Synthetic and cytotoxic and antimicrobial activity studies on annomuricatin B

Article abstract of DOI:10.1515/znb-2009-0215

The first total synthesis of annomuricatin B (8) is described via coupling of the tripeptide Boc-L-asparaginyl(benzhydryl)-L-alanyl-L-tryptophan-OH and the tetrapeptide L-leucyl-glycyl-L-thryl-L-proline-OMe followed by cyclization of the linear heptapeptide fragment. On pharmacological investigation, it was observed that the cycloheptapeptide 8 displays moderate cytotoxicity against Datton's lymphoma ascites and Ehrlich's ascites carcinoma cell lines with cytotoxic inhibitory concentration (50%) values of 11.6 and 14.1 μM, in addition to potent antidermatophyte activity against Trichophyton mentagrophytes and Microsporum audouinii with a minimum inhibitory concentration of 6 μg mL^-1. Moreover, Gram-negative bacteria and Candida albicans were found to be moderately sensitive towards the newly synthesized peptide.

Full text of DOI:10.1515/znb-2009-0215

Synthetic and Cytotoxic and Antimicrobial Activity Studies
on Annomuricatin B
Rajiv Dahiyaa, Monika Maheshwarib, and Rakesh Yadavb
a Department of Pharmaceutical Chemistry, NRI Institute of Pharmacy, Bhopal - 462022,
Madhya Pradesh, India
b Department of Pharmaceutical Chemistry, Rajiv Academy for Pharmacy, Mathura – 281 001,
Uttar Pradesh, India
Reprint requests to Dr. Rajiv Dahiya. E-mail: rajivdahiya02@yahoo.com or
rajivdahiya77@rediffmail.com
Z. Naturforsch. 2009, 64b, 237 – 244; received August 21, 2008
The first total synthesis of annomuricatin B (8) is described via coupling of the tripeptide Boc-
L-asparaginyl(benzhydryl)-L-alanyl-L-tryptophan-OH and the tetrapeptide L-leucyl-glycyl-L-thryl-
L-proline-OMe followed by cyclization of the linear heptapeptide fragment. On pharmacological
investigation, it was observed that the cycloheptapeptide 8 displays moderate cytotoxicity against
Dalton’s lymphoma ascites and Ehrlich’s ascites carcinoma cell lines with cytotoxic inhibitory con-
centration (50 %) values of 11.6 and 14.1 µM, in addition to potent antidermatophyte activity against
Trichophyton mentagrophytes and Microsporum audouinii with a minimum inhibitory concentration
of 6 µg mL−1. Moreover, Gram-negative bacteria and Candida albicans were found to be moderately
sensitive towards the newly synthesized peptide.
Key words: Annomuricatin B, Cycloheptapeptide, Solution-phase Synthesis, Macrocyclization,
Pharmacological Activity
Introduction
fungal activities of the synthesized peptide were also
evaluated.
During past years, a lot of work has been reported
by various scientists which demonstrates the potential
of higher plants to produce a wide array of natural
products with interesting bioactivities [1 – 5]. Among
Results and Discussion
The cycloheptapeptide molecule was split into three
these, cyclopolypeptides and related congeners [6] are dipeptide units Boc-L-Leu-Gly-OMe (1), Boc-L-Thr-
emerging as novel organic compounds with unique L-Pro-OMe (2) and Boc-L-Ala-L-Trp-OMe (3) and
structures and a wide pharmacological profile that may a single amino acid unit Boc-L-Asn-OH (4). Dipep-
prove better candidates to overcome the problem of tide units 1 – 3 were prepared by coupling of Boc-
resistance towards conventional drugs. Plant-derived amino acids such as Boc-L-Leu, Boc-L-Thr and Boc-
cyclic peptides possess a variety of biological activ- L-Ala with the corresponding amino acid methyl
ities including antitumor [7], vasorelaxant [8], im- ester hydrochlorides such as Gly-OMe·HCl, L-Pro-
munosuppressive [9], tyrosinase and cyclooxygenase OMe·HCl and L-Trp-OMe·HCl by following the mod-
inhibitory [10, 11], antimalarial [12], and estrogen-like ified Bodanzsky and Bodanzsky method [16]. The
activity [13, 14]. A novel cyclic heptapeptide, anno- carboxamide side chain of amino acid unit 4 was
muricatin B was isolated by column chromatography protected using benzhydrol to get Boc-L-Asn(bzh)-
from seeds of Annona muricata (Annonaceae), and OH (4a). After deprotection at the carboxy termi-
the structure was elucidated by chemical and spectral nus, dipeptide 1 was coupled with dipeptide 2 de-
methods [15].
protected at the amino terminus, to get the tetrapep-
Prompted by the medicinal properties of plant- tide unit Boc-L-Leu-Gly-L-Thr-L-Pro-OMe (5). The
derived cyclopolypeptides as well as to obtain the nat- Boc group of dipeptide 3 was removed using tri-
ural peptide in good yield, the present study aimed at fluoroacetic acid (TFA), and the deprotected pep-
the synthesis of annomuricatin B employing solution- tide was coupled with the benzhydryl-protected amino
phase chemistry. The cytotoxic, antibacterial and anti- acid unit 4a utilizing three different carbodiimides to
c
0932–0776 / 09 / 0200–0237 $ 06.00 ꢀ 2009 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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238
R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
Scheme 1. Synthetic route to the cycloheptapeptide (annomuricatin B) 8. Reaction conditions: a = LiOH·H2O (1 : 1), r. t., 1 h;
b = TFA, CHCl3, r. t., 1 h; c = benzhydrol, GAA, H2SO4, r. t., 30 min; d = EDC·HCl or DIPC, TEA or NMM, THF or DMF,
r. t., 24 – 36 h; e = DIPC, pnp/pfp, r. t., 12 h; f = TEA or NMM or pyridine, 7 d, 0 ◦C.
get the tripeptide unit Boc-L-Asn(bzh)-L-Ala-L-Trp- ear peptide fragment was replaced by p-nitrophenyl
OMe (6). After removal of the ester group of tripep- or pentafluorophenyl (Pnp or Pfp) ester groups. The
tide 6 and the Boc group of tetrapeptide 5, the depro- Boc and Bzh groups of the resulting compound were
tected units were coupled to get the linear heptapep- removed using TFA, and the deprotected linear frag-
tide unit Boc-L-Asn(bzh)-L-Ala-L-Trp-L-Leu-Gly-L- ment was now cyclized by keeping the whole contents
Thr-L-Pro-OMe (7). The methyl ester group of the lin- at 0 ◦C for 7 d in the presence of catalytic amounts of
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R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
239
Table 1. Cytotoxic activity data.
