Synthesis of the key precursor of Hirsutellide A

Article abstract of DOI:10.3390/10010259

Hexadepsipeptide 2, the precursor of Hirsutellide A (1), was synthesized in an overall yield of 45% from N-Boc-Me-Gly via three coupling reactions using dicyclohexylcarbodiimide (DCC), O-(7-azabenzotriazol-1-yl)-N,N,N′, N′-tetramethyluronium hexafluorophosphate (HATU) and bis(2-oxo-3- oxazolidinyl)phosphinic chloride (BOP-Cl), respectively.

Full text of DOI:10.3390/10010259

Molecules 2005, 10, 259-264
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of the Key Precursor of Hirsutellide A
Yanjie Xu 1, Xuemin Duan 1, Meiling Li 1, Liqin Jiang 1, Guangle Zhao 1, Yi Meng 2 and Ligong
Chen 1,*
1
College of Pharmaceuticals and Biotechnology, Tianjin University, Tianjin, 300072, P.R. China
Shenzhen Graduate School of Peking University, Shenzhen University Town, Shenzhen, 518055,
2
P.R. China
* Author to whom correspondence should be addressed; Email: lgchen@tju.edu.cn; Telephone:
(+86)-22-27891034; Fax: (+86)-22-27406314
Received: 27 February 2004 / Accepted: 19 March 2004 / Published: 31 January 2005
__________________________________________________________________________________
Abstract: Hexadepsipeptide 2, the precursor of Hirsutellide A (1), was synthesized in an
overall yield of 45% from N-Boc-Me-Gly via three coupling reactions using
dicyclohexylcarbodiimide
(DCC),
O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyl-
uronium hexafluorophosphate (HATU) and bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(BOP-Cl), respectively.
Keywords: Hirsutellide A, linear hexadepsipeptide, coupled reagent, diazotization
hydrolysis, Boc protection
__________________________________________________________________________________
Introduction
Hirsutellide A (1) is a new antimycobacterial cyclohexadepsipeptide from the cell extracts of
Hirsutella kobayasii BCC 1660. It exhibited antimycobacterial activity with a MIC (minimum
inhibitory concentration) of 6-12 µg/mL with no cytotoxic effect towards Vero cells at 50 µg/mL.
Additionally, compound 1 also possessed weak in vitro antimalarial activity, with an IC50 value of 2.8
µg/mL [1]. With the rapidly increasing worldwide incidence of tuberculosis, particularly among those
associated with HIV infection, studies on new drugs against the emerging drug-resistant strains of
turberculosis are urgently needed.
Our retrosynthetic analysis of hirsutellide A (1) is shown in Figure 1. Recognizing the importance
of structure 2, we divided it into two tridepsipeptides units. A further division led to three subunits,
2-hydroxy-3-phenylpropanoic acid benzyl ester (4), (tert-butoxycarbonylmethylamino)acetic acid (5)
and 2-tert-butoxycarbonylamino-3-methylpentanoic acid (6).

