Cytotoxic 1,3-thiazole and 1,2,4-thiadiazole alkaloids from Penicillium oxalicum: Structural elucidation and total synthesis

Article abstract of DOI:10.3390/molecules21030232

Two new thiazole and thiadiazole alkaloids, penicilliumthiamine A and B (2 and 3), were isolated from the culture broth of Penicillium oxalicum, a fungus found in Acrida cinerea. Their structures were elucidated mainly by spectroscopic analysis, total synthesis and X-ray crystallographic analysis. Biological evaluations indicated that compound 1, 3a and 3 exhibit potent cytotoxicity against different cancer cell lines through inhibiting the phosphorylation of AKT/PKB (Ser 473), one of important cancer drugs target.

Full text of DOI:10.3390/molecules21030232

molecules
Article
Cytotoxic 1,3-Thiazole and 1,2,4-Thiadiazole
Alkaloids from Penicillium oxalicum: Structural
Elucidation and Total Synthesis
Zheng Yang 1,†, Nianyu Huang 1,†, Bang Xu 1,2, Wenfeng Huang 3, Tianpeng Xie 1, Fan Cheng 1,
*
and Kun Zou 1,
*
1
Hubei Key Laboratory of Natural Products Research and Development, College of Biological and
Pharmaceutical Sciences, China Three Gorges University, Yichang 443002, China; yangzctgu@sina.com (Z.Y.);
hny115@126.com (N.H.); xubang08@163.com (B.X.); sxdxxtp@foxmail.com (T.X.)
China Three Gorges University People’s Hospital & Yichang First People’s Hospital, Yichang 443000, China
Hubei Key Laboratory of Natural Products Research and Development, Medical College,
China Three Gorges University, Yichang 443002, China; xyyxy1999@aliyun.com
Correspondence: fancy1351@163.com (F.C.); kzou@ctgu.edu.cn (K.Z.);
2
3
*
Tel.: +86-717-639-7478 (F.C.); +86-717-639-2286 (K.Z.)
†
These authors contributed equally to this work.
Academic Editor: Derek J. McPhee
Received: 25 January 2016 ; Accepted: 15 February 2016 ; Published: 26 February 2016
Abstract: Two new thiazole and thiadiazole alkaloids, penicilliumthiamine A and B (2 and 3),
were isolated from the culture broth of Penicillium oxalicum, a fungus found in Acrida cinerea. Their
structures were elucidated mainly by spectroscopic analysis, total synthesis and X-ray crystallographic
analysis. Biological evaluations indicated that compound 1, 3a and 3 exhibit potent cytotoxicity
against different cancer cell lines through inhibiting the phosphorylation of AKT/PKB (Ser 473), one
of important cancer drugs target.
Keywords: Penicillium oxalicum; alkaloids; total synthesis; structure elucidation; cytotoxicity
1. Introduction
Numerous natural products with novel structures and distinct biological activities have been
discovered as secondary metabolites of insect-derived microbes [
1,2]. Penicillium oxalicum is one
of the most ubiquitous toxigenic fungi found in soil and musty cereal, with many biologically and
structurally novel secondary metabolites, such as secalonic acid D [
peroxide [5], have been isolated from this fungus.
3], oxalicine [4], as well as ergosterol
Heterocyclic compounds are attractive to medicinal chemists because of their unique chemical
properties and wide-ranging biological activities. As one of basic five-membered heterocycles, the
thiazole substructure is widely found in many bioactive natural products including the cytotoxic
compound myxothiazol [6–9], the sodium channel activator hoiamides A [10,11], and the orally active
peptide sanguinamide A [12]. Moreover, the thiadiazole ring has also received increasing attention in
recent decades because of its broad-spectrum activities, together with many important therapeutic
applications [13,14]. For example, the polycarpathiamines A and B showed significant cytotoxic activity
against L5178Y murine lymphoma cells [15], while indole alkaloids containing 1,2,4-thiadiazole rings
exhibit anti-viral antiviral activity against the herpes simplex virus 1 (HSV-1) [16].
The literature reports different approaches to construct the thiazole and thiadiazole skeleton.
In general, the Hantzsch procedure employing thioamides [17
oxidation reaction between cysteine esters and N-protected iminoesters [19
,
18] or using the condensation and
20] has become the classic
,
Molecules 2016, 21, 232; doi:10.3390/molecules21030232
www.mdpi.com/journal/molecules

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method for the synthesis of thiazoles, and intramolecular or intermolecular cyclization strategies [21
are widely used for the preparation of thiadiazoles. In order to determine the final structures for two
novel compounds and recently isolated from the secondary metabolites of Penicillium oxalicum,
two pairs of thiazoles and
,22]
2
3
1
2
and thiadiazoles
3
and
4
(Figure 1) were designed and systemically
synthesized in this work.
