Synthesis and biological activity of Citridone A and its derivatives

Article abstract of DOI:10.1038/ja.2014.14

Citridone A (1), originally isolated as a potentiator of antifungal miconazole activity from a fungal culture broth, has a phenyl-R-furopyridone structure. Because of its unique ring structure, 11 derivatives were chemically synthesized and their biological activity was evaluated. Derivatives 17, 20 and 21 potentiated miconazole activity against Candida albicans. Furthermore, 1, 14, 20 and 21 were found to inhibit yellow pigment production in methicillin-resistant Staphylococcus aureus.

Full text of DOI:10.1038/ja.2014.14

The Journal of Antibiotics (2014) 67, 445–450
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
www.nature.com/ja
&
ORIGINAL ARTICLE
Synthesis and biological activity of Citridone A
and its derivatives
Takashi Fukuda, Kenta Shimoyama, Tohru Nagamitsu and Hiroshi Tomoda
Citridone A (1), originally isolated as a potentiator of antifungal miconazole activity from a fungal culture broth, has a
phenyl-R-furopyridone structure. Because of its unique ring structure, 11 derivatives were chemically synthesized and their
biological activity was evaluated. Derivatives 17, 20 and 21 potentiated miconazole activity against Candida albicans.
Furthermore, 1, 14, 20 and 21 were found to inhibit yellow pigment production in methicillin-resistant Staphylococcus aureus.
The Journal of Antibiotics (2014) 67, 445–450; doi:10.1038/ja.2014.14; published online 19 March 2014
Keywords: 3-phenyl-4-hydroxy-2-pyridone; fungal metabolite; miconazole potentiator; MRSA; staphyloxanthin inhibitor
INTRODUCTION
derivatives 16 and 19, respectively. In addition, 10, which was
Citridone A (1) (Figure 1), a potentiator of miconazole activity synthesized by regioselective iodocyclization according to our
against Candida albicans, was originally isolated from the culture established method,4 was converted to regioisomer 20 (Chart 1).
broth of Penicillium sp. FKI-1938.1–3 Compound 1 has a rare phenyl- Derivatives 27 and 29 were also prepared as shown in Chart 2. The
R-furopyridone skeleton (6-6/5/5 ring system) and 1 was the only Pd(0)-catalyzed coupling reaction between 22 and 234 followed by
natural compound having this ring system. Structurally related heating at 210 1C afforded pyridone 25. Regioselective iodocyclization
citridones 2–4 (Figure 1) were also isolated from the fungus, but under different conditions produced the corresponding iodides 26
they showed very weak miconazole-potentiating activity.3 Because of and 28, from which E2 elimination then gave the desired derivatives
its unique structure and biological properties, two groups have 27 and 29, respectively.
already completed its total synthesis.4,5 These findings prompted us
Potentiation of miconazole activity against C. albicans in combina-
to synthesize new citridone A derivatives to understand the structure– tion with citridones and their derivatives (Figure 1) was assayed by
activity relationship. Furthermore, the biological activity of the the conventional method using paper disks.1 None of the citridones
derivatives was re-evaluated in 15 in-house assay systems. Among themselves showed any inhibition against C. albicans at up to 20mg
11 derivatives synthesized in this study (Figure 1), 3 derivatives, 17, per disk on plate A (without miconazole). Citridone A (1) and
20 and 21, potentiated miconazole activity against C. albicans. derivatives 17, 20 and 21 (20 mg per 6 mm disk) were found to
Interestingly, 1, 14, 20 and 21 were found to inhibit yellow pigment potentiate miconazole activity by forming inhibitory zones around
production in methicillin-resistant Staphylococcus aureus (MRSA).
the paper disks on plate B in contact with a small amount of
In this study, we described the synthesis of citridone derivatives miconazole (60 nM, which, at this level, had no effect on the growth of
and their biological activity, including the potentiating activity of C. albicans; 21, 15, 16, 21mm); however, the other compounds (20 mg
antifungal miconazole and inhibitory activity of yellow pigment per 6 mm disk) showed no potentiation activity. Citridone A and its
production in MRSA.
enantiomer (21) showed the largest inhibition zone on plate B
(Table 1).