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260
Figure 1. Retrosynthetic analysis of Hirsutellide A (1)
O
OBn
O
O
O
N
O
O
N
H
OBn
NHBoc
O
N
O
N
O
O
N
O
O
Me
Me
N
Me
Me
O
O
O
O
H
H
N
N
NHBoc
NHBoc
3
O
O
O
O
2
1
O
OH
OH
BocN
OBn
+
+
Me
O
OH
O
5
4
6
Results and Discussion
Compound 4 was obtained as a colorless oil from D-phenylalanine through the diazotization,
hydrolysis [2] and esterification by benzyl alcohol [3] (Scheme 1).
Scheme 1
O
O
O
NaNO2
H2SO4
BnOH
TsOH
OH
OH
OBn
OH
NH2
OH
7
4
D-Phenylalanine
Subunit 5 was obtained from N-Boc-Gly-OH (8) through N-methylation with CH3I (Scheme 2).
After adding the first portion of CH3I (4.0 equiv.) and NaH (2.5 equiv.) to the reaction mixture, CH3I
(2.0 equiv.) and NaH (1.0 equiv.) were added again to drive the N-methylation to completion. Unit 6
was N-Boc-Ile.
Scheme 2
OH
1) CH3I, NaH, THF
2) HCl
OH
BocHN
BocN
Me
O
O
8
5
The synthetic sequence leading to compound 2 is shown in Scheme 3. Esterification of 5 with 4
using DCC provided the fully protected didepsipeptide 9. Removal of the Boc protecting group by
treatment with TFA afforded compound 10. Then the TFA amine salt 10 was coupled with unit 6 using
HATU-promoted reagent to yield the corresponding protected tridepsipeptide 3 in 81% yield compared
with PyBOP-promoted in 60% yield and DCC in lower yield. Cleavage of the benzyl ester and the Boc
group gave two deprotected precursors 11 and 12, respectively. Subsequently, the TFA amine salt 12
and free acid residue 11 were coupled separately with HATU, PyBOP and BOP-Cl promoting to
synthesize the linear hexadepsipetide 2. Only the reaction with BOP-Cl reagent worked in 77% yield
to afford the product 2 whose structure was fully characterized by [α]D, IR, NMR and MS.

Molecules 2005, 10
261
Scheme 3
O
O
O
OBn
Me
OR1
OH
DCC
OBn
NR
N
Boc
O
6
O
O
N
+
O
OH
HATU
O
Et3N
5
O
4
9: R=Boc
10: R=H
NHR2
TFA
3: R1=Bn,
5%Pd/C, H2
TFA
2
1
2
R =Boc
11: R =H, R =Boc
12: R1=Bn, R2=H
O
O
O
N
H
N
O
O
BOP-Cl
Et3N
11 +
Me
12
Me
O
O
Boc
HN
N
O
OBn
2
Reagents: DCC: Dicyclohexyl carbodiimide; TFA: Trifluoroacetic acid; HATU: O-(7-Azabenzo-
triazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; PyBOP: Benzotriazole-1-yl-
oxy-tris-pyrrolidinophosphonium hexafluorophosphate; BOP-Cl: Bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride.
Conclusions
The hexadepsipeptide 2, a key hirsutellide A (1) precursor, was successfully synthesized in an
overall yield of 45% based on N-Boc-Me-Gly using three coupling reactions with DCC, HATU, and
BOP-Cl, respectively.
Experimental
General
Melting points were determined by the capillary tube method, and are uncorrected. IR spectra
(KBr pellets) were recorded on a Bio-Rad FTS 135 spectrophotometer. Optical rotations were
1
measured on a WZZ-3 digital polarimeter with a sodium lamp (λ=589nm, D line). H- and 13C-NMR
spectra were recorded on a Bruker AV400 instrument. Chemical shifts (δ) are reported in ppm relative
to TMS used as internal standard. Mass spectra were recorded on a LCQ analytical Advantage MAX
spectrometer. Organic solvents were dried by standard methods when necessary. Thin-layer
chromatography (TLC) was performed on glass sheets coated with silica gel (type GF254, Qingdao
Haiyang). Flash column chromatography was carried out using 200-300 mesh silica gel.
(R)-2-hydroxy-3-phenylpropanoic acid (7).
A solution of 40% sodium nitrite (12 mL) was added into a solution of D-phenylalanine (6.60 g, 40
mmol) in H2SO4 (2 mol/L, 45 mL) over 2 h under ice cooling. The mixture was stirred for 6h at 0°C,

