atmosphere and dissolved in THF (10 mL);
then triphenylphosphine (PPh3) (3.3 mmol) and 4-hydroxybenzalde-
hyde (3.3 mmol) were added. The reaction was kept under magnetic
stirring; then diethyl azodicarboxylate (4.2 mmol) was added. Stirring
was continued for 12 h at room temperature. The reaction was
monitored to completion by TLC analysis. The solution was filtered
and evaporated to dryness under reduced pressure. The crude product
(ca. 2.0 g) was purified by column chromatography using silica gel
(230−400 mesh) as stationary phase and CHCl3 as eluent.
(S)-Methyl-2-((2S,3S)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-4-
methylpentanoate (17a): yield 37%; yellowish, viscous mass; 1H
NMR (CDCl3) δ 9.83 (1H, s), 7.65 (2H, d, J = 8.6 Hz), 7.60 (1H, d,
overlap), 7.30 (2H, m, overlap), 7.26 (2H, m, overlap), 7.23 (1H, m,
overlap), 7.02 (1H, d, J = 7.9 Hz), 6.94 (2H, d, J = 8.6 Hz), 5.70 (1H,
d, J = 9.0 Hz, H-3), 4.78 (1H, d, J = 9.0 Hz, H-4), 4.34 (1H, d, J = 6.2
Hz), 4.25 (1H, m), 3.68 (3H, s), 3.61 (1H, d, J = 6.2 Hz), 2.75 (1H, t,
J = 9.5 Hz, H-23), 2.20 (6H, s), 1.57 (1H, m), 1.49 (1H, m), 1.09/0.98
(2H, m), 0.83 (3H, d, J = 6.3 Hz), 0.76 (3H, d, J = 6.0 Hz), 0.74 (3H,
d, J = 6.0 Hz), 0.67 (3H, d, J = 6.3 Hz); 13C NMR (CDCl3) δ 191.3,
173.4, 172.4, 171.2, 163.2, 138.7, 131.9, 129.4, 128.9, 127.5, 114.9, 80.2
(CH, C-3), 67.2 (CH, C-23), 58.4 (CH, C-4), 51.9 (CH, C-7), 50.8,
42.7, 40.5, 34.8, 25.6, 24.7, 23.3, 22.7, 21.7, 21.6; HRESIMS m/z
554.3213 [M + H]+ (calcd for C31H43N3O6 554.33230).
(S)-Methyl-2-((2R,3R)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-4-
methylpentanoate (17b): yield 45%; yellow semisolid mass; 1H NMR
(CDCl3) δ 9.79 (1H, s), 7.70 (2H, d, J = 8.0 Hz), 7.58 (1H, d, J = 8.0
Hz), 7.53 (2H, m), 7.45 (2H, m), 7.27 (1H, m), 6.87 (2H, d, J = 8.0
Hz), 6.48 (1H, d, J = 8.2 Hz), 5.79 (1H, d, J = 5.8 Hz, H-3), 5.00 (1H,
dd, J = 8.0, 5.8 Hz, H-4), 4.45 (1H, dd, J = 8.2, 4.8 Hz), 3.70 (3H, s),
2.68 (1H, d, J = 8.5, 4.4 Hz, H-23), 2.19 (6H, s), 1.75 (1H, m), 1.43
(1H, m), 1.33/1.03 (2H, m), 1.10/0.98 (2H, m), 0.78 (3H, t, J = 6.7
Hz), 0.75 (3H, d, J = 6.7 Hz), 0.84 (3H, d, J = 6.1 Hz), 0.74 (3H, d, J
= 6.1 Hz); 13C NMR (CDCl3) δ 190.6, 173.9, 171.5, 168.2, 162.2,
135.3, 131.6, 129.8, 128.9, 128.5, 127.0, 116.0, 79.2 (CH, C-3), 66.5
(CH, C-23), 57.4 (CH, C-4), 52.3 (CH, C-7), 51.0, 42.6, 40.8, 35.1,
25.8, 24.7, 23.3, 22.7, 21.7, 21.4; HRESIMS m/z 554.3213 [M + H]+
(calcd for C31H43N3O6 554.33230).
