Synthesis and structure-activity relationship study of NBRI16716B, an antitumor natural product

Article abstract of DOI:10.1248/cpb.c15-00200

The total synthesis of NBRI16716B (2), a naturally occurring modulator of tumor-stroma interactions, was successfully achieved. Using this synthetic route, a dehydroxy analogue (21) and a derivative lacking the 5-hydroxy-3-methylpentenoyl side chain (22) became accessible. A preliminary structure-activity relationship study to unveil the structural requirements for selective inhibition of tumor cells cocultured with stromal cells revealed that both of the hydroxamate structures of 2 are indispensable, whereas the 5-hydroxy- 3-methylpentenoyl side chain is not essential.

Full text of DOI:10.1248/cpb.c15-00200

Vol. 63, No. 6
Chem. Pharm. Bull. 63, 463–468 (2015)
463
Regular Article
Synthesis and Structure–Activity Relationship Study of NBRI16716B,
an Antitumor Natural Product
a
a
b
a
,a
Hikaru Abe, Chiharu Sakashita, Manabu Kawada, Akio Nomoto, Takumi Watanabe,* and
,
a
Masakatsu Shibasaki*
a
Institute of Microbial Chemistry (BIKAKEN), Tokyo; 3–14–23 Kamiosaki, Shinagawa-ku, Tokyo 141–0021,
b
Japan: and Institute of Microbial Chemistry (BIKAKEN), Numazu; 18–14 Miyamoto, Numazu, Shizuoka 410–0301,
Japan.
Received March 5, 2015; accepted March 14, 2015
The total synthesis of NBRI16716B (2), a naturally occurring modulator of tumor–stroma interactions,
was successfully achieved. Using this synthetic route, a dehydroxy analogue (21) and a derivative lacking
the 5-hydroxy-3-methylpentenoyl side chain (22) became accessible. A preliminary structure–activity rela-
tionship study to unveil the structural requirements for selective inhibition of tumor cells cocultured with
stromal cells revealed that both of the hydroxamate structures of 2 are indispensable, whereas the 5-hydroxy-
3
-methylpentenoyl side chain is not essential.
Key words antitumor agent; natural product; diketopiperazine; tumor–stroma interaction; structure–activity
relationship
Tumor tissues comprise not only of tumor cells, but also discovered from the fermentation broth of fungal strain Peri-
1)
the surrounding stroma made up of normal cells, including sporiopsis melioloidesꢀ Mer-f16716ꢀ displayingꢀ moreꢀ potentꢀ
endothelialꢀ cells,ꢀ fibroblast-likeꢀ cellsꢀ (termedꢀ stromalꢀ cells),ꢀ anti-proliferativeꢀactivityꢀagainstꢀDU-145ꢀprostateꢀcancerꢀcellsꢀ
2)
and extracellular matrix. Stromal cells regulate the growth cocultured with PrSCs human prostate stromal cells than to-
of adjacent tumor cells either positively or negatively via di- wardꢀmonoculturedꢀDU-145ꢀcells.ꢀIndeed,ꢀevidenceꢀsupportsꢀaꢀ
rectꢀ orꢀ indirectꢀ communicationꢀ influencedꢀ byꢀ cellꢀ adhesionꢀ close relationship between the prostate stroma and the growth
3
–6)
16,17)
or secreted factors (tumor–stroma interactions).
Signals and metastasis of surrounding tumor cells.
Of the three
transmitted from stromal cells are particularly interesting as compounds, NBRI16716B had the most potent and selective in
the machineries come from normal cells in which mutations vitro activity under the cocultured conditions described above,
of participating molecules occur less frequently than those whereas NBRI16716C exhibited weak activity, indicating that
2
from tumor cells. Hence, the signal molecules from stromal the OH group at the R position (Fig. 1) is crucial. Notably,
cells responsible for controlling tumor growth could be novel NBRI16716A and B displayed in vivo antitumor activity to-
molecular targets of antitumor agents with a lower tendency to wardꢀ tumorꢀ modelꢀ miceꢀ inoculatedꢀ withꢀ DU-145ꢀ andꢀ PrSCsꢀ
develop resistance, which prompted us to screen for modula- cells, which renders these compounds potential candidates
7
)
tors of tumor–stroma interactions.
ofꢀ leadꢀ forꢀ anticancerꢀ drugs.ꢀ Althoughꢀ insulin-likeꢀ growthꢀ
Toward this end, we constructed an assay system to select factor-Iꢀ secretedꢀ byꢀ humanꢀ prostateꢀ stromalꢀ cellsꢀ areꢀ knownꢀ
8
,18)
molecules that inhibit the growth of tumor cells cocultured to promote the growth of human prostate cancer cells,
with stromal cells more potently than that of monocultured no evidence showing that it is the direct molecular target of
8
,9)
tumor cells. ꢀSinceꢀtumor-stromaꢀinteractionsꢀareꢀaꢀrelativelyꢀ NBRI16716s has been obtained. Herein we disclose a synthetic
newꢀresearchꢀtopicꢀinꢀtheꢀfieldꢀofꢀoncology,ꢀkeyꢀsignalingꢀmol- route to NBRI16716B (2), with which structurally related de-
ecules involved in these interactions are currently under active rivatives are accessible, thereby paving the way to preliminary
investigation. A phenotypic assay system of this type would structure–activity relationship (SAR) studies.
require identifying the molecular targets of hit compounds,
which would provide insight into the communication between Results and Discussion
tumor and stroma cells. Natural products were used as the
Total Synthesis of NBRI16716B (2) NBRI16716B (2)
major source for our screening platform; the structural variety has been reported as a degradation product of isotriornitin, a
of natural products is expected to bait a diverse array of coun-
terpart proteins. In fact, several natural products exhibit the
1
0)
desired activity and selectivity, including phthoxazolin A,
11)
12)
13,14)
NBRI23477 A and B, leucinostatins, and intervenolin.
15)
Two of NBRI16716s (Fig. 1), having a diketopiperazine moi-
ety as the core structure, are also among this class.
The structures of NBRI16716A (1), B (2), and C (3) share
an identical molecular framework, but differ in OH func-
tionality at the amide nitrogen. These natural products were
AkioꢀNomotoꢀ(DeceasedꢀonꢀNovemberꢀ13,ꢀ2014.)
Fig. 1. Structure of NBRI16716s
*
ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀtwatanabe@bikaken.or.jp;ꢀmshibasa@bikaken.or.jp
©
2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 6 (2015)
Reagentsꢀandꢀconditions:ꢀ(a)ꢀBocHNOAc,ꢀDEAD,ꢀPPh , toluene rt, 20h, 72%; (b) H , 10% Pd/C, MeOH, rt, 1h; (c) 7,ꢀWSC·HCl,ꢀHOBt,ꢀDIPEA,ꢀCH Cl , rt, 4h, 41% for
3
2
2
2
2
steps; (d) H ,ꢀ10%ꢀPd/C,ꢀMeOH,ꢀrt,ꢀ3ꢀh;ꢀ(e)ꢀDIPEA,ꢀMeOH,ꢀrt,ꢀ48ꢀh,ꢀ24%ꢀforꢀ2ꢀsteps;ꢀ(f)ꢀTFA,ꢀCH Cl , 0°C, 1.5h; (g) 12,ꢀCOMU,ꢀDIPEA,ꢀDMF,ꢀrt,ꢀ3.5ꢀh,ꢀ21%ꢀforꢀ2ꢀsteps.