Molecules 2005, 10
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and then at room temperature for 12 h. Additional H2SO4 (2 mol/L, 10 mL) and sodium nitrite (40%, 6
mL) were added as above. The reaction mixture was saturated by NaCl, followed by extraction with
ethyl acetate (3×60 mL). The combined extracts were washed with brine (200 mL), dried (MgSO4),
filtered and evaporated to give a white solid which upon recrystallization from diethyl ether, gave the
20
product 7 (6.54g, 98.5%), m.p. 125-127°C (lit.[2], 123-124°C), [α] =+38.6°(c 1.00, DMF) [lit.[2],
D
25
[α] =+26.7°(c 3.80, acetone)]
D
(R)-benzyl-2-hydroxy-3-phenylpropanoate (4).
Into a 100-mL round-bottom flask equipped with a Stark and Dean distilling receiver were placed
compound 7 (1.66 g, 10 mmol), p-toluenesulfonic acid monohydrate (0.02 g, 0.12 mmol), benzyl
alcohol (1.6 mL, 15 mmol), and toluene (30 mL). Then the mixture was heated under reflux. When
water was no longer distilled off (about 4 h), the reaction mixture was permitted to cool to room
temperature. Then ether (30 mL) and saturated aqueous Na2CO3 (50 mL) were added. The two layers
were separated, and the water layer was extracted with ether (2×30mL). The combined extracts were
washed with brine (100 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 10:1) to
20
25
give 4 (1.45g, 56.6%). [α] =+54.9°(c 1.5, CH2Cl2) [lit.[2], [α] =+55.2°(c 1.88, CH2Cl2)]; IR (KBr):
D
D
3484, 3034, 2928, 1737, 1495, 1450, 1380, 1264, 1189, 1094, 745, 698 cm-1; H-NMR (400 MHz,
CDCl3) δ: 7.18～7.41 (m, 10H), 5.20 (s, 2H), 4.51 (dd, 1H, J1=4.8 Hz, J2=6.4 Hz), 3.15 (dd, 1H,
J1=4.8 Hz, J2=13.6 Hz), 3.01 (dd, 1H, J1=6.4 Hz, J2=13.6 Hz), 2.92 (br, OH); 13C-NMR (100 MHz,
CDCl3) δ: 173.85, 136.09, 134.94, 129.40, 128.50, 128.44, 128.23, 126.67, 71.17, 67.18, 40.34.
1
(tert-Butoxycarbonylmethylamino)acetic acid (5).
To a cooled (0°C) solution of Boc-Gly-OH 8 (7.33 g, 41.9 mmol) and MeI (10.6 mL, 170 mmol) in
dry THF (200 mL), was added NaH (5.23g, 120 mmol, 55% in mineral oil) cautiously and the mixture
was with stirred gently overnight. CH3I (5.3 mL) and NaH (1.75 g) were added in again by the above
procedure to push the reaction to completion. Ethyl acetate (100 mL) was then added, followed by
water (15 mL) added dropwise to destroy the excess NaH. The solution was evaporated to dryness, and
the oily residue partitioned between ether (60 mL) and water (100 mL). The ether layer was washed
with saturated aqueous NaHCO3 (50 mL), and the combined aqueous extracts acidified to pH 2 with 1
mol/L HCl under ice cooling. The product was extract into ethyl acetate (2×100 mL). Then the
extracts were washed with water (100 mL), 5% aqueous sodium thiosulfate (2×100 mL, to remove
iodine), and brine (100 mL), dried (MgSO4), filtered, and evaporated to give a pale white oil 5 (7.32g,
92.5%).
2-[2-(tert-Butoxycarbonylmethylamino)acetoxy]-3-phenylpropionic acid benzyl ester (9).
Compound 4 (1.29 g, 5.0 mmol) was dissolved in dry CH2Cl2 (10 mL) and cooled to 0°C. A
solution of N-Me-Boc-Gly-OH 5 (0.95 g, 5.0 mmol) in CH2Cl2 (10 mL) was added in one portion
followed by HOBt (0.73 g, 5.4 mmol) and DMAP (0.66 g, 5.4 mmol) and the resulting suspension was

