New alkaloidal metabolites from cultures of entomopathogenic fungus Cordyceps takaomontana NBRC 101754

Article abstract of DOI:10.1016/j.fitote.2019.104364

Two new alkaloidal metabolites, cordytakaoamides A (1) and B (2), as well as, 2-[(2-hydroxyethyl) amino] benzoic acid (3) and 2E-decenamide (4), and three known compounds (5–7) were isolated from ethyl acetate and n-butanol soluble portions of the entomopathogenic fungus, Cordyceps takaomontana NBRC 101754. Compounds 3 and 4 were isolated here for first time from natural resources. The chemical structures were established depending upon spectroscopic techniques such as 1D, 2D NMR, and HRMS. The absolute configuration of 1 and 2 was elucidated via the total synthesis of 1 as well as the experimental circular dichroism. Compound 3 was confirmed by a signal crystal X-ray analysis.

Full text of DOI:10.1016/j.fitote.2019.104364

Fitoterapia139(2019)104364
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New alkaloidal metabolites from cultures of entomopathogenic fungus
Cordyceps takaomontana NBRC 101754
⁎
Maichi Hamaa, Abdelsamed I. Elshamya,b, , Tatsuro Yoneyamaa, Yusuke Kasaia,
Hirohumi Yamamotoa, Kana Tanigawaa, Ayari Oshiroa, Masaaki Nojia, Sayaka Banc,
Hiroshi Imagawaa, Akemi Umeyamaa
a Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
b Chemistry of Natural Compounds Department, National Research Centre, Dokki, 12622 Giza, Egypt
c Medical Mycology Research Center, Chiba University, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two new alkaloidal metabolites, cordytakaoamides A (1) and B (2), as well as, 2-[(2-hydroxyethyl) amino]
benzoic acid (3) and 2E-decenamide (4), and three known compounds (5–7) were isolated from ethyl acetate
and n-butanol soluble portions of the entomopathogenic fungus, Cordyceps takaomontana NBRC 101754.
Compounds 3 and 4 were isolated here for first time from natural resources. The chemical structures were
established depending upon spectroscopic techniques such as 1D, 2D NMR, and HRMS. The absolute config-
uration of 1 and 2 was elucidated via the total synthesis of 1 as well as the experimental circular dichroism.
Compound 3 was confirmed by a signal crystal X-ray analysis.
Cordyceps takaomontana
Cordytakaoamides
Alkaloidal metabolites
Total synthesis
X-ray
1. Introduction
constituents of the ethyl acetate and n-butanol soluble portions of en-
tomopathogenic fungi, Cordyceps takaomontana NBRC 101754 for the
The entomopathogenic fungi were reported as promising resources
of several novel and bioactive metabolites especially nitrogenous
compounds and alkaloids [1]. Recently, several scientists and re-
searchers pay attention to entomopathogenic fungal resources of novel
bioactive compounds [2].
first time (ii) identification of rare types of alkaloids depending upon
NMR techniques, (iii) confirmation of the absolute configuration of 1
and 2 by total synthesis of 1, as well as circular dichroism, respectively,
and (iv) isolation of two nitrogenated compounds (3 and 4) for the first
time from natural resource.
The Ophiocordyceps sinensis (synonym: Cordyceps sinensis) that be-
longing to the Cordyceps genus (Ascomycota) is an important medicinal
kind of entomopathogenic fungi and widely used in traditional Chinese
herb from ancient times in treatment of heart, liver, kidney and lung
diseases, hyperlipidemia, hyperglycemia, renal dysfunction and failure,
and arrhythmias [2,3]. About 400 species belonging to Cordyceps were
documented [2]. The fungi belonging to genus Cordyceps are very rich
resources for rare and novel secondary metabolites such as cordycepin
and cyclodepsipeptides, polyketide [2,4–6], in addition to cerebrosides,
steroids, mannitol, adenosine and polysaccharides [7,8]. Several
bioactivities were reported such as antiapoptotic [7], anticancer [7],
cardiovascular [7,8], anti-inflammatory [8], immunologic [9–11], an-
titrypanosomal [5], multiple sclerosis diseases of the nervous systems
[2,3], hepatoprotection, nephroprotective, and antioxidant activities
[10,11].
