Cytostatic tetrazole-butenolide conjugates: Linking tetrazole and butenolide rings via stille coupling and biological activity of the target substances

Article abstract of DOI:10.1135/cccc2009040

A series of tetrazoles linked to the butenolide core via benzene rings were prepared by the Stille coupling reaction of α-(tributylstannyl) butenolides and 5-(alkylsulfanyl)-1-(4-iodophenyl)tetrazoles, and the compounds were tested for antifungal and cytostatic activity. Interesting antifungal activities against the filamentous strain Absidia corymbifera, and cytostatic activities against leukemic cells HL-60 and CCRF-CEM were found. The cytostatic activity requires the presence of both the butenolide ring and the alkylsulfanyl group bound to tetrazole ring. In addition, the feasibility of Pd-coupling reactions with 5-iodotetrazoles was evaluated.

Full text of DOI:10.1135/cccc2009040

Cytostatic Tetrazole–Butenolide Conjugates
1161
CYTOSTATIC TETRAZOLE–BUTENOLIDE CONJUGATES:
LINKING TETRAZOLE AND BUTENOLIDE RINGS VIA
STILLE COUPLING AND BIOLOGICAL ACTIVITY OF
THE TARGET SUBSTANCES
a
b3
Vojtěch BALŠÁNEK , Lucie TICHOTOVÁb1, Jiří KUNEŠb2, Marcel ŠPULÁK
,
Milan POURb4,*, Ivan VOTRUBA and Vladimír BUCHTA
c
d
a
RE&D VUFB Ltd., Poděbradská 56/186, 180 66 Prague 9, Czech Republic;
e-mail: balsanek@readvufb.cz
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 03 Hradec Králové, Czech Republic; e-mail: lucie.tichotova@faf.cuni.cz,
jiri.kunes@faf.cuni.cz, marcel.spulak@faf.cuni.cz, milan.pour@faf.cuni.cz
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: votruba@uochb.cas.cz
Department of Clinical Microbiology, University Hospital, Sokolská 581, 500 05 Hradec Králové,
Czech Republic; e-mail: buchta@fnhk.cz
b
1
2
3
4
c
d
Received March 18, 2009
Accepted May 23, 2009
Publish ed on lin e July 24, 2009
Dedicated to Dr. Alfred Bader on the occasion of his 85th birthday.
A series of tetrazoles lin ked to th e buten olide core via ben zen e rin gs were prepared by th e
Stille couplin g reaction of α-(tributylstan n yl)buten olides an d 5-(alkylsulfan yl)-1-(4-iodo-
ph en yl)tetrazoles, an d th e com poun ds were tested for an tifun gal an d cytostatic activity.
In terestin g an tifun gal activities again st th e filam en tous strain Absidia corymbifera, an d
cytostatic activities again st leukem ic cells HL-60 an d CCRF-CEM were foun d. Th e cytostatic
activity requires th e presen ce of both th e buten olide rin g an d th e alkylsulfan yl group boun d
to tetrazole rin g. In addition , th e feasibility of Pd-couplin g reaction s with 5-iodotetrazoles
was evaluated.
Keyw ord s: Pd catalysis; Cross-couplin g reaction s; Heterocycles; Tetrazoles; Buten olides;
Dih ydrofuran es; An tifun gals; Biological activity.
Tetrazoles, a class of n itrogen -con tain in g h eterocycles, attract con siderable
atten tion of m edicin al ch em ists. Th e tetrazole rin g can serve as a m etaboli-
cally stable surrogate for carboxylic group; tetrazole-con tain in g an ti-
h yperten sives of th e sartan type (such as 1) are good exam ples1 of a success-
ful em ploym en t of tetrazoles as parts of m edicin al agen ts (Ch art 1). Even
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© 2009 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2009040