Molecules 2005, 10
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then stirred for 15 min. A solution of DCC (1.12 g, 5.4 mmol) in dry CH2Cl2 (10 mL) was added to the
suspension over 10 min and the mixture was slowly warmed to room temperature and stirred for 24 h.
The mixture was evaporated in vacuo to leave a solid and then taken up in ethyl acetate (50 mL),
filtered to remove DCU (by product). The filtrate was washed with saturated aqueous NaHCO3 (2×50
mL) and brine (50 mL). The organic layer was dried (MgSO4) and evaporated in vacuo to leave a
residue and then purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 10:1)
20
to yield title compound 9 (1.56 g, 72.5%). [α] ＝＋16.3°(c 1.0, CHCl3); IR (KBr): 2970, 2932, 1754,
D
1
1703, 1453, 1381, 1243, 1179, 1160, 1070, 746, 703cm-1; H-NMR (400 MHz, CDCl3) δ (rotamer):
7.14～7.32 (m, 10H), 5.30～5.32 (m, 1H), 5.08～5.15 (m, 2H), 3.91～4.05 (m, 2H), 3.10～3.18 (m,
2H), 2.79, 2.78 (s, 3H, rotamer), 1.26～1.46(m, 9H); 13C-NMR (100 MHz, CDCl3) δ (rotamer): 169.05,
168.64, 155.60, 154.95, 135.20, 135.13, 134.84, 129.16, 129.06, 128.31, 128.28, 128.26, 128.19,
128.09, 128.05, 126.81, 79.83, 79.77, 73.09, 73.01, 66.86, 50.39, 49.68, 36.98, 34.93, 28.06, 27.87.
2-{2-[(2-tert-Butoxycarbonylamino-3-methylpentanoyl)methylamino]acetoxy}-3-phenylpropionic acid
benzyl ester (3).
To a solution of 9 (1.70 g, 4.0 mmol) in dry CH2Cl2 (6 mL) was added TFA (2 mL, 26.0 mmol)
dropwise at 0°C with stirring. The mixture then was slowly warmed to room temperature and stirred
for 5 h. The reaction mixture was concentrated, the residue dried under high vacuum and dissolved in
dry CH2Cl2 (10 mL). A solution of N-Boc-Ile-OH 6 (1.01 g, 4.4 mmol) in dry CH2Cl2 (10 mL) was
added in one portion followed by HATU (99%) (1.84 g, 4.8 mmol). Triethylamine (5.6 mL, 39.8 mmol)
was added to the mixture dropwise under ice cooling. Then the mixture was warmed to room
temperature slowly and stirred for 12 h. The reaction was quenched by adding saturated aqueous
NH4Cl (2mL) dropwise and evaporated in vacuo followed by dissolving in ethyl acetate (150 mL). The
mixture was washed with saturated aqueous NaHCO3 (2×100 mL) and brine (100 mL), dried
(MgSO4), filtered and evaporated in vacuo to leave a residue which was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate, 5:1) to afford tridepsipeptide 3 (1.74 g,
20
80.9%). [α] ＝＋6.3° (c 1.0 CHCl3); IR (KBr): 3409, 3316, 2969, 1753, 1708, 1652, 1497, 1402,
D
1377, 1246, 1176, 1075, 1031, 745, 703cm-1; 1H-NMR (400 MHz, CDCl3) δ (rotamer): 7.10～7.34 (m,
10H), 5.31 (dd, 1H, J1=5.2 Hz, J2=7.6 Hz), 5.04～5.17 (m, 3H), 4.51 (br, NH), 4.53 (d, 1H, J=17.6
Hz), 3.80 (d, 1H, J=17.6 Hz), 3.16(dd, 1H, J1=5.2 Hz, J2=14.4 Hz), 3.10 (dd, 1H, J1=7.6 Hz, J2=14.4
Hz), 3.00 (s, 3H), 1.66～1.71 (m, 1H), 1.40 (s, 9H), 0.88～0.96 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ (rotamer): 173.12, 168.95, 168.38, 155.83, 135.23, 134.97, 129.33, 128.56, 128.52, 128.49, 128.42,
128.36, 127.13, 79.48, 73.43, 67.23, 54.22, 49.16, 38.03, 37.15, 36.36, 28.32, 23.91, 15.55, 11.29.
2-(2-{[2-(2-{2-[(2-tert-Butoxycarbonylamino-3-methylpentanoyl)methylamino]acetoxy}-3-phenyl-
propionylamino)-3-methylpentanoyl]methylamino}acetoxy)-3-phenylpropionic acid benzyl ester (2).
A solution of 3 (0.26 g, 0.5 mmol) in ethyl acetate (30 mL) was treated with Pd-C (5%, 0.52 g), and
the black suspension was stirred at room temperature under an atmosphere of H2 for 12 h. The catalyst
was removed by filtration, and the filtrate was concentrated in vacuo to give crude 11 (used in the next
step without further purification). To a solution of 3 (0.20 g, 0.4 mmol) in dry CH2Cl2 (2 mL) was
added TFA (0.5 mL, 6.5 mmol) dropwise at 0°C with stirring. The mixture then was slowly warmed to