2. Experimental
2.1. General experimental procedures
Optical rotations were recorded on a JASCO P-2300 polarimeter
(Tokyo, Japan). IR spectra were measured on a Shimazu FTIR-8400S
instrument (Columbia MD 21046, USA). 1D and 2D NMR spectra were
recorded on a Bruker 600 and or 500 NMR spectrometer (MA, USA).
The chemical shifts were given in δ (ppm), and coupling constants were
reported in Hz. HR-MS spectra were obtained on a JEOL JMS-700 in-
strument (Tokyo, Japan). Electronic circular dichroism (ECD) was dis-
played using CD Spectra: JASCO 810 spectropolarimeter. Silica gel 60
(Merck, 230–400 mesh, Merck, Darmstadt, Germany) was used for
column chromatography. Precoated silica gel plates (Merck, Kieselgel
60 F254, 0.25 mm, Merck, Darmstadt, Germany) was used for TLC
Herein, we reported (i) the isolation and identification of chemical
⁎ Corresponding author.
E-mail address: elshamynrc@yahoo.com (A.I. Elshamy).
https://doi.org/10.1016/j.fitote.2019.104364
Received 27 August 2019; Received in revised form 24 September 2019; Accepted 28 September 2019
Availableonline17October2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

M. Hama, et al.
Fitoterapia139(2019)104364
analysis. Isolera-one flash chromatography (Biotage; Suite C Charlotte,
NC; USA) was used for flash chromatography and purification. High-
performance liquid chromatography (HPLC) was performed on a Jasco
PU-980 pump intelligent HPLC pump equipped with a Jasco UV-970
intelligent UV/VIS detector at 210 nm. A semi preparative reversed-
phase column (Cosmosil C18 column 250 × 10 mm, 5 μm) was used for
HPLC.
recrystallized from a n-butanol solution of 3, were selected and fitted
onto a glass fiber and measured at −173 °C with a Bruker Apex II ultra-
diffractometer using MoKα radiation. Data correction and reduction
were performed with the crystallographic package Apex3. The structure
was solved and refined with the Bruker SHELXTL software package. The
final anisotropic full-matrix least-squares refinement on F2 with 124
variables converged at R1 = 7.49%, for the observed data and
wR2 = 21.27% for all data. The ORTEP plot was obtained by the pro-
gram PLATON (A. L. Spek, 2009). Crystal data: C9H11NO3, MW = 181,
2.2. Fungal material
Monoclinic, space group
b = 4.857(4) Å, c = 21.575(19)
lume = 890.3(14) Å3, GOF = 1.068.
Crystallographic data of 3 have been deposited at the Cambridge
Crystallographic Data Centre as supplementary publication numbers
CCDC1904434. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ UK (Fax:
+44(0)-1223–336,033 or e-mail: deposit@ccdc.cam.ac.uk).
P
1
21/n 1, Z = 2, a = 8.566(8) Å,
Å, β = 97.314(13)°, vo-
The entomopathogenic fungus C. takaomontana NBRC 101754 was
derived from single-ascospore of stroma that occurring on an uni-
dentified pupa. The voucher strain and specimen (NBRC 101754) was
deposited at the Biological Resource Center, NITE (NBRC), Kazusa
Kamatari 2-5-8, Kizarashi, Chiba, Japan.