Compd.
Conc.
— DLA cells —
No. of dead
% GIa
— EAC cells —
(µg mL−1
)
Live cells
counted
CTC50
Live cells
counted
No. of dead
% GI
CTC50
(µM)
cells
38
32
22
12
7
(µM)b
cells
28
24
13
5
1
62.5
0
6
16
26
31
100.0
84.21
57.89
31.58
18.42
0
4
15
23
27
100.0
83.33
46.43
17.86
3.57
31.25
15.63
7.81
12.8
11.6
–
16.5
14.1
–
3.91
1
2
62.5
0
2
10
20
24
38
36
28
18
14
100.0
94.74
73.68
47.37
36.84
0
1
8
17
22
28
27
20
11
6
100.0
96.43
71.43
39.28
21.43
31.25
15.63
7.81
3.91
Control
62.5
38
38
38
38
38
0
0
0
0
0
–
–
–
–
–
28
28
28
28
28
0
0
0
0
0
–
–
–
–
–
31.25
15.63
7.81
3.91
Standard
(5-FU)
62.5
0
0
10
13
22
38
38
28
25
16
100.0
100.0
73.68
65.79
42.11
0
0
11
19
23
28
28
17
9
100.0
100.0
60.71
32.14
17.86
31.25
15.63
7.81
37.4
90.6
3.91
5
a % Growth inhibition (GI) = 100−[{(Celltotal −Celldead)×100}/Celltotal]; b CTC50 = cytotoxic concentration inhibiting 50 % of percentage
growth.
Table 2. Antifungal activity dataa.
TEA or NMM or pyridine to get the cyclic product 8
(Scheme 1).
Compd.
Diameter of zone of inhibition (mm)
The structure of the newly synthesized cyclic hep-
tapeptide as well as that of the intermediate tri/tetra/
Fungal strains
C. albi- A. niger
cans
Gano-
M.
T. menta-
1
derma sp. audouinii grophytes
heptapeptides were confirmed by FTIR and H NMR
7
8
12(6)
14(6)
–
9(50)
13(50)
–
11(25)
14(25)
–
19(6)
23(6)
–
22(6)
26(6)
–
spectroscopy, and elemental analysis. In addition,
13C NMR and mass spectra were recorded for the lin-
ear as well as for the cyclic heptapeptide.
Control
Griseofulvin 20(6) 18(12.5)
22(6)
17(6)
19(6)
a
The synthesis of cyclopeptide 8 was accomplished
with 85 % yield, and pyridine proved to be an ef-
fective base for cyclization of the linear heptapep-
tide fragment. Cyclization of the linear peptide was
indicated by the disappearance of absorption bands
at 1748, 1270 and 1388, 1371 cm−1 (C–Ostr, ester and
C–Hdef, tert-butyl groups) and the presence of addi-
tional amide I and II bands of the -CO–NH- moiety
at 1637– 1634 cm−1 and 1527– 1525 cm−1 in the IR
spectrum of 8. Deprotection of asparagine was con-
firmed by the presence of amide I and II bands (1653,
1628 cm−1) and bands at 3350, 3178 and 1405 cm−1
due to N–Hstr and C–Nstr of the -CONH2 moiety, and
the disapppearance of strong out-of-plane deformation
bands at 733 – 731 cm−1 and 699 – 694 cm−1 due to
the aromatic rings of the bzh group, in the IR spectra
and the disapppearance of the multiplet at 7.23– 7.16
and 7.09 – 7.03 ppm due to 10 protons of phenyl rings
Values in parentheses are MIC values (µg mL−1).
1
of the benzhydryl (Bzh) group in the H NMR spec-
trum of 8. The formation of the cyclopeptide was fur-
ther confirmed by the disappearance of singlets at 3.63
and 1.54 ppm corresponding to three protons of the
methyl ester group and nine protons of the tert-butyl
1
group of Boc in the H NMR spectrum and the dis-
appearance of the singlets at 154.6, 79.9 and 53.9,
28.3 ppm corresponding to carbon atoms of ester and
tert-butyl groups in the 13C NMR spectrum of 8. Fur-
thermore, the H NMR and 13C NMR spectra of the
1
synthesized cyclic heptapeptide showed characteristic
peaks confirming the presence of all the 49 protons
and 35 carbon atoms. The appearance of the pseu-
domolecular ion peak [M + 1]+ at m/z = 740 cor-
responding to the molecular formula C35H49N9O9 in
the mass spectrum of 8, along with other fragment
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Compd.
R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
Table 3. Antibacterial activ-
ity dataa.
Diameter of zone of inhibition (mm)
Bacterial strains
C. pyogenes
S. aureus
B. subtilis
K. pneumoniae
P. aeruginosa
E. coli
7
8
10(50)
14(50)
–
12(25)
17(25)
–
9(50)
13(50)
–
16(6)
20(6)
–
13(6)
19(6)
–
11(6)
15(6)
–
a
Control
Gatifloxacin
Values in parentheses are MIC
values (µg mL−1).