2 2 2
Chart 1. Total Synthesis of NBRI16716B
19)
natural product produced by Epicoccus purpurascens. It was cal data and biological activity (vide infra) of the synthetic
15)
also found as a natural product by us, however, no synthetic sample were identical to those of the natural product.
study has been reported to date. The synthetic strategy in Synthesis of NBRI16716B Derivatives To assess the
thisꢀ studyꢀ isꢀ straightforwardꢀ (Chartꢀ 1):ꢀ theꢀ diketopiperazineꢀ importance of the OH groups on the nitrogen of the amide
skeleton was constructed by dimerization of suitably protected functionalities,ꢀ andꢀ theꢀ 5-hydroxy-3-methylpentenoylꢀ side-
N-hydroxyornithineꢀ derivativesꢀ andꢀ subsequentꢀ cyclization.ꢀ chain, we performed additional SAR studies taking advan-
The two hydroxylamine moieties were then differentially tage of the synthetic method to access the mother compound
acylated to form the whole framework of NBRI16716B (2). NBRI16716B.ꢀ Atꢀ first,ꢀ theꢀ dehydroxyꢀ derivativeꢀ 21 was pre-
Toward this end, the hydroxyl group of commercially avail- pared as illustrated in Chart 2. Initially, the hydroxyl group
able N-Cbz-norvalineꢀ (4) was converted to the N-Boc-O-Ac- ofꢀ Fmoc-norvalinꢀ methylꢀ esterꢀ 13 was substituted with the
hydroxyamino functionality by the Mitsunobu reaction to N-Cbz-O-tert-butyldimethylsilylꢀ (TBS)-hydroxyaminoꢀ groupꢀ
give 5. The Cbz group was then removed by conventional by a Mitsunobu reaction to give 14, from which the Fmoc
methods to afford a free amino acid 6, which was condensed protecting group was removed by treatment with piperidine
with the norvaline derivative 7ꢀusingꢀWSC·HClꢀ(1-ethyl-3-(3- to afford an intermediate with an unmasked amino group
dimethylaminopropyl)carbodiimide hydrochloride) as a cou- (15). Next, the amide linkage between the free primary amino
pling reagent. The coupling product was obtained with many group of 15 and the carboxyl group of a known ornithine
unidentifiedꢀbyproductsꢀtoꢀresultꢀinꢀmoderateꢀyield.ꢀTheꢀdipep- derivative 16 was formed with WSC·HCl. The resulting fully
tide 8 obtained as described above was subjected to standard protected dipeptide 17 was subjected to detachment of the
hydrogenolysis conditions, resulting in the diketopiperazine FmocꢀgroupꢀusingꢀEtNH with subsequent cyclization to yield
2
precursor 9ꢀwithoutꢀanyꢀdifficulty.ꢀSubsequentꢀtreatmentꢀofꢀ 9 the diketopiperazine compound 18. Hydrogenolysis removed
with N,N-diisopropylethylamineꢀ(DIPEA)ꢀledꢀtoꢀtheꢀformationꢀ the Cbz group to give 19, to which side chain precursor 20
of a diketopiperazine framework with concomitant removal of was attached with concomitant removal of the TBS groups
Ac protecting groups on the N-hydroxylꢀ moieties,ꢀ affordingꢀ to afford the desired dehydroxy analog 21. Regarding length
1
0. The uncyclized deacetyl compounds were found as by- and chemical yield of each step, the synthetic route in Chart 2
products.ꢀ Followingꢀ theꢀ removalꢀ ofꢀ Bocꢀ usingꢀ trifluoroaceticꢀ moreꢀefficientꢀthanꢀtheꢀoneꢀinꢀChartꢀ1.ꢀHowever,ꢀtheꢀsameꢀsetꢀ
acidꢀ (TFA),ꢀ introductionꢀ ofꢀ theꢀ 5-hydroxy-3-methylpentenoylꢀ of protecting groups in Chart 2 could not be employed upon
®
sideꢀchainꢀwasꢀachievedꢀusingꢀCOMU ꢀ ((1-cyano-2-ethoxy-2- synthesis of 2ꢀbecauseꢀofꢀextremelyꢀlowꢀconversionꢀofꢀtheꢀfirstꢀ
oxoethylidenaminooxy) ꢀd imethylamino-morpholino-carbeniumꢀ amide bond formation.
2
0)
hexafluorophosphate) as a coupling reagent, while at the
To evaluate the importance of the side chain moiety, the
same time the tert-butyldiphenylsilylꢀ (TBDPS)ꢀ groupꢀ wasꢀ diacetyl analog 22 was also synthesized by simple acetylation
uninstalled to accomplish the total synthesis of NBRI16716B of 11 (Chart 3).
(2).ꢀTheꢀhighꢀwaterꢀsolubilityꢀcausedꢀdifficultyꢀinꢀworkꢀupꢀtoꢀ
Biological Activity of NBRI16716B Derivatives In the
ended up with the moderate isolated yield. The physicochemi- next study, the selective inhibitory activity of the synthesized

Vol. 63, No. 6 (2015)
Chem. Pharm. Bull.
465
Reagentsꢀandꢀconditions:ꢀ(a)ꢀCbzHNOTBS,ꢀDEAD,ꢀPPh ,ꢀtoluene,ꢀTHF,ꢀrt,ꢀ18ꢀh,ꢀ51%;ꢀ(b)ꢀPiperidine,ꢀDMF,ꢀrt,ꢀ2.5ꢀh;ꢀ(c)ꢀ16,ꢀWSC·HCl,ꢀHOBt,ꢀDIPEA,ꢀCH Cl , rt, 6h,
3
2
2
3
8%ꢀforꢀ2ꢀsteps;ꢀ(d)ꢀEt NH, THF, rt for 5h, 40°C for 12h, 71%; (e) H , 10% Pd/C, MeOH, rt, 1h; (f) 20,ꢀHATU,ꢀHOBt,ꢀDIPEA,ꢀDMF,ꢀ0°C,ꢀ10ꢀh,ꢀ30%ꢀforꢀ2ꢀsteps.
2
2
Chartꢀ 2.ꢀ SynthesisꢀofꢀaꢀDehydroxyꢀDerivativeꢀofꢀNBRI16716Bꢀ(21)
Reagentsꢀandꢀconditions:ꢀ(a)ꢀAcCl,ꢀNaHCO , CH CN, 0°C, 1h, 79%.
