Penicacids A-C, three new mycophenolic acid derivatives and immunosuppressive activities from the marine-derived fungus Penicillium sp. SOF07

Article abstract of DOI:10.1016/j.bmcl.2012.02.106

Three new mycophenolic acid derivatives, penicacids A-C (1-3), together with two known analogues, mycophenolic acid (MPA, 4) and 4′-hydroxy-MPA (5), were isolated from a fungus Penicillium sp. SOF07 derived from a South China Sea marine sediment. The structures of compounds 1-3 were elucidated on the basis of MS and NMR (¹H, ¹³C, HSQC and HMBC) data analyses and comparisons with the known compounds. Structure-activity relationship studies of compounds 1-5 focused on inosine-monophosphate dehydrogenase inhibition revealed that hydroxylation at C-4′, methylation at C-7-OH, dual hydroxylation at C-2′/C-3′ double bond of MPA diminished bioactivity whereas glucosyl hydroxylation at C-4′ correlated to bioactivity comparable to that observed for MPA.

Full text of DOI:10.1016/j.bmcl.2012.02.106

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3332–3335
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Penicacids A–C, three new mycophenolic acid derivatives and immunosup-
pressive activities from the marine-derived fungus Penicillium sp. SOF07
Ziming Chen a,b,c, Zhihui Zheng d, Hongbo Huang a,b,c, Yongxiang Song a,b,c, Xuelian Zhang d,
Junying Ma a,b,c, Bo Wang a,b,c, Changsheng Zhang a,b,c, Jianhua Ju a,b,c,
⇑
a Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
b Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
c RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
d New Drug Research Center, North China Pharmaceutial Group Corporation, National Microbial Medicine Engineering and Research Center, Shijiazhuang 050015, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new mycophenolic acid derivatives, penicacids A–C (1–3), together with two known analogues,
mycophenolic acid (MPA, 4) and 40-hydroxy-MPA (5), were isolated from a fungus Penicillium sp.
SOF07 derived from a South China Sea marine sediment. The structures of compounds 1–3 were eluci-
dated on the basis of MS and NMR (1H, 13C, HSQC and HMBC) data analyses and comparisons with the
known compounds. Structure–activity relationship studies of compounds 1–5 focused on inosine-mono-
phosphate dehydrogenase inhibition revealed that hydroxylation at C-40, methylation at C-7-OH, dual
hydroxylation at C-20/C-30 double bond of MPA diminished bioactivity whereas glucosyl hydroxylation
at C-40 correlated to bioactivity comparable to that observed for MPA.
Received 27 November 2011
Revised 13 February 2012
Accepted 28 February 2012
Available online 11 March 2012
Keywords:
Marine fungi
Penicillium sp.
Penicacid A–C
Ó 2012 Elsevier Ltd. All rights reserved.
Inosine-monophosphate dehydrogenase
Structure–activity relationship
Marine-derived fungi are prolific producers of new, structurally
intriguing and bioactive compounds as reflected by the fact that, at
present, more than 1000 new compounds have been isolated and
identified from marine fungi.1 In our efforts to discover new bioac-
tive natural products from marine fungi originating from the South
China Sea, we have isolated new cytotoxic cytochalasins from the
marine-derived fungus Xylaria sp. SCSIO 156.2 Another strain, Pen-
icillium sp. SOF07, attracted our attention because a crude extract
of this fungal culture was lethal to brine shrimp (Artemia salina)
and displayed cytotoxicity against human cancer cell lines A549,
HCT15 and HEP3B. HPLC-(DAD)-UV analysis of this extract un-
veiled an array of structurally related secondary metabolites (1–
5, Fig. 1) with similar characteristic UV absorptions; subsequent
structure elucidation efforts revealed a conserved mycophenolic
acid (MPA, 4)-based structural scaffold.3
ing and glycosylation.9 Consequently, IMPDH is an important drug
target for immunosuppressive, antiviral and cancer chemother-
apy.10 Mycophenolate mofetil (MMF) is the 2-morpholinoethyl
ester prodrug of MPA and has been widely used as an immunosup-
pressant in kidney, heart, and liver transplantation procedures.