1162
Balšánek et al.:
th ough th e tetrazole m oiety as such is n ot associated with an y specific bio-
logical activity, several recen t reports h ave described an tituberculous activ-
ity of 1-aryl-5-(ben zylsulfan yl)tetrazoles2,3. Several years ago, we described
th e an tifun gal activity of a wide variety of 3,5-disubstituted-2,5-dih ydro-
furan -2-on es4–7, in cludin g th ose bearin g a h eteroaryl substituen t8 at C3 of
th e furan on e rin g (2). In con jun ction with th ese studies, we were also in ter-
ested in substitutin g th e 2,5-dih ydrofuran on e rin g with a tetrazolyl m oiety
in position 3 (3), an d evaluatin g an tifun gal properties of th e com poun ds.
Given th e structure of th e target com poun ds, a Pd-m ediated couplin g
process was an obvious ch oice of strategy. Sin ce th e couplin g reaction s
of tetrazoles h ave n ot been system atically in vestigated, preparation of
tetrazolyl-substituted buten olides would also con tribute to exploration of
th e poten tial of Pd-catalyzed procedures in th e syn th esis of 1,5-disub-
stituted tetrazoles. An altern ative strategy for th e preparation of com -
poun ds 3, i.e. buildin g th e tetrazole rin g via a 1,3-dipolar addition of azide
an ion across th e cyan o group appears less advan tageous, sin ce th is would
m ean th e n ecessity of preparation of α-cyan obuten olides an d treatm en t of
th e com poun ds (good Mich ael acceptors) with a stron gly n ucleoph ilic azide
an ion . In th is report, we wish to give an accoun t of our work in cludin g th e
biological activities of th e substan ces prepared.
CHART 1
RESULTS AND DISCUSSION
An obvious process to be attem pted first was th e Stille couplin g reaction of
a 5-iodotetrazole derivative with th e previously prepared8 tributylstan n yl
lacton e 8. Th e ch oice of a 5-h alotetrazole as couplin g com pon en t was dic-
tated by th e in stability of 5-lith iated tetrazoles9, an d by easy availability of
α-(tributylstan n yl)lacton es in gram quan tities. Th us, 1-ph en yl-5-sulfan yl-
tetrazole (6), easily available from an ilin e in two steps10, was con verted
in to 5-iodo-1-ph en yltetrazole (7) via oxidative desulfurization followed by
m ercuration an d decom position of th e organ om ercury in term ediate with
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Cytostatic Tetrazole–Butenolide Conjugates
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iodin e11. Sim ilarly to our previous results8 on couplin g α-(tributylstan n yl)-
buten olide 8 with 2-iodofuran , Pd-m ediated couplin g failed upon usin g a
n um ber of establish ed procedures, in cludin g variation of catalysts, solven ts
an d reaction con dition s. A reaction was en forced em ployin g th e protocol
proposed by Farin a for difficult cases12; h owever, apart from a recovered
startin g m aterial (26%), th e reaction afforded iodolacton e 9 an d th e prod-
13
uct of deiodin ation of th e tetrazole rin g 10 as th e on ly isolable products
(Sch em e 1).
SCHEME 1
Sin ce th e above couplin g con dition s were rath er h arsh , we suspected th at
th e low stability of th e product m ay h ave been on e of th e reason s wh y th e
isolation of th e desired product was un successful. Th us, furth er elaboration
of th e acetic acid m oiety in th e tetrazole-substituted com poun ds 11a14 an d
11b was an oth er strategy we em ployed (Sch em e 2). Th e reaction sequen ce
em ployin g gen erally m ild con dition s h as been used before by us4–6 an d
oth ers15. Th e acids were con verted in to th e correspon din g m eth yl esters,
deproton ated an d th e en olates treated with allyl brom ide to afford in ter-
m ediates 12a an d 12b. Th e carboxylic groups were th en liberated by h ydro-
lysis an d cyclized on to th e term in al double bon d. Fin ally, th e saturated
lacton es 13a an d 13b were en olized with LDA an d th e en olates quen ch ed
with Ph SeCl. Th e in term ediate 3-(ph en ylselan yl) derivatives were, due to
th eir lim ited stability, im m ediately oxidized to furn ish th e correspon din g
selen oxides, wh ich un derwen t a spon tan eous syn-elim in ation in troducin g
th e double bon d. However, due to th e electron -with drawin g properties of
th e tetrazolyl m oiety, th e double bon d of th e lacton e rin g was extrem ely
pron e to a n ucleoph ilic attack. Hen ce, in case of th e 1-ph en yltetrazol-5-yl
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1164
Balšánek et al.:
derivative 14a, on ly a n egligible yield of epoxide 15 was isolated from a
com plex reaction m ixture after oxidation of th e in term ediate ph en ylselan yl
derivative with H2O2. In order to suppress or avoid epoxide form ation ,
NaIO4 was em ployed for th e oxidation of 14b in stead of H2O2 an d deriva-
tive 16 with th e tetrazole rin g lin ked directly to th e buten olide rin g was ob-
tain ed in a low, 19% yield. Th e structure of com poun d 16 was eviden t from
th e 1H NMR spectrum of th e crude reaction m ixture, wh ich con tain ed
buten olide 16, th e sign als of wh ich correspon ded very well to th e spectra of
a n um ber of related com poun ds as th e m ajor product. However, th is com -
