Cytotoxic 1,3-Thiazole and 1,2,4-Thiadiazole Alkaloids from Penicillium oxalicum: Structural Elucidation and Total Synthesis

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.


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 [3], oxalicine [4], as well as ergosterol peroxide [5], have been isolated from this fungus.
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][7][8][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 [17,18] or using the condensation and oxidation reaction between cysteine esters and N-protected iminoesters [19,20] has become the classic method for the synthesis of thiazoles, and intramolecular or intermolecular cyclization strategies [21,22] are widely used for the preparation of thiadiazoles.In order to determine the final structures for two novel compounds 2 and 3 recently isolated from the secondary metabolites of Penicillium oxalicum, two pairs of thiazoles 1 and 2 and thiadiazoles 3 and 4 (Figure 1) were designed and systemically synthesized in this work.
Molecules 2016, 21, 232 2 of 11 classic method for the synthesis of thiazoles, and intramolecular or intermolecular cyclization strategies [21,22] are widely used for the preparation of thiadiazoles.In order to determine the final structures for two novel compounds 2 and 3 recently isolated from the secondary metabolites of Penicillium oxalicum, two pairs of thiazoles 1 and 2 and thiadiazoles 3 and 4 (Figure 1) were designed and systemically synthesized in this work.
Penicilliumthiamine B (3) 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 3 closely resembled those of 1 and 2, except for two sets of 4-hydroxybenzene groups and a thiadiazole
Penicilliumthiamine B (3) was obtained as a white and amorphous powder.Its molecular formula of C 16 H 14 N 2 O 2 S (suggesting eleven degrees of unsaturation) was determined by HRESIMS (m/z 299.0853 [M + H] + , calcd.for C 16 H 15 N 2 O 2 S 299.0849).The characteristic NMR data of 3 closely 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.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.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 1-4, not only to facilitate the unambiguous confirmation of their initially uncertain structures, but also to support further biological evaluations and to enable structure-activity studies.
Compound  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 1-4, not only to facilitate the unambiguous confirmation of their initially uncertain structures, but also to support further biological evaluations and to enable structure-activity studies.
Compound 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 1 was determined by spectroscopic analysis, it was regrettable that both its HPLC retention time and 13 C-NMR signals were not consistent with those of penicilliumthiamines A.
Compound 2 was also prepared from 4-methoxyphenylacetic acid (1a) and 2-bromo-1phenylethanone (1e).[23,24].Finally, complete removal of the methyl protection was affected with BBr3 at −78 °C to produce the target compound 2 in total 34% yield (Scheme 2).The structure of 2 was established by the spectroscopic 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.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 1 was determined by spectroscopic analysis, it was regrettable that both its HPLC retention time and 13 C-NMR signals were not consistent with those of penicilliumthiamines A.
Compound 2 was also prepared from 4-methoxyphenylacetic acid (1a) and 2-bromo-1phenylethanone (1e).[23,24].Finally, complete removal of the methyl protection was affected with BBr 3 at ´78 ˝C to produce the target compound 2 in total 34% yield (Scheme 2).The structure of 2 was established by the spectroscopic 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.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 1 was determined by spectroscopic analysis, it was regrettable that both its HPLC retention time and 13 C-NMR signals were not consistent with those of penicilliumthiamines A.
Compound 2 was also prepared from 4-methoxyphenylacetic acid (1a) and 2-bromo-1phenylethanone (1e).[23,24].Finally, complete removal of the methyl protection was affected with BBr3 at −78 °C to produce the target compound 2 in total 34% yield (Scheme 2).The structure of 2 was established by the spectroscopic 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.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 BBr 3 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 4 was obtained with four steps in total 49% yield (Scheme 4).After characterizing the structures, compound 3 was found to be identical to penicilliumthiamine B through comparison of the corresponding HPLC retention time and NMR signals.After deprotection of the methyl group, the compound 4 was obtained with four steps in total 49% yield (Scheme 4).After characterizing the structures, compound 3 was found to be identical to penicilliumthiamine B through comparison of the corresponding HPLC retention time and NMR signals.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 4 was obtained with four steps in total 49% yield (Scheme 4).After characterizing the structures, compound 3 was found to be identical to penicilliumthiamine B through comparison of the corresponding HPLC retention time and NMR signals.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).
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 IC 50 values of 12 h, 24 h, and 48 h for compound 1 in MDA-MB-231 cell were 37.16 µM, 22.36 µM, and 15.29 µM, respectively.Compounds 3a and 3 showed certain inhibitory effect against MDA-MB-231 cell and HGC-27 cells, respectively.The IC 50 values of compound 3a were 199.30µM and 51.80 µM against MDA-MB-231 cells for 24 h and 48 h.The IC 50 values of 24 h and 48 h were 183.82 µM and 172.30µM for compound 3 against HGC-27 cells, respectively (Figure 4).These results demonstrated that these 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].for compound 3 against HGC-27 cells, respectively (Figure 4).These results demonstrated that these 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].

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  for compound 3 against HGC-27 cells, respectively (Figure 4).These results demonstrated that these 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].

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

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.

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 C 18 HPLC (250 ˆ10 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).

General Procedure for the Demethylation Reaction
A cooled solution of the methyl ether (1f, 2c, 3a or 4b, 0.5 mmol) in dichloromethane (CH 2 Cl 2 , 5.0 mL) was treated with BBr 3 (1.0 mmol BBr 3 in 2.0 mL CH 2 Cl 2 ), 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% NaHCO 3 (30.0mL), and extracted with CH 2 Cl 2 (3 ˆ20.0 mL).The organic layers were combined, dried over anhydrous Na 2 SO 4 , 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).

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).

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 3) from the extract of the Penicillium oxalicum.Compounds 1, 3a and 3 showed different cytotoxicity 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.

1 Figure 1 .
Figure 1.Chemical structures for the two pairs of isomers.

Figure 1 .
Figure 1.Chemical structures for the two pairs of isomers.
Scheme 1.The synthetic route to 1.

Figure 4 .
Figure 4. MTT assays showed the cytotoxicity on cancer cell lines.

Figure 4 .
Figure 4. MTT assays showed the cytotoxicity on cancer cell lines.

Figure 4 .
Figure 4. MTT assays showed the cytotoxicity on cancer cell lines.