2.3. Fungal culture, fermentation, extraction and isolation
2.4.4. E-dec-2-enamide (4)
The fungus C. takaomontana NBRC 101754 was cultured in potato
sucrose medium (at 25 °C, 21 days). Mycelia were separated from the
broth by filtration and subsequently extracted with MeOH (2 times,
60 min) in an ultrasonic bath. After concentrating under vacuum, the
MeOH crude extract (13.2 g) was then dissolved in distilled H2O and
fractionated with EtOAc and then n-butanol (500 ml × 3 times). The
EtOAc and n-butanol soluble portions were concentrated under vacuum
to afford a black gum (6.8 g and 3.7 g, respectively). The EtOAc extract
was subjected to silica gel flash CC by using a gradient mobile phase, n-
hexan-CHCl3-MeOH, to yield 12 fractions that collected to 5 major
fractions (CTE-1:CTE-5) after TLC examination. Fraction CTE-2 (1.27 g)
was subjected to Sephadex LH-20 CC using CHCl3-MeOH (1:1, v/v) as a
solvent system and followed by RP-18 HPLC (MeOH−H2O, 7:3) af-
forded 1 (9.4 mg) and 7 (5.3 mg). Fraction CTE-3 (869 mg) was sub-
jected to the same steps of chromatography with RP-18 HPLC solvent
systems (MeOH−H2O, 1:1) afforded, 5 (3.2 mg) and 6 (4.7 mg). The n-
butanol soluble portion was further chromatographed and fractionated
via silica gel CC by using a n-hexan-CHCl3-MeOH as elution system step
gradient afforded 4 major fractions (CTB-1: CTB-4) after TLC ex-
amination. Fraction CTB-2 (931.2 mg) was eluted by CHCl3-MeOH (1:1,
v/v) over Sephadex LH-20 CC and followed by RP-18 HPLC
(MeOH−H2O, 3:2) afforded 4 (4.1 mg). By the same, the two sub-
fractions (CTB-3A and CTB-3B) were obtained from chromatography of
fraction CTB-3 (1.12 g) over Sephadex LH-20 CC eluted with CHCl3-
MeOH (1:1, v/v). Sub-fraction CTB-3A (142.3 mg) was subjected to RP-
18 HPLC (MeOH−H2O, 3:2) afforded 2 (2.1 mg) and 3 (3.3 mg).
White powder, FT-IR: 1619, 3378 cm−1 1H and 13C NMR (see
;
Table 2); HR-EIMS, m/z 169.1485 [(M)+; C10H19NO; calcd; 169.1467].
2.5. Total synthesis of 1S
2.5.1. Synthesis of (N-acryloyl)-L-phenylalaninol (1a, Scheme 1)
To a suspension of NaBH4 (1.17 g, 30.88 mmol), L-phenylalanine
(1.99 g, 12.05 mmol) in THF (40 ml) was slowly added a solution of I2
(3.15 g, 12.41 mmol) in THF (7 ml) at 0 °C. The mixture was warmed to
room temperature, stirred for 30 min, and then heated to reflux for 21 h
before adding MeOH cautiously at room temperature. The organic
solvent was removed under reduced pressure and the resulting white
solid was dissolved with CHCl3. The organic layer was washed with
20% aq KOH, dried over Na2SO4, filtered, and concentrated under re-
duced pressure. The resulting white solid was recrystallized from to-
luene to obtain L-phenylalaninol (1.06 g, 7.01 mmol, 58%) as colorless
needles. The spectral data was identical with the literature data [12].
To a stirred solution of L-phenylalaninol (50.3 mg, 0.332 mmol) in
THF/H2O (4:1, 2.0 ml) was added magnesium oxide (68.0 mg,
1.687 mmol), acryloyl chloride (60 ml, 0.742 mmol) at room tempera-
ture. After stirring for 4 h, the suspension was filtered through celite
and washed with EtOAc. The filtrate was washed successively with
saturated aqueous NH4Cl and saturated aqueous NaCl, dried over
Na2SO4, filtered, and concentrated under reduced pressure to furnish
(N-acryloyl)-L-phenylalaninol (1a, 60.7 mg, 0.296 mmol, 89%) as a
25
white solid: [α]D −23.5 (c 1.05, CHCl3); IR (film) 3294, 3067, 3027,
2930, 2862, 1659, 1624, 1541 cm−1 1H NMR (300 MHz, CDCl3) δ
;
2.4. Spectroscopic data of 1–4
7.31–7.19 (m, 5H), 6.29 (br d, J = 8.3 Hz, 1H), 6.23 (dd, J = 17.0,
1.3 Hz, 1H), 6.06 (dd, J = 17.0, 10.3 Hz, 1H), 5.61 (dd, J = 10.3,
1.3 Hz, 1H), 4.23 (m, 1H), 3.71–3.55 (m, 2H), 3.46 (m, 1H), 2.91 (d,
J = 7.2 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 166.0, 137.6, 130.6,
129.2 (2C), 128.6 (2C), 126.9, 126.6, 63.5, 52.8, 36.8; HRMS (CI) calcd
for C12H16NO2 [(M + H)+] 206.1176, found 206.1184.
