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, São Cristóvão, 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, Florianópolis, 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 febrif uga, 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.
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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]. 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 Ndimethylated 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 was proposed using 1H and 13C NMR data. In 1997 Han et al. reported that frangufoline, a sedative 14membered 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-
yclopeptide alkaloids are polyamide bases composed of 13-, 14-, or 15-membered cyclic ethers, found mainly in 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. 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 Gramnegative 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-phenylserine) 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 © XXXX American Chemical Society and American Society of Pharmacognosy
Received: April 15, 2013
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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
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.
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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. According to the literature,9 in the 1H 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 3hydroxyleucine 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-OHamino acid). However, the 13C NMR data provide information on the absolute configuration of this unit.9,11
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 Derythro 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-LeuD,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. 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
Figure 1. The Karplus relationship for 3JH3−H4 coupling constants in alkaloids 1−3. B
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Scheme 1. Synthetic Route to Tripeptide Fragments 5−16a
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.
Scheme 2. Synthetic Route to Tripeptide Fragments 17a,b−22a,ba
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-3phenylserine. Thus, the tripeptides 11−16 were reacted with 4hydroxybenzaldehyde, triphenylphosphine (PPh3), and diethyl azodicarboxylate (DEAD) at room temperature, efficiently producing compounds 17a,b−22a,b (a = L erythro/threo 3phenylserine, and b = D erythro/threo 3-phenylserine), which contain the new linear alkyl−aryl ether bond, also present in the alkaloids. Confirmation of the inversion of configuration at C-3 of the 3-phenylserine was obtained through 1H 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-phenylserine in the form of the erythro alkaloid discarines C and D and myrianthine A, made previously from NMR data.
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 inactive). Unfortunately 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-PheOMe (9a) was more active (3.1 μg) than the derivative H2N-LLeu-D-threo-Phe(βOH)-L-Phe-OMe (9b), which was considered 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 C
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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 Derythro 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 nonproteinogenic 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 (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.
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been previously reported.12 It is described again in Supporting Information. 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-methylpentanamido)-3-hydroxy-3-phenylpropanamido)-4-methylpentanoate (11a): yield 66%; yellowish, viscous mass; 1H 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, C4), 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-methylpentanamido)-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-methylpentanamido)-3-hydroxy-3-phenylpropanamido)-3-phenylpropanoate (12a): yield 73%; white solid; mp 99−101 °C; 1H NMR (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)+. (S)-Methyl-2-((2R,3S)-2-((S)-2-(dimethylamino)-4-methylpentanamido)-3-hydroxy-3-phenylpropanamido)-3-phenylpropanoate (12b): yield 70%; white solid; mp 97−98 °C; 1H 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)+. (2S,3S)-Methyl-2-((2S,3R)-2-((S)-2-(dimethylamino)-4-methylpentanamido)-3-hydroxy-3-phenylpropanamido)-3-methylpentanoate (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, H7), 3.75 (3H, s), 2.80 (1H, m, H-23), 2.04 (6H, s), 1.90 (1H, m), 1.39
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined with a MQAPF-301 apparatus and are uncorrected. 1H 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), otoluidine, 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. 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 D
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Journal of Natural Products
Article
(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)+. (2S,3S)-Methyl-2-((2R,3S)-2-((S)-2-(dimethylamino)-4-methylpentanamido)-3-hydroxy-3-phenylpropanamido)-3-methylpentanoate (13b): yield 66%; yellowish semisolid mass; 1H 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-((2S,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-oxoimidazolidin-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, C23), 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-oxoimidazolidin-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)+. (S)-Methyl-2-((2S,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-phenylpropanoate (15a): :yield 25%; yellow, viscous mass; 1H 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, C23), 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-oxoimidazolidin-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,
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-oxoimidazolidin-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, C23), 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)+. (S)-Methyl-2-((2R,3R)-3-hydroxy-2-((S)-4-isobutyl-3-methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)isomethylpentanoate (16b): yield 29%; yellowish, viscous mass; 1H NMR (CDCl3) δ 7.30 (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, H23), 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-hydroxybenzaldehyde (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-methylpentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-4methylpentanoate (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-methylpentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-4methylpentanoate (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). 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-methylpentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3-phenylpropanoate (18a): yield 36%; yellowish semisolid mass; 1H 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 = 8.8, 6.4 Hz, H-4), 4.81 (1H, dd, J = 7.8, 6.2, 5.9H ̀ z, 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); 13 C 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-methylpentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3phenylpropanoate (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-methylpentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3methylpentanoate (19a): yield 37%; yellow, viscous 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 = 8.5, 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-methylpentanamido)-3-(4-formylphenoxy)-3-phenylpropanamido)-3methylpentanoate (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-3methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-methylpentanoate (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,
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). (S)-Methyl-2-((2R,3R)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-methylpentanoate (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, C7), 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-3methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-phenylpropanoate (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). (S)-Methyl-2-((2R,3R)-3-(4-formylphenoxy)-2-((S)-4-isobutyl-3methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3-phenylpropanoate (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-3methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3isomethylpentanoate (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, C23), 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-3methyl-5-oxoimidazolidin-1-yl)-3-phenylpropanamido)-3isomethylpentanoate (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 Compound concentrations from 50 to 1.56 μg were applied to precoated TLC plates. Muller Hinton agar and Sabouraud agar media inoculated with microorganism suspensions (105 cfu mL−1) were distributed over TLC plates. The plates were incubated for 24 h at 37 °C. The results were stained with an aqueous solution of 2,3,5triphenyltetrazolium chloride (1 mg mL−1). The appearance of inhibition zones was used to determine the lowest sample amount capable of inhibiting microbial growth. Samples were tested in triplicate.
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ASSOCIATED CONTENT
* Supporting Information S
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.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: (+55) 55-3220-8869. Fax: (+55) 55-3220-8031. Author Contributions
⊥ M. A. Mostardeiro and V. Ilha contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank CNPq (Conselho Nacional de Desenvolví mento Cientifico e Tecnológico) and FAPERGS (Fundaçaõ de Amparo a Pesquisa do Estado do Rio Grande do Sul)PRONEX for financial support of this work. G
dx.doi.org/10.1021/np400313w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
(19) The dipeptide Z-L-Leu-D,L-threo-Phe(βOH)-OH (4) was obtained from the basic hydrolysis of its methyl ester (L-leucylcarbobenziloxy-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).
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dx.doi.org/10.1021/np400313w | J. Nat. Prod. XXXX, XXX, XXX−XXX