20(12.5)
28(6)
18(12.5)
25(6)
24(6)
19(6)
ion peaks resulting from cleavage at ‘Gly-Thr’, ‘Thr- one receptor molecule, resulting in undesirable adverse
Pro’ and ‘Pro-Asn’ amide bonds showed the exact se- effects.
quence of the attachment of all the seven amino acid
Conclusion
moieties in a chain. In addition, the presence of the
immonium ion peaks at m/z = 159 (Trp), 87 (Asn),
86 (Leu), 74 (Thr), 70 (Pro), 44 (Ala), and 30 (Gly)
further confirmed all the seven amino acid moieties
in the cyclopeptide structure. Furthermore, the ele-
mental analysis of 8 afforded values with tolerances
of 0.03 in strict accordance with the molecular com-
position.
The present study presents the successful synthe-
sis of the natural peptide annomuricatin B (8) in good
yield via coupling reactions utilizing different carbodi-
imides. The DIPC/TEA coupling method proved to
be yield-effectice, in comparison to methods utilizing
EDC·HCl and NMM, providing 10 – 12 % additional
yield. The pentafluorophenyl ester was shown to be
better for the activation of the acid functionality of
the linear heptapeptide unit when compared to the p-
nitrophenyl ester. Pyridine was found to be a good base
for the intramolecular cyclization of the linear peptide
fragment in comparison to TEA or NMM. The syn-
thesized cycloheptapeptide displayed moderate cyto-
toxicity as well as potent antidermatophyte activity. In
comparison, Gram-negative bacteria were found to be
more sensitive than Gram-positive bacteria towards the
newly synthesized peptide. On passing toxicity tests,
cyclopeptide 8 may prove as a good candidate for clin-
ical studies and can be a new antifungal and cytotoxic
drug of future.
The synthesized cyclopeptide 8 exhibited mod-
erate cytotoxic activity against Dalton’s lymphoma
ascites (DLA) and Ehrlich’s ascites carcinoma
(EAC) cell lines with CTC50 values of 11.6 and
14.1 µM, respectively, in comparison to the stan-
dard drug 5-fluorouracil (5-FU) (CTC50 values –
37.4 and 90.6 µM) (Table 1). The possible mechanism
of the cytotoxic action of 8 might be through apopto-
sis via induction of early cell death, nuclear fragmen-
tation and internucleosomal DNA scission. Compari-
son of the antifungal activity data suggested that 8 pos-
sessed potent bioactivity against dermatophytes M. au-
douinii and T. mentagrophytes and moderate antifun-
gal activity against pathogenic Candida albicans with
MIC values of 6 µg/mL when compared to the refer-
ence drug griseofulvin (Tables 2 and 3). Moreover, a
moderate level of activity was observed against Gram-
negative bacteria K. pneumoniae, P. aeruginosa and E.
coli for the newly synthesized cyclopeptide, in com-
parison to the standard drug gatifloxacin. However, 8
displayed no significant activity against neither Gram-
positive bacteria nor pathogenic Ganoderma sp. and
Aspergillus niger. In addition, the analysis of the phar-
macological activity data revealed that cycloheptapep-
tide 8 displayed a higher bioactivity against pathogenic
microbes and cell lines than its linear form 7. This is
possibly because cyclization of peptides reduces the
degree of freedom for each constituent within the ring
and thus substantially leads to reduced flexibility, in-
creased potency and selectivity of cyclic peptides. Fur-
ther, the inherent flexibility of linear peptides leads to
Experimental Section
General methods
Melting points were determined using a Jindal Scien-
tific melting point apparatus (Jindal, Delhi, India) by the
open capillary method and are uncorrected. IR spectra were
recorded on an FTIR-8400S Fourier transform spectropho-
tometer (Shimadzu, Kyoto, Japan) using a thin film sup-
ported on KBr pellets for solids and CHCl3 as solvent for
intermediate semisolids. 1H and 13C NMR spectra were
recorded on a Bruker AC 300 spectrometer at 300 MHz
(Bruker, IL, USA) using CDCl3 as solvent and tetramethyl-
silane as internal standard. Mass spectra were recorded on
a JMS-DX 303 spectrometer (Jeol, Tokyo, Japan) operating
at 70 eV using the fast atom bombardment technique. Ele-
mental analyses of the cyclopeptide as well as of the interme-
diates were performed on a Vario EL III elemental analyzer
(Elementar Vario EL III, Hanau, Germany). Optical rotation
different conformations which can bind to more than of the synthesized peptides was measured on an Optics Tech-
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R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
241
nology automatic polarimeter (OpticsTech, Delhi, India) in v = 3492 (N–Hstr, ring), 3127, 3122 (N–Hstr, amide), 3077,
◦
a 2 dm tube at 25 C using a sodium lamp and methanol 3056 – 3052 (C–Hstr, rings), 2969, 2925 (C–Hstr, asym, CH3
as solvent. Purity of the synthesized cyclopeptide and the and CH2), 2872, 2846 (C–Hstr, sym, CH3 and CH2), 1744
intermediates was checked by TLC on precoated silica gel (C=Ostr, ester), 1646, 1640 (C=Ostr, amide), 1575 – 1569,
G plates (Kieselgel 0.25 mm, 60G F254, Merck, Germany) 1480 – 1476 (skeletal bands), 1537, 1532 (N–Hdef, amide),
utilizing CHCl3·MeOH as developing solvent in different ra- 1387, 1372 (C–Hdef, tert-butyl), 1267 (C–Ostr, ester), 739 –
tios. Brown spots were detected on exposure to iodine vapors 732, 697 – 693, 675 (C–Hdef, out-of-plane, rings) cm−1. –
in a tightly closed chamber.