2.4.1. (S,E)-8-((1-hydroxy-3-phenylpropan-2-yl) amino)-8-oxooct-6-enoic
acid (cordytakaoamide A; 1)
25
White powder, [α]D – 15.38° (c 0.1, MeOH); FT-IR: 1511, 1669,
and 3397 cm−1 1H and 13C NMR (see Table 1); HR-CIMS, m/z
;
306.1668 [(M + H)+; C17H24NO4; calcd; 306.1705], Positive-TOF-
ESIMS, m/z 328.1527 [(M + Na)+; C17H23NO4Na; calcd; 328.1525],
and Negative-TOF-ESIMS, m/z 304.1517 [(M-H)−;C17H22NO4; calcd;
304.1549].
2.5.2. Synthesis of 6-heptenoic acid (1b, Scheme 1)
To a solution of LDA (prepared in situ from iPr2NH (0.90 ml,
6.404 mmol) and nBuLi (1.6 M in hexane, 3.60 ml)) in THF (17.0 ml)
were added AcOH (0.16 ml, 2.790 mmol) and DMPU (8.5 ml) at 0 °C.
After cooling to −78 °C, 5-bromo-1-pentene (0.3 ml, 2.536 mmol) was
added and then the solution was warmed gently to room temperature.
The solution was stirred for 5 h before addition of 1 M aqueous HCl. The
organic layer was extracted with Et2O, dried over MgSO4, filtered
through celite, concentrated under reduced pressure. Purification of the
residue by column chromatography (silica gel, 0% to 30% EtOAc/
hexane) gave 6-heptenoic acid (1b, 64.8 mg, 0.506 mmol, 20%) as a
colorless oil: The spectral data was identical with the literature data
[13].
2.4.2. (S,E)-10-((1-hydroxy-3-(2-hydroxyphenyl)propan-2-yl)
amino)-
10-oxodec-8- enoic acid (cordytakaoamide B; 2)
25
White powder, [α]D – 23.22° (c 0.1, MeOH); FT-IR: 1593, 1661,
and 3402 cm−1 1H and 13C NMR (see Table 1); HR-EIMS, m/z
;
349.1883 [(M)+; C19H27NO5; calcd; 349.1889].
2.4.3. 2-((2-hydroxyethyl)amino)benzoic acid (3): FT-IR: 1735 cm−1
,
3404 cm−1 1H and 13C NMR (see Table 2)
;
White crystals, HR-EIMS, m/z 181.0769 (M)+; C9H11NO3; calcd;
181.0793). X-ray Crystallographic Procedure: Single crystals of 3,
2

M. Hama, et al.
Fitoterapia139(2019)104364
Table 1
1H and 13C NMR (MeOH-d4, 600 Hz) of cordytakaoamides A (1) and B (2).
No
Cordytakaoamide A (1)
Cordytakaoamide A (2)
1H NMR (J Hz)
13C NMR
1H NMR (J Hz)
13C NMR
1
1S
1
1S
1
2
3
4
5
6
7
8
–
–
178.1, s
31.6, t
25.0, t
28.9, t
32.7, t
145.2, d
125.0, d
167.8, s
–
177.4, s
34.6, t
25.5, t
28.8, t
32.6, t
145.1, d
125.1, d
168.5, s
–
–
178.1, s
35.2, t
26.1, t
29.8, t
30.0, t
29.2, t
32.9, t
145.5, d
124.9, d
168.7, s
53.7, d
64.3, t
2.31 m
1.61 m
2.29 t (7.4)
1.61 m
1.48 m
2.19 m
6.69 dt (15.4, 7.0)
5.90 dt (15.4, 1.5)
–
–
2.26 t (7.4)
1.60 m
1.34 m
1.34 m
1.45 m
1.34 m
2.19 ma
6.70 dt (15.1, 7.3)
5.90 br d (15.1)
–
–
2.17 m
6.70 dt (15.4, 7.0)
5.89 dt (15.4, 1.4)
–
9
10
1′
2′a
2′b
1″
2″
3″
4″
5″
6″
7″
–
–
–
–
4.15 m
3.52 m
4.15 m
3.52 m
54.2, d
64.2, t
54.2, d
64.1, t
4.16 m
3.57 dd (11.3, 4.8)
3.54 dd (11.3, 5.4)
–
–
139.8, s
130.3, d
129.3, d
127.3, d
130.3, d
129.3, d
38.0, t
139.8, s
130.3, d
129.3, d
127.3, d
129.3, d
127.3, d
38.0, t
125.9, s
156.6, s
116.0, d
128.7, d
120.6, d
132.2, d
32.4, t
7.21–7.26 ma
7.21–7.26 ma
7.16 m
7.21–7.26 ma
7.21–7.26 ma
7.16 m
–
6.75 dd (8.1, 1.0)
7.01 ddd (8.1, 8.1, 1.6)
6.73 ddd (8.1, 7.4, 1.0)
7.08 dd (7.4, 1.6)
2.77 dd (13.6, 7.7)
2.89 dd (13.6, 6.6)
7.21–7.26 ma
7.21–7.26 ma
2.74 dd (13.7, 8.0)
2.92 dd (13.7, 6.3)
7.21–7.26 ma
7.21–7.26 ma
2.74 dd (13.9, 8.2)
2.92 dd (13.9, 6.3)
a
overlapped; s, quaternary, d, methine, and t, methylene; Multiplicity of carbons was determined by DEPT and HMQC experiments.