The fungal metabolite 4 displays a wide range of biological
activities including antiviral, antitumor, and RNA capping inhibi-
tory activity.4–7 MPA is also a potent inhibitor of inosine 50-mono-
phosphate dehydrogenase (IMPDH), an essential rate-limiting
enzyme in the purine metabolic pathway.8 IMPDH controls the size
of the guanine nucleotide pool which, in turn, controls many
physiological processes including replication, transcription, signal-
⇑
Corresponding author. Tel.: +86 20 89023028.
E-mail address: jju@scsio.ac.cn (J. Ju).
Figure 1. Structures of compounds 1–5.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.02.106

Z. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3332–3335
3333
Strain SOF07 was isolated from a sediment collected in the
South China Sea at a depth of 675 m. The strain was identified as
Penicillium sp. SOF07 on the basis of morphology and analysis of
the ITS region sequence. The voucher specimen was deposited in
RNAM Center for Marine Microbiology, South China Sea Institute
of Oceanology, Chinese Academy of Sciences (Guangzhou, China).
Strain SOF07 was fermented in rice solid medium supple-
mented with 3% sea salt and the harvested whole culture broth
extracted with methylethylketone to afford a residue after solvent
evaporation. The residue was subjected to chromatography over
normal phase silica gel and reverse phase C18 silica gel chromatog-
raphy using MPLC and semi-preparative HPLC, respectively, to
yield compounds 1–5 (Fig. 1).11 Compounds 4 and 5 were identi-
fied as MPA and 40-hydroxy-MPA, respectively, by comparing their
MS, 1H and 13C NMR spectroscopic data with those previously re-
ported.12–14 Compounds 1–3 were elucidated as new compounds,
designated penicacids A–C, on the basis of MS, 1D and 2D NMR
data analyses and comparisons with known compounds.
Penicacid A (1) was isolated as an amorphous solid. Its molecu-
lar formula was established as C18H22O7 by HRESIMS which dis-
played a quasimolecular peak at m/z 349.1521 [MꢀH]ꢀ.15a The 1H
NMR spectrum of 1 was characterized by signals corresponding
to a methyl group at dH 1.81 (3H, s, 30-Me), an aromatic methyl
group at dH 2.14 (3H, s, C-8), two methoxy moieties at dH 3.75
(3H, s, 9-OMe) and dH 3.66 (3H, s, 7-OMe), and an oxygen-bearing
methylene singlet at dH 5.19 ppm. Detailed comparisons of the 1H
and 13C NMR data for 1 with those of the known compound 5
showed a high degree of similarity although the hydroxy group
of 5 was replaced in 1 with a methoxy group (Table 1). HMBC cor-
relations involving C-7 and this OMe moiety clearly established
OMe connectivity to C-7 of the MPA-based scaffold of 1. Other
HMBC correlations in 1 confirmed the highly conserved nature of
1 with 5; numerous structural features of 1 and 5 were found to
be identical. Thus, 1 was identified as 7-O-methyl-40-hydroxy-
MPA, a new compound.
Penicacid B (2) was obtained as an amorphous solid and its
molecular formula determined to be C23H30O12 on the basis of
the pseudomolecular ion in the HRESIMS at m/z 497.1624
[MꢀH]ꢀ.15b The molecular formula of 2 suggested the presence of
nine degrees of unsaturation. The UV spectrum of 2 displayed char-
acteristic absorption maxima for the MPA chromophore at 216,
250 and 304 nm. The 1H, 13C and DEPT NMR data for 2 strongly
resembled those of 5 with the exception of an additional set of sig-
nals consistent with the presence of a sugar unit. The 1H NMR data
for 2 showed two methyl groups at dH 1.86 (3H, s, 30-Me) and dH
2.18 (3H, s, C-8), and one methoxy group at dH 3.80 (3H, s, 9-
OMe). A set of 2D NMR spectra (1H–1H COSY, HSQC and HMBC)
of 2 was acquired allowing full assignment of all its 1H and 13C sig-
nals (Table 1). Examination of the COSY and HMBC correlations
observed in 2 confirmed the presence of 40-hydroxy-MPA (5) as
the aglycone. Further HMBC correlation of the anomeric H-100 to
C-40 confirmed the sugar connectivity to C-40 of aglycone core 5
(Fig. 2). The sugar moiety was identified as glucopyranose on the
basis of 1H and 13C NMR chemical shifts. The coupling constant
of the anomeric proton obtained in DMSO-d6 (J = 4.0 Hz) suggested
the
a
linkage in the molecule. The optical rotation (½a D25
ꢁ
+48 (c 0.41,
H2O)) of the sugar obtained through acid hydrolysis of 2, and the
1H NMR data of the acetylated sugar confirmed that the sugar
moiety was
-glucopyranosyl-40-O-MPA.