3
3
Chartꢀ 3.ꢀ SynthesisꢀofꢀaꢀDiacetylꢀDerivativeꢀofꢀNBRI16716Bꢀ(22)
compounds against cancer cells cocultured with stromal cells
was examined. Originally, NBRI16716B (2) was discovered
by the assay system using human prostate cancer and stromal
cells,ꢀDU-145ꢀandꢀPrSCꢀcells,ꢀrespectively.ꢀCoculture-selectiveꢀ
growth inhibition of NBRI16716s was recently reported to be
more distinct for the combination of lung cancer and stromal
cells,ꢀA549ꢀandꢀnormalꢀhumanꢀlungꢀfibroblastꢀ(NHLF).ꢀInꢀtheꢀ
Fig.ꢀ 2.ꢀ Effectꢀ ofꢀ NBRI16716Bꢀ (2)ꢀ andꢀ Itsꢀ Derivativesꢀ (21 and 22) on
Coculture of Tumor Cells and Stromal Cells
Theꢀgrowthꢀofꢀtumorꢀcellsꢀcoculturedꢀwithꢀstromalꢀcellsꢀ(filled)ꢀorꢀthatꢀofꢀtumorꢀ
cells alone (open) in the presence of the indicated concentrations of the test com-
present study, the in vitro biological activity of the two ana- pounds (2ꢀ synthetic:ꢀ circle,ꢀ 2ꢀ natural:ꢀ square,ꢀ 21:ꢀ triangle,ꢀ 22:ꢀ invertedꢀ triangle)ꢀ
wasꢀ determinedꢀ byꢀ measuringꢀ fluorescenceꢀ intensityꢀ ofꢀ greenꢀ fluorescentꢀ proteinꢀ
logs (21, 22)ꢀwasꢀevaluatedꢀusingꢀtheꢀA549-NHLFꢀsystem,ꢀandꢀ
(GFP).ꢀA549ꢀhumanꢀlungꢀcancerꢀcellꢀlinesꢀwereꢀcoculturedꢀwithꢀNHLFꢀlungꢀstro-
compared with that of NBRI16716B alone (Fig. 2).
malꢀcells.ꢀDataꢀareꢀmeansꢀofꢀduplicateꢀdetermination.ꢀS.E.ꢀisꢀlessꢀthanꢀ10%.
The synthetic sample of NBRI16716B (2) displayed identical
coculturedꢀconditionsꢀ(withꢀNHLF)ꢀselectiveꢀgrowthꢀinhibitionꢀ Conclusion
towardꢀ A-549ꢀ (IC ;ꢀ synthetic:ꢀ 23.6ꢀµg/mLꢀ forꢀ co-cultured,ꢀ The total synthesis of NBRI16716B (2), a naturally occur-
6.5µg/mLꢀ forꢀ mono-cultured;ꢀ natural:ꢀ 22.7ꢀµg/mLꢀ forꢀ co- ring modulator of tumor–stroma interactions, was successfully
5
0
4
cultured, 41.8µg/mLꢀ forꢀ mono-cultured).ꢀ Diacetylꢀ analogꢀ 22 developed. This synthetic route rendered analogs 21 and 22
retainedꢀ theꢀ growth-inhibitoryꢀ activityꢀ andꢀ alsoꢀ exhibitedꢀ accessible, allowing for preliminary SAR studies to unveil the
substantially lower IC50 values in the presence of stromal cells structural requirements for selective inhibition of tumor cells
(51.7 µg/mL)ꢀ thanꢀ inꢀ theirꢀ absenceꢀ (79.2ꢀµg/mL),ꢀ suggestingꢀ cocultured with stromal cells. Further SAR studies using the
thatꢀ theꢀ 5-hydroxy-3-methylpentenoylꢀ sideꢀ chainꢀ moietyꢀ isꢀ aꢀ synthetic method described here as a key technique to develop
targetꢀ forꢀ structuralꢀ modificationꢀ inꢀ furtherꢀ SARꢀ studies.ꢀ Inꢀ anticancer leads and molecular probes to identify the primary
contrast, dehydroxy derivative 21 completely lost the activity. target of NBRI16716B are ongoing.
Thisꢀ finding,ꢀ togetherꢀ withꢀ theꢀ observationꢀ thatꢀ NBRI16716Cꢀ
lackingꢀ theꢀ OHꢀ groupꢀ onꢀ theꢀ nitrogenꢀ inꢀ theꢀ 5-hydroxy- Experimental
3
-methylpentenoylꢀsideꢀchainꢀdidꢀnotꢀinhibitꢀtheꢀgrowthꢀofꢀtheꢀ
General Remarks The reactions were performed in an
cells examined, indicates that the hydroxamate structures in oven-driedꢀ testꢀ tubeꢀ orꢀ roundꢀ bottomꢀ flaskꢀ withꢀ aꢀ Teflon-
both of the side chains are indispensable.
coated magnetic stirring bar unless otherwise noted. All
work-upꢀ andꢀ purificationꢀ proceduresꢀ wereꢀ carriedꢀ outꢀ withꢀ
reagent-gradeꢀ solventsꢀ underꢀ ambientꢀ atmosphere.ꢀ Infraredꢀ

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Chem. Pharm. Bull.
Vol. 63, No. 6 (2015)
(
IR) spectra were recorded on a JASCO FT/IR 4100 Fourier DIPEAꢀ(14.9ꢀµL,ꢀ85.5ꢀµmol) were added to the solution at 0°C,
transform infrared spectrophotometer. NMR was recorded on and the mixture was stirred at room temperature for 4h. The
1
13
JEOLꢀ ECS-400ꢀ ( H-NMR:ꢀ 400ꢀMHz,ꢀ C-NMR:ꢀ 100ꢀMHz)ꢀ reaction mixture was concentrated in vacuo, after which the
13
orꢀ onꢀ JEOLꢀ ECA-600ꢀ ( C-NMR:ꢀ 150ꢀMHz)ꢀ orꢀ onꢀ Brukerꢀ resultantꢀ residueꢀ wasꢀ dissolvedꢀ inꢀ AcOEt,ꢀ washedꢀ withꢀ 1ꢀM
AVANCEꢀ 500ꢀ ( C-NMR:ꢀ 125ꢀMHz)ꢀ spectrometers.ꢀ Chemicalꢀ HCl, saturated NaHCO , and brine successively. The organic
13
3
shiftsꢀ forꢀ protonꢀ areꢀ reportedꢀ inꢀ partsꢀ perꢀ millionꢀ downfieldꢀ layer was died over Na SO , and was concentrated to dry-
2
4
from tetramethylsilane and are referenced to residual protium ness.ꢀTheꢀresidueꢀwasꢀpurifiedꢀwithꢀpreparativeꢀTLCꢀ(CHCl3/
inꢀtheꢀNMRꢀsolventꢀ(CDCl :ꢀδꢀ7.26ꢀppm,ꢀCD OD:ꢀδ 3.30ppm, MeOH=10/1) to give 8 as a colorless oil (15.2mg, 23.3µmol)
3
3
13
26
DMSO-d :ꢀ2.49).ꢀForꢀ C-NMR,ꢀchemicalꢀshiftsꢀwereꢀreportedꢀ in 41% yield over 2 steps; [α]D −10.9 (c=1.43, MeOH); IR
inꢀ theꢀ scaleꢀ relativeꢀ toꢀ NMRꢀ solventꢀ (CDCl :ꢀ δ 77.0ppm, (neat) ν 3310, 2937, 1793, 1716, 1671, 1526, 1368cm ;ꢀHR-MSꢀ
CD OD:ꢀ δꢀ 49.0ꢀppm,ꢀ DMSO-d :ꢀ 39.7ꢀppm)ꢀ asꢀ anꢀ internalꢀ ref- (ESI)ꢀ Anal. Calcd for C H N NaO m/z 675.2853 [M+Na] ,
erence.ꢀ NMRꢀ dataꢀ areꢀ reportedꢀ asꢀ follows:ꢀ chemicalꢀ shifts,ꢀ Found 675.2837; H-NMRꢀ (400ꢀMHz,ꢀ CD OD)ꢀ δ:ꢀ 7.37–7.25ꢀ
6
−1
3
+
3
6
30 44
4
12
1
3
multiplicityꢀ (s:ꢀ singlet,ꢀ d:ꢀ doublet,ꢀ t:ꢀ triplet,ꢀ m:ꢀ multiplet,ꢀ br:ꢀ (m, 5H), 5.06 (2H, s), 4.42 (1H, dd, J=9.1, 4.7Hz), 4.14 (1H,
broad signal), coupling constant (Hz), and integration. Optical m), 3.78–3.50 (4H, m), 3.68 (3H, s), 2.18 (3H, s), 2.10 (3H,
13
rotationꢀ wasꢀ measuredꢀ usingꢀ aꢀ 2ꢀmLꢀ cellꢀ withꢀ aꢀ 1.0ꢀ dmꢀ pathꢀ s), 1.98–1.58 (8H, m), 1.96 (3H, s), 1.43 (9H, s); C-NMRꢀ
lengthꢀonꢀaꢀJASCOꢀpolarimeterꢀP-1030.ꢀHigh-resolutionꢀmassꢀ (100ꢀMHz,ꢀCD OD)ꢀ δ:ꢀ174.7,ꢀ173.7,ꢀ170.2,ꢀ158.3,ꢀ156.2,ꢀ138.2,ꢀ
3
spectraꢀ (HR-MS)ꢀ (electrosprayꢀ ionizationꢀ (ESI)-Orbitrap)ꢀ 129.5, 129.0, 128.8, 83.6, 67.6, 55.6, 53.1, 52.7, 50.5, 30.4, 29.4,
wereꢀmeasuredꢀonꢀThermoFisherꢀScientificꢀLTQꢀOrbitrapꢀXL.ꢀ 28.4, 24.6, 24.2, 20.2, 18.3, 18.2.