2.5.3. Synthesis of cordytakaoamide A (1S, Scheme 1)
solution of (N-acryloyl)-L-phenylalaninol 1a (26.0 mg,
Table 2
1H and 13C NMR of 3 (MeOH-d4, 600 Hz) and 4 (CDCl3, 500 Hz).
A
0.127 mmol), 6-heptenoic acid 1b (15.7 mg, 0.122 mmol) and
Hoveyda–Grubbs 2nd (5.2 mg, 0.00829 mmol) in CH2Cl2 (1.2 ml) was
refluxed for 13 h before removal of solvent under reduced pressure.
Purification of the residue by column chromatography (silica gel, 1st:
CHCl3/acetone = 4/1 to 2/1, 2nd: CHCl3/MeOH = 1/0 to 10/1) gave
cordytakaoamide A (1S, 19.1 mg, 0.0625 mmol, 51%) as a colorless oil:
[α]D25 −31.2 (c 0.85, MeOH); IR (film) 3430, 3028, 2944, 2860, 1734,
No
3
No
4
1H NMR (J Hz)
13C
1H NMR (J Hz)
13C
1
2
3
4
5
6
7
1′
2′
–
–
110.2, s
151.3, s
110.9, d
134.2, d
114.2, d
131.9, d
170.6, s
44.5, t
1
2
3
4
5
6
7
8
9
–
168.8, s
122.5, d
146.5, d
31.9, t
28.0, d
28.9, d
29.0. d
31.6, d
22.5, d
13.9, q
5.85 d (15.6)
6.84 m
2.18 m
6.74 d (8.5)
7.34 m
6.57 t (14.9)
7.90 d (7.9)
–
1671, 1541, 1458 cm−1 1H and 13C NMR (600 MHz, CD3OD); see
;
1.44 m
1.31 ma
1.31 ma
1.31 ma
1.31 ma
0.88 t (7.2)
Table 1; HRCIMS: m/z 306.1696; (calcd: 306.1700 for C17H24NO4
[(M + H)+].
3.34 t (11.4)
3.78 t (11.4)
60.1, t
10
3. Results and discussion
a
overlapped; s, quaternary, d, methine, t, methylene, and q, methyl;
The chemical characterization of the EtOAc and n-butanol soluble
portions of fungus, C. takaomontana NBRC 101754, afforded two new
alkaloidal compounds namely, cordytakaoamide A (1) and B (2), as
well as two compounds isolated here for the first time from natural
Multiplicity of carbons was determined by DEPT and HMQC experiments.
Scheme 1. Total synthesis of cordytakaoamide A (1S).
3

M. Hama, et al.
Fitoterapia139(2019)104364
Fig. 1. Isolated compounds from C. takaomontana.
Fig. 2. Selected 1H-1H COSY and key HMBC of 1–4.
resource, 2-((2-hydroxyethyl)amino)benzoic acid (3) and E-dec-2-en-
amide (4), and three known compound, TK-57-164B (5) [14], one
depsipeptide, beauvericin (6) [15,16] and one steroid, 5α,6α-epox-
yergosta-8(14),22-diene-3β,7α-diol (7) [17,18] (Fig. 1).