Penicacid C (3) was isolated as an amorphous solid and its
D
-glucose.16 Thus, compound 2 was elucidated to be
a-D
molecular formula determined to be C17H22O9 by the HRESIMS
[(MꢀH2O)ꢀH]ꢀ signal at m/z 351.1018, suggesting seven degrees
of unsaturation.15c The 1H NMR data of 3 revealed a methyl singlet
at dH 1.47 (3H, s, 30-Me), an aromatic methyl singlet at dH 2.19 (3H,
s, C-8), and a OMe moiety at dH 3.84 (3H, s, 9-OMe). The 13C NMR of
3 was characterized by the absence of two olefinic carbons (C-20
and C-30) readily found in 5. In turn, two oxygen-bearing carbons
appeared at dC 72.9 [methine, dH 4.33 (1H, br s, H-20), C-20] and
dC 91.4 (quaternary carbon, C-30), and one of the methyl groups
Table 1
Summay of 1H (500 MHz) and 13C (125 MHz) NMR spectroscopic data for compounds 1–3
Position
1a
2b
3b
dC
dH (mult.; J Hz)
dC
dH (mult.; J Hz)
dC
dH (mult.; J Hz)
1
3
172.9
70.1
173.8
70.9
173.5
70.6
5.19 (2H, s)
5.27 (2H, s)
5.27 (2H, s)
3a
4
5
6
7
7a
7-OMe
8
144.2
116.8
163.7
124.1
153.7
106.4
51.8
11.6
61.0
22.3
146.9
117.9
164.9
123.1
154.8
107.8
147.9
117.9
165.3
121.9
156.0
108.3
3.66 (3H, s)
2.14 (3H, s)
3.75 (3H, s)
3.41 (2H, d, 6.5)
11.5
61.8
23.4
2.18 (3H, s)
3.80 (3H, s)
3.45 (2H, m)
11.5
61.8
27.4
2.19 (3H, s)
3.84 (3H, s)
9-OMe
10
2.98 (1H, dd, 12.5, 3.0)
3.11 (1H, d, 12.5)
4.33 (1H, br s)
20
30
40
50
121.6
136.0
73.2
5.55 (1H, t, 7.0)
127.6
135.8
83.5
5.59 (1H, t, 6.5)
72.9
91.4
74.0
39.4
4.42 (1H, dd, 9.0, 3.5)
2.54 (2H, m)
4.35 (1H, dd, 11.5, 4.0)
2.45 (1H, dd, 16.0, 8.0)
2.74 (1H, dd, 16.0, 9.0)
4.35 (1H, d, 3.0)
2.48 (1H, d, 18.0)
3.17 (1H, dd, 18.0, 5.5)
40.0
40.6
60
173.0
12.1
176.4
12.0
100.2
74.0
75.4
71.1
74.1
61.9
178.0
18.1
30-Me
100
1.81 (3H, s)
1.86 (3H, s)
1.47 (3H, s)
4.66 (1H, d, 4.0)c
3.51 (1H, m)
200
300
3.59 (1H, m)
3.34 (1H, m)
3.31 (1H, m)
3.59 (1H, m); 3.51 (1H, m)
400
500
600
a
b
c
Recorded in CDCl3.
Recorded in MeOH-d4.
Recorded in DMSO-d6.