As the structures of citridone derivatives are unique as small
molecules, other biological activities of the derivatives were evaluated.
RESULTS AND DISCUSSION
Citridone A (1) and its 11 derivatives (7, 11, 14, 15, 16, 17, 19, 20, 21,
27 and 29) were synthesized.4 Derivatives 11, 17 and 21 (enantiomer Inhibitory activity of yellow pigment production in MRSA was
assayed by our established method using paper disks.9 Compounds
of 1) were prepared as shown in Chart 1, according to the total
synthesis of 1 we achieved previously.4 Intermediate 5 was exposed to
tetra-n-butylammonium fluoride to afford 7, a new derivative
without a dihydrofuran ring. The reactions of other intermediates
11 and 12 with t-BuOK proceeded via cyclobutane formation,
followed by novel pyrolysis6–8 to give alkenes 14 and 15.
Deprotection of 15 and reduction of aldehyde 17 gave the new
1, 14, 20 and 21 (20 mg per 6 mm disk) were found to inhibit
yellow pigment production by forming white zones around the paper
disks (16, 13, 15, 15mm); however, the other compounds (20mg per
6 mm disk) showed no activity. Similar results were obtained when
using methicillin-sensitive S. aureus (MSSA) instead of MRSA
(Table 1).
Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan
Correspondence: Professor H Tomoda, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Tokyo 108-8641, Japan.
E-mail: tomodah@pharm.kitasato-u.ac.jp
Received 16 December 2013; revised 22 January 2014; accepted 27 January 2014; published online 19 March 2014

Synthesis and biological activity of Citridone A
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Natural
Synthetic
Figure 1 Structures of natural and synthetic citridones.
To confirm the inhibition of MRSA yellow pigment production by
Citridone A and derivatives 20 and 21, having a common
these compounds, they were evaluated by the liquid culture method.9 4,5,6a-trimethyl-4,6a-dihydro-3aH-cyclopentafuran skeleton, showed
As summarized in Table 1, compounds 1, 14, 20 and 21 inhibited both biological activities. It is very difficult to imagine that both
yellow pigment production with IC50 values of 11.1mg mlꢀ1
30.0mg mlꢀ1, 22.5mg mlꢀ1 and 22.7mg mlꢀ1, respectively, without precise analysis is necessary to demonstrate the mechanisms.
any effect on the growth of MRSA at 30mg mlꢀ1
,
activities are derived from the same mechanism of action. Further
.
It is well known that S. aureus produces a yellow pigment called
staphyloxanthin.10,11 Recently, several research groups reported
that staphyloxanthin is one of the important virulent factors of
S. aureus.12,13 Staphyloxanthin acts as an antioxidant with its
numerous conjugated double bonds, which enable S. aureus to
survive by detoxification of host-generated reactive oxgen species
such as O2 ꢀ, H2O2 and HOCl.14,15 Staphyloxanthin develops in the
cell membrane of S. aureus and is associated with enhancing S. aureus
survival and infection.16
MATERIALS AND METHODS
General experimental procedures
Ultraviolet spectra were recorded on a spectrophotometer (8453 UV–Visible
spectrophotometer; Agilent Technologies Inc., Santa Clara, CA, USA).