(S)-Methyl-2-((2S,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-ox-
oimidazolidin-1-yl)-3-phenylpropanamido)-3-phenylpropanoate
1
(15a): :yield 25%; yellow, viscous mass; H NMR (CDCl3) δ 7.31
(1H, d, J = 6.8 Hz), 7.29−7.27 (2H, m), 7.26−7.20 (2H, overlap), 7.25
(1H, m), 7.21 (1H, m), 7.11 (1H, d, J = 6.8 Hz), 6.98 (1H, d, J = 7.6
Hz), 5.40 (1H, d, J = 4.4 Hz, H-3), 4.85 (d, 1H, J = 2.0 Hz, H-4), 4.79
(1H, dt, J = 7.6, 5.2 Hz, H-7), 4.47 (1H, d, J = 7.2 Hz), 3.69 (3H, s),
3.54 (1H, d, J = 7.2 Hz), 3.21 (1H, dd, J = 14.0, 7.6 Hz), 2.96 (1H, dd,
J = 14.0, 5.2 Hz), 2.67 (1H, t, J = 6.4 Hz, H-23), 2.10 (3H, s), 1.60
(1H, m), 1.05/0.95 (2H, m), 0.79 (3H, d, J = 6.4 Hz), 0.76 (3H, d, J =
6.4 Hz); 13C NMR (CDCl3) δ 175.3, 171.2, 168.1, 139.0, 135.7, 129.1,
128.4, 128.1, 127.5, 126.9, 125.7, 71.7 (CH, C-3), 68.8, 64.9 (CH, C-
23), 59.3 (CH, C-4), 53.2 (CH, C-7), 52.3, 41.2, 37.8, 37.2, 24.4, 23.6,
22.1; ESIMS m/z 482 (M + H)+.
(S)-Methyl-2-((2R,3S)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-ox-
oimidazolidin-1-yl)-3-phenylpropanamido)-3-phenylpropanoate
(15b): yield 26%; yellow, viscous mass; 1H NMR (CDCl3) δ 7.40 (2H,
m), 7.33 (2H, m), 7.29 (1H, m), 7.29 (2H, m), 7.26 (1H, m), 7.26
(2H, m), 6.08 (1H, sb), 5.44 (1H, d, J = 2.4 Hz, H-3), 4.85 (1H, dd, J
= 7.8, 2.4 Hz, H-4), 4.82 (1H, dt, J = 6.6, 5.7 Hz, H-7), 4.37 (1H, d, J =
6.0 Hz), 3.92 (1H, d, J = 6.0 Hz), 3.71 (3H, s), 3.08 (1H, dd, J = 14.0,
5.7 Hz), 3.06 (1H, dd, J = 14.0, 6.6 Hz), 2.61 (1H, t, J = 5.9 Hz, H-23),
2.18 (3H, s), 1.78 (1H, hept, J = 6.6 Hz), 1.40/1.29 (2H, m), 0.87
(3H, d, J = 6.6 Hz), 0.84 (3H, d, J = 6.6 Hz); 13C NMR (CDCl3) δ
171.2, 171.2, 169.1, 139.1, 135.4, 129.1, 128.7, 128.3, 127.7, 126.9,
E
dx.doi.org/10.1021/np400313w | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(S)-Methyl-2-((2S,3S)-2-((S)-2-(dimethylamino)-4-methylpenta-
namido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3-phenyl-
172.6, 167.2, 162.1, 135.7, 131.7, 129.3, 128.9, 127.3, 116.5, 77.7 (CH,
C-3), 69.2, 65.4 (CH, C-23), 60.3 (CH, C-4), 52.3, 50.9 (CH, C-7),
41.2, 38.2, 24.8, 24.5, 22.8, 22.7, 22.5, 21.7; HRESIMS m/z 552.2995
[M + H]+ (calcd for C31H41N3O6 552.2995).