3334
Z. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3332–3335
Figure 2. Key COSY and HMBC correlations of 2 and 3.
attention due to their vast array of biological activities. Among
these activities, the MPA scaffold was associated with antiviral
and immunosuppressive activities. However, there have been very
few reports on the isolation of MPA and derivatives from natural
sources12,13,19 and even fewer investigations aimed at understand-
ing the SAR of these compounds.20 We aim in this report to recon-
cile, in part, this scarcity of information by reporting three new
MPA analogues from a marine-derived fungus. Moreover, the 1–3
producer Penicillium sp. SOF07, is a previously unrecognized pro-
ducer of known compounds 4 and 5. On the basis of bioassays
exploiting isolated enzyme and intact cells we provide further in-
sight into MPA scaffold characteristics that may be applied to the
design of new MPA derivatives as drug leads particularly in the
context of immunosuppressives.
Table 2
Summary of IMPDH and mouse splenocyte proliferation inhibition assays for
compounds 1–5
a
a
Compounds IC50
(lM)
IC50 (lM)
Inosine monophosphate
dehydrogenase
Mouse splenocyte
proliferation
1
2
3
4
5
28.86 2.50
6.43 1.10
73.24 6.40
0.63 0.09
1.79 0.13
2.46 0.32
>20
>30
0.32 0.06
1.10 0.21
a
Data represent the mean SD of three triplicate experiments.
(30-Me) characteristic of 1, 2, 4 and 5 was down shifted to dC
18.1 ppm. H–1H COSY data revealed a H-10/H-20 spin system and
1
Acknowledgments
HMBC experiments revealed correlations of H-20/C-6, 30-Me/C-30,
C-20 (Fig. 2). Thus, the two olefinic side chain carbons characteristic
of the other penicacids are replaced in 3 with the oxygen-bearing
carbons C-20 and C-30. Limited amounts of 3, coupled with our
inability to obtain diffraction quality crystals have, thus far,
abrogated stereochemical assignments for C-20, C-30 and C-40
though efforts in this direction are ongoing. Consequently, 3 is
assigned as 20,30-dihydroxy-40-hydroxy-MPA.
We thank the analytical facility center of South China Sea Insti-
tute of Oceanology for recording NMR data. This work is supported,
in part, by grants from the Knowledge Innovation Programs of the
Chinese Academy of Sciences (KZCX2-YW-JC202, KZCX2-EW-G-12
and KSCX2-YW-G-065), ‘863’ Project (2012AA092104), Science
and Technology Planning Project of Guangdong Province
(2010B030600010), and Scientific Research Foundation for the Re-
turned Overseas Chinese Scholars of the State Education Ministry.
J.J. is a scholar of the ‘100 Talents Project’ of Chinese Academy of
Sciences (08SL111001).
To assess the biological activity of the penicacids we first eval-
uated 1–5 for their ability to inhibit IMPDH (type II) activity using
the method reported by Magasanik et al.17 Enzyme assays, in the
presence or absence of varying concentrations of 1–5, were carried
out in 96-well microtiter plates and rates of reaction determined
by monitoring absorbance at 340 nm resulting from enzymatic
NADH production. Compounds 1–5 were dissolved in DMSO and
serially diluted before being added to the initial assay mixture
for pre-incubation with the enzyme. Concentrations of DMSO to
dissolve compounds in assay mixtures were not allowed to exceed
2% (v/v). Data acquisition and processing revealed that 1–5 inhib-
ited IMPDH with IC50 values of 28.86, 6.43, 73.24, 0.63, and
Supplementary data
Supplementary data (1H and 13C NMR spectra of compounds 1–
3) associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.02.106.
References and notes
1.79 lM, respectively. These data suggest that: (i) hydroxylation
1. Rateb, M. E.; Ebel, R. Nat. Prod. Rep. 2011, 28, 290.
at C-40, methylation at C-7-OH, and dual hydroxylation at the C-
20/C-30 olefin of 4 all decrease IMPDH inhibitory activity; (ii) C-40
glycosylation of 4 (yielding 2) minimally affects IMPDH inhibition,
and (iii) the parent compound 4 remains the best inhibitor of
IMPDH.