Cordytakaoamide A (1, Fig. 1) was isolated as a white solid with a
25
negative optical rotation in methanol ([α]D – 15.38°). The HR-CIMS
and TOF-ESIMS exhibited molecular ion peak at m/z HR-CIMS, m/z
306.1668 [(M + H)+; C17H24NO4; calcd; 306.1705], positive-TOF-
4

M. Hama, et al.
Fitoterapia139(2019)104364
Fig. 3. Experimental CD of 1S, 1, and 2.
Fig. 4. X-ray crystallographic structure of 3.
ESIMS, m/z 328.1527 [(M + Na)+; C17H23NO4Na; calcd; 328.1525],
and negative-TOF-ESIMS, m/z 304.1517 [(M-H)−; C17H22NO4; calcd;
304.1549], respectively displaying seven degrees of unsaturation. The
FT-IR of 1 exhibited absorption bands at 3397, 1669, and 1511 cm−1
corresponding to hydroxy and/or nitrogen, amide carbonyl and olefinic
groups, respectively. The 1H NMR spectra data (Table 1) of 1 exhibited
five aromatic olefinic protons [at δH 7.16, 7.21–7.26 m], two aliphatic
olefinic protons [at δH 5.90 br d (J = 15.1 Hz) and δH 6.70 dt (J = 15.1,
7.3 Hz)], one hydroxylated methylene protons [at δH 3.52 m], one
methine proton attached with nitrogen [at δH 4.15 m] in addition to
five aliphatic methylenes [at δH 1.34 m, δH 1.61 m, δH 2.19 m, δH
2.31 m, and δH 2.74 dd (J = 13.7, 8.0 Hz); 2.92 dd (J = 13.7, 6.3 Hz)].
Seventeen carbon resonances were assigned by 13C NMR spectrum that
characterized with DEPT and HMQC to, three quaternary carbons (in-
cluding one carboxylic carbon at δC 178.1, one amide carbonyl carbon
at δC 167.3, and one aromatic carbon at δC 139.8), five aromatic me-
thines (at δC 127.3, 129.3 (2 x C) and 130.3 (2 x C)), two olefinic
methines at δC 125.0, and 145.2, one methine attached to nitrogen at δC
54.2, alongside six aliphatic methylenes at δC 25.0, 28.9, 31.6, 32.7,
38.0, and oxyhenated one at δC 64.2. The complete analysis of 1D NMR
followed by 2D NMR especially 1H-1H COSY and HMBC revealed that 1
include three internal structural part (i) monosubstituted benzene ring
at the quaternary carbon at δC 139.8, (ii) CH2-CH(NHCOR)-CH2OH, and
(iii) R-CH=CH-(CH2)4-COOH. The monosubstituted benzene ring was
established via 1H-1H COSY correlations (Fig. 2) of the aromatic pro-
tons, H-2″ and/or H-6″ (δH 7.21–7.26 m)/H-3″ and/or H-5″ (δH
7.21–7.26 m), and H-3”/H-4″ (δH 7.16 m), as well as HMBC correlations
(Fig. 2) of H-2″ and/or H-6”/C-4″ (δC 127.3, J3), alongside H-3″ and/or
5″/ C-1″ (δC 139.8, J3). The 1H-1H COSY correlations (Fig. 2) of H-7”/H-
1′, H-1’/H-2′, in addition to HMBC correlations of H-1’/H-8 (amide
carbonyl carbon at δC 167.8, J3), H-7”/H-2′ (J3), and H-1’/H-2′ (J2)
deduced the 2nd internal structural part. The continuous of 1H-1H
COSY sequence of H-2/H-3 until H-5/H-6 (δH 6.70 dt (J = 15.1,
7.3 Hz)) and H-6/H-7 (5.90 br d, J = 15.1 Hz) alongside the HMBC
correlations of H-3/C-1 (carboxyl group at δC 178.1, J3), and H-5/C-7
(at δC 125.0, J3) deduced the 3th structural part of 1. The strong HMBC
correlations of H-7″/C-2″ and/or C-6″ (at δC 130.3, J3), and H-1′’/C-1″
(at δC 139.8, J3) assigned the connectivity of partial structure (ii) to the
5

M. Hama, et al.
Fitoterapia139(2019)104364
monosubstituted benzene ring (i) via the quaternary C-1″ constructing
phenylalaninol moiety. By the same, the clear HMBC correlations of H-
6/C-8 (amide carbonyl carbon at δC 167.8, J3), and H-7/C-8 (J2) con-
firmed the connectivity of partial structure (ii) and (iii) via secondary
amide linkage (-NHCO-). From above described 1D and 2D NMR data, 1
was established as 8-((1-hydroxy-3-phenyl-propan-2-yl)-amino)-8-ox-
ooct-6-enoic acid. Compound 1 was characterized here with very rare
alkaloidal skeleton that may be biosynthesized from the enzymatic re-
action of L-phenylalaninol and 2-octenedioic acid. Recently, only one
compound with similar alkaloidal skeleton namely, farinosone C, was
reported from Paecilomyces farinosus [1].