Centrifugal liquid–liquid partition chromatography (CPC)
Methyl (S)-5-(Acetoxy(tert-butoxycarbonyl)amino)-2-((S)-
wasꢀperformedꢀwithꢀaꢀCPC240ꢀsystemꢀ(SenshuꢀScientificꢀCo.,ꢀ 5-(N-acetoxyacetamido)-2-aminopentanamido)pentanoate
Ltd.).ꢀUnlessꢀotherwiseꢀnoted,ꢀmaterialsꢀwereꢀpurchasedꢀfromꢀ (9) To a solution of 8 (19.9mg, 30.5µmol)ꢀ inꢀ 0.44ꢀmLꢀ ofꢀ
commercialꢀsuppliersꢀandꢀwereꢀusedꢀwithoutꢀpurification.ꢀForꢀ MeOH was added 10% Pd/C (2mg), and the mixture was
reaction, tetrahydrofuran (THF), N,N-dimethylformamideꢀ stirred at room temperature for 3h under atmospheric pressure
(
DMF),ꢀCH CN,ꢀtoluene,ꢀAcOEt,ꢀandꢀCH Cl ꢀwereꢀpurifiedꢀbyꢀ of H .ꢀTheꢀcatalystꢀwasꢀfilteredꢀoffꢀwithꢀaꢀpadꢀofꢀCelite,ꢀandꢀ
3 2 2 2
passingꢀthroughꢀaꢀsolventꢀpurificationꢀsystemꢀ(GlassꢀContour).ꢀ theꢀfiltrateꢀwasꢀconcentratedꢀunderꢀreducedꢀpressureꢀtoꢀgiveꢀaꢀ
Dryꢀ 1,4-dioxane,ꢀ MeOH,ꢀ dimethylꢀ sulfoxideꢀ (DMSO),ꢀ andꢀ crude material containing 9, which was used for the succeed-
pyridine were purchased from Wako Pure Chemical Indus- ingꢀstepꢀwithoutꢀfurtherꢀpurification.
tries,ꢀLtd.ꢀ(Osaka,ꢀJapan)ꢀandꢀusedꢀasꢀreceived.
tert-Butyl Hydroxy(3-((2S,5S)-5-(3-(N-hydroxyacetami-
Methyl (S)-5-(Acetoxy(tert-butoxycarbonyl)amino)-2-(((ben- do)propyl)-3,6-dioxopiperazin-2-yl)propyl)carbamate (10)
zyloxy)carbonyl)amino) Pentanoate (5) To a solution of To a solution of 9ꢀ(allꢀtheꢀmaterialꢀobtainedꢀabove)ꢀinꢀ0.15ꢀmLꢀ
4
ꢀ (125ꢀmg,ꢀ 0.444ꢀmmol)ꢀ inꢀ 4.44ꢀmLꢀ ofꢀ tolueneꢀ wereꢀ addedꢀ ofꢀMeOHꢀwasꢀaddedꢀDIPEAꢀ(8.0ꢀµL,ꢀ45.9 ꢀµ mol). The mixture
BocNHOAc (84.8mg, 0.484mmol), PPh (150mg, 0.572mmol), was stirred at room temperature for 48h. The resultant white
3
andꢀ DEADꢀ (diethylꢀ azodicarboxylate,ꢀ 2.2ꢀmol/Lꢀ inꢀ toluene,ꢀ precipitates were collected, and washed with MeOH thorough-
2
50µL,ꢀ0.550ꢀmmol)ꢀatꢀ0°C.ꢀTheꢀmixtureꢀwasꢀstirredꢀatꢀroomꢀ ly to give 10 (2.9mg, 7.21µmol, 24% yield over 2 steps) as a
2
5
temperature for 20h and concentrated in vacuo. The resul- white powder; mp 174–176°C; [α]D –13.2 (c=0.155,ꢀ DMSO);ꢀ
tantꢀ residueꢀ wasꢀ purifiedꢀ withꢀ silicaꢀ gelꢀ columnꢀ chromatog- IR (neat) ν 3199, 2938, 1793, 1684, 1608, 1457, 1169cm ;
raphy (n-hexane/AcOEt=2/1 to 3/2) to give 5 as a colorless HR-MSꢀ (ESI)ꢀ Anal. Calcd for C H N NaO m/z 425.2012
oil (140mg, 0.319mmol) in 72% yield; [α] 6.75 (c=0.150, [M+Na] , Found 425.1997; H-NMRꢀ(600ꢀMHz,ꢀDMSO-d ) δ:ꢀ
CHCl ; IR (neat) ν 3349, 2978, 1792, 1716, 1527, 1455, 1366, 9.64 (1H, s), 9.11 (1H, s), 8.09 (1H, s), 8.08 (1H, s), 3.78 (2H,
−1
17
30
4
7
2
6
+
1
D
6
3
−1
1
4
254cm ;ꢀ HR-MSꢀ (ESI)ꢀ Anal. Calcd for C H N NaO m/z m), 3.43 (2H, t, J=6.5Hz), 3.30 (2H, t, J=6.6Hz), 1.93 (3H,
21 30 2 8
1 13
+
61.1900 [M+Na] , Found 461.1894; H-NMRꢀ (400ꢀMHz,ꢀ s), 1.69–1.50 (6H, m), 1.37 (9H, s) 1.23 (2H, m); C-NMRꢀ
CD OD)ꢀ δ:ꢀ 7.37–7.26ꢀ (m,ꢀ 5H),ꢀ 6.55ꢀ (1H,ꢀ d,ꢀ J=16.0Hz), 5.09 (150ꢀMHz,ꢀ DMSO-d ) δ:ꢀ 170.8,ꢀ 168.4,ꢀ 156.4,ꢀ 79.9,ꢀ 54.4,ꢀ 54.3,ꢀ
3
6
(
2H, s), 4.21 (1H, dd, J=8.6, 5.1Hz), 3.71 (3H, s), 3.58 (2H, 50.6, 47.3, 30.9, 28.6, 22.8, 22.6, 20.9.