2. Chen, Z. M.; Huang, H. B.; Chen, Y. C.; Wang, Z. W.; Ma, J. Y.; Wang, B.; Zhang,
W. M.; Zhang, C. S.; Ju, J. Helv. Chim. Acta 2011, 94, 1671.
3. Bentley, R. Chem. Rev. 2000, 100, 3801.
4. Robertson, C. M.; Hermann, L. L.; Coombs, K. M. Antiviral Res. 2004, 64, 55.
5. Ui, H.; Asanuma, S.; Chiba, H.; Takahashi, A.; Yamaguchi, Y.; Masuma, R.;
Omura, S.; Tanake, H. J. Antibiot. 2005, 58, 514.
6. Domhan, S.; Muschal, S.; Schwager, C.; Morath, C.; Wirkner, U.; Ansorge, W.;
Maercker, C.; Zeier, M.; Huber, P. E.; Abdollahi, A. Mol. Cancer Ther. 2008, 7,
1656.
7. Tremblay-Létourneau, M.; Despins, S.; Bougie, I.; Bisaillon, M. PLoS One 2011, 6,
e24806.
Inspired to further dissect the impact of the subtle structural
differences among 1–5 compounds were then subjected to a
splenocyte T lymphocyte proliferation assay,18 the results of which
are shown in Table 2. With the exception of glycosylated 2, the
immunosuppresive activities of 1–5 at the cellular level paralleled
their IMPDH inhibitory activities. The collective SAR data from
both bioassays reveal the importance of the C-7 OH, the C-20/C-30
olefin, and the absence of the C-40 OH in the immunosuppresive
activities displayed by the MPA scaffold at both the enzymatic
and cellular levels.
8. Jackson, R. C.; Weber, G.; Morris, H. P. Nature 1975, 256, 331.
9. Weber, G.; Nakamura, H.; Natsumeda, Y.; Szekeres, T.; Nagai, M. Adv. Enzyme
Regul. 1992, 32, 57.
10. Shu, Q.; Nair, V. Med. Res. Rev. 2008, 28, 219.
11. The strain Penicillium sp. SOF07 was cultured on PDA medium supplemented
with 3% sea salt at 25 °C for a week. The agar plugs containing strain SOF07
were inoculated into Erlenmeyer flasks containing 50 mL potato dextrose
liquid broth supplemented with 3% sea salt. Flask cultures were incubated at
28 °C on a rotary shaker at 200 rpm for 2 d as seed culture. Ten milliliter of the
seed culture was then inoculated to autoclaved solid medium (100 g rice,
MPA was discovered in 1893 by the Italian physician Bartolo-
meo Gosio.3 MPA and related derivatives attracted a great deal of

Z. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3332–3335
3335
100 mL H2O, 3 g sea salt). Incubation was carried out in twenty 500 mL
Fernbach flasks each containing solid medium at 25 °C for 21 days. The
fermented rice substrate was extracted with methylethylketone (3 ꢂ 4 L) to
afford 40.45 g of crude residue. This extract was subjected to column
chromatography (CC) on silica gel eluted with a gradient of CHCl3–MeOH
(100:0–0:100, v/v) to afford fractions 1–5. Fraction 2 was further purified on
silica gel CC by gradient elution using petroleum–EtOAc (90:10–0:100, v/v) to
get subfractions 1–7. Subfractions 2 and 4 were further purified by MPLC (ODS)
with H2O/MeOH gradient elution to yield compound 1 (11.0 mg), 4 (13.0 mg),
and 5 (1.25 g), respectively. Fraction 3 was further refined by semi-preparative
([(MꢀH2O)+Na]+); HR-ESI-MS (negative) m/z: 351.1018 [(MꢀH2O)ꢀH]ꢀ, calcd
for C17H22O9.