OH
3''
4''
5'
4'
HO
6'
2''
4
4
3
5''
N
5'
HO
4'
2''
3'
5
3''
4''
1''
6''
1'
1'' 6''
5
6'
3
2'
2
N
7'
S
1
3'
OH
OH
1'
2'
5''
2
7'
S
1
1
2
2''
3''
4''
5'
3''
HO
HO
5'
4'
4'
7"
5
1''
4''
3
4
2''
7"
6'
3
6'
4
OH
N N
N
5''
3'
3'
6''
5''
1'
1''
6''
1'
2'
N
2
5
2'
2
7'
S
7'
S
1
1
3
4
Figure 1. Chemical structures for the two pairs of isomers.
2. Results and Discussion
Repeated separation of a 40 L culture extract from Penicillium oxalicum using silica gel column
chromatography yielded compounds and . Penicilliumthiamine A ( ) was obtained as a white
2
3
2
amorphous powder. Its molecular formula of C16H13NO2S (indicating eleven degrees of unsaturation)
was determined by HRESIMS (m/z 284.0746 [M + H]+, calcd. for C16H14NO2S, 284.0740). The 1H-NMR
spectrum of
2 exhibited two hydroxyl protons (δH 9.70 and 9.36, which disappeared on exchange
with D2O), two sets of AA’BB’ spin systems of the para-substituted benzene ring at δH 7.13 (2H, d,
J = 8.6 Hz, H-2’, 6’), δH 6.72 (2H, d, J = 8.6 Hz, H-3’, 5’) and δH 7.37 (2H, d, J = 8.7 Hz, H-2”, 6”), δH
6.77 (2H, d, J = 8.7 Hz, H-3”, 5”), one olefinic singlet at δH 7.84 (1H, s, H-4), one benzylic methylene
singlet at δH 4.15 (2H, s, H-7’). The 13C-NMR spectrum gave the corresponding resonances. One and
two-dimensional NMR techniques (DEPT, 1H-1H COSY, HSQC and HMBC) permitted assignment of
all the 1H- and 13C-NMR signals for
δH 9.36 to C-4’ (δC 156.29), H-2’/6’ (δH 7.13) to C-1’ (δC 128.36), C-3’/5’ (δC 115.38), C-4’ (δC 156.29), C-7’
δC 38.03), and H-3’/5’ (δH 6.72) to C-1’ (δC 128.36), C-2’/6’ (δC 129.99), C-4’ (δC 156.29), revealed the
2 (Table 1, Figure 2). In the HMBC spectrum of 2, correlations from
(
presence of a 4-hydroxybenzyl group; correlations from δH 9.70 to C-4” (δC 157.54), H-2”/6” (δH 7.37)
to C-1” (δC 121.92), C-3”/5” (δC 115.89), C-4” (δC 157.54), and H-3”/5” (δH 6.77) to C-1” (δC 121.92),
C-2”/6” (δC 127.63), C-4” (δC 157.54), revealed the presence of a 4-hydroxybenzene group. Apart
from this unit, one olefinic carbon (δC 136.43), two quaternary carbon δC (138.76 and 168.66), one
sulfur, and one nitrogen atoms remained to be assigned according to the molecular formula. It was
clear that eight of eleven degrees of unsaturation came from two phenyl groups, the remaining three
degrees of unsaturation had to originate from the -C3HNS-moiety, so it had to be a thiazole group.
The 4-hydroxybenzyl group attached at the C-2 position was confirmed by the HMBC correlations
from H-7’ (δH 4.15) to C-2 (δC 168.66). Furthermore, a 4-hydroxybenzene group could be located at C-4
or C-5, which was deduced by the HMBC correlations from H-4 (δH 7.84) to C-5 (δC 138.76), C-1’ (δC
128.36), together with the cross-peaks between H-4 (δH 7.84) with H-2”/6” (δH 7.37) in the NOESY
spectrum of
heterocyclic ring structure, thus two possible structures 1 or 2 remained as options.
Penicilliumthiamine B ( ) was obtained as a white and amorphous powder. Its molecular
formula of C16H14N2O2S (suggesting eleven degrees of unsaturation) was determined by HRESIMS
(m/z 299.0853 [M + H]+, calcd. for C16H15N2O2S 299.0849). The characteristic NMR data of
closely
2. However, it was difficult to assign the positions of the 4-hydroxy- benzene group in this
3
3

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resembled those of
1
and
2, except for two sets of 4-hydroxybenzene groups and a thiadiazole group
in 3 (Table 1, Figure 2). The locations of heteroatoms (S and N) could not be exactly assigned in these
isomers, thus two possible structures 3 or 4 were also possible as the exact structure.