1H NMR (CDCl3): δ = 8.95 (br. s, 1 H, NH, indole), 8.83
(br. s, 1 H, NH), 8.52 (br. s, 1 H, NH), 7.53 (d, J = 7.85 Hz,
1 H, β-H, indole), 7.25 – 7.18 (m, 6 H, m-Hꢀs and p-Hꢀs,
phenyl rings, Bzh), 7.15 – 7.06 (m, 4 H, δ–η-Hꢀs, indole),
7.05 – 6.98 (m, 4 H, o-Hꢀs, phenyl rings, Bzh), 6.89 (br. s,
1 H, δ-NH, Asn), 6.77 (br. s, 1 H, NH), 5.98 (d, J = 5.45 Hz,
1 H, α-H, Bzh), 4.46 – 4.42 (m, 1 H, α-H, Asn), 4.41 – 4.37
(m, 1 H, α-H, Ala), 4.24 – 4.18 (m, 1 H, α-H, Trp), 3.56 (s,
3 H, OCH3), 3.22 (d, J = 4.85 Hz, 2 H, β-Hꢀs, Asn), 2.98 (d,
J = 5.65 Hz, 2 H, β-Hꢀs, Trp), 1.48 (d, J = 5.9 Hz, 3 H, β-Hꢀs,
Ala), 1.55 (s, 9H, tert-butyl). – C37H43N5O7 (669.77): calcd.
C 66.35, H 6.47, N 10.46; found C 66.38, H 6.45, N 10.45.
Protection of the carboxamide side chain of Boc-L-aspara-
gine (4)
To the solution of Boc-L-asparagine (2.32 g, 0.01 mol)
in glacial acetic acid (GAA, 25 mL), benzhydrol (1.84 g,
0.01 mol) was added with stirring at room temperature (r. t.)
for a time period of 30 min. Concentrated sulfuric acid
(0.05 mL) was added to the mixture which was allowed to
stand overnight. The reaction mixture was poured in water
(75 mL). An oily product separated which soon solidified.
The crude product was finally purified with ethyl acetate
to provide 3 g (72 %) of the pure product as a white solid.
tert-Butyloxycarbonyl-L-leucyl-glycyl-L-thryl-L-proline
methyl ester (6)
◦
M. p. 145 – 146 C. – [α]D = −11.7◦ (c = 0.2, MeOH). –
IR (KBr): v = 3295 – 2515 (O–Hstr, COOH), 3134 (N–Hstr
,
,
Off-white solid. M. p. 112 – 113 ◦C. Yield 75 %. – [α]D
+13.4◦ (c = 0.25, MeOH). Rf = 0.73 (CHCl3-MeOH =
8 : 2). – IR (KBr): v = 3342 (O–Hstr), 3129, 3124 (N–Hstr
=
amide), 3075, 3044 (C–Hstr, rings), 2926, 2853 (C–Hstr
CH2), 1710 (C=Ostr, COOH), 1642 (C=Ostr, 2◦ amide),
1535 (N–Hdef, 2◦ amide), 1574, 1482, 1477 (skeletal bands),
1409 (C–OHdef, COOH), 1395, 1372 (C–Hdef, tert-butyl),
738, 733, 695 – 692 (C–Hdef, out-of-plane, rings) cm−1. –
1H NMR (CDCl3): δ = 11.25 (br. s, 1 H, OH), 7.82 (br. s,
1 H, α-NH), 7.22 – 7.10 (m, 10 H, o-Hꢀs, m-Hꢀs and p-Hꢀs,
phenyl rings), 6.75 (br. s, 1 H, δ-NH), 6.02 (d, J = 5.5 Hz,
1 H, α-H, Bzh), 4.37 – 4.32 (m, 1 H, α-H, Asn), 2.95 (d, J =
4.9 Hz, 2 H, β-Hꢀs, Asn), 1.55 (s, 9 H, tert-butyl).
,
amide), 2998 – 2992 (C–Hstr, CH2, Pro), 2968, 2927, 2923
(C–Hstr, asym, CH3 and CH2), 2854, 2849 (C–Hstr, sym,
CH2), 1747 (C=Ostr, ester), 1665, 1647 – 1642 (C=Ostr, 3◦
and 2◦ amide), 1538 – 1533 (N–Hdef, 2◦ amide), 1389, 1370
(C–Hdef, tert-butyl), 1383, 1362 (C–Hdef, iso-propyl), 1269,
1095 (C–Ostr, ester and C–OH) cm−1. – 1H NMR (CDCl3):
δ = 9.35 (br. s, 1 H, NH), 8.25 (br. s, 1 H, NH), 6.52 (br. s,
1 H, NH), 4.49 (br. s, 1 H, OH), 4.28 (dd, J = 6.2 Hz, 4.9 Hz,
1 H, α-H, Thr), 4.13 (d, J = 5.45 Hz, 2 H, α-H, Gly), 3.93
(t, 1 H, J = 6.85 Hz, α-H, Pro), 3.89 – 3.85 (m, 1 H, β-H,
Thr), 3.84 – 3.78 (m, 1 H, α-H, Leu), 3.62 (s, 3 H, OCH3),
3.40 (t, 2 H, J = 7.2 Hz, δ-H, Pro), 2.05 – 1.98 (m, 4 H, β-H
and γ-H, Pro), 1.89 (t, 2 H, J = 5.9 Hz, β-H, Leu), 1.66 –
1.59 (m, 1 H, γ-H, Leu), 1.56 (s, 9 H, tert-butyl), 1.27 (d,
3 H, J = 5.8 Hz, γ-H, Thr), 0.99 (d, 6 H, J = 6.25 Hz, δ-
H, Leu). – C23H40N4O8 (500.59): calcd. C 55.19, H 8.05,
N 11.19; found C 55.22, H 8.03, N 11.20.
General procedure for the synthesis of linear tri/tetrapeptide
segments 5 and 6
To the solution of the amino acid methyl ester hydrochlo-
ride or dipeptide methyl ester (0.01 mol) in DMF (25 mL),
NMM (0.021 mol) was added at 0 ◦C, and the reaction mix-
ture was stirred for 15 min. The Boc-dipeptide (0.01 mol) in
DMF (25 mL) and 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDC·HCl, 0.01 mol) were added
◦
with stirring. Stirring was first done for 1 h at 0 – 5 C and
then for another 24 – 36 h at r. t. After the completion of
the reaction, the reaction mixture was diluted with an equal
amount of water. The precipitated solid was filtered, washed
with water and recrystallized from ◦a mixture of chloroform
and petroleum ether (b. p. 40 – 60 C) followed by cooling
at 0 ◦C to get the title compounds.