Molecules 2005, 10
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room temperature and stirred for 5 h. The reaction mixture was concentrated, and the residue dried
under high vacuum to give crude 12 (used directly in the next step). TFA salt 12 was dissolved in dry
CH2Cl2 (5 mL). A solution of 11 in dry CH2Cl2 (5 mL) was added in one portion with stirring and
cooled to 0°C. BOP-Cl (0.13 g, 0.5 mmol) was added at 0°C followed by triethylamine (0.5 mL, 3.7
mmol). The mixture was then warmed to room temperature and stirred for 12 h. The reaction was
quenched by adding saturated aqueous NH4Cl (2 mL) dropwise and evaporated in vacuo followed by
dissolving in ethyl acetate (50 mL). The mixture was washed with saturated aqueous NaHCO3 (2×50
mL) and brine (50 mL), dried (MgSO4), filtered and evaporated in vacuo to leave a residue which was
purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 2:1) to give
20
hexadepsipeptide 2 (0.16g, 76.6%). [α] =+1.06º(c 3.16, CHCl3); IR (KBr): 3309, 2963, 2931, 1753,
D
1705, 1645, 1501, 1465, 1406, 1376, 1250, 1176, 1071, 745, 707cm-1; 13C-NMR (100 MHz, CDCl3) δ:
174.24, 172.53, 168.84, 168.67, 168.19, 167.47, 156.06, 136.29, 135.28, 134.90, 130.83, 129.32,
129.29, 129.25, 128.56, 128.53, 128.48, 128.45, 128.40, 128.39, 128.35, 128.28, 127.07, 126.90,
126.79, 79.19, 74.79, 73.42, 67.21, 65.47, 54.00, 52.38, 49.06, 38.00, 37.27, 36.83, 36.28, 30.51, 29.61,
28.40-28.38 (3C), 24.27, 24.24, 15.37, 15.20, 10.84, 10.57; MS: m/z= 895.5 (M+Na)+; Anal. Calcd for
C48H64N4O11: C, 66.04; H, 7.39; N, 6.42; O, 20.16. Found: C, 66.18; H, 7.36; N, 6.40; O, 20.06.
References
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Thebtaranonth, Y. HirsutellideA, a new Antimycobacterial Cyclohexadepsipeptide from the
Entomopathogenic Fungus Hirsutella kobayasii. J. Nat. Prod. 2002, 65, 1346.
2. Degerbeck, F.; Fransson, B.; Grehn, L.; Ragnarsson, U. Synthesis of 15N-labelled Chiral
Boc-amino Acids from Triflates: Enantiomers of Leucine and Phenylalanine. J. Chem. Soc. Perkin
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Sample Availability: Available from the authors.
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