1
propanoate (18a): yield 36%; yellowish semisolid mass; H NMR
(CDCl3) δ 9.13 (1H, s), 7.50 (1H, d, J = 8.8 Hz), 7.34−7.20 (5H, m),
7.19−6.80 (5H, m), 6.81 (1H, d, J = 8.8 Hz), 6.36 (1H, d, J = 7.8 Hz),
6.20 (2H, d, J = 8.8 Hz), 5.73 (1H, d, J = 6.4 Hz, H-3), 5.03 (1H, dd, J
(S)-Methyl-2-((2R,3R)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3-
methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-methyl-
pentanoate (20b): yield 31%; yellowish, viscous mass; 1H NMR
(CDCl3) δ 9.76 (1H, s), 7.72 (1H, bs), 7.88 (2H, d, J = 8.4 Hz),7.45−
7.19 (5H, m), 7.37−7.24 (5H, m), 7.18 (2H, d, J = 8.4 Hz), 5.73 (1H,
d, J = 9.3 Hz, H-3), 4.81 (1H, d, J = 9.3 Hz, H-4), 4.21 (1H, J = 5.2
Hz, H-7), 4.34 (1H, d, J = 6.2 Hz), 4.25 (1H, m), 3.68 (3H, s), 3.62
(1H, d, J = 6.2 Hz), 2.56 (1H, t, J = 6.0 Hz, H-23), 2.26 (3H, s), 1.57/
1.49 (2H, m), 1.49 (1H, m), 1.09/0.98 (2H, m), 0.81 (3H, d, J = 6.0
Hz), 0.79 (3H, d, J = 6.0 Hz); 13C NMR (CDCl3) δ 191.0, 175.0,
172.9, 172.7, 166.2, 162.4, 130.4, 131.9, 128.9, 128.5, 127.3, 115.5, 77.7
(CH, C-3), 73.1, 69.2 (CH, C-23), 60.6 (CH, C-4), 52.0, 50.8 (CH, C-
7), 43.0, 41.6, 41.6, 36.7, 25.0, 24.8, 22.9, 22.8, 22.5, 21.8; HRESIMS
m/z 552.2990 [M + H]+ (calcd for C31H41N3O6 552.2995).
(S)-Methyl-2-((2S,3S)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3-
methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-phenyl-
propanoate (21a): yield 28%; yellow semisolid mass; 1H NMR
(CDCl3) δ 9.80 (1H, s), 7.68 (2H, d, J = 8.4 Hz), 7.39−7.21 (5H, m),
730−7.09 (5H, m), 7.02 (1H, d, J = 8.0 Hz), 6.90 (2H, d, J = 8.4 Hz),
5.71 (1H, d, J = 9.6 Hz, H-3), 4.89 (1H, d, J = 9.6 Hz, H-4), 4.88 (1H,
overlap), 4.30 (1H, d, J = 6.0 Hz), 3.69 (3H, s), 3.49 (1H, d, J = 6.0
Hz), 3.17 (1H, dd, J = 14.0, 5.2 Hz), 2.99 (1H, dd, J = 14.0, 5.2 Hz),
2.64 (1H, t, J = 6.0 Hz, H-23), 2.18 (3H, s), 1.51 (1H, hept, J = 6.4
Hz), 1.16 (1H, m), 1.04 (1H, m), 0.79 (3H, d, J = 6.4 Hz), 0.71 (3H,
d, J = 6.4 Hz); 13C NMR (CDCl3) δ 190.6, 174.2, 171.3, 167.0, 162.0,
135.1, 131.6, 130.5, 129.2, 129.1, 128.8, 128.5, 127.3, 127.1, 116.5, 77.8
(CH, C-3), 68.6, 64.9 (CH, C-23), 59.6 (CH, C-4), 53.3 (CH, C-7),
52.4, 40.7, 38.2, 37.9, 24.4, 22.8, 22.5; HRESIMS m/z 586.2916 [M +
H]+ (calcd for C34H39N3O6 586.2917).
