Synthesis and biological activity of citridone a and its derivatives

ABSTRACT 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_. SIMILAR CONTENT BEING VIEWED BY OTHERS SYNTHESIS AND

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ACTINOMYCETE STRAIN TMPU-A0287 Article 02 December 2022 INTRODUCTION Citridone A (1) (Figure 1), a potentiator of miconazole activity against _Candida albicans_, was originally isolated from

the culture broth of _Penicillium_ sp. FKI-1938.1, 2, 3 Compound 1 has a rare phenyl-_R_-furopyridone skeleton (6-6/5/5 ring system) and 1 was the only natural compound having this ring

system. Structurally related citridones 2–4 (Figure 1) were also isolated from the fungus, but they showed very weak miconazole-potentiating activity.3 Because of its unique structure and

biological properties, two groups have already completed its total synthesis.4, 5 These findings prompted us to synthesize new citridone A derivatives to understand the structure–activity

relationship. Furthermore, the biological activity of the derivatives was re-evaluated in 15 in-house assay systems. Among 11 derivatives synthesized in this study (Figure 1), 3 derivatives,

17, 20 and 21, potentiated miconazole activity against _C. albicans_. Interestingly, 1, 14, 20 and 21 were found to inhibit yellow pigment production in methicillin-resistant

_Staphylococcus aureus_ (MRSA). In this study, we described the synthesis of citridone derivatives and their biological activity, including the potentiating activity of antifungal miconazole

and inhibitory activity of yellow pigment production in MRSA. 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 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, 7, 8 to give alkenes 14 and 15. Deprotection of 15 and reduction of aldehyde 17 gave the new derivatives 16 and 19, respectively. In addition, 10, which was

synthesized by regioselective iodocyclization according to our established method,4 was converted to regioisomer 20 (Chart 1). Derivatives 27 and 29 were also prepared as shown in Chart 2.

The Pd(0)-catalyzed coupling reaction between 22 and 234 followed by heating at 210 °C afforded pyridone 25. Regioselective iodocyclization under different conditions produced the

corresponding iodides 26 and 28, from which E2 elimination then gave the desired derivatives 27 and 29, respectively. Potentiation of miconazole activity against _C. albicans_ in combination

with citridones and their derivatives (Figure 1) was assayed by the conventional method using paper disks.1 None of the citridones themselves showed any inhibition against _C. albicans_ at

up to 20 μg per disk on plate A (without miconazole). Citridone A (1) and derivatives 17, 20 and 21 (20 μg per 6 mm disk) were found to potentiate miconazole activity by forming inhibitory

zones around the paper disks on plate B in contact with a small amount of miconazole (60 nM, which, at this level, had no effect on the growth of _C. albicans_; 21, 15, 16, 21 mm); however,

the other compounds (20 μg per 6 mm disk) showed no potentiation activity. Citridone A and its 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. Inhibitory activity of yellow pigment production in MRSA was assayed by

our established method using paper disks.9 Compounds 1, 14, 20 and 21 (20 μg per 6 mm disk) were found to inhibit yellow pigment production by forming white zones around the paper disks (16,

13, 15, 15 mm); however, the other compounds (20 μg per 6 mm disk) showed no activity. Similar results were obtained when using methicillin-sensitive _S. aureus_ (MSSA) instead of MRSA

(Table 1). To confirm the inhibition of MRSA yellow pigment production by these compounds, they were evaluated by the liquid culture method.9 As summarized in Table 1, compounds 1, 14, 20

and 21 inhibited yellow pigment production with IC50 values of 11.1 μg ml−1, 30.0 μg ml−1, 22.5 μg ml−1 and 22.7 μg ml−1, respectively, without any effect on the growth of MRSA at 30 μg 

ml−1. 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 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. Citridone A and derivatives 20 and 21, having a common 4,5,6a-trimethyl-4,6a-dihydro-3a_H_-cyclopentafuran skeleton, showed both

biological activities. It is very difficult to imagine that both activities are derived from the same mechanism of action. Further precise analysis is necessary to demonstrate the

mechanisms. 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 a mass spectrometer (JMS-T100LP;

JEOL, Tokyo, Japan). Various NMR spectra were measured with a spectrometer (XL-400; Varian, Inc., Palo Alto, CA, USA). 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.

(5A_S_,8_R_,8A_S_)-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) [α]21D –76.9 (_c_ 1.0, CH3OH). IR (KBr) cm–1: 2926, 1653, 1431,

1231, 1030, 763, 697. 1H-NMR (300 MHz, CDCl3) _δ_: 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_=1.7 Hz, 1H), 3.27 (d, _J_=1.7 Hz, 1H), 2.91 (bq,

_J_=7.2 Hz, 1H), 1.73 (s, 3H), 1.65 (s, 3H), 1.30 (d, _J_=7.9 Hz, 3H). 13C-NMR (75 MHz, CDCl3) _δ_: 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) [M+Na]+ calcd for C19H19NNaO2=316.1314, found=316.1331.