16. Penicacid B (2, 2.0 mg) was treated with 1 N aqueous HCl (2 mL) at 95 °C for
2 h. The acidic aqueous mixture was extracted with EtOAc (3 ꢂ 2 mL), the
aqueous fraction was evaporated to dryness. The optical rotation of the dried
residue was determined (½a D25
ꢁ
+48 (c 0.41, H2O)). The residue of sugar fraction
L), Ac2O (100 L) at room temperature for
was treated with pyridine (100
l
l
24 h. The reaction mixture was subjected to column chromatography on silica
gel, eluted with a gradient of CHCl3–MeOH (100:0–90:10) to yield compound
6. The 1H NMR of 6 was in agreement with that of an authentic
acetate (7) (see Figures S8 and S9).
D-glucose penta
HPLC (YMC-pack ODS-A, 5
(4.2 mg).
lm; 10 ꢂ 250 mm) to render 2 (5.8 mg) and 3
17. Magasanik, B.; Moyed, H. S.; Gehring, L. B. J. Biol. Chem. 1957, 226, 339.
12. Lu, X. H.; Zheng, Z. H.; Zhang, H.; Huo, C. H.; Dong, Y. S.; Ma, Y.; Ren, X.; Ke, A.
B.; He, J. G.; Gu, Y. C.; Shi, Q. W. J. Antibiot. 2009, 62, 527.
13. Habib, E.; Leon, F.; Bauer, J. D.; Hill, R. A.; Carvalho, P.; Cutler, H. G.; Cutler, S. J. J.
Nat. Prod. 2008, 71, 1915.
18. C57BL/6 mice were killed by cervical dislocation, the spleens isolated, mashed
and passed aseptically through
a nylon wool sieve to give a single cell
suspension. Cells were centrifuged at 1000 rpm for 5 min and the pelleted cells
were resuspended in Tris-buffered 0.83% ammonium chloride for lysing
erythrocytes. The total number of cells was calculated after two washes with
RPMI1640 and resuspended at 106/mL in RPMI11640 containing 30 mM
14. Haishi, T.; Furuya, K.; Nakajima, M.; Kinoshita, T.; Kagazaki, T.; Sakaida, Y.
Japan Patent 01290667, 1989.
15. Physicochemical properties of 1–3. (a) Compound 1: amorphous solid; ½a D25
ꢁ
HEPES, 100 U of penicillin/mL, 100 lg of streptomycin/mL, 2 mM glutamine,
ꢀ14 (c, 0.65, CHCl3); UV (MeOH) kmax (log
e
): 216 (4.71), 250 (4.18), 304
and 10% FCS (complete medium). 5 ꢂ 105 cells/well were plated in 96-well flat-
(3.92) nm; 1H and 13C NMR, see Table 1; ESI-MS (negative) m/z: 349.2
([MꢀH]ꢀ); HR-ESI-MS (negative) m/z: 349.1521, calcd for C18H22O7. (b)
bottom plates and stimulated with 2 lg/mL ConA. Compounds 1–5 or 0.5%
DMSO were incubated with cells for 72 h, and the proliferation of cells was
then assessed with MTT method.
Compound 2: amorphous solid; ½a D25
ꢁ
74 (c, 0.5, MeOH); UV (MeOH) kmax
(log
e
): 216 (4.70), 250 (4.15), 304 (3.88) nm; 1H and 13C NMR, see Table 1; ESI-
19. Muroi, M.; Sano, K.; Okada, G.; Koshino, H.; Oku, T.; Takatsuki, A. J. Antibiot.
2001, 54, 489.
20. Mitsuhashi, S.; Takenaka, J.; Iwamori, K.; Nakajima, N.; Ubukata, M. Bioorg.
Med. Chem. 2010, 18, 8106.
MS (negative) m/z: 497.6 ([MꢀH]ꢀ); HR-ESI-MS (negative) m/z: 497.1624, calcd
for C23H30O12. (c) Compound 3: amorphous solid; ½a D25
ꢁ
25 (c, 0.28, MeOH); UV
(MeOH) kmax (loge
): 216 (4.68), 249 (4.10), 304 (3.79) nm; 1H and 13C NMR, see
Table 1; ESI-MS (negative) m/z: 369.3 ([MꢀH]ꢀ); ESI-MS (positive) m/z: 375.2