= 8.8, 6.4 Hz, H-4), 4.81 (1H, dd, J = 7.8, 6.2, 5.9Hz, H-7), 3.56 (3H,
̀
s), 2.00 (6H, s), 3.03 (1H, dd, J = 8.6, 5.1 Hz, H-23), 3.17 (1H, dd, J =
11.0, 5.9 Hz), 2.94 (1H, dd, J = 11.0, 6.2 Hz), 1.81 (1H, m), 1.40 (1H,
m), 0.80 (3H, d, J = 6.5 Hz), 1.27 (1H, m), 0.75 (3H, d, J = 6.5 Hz);
13C NMR (CDCl3) δ 190.7, 173.8, 171.0, 168.3, 162.1, 135.8, 135.4,
131.7, 130.4, 129.0, 128.9, 128.5, 127.1, 127.0, 116.2, 79.3 (CH, C-3),
67.4 (CH, C-23), 57.1 (CH, C-4), 53.3 (CH, C-7), 52.3, 42.0, 37.8,
35.5, 25.7, 23.4, 21.9; HRESIMS m/z 588.3079 [M + H]+ (calcd for
C34H41N3O6 588.3054).
(S)-Methyl-2-((2R,3R)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3-
phenylpropanoate (18b): yield 43%; yellow semisolid mass; 1H NMR
(CDCl3) δ 9.77 (1H, s), 7.70 (2H, d, J = 8.4 Hz), 7.56 (1H, d, J = 8.2
Hz), 7.48 (2H, m), 7.40 (2H, m), 7.27 (1H, m), 6.84 (2H, d, J = 8.4
Hz), 6.57 (1H, d, J = 8.3 Hz), 5.79 (1H, d, J = 5.0 Hz, H-3), 4.95 (1H,
dd, J = 8.2, 5.0 Hz, H-4), 4.50 (1H, dd, J = 8.3, 5.0 Hz, H-7), 3.69 (3H,
s), 2.84 (1H, d, J = 9.0, 4.5 Hz, H-23), 2.20 (6H, s), 1.83 (1H, m), 1.54
(1H, m), 1.50/1.30 (2H, m), 1.40/1.15 (2H, m), 0.96 (3H, t, J = 7.4
Hz), 0.91 (3H, d, J = 6.9 Hz), 0.88 (3H, d, J = 6.0 Hz), 0.87 (3H, d, J
= 6.0 Hz); 13C NMR (CDCl3) δ 191.0, 174.0, 171.5, 169.3, 162.1,
134.7, 134.4, 131.7, 130.4, 128.7, 128.5, 127.8, 127.1, 127.0, 115.9, 80.3
(CH, C-3), 66.2 (CH, C-23), 56.8 (CH, C-4), 52.7 (CH, C-7), 52.3,
42.6, 37.5, 34.9, 26.3, 23.4, 21.9; HRESIMS m/z 588.3078 [M + H]+
(calcd for C34H41N3O6 588.3054).
(2S,3S)-Methyl-2-((2S,3S)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3-
1
methylpentanoate (19a): yield 37%; yellow, viscous mass; H NMR
(CDCl3) δ 9.82 (1H, s), 7.73 (2H, d, J = 8.8 Hz), 7.60 (1H, d, J = 8.4
Hz), 7.43 (2H, m), 7.35 (2H, m), 7.31 (1H, m), 6.96 (2H, d, J = 8.8
Hz), 6.48 (1H, d, J = 8.5 Hz), 5.85 (1H, d, J = 5.6 Hz, H-3), 5.06 (1H,
dd, J = 8.4, 5.6 Hz, H-4), 4.54 (1H, dd, J = 8.5, 5.0 Hz, H-7), 3.67 (3H,
s), 2.76 (1H, d, J = 9.5, 4.4 Hz, H-23), 2.16 (6H, s), 1.80 (1H, m), 1.51
(1H, m), 1.47/1.34 (2H, m), 1.37/1.10 (2H, m), 0.92 (3H, t, J = 7.4
Hz), 0.91 (3H, d, J = 6.9 Hz), 0.88 (3H, d, J = 6.1 Hz), 0.85 (3H, d, J
= 6.1 Hz); 13C NMR (CDCl3) δ 190.6, 173.9, 171.5, 168.2, 162.2,
135.7, 131.8, 130.4, 128.9, 128.5, 126.8, 116.0, 79.2 (CH, C-3), 67.2
(CH, C-23), 57.4 (CH, C-4), 56.7 (CH, C-7), 52.0, 42.1, 37.8, 37.1,
25.6, 25.0, 25.0, 23.2, 15.2, 11.4; HRESIMS m/z 554.3215 [M + H]+
(calcd for C31H43N3O6 554.3230).