Optical rotations were measured with a digital polarimeter (DIP-1000;
JASCO, Tokyo, Japan). HR-ESI-TOF-mass spectra were recorded on
mass spectrometer (JMS-T100LP; JEOL, Tokyo, Japan). Various NMR spectra
a
were measured with
CA, USA).
a spectrometer (XL-400; Varian, Inc., Palo Alto,
Staphyloxanthin is composed of a glucose core to which a prenyl
residue and a fatty acyl residue are attached. The biosynthetic pathway
of staphyloxanthin was reported previously.11 Importantly, a CrtM-
deficient mutant, which lacks an enzyme involved in synthesis of the
prenyl residue and cannot produce staphyloxanthin, was reported to
fail to survive in the host mouse.17,18 Recently, several squalene
synthase inhibitors, BPH-652,19 zaragozic acid,20 rhodomyrtone21 and
tylopilusins,22 were found to inhibit staphyloxanthin production in
S aureus. Furthermore, BPH-652 was demonstrated to block infection
of the host mouse with S. aureus.19 Therefore, the staphyloxanthin
biosynthetic pathway of S aureus is expected to offer a new potential
target to combat MSSA and MRSA infection.
Experimental procedures and characterization data
Stocked natural citridones B to D (2–4) used for this investigation were
isolated from a culture broth of Penicillium sp. FKI-1938.1,3
All experimental procedures for the synthesis of compounds (7, 11, 14, 15,
16, 17, 19, 20, 21, 27 and 29), including citridone A (1), are summarized in
Supplementary Information.
(5aS,8R,8aS)-5a,7,8-Trimethyl-4-phenyl-2,5a,8,8a-tetrahydro-1H-cyclopenta[4,5]
furo[3,2–c]pyridin-1-one (citridone A, 1). [a]2D1 –76.9 (c 1.0, CH3OH). IR
(KBr) cm–1: 2926, 1653, 1431, 1231, 1030, 763, 697. 1H-NMR (300 MHz,
CDCl3) d: 7.55–7.51 (m, 2H), 7.45 (s, 1H), 7.42–7.36 (m, 2H), 7.33–7.27
(m, 1H), 5.39 (dq, J ¼ 1.7 Hz, 1H), 3.27 (d, J ¼ 1.7 Hz, 1H), 2.91 (bq, J ¼ 7.2
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Chart 1 Preparation of compounds 7, 11, 14, 15, 16, 17, 19, 20 and 21. Reagents and conditions: (a) tetra-n-butylammonium fluoride, tetrahydrofuran,
room temperature, 88%; (b) N-iodosuccinimide, pyridine, CH2Cl2, room temperature*; (c) I2, CH2Cl2, room temperature*; (d) tetra-n-butylammonium
fluoride, tetrahydrofuran, room temperature, 88% for 11*, 89% for 12, 77% for 13; (e) t-BuOK, N,N-dimethylformamide. 1001C, 88% for 14,
77% for 15; (f) trifluoroacetic acid, CH2Cl2, 0 1C, 92%; (g) Dess-Martin Periodinane, CH2Cl2, dimethyl sulfoxide, pyridine, room temperature;
(h) 1,8-diazabicyclo[5.4.0]undec-7-ene, CH2Cl2, room temperature, 99% (2 steps) for 17*, 65% (2 steps) for 18; (i) NaBH4, CeCl3 ꢁ 7H2O,
MeOH, ꢀ781C, 77%; (j) (TMSSCH2)2, ZnI2, CH2Cl2, Et2O; (k) Bu3SnH, azobisisobutyronitrile, benzene, 55% (2 steps) for 1*, 40% (2 steps) for 20.
*See, ref. 1.
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Chart 2 Preparation of compounds 27 and 29. Reagents and conditions: (a) diisobutylaluminium hydride, CH2Cl2, ꢀ781C; (b) ClCO2Me, N,N,N0,N0-
tetramethylethylenediamine, CH2Cl2, 0 1C, 92% (2 steps); (c) (allylPdCl)2, 1,10-bis(diphenylphosphino)ferrocene, NaH, tetrahydrofuran, room temperature,
quant.; (d) Ph2O, 210 1C, 86%; (e) N-iodosuccinimide, pyridine, CH2Cl2, room temperature, 61%; (f) I2, CH2Cl2, room temperature, 83%; (g) t-BuOK,
N,N-dimethylformamide, 1001C, 95% for ( )-27, 93% for ( )-29.