13
m), 1.65 (1H, m), 1.74–1.58 (3H, m), 1.41 (9H, s); C-NMRꢀ
N-Hydroxy-N-(3-((2S,5S)-5-(3-(hydroxyamino)propyl)-
(
100ꢀMHz,ꢀCD OD)ꢀ δ:ꢀ174.4,ꢀ170.1,ꢀ158.6,ꢀ156.2,ꢀ138.2,ꢀ129.5,ꢀ 3,6-dioxopiperazin-2-yl)propyl)acetamide (11) To a mix-
3
129.0, 128.8, 83.7, 67.6, 54.9, 52.7, 50.6, 29.6, 28.4, 24.6, 18.2.
tureꢀ ofꢀ 0.5ꢀmLꢀ ofꢀ TFAꢀ andꢀ 0.5ꢀmLꢀ ofꢀ CH Cl was added 10
2 2
Methyl (S)-5-(Acetoxy(tert-butoxycarbonyl)amino)-2-amino- (20.0mg, 49.7µmol), and the solution was stirred at room tem-
pentanoate (6) To a solution of 5 (25.0mg, 57.0µmol) in perature for 1.5h at 0°C and was concentrated to dryness. The
.2ꢀmLꢀ ofꢀ MeOHꢀ wasꢀ addedꢀ 10%ꢀ Pd/Cꢀ (2.5ꢀmg),ꢀ andꢀ theꢀ residue was suspended in toluene and concentrated, which was
1
mixture was stirred at room temperature for 4h under atmo- repeated 5 times. The resultant residue was used for the next
spheric pressure of H .ꢀ Theꢀ catalystꢀ wasꢀ filteredꢀ offꢀ withꢀ aꢀ reactionꢀwithoutꢀfurtherꢀpurification.
2
padꢀofꢀCelite,ꢀandꢀtheꢀfiltrateꢀwasꢀconcentratedꢀunderꢀreducedꢀ
pressure to give a crude material containing 6, which was inꢀ0.25ꢀmLꢀofꢀDMFꢀwereꢀaddedꢀDIPEAꢀ(28.0ꢀµL,ꢀ0.159ꢀmmol)ꢀ
NBRI16716B (2) To a solution of 12 (36.6mg, 99.4µmol)
®
usedꢀforꢀtheꢀsucceedingꢀreactionꢀwithoutꢀfurtherꢀpurification.
andꢀ COMU (43.0mg, 99.4µmol) at 0°C, and the resultant
Methyl (S)-5-(Acetoxy(tert-butoxycarbonyl)amino)-2-((S)-5- mixture was stirred at the same temperature for 5min. Then,
N-acetoxyacetamido)-2-(((benzyloxy)carbonyl)amino)pen- 10 obtained from the previous step was added to the solu-
(
tanamido)pentanoate (8) To a solution of 7 (25.0mg, tion and stirring continued at room pemperature for 3.5h.
8.2µmol)ꢀinꢀ0.5ꢀmLꢀofꢀCH Cl ꢀwasꢀaddedꢀHOBtꢀ(1-hydroxy- After evaporation of the solvent, the residue was partitioned
6
2
2
benzotriazole, 11.5mg, 85.1µmol) at 0°C, and the mixture between H Oꢀ andꢀ AcOEt.ꢀ Afterꢀ concentratingꢀ theꢀ aqueousꢀ
2
was stirred for 30min at room temperature. Then, 6 (all the layer,ꢀtheꢀcrudeꢀsampleꢀthusꢀobtainedꢀwasꢀpurifiedꢀwithꢀsilicaꢀ
material obtained above), WSC·HCl (13.1mg, 68.3µmol), gel column chromatography (CHCl /MeOH=6/1) and the suc-
3

Vol. 63, No. 6 (2015)
Chem. Pharm. Bull.
467
2
6
ceeding CPC (CHCl /MeOH/H O=5/6/4, ascending mode) 2 steps; [α]D 6.85 (c=0.18, CHCl ); IR (KBr) ν 3424, 2953,
3
2
3
−1
to give 2 (4.4mg, 21% yield over 2 steps) as a white powder; 1708, 1658, 1532, 1450, 1253cm ;ꢀHR-MSꢀ(ESI)ꢀAnal. Calcd
mp 156–158°C; [α] −29.0 (c=0.11, MeOH) (lit. [α] −21.3 for C H N O Si m/z 789.3895 [M+H] , Found 789.3889;
c=0.4, MeOH)); IR (KBr) ν 3421, 1684, 1457cm ;ꢀ HR-MSꢀ H-NMRꢀ (400ꢀMHz,ꢀ CDCl ) δ:ꢀ 7.75ꢀ (2H,ꢀ d,ꢀ J=7.5Hz), 7.59
ESI)ꢀ Anal. Calcd for C H N NaO m/z 437.2012 [M+Na] , (2H, d, m), 7.40–7.16 (9H, m), 5.80 (1H, br), 5.65 (1H, brd,
1
9
+
D
D
42 57
4
9
−1
1
(
3
+
(
18
30
4
7
1
Found 425.2007; H-NMRꢀ (400ꢀMHz,ꢀ CD OD)ꢀ δ:ꢀ 6.28ꢀ (1H,ꢀ J=8.0Hz), 5.12 (2H, s), 4.54–4.46 (3H, m), 4.36 (1H, d,
3
s), 3.99 (2H, t, J=5.2Hz), 3.68 (2H, t, J=6.6Hz), 3.67–3.60 J=6.7Hz), 4.20 (1H, t, J=7.1Hz), 3.68 (3H, s), 3.68–3.48 (3H,
(
1
6H, m), 2.34 (2H, t, J=6.4Hz), 2.08 (3H, s), 2.05 (2H, br), m), 3.11 (2H, m), 1.97 (3H, s), 1.93–1.55 (8H, m), 0.87 (9H,
1
3
1
3
.85–1.68 (8H, m); C-NMRꢀ (150ꢀMHz,ꢀ CD OD)ꢀ δ:ꢀ 172.4,ꢀ s), 0.07 (3H, s); C-NMRꢀ (125ꢀMHz,ꢀ CDCl ) δ:ꢀ 172.6,ꢀ 172.0,ꢀ
3
3
1
69.0, 168.2, 151.2, 116.5, 59.6, 54.4, 43.5, 31.0, 30.9, 29.3, 171.1, 158.3, 156.2, 144.0, 143.8, 141.3, 135.9, 128.5, 128.3,
2.2, 22.0, 18.9, 17.5. 127.7, 127.1, 125.2, 120.0, 67.9, 67.0, 52.9, 52.3, 52.1, 51.5, 47.2,
NMRꢀ dataꢀ fromꢀ aꢀ naturalꢀ sample:ꢀ H-NMRꢀ (400ꢀMHz,ꢀ 37.9, 30.9, 28.8, 25.6, 23.3, 22.5, 17.8, −5.1.