(2S,3S)-Methyl-2-((2R,3R)-2-((S)-2-(dimethylamino)-4-methyl-
pentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3-
methylpentanoate (19b): yield 34%; yellow semisolid mass; 1H NMR
(CDCl3) δ 9.82 (1H, s), 7.73 (2H, d, J = 8.8 Hz), 7.60 (1H, d, J = 8.4
Hz), 7.43 (2H, m), 7.35 (2H, m), 7.31(1H, m), 6.96 (2H, d, J = 8.8
Hz), 6.48 (1H, d, J = 8.5 Hz), 5.85 (1H, d, J = 5.6 Hz, H-3), 5.06 (1H,
dd, J = 8.4, 5.6 Hz, H-4), 4.54 (1H, dd, J = 8.0, 5.0 Hz, H-7), 3.67 (3H,
s), 2.76 (1H, d, J = 9.5, 4.4 Hz, H-23), 2.16 (6H, s), 1.80 (1H, m), 1.51
(1H, m), 1.47/1.34 (2H, m), 1.37/1.10 (2H, m), 0.92 (3H, t, J = 7.4
Hz), 0.91 (3H, d, J = 6.9 Hz), 0.88 (3H, d, J = 6.1 Hz), 0.85 (3H, d, J
= 6.1 Hz); 13C NMR (CDCl3) δ 190.6, 173.9, 171.5, 168.2, 162.2,
135.7, 131.8, 130.4, 128.9, 128.5, 126.8, 116.0, 79.2 (CH, C-3), 67.2
(CH, C-23), 57.4 (CH, C-4), 56.7 (CH, C-7), 52.0, 42.1, 37.8, 37.1,
25.6, 25.0, 25.0, 23.2, 15.2, 11.4; HRESIMS m/z 554.3215 [M + H]+
(calcd for C31H43N3O6 554.3230).
(S)-Methyl-2-((2S,3S)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3-
methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-methyl-
pentanoate (20a): yield 30%; yellow, viscous mass; 1H NMR
(CDCl3) δ 9.73 (1H, s), 7.63 (2H, d, J = 8.8 Hz), 7.38 (2H, d, J =
7.4 Hz), 7.28 (2H, m, overlap), 7.25 (1H, m, overlap), 7.02 (1H, d, J =
7.9 Hz), 6.91 (2H, d, J = 8.8 Hz), 5.75 (1H, d, J = 9.7 Hz, H-3), 4.81
(1H, d, J = 9.7 Hz, H-4), 4.34 (1H, d, J = 6.2 Hz), 4.25 (1H, m, H-7),
3.61 (1H, d, J = 6.2 Hz), 3.60 (3H, s), 2.65 (1H, t, J = 6.0 Hz, H-23),
2.16 (3H, s), 1.57/1.49 (2H, m), 1.49 (1H, m), 1.09/0.98 (2H, m),
0.67 (3H, d, J = 6.7 Hz), 0.73 (3H, d, J = 6.7 Hz), 0.79 (3H, d, J = 6.0
Hz), 0.77 (3H, d, J = 6.0 Hz); 13C NMR (CDCl3) δ 190.7, 174.7,
(S)-Methyl-2-((2R,3R)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3-
methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-phenyl-
propanoate (21b): yield 27%; yellow, viscous mass; 1H NMR
(CDCl3) δ 9.88 (1H, s), 8.32 (1H, d, J = 8.0 Hz), 7.88 (2H, d, J = 8.4
Hz), 7.40−7.29 (5H, m), 7.38−7.36 (5H, m), 7.18 (2H, d, J = 8.4 Hz),
5.72 (1H, d, J = 9.6 Hz, H-3), 5.21 (1H, d, J = 9.6 Hz, H-4), 4.55 (1H,
J = 5.2 Hz, H-7), 4.32 (1H, d, J = 6.0 Hz), 4.22 (1H, d, J = 6.0 Hz),
3.68 (3H, s), 3.56 (1H, d, J = 6.4 Hz, H-23), 3.23/2.98 (2H, dd, J =
14.0, 5.2 Hz), 2.26 (3H, s), 1.58 (2H, hept, J = 6.4 Hz), 1.39 (1H, m),
0.91 (3H, d, J = 6.3 Hz), 0.81 (3H, d, J = 6.3 Hz); 13C NMR (CDCl3)
δ 191.0, 178.5, 171.7, 171.5, 163.2, 140.6, 136.6, 131.9, 128.9, 128.6,
128.5, 127.7, 127.6, 127.1, 125.9, 114.9, 77.7 (CH, C-3), 73.1, 69.2
(CH, C-23), 60.6 (CH, C-4), 53.0 (CH, C-7), 51.9, 43.0, 36.8, 34.7,
25.0, 22.5; HRESIMS m/z 586.2914 [M + H]+ (calcd for C34H39N3O6
586.2917).