55.2, 51.9, 41.2, 20.9, 15.1. HRMS (ESI) [Mþ Na]þ calcd for
Table 1 Summary of biological activity of citridone derivatives
C19H21NNaO3 ¼ 334.1521, found¼ 334.1518.
Miconazole-potentiating
activity
Inhibition of yellow
pigment production
(5aR,6R,7R,8R,8aS)-7-(Hydroxymethyl)-6-iodo-5a,8-dimethyl-4-phenyl-2,5a,6,7,
8,8a-hexahydro-1H-cyclopenta[4,5]furo[3,2–c]pyridin-1-one (11). [a]2D0 –56.0
(c 1.0, CH3OH). IR (KBr) cm–1: 3321, 2960, 2872, 1656, 1429, 1379, 1033.
1H-NMR (400MHz, CD3OD) d: 7.53–7.50 (m, 2H), 7.41 (1H), 7.40–7.36
(m, 2H), 7.33–7.28 (m, 1H), 4.47 (d, J ¼ 14.3Hz, 1H), 3.76 (dd, J ¼ 11.8,
2.6Hz, 1H), 3.72 (dd, J ¼ 11.8, 2.3 Hz, 1H), 3.14 (d, J ¼ 9.8 Hz, 1H), 2.10–2.00
(m, 1H), 1.73–1.63 (m, 1H), 1.67 (s, 3H), 1.40 (d, J ¼ 6.7 Hz, 3H). 13C-NMR
(100 MHz, CD3OD) d: 166.1, 163.3, 135.6, 134.2, 129.7, 128.7, 128.5, 114.6,
112.9, 99.0, 58.8, 57.0, 56.5, 41.6, 39.9, 29.6, 19.6. HRMS (ESI) [Mþ H]þ
calcd for C19H21INO3 ¼ 438.0566, found ¼ 438.0551.
Inhibition zone against
C. albicans
MSSA
MRSA
Plate
Aa
Plate B
White
zonea
White
Compound
(60nM MZ)a
zonea Growthb Y. P. prod.b
(mm)
—
(mm)
21
(mm)
17
(mm)
16
(IC50 mg mlꢀ1
)
1
430
430
430
430
430
11.1
30.0
(5aS,8S,8aS)-5a,8-Dimethyl-4-phenyl-2,5a,8,8a-tetrahydro-1H-cyclopenta[4,5]
14
17
20
21
—
—
15
13
furo[3,2–c]pyridin-1-one (14). [a]2D7 –129.8 (c 1.0, CHCl3). IR (KBr) cm–1
:
—
15
—
—
430.0
22.5
2926, 1739, 1655, 1501, 1459, 1428, 1369, 1263, 1227, 1150, 1096. 1H-NMR
(300 MHz, CDCl3) d: 7.54–7.50 (m, 3H), 7.43–7.36 (m, 1H), 7.34–7.28
(m, 2H), 5.95 (dd, J ¼ 5.6, 2.1 Hz, 1H), 5.73 (dd, J ¼ 5.4, 1.5 Hz, 1H), 3.25
(s, 1H), 3.14-3.12 (m, 1H), 1.69 (s, 3H), 1.29 (d, J ¼ 7.5 Hz, 3H). 13C-NMR
(75.5MHz, CDCl3) d: 141.4, 134.5, 133.2, 131.1, 128.5, 127.6, 127.4, 127.4,
104.8, 55.5, 46.3, 29.5, 26.0, 21.6. HRMS (ESI) [Mþ Na] þ calcd for
C18H17NNaO2 ¼ 302.1157, found¼ 302.1142.
—
16
15
15
—
21
15
15
22.7
Abbreviations: -, No inhibition zone or no white zone; MRSA, methicillin-resistant
Staphylococcus aureus; MZ, miconazole; Y. P. prod., yellow pigment production.
aPaper disk method (20 mg per sample per 6 mm disk).
bLiquid culture method.