CD OD)ꢀ δ:ꢀ 6.28ꢀ (1H,ꢀ s),ꢀ 3.99ꢀ (2H,ꢀ t,ꢀ J=5.2Hz), 3.68 (2H, t, Benzyl (3-((2S,5S)-5-(3-Acetamidopropyl)-3,6-dioxopi-
J=6.6Hz), 3.67–3.60 (6H, m), 2.34 (2H, t, J=6.5Hz), 2.09 perazin-2-yl)propyl)((tert-butyldimethylsilyl)oxy)carbamate
2
1
3
13
(
3H, s), 2.05 (2H, br), 1.85–1.68 (8H, m); C-NMRꢀ(150ꢀMHz,ꢀ (18) To a solution of 17 (10.0mg, 12.7µmol)ꢀ inꢀ 0.5ꢀmLꢀ ofꢀ
CD OD)ꢀ δ:ꢀ 172.5,ꢀ 169.1,ꢀ 168.3,ꢀ 151.3,ꢀ 116.5,ꢀ 59.6,ꢀ 54.4,ꢀ 43.5,ꢀ THFꢀwasꢀaddedꢀ0.2ꢀmLꢀofꢀEt NH. The mixture was stirred at
3
2
31.0, 30.9, 29.3, 22.2, 22.0, 18.9, 17.5.
room temperature for 5h and at 40°C for 12h. The solution
Methyl (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)- was concentrated in vacuo,ꢀandꢀEt O and hexane was added to
2
5
-(((benzyloxy)carbonyl)((tert-butyldimethylsilyl)oxy)- the resultant residue to give a white solid. After through wash
amino)pentanoate (14) To
.35ꢀmmol)ꢀinꢀ10ꢀmLꢀofꢀtolueneꢀandꢀ2ꢀmLꢀofꢀTHFꢀwereꢀaddedꢀ phous (4.8mg, 9.00µmol) in 71% yield; [α]D −35.7 (c=0.23,
CbzNHOTBS (381mg, 1.35mmol), PPh (461mg, 1.76mmol), CHCl ); IR (KBr) ν 3209, 3069, 2954, 1672, 1456, 1338,
a solution of 13 (500mg, withꢀEt O and n-hexane,ꢀ18 was obtained as a colorless amor-
2
2
6
1
3
3
−1
andꢀ DEADꢀ (0.78ꢀmL,ꢀ 1.69ꢀmmol),ꢀ atꢀ roomꢀ temperatureꢀ suc- 1258cm ;ꢀ HR-MSꢀ (ESI)ꢀ Anal. Calcd for C H N NaO Si
cessively. The mixture was stirred at the temperature for 18h, m/z 557.2771 [M+Na] , Found 557.2766; H-NMRꢀ (400ꢀMHz,ꢀ
andꢀ concentrated.ꢀ Theꢀ resultantꢀ residueꢀ wasꢀ purifiedꢀ withꢀ CDCl ) δ:ꢀ7.36–7.30ꢀ(5H,ꢀm),ꢀ6.78ꢀ(1H,ꢀbr),ꢀ6.45ꢀ(1H,ꢀbr),ꢀ6.11ꢀ
2
6
42
4
6
+
1
3
silica gel column chromatography (n-hexane/AcOEt=1/1) to (1H, br), 5.13 (2H, s), 4.03 (1H, m), 3.96 (1H, m), 3.52 (2H, t,
give 14 as a colorless oil (437mg, 0.691mmol) in 51% yield; J=6.4Hz), 3.22 (2H, m), 1.96 (3H, s), 1.89–1.53 (8H, m), 0.88
2
6
13
[
1
α] 8.22 (c=0.13, MeOH); IR (KBr) ν 3339, 2952, 1725, (9H, s), 0.08 (3H, s); C-NMRꢀ (125ꢀMHz,ꢀ CDCl ) δ:ꢀ 170.6,ꢀ
D 3
1
−
522, 1450, 1252, 1213cm ;ꢀ HR-MSꢀ (ESI)ꢀ Anal. Calcd 167.92, 167.88, 158.5, 135.8, 128.6, 1287.5, 128.4, 68.1, 54.6,
for C H N O Si m/z 633.2996 [M+H] , Found 633.2991; 54.4, 51.4, 38.9, 31.0, 30.8, 25.7, 24.8, 23.2, 21.8, 17.8, −5.1.
+
35
45
2
7
1
H-NMRꢀ (400ꢀMHz,ꢀ CDCl ) δ:ꢀ 7.77ꢀ (2H,ꢀ d,ꢀ J=7.3Hz), 7.60
N-(3-((2S,5S)-5-(3-(((tert-Butyldimethylsilyl)oxy)amino)-
2H, d, J=6.9Hz), 7.42–7.30 (9H, m), 5.32 (1H, d, J=7.8Hz), propyl)-3,6-dioxopiperazin-2-yl)propyl)acetamide (19) To
.15 (2H, s), 4.44–4.34 (3H, m), 4.23 (1H, t, J=6.9Hz), 3.73 a solution of 18 (40mg, 74.8µmol)ꢀ inꢀ 1ꢀmLꢀ ofꢀ MeOHꢀ wasꢀ
3H, s), 3.53 (2H, m), 1.88–1.60 (4H, m), 0.90 (9H, s), 0.10 (3H, added 10% Pd/C (4.0mg), and the mixture was stirred at room
3
(
5
(
1
3
s); C-NMRꢀ (100ꢀMHz,ꢀ CDCl ) δ:ꢀ 172.8,ꢀ 158.4,ꢀ 156.0,ꢀ 144.0,ꢀ temperature for 1h under atmospheric pressure of H . The cat-
3
2
1
1
−
43.8, 141.4, 136.0, 128.6, 128.5, 128.4, 127.8, 127.2, 125.2, alystꢀwasꢀfilteredꢀoffꢀwithꢀaꢀpadꢀofꢀCelite,ꢀandꢀtheꢀfiltrateꢀwasꢀ
20.1, 68.0, 67.2, 53.7, 52.5, 51.6, 47.2, 29.7, 25.8, 22.2, 17.9, concentrated under reduced pressure to give a crude material
5.0. containing 19, which was used for the succeeding step without
Methyl (S)-2-Amino-5-(((benzyloxy)carbonyl)((tert-butyl- furtherꢀpurification.
dimethylsilyl)oxy)amino)pentanoate (15) To a solution of (E)-N-(3-((2S,5S)-5-(3-Acetamidopropyl)-3,6-dioxopiper-
4ꢀ (96.0ꢀmg,ꢀ 0.152ꢀmmol)ꢀ inꢀ 1ꢀmLꢀ ofꢀ DMFꢀ wasꢀ addedꢀ piperi- azin-2-yl)propyl)-N,5-dihydroxy-3-methylpent-2-enamide
1
dine (17.0µL,ꢀ 0.167ꢀmmol).ꢀ Theꢀ mixtureꢀ wasꢀ stirredꢀ atꢀ roomꢀ (21) To a solution of 20 16.1mg, 65.9µmol)ꢀ inꢀ 0.5ꢀmLꢀ ofꢀ
temperature for 2.5h. Then the mixture was concentrated to DMFꢀwereꢀaddedꢀDIPEAꢀ12.5ꢀµL,ꢀ71.8 ꢀµ mol)ꢀandꢀHATUꢀ(O-7-
dryness to give a crude material containing 15, which was azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumꢀ hexafluo-
used for the succeeding step after short column chromatogra- rophosphate, 27.3mg, 71.8µmol) at 0°C, and the mixture
phy (CHCl /MeOH=10/1).