(S)-Methyl-2-((2S,3S)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3-
methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-
isomethylpentanoate (22a): yield 25%; yellow, viscous mass; 1H
NMR (CDCl3) δ 9.81 (1H, s), 7.71 (2H, d, J = 8.7 Hz), 7.45 (2H, m),
7.31 (2H, m, overlap), 7.31 (1H, m, overlap), 7.13 (1H, d, J = 8.6 Hz),
6.98 (2H, d, J = 8.7 Hz), 5.83 (1H, d, J = 9.6 Hz, H-3), 4.88 (1H, d, J =
9.6 Hz, H-4), 4.52 (1H, dd, J = 8.4, 5.0 Hz, H-7), 4.41 (1H, d, J = 6.1
Hz), 3.70 (1H, d, J = 6.1 Hz), 3.69 (3H, s), 2.73 (1H, m), 2.23 (3H,
s), 1.87 (1H, m), 1.57 (1H, m), 1.38 (2H, m), 1.38 (2H, m), 0.85 (3H,
t, J = 7.4 Hz), 0.84 (3H, d, J = 6.8 Hz), 0.81 (3H, d, J = 6.6 Hz), 0.74
(3H, d, J = 6.6 Hz); 13C NMR (CDCl3) δ 190.7, 174.7, 171.6, 168.9,
162.1, 135.7, 132.1, 131.9, 127.3, 77.8 (CH, C-3), 69.2, 65.4 (CH, C-
23), 60.5 (CH, C-4), 56.7 (CH, C-7), 52.1, 41.2, 38.2, 37.7, 25.0, 24.5,
22.8, 22.5, 15.5, 11.4; HRESIMS m/z 552.2991 [M + H]+ (calcd for
C31H41N3O6 552.2995).
(S)-Methyl-2-((2R,3R)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3-
methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-
isomethylpentanoate (22b): yield 29%; yellow, viscous mass; 1H
NMR (CDCl3) δ 9.81 (1H, s), 7.70 (2H, d, J = 8.6 Hz), 7.45 (2H, m),
7.31 (2H, overlap), 7.31 (1H, overlap), 7.00 (2H, d, J = 8.6 Hz), 5.91
(1H, d, J = 9.0 Hz, H-3). 4.97 (1H, d, J = 9.0 Hz, H-4), 4.33 (1H, d, J
= 6.4 Hz), 4.52 (1H, dd, J = 8.6, 5.0 Hz, H-7), 3.72 (1H, d, J = 6.4
Hz), 3.64 (3H, s), 2.63 (1H, t, J = 6.4 Hz, H-23), 2.12 (3H, s), 1.87
F
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(1H, m), 1.80 (1H, hept, J = 6.9 Hz), 1.40 (2H, m), 1.39/1.15 (2H,
m), 0.88 (3H, overlap), 0.88 (3H, overlap), 0.86 (3H, overlap), 0.86
(3H, overlap); 13C NMR (CDCl3) δ 190.6, 174.9, 171.4, 167.1, 162.0,
131.8, 130.5, 129.2, 128.7, 127.6, 116.4, 77.9 (CH, C-3), 68.8, 65.2
(CH, C-23), 58.4 (CH, C-4), 56.8 (CH, C-7), 51.9, 41.8, 38.1, 37.3,
24.9, 24.7, 22.9, 22.0, 15.4, 11.4; HRESIMS m/z 552.2993 [M + H]+
(calcd for C31H41N3O6 552.2995).