(5aS,8S,8aS)-4-(4-((2-Methoxyethoxy)methoxy)phenyl)-5a,8-dimethyl-2,5a,8,8a-
Hz, 1H), 1.73 (s, 3H), 1.65 (s, 3H), 1.30 (d, J ¼ 7.9Hz, 3H). 13C-NMR
(75 MHz, CDCl3) d: 164.9, 163.1, 150.5, 133.9, 133.6, 128.5, 127.6, 127.2,
126.3, 113.4, 111.1, 103.8, 56.7, 49.0, 26.3, 20.3, 14.8. high resolution mass
spectrum (HRMS) (ESI) [Mþ Na]þ calcd for C19H19NNaO2 ¼ 316.1314,
found ¼ 316.1331.
tetrahydro-1H-cyclopenta[4,5]furo[3,2–c]pyridin-1-one
(15). [a]2D5 –59.2
(c 1.0, CHCl3). IR (KBr) cmꢀ1: 3401, 2925, 1652, 1614, 1514, 1445, 1228,
1103, 1000, 833. 1H-NMR (400MHz, CDCl3) d: 7.58–7.56 (m, 1H), 7.45–7.41
(m, 2H), 7.09–7.06 (m, 2H), 5.94 (dd, J ¼ 7.6, 1.8 Hz, 1H), 5.72 (dd, J ¼ 7.6,
1.8Hz, 1H), 5.29 (s, 2H), 3.85–3.82 (m, 2H), 3.58–3.55 (m, 2H), 3.38 (s, 3H),
3.24 (s, 1H), 3.13–3.08 (m, 1H), 1.69 (s, 3H), 1.27 (d, J ¼ 6.8Hz, 3H).
13C-NMR (150 MHz, CDCl3) d: 165.5, 161.2, 156.7, 141.3, 133.9, 131.0, 128.8,
126.5, 116.3, 104.9, 93.4, 71.6, 67.6, 59.0, 55.4, 46.4, 26.2, 21.5. HRMS (ESI)
[Mþ H]þ calcd for C22H26NO5 ¼ 384.1811, found¼ 384.1824.
4-Hydroxy-3-((1R,4S,5R)-4-(hydroxymethyl)-2,5-dimethylcyclopent-2-en-1-yl)-5-
phenylpyridin-2(1H)-one (7). [a]2D7 þ 39.2 (c 0.5, CHCl3). IR (KBr) cm–1
:
3373, 2925, 2361, 1639, 1433, 1214. 1H-NMR (400MHz, CDCl3) d: 7.54–7.34
(m, 6H), 5.53 (s, 1H), 4.04 (bd, J ¼ 6.8 Hz, 1H), 3.87 (dd, J ¼ 12.0, 2.2 Hz,
(5aS,8S,8aS)-4-(4-Hydroxyphenyl)-5a,8-dimethyl-2,5a,8,8a-tetrahydro-1H-cyclo-
1H), 3.67 (dd, J ¼ 12.0, 2.2Hz, 1H), 2.61–2.57 (m, 1H), 2.35–2.29 (m, 1H), penta[4,5]furo[3,2–c]pyridin-1-one (16). [a]2D5 –9.91 (c 0.5, CH3OH). IR
1.64 (s, 3H), 1.28 (d, J ¼ 6.8 Hz, 3H). 13C-NMR (150MHz, CDCl3) d: 166.7,
(KBr) cm–1: 3435, 1646, 1516, 1430, 1270, 1108, 1042, 834, 786, 630.
160.8, 140.2, 132.9, 131.6, 129.8, 129.3, 128.8, 128.6, 122.5, 112.3, 63.8, 1H-NMR (400 MHz, CD3OD) d: 7.35–7.30 (m, 3H), 6.81–6.77 (m, 2H),
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5.98 (dd, J ¼ 7.6, 2.4 Hz, 1H), 5.73 (dd, J ¼ 7.6, 1.8 Hz, 1H), 3.18 (d, J ¼ 1.6 Hz, Assay for miconazole-potentiating activity against C. albicans using
1H), 3.04–2.97 (m, 1H), 1.67 (s, 3H), 1.25 (d, J ¼ 7.2 Hz). 13C-NMR
paper disks
(100 MHz, CD3OD) d: 166.8, 163.0, 158.0, 133.9, 132.0, 129.7, 125.3, 116.1,
114.5, 113.0, 105.4, 56.7, 26.2, 21.6. HRMS (ESI) [Mþ Na]þ calcd for
C18H17NNaO3 ¼ 318.1106, found ¼ 318.1108.