1
Table 1. H-NMR (400 MHz) and 13C-NMR (100 MHz) data of 2 and 3 (DMSO-d6).
2
3
No
δ
(J in Hz)
δC
No
δ
(J in Hz)
H
δC
H
1
2
3
-
-
-
-
1
2
3
-
-
-
-
168.66
-
193.51
-
4
5
7’
-
7.84, s
-
4.15, s
136.43
138.76
38.03
4
5
-
-
175.75
-
7’
7”
1’
2’
3’
4’
5’
6’
1”
2”
3”
4”
5”
6”
4.28, s
4.09, s
-
35.79
37.63
126.89
130.21
115.54
156.64
115.54
130.21
127.42
129.86
115.14
155.95
115.14
129.86
1’
2’
3’
4’
5’
6’
1”
2”
3”
4”
5”
6”
-
128.36
129.99
115.38
156.29
115.38
129.99
121.92
127.63
115.89
157.54
115.89
127.63
7.13, d (8.6)
6.72, d (8.6)
-
6.72, d (8.6)
7.13, d (8.6)
-
7.37, d (8.7)
6.77, d (8.7)
-
6.77, d (8.7)
7.37, d (8.7)
7.16, d (8.4)
6.73, d (8.4)
-
6.73, d (8.4)
7.16, d (8.4)
-
7.07, d (8.5)
6.67, d (8.5)
-
6.67, d (8.5)
7.07, d (8.5)
Figure 2. Selected 1H-1H COSY, HMBC and NOESY correlations of 2 and 3.
As limited quantities of penicilliumthiamine A and B were accessible through isolation from
the organism, this piqued our interest in developing a total synthesis of the two pairs of thiazoles
and thiadiazoles
structures, but also to support further biological evaluations and to enable structure-activity studies.
Compound was prepared from the commercial availably 2-(4-methoxyphenyl)acetic acid (1a
1–4, not only to facilitate the unambiguous confirmation of their initially uncertain
1
)
and 1-(4-methoxyphenyl)ethanone (1e). Compound 1a was halogenated, aminated and thiolated
to generate the 2-(4-methoxyphenyl)ethanethioamide (1d) with Lawesson’s reagent (LR), which
was cyclized with 2-bromo-1-(4-methoxyphenyl)ethanone (1f) to give 2-(4-methoxybenzyl)-4-(4-
methoxyphenyl)thiazole (1g) according to the classical Hantzsch thiazole synthesis procedure. Finally,
the methyl ether was successfully removed by boron tribromide (BBr3) as deprotection reagent,
and 4-(2-(4-hydroxybenzyl)thiazol-4-yl)phenol (1) was successful synthesized in a total yield of 58%
(Scheme 1).

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Scheme 1. The synthetic route to 1.
All the important intermediates and products were confirmed by spectroscopic analysis
with satisfactory spectral data. The important intermediate 1g was also confirmed by the X-ray
crystallographic analysis (CCDC number: 1434555). The ORTEP drawing of 1g with common atom
numbering scheme was shown in Figure S21, Supplementary Materials. Although the exact structure
of
and 13C-NMR signals were not consistent with those of penicilliumthiamines A.
Compound was also prepared from 4-methoxyphenylacetic acid (1a) and 2-bromo-1-
1 was determined by spectroscopic analysis, it was regrettable that both its HPLC retention time
2
phenylethanone (1e). Firstly, compound 1e was aminated through a Delepine reaction to generate
the 2-amino-1-(4-methoxyphenyl)ethanone (2a), which was acylated by 2-(4-methoxy-phenyl)acetyl
chloride (1b) to give 2-(4-methoxyphenyl)-N-(2-(4-methoxyphenyl)-2-oxoethyl)-acetamide (2b). Then
the amide 2b smoothly underwent thiolation and subsequent cyclization by the action of Lawesson’s
reagent in refluxing toluene to lead to thiazole 2c by referring to recent literature [23
complete removal of the methyl protection was affected with BBr3 at
78 ˝C to produce the target
compound in total 34% yield (Scheme 2). The structure of was established by the spectroscopic
,24]. Finally,
´
2
2
analysis and X-ray crystallographic analysis (CCDC number: 1434559) conducted with colorless
crystals grown from ethyl acetate (Figure S22, Supplementary Materials). It was exciting that the HPLC
retention time and all the NMR signals of
2 completely matched with those of penicilliumthiamine A,
therefore the structure of penicilliumthiamine A was finally confirmed.
Scheme 2. The synthetic route to 2.