4-HYDROXY-3-((1_R_,4_S_,5_R_)-4-(HYDROXYMETHYL)-2,5-DIMETHYLCYCLOPENT-2-EN-1-YL)-5-PHENYLPYRIDIN-2(1_H_)-ONE (7) [α]27D +39.2 (_c_ 0.5, CHCl3). IR (KBr) cm–1: 3373, 2925, 2361, 1639, 1433,

1214. 1H-NMR (400 MHz, CDCl3) _δ_: 7.54–7.34 (m, 6H), 5.53 (s, 1H), 4.04 (bd, _J_=6.8 Hz, 1H), 3.87 (dd, _J_=12.0, 2.2 Hz, 1H), 3.67 (dd, _J_=12.0, 2.2 Hz, 1H), 2.61–2.57 (m, 1H), 2.35–2.29

(m, 1H), 1.64 (s, 3H), 1.28 (d, _J_=6.8 Hz, 3H). 13C-NMR (150 MHz, CDCl3) _δ_: 166.7, 160.8, 140.2, 132.9, 131.6, 129.8, 129.3, 128.8, 128.6, 122.5, 112.3, 63.8, 55.2, 51.9, 41.2, 20.9,

15.1. HRMS (ESI) [M+Na]+ calcd for C19H21NNaO3=334.1521, found=334.1518.

(5A_R_,6_R_,7_R_,8_R_,8A_S_)-7-(HYDROXYMETHYL)-6-IODO-5A,8-DIMETHYL-4-PHENYL-2,5A,6,7,8,8A-HEXAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDIN-1-ONE (11) [α]20D –56.0 (_c_ 1.0, CH3OH). IR (KBr)

cm–1: 3321, 2960, 2872, 1656, 1429, 1379, 1033. 1H-NMR (400 MHz, CD3OD) _δ_: 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_=14.3 Hz, 1H), 3.76 (dd,

_J_=11.8, 2.6 Hz, 1H), 3.72 (dd, _J_=11.8, 2.3 Hz, 1H), 3.14 (d, _J_=9.8 Hz, 1H), 2.10–2.00 (m, 1H), 1.73–1.63 (m, 1H), 1.67 (s, 3H), 1.40 (d, _J_=6.7 Hz, 3H). 13C-NMR (100 MHz, CD3OD) _δ_:

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) [M+H]+ calcd for C19H21INO3=438.0566, found=438.0551.

(5A_S_,8_S_,8A_S_)-5A,8-DIMETHYL-4-PHENYL-2,5A,8,8A-TETRAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDIN-1-ONE (14) [α]27D –129.8 (_c_ 1.0, CHCl3). IR (KBr) cm–1: 2926, 1739, 1655, 1501, 1459,

1428, 1369, 1263, 1227, 1150, 1096. 1H-NMR (300 MHz, CDCl3) _δ_: 7.54–7.50 (m, 3H), 7.43–7.36 (m, 1H), 7.34–7.28 (m, 2H), 5.95 (dd, _J_=5.6, 2.1 Hz, 1H), 5.73 (dd, _J_=5.4, 1.5 Hz, 1H), 3.25

(s, 1H), 3.14-3.12 (m, 1H), 1.69 (s, 3H), 1.29 (d, _J_=7.5 Hz, 3H). 13C-NMR (75.5 MHz, CDCl3) _δ_: 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) [M+Na]+ calcd for C18H17NNaO2=302.1157, found=302.1142.

(5A_S_,8_S_,8A_S_)-4-(4-((2-METHOXYETHOXY)METHOXY)PHENYL)-5A,8-DIMETHYL-2,5A,8,8A-TETRAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDIN-1-ONE (15) [α]25D –59.2 (_c_ 1.0, CHCl3). IR (KBr) cm−1:

3401, 2925, 1652, 1614, 1514, 1445, 1228, 1103, 1000, 833. 1H-NMR (400 MHz, CDCl3) _δ_: 7.58–7.56 (m, 1H), 7.45–7.41 (m, 2H), 7.09–7.06 (m, 2H), 5.94 (dd, _J_=7.6, 1.8 Hz, 1H), 5.72 (dd,

_J_=7.6, 1.8 Hz, 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_=6.8 Hz, 3H). 13C-NMR (150 MHz, CDCl3) _δ_:

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) [M+H]+ calcd for C22H26NO5=384.1811, found=384.1824.