was stirred for 30min at room temperature. Then, 19 (all
(S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)car- the material obtained above), was added to the solution at
3
Methyl
bonyl)amino)-5-acetamidopentanamido)-5-(((benzyloxy)- 0°C, and the mixture was stirred at room temperature for
carbonyl)((tert-butyldimethylsilyl)oxy)amino)pentanoate 10h. The reaction mixture was concentrated in vacuo, and
(17) To a solution of 16ꢀ (72.3ꢀmg,ꢀ 0.182ꢀmmol)ꢀ inꢀ 0.9ꢀmLꢀ theꢀ residueꢀ wasꢀ purifiedꢀ withꢀ silicaꢀ gelꢀ columnꢀ chromatogra-
of CH Cl was added HOBt (31.0mg, 0.229mmol) at 0°C, phy (CHCl /MeOH=3/1) and repeated CPC (ascending mode,
2
2
3
and the solution was stirred at room temperature for 0.5h. CHCl /MeOH/H O=5/6/4; descending mode, CHCl /MeOH/
3
2
3
After successive addition of 15 (all the material obtained H O=5/6/4; descending mode, CHCl /MeOH/H O=2/2/1) to
2
3
2
above),ꢀWSC·HClꢀ(35.0ꢀmg,ꢀ0.182ꢀmmol),ꢀandꢀDIPEAꢀ(32.0 ꢀµ L,ꢀ give 21 as a white powder (7.2mg, 18.1µmol) in 30% yield
2
3
0
.184mmol) at 0°C, the mixture was stirred at room tem- over 2 steps; mp 149–151°C; [α] −16.0 (c=0.165, MeOH);
D
−
1
perature for 6h. Then, the mixture was diluted with CH Cl2 IR (KBr) ν 3242, 1654, 1457cm ;ꢀHR-MSꢀ(ESI)ꢀAnal. Calcd
2
+
and washed with 1M HCl, saturated NaHCO , and brine. The for C H N NaO m/z 421.2063 [M+Na] , Found 421.2058;
3
18 30
4
6
1
organic layer was dried over Na SO , and was concentrated
H-NMRꢀ (400ꢀMHz,ꢀ CD OD)ꢀ δ:ꢀ 6.29ꢀ (1H,ꢀ br),ꢀ 3.98ꢀ (2H,ꢀ m),ꢀ
2
4
3
in vacuo.ꢀ Theꢀ resultantꢀ residueꢀ wasꢀ purifiedꢀ withꢀ silicaꢀ gelꢀ 3.70–3.64 (2H, m), 3.67–3.61 (2H, m), 3.17 (2H, m), 2.34 (2H,
column chromatography (n-hexane/AcOEt=1/1) to give 17 as t, J=6.5Hz), 2.05 (2H, br), 1.91 (3H, s), 1.84–1.50 (8H, m);
13
a colorless amorphous (41.3mg, 52.3µmol) in 38% yield over
C-NMRꢀ (125ꢀMHz,ꢀ CD OD)ꢀ δ:ꢀ 173.4,ꢀ 170.4,ꢀ 169.6,ꢀ 152.6,ꢀ
3

4
68
Chem. Pharm. Bull.
Vol. 63, No. 6 (2015)
120.9, 117.8, 60.9, 55.7, 44.8, 44.6, 40.0, 32.5, 25.9, 23.4, 22.6,
Acknowledgment Weꢀ thankꢀ Dr.ꢀ Ryuichiꢀ Sawa,ꢀ Ms.ꢀ Yu-
18.8.
mikoꢀKubota,ꢀandꢀMs.ꢀYukoꢀTakahashiꢀ(BIKAKEN)ꢀforꢀcol-
N,N′-(((2S,5S)-3,6-Dioxopiperazine-2,5-diyl)bis(pro- lection of spectral data.
21)
pane-3,1-diyl))bis(N-hydroxyacetamide) (22)
To a mix-
tureꢀ ofꢀ TFAꢀ (0.55ꢀmL)ꢀ inꢀ CH Cl ꢀ (0.55ꢀmL)ꢀ wasꢀ addedꢀ 11
Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
2
2
(
2
20.5mg, 54.7µmol) at 0°C, then the solution was stirred for interest.
h at the temperature. A crude mixture containing 21 was
obtained after concentration, which was used for the next re-
Supplementary Materials The online version of this ar-
actionꢀwithoutꢀfurtherꢀpurification.ꢀTheꢀresidueꢀwasꢀdissolvedꢀ ticle contains supplementary materials.
inꢀ 0.5ꢀmLꢀ ofꢀ CH CN, and NaHCO (18.4mg, 219µmol) AcCl
3
3
(
3.9 µL,ꢀ 54.7ꢀµmol) was added to the solution at 0°C, then References
the solution was stirred for 1h at the temperature. A white
powder of 22 was precipitated out as the reaction proceeded.
The white powder was corrected on funnel, which was thor-
oughly washed with CH CN and MeOH to give pure sample
of 22 (14.9mg, 43.3µmol) in 79% yield; mp 207–209°C;
1) Wernert N., Virchows Arch., 430, 433–443 (1997).
ꢀ
2)ꢀ TuxhornꢀJ.ꢀA.,ꢀAyalaꢀG.ꢀE.,ꢀSmithꢀM.ꢀJ.,ꢀSmithꢀV.ꢀC.,ꢀDangꢀT.ꢀD.,ꢀ
RowleyꢀD.ꢀR.,ꢀClin. Cancer Res., 8, 2912–2923 (2002).
3
4)
)
Wiseman B. S., Werb Z., Science, 296, 1046–1049 (2002).
Hwang R. F., Moore T., Arumugam T., Ramachandran V., Amos K.
D.,ꢀRiveraꢀA.,ꢀJiꢀB.,ꢀEvansꢀD.ꢀB.,ꢀLogsdonꢀC.ꢀD.,ꢀCancer Res., 68,
3
2
5
[
1
α] −12.3 (c=0.165, MeOH); IR (KBr) ν 3430, 3191, 2937,
D
1
9
18–926 (2008).
−
682, 1598, 1468, 1444, 1206cm ;ꢀHR-MSꢀ(ESI)ꢀAnal. Calcd
ꢀ
5)ꢀ Picardꢀ O.,ꢀ Rollandꢀ Y.,ꢀ Pouponꢀ M.ꢀ F.,ꢀ Cancer Res., 46, 3290–3294
+
for C H N NaO m/z 367.1594 [M+Na] , Found 367.1588;
1
4
24
4
6
(1986).
1
H-NMRꢀ (600ꢀMHz,ꢀ DMSO-d ) δ:ꢀ 9.71ꢀ (2H,ꢀ s),ꢀ 8.09ꢀ (2H,ꢀ s),ꢀ ꢀ 6)ꢀ Campsꢀ J.ꢀ L.,ꢀ Changꢀ S.-M.,ꢀ Hsuꢀ T.ꢀ C.,ꢀ Freemanꢀ M.ꢀ R.,ꢀ Hongꢀ S.-J.,ꢀ
6
3
.78 (2H, m), 3.43 (4H, t, J=6.4Hz), 1.93 (6H, s), 1.68–1.51
ZhauꢀH.ꢀE.,ꢀvonꢀEschenbachꢀA.ꢀC.,ꢀChungꢀL.ꢀW.,ꢀProc. Natl. Acad.
Sci. U.S.A., 87, 75–79 (1990).
Kawada M., Sakamoto S., Nomoto A., For. Immunopathol. Dis.
Therap., 4, 53–62 (2013).
13
(8H, m); C-NMRꢀ(150ꢀMHz,ꢀDMSO-d ) δ:ꢀ170.7,ꢀ168.4,ꢀ54.3,ꢀ
6
7
)
47.3, 30.8, 22.5, 20.9.