General Procedure for Determination of Amino Acid
Configurations. Hydrolysis of cyclopeptide alkaloids 1, 2, and 3
and of compounds 5−22 (2 mg of each) was performed by heating in
a sealed tube at 110 °C with 6 M HCl for 12 h. The acid solution was
removed under vacuum, and the residues were treated as described for
amino acids.7
Acid-catalyzed esterification was carried out by addition of 1.6 M
anhydrous HCl (gas) in MeOH and leaving the mixture at room
temperature for 30 min.7 After removal of the reagent in a stream of
dry nitrogen, the samples were taken up in CH2Cl2 (200 μL) and
trifluoroacetic anhydride (50 μL); the mixture was allowed to stand at
rt for 30 min, and the reagent was removed in a stream of dry nitrogen.
The derivatized amino acids were analyzed by CPGC, employing
modified cyclodextrin 2,6-Me-3-Pe-β-CD as the chiral stationary
phase, and by co-injection with authentic samples.7
Antimicrobial Activity. The antimicrobial activity of all
compounds was assayed using the bioautography method.16 Eleven
microorganisms were used: three Gram-positive bacteria, Micrococcus
luteus (ATCC 9341), Staphylococcus aureus (ATCC 6538p), and
Staphylococcus epidermidis (ATCC 12228), and three Gram-negative
bacteria, Klebsiella pneumoniae (ATCC 10031), Escherichia coli (ATCC
11103), and Salmonella setubal (ATCC 19196), and five yeasts,
Saccharomyces cerevisiae (ATCC 2601), Candida albicans (ATCC
10231), Cryptococcus neoformans (ATCC 28952), Candida tropicalis
(ATCC 750), and Candida dubliniensis (SM 26, clinical isolation
provided by the Laboratory of Mycology of the Department of
Pharmacy). Standard antibiotics chloramphenicol and nystatin were
used in order to control microbial test sensitivity.18
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ASSOCIATED CONTENT
* Supporting Information
1D and 2D NMR spectra, CPGC analysis, and biological
activity for compounds 1−22. Supplementary data associated
with this article are available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: afmorel@base.ufsm.br. Tel: (+55) 55-3220-8869. Fax:
(+55) 55-3220-8031.
(11) Maldaner, G.; Marangon, P.; Ilha, V.; Caro, M. S. B.; Burrow, R.
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Author Contributions
⊥M. A. Mostardeiro and V. Ilha contributed equally to this
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank CNPq (Conselho Nacional de Desenvolvi-
(17) Shaw, K. N. F.; Fox, S. W. J. Am. Chem. Soc. 1953, 75, 3417−
3421.
(18) Food and Drug Administration. Code of Federal Regulation 1991,
́
mento Cientifico e Tecnolog
́
ico) and FAPERGS (Fundaca
̧
o de
̃
Amparo a Pesquisa do Estado do Rio Grande do Sul)-
PRONEX for financial support of this work.
21, 300.
G
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(19) The dipeptide Z-L-Leu-D,L-threo-Phe(βOH)-OH (4) was
obtained from the basic hydrolysis of its methyl ester (L-leucyl-
carbobenziloxy-D,L-threo-3-phenylserine-OMe),12 by dissolution of
29.4 mmol (13.1 g) in 60 mL of MeOH, cooled in an ice bath, and
then addition of 65 mL of 1 M NaOH under magnetic stirring. The
mixture was left to stand at 25 °C for 2 h. After that, 30 mL of a
solution of 1 M HCl was added, and the MeOH was removed under
vacuum. The aqueous solution was cooled and acidified to pH 2−3
with 2 M HCl solution. The reaction, stored in a refrigerator for 14 h,
formed an oily mass, which was decanted and dissolved in CHCl3 and
dried with anhydrous Na2SO4. The solvent was evaporated, yielding a
yellowish oil (66% yield).
H
dx.doi.org/10.1021/np400313w | J. Nat. Prod. XXXX, XXX, XXX−XXX