C. albicans ATCC 64548 was inoculated into a 50-ml test tube containing 10ml
seed medium (potato extract containing peptone 0.50% and glucose 1.0%),
and was grown for 24 h on a rotary shaker. In Method A, the seed culture of
C. albicans (0.10%, v/v) was transferred to two different agar plates, GY agar
(glucose 1.0%, yeast extract 0.50% and agar 0.80%; plate A) and GY agar plus
miconazole (60 nM; plateB). The concentration (60 nM) of miconazole is one-
fourth of the MIC value against C. albicans, and showed no effect on the
growth of C. albicans. Paper disks (6mm; ADVANTEC, Tokyo, Japan)
containing a 20-mg sample were placed on plates A and B, which were
incubated at 271C for 24h. Samples showing inhibition zones selectively on
plate B were selected as potentiators of miconazole activity against C. albicans.
(5aS,8R,8aS)-5a,8-Dimethyl-1-oxo-4-phenyl-2,5a,8,8a-tetrahydro-1H-cyclopenta
[4,5]furo[3,2–c]pyridine-7-carbaldehyde (17). [a]2D0 –191.0 (c 1.0, CHCl3). IR
(KBr) cm–1: 2969, 1689, 1652, 1456, 1430, 1373, 1227. 1H-NMR (400MHz,
CDCl3) d: 9.83 (s, 1H), 7.53–7.30 (m, 6H), 6.58 (s, 1H), 3.48 (q, J ¼ 7.2 Hz,
1H), 3.41 (s, 1H), 1.80 (s, 3H), 1.38 (d, J ¼ 7.1 Hz, 3H). 13C-NMR (100MHz,
CDCl3) d: 190.0, 164.3, 162.9, 151.8, 147.2, 134.7, 133.0, 128.5, 127.5, 127.3,
112.3, 110.8, 101.3, 56.8, 42.4, 25.1, 20.4. HRMS (ESI) [Mþ Na] þ calcd for
C19H17NNaO3 ¼ 30.1106, found ¼ 330.1077.
Assay for inhibition of yellow pigment production in MRSA using
paper disks
(5aS,8R,8aS)-7-(Hydroxymethyl)-5a,8-dimethyl-4-phenyl-2,5a,8,8a-tetrahydro-1H-
cyclopenta[4,5]furo[3,2–c]pyridin-1-one (19). [a]2D9 –51.7 (c 0.5, CH3OH). IR
(KBr) cm–1: 3460, 2849, 2150, 1650, 1453, 1430, 1112. 1H-NMR (400MHz,
CD3OD) d: 7.50 (d, J ¼ 8.4Hz, 2H), 7.44 (s, 1H), 7.37 (t, J ¼ 8.4 Hz, 2H),
7.31–7.27 (m, 1H), 5.65 (s, 1H), 4.18 (d, J ¼ 15.6 Hz, 1H), 4.10 (d, J ¼ 15.6 Hz,
1H), 3.28 (d, J ¼ 0.8 Hz, 1H), 3.01 (q, J ¼ 7.2 Hz, 1H), 1.68 (s, 3H), 1.30
(d, J ¼ 7.2 Hz, 3H). 13C-NMR (150 MHz, CD3OD) d: 166.5, 163.3, 155.5,
135.1, 129.5, 128.7, 128.5, 128.4, 126.3, 114.8, 113.0, 104.7, 60.2, 58.0, 47.0,
26.3, 20.8. HRMS (ESI) [Mþ Na]þ calcd for C19H19NNaO3 ¼ 332.1262,
found ¼ 332.1276.