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Compound
3
was synthesized by the oxidative dimerization of thioamides according to the Patil
method (Scheme 3) [25]. The thioamide 1d underwent oxidative dimerization by hypervalent iodine
(V)-containing reagents, o-iodoxybenzoic acid (IBX) in the presence of tetraethylammonium bromide
(TEAB) to generate the thiadiazole skeleton 3a. The demethylation reaction was conducted by treating
3a with BBr3 to give one target compound 3 in a total yield of 65% (Scheme 3). Compound 4 isomer
was prepared by amination of 1a with hydrazine, and the product bisacylhydrazine 4a was cyclized to
form the 1,3,4-thiadiazole core 4b by Gierczyk’s method [26].
Scheme 3. The synthetic route to 3.
After deprotection of the methyl group, the compound
total 49% yield (Scheme 4). After characterizing the structures, compound
4
was obtained with four steps in
was found to be
3
identical to penicilliumthiamine B through comparison of the corresponding HPLC retention time and
NMR signals.
Scheme 4. The synthetic route to 4.
All the compounds were firstly used to test whether they could inhibit the phosphorylation of
AKT/PKB (Ser 473) under the stimulus of the fetal calf serum. The results showed that compounds 1
and 3a could inhibit the phosphorylation of AKT/PKB (Ser 473) in the MDA-MB-231 cell while
compound
3 inhibited the phosphorylation of AKT/PKB (Ser 473) in the HGC-27 cells (Figure 3).

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MTT experimental results showed that compound
1
exhibited the moderate growth inhibitory effect
against MDA-MB-231 cell, which was in a dose- and time-dependent manner. The IC50 values of
12 h, 24 h, and 48 h for compound in MDA-MB-231 cell were 37.16 M, 22.36 M, and 15.29 M,
respectively. Compounds 3a and showed certain inhibitory effect against MDA-MB-231 cell and
HGC-27 cells, respectively. The IC50 values of compound 3a were 199.30 M and 51.80 M against
MDA-MB-231 cells for 24 h and 48 h. The IC50 values of 24 h and 48 h were 183.82 M and 172.30
for compound against HGC-27 cells, respectively (Figure 4). These results demonstrated that these
1
µ
µ
µ
3
µ
µ
µ
µM
3
cytotoxic compounds were cell-selective, and might target the phosphorylation of AKT/PKB (Ser 473),
a key signaling component of one of the most frequently activated pathways in cancer and a major
target of cancer drug development [27,28].
Figure 3. The phosphorylation of AKT/PKB (Ser 473) could be inhibited by compound
Representative western blot bands of P-AKT in MDA-MB-231 cells, ( ) Representative western blot
bands of P-AKT in HGC-27 cells; 1: Control, 2: LY294002 stimulated by 20 µmol/L 20 min before FBS
1, 3a and 3 (A)
B
20 min, 3: FBS incubated at FBS 20 min, 4–6: Compound 3a, 1 and 3 stimulated by 10 µmol/L 2 min
before FBS 20 min, respectively).
Figure 4. MTT assays showed the cytotoxicity on cancer cell lines.
3. Experimental Section
3.1. General Procedures
UV spectra were run as methanol solutions on a Shimadzu UV-2550 spectrophotometer (Shimadzu,
Kyoto, Japan). IR spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Fisher
Scientific, Waltham, MA, USA). NMR spectra were recorded on Bruker AVANCE III 400 MHz Plus
NMR spectrometer (Bruker, Bremen, Germany) using TMS as internal standard, Chemical shifts
are reported as values and the coupling constants (J) are in Hz. HRESIMS spectra were got on a
microTOF-QII mass spectrometer (Bruker, Bremen, Germany). Dionex UltiMate 3000 Rapid Separation
LC Systems (Thermo Fisher Scientific, Waltham, MA, USA). A Cosmosil MS-II C18 preparative HPLC
column (250
ˆ
10 mm, 5 µm) was used. Column chromatography was carried out with silica gel
(Qingdao Ocean Chemical Croup Co., Qingdao, China; 200–300 mesh), RP-C18 silica gel (YMC, Kyoto,
Japan; 100–200 mesh), and Sephadex LH-20 (Amersham Biosciences, GE Healthcare Life Science,
Santa Clara, CA, USA). The single-crystal X-ray diffraction analysis was performed on a Rigaku
Mecury CCD diffractometer (Rigaku, Tokyo, Japan).