Deprotection of the tripeptide unit at the carboxyl terminal
To a solution of the tripeptide 5 (6.7 g, 0.01 mol) in THF-
H2O (1 : 1, 36 mL), LiOH (0.36 g, 0.015 mol) was added
at 0 ◦C. The mixture was stirred at r. t. for 1 h and then acid-
ified to pH = 3.5 with 1 N H2SO4. The aqueous layer was
extracted with Et2O (3×25 mL). The combined organic ex-
tracts were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude product was finally crys-
tert-Butyloxycarbonyl-L-asparaginyl(bzh)-L-alanyl-L-trypto-
phan methyl ester (5)
Semisolid mass. Yield 79 %. – [α]D = −93.4◦ (c = 0.25, tallized from methanol and ether to get the pure deprotected
MeOH). – Rf = 0.59 (CHCl3-MeOH = 8 : 2). – IR (CHCl3): compound 5a.
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242
R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
tert-Butyloxycarbonyl-L-asparaginyl(bzh)-L-alanyl-L-trypto-
phan (5a)
3 H, J = 5.75 Hz, γ-H, Thr), 1.15 – 1.07 (m, 1 H, γ-H, Leu),
0.98 (d, 6 H, J = 6.2 Hz, δ-H, Leu), 0.11 (br. s, 3 H, OH
and NH2). – C18H32N4O6 (400.47): calcd. C 53.99, H 8.05,
N 13.99; found C 54.01, H 8.02, N 14.02.
White solid. M. p. 91 – 93 ◦C. Yield 87 %. – [α]D
=
−69.7◦ (c = 0.25, MeOH). Rf = 0.77 (CHCl3-MeOH =
8 : 2). – IR (KBr): v = 3489 (N–Hstr, ring), 3297 – 2509
(O–Hstr, COOH), 3129, 3126 (N–Hstr, amide), 3075, 3055 –
3052 (C–Hstr, rings), 2968, 2927 (C–Hstr, asym, CH3 and
CH2), 2874, 2845 (C–Hstr, sym, CH3 and CH2), 1713
(C=Ostr, COOH), 1645, 1639 (C=Ostr, amide), 1576 – 1568,
1482 – 1478 (skeletal bands), 1538 – 1533 (N–Hdef, amide),
1386, 1370 (C–Hdef, tert-butyl), 737 – 732, 698 – 692, 678
(C–Hdef, out-of-plane, rings) cm−1. – 1H NMR (CDCl3):
δ = 10.48 (br. s, 2 H, NH, indole and OH, COOH), 8.87
(br. s, 1 H, NH), 8.50 (br. s, 1 H, NH), 7.55 (d, J = 7.9 Hz,
1 H, β-H, indole), 7.52 (br. s, 1 H, NH), 7.22 – 7.17 (m, 6 H,
m-Hꢀs and p-Hꢀs, phenyl rings, Bzh), 7.16 – 7.08 (m, 4 H,
δ–η-Hꢀs, indole), 7.06 – 6.99 (m, 4 H, o-Hꢀs, phenyl rings,
Bzh), 6.87 (br. s, 1 H, δ-NH, Asn), 5.99 (d, J = 5.5 Hz, 1 H,
α-H, Bzh), 5.43 – 5.37 (m, 1 H, α-H, Ala), 4.48 – 4.43 (m,
1 H, α-H, Trp), 4.42 – 4.38 (m, 1 H, α-H, Asn), 3.28 (d, J =
5.7 Hz, 2 H, β-Hꢀs, Trp), 3.02 (d, J = 4.9 Hz, 2 H, β-Hꢀs,
Asn), 1.53 (s, 9H, tert-butyl), 1.49 (d, J = 5.85 Hz, 3 H, β-
Hꢀs, Ala). – C36H41N5O7(655.75): calcd. C 65.94, H 6.30,
N 10.68; found C 65.95, H 6.27, N 10.70.
Procedure for the synthesis of the linear heptapeptide unit 7
Compound 6a (4.0 g, 0.01 mol) was dissolved in THF
(35 mL). To this solution, TEA (2.8 mL, 0.021 mol)
was added at 0 ◦C, and the resulting mixture was stirred
for 15 min. Compound 5a (6.6 g, 0.01 mol) was dissolved
in THF (35 mL), and DIPC (1.26 g, 0.01 mol) was added to
the mixture with stirring. Stirring was continued for 24 h, af-
ter which the reaction mixture was filtered, and the filtrate
was washed with 5 % NaHCO3 and saturated NaCl solu-
tions (30 mL each). The organic layer was dried over anhy-
drous Na2SO4, filtered and evaporated in vacuum. The crude
product was recrystallized from a mixture of chloroform and
petroleum ether (b. p. 40 – 60 ◦C) followed by cooling at 0 ◦C.