MRSA K-24 strain, a clinical isolate, was used as a yellow pigment-producing
strain. MRSA was cultured in Mueller-Hinton broth at 371C for 20h and
adjusted to 1 ꢂ 108 CFU per ml. The inoculum (100ml) was spread on 25ml
TYB agar (tryptone 1.7%, yeast extract 1.0%, NaCl 0.5%, K2HPO4 0.25%, agar
1.5% and glycerol monoacetate 1.5%) on a plate (100ꢂ 140 mm). Paper disks
(6mm i.d.) containing a 20-mg sample were placed on the plate and incubated
at 371C for 72h. Inhibition of the production of yellow pigments by a sample
is expressed as the diameter (mm) of the white zone on the plate.
Assay for growth and yellow pigment production in MRSA by
liquid culture
(4bS,5R,7aS)-5,6,7a-Trimethyl-3-phenyl-4b,5-dihydro-1H-cyclopenta[4,5]furo[2,
3–b]pyridin-4(7aH)-one (20). [a]2D5 þ 107.6 (c 1.0, CH3OH). IR (KBr) cm–1
:
A mixture containing TYB (980ml), a sample (10 ml) and MRSA (10 ml, at a
final concentration of 1 ꢂ 107 CFU per ml) in a total volume of 1000ml was
incubated on a rotary shaker at 210 r.p.m. for 72h at 37 1C. (1) MRSA growth:
the culture’s turbidity was determined at 600 nm using a Power Wave x 340
(BIO-TEK Instruments Inc., Winooski, VT, USA). (2) Yellow pigment
production: after the culture was centrifuged, yellow pigments in MRSA
mycelia were extracted with methanol (500ml) at 601C for 2 h in the dark. The
absorbance of yellow pigments was determined at 450 nm using a Power
Wave ꢂ 340. Inhibition of MRSA growth and yellow pigment production by
a sample (% of control) is defined as (absorbance-sample/absorbance-
control) ꢂ 100. The IC50 values are defined as the sample concentrations that
cause 50% inhibition of MRSA growth and yellow pigment production.
2965, 1653, 1454, 1432, 1217, 1041. 1H-NMR (400MHz, CDCl3) d: 7.54–7.51
(m, 3H), 7.41–7.37 (m, 2H), 7.32–7.28 (m, 1H), 5.40 (bs, 1H), 3.28 (d,
J ¼ 1.0 Hz, 1H), 2.90 (bq, J ¼ 7.0Hz, 1H), 1.73 (s, 3H), 1.67 (s, 3H), 1.30
(d, J ¼ 7.0 Hz, 3H). 13C-NMR (100 MHz, CDCl3) d: 165.4, 162.7,
150.7, 134.3, 133.5, 128.7, 127.7, 127.4, 126.4, 111.8, 104.3, 56.6, 49.2, 26.4,
20.4, 14.9. HRMS (ESI) [M þ H]þ calcd for C19H20NO2 ¼ 294.1494,
found ¼ 294.1485.
(5aR,8S,8aR)-5a,7,8-Trimethyl-4-phenyl-2,5a,8,8a-tetrahydro-1H-cyclopenta[4,5]
furo[3,2–c]pyridin-1-one (ent-Citridone A, 21). [a]2D3 þ 77.6 (c 0.1, CH3OH).
IR (KBr) cm–1: 2924, 1652, 1431, 1204, 1034, 765, 697. 1H-NMR (400 MHz,
CDCl3) d: 7.54–7.51 (m, 2H), 7.47–7.34 (m, 4H), 5.43 (dq, J ¼ 1.4 Hz, 1H),
3.33 (bs, 1H), 2.88 (bq, J ¼ 7.0 Hz, 1H), 1.74 (s, 3H), 1.70 (s, 3H), 1.30
(d, J ¼ 7.2 Hz, 3H). 13C-NMR (100 MHz, CDCl3) d: 164.1, 163.1, 150.7, 133.7,
133.4, 128.7, 127.8, 127.5, 126.4, 113.1, 111.9, 104.2, 56.6, 49.2, 26.4,
20.4, 14.9. HRMS (ESI) [M þ H]þ calcd for C19H20NO2 ¼ 294.1494,
found ¼ 294.1488.