In out trying for establishment of the absolute configuration at C-1′,
1S was totally synthesized using L-phenylalanine as the starting mate-
rial. The optically active 1S synthesized with olefin metathesis as a key
step exhibited a positive cotton effect at 258 and this is totally agree-
ment with naturally isolated one with a positive cotton effect at 251
(Fig. 3). Thus, from above mentioned data, 1 was established as (S, E)-
J = 7.9 Hz), as well as H2–2′ (at δH 3.78 t, J = 11.4 Hz)/H2–1′ (at δH
3.34 t, J = 11.4 Hz) in addition to HMBC (Fig. 2) correlations of H2–1’/
C-2 (at δC 151.3, J3) and H-6/C-7 (Carboxylic acid at δC 170.6, J3). Thus
3 was established as 2-[(2-hydroxyethyl)-amino] benzoic acid. 3 was
already known synthetic compound [19], but it was reported here for
the first time from natural resource. The structure of 3 was confirmed
by a single-crystal X-ray analysis (Fig. 4).
E-Dec-2-enamide (4, Fig. 1) was isolated as white solid. HR-EIMS of
4 showed a molecular ion peak at m/z 169.1485 [(M)+ as C10H19NO
(calcd;169.1467]. FT-IR of 4 displayed nitrogen at 3378 cm−1 and an
amide carbonyl group at 1619 cm−1. The 1D NMR of 4 (Table 2) ex-
hibited one methyl at C-10, at [δH 0.88 (t, J = 7.2); δC 13.9)], two
olefinic carbons at C-2 and C-3 [at δH 5.85 (d, J = 15.6); δC 122.5 and
δH 6.84 m; δC 146.5; respectively], and six aliphatic methylenes [at δH
1.31 m, 1.44 m, 2.18 m; and at δC 22.5–31.9]. 1H-1H COSY sequence
(Fig. 2) of H-2/H-3, H-3/H-4, until H-9/H-10 as well as HMBC corre-
lations (Fig. 2) of H-3/C-1 (amide carbonyl, δC 168.8, J3), and H-4/C-2
(δC 146.5, J3) were clearly observed. The E-geometry of Δ2,3 was de-
termined via the vicinal coupling constant J = 15.6 Hz (Concellon et al.,
2010). From above described data, 4 was constructed as E-dec-2-en-
amide. Although this compound was described before as a synthetic
compound [20], but herein is the first time for isolation and identifi-
cation of it from natural resource.
8-((1-hydroxy-3-phenyl-propan-2-yl)-amino)-8-oxooct-6-enoic
acid
(cordytakaoamide A; 1).
Cordytakaoamide B (2, Fig. 1) was isolated as a white solid with a
25
negative optical rotation in methanol ([α]D – 23.22°). The HR-EIMS
exhibited a molecular ion peak at m/z 349.1883 (M)+, indicating the
molecular formula C19H27NO5 (calcd; 349.1889) revealing seven de-
grees of unsaturation. Absorption bands at 3402, 1661, and 1593 cm−1
were identified in FT-IR of 2 corresponding to hydroxy and/or nitrogen,
amide carbonyl and olefinic groups, respectively. 1D NMR (Table 1)
analysis deduced that 2 have very closed structure to the alkaloidal
compound 1, with exceptions of the (i) presence of hydroxylated aro-
matic quaternary carbon at δC 156.6 in the aromatic ring, and (ii) the
difference of the numbers of methylenes in aliphatic chain. The HMBC
correlations (Fig. 2) of H-7″ [at δH 2.77 dd (J = 13.6, 7.7 Hz); 2.89 dd
(J = 13.6, 6.6 Hz)]/C-2″ (δC 156.6, J3), H-4″ [at δH 7.01 ddd (J = 8.1,
8.1, 1.6 Hz)]/C-2″ (J3), and H-6″ [at δH 7.08 dd, J = 7.4, 1.6 Hz]/C-2″
(J3), confirmed the hydroxylation of C-2″. Also, the 1H-1H COSY of H-2
[δH 2.26 t (J = 7.4 Hz)]/H-3 [δH 1.60 m] until H-8 [δH 6.70 dt,
J = 15.4, 7.0 Hz]/H-9 [δH 5.89 dt, J = 15.4, 1.4 Hz)] alongside the
HMBC of H-3/C-1 (δC 178.1, J2) deduced that the aliphatic chain is
4. Conclusion
From the entomopathogenic fungus, C. takaomontana NBRC
101754, seven compounds were isolated and identified including two
new rare alkaloidal metabolites, cordytakaoamides A (1) and B (2), as
well as two compounds 2-[(2-hydroxyethyl) amino] benzoic acid (3)
and 2E-decenamide (4), isolated for the first from natural resources.