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3.2. Fungal Material
The fungus Penicillium oxalicum was isolated from Acrida cinerea gut collected in July 2012 from
the Chinese Big-Nine-Lake National Wetland Park in Hubei Province. The procedures of isolation and
identification of the fungal strain used in this experiment were described in an earlier study [29]. The
fungus was identified using a molecular biological protocol by DNA amplification and sequencing of
the ITS region, as described in an earlier study [30]. The BLAST results indicated the sequence was
the most similar (99%) to the sequence of Penicillium oxalicum. The strain was kept in the Hubei Key
Laboratory of Natural Products Research and Development, China Three Gorges University.
3.3. Fermentation, Extraction and Isolation
The fermentation was carried out dynamically in a SD medium (consisting of 40 g glucose, 10 g
peptone in 1 L of distilled water) in 500 mL Erlenmeyer flasks for 20 days at room temperature.
The fermented liquids substrate (200 flasks) was extracted repeatedly with ethyl acetate, and the
organic layers were combined and evaporated to dryness under vacuum to afford an extract (13.0 g),
which was fractionated by silica gel chromatography using chloroform–methanol (100:0–50:50, v/v)
gradient elution to produce five portions (Fr. I–Fr. V). Fractions III were combined and subjected to
silica gel column chromatography, Sephadex LH-20 gel, and preparative reverse-phase C18 HPLC
(250
ˆ
10 mm i.d., Cosmosil MS-II) using an acetonitrile–water system (27:73, v/v) to yield compound 1
or 2 (4.1 mg) and 3 or 4 (5.5 mg).
3.4. Synthesis
2-(4-Methoxyphenyl)ethanethioamide (1d). In a round bottomed flask equipped with a magnetic stirring
bar and argon gas inlet, 2-(4-methoxyphenyl)acetic acid (1a, 5.0 mmol) was dissolved in thionyl
chloride (10.0 mL). The mixture was allowed to heat to reflux for 3 h, which was concentrated to
remove the additional thionyl chloride under reduced pressure to give the 2-(4-methoxyphenyl)acetyl
chloride (1b). Then ammonia solution (5.0 mmol ammonia gas in 5.0 mL water) was added into the
acyl chloride in ethyl acetate (5.0 mL), and the 2-(4-methoxyphenyl)acetamide (1c) was crystallize
form the mixture in 95% yield as˝white solids; Its spectral data were identical with those reported
˝
with the melting point of 169–170 C (lit. [31] 163–165 C). The 2-(4-methoxyphenyl)ethanethioamide
(
1d, 0.45 g) was synthesized and isolated by using Lawesson's reagent (5.0 mmol) in toluene (20.0 mL)
˝
under 50 C for 2 h, and the residue was purified by flash column chromatography on silica gel
(EtOAc/hexane, 1:1) to afford yellow solids with the yield of 85%. H-NMR (400 MHz, DMSO-d6,
1
δ ppm): 7.66 (s, 1H), 7.26–7.17 (m, 2H), 6.92–6.71 (m, 2H), 6.70 (s, 1H), 4.05 (s, 2H), 3.81 (s, 3H).
2-Bromo-1-(4-methoxyphenyl)ethanone (1f). 1-(4-Methoxyphenyl)ethanone (1e
,
5.0 mmol)˝ and
N-bromosuccinimide (NBS, 5.0 mmol) were stirred in carbon tetrachloride (CCl4, 20.0 mL) at 50 C for
2 h. After 1e was completely consumed, the succinimide was removed by filtration. The organic phase
was washed with water (10 mL), dried over Na2SO4 and solvent evaporated under reduced pressure
˝
to give the 2-bromo-1-(4-methoxyphenyl)ethanone (1f). White solid, yield 90%, mp 70–72 C (lit. [32
]
˝
69–71 C). 1H-NMR (CDCl3,
δ
ppm): 7.97 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 4.40 (s, 2H), 3.89
(s, 3H). 13C-NMR (CDCl3, δ ppm): 189.94, 164.11, 131.34, 126.87, 114.04, 55.55, 30.69.
2-(4-Methoxybenzyl)-4-(4-methoxyphenyl)thiazole (1g). Compound 1f (2.0 mmol) was heated with 1d
1
˝
(2.0 mmol) in N,N -dimethylformide (DMF, 20.0 mL) at 100 C for 5 h under the protection of nitrogen
atmosphere. The progress of the reaction was monitored by thin-layer chromatography (TLC). After
disappearance of starting materials, the solution was cooled to room temperature, and 10% NaCl
solution (80.0 mL) was added, and then the product was extracted with dichloromethane (40.0 mL ˆ 3).