(5A_S_,8_S_,8A_S_)-4-(4-HYDROXYPHENYL)-5A,8-DIMETHYL-2,5A,8,8A-TETRAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDIN-1-ONE (16) [α]25D –9.91 (_c_ 0.5, CH3OH). IR (KBr) cm–1: 3435, 1646, 1516,

1430, 1270, 1108, 1042, 834, 786, 630. 1H-NMR (400 MHz, CD3OD) _δ_: 7.35–7.30 (m, 3H), 6.81–6.77 (m, 2H), 5.98 (dd, _J_=7.6, 2.4 Hz, 1H), 5.73 (dd, _J_=7.6, 1.8 Hz, 1H), 3.18 (d, _J_=1.6 Hz,

1H), 3.04–2.97 (m, 1H), 1.67 (s, 3H), 1.25 (d, _J_=7.2 Hz). 13C-NMR (100 MHz, CD3OD) _δ_: 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) [M+Na]+ calcd for C18H17NNaO3=318.1106, found=318.1108. (5A_S_,8_R_,8A_S_)-5A,8-DIMETHYL-1-OXO-4-PHENYL-2,5A,8,8A-TETRAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDINE-7-CARBALDEHYDE

(17) [α]20D –191.0 (_c_ 1.0, CHCl3). IR (KBr) cm–1: 2969, 1689, 1652, 1456, 1430, 1373, 1227. 1H-NMR (400 MHz, CDCl3) _δ_: 9.83 (s, 1H), 7.53–7.30 (m, 6H), 6.58 (s, 1H), 3.48 (q, _J_=7.2 Hz,

1H), 3.41 (s, 1H), 1.80 (s, 3H), 1.38 (d, _J_=7.1 Hz, 3H). 13C-NMR (100 MHz, CDCl3) _δ_: 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) [M+Na]+ calcd for C19H17NNaO3=30.1106, found=330.1077.

(5A_S_,8_R_,8A_S_)-7-(HYDROXYMETHYL)-5A,8-DIMETHYL-4-PHENYL-2,5A,8,8A-TETRAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDIN-1-ONE (19) [α]29D –51.7 (_c_ 0.5, CH3OH). IR (KBr) cm–1: 3460, 2849,

2150, 1650, 1453, 1430, 1112. 1H-NMR (400 MHz, CD3OD) _δ_: 7.50 (d, _J_=8.4 Hz, 2H), 7.44 (s, 1H), 7.37 (t, _J_=8.4 Hz, 2H), 7.31–7.27 (m, 1H), 5.65 (s, 1H), 4.18 (d, _J_=15.6 Hz, 1H), 4.10

(d, _J_=15.6 Hz, 1H), 3.28 (d, _J_=0.8 Hz, 1H), 3.01 (q, _J_=7.2 Hz, 1H), 1.68 (s, 3H), 1.30 (d, _J_=7.2 Hz, 3H). 13C-NMR (150 MHz, CD3OD) _δ_: 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) [M+Na]+ calcd for C19H19NNaO3=332.1262, found=332.1276.

(4B_S_,5_R_,7A_S_)-5,6,7A-TRIMETHYL-3-PHENYL-4B,5-DIHYDRO-1_H_-CYCLOPENTA[4,5]FURO[2,3–B]PYRIDIN-4(7A_H_)-ONE (20) [α]25D +107.6 (_c_ 1.0, CH3OH). IR (KBr) cm–1: 2965, 1653, 1454, 1432,

1217, 1041. 1H-NMR (400 MHz, CDCl3) _δ_: 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_=1.0 Hz, 1H), 2.90 (bq, _J_=7.0 Hz, 1H), 1.73 (s, 3H), 1.67 (s,

3H), 1.30 (d, _J_=7.0 Hz, 3H). 13C-NMR (100 MHz, CDCl3) _δ_: 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+H]+

calcd for C19H20NO2=294.1494, found=294.1485. (5A_R_,8_S_,8A_R_)-5A,7,8-TRIMETHYL-4-PHENYL-2,5A,8,8A-TETRAHYDRO-1_H_-CYCLOPENTA[4,5]FURO[3,2–C]PYRIDIN-1-ONE (_ENT_-CITRIDONE A, 21) [α]23D

+77.6 (_c_ 0.1, CH3OH). IR (KBr) cm–1: 2924, 1652, 1431, 1204, 1034, 765, 697. 1H-NMR (400 MHz, CDCl3) _δ_: 7.54–7.51 (m, 2H), 7.47–7.34 (m, 4H), 5.43 (dq, _J_=1.4 Hz, 1H), 3.33 (bs, 1H),

2.88 (bq, _J_=7.0 Hz, 1H), 1.74 (s, 3H), 1.70 (s, 3H), 1.30 (d, _J_=7.2 Hz, 3H). 13C-NMR (100 MHz, CDCl3) _δ_: 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+H]+ calcd for C19H20NO2=294.1494, found=294.1488.