tert-Butyloxycarbonyl-L-asparaginyl(bzh)-L-alanyl-L-
tryptophyl-L-leucyl-glycyl-L-thryl-L-proline methyl ester
(7)
Semisolid mass. Yield 78 %. – [α]D = −51.8◦ (c = 0.35,
MeOH). Rf = 0.63 (CHCl3-MeOH = 9 : 1). – IR (CHCl3):
Deprotection of the tetrapeptide unit at the amino terminal
v = 3486 (N–Hstr, ring), 3374 (O–Hstr), 3132 – 3125 (N–Hstr
,
amide), 2998 – 2991 (C–Hstr, CH2, Pro), 3077, 3056, 3053
(C–Hstr, rings), 2969, 2928, 2922 (C–Hstr, asym, CH3 and
CH2), 2875, 2847, 2844 (C–Hstr, sym, CH3 and CH2),
1748 (C=Ostr, ester), 1669, 1646 – 1639 (C=Ostr, amide),
1578 – 1569, 1484 – 1479 (skeletal bands), 1539, 1535, 1530
(N–Hdef, amide), 1388, 1371 (C–Hdef, tert-butyl), 1381,
1363 (C–Hdef, iso-propyl), 1270, 1094 (C–Ostr, ester and
C–OH), 736 – 731, 699 – 694, 676 (C–Hdef, out-of-plane,
rings) cm−1. – 1H NMR (CDCl3): δ = 8.90 (br. s, 1 H, NH),
8.75 (br. s, 1 H, NH), 8.63 (br. s, 1 H, NH), 8.50 (br. s, 1 H,
NH), 8.19 (br. s, 1 H, δ-NH, Asn), 8.16 (br. s, 1 H, NH),
Tetrapeptide 6 (5.0 g, 0.01 mol) was dissolved in CHCl3
(15 mL) and treated with CF3COOH (2.28 g, 0.02 mol). The
resulting solution was stirred at r. t. for 1 h and washed with
a saturated NaHCO3 solution (25 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude product was purified by crystal-
lization from CHCl3 and petroleum ether (b. p. 40 – 60 ◦C) to
get the pure deprotected compound 6a.
L-Leucyl-glycyl-L-thryl-L-proline methyl ester (6a)
Semisolid mass. Yield 83 %. – [α]D = +2.8◦ (c = 0.25, 7.82 (br. s, 2 H, NH, indole and OH, Thr), 7.69 (br. s, 1 H,
MeOH). Rf = 0.55 (CHCl3-MeOH = 8 : 2). – IR (KBr): NH), 7.25 (d, J = 7.85 Hz, 1 H, β-H, indole), 7.23 – 7.16 (m,
v = 3379, 3268 (N–Hstr, amine), 3371 (O–Hstr), 3133, 3126 6 H, m-Hꢀs and p-Hꢀs, phenyl rings, Bzh), 7.15 – 7.10 (m,
(N–Hstr, amide), 2997 – 2992 (C–Hstr, CH2, Pro), 2966, 4 H, δ–η-Hꢀs, indole), 7.09 – 7.03 (m, 4 H, o-Hꢀs, phenyl
2929, 2922 (C–Hstr, asym, CH3 and CH2), 2855, 2848 rings, Bzh), 5.98 (d, J = 5.45 Hz, 1 H, α-H, Bzh), 4.73 – 4.68
(C–Hstr, sym, CH2), 1748 (C=Ostr, ester), 1667, 1649 – 1644 (m, 1 H, α-H, Ala), 4.45 – 4.41 (m, 1 H, α-H, Asn), 4.24 –
(C=Ostr, 3◦ and 2◦ amide), 1624 (N–Hdef, amine), 1539, 4.19 (m, 1 H, α-H, Trp), 4.09 (dd, J = 6.2 Hz, 4.85 Hz, 1 H,
1536, 1533 (N–Hdef, 2◦ amide), 1382, 1364 (C–Hdef, iso- α-H, Thr), 4.02 (d, J = 5.45 Hz, 2 H, α-Hꢀs, Gly), 3.90 (t,
propyl), 1272, 1092 (C–Ostr, ester and C–OH), 1139 (C–Nstr
,
J = 6.85 Hz, 1 H, α-H, Pro), 3.77 – 3.72 (m, 1 H, β-H, Thr),
amine) cm−1. – 1H NMR (CDCl3): δ = 9.33 (br. s, 1 H, 3.63 (s, 3 H, OCH3), 3.52 – 3.48 (m, 1 H, α-H, Leu), 3.45
NH), 8.19 (br. s, 1 H, NH), 4.30 (dd, J = 6.15 Hz, 4.85 Hz, (t, J = 7.2 Hz, 2 H, δ-Hꢀs, Pro), 3.22 (d, J = 4.85 Hz, 2 H,
1 H, α-H, Thr), 4.07 (d, J = 5.5 Hz, 2 H, α-H, Gly), 3.92 (t, β-Hꢀs, Asn), 3.18 (d, J = 5.65 Hz, 2 H, β-Hꢀs, Trp), 2.06 –
1 H, J = 6.9 Hz, α-H, Pro), 3.88 – 3.83 (m, 1 H, β-H, Thr), 1.98 (m, 4 H, β-Hꢀs and γ-Hꢀs, Pro), 1.71 (t, J = 5.9 Hz, 2 H,
3.64 (s, 3 H, OCH3), 3.42 (t, 2 H, J = 7.15 Hz, δ-H, Pro), β-Hꢀs, Leu), 1.54 (s, 9 H, tert-butyl), 1.50 – 1.43 (m, 1 H,
3.30 – 3.26 (m, 1 H, α-H, Leu), 2.07 – 1.99 (m, 4 H, β-H γ-H, Leu), 1.32 (d, J = 5.8 Hz, 3 H, γ-Hꢀs, Thr), 1.22 (d,
and γ-H, Pro), 1.82 (t, 2 H, J = 5.95 Hz, β-H, Leu), 1.25 (d, J = 5.8 Hz, 3 H, β-Hꢀs, Ala), 0.99 (d, J = 6.15 Hz, 6 H, δ-
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R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
243
Hꢀs, Leu). – 13C NMR (CDCl3): δ = 173.8 (γ-C=O, Asn), natural annomuricatin B [15]). Rf = 0.81 (CHCl3-MeOH =
172.1 (C=O, Pro), 170.5 (C=O, Ala), 169.8 (C=O, Asn), 9 : 1). – IR (KBr): v = 3488 (N–Hstr, ring), 3372 (O–Hstr),