The organic phase was separated, washed with saturated NaCl solution (50 mL
ˆ
2) and dried over
anhydrous sodium sulphate. Removal of the solvent on a rotary evaporator under high vacuum
gave a viscous brown oil, which was purified by flash column chromatography on silica gel (eluent:
n-hexane/EtOAc = 5:1, v/v) to yield compound 1g as a white solid in 80% yield, mp 118–120 ˝C. IR

Molecules 2016, 21, 232
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(KBr, cm´1): = 3107, 2956, 2937, 2838, 1687, 1608, 1582, 1530, 1511, 1492, 1466, 1453, 1440, 1419, 1323,
1
1299, 1275, 1252, 1207, 1171, 1109, 1055, 1028, 989, 850, 835. H-NMR (DMSO-d6,
δ ppm): 7.82 (dd,
J = 6.8, 2Hz, 2H), 7.29–7.26 (t, J =8.8, 4Hz, 2H), 7.19 (s, 1H), 6.94 (dd, J = 6.8, 2.0 Hz, 2H), 6.88 (dd,
J = 6.4, 2.0 Hz, 2H), 4.31 (s, 2H), 3.85 (s, 3H), 3.81 (s, 3H). ESIMS m/z: 312 [M + H]+.
2-(4-Methoxyphenyl)-2-oxoethanaminium bromide (2a). 2-Bromo-1-(4-methoxyphenyl)ethanone (1e
,
10.0 mmol) was added to a solution of hexamethylenetetramine (1.40 g, 10.0 mmol) in chloroform
(40.0 mL), and the resulting mixture was heated at 50 ˝C for 3 h. The mixture was cooled to room
temperature and the white precipitate was collected by filtration, washed with CHCl3, and dried
˝
˝
1
in vacuo to afford 2a as white solids, yield 90%, mp 191–193 C (lit. [33] 195–197 C). H-NMR
(DMSO-d6,
δ ppm): 8.46 (br s, 3H), 7.99 (dd, J = 9.2, 2.0 Hz, 2H), 7.09 (dd, J = 9.2, 2.0 Hz, 2H),
4.49 (s, 2H), 3.86 (s, 3H). 13C-NMR (DMSO-d6, δ ppm): 191.1, 146.1, 130.6, 126.6, 114.2, 55.7, 44.3.
2-(4-Methoxyphenyl)-N-(2-(4-methoxyphenyl)-2-oxoethyl)acetamide (2b). 2-(4-Methoxyphenyl)acetyl
chloride (1b) was dissolved in anhydrous ethyl acetate (20.0 mL) under 0 ˝C, then 2a (5.0 mmol) was
added and the amide 2b was quickly precipitated in 5 min. The yellow solids were collected by
filtration, washed with ethyl acetate (2
product in 50% yield, as a white solid, mp 83–86 ˝C. 1H-NMR (DMSO-d6,
2H), 7.24 (d, J = 8.4 Hz, 2H), 6.95–6.90 (m, 4H), 6.58 (s, 1H), 4.67 (d, J = 4.0 Hz, 2H), 3.87 (s, 3H), 3.81 (s,
2H), 3.61 (s, 2H). 13C-NMR (DMSO-d6,
ppm): 192.37, 171.54, 164.23, 158.86, 130.49, 130.17, 127.27,
ˆ
8.0 mL) and dried under vacuum to afford analytically pure
δ
ppm): 7.91 (d, J = 8.8 Hz,
δ
126.51, 114.40, 114.07, 55.52, 55.23, 46.03, 42.71.
2-(4-Methoxybenzyl)-5-(4-methoxyphenyl)thiazole (2c). To a solution of the above amide 2b (2.0 mmol)
in toluene (20.0 mL) was added Lawesson’s reagent (10.0 mmol), and the mixture was heated under
reflux for 2 h. The solvent was evaporated under reduced pressure, and the residue was purified by
silica gel column chromatography (petroleum ether: ethyl acetate = 1:1, v/v) to give the thiazole 2c as
a white solid in 85% yield, mp 111–112 ˝C. IR (KBr, cm´1): 2963, 2836, 1609, 1536, 1514, 1501, 1466,
1
1424, 1307, 1302, 1283, 1249, 1212 1183, 1174, 1108, 1071, 1030, 843, 825. H-NMR (CDCl3,
δ ppm): 7.72
(s, 1H), 7.41–7.39 (m, 2H), 7.28-7.26 (m, 2H), 6.90–6.87 (m, 4H), 4.25 (s, 2H), 3.82 (s, 3H), 3.80 (s, 3H).
13C-NMR (CDCl3,
δ ppm): 169.58, 159.51, 158.70, 139.18, 136.76, 130.07, 130.01, 127.83, 124.15, 114.35,
114.17, 55.32, 55.24, 51.59, 39.03, 30.90. ESIMS m/z = 312 [M + H]+.