ACKNOWLEDGEMENTS
We thank Ms Noriko Sato and Dr Kenichiro Nagai (School of Pharmaceutical
Sciences, Kitasato University) for measuring NMR spectra and MS data. We
thank Ms Minori Shinkai and Ms Eri Sasaki for measuring the activity of
citridones. This work was supported by Takeda Science Foundation and JSPS
KAKENHI Grant Number 25870704.
(5aS*,8aS*)-4-Phenyl-2,5a,8,8a-tetrahydro-1H-cyclopenta[4,5]furo[3,2–c]pyridin-
1-one (27). IR (KBr) cm–1: 3055, 2929, 2360, 2340, 1649, 1598, 1230, 1062,
1197, 762. 1H-NMR (400 MHz, CDCl3) d: 7.65–7.61 (m, 1H), 7.52–7.50
(m, 2H), 7.42–7.38 (m, 2H), 7.34–7.30 (m, 1H), 6.15 (d, J ¼ 5.2 Hz, 1H), 6.05
(d, J ¼ 5.2 Hz, 1H), 5.89 (dd, J ¼ 5.2, 2.4 Hz, 1H), 4,15 (t, J ¼ 7.8 Hz, 1H), 2.90
(d, J ¼ 7.8 Hz, 1H), 2.85 (t, J ¼ 2.4 Hz, 1H). 13C-NMR (150 MHz, CDCl3)
d: 166.2, 162.1, 137.4, 134.5, 132.9, 128.6, 128.0, 127.6, 127.6, 113.3, 112.2,
95.7, 40.9, 38.2. HRMS (ESI) [Mþ Na]þ calcd for C16H13NNaO2 ¼ 274.0844,
found ¼ 274.0841.
¯
1
Fukuda, T., Yamaguchi, Y., Masuma, R., Tomoda, H. & Omura, S. Citridones, new
potentiators of antifungal miconazole activity, produced by Penicillium sp. FKI-1938 I.
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¯
2
3
Fukuda, T., Tomoda, H. & Omura, S. Citridones, new potentiators of antifungal
miconazole activity, produced by Penicillium sp. FKI-1938 II. Structure elucidation.
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¯
Fukuda, T., Sakabe, Y., Tomoda, H. & Omura, S. Fungal citridone D having a novel
phenylfuropyridine skeleton. Chem. Pharm. Bull. 54, 1659–1661 (2006).
4
5
Miyagawa, T. et al. Total synthesis of citridone A. Org. Lett. 13, 1158–1161 (2011).
(4bS*,7aS*)-3-Phenyl-4b,5-dihydro-1H-cyclopenta[4,5]furo[2,3–b]pyridin-4(7aH)-
one (29). IR (KBr) cm–1: 3020, 2929, 2857, 2400, 1642, 1597, 1477, 1216.
1H-NMR (400MHz, CD3OD) d: 7.48–7.45 (m, 3H), 7.39–7.35 (m, 2H),
7.32–7.28 (m, 1H), 6.20–6.17 (m, 1H), 5.98 (d, J ¼ 8.0Hz, 1H), 5.92-5.89
(m, 1H), 4.14 (td, J ¼ 8.0, 2.4 Hz, 1H), 2.89 (ddt, J ¼ 17.6, 8.0, 2.4 Hz, 1H),
2.69 (dt, J ¼ 17.6, 2.4Hz, 1H). 13C-NMR (150 MHz, CD3OD) d: 169.4, 163.9,
138.8, 136.5, 130.4, 129.2, 129.0, 128.2, 111.1, 95.6, 41.8, 39.7. HRMS (ESI)
[Mþ H]þ calcd for C16H14NO2 ¼ 252.1024, found¼ 252.1012.
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
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