(5A_S_*,8A_S_*)-4-PHENYL-2,5A,8,8A-TETRAHYDRO-1_H_-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) _δ_: 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_=5.2 Hz, 1H), 6.05 (d, _J_=5.2 Hz, 1H), 5.89 (dd, _J_=5.2, 2.4 Hz, 1H), 4,15 (t, _J_=7.8 

Hz, 1H), 2.90 (d, _J_=7.8 Hz, 1H), 2.85 (t, _J_=2.4 Hz, 1H). 13C-NMR (150 MHz, CDCl3) _δ_: 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) [M+Na]+ calcd for C16H13NNaO2=274.0844, found=274.0841. (4B_S_*,7A_S_*)-3-PHENYL-4B,5-DIHYDRO-1_H_-CYCLOPENTA[4,5]FURO[2,3–B]PYRIDIN-4(7A_H_)-ONE (29) IR (KBr) cm–1: 3020, 2929, 2857,

2400, 1642, 1597, 1477, 1216. 1H-NMR (400 MHz, CD3OD) _δ_: 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_=8.0 Hz, 1H), 5.92-5.89 (m, 1H), 4.14 (td,

_J_=8.0, 2.4 Hz, 1H), 2.89 (ddt, _J_=17.6, 8.0, 2.4 Hz, 1H), 2.69 (dt, _J_=17.6, 2.4 Hz, 1H). 13C-NMR (150 MHz, CD3OD) _δ_: 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) [M+H]+ calcd for C16H14NO2=252.1024, found=252.1012. ASSAY FOR MICONAZOLE-POTENTIATING ACTIVITY AGAINST _C. ALBICANS_ USING PAPER DISKS _C. albicans_ ATCC 64548

was inoculated into a 50-ml test tube containing 10 ml 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 (6 mm; ADVANTEC,

Tokyo, Japan) containing a 20-μg sample were placed on plates A and B, which were incubated at 27 °C for 24 h. Samples showing inhibition zones selectively on plate B were selected as

potentiators of miconazole activity against _C. albicans_. ASSAY FOR INHIBITION OF YELLOW PIGMENT PRODUCTION IN MRSA USING PAPER DISKS MRSA K-24 strain, a clinical isolate, was used as a

yellow pigment-producing strain. MRSA was cultured in Mueller-Hinton broth at 37 °C for 20 h and adjusted to 1 × 108 CFU per ml. The inoculum (100 μl) was spread on 25 ml 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 (100 × 140 mm). Paper disks (6 mm i.d.) containing a 20-μg sample were placed on the

plate and incubated at 37 °C for 72 h. 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 A mixture containing TYB (980 μl), a sample (10 μl) and MRSA (10 μl, at a final concentration of 1 × 107 CFU per ml) in a total volume of

1000 μl was incubated on a rotary shaker at 210 r.p.m. for 72 h at 37 °C. (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 (500 μl) at 60 °C for 2 h in the

dark. The absorbance of yellow pigments was determined at 450 nm using a Power Wave × 340. Inhibition of MRSA growth and yellow pigment production by a sample (% of control) is defined as

(absorbance-sample/absorbance-control) × 100. The IC50 values are defined as the sample concentrations that cause 50% inhibition of MRSA growth and yellow pigment production. REFERENCES *

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2228–2231 (2012). Article  CAS  PubMed  Google Scholar  Download references 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan Takashi Fukuda, Kenta

Shimoyama, Tohru Nagamitsu & Hiroshi Tomoda Authors * Takashi Fukuda View author publications You can also search for this author inPubMed Google Scholar * Kenta Shimoyama View author

publications You can also search for this author inPubMed Google Scholar * Tohru Nagamitsu View author publications You can also search for this author inPubMed Google Scholar * Hiroshi

Tomoda View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Hiroshi Tomoda. ADDITIONAL INFORMATION Supplementary

Information accompanies the paper on The Journal of Antibiotics website SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION (DOC 565 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Fukuda, T., Shimoyama, K., Nagamitsu, T. _et al._ Synthesis and biological activity of Citridone A and its derivatives. _J Antibiot_ 67, 445–450 (2014).

https://doi.org/10.1038/ja.2014.14 Download citation * Received: 16 December 2013 * Revised: 22 January 2014 * Accepted: 27 January 2014 * Published: 19 March 2014 * Issue Date: June 2014 *

DOI: https://doi.org/10.1038/ja.2014.14 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * 3-phenyl-4-hydroxy-2-pyridone * fungal metabolite *

miconazole potentiator * MRSA * staphyloxanthin inhibitor