3,5-Bis(4-methoxybenzyl)-1,2,4-thiadiazole (3a). To a stirred suspension of IBX (3.5 mmol) and TEAB
(3.5 mmol) in acetonitrile (20.0 mL) was added 2-(4-methoxyphenyl)ethanethioamide (1d) in 20 min
˝
at 10 C. Consumption of starting material was observed by TLC. After completion of reaction,
acetonitrile was removed under reduced pressure and the resultant residue was washed with ethyl
acetate (25.0 mL) followed by 10% sodium bisulfite solution (30.0 mL), saturated sodium carbonate
(30.0 mL), and brine (30.0 mL). The organic layer was dried over anhydrous sodium sulfate and
concentrated under reduced pressure to give crude product. Pure product was isolated after column
chromatography on silica gel mesh (eluent: petroleum ether/ethyl acetate = 2/1, v/v) to yield the
˝
compound 3a as white solids in 90% yield, mp 69–71 C. IR (KBr, cm´1): 2933, 2840, 1609, 1582, 1512,
1
1491, 1456, 1444, 1432, 1301, 1283, 1247, 1221, 1178, 1146, 1116, 1087, 1026, 837, 814. H-NMR (CDCl3,
ppm): 7.27–7.21 (m, 2H), 6.89 (m, 2H), 4.27 (s, 2H), 4.23 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H). 13C-NMR
δ
(CDCl3,
δ ppm): 193.25, 175.99, 159.13, 158.40, 130.20, 130.06, 129.21, 128.19, 114.41, 113.99, 55.26, 55.21,
38.44, 37.01. ESIMS m/z = 327 [M + H]+.
2-(4-Methoxyphenyl)-N1-(2-(4-methoxyphenyl)acetyl)acetohydrazide
chloride (1b, 5.0 mmol) was dissolved in anhydrous ethyl acetate (20.0 mL) under 0 C, then 80%
hydrazine was added. Then the bisacetohydrazide 4a was immediately precipitated within 5 min. The
(4a). 2-(4-Methoxyphenyl)acetyl
˝
white solids were collected by filtration, washed with ethyl acetate˝(2
ˆ
10.0 mL) and dried under
ppm):
vacuum to afford analytically pure product in 90% yield, mp 108–109 C. 1H-NMR (DMSO-d6,
δ
7.16 (br s, 2H), 7.19 (d, J = 8.4 Hz, 4H), 6.85 (d, J = 8.4 Hz, 4H), 3.71 (s, 6H), 3.37 (s, 4H). 13C-NMR
(DMSO-d6, δ ppm): 169.22, 157.96, 130.00, 127.63, 113.62, 55.02, 39.21.

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2,5-Bis(4-methoxybenzyl)-1,3,4-thiadiazole (4b). To a solution of the above bisacetohydrazide 4a
(2.0 mmol) in toluene (20.0 mL) was added Lawesson’s reagent (10.0 mmol), and the mixture was
heated at reflux for 4 h. The solvent was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1, v/v) to give the
thiadiazole 4b as a white solid in 60% yield, mp 108–110 ˝C. IR (KBr, cm´1): 2960, 2916, 2837, 2360,
1
2341, 1611, 1584, 1513, 1456, 1442, 1426, 1301, 1251, 1214, 1175, 1140, 1118, 1032, 837, 825. H-NMR
(CDCl3,
δ
ppm): 7.16 (d, J = 8.4 Hz, 4H), 6.83 (d, J = 8.4 Hz, 4H), 4.27 (s, 4H), 3.77 (s, 6H). 13C-NMR
(CDCl3, δ ppm): 171.39, 158.86, 129.86, 129.20, 114.29, 55.23, 35.69. ESIMS m/z = 327 [M + H]+.
3.5. General Procedure for the Demethylation Reaction
A cooled solution of the methyl ether (1f, 2c, 3a or 4b, 0.5 mmol) in dichloromethane (CH2Cl2,
5.0 mL) was treated with BBr3 (1.0 mmol BBr3 in 2.0 mL CH2Cl2), and then the mixture was allowed to
stand at –78 ˝C for 3 h until the methyl ether was consumed completely (monitored by TLC). Finally
the solution was diluted with 1% NaHCO3 (30.0 mL), and extracted with CH2Cl2 (3
ˆ
20.0 mL). The
organic layers were combined, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to
give a yellow residue. The crude product was purified by flash chromatography on silica gel to yield
the target compound 1–4 (eluent: petroleum ether/ethyl acetate = 2/1, v/v).
˝
4-(2-(4-Hydroxybenzyl)thiazol-4-yl)phenol (1). White and amorphous powder, yield 86%, mp 200–201 C.