168.7 (C=O, Leu), 167.3 (C=O, Trp), 166.5 (C=O, Gly), 3350, 3178 (N–Hstr, 1◦ amide), 3136 – 3127 (N–Hstr, 2◦
163.3 (C=O, Thr), 154.6 (C=O, Boc), 151.4 (2 C, β-Cꢀs, amide), 2998 – 2993 (C–Hstr, CH2, Pro), 3074 (C–Hstr, ring),
Bzh), 137.7 (2ꢀ-C, indole), 129.6 (2 C, o-Cꢀs, Ph-1, Bzh), 2967, 2929, 2920 (C–Hstr, asym, CH3 and CH2), 2878,
128.3 (2 C, o-Cꢀs, Ph-2, Bzh), 127.8 (3ꢀ-C, indole), 127.2 2844, 2842 (C–Hstr, sym, CH3 and CH2), 1667, 1653, 1645 –
(2 C, m-Cꢀs, Ph-1, Bzh), 126.5 (2 C, p-Cꢀs, Bzh), 125.9 (2 C, 1634 (C=Ostr, amides), 1628 (NH2(def), 1◦ amide), 1572,
m-Cꢀs, Ph-2, Bzh), 125.0 (5-C, indole), 124.4 (6-C, indole), 1480 (skeletal bands), 1538 – 1532, 1529 – 1525 (N–Hdef
,
122.9 (2-C, indole), 119.4 (4-C, indole), 111.6 (7-C, indole), amide), 1405 (C–Nstr, 1◦ amide), 1383, 1362 (C–Hdef, iso-
110.7 (3-C, indole), 79.9 (α-C, tert-butyl), 72.2 (α-C, Asn), propyl), 1095 (C–Ostr, C–OH), 735, 677 (C–Hdef, out-of-
69.5 (β-C, Thr), 66.8 (α-C, Thr), 60.3 (α-C, Pro), 56.8 (α-C, plane, ring) cm−1. – 1H NMR (CDCl3): δ = 11.78 (br. s,
Bzh), 54.5 (α-C, Trp), 53.9 (OCH3), 53.2 (α-C, Leu), 51.8 1 H, NH), 8.25 (br. s, 2 H, NH, indole and OH, Thr), 8.20
(α-C, Ala), 50.4 (β-C, Asn), 45.8 (δ-C, Pro), 44.5 (β-C, (br. s, 1 H, NH), 8.05 (br. s, 1 H, NH), 7.93 (br. s, 1 H,
Leu), 42.7 (α-C, Gly), 34.4 (β-C, Trp), 29.0 (β-C, Pro), 28.3 NH), 7.28 (d, J = 7.9 Hz, 1 H, β-H, indole), 7.25 (d, J =
(3 C, β-Cꢀs, tert-butyl), 26.6 (γ-C, Leu), 24.8 (γ-C, Pro), 7.65 Hz, 1 H, δ-H, indole), 7.14 (br. s, 1 H, NH), 7.13 – 7.10
23.2 (2 C, δ-Cꢀs, Leu), 22.0 (γ-C, Thr), 18.3 (β-C, Ala). – (m, 3 H, ε–η-Hꢀs, indole), 6.97 (br. s, 1 H, NH), 6.75 (br.
C54H71N9O12 (1038): calcd. C 62.47, H 6.89, N 12.14; s, 2 H, NH2, Asn), 6.28 – 6.24 (m, 1 H, α-H, Leu), 5.98 –
found C 62.49, H 6.92, N 12.15.
5.93 (m, 1 H, α-H, Ala), 5.78 (dd, J = 6.15 Hz, 4.9 Hz,
1 H, α-H, Thr), 5.74 – 5.69 (m, 1 H, α-H, Trp), 5.28 (d, J =
5.5 Hz, 2 H, α-Hꢀs, Gly), 4.99 – 4.95 (m, 1 H, α-H, Asn),
Synthesis of the cyclic heptapeptide – annomuricatin B (8)
To synthesize compound 8, the linear heptapeptide unit 7 3.94 (t, J = 6.9 Hz, 1 H, α-H, Pro), 3.81 – 3.75 (m, 1 H, β-
(5.2 g, 0.005 mol) was deprotected at the carboxyl end H, Thr), 3.26 (t, J = 7.15 Hz, 2 H, δ-Hꢀs, Pro), 2.95 (d, J =
using LiOH (0.18 g, 0.0075 mol) to get Boc-L-Asn(bzh)- 4.9 Hz, 2 H, β-Hꢀs, Asn), 2.88 (d, J = 5.7 Hz, 2 H, β-Hꢀs,
L-Ala-L-Trp-L-Leu-Gly-L-Thr-L-Pro-OH. The deprotected Trp), 2.67 – 2.63 (m, 2 H, β-Hꢀs, Pro), 1.88 (t, J = 5.85 Hz,
heptapeptide unit (5.12 g, 0.005 mol) was now dissolved 2 H, β-Hꢀs, Leu), 1.85 – 1.79 (m, 2 H, γ-Hꢀs, Pro), 1.47 (d,
in CHCl3 (50 mL) at 0 ◦C. To this solution, 0.0067 mol J = 5.75 Hz, 3 H, β-Hꢀs, Ala), 1.42 (d, J = 5.7 Hz, 3 H,
of p-nitrophenol or pentafluorophenol (0.94 g or 1.23 g) γ-Hꢀs, Thr), 0.98 (d, J = 6.2 Hz, 6 H, δ-Hꢀs, Leu), 0.86 –
and DIPC (0.63 g, 0.005 mol) were added, and stirring 0.78 (m, 1 H, γ-H, Leu). – 13C NMR (CDCl3): δ = 175.2
was done at r. t. for 12 h. The reaction mixture was fil- (γ-C=O, Asn), 174.8 (C=O, Trp), 174.0 (C=O, Thr), 172.6
tered, and the filtrate was washed with 10 % NaHCO3 (C=O, Ala), 171.4 (C=O, Asn), 169.8 (C=O, Gly), 168.9
solution (3 × 25 mL) and finally with 5 % HCl (2 × (C=O, Pro), 165.7 (C=O, Leu), 137.3, 129.6 (2 C, 2ꢀ-C and
30 mL) to get the corresponding p-nitrophenyl or pentaflu- 3ꢀ-C, indole), 125.5 (5-C, indole), 122.3 (2-C, indole), 119.2
orophenyl esters Boc-L-Asn(bzh)-L-Ala-L-Trp-L-Leu-Gly- (4-C, indole), 117.4 (6-C, indole), 112.0 (7-C, indole), 110.3
L-Thr-L-Pro-Opnp or Boc-L-Asn(bzh)-L-Ala-L-Trp-L-Leu- (3-C, indole), 68.4 (β-C, Thr), 59.7 (α-C, Pro), 57.2 (α-C,
Gly-L-Thr-L-Pro-Opfp. To this compound (4.58 g or 4.76 g, Thr), 56.0 (α-C, Asn), 55.7 (α-C, Trp), 50.2 (α-C, Ala), 49.5