The absolute stereochemistry of 1 and 2 was established by the total
synthesis of 1 in addition to experimental CD. 3 was confirmed by a
signal crystal X-ray analysis.
Acknowledgements
C
10H16NO3 in 2 instead of C8H12NO3 in 1. Compound 2 exhibited a
Dr. Elshamy wishes to gratefully acknowledge Takeda Science
Foundation, Japan for the financial support. Also, this work was sup-
ported by Tokushima Bunri University, Japan and National Research
Centre, Egypt.
positive cotton effect at 249 that completely agreement with 1 and 1S
(Fig. 3). Thus, from above mentioned data, 2 was assigned (S,E)-10-((1-
hydroxy-3-(2-hydroxyphenyl)-propan-2-yl)-amino)-10-oxodec-8-enoic
acid (cordytakaoamide B). The biosynthesis of this compound might be
performed via two main steps, (i) the enzymatic hydroxylation of L-
phenylalaninol by involvement of the enzyme P450 that afforded 2-
hydroxy-phenylalaninol, followed by (ii) the 2-hydroxy- phenylalaninol
then enzymatically react with 2-decenedioic acid.
Declaration of Competing Interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
2-[(2-Hydroxyethyl)-amino] benzoic acid (3, Fig. 1) was isolated as
white crystals. The HR-EIMS exhibited a molecular ion peak at m/z
181.0769 (M)+ indicating the molecular formula C9H11NO3 (calcd;
181.0739). FT-IR of 3 exhibited absorption bands corresponding to
hydroxy and/or nitrogen, and carboxylic carbonyl at 3404, and
1735 cm−1, respectively. 1H NMR (Table 2) exhibited four aromatic
protons at δH 6.74 d (J = 8.5 Hz), δH 7.34 m, δH 6.57 t (J = 14.9 Hz),
and δH 7.90 d (J = 7.9 Hz), in addition to one nitrogenated methylene
at δH 3.34 (t, J = 11.4 Hz), as well as one oxygenated methylene at δH
3.78 (t, J = 11.4 Hz). 13C NMR (Table 2) exhibited nine carbon signals
that categorized depending upon DEPT-135 and HSQC to, three quin-
tenary carbons (one carboxylic carbonyl at δC 170.6, nitrogenated
aromatic one at δC 151.1 and aromatic at δC 110.2), two alphatic me-
thylenes (one hydroxylated at δC 60.1 and one nitrogenated at δC 44.5),
and four aromatic methines (at δC 110.9, 114.2, 131.9 and 134.2). The
complete analysis of 1D (Table 2) and 2D NMR (Fig. 2) suggested that 3
is O-disubstituted benzene ring with carnoxylic acid and aminoethane-
2-ol moiety. This structure was deduced by 1H-1H COSY (Fig. 2) se-
quence of the aromatic ring, H-3 (at δH 6.74 d, J = 8.5 Hz)/H-4 (at δH
7.34 m), H-4/H-5 (at δH 6.57 t, J = 14.9 Hz), and H-5/H-6 (at δH 7.90 d,
LR and HR-MS,1H, 13C NMR, DEPT-135, HSQC, HMBC, 1H 1H COSY
and NOESY spectral data of 1–4 (S1–S47) are found in supplementary
data. Supplementary data to this article can be found online.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fitote.2019.104364.
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