IR (KBr, cm´1): 3389, 3103, 3009, 2951, 2792, 1610, 1594, 1515, 1489, 1438, 1375, 1273, 1244, 1213, 1182,
1
1170, 1133, 839. H-NMR (DMSO-d6,
δ
ppm): 9.60 (br s, 1H), 9.37 (br s, 1H), 7.75-7.73 (m, 2H), 7.65 (s,
1H), 7.15 (d, J = 8.4 Hz, 2H), 6.80-6.79 (m, 2H), 6.74–6.71 (m, 2H), 4.21 (s, 2H). 13C-NMR (DMSO-d6,
δ
ppm): 170.7, 157.3, 156.3, 154.3, 130.0, 128.3, 127.3, 125.6, 115.4, 115.3, 111.0, 49.1, 39.9, 39.7, 39.5, 39.3,
39.1, 38.1, 38.0. HRESIMS, calcd. for C16H14NO2S [M + H]+ 284.0745, found 284.0749.
Penicilliumthiamine A (2
). White and amorphous powder, yield 90%, mp 189–190 ˝C. IR (KBr, cm´1):
3238, 3018, 2961, 2925, 2668, 1609, 1596, 1586, 1515, 1504, 1447, 1422, 1375, 1278, 1244, 1176, 1103,
1
1082, 1030, 852, 832. H- and 13C-NMR data, see Table 1. HRESIMS, calcd. for C16H14NO2S [M + H]+
284.0745, found 284.0746.
Penicilliumthiamine B (3
). White and amorphous powder, yield 90%, mp 180–181 ˝C. IR (KBr, cm´1):
3520, 3158, 1614, 1593, 1516, 1496, 1449, 1434, 1359, 1336, 1311, 1265, 1232, 1209, 1173, 1102, 842,
1
833. H- and 13C-NMR data, see Table 1. HRESIMS, calcd. for C16H15N2O2S [M + H]+ 2899.0854,
found 299.0853.
1
4,4 -((1,3,4-Thiadiazole-2,5-diyl)bis(methylene))diphenol (4). White and amorphous powder, yield 90%, mp
˝
209–210 C. IR (KBr, cm´1): 3520, 3158, 1614, 1593, 1516, 1496, 1449, 1434, 1311, 1265, 1232, 1209, 1173,
1
1102, 816 cm-1. H-NMR (DMSO-d6,
δ ppm): 9.37 (br s, 2H), 7.07 (d, J = 8.4 Hz, 4H), 6.71–6.67 (m, 4H),
4.20 (s, 4H). 13C-NMR (DMSO-d6,
δ ppm): 171.07, 156.42, 129.83, 127.84, 115.51, 34.45. HRESIMS, calcd.
for C16H15N2O2S [M + H]+ 2899.0854, found 299.0859.
3.6. Biological Evaluation
Each compound was dissolved in distilled water. The filtered stock compound solution was
˝
separated into individual aliquots which were kept at
´
20 C until further use. Human breast cancer
MDA-MB-231 cells and human gastric cancer HGC-27 cells were from the Institute of Molecular
Biology, China Three Gorges University. Cancer cells were maintained in RPMI 1640 culture medium
supplemented with 10% fetal bovine serum and antibiotics in a 5% carbon dioxide incubator at 37 C.
˝
All the cells were firstly starved for 12 h and stimulated by FBS 20 min before adding the drugs
for 2 min. A western blot assay was used to detect the phosphorylation of PKB/AKT (Ser473) kinase.
Then analyze the cytotoxicity of positive compounds on cancer cell lines, cells were treated with
different concentrations at different time points respectively (MTT assay).

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4. Conclusions
In summary, in this work four total synthetic routes were designed to prepare thiazoles and
thiadiazoles using commercial 1-(4-methoxyphenyl)-ethanone and 4-methoxyphenylacetic acid as the
starting materials, and two of them were confirmed as penicilliumthiamines A and B (compounds
2
and ) from the extract of the Penicillium oxalicum. Compounds 3a and showed different cytotoxicity
3
1,
3
activities with cell selectivity, which might all target AKT/PKB. This is the first report on the anticancer
activities of these new molecules, which could be the starting point for further development of drug
candidates with potential in the treatment of cancer.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/3/232/s1.
Acknowledgments: We are thankful for the finance supported by the National Natural Science Foundation
of China (No. 21272136), Natural Science Foundation of Hubei Province (No. 2014CFB692) and Youth Talent
Development Foundation of China Three Gorges University.
Author Contributions: Fan Cheng and Kun Zou conceived and designed the experiments; Zheng Yang, Bang Xu
and Wengfeng Huang performed the experiments; Nianyu Huang and Tianpeng Xie analyzed the data; Fan Cheng
and Nianyu Huang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).