0.004 mol) dissolved in CHCl3 (35 mL), CF3COOH (0.91 g, (α-C, Leu), 48.8 (δ-C, Pro), 44.5 (α-C, Gly), 43.6 (β-C,
0.008 mol) was added, and the mixture was stirred at r. t. Asn), 39.2 (β-C, Leu), 33.0 (β-C, Pro), 30.3 (γ-C, Leu),
for 1 h and washed with 10 % NaHCO3 solution (2×25 mL). 26.8 (β-C, Trp), 25.0 (γ-C, Pro), 23.7 (2 C, δ-Cꢀs, Leu),
The organic layer was dried over anhydrous Na2SO4 to get 23.1 (γ-C, Thr), 18.9 (β-C, Ala). – MS (FAB, 70 eV): m/z
L-Asn-L-Ala-L-Trp-L-Leu-Gly-L-Thr-L-Pro-Opnpor L-Asn- (%) = 740 (100) [M + 1]+, 712 (11) [740–CO]+, 683 (72)
L-Ala-L-Trp-L-Leu-Gly-L-Thr-L-Pro-Opfp which was dis- [Thr-Pro-Asn-Ala-Trp-Leu]+, 582 (39) [Pro-Asn-Ala-Trp-
solved in CHCl3 (25 mL), and TEA/NMM/C5H5N (2.8 mL Leu]+, 570 (27) [Thr-Pro-Asn-Ala-Trp]+, 554 (14) [582–
or 2.21 mL or 1.61 mL, 0.021 mol) was added. Then the CO]+, 542 (18) [570–CO]+, 485 (48) [Asn-Ala-Trp-Leu]+,
mixture was kept at 0 ◦C for 7 d. The reaction mixture 384 (55) [Thr-Pro-Asn-Ala]+, 372 (29) [Asn-Ala-Trp]+, 356
was washed with 10 % NaHCO3 (3 × 25 mL) and 5 % HCl (14) [384–CO]+, 344 (10) [372–CO]+, 313 (32) [Thr-Pro-
(2 × 30 mL) solutions. The organic layer was dried over Asn]+, 283 (19) [Pro-Asn-Ala]+, 199 (39) [Thr-Pro]+, 171
anhydrous Na2SO4, and the crude product was crystallized (10) [199–CO]+, 159 (16) [C10H11N2]+, 130 (9), 116 (11),
from CHCl3-n-hexane to get the pure cyclic product 8.
115 (8) [Asn]+, 102 (15) [Thr]+, 98 (13) [Pro]+, 87 (11)
[C3H7N2O]+, 86 (16) [C5H12N]+, 74 (14) [C3H8NO]+, 70
(10) [C4H8N]+, 58 (7), 57 (13), 45 (8), 44 (8) [C2H6N]+,
Cyclo (L-Asn-L-Ala-L-Trp-L-Leu-Gly-L-Thr-L-Pro) (8)
White crystals. M. p. 212 – 213 ◦C (213 ◦C for natural an- 43 (16), 42 (12), 30 (9) [CH4N]+, 17 (4), 15 (6) [CH3]+. –
nomuricatin B [15]). Yield 85 % (C5H5N), 78 % (NMM), C35H49N9O9(739): calcd. C 56.82, H 6.68, N 17.04; found
69 % (TEA). – [α]D = −37.3◦ (c = 0.5, MeOH) (−37.25◦ for C 56.79, H 6.70, N 17.05.
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244
R. Dahiya et al. · Synthetic and Biological Studies on Annomuricatin B
Pharmacological activity studies
Cytotoxicity screening
six bacterial strains Corynebacterium pyogenes (MUMC
73), Staphylococcus aureus (MUMC 377), Bacillus sub-
tilis (MUMC 408), Klebsiella pneumoniae (MUMC 95),
The synthesized linear and cyclic heptapeptides (7, 8)
were subjected to a short term in vitro cytotoxicity
study [17] against Dalton’s lymphoma ascites (NCRC 101)
and Ehrlich’s ascites carcinoma (NCRC 69) cell lines
at 62.5 – 3.91 µg mL−1 using 5-fluorouracil (5-FU) as ref-
erence compound. The activity was assessed by determining
the percentage inhibition of DLA and EAC cells. CTC50 val-
ues were determined by the graphical extrapolation method
(Table 1).
Pseudomonas aeruginosa (MUMC 266) and Escherichia
coli (MUMC 106), and five fungal strains Candida albi-
cans (MUMC 29), Aspergillus niger (MUMC 77), Gano-
derma sp. (MUMC 218), Microsporum audouinii (MUMC
545) and Trichophyton mentagrophytes (MUMC 665)
at 50 – 6 µg mL−1 using the Kirby-Bauer disk diffusion
method [18]. MIC values of the test compounds were deter-
mined by the tube dilution technique. The solvents DMF and
DMSO were used as negative controls, and gatifloxacin and
griseofulvin were used as antibacterial and antifungal stan-
dards (Tables 2 and 3).
Antimicrobial screening
Experimental details of the biological activity test proce-
dures are given in our previously published reports [19].
The synthesized linear and cyclic heptapeptides (7, 8)
were evaluated for their antimicrobial activity against
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Unauthenticated
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