Cells and Reagents Human lung cancer cell line A549
2
2)
ꢀ 8)ꢀ KawadaꢀM.,ꢀYoshimotoꢀY.,ꢀMinamiguchiꢀK.,ꢀKumagaiꢀH.,ꢀSomenoꢀ
was obtained as described. ꢀNHLFꢀnormalꢀhumanꢀlungꢀfibro-
blasts were obtained from BioWhittaker. The stromal cells
wereꢀ maintainedꢀ inꢀ Dulbecco’sꢀ modifiedꢀ Eagle’sꢀ mediumꢀ
T.,ꢀ Masudaꢀ T.,ꢀ Ishizukaꢀ M.,ꢀ Ikedaꢀ D.,ꢀ Anticancer Res., 24 (3a),
1
561–1568 (2004).
9)ꢀ KawadaꢀM.,ꢀInoueꢀH.,ꢀMasudaꢀT.,ꢀIkedaꢀD.,ꢀCancer Res., 66, 4419–
425 (2006).
0)ꢀ Kawadaꢀ M.,ꢀ Inoueꢀ H.,ꢀ Usamiꢀ I.,ꢀ Ikedaꢀ D.,ꢀ Cancer Sci., 100, 150–
57 (2009).
andꢀ5ꢀng/mLꢀbasic-FGFꢀ(PeproꢀTech,ꢀNJ,ꢀU.S.A.)ꢀatꢀ37°Cꢀwithꢀ 11)ꢀ KawadaꢀM.,ꢀMomoseꢀI.,ꢀSomenoꢀT.,ꢀTsujiuchiꢀG.,ꢀIkedaꢀD.,ꢀJ. Anti-
ꢀ
(
1
DMEM)ꢀ supplementedꢀ withꢀ 10%ꢀ fetalꢀ bovineꢀ serumꢀ (FBS),ꢀ
00ꢀunits/mLꢀpenicillinꢀG,ꢀ100ꢀµg/mLꢀstreptomycin,ꢀITHꢀ(5ꢀµg/
mLꢀ insulin,ꢀ 5ꢀµg/mLꢀ transferrin,ꢀ andꢀ 1.4ꢀµM hydrocortisone),
4
1
1
8)
5% CO as described.
biot., 62, 243–246 (2009).
2
Cell Growth and Coculture Experiment For coculture 12)ꢀ Kawadaꢀ M.,ꢀ Inoueꢀ H.,ꢀ Ohbaꢀ S.,ꢀ Masudaꢀ T.,ꢀ Momoseꢀ I.,ꢀ Ikedaꢀ D.,ꢀ
Int. J. Cancer, 126, 810–818 (2010).
experiment,ꢀ stromalꢀ cellsꢀ wereꢀ firstꢀ inoculatedꢀ inꢀ 96-wellꢀ
plates at 5×10 ꢀcellsꢀperꢀwellꢀinꢀ0.1ꢀmLꢀofꢀDMEMꢀsupplement-
3
13) Kawada M., Inoue H., Ohba S., Hatano M., Amemiya M., Hayashi
C.,ꢀUsamiꢀI.,ꢀAbeꢀH.,ꢀWatanabeꢀT.,ꢀKinoshitaꢀN.,ꢀIgarashiꢀM.,ꢀMa-
sudaꢀT.,ꢀIkedaꢀD.,ꢀNomotoꢀA.,ꢀJ. Antibiot., 66, 543–548 (2013).
4) Abe H., Kawada M., Inoue H., Ohba S., Nomoto A., Watanabe T.,
Shibasaki M., Org. Lett., 15, 2124–2127 (2013).
5) Kawada M., Someno T., Inoue H., Ohba S., Masuda T., Kato T.,
ed with 1% D-FBSꢀandꢀITH.ꢀTestꢀsamplesꢀwereꢀaddedꢀintoꢀtheꢀ
well and the stromal cells were cultured for 2d. Then 10µLꢀ
1
3
of cancer cell suspension (5×10 )ꢀinꢀserum-freeꢀDMEMꢀwereꢀ
inoculated onto a monolayer of the stromal cells and the cells
were further cultured for 3d. For monoculture of cancer cells,
1
IkedaꢀD.,ꢀJ. Antibiot., 63, 319–323 (2010).
onlyꢀ assayꢀ mediumꢀ withꢀ testꢀ samplesꢀ wasꢀ firstꢀ incubatedꢀ forꢀ 16)ꢀ GrossfeldꢀG.ꢀD.,ꢀHaywardꢀS.ꢀW.,ꢀTlstyꢀT.ꢀD.,ꢀCunhaꢀG.ꢀR.,ꢀEndocr.
d, and then cancer cells were inoculated as described above
Relat. Cancer, 5, 253–270 (1998).
and further cultured for 3d. The growth of the cancer cells 17)ꢀ TuxhornꢀJ.ꢀA.,ꢀAyalaꢀG.ꢀE.,ꢀRowleyꢀD.ꢀR.,ꢀJ. Urol., 166, 2472–2483
2
8
)
(
2001).
8)ꢀ Kawadaꢀ M.,ꢀ Inoueꢀ H.,ꢀ Arakawaꢀ M.,ꢀ Ikedaꢀ D.,ꢀ Anticancer Res., 28
2A), 721–730 (2008).
9)ꢀ Frederickꢀ C.ꢀ B.,ꢀ Bentleyꢀ M.ꢀ D.,ꢀ Shiveꢀ W.,ꢀ Biochem. Biophys. Res.
was determined using rhodanile blue dye as described. Cells
wereꢀ fixedꢀ forꢀ 15ꢀminꢀ byꢀ addingꢀ 50 ꢀµ Lꢀ ofꢀ 5%ꢀ glutaraldehydeꢀ
inꢀ phosphate-bufferedꢀ saline.ꢀ Afterꢀ washingꢀ threeꢀ timesꢀ withꢀ
tap water, the plate was dried. Then the cells were stained for
1
(
1
Commun., 105, 133–138 (1982).
0)ꢀ El-FahamꢀA.,ꢀSubirósꢀFunosasꢀR.,ꢀProhensꢀR.,ꢀAlbericioꢀF.,ꢀ Chem.
Eur. J., 15, 9404–9416 (2009).
1
5min by adding 10µLꢀofꢀ0.2%ꢀrhodanileꢀblueꢀdyeꢀinꢀdistilledꢀ
2
water. After washing seven times with tap water and drying,
the dye was eluted with 100µLꢀ ofꢀ 50%ꢀ ethanolꢀ andꢀ absor-
bance at 550nm measured using a microplate reader. The ab-
2
1)ꢀ Isowaꢀ Y.,ꢀ Takashimaꢀ T.,ꢀ Ohmoriꢀ M.,ꢀ Kuritaꢀ H.,ꢀ Satoꢀ M.,ꢀ Moriꢀ K.,ꢀ
Bull. Chem. Soc. Jpn., 45, 1467–1471 (1972).
sorbanceꢀatꢀ550ꢀnmꢀofꢀtheꢀmediumꢀaloneꢀandꢀtheꢀNHLFꢀaloneꢀ 22)ꢀ NakatsuꢀN.,ꢀNakamuraꢀT.,ꢀYamazakiꢀK.,ꢀSadahiroꢀS.,ꢀMakuuchiꢀH.,ꢀ
was subtracted from the values in monoculture and coculture,
respectively, as a background value.
KannoꢀJ.,ꢀYamoriꢀT.,ꢀMol. Pharmacol., 72, 1171–1180 (2007).