poun d was quickly decom posin g durin g flash ch rom atograph y, an d its lim -
ited stability was apparen t even at low tem peratures.
SCHEME 2
We suspected th at th e electron -with drawin g properties of th e tetrazole
rin g facilitatin g Mich ael addition to th e β-carbon of th e furan on e could
h ave been th e m ajor reason for th e in stability of th e com poun ds. Th is as-
sum ption was easily verified by th e preparation of an alogous buten olides
bearin g pyridin -3-yl6 an d pyrim idin -5-yl m oieties with differen t with draw-
in g powers in th e α-position . Th e com poun ds were prepared via Stille cou-
plin g of th e correspon din g h eteroaryl iodides with tributylstan n yl lacton e 8
(Sch em e 3). As expected, wh ile th e pyridyl an alogue 17 was sufficien tly sta-
ble, th e pyrim idin yl-substituted com poun d 18, th ough obtain ed in a re-
spectably h igh yield, was quickly decom posin g.
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Cytostatic Tetrazole–Butenolide Conjugates
1165
SCHEME 3
Sin ce th e target com poun ds were sh own to be preparable, but in h eren tly
un stable, we m ade an oth er attem pt to syn th esize com poun ds th at would
com bin e th e tetrazole an d buten olide ph arm acoph oric substructures. As
th e stron g electron -with drawin g properties of th e tetrazolyl m oiety were
clearly on e of th e m ajor reason s for th e in stability of th e directly con ju-
gated com poun ds, we reason ed th at m ore stable com poun ds could possibly
be obtain ed if a suitable lin ker was in serted between both rin gs. Havin g in
m in d th e plan n ed exploration of th eir biological activity, we used th e ben -
zen e rin g to join th e two un its with lim ited flexibility an d to preserve th e
possibility of tran sm ission of electron ic effects between th em . Hen ce,
1-(4-iodoph en yl)-5-sulfan yltetrazole (19a) was prepared from 4-iodoan ilin e
via stan dard procedure16 (Sch em e 4) as a com pon en t for th e Pd-couplin g re-
action with tributylstan n yl lacton e 8. Com poun d 19a was furth er con -
verted in to th e product of oxidative desulfurization 19b an d 5-alkylsulfan yl
derivatives 19c–19f.
SCHEME 4
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1166
Balšánek et al.:
It sh ould also be n oted th at an oth er reason for th e preparation of th e
alkylsulfan yl com poun ds was th e fin din g of Waisser et al.17 th at an alkyl-
sulfan yl group attach ed to an electron -deficien t carbon atom is a ph arm a-
coph ore of an titubercular activity. Hen ce, th e in clusion of th e alkylsulfan yl
group would add an oth er dim en sion of biological activity to th e target
com poun ds. All aryl iodides 19b–19f sm ooth ly coupled with lacton e 8 un -
der th e above con dition s to yield th e desired products 20b–20f as stable
crystallin e com poun ds in good to excellen t yields (Sch em e 5).
SCHEME 5
An tifun gal activity screen in g of 20b–20f again st a pan el of yeasts an d
filam en tous fun gi (Table I) revealed th at th e ben zylsulfan yl derivative 20f
was practically in active, an d com poun d 20b with out a sulfan yl group was
m argin ally active again st Absidia corymbifera, Trichophyton mentagrophytes
an d Microsporum gypseum. Som e activity again st both yeasts an d filam en -
tous strain s was detected on ly with th e alkylsulfan yl derivatives 20c–20e.
Sin ce th e i-PrS-substituted derivative 20d was th e least active of th e th ree
substan ces, bran ch in g h ad a n egative in fluen ce on th e activity. Wh ile th e
activities of th e oth er two com poun ds again st yeasts can be described as
m oderate, th eir MICs (in particular th ose of th e eth ylsulfan yl derivative
20c) again st Aspergillus fumigatus an d Absidia corymbifera are of special in -
terest sin ce both are dan gerous path ogen s wh ose attacks often lead to leth al
in fection s. Th e MICs are rough ly com parable to th at of ketocon azole
again st A. fumigates, an d lower th an th at of th is stan dard azole drug again st
A. corymbifera. For th e sake of fair com parison , h owever, it sh ould be stated
th at even th e relatively low MICs again st A. corymbifera would n ot be lower
th an th at of th e polyen e an tibiotic am ph otericin B.
Th e target com poun ds 20 (except 20d ) were also evaluated for cytostatic
activity again st th ree leukem ic an d on e solid tum or lin e. Cell growth in h i-
bition at 10 µm ol/l sh owed th at 20b was in active at th is con cen tration ,
wh ile th e oth er derivatives exh ibited sign ifican t growth in h ibition . Th e re-
sults of th e subsequen t IC50 determ in ation are sum m arized in Table II.
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Cytostatic Tetrazole–Butenolide Conjugates
1167
TABLE I
An tifun gal activity of com poun ds 20b–20f an d ketocon azole (KET)
MICn, µm ol/l
Tim e
Strain
h
KET
20b
20c
20d
20e
20f
CA1a
CA2b
CPc
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
72
120
72
120
62.5
7.81
15.63
7.81
31.25
125
7.81
31.25
7.81
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
0.11
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
31.25
31.25
62.5
0.11
≤0.06
≤0.06
0.11
125
15.63
31.25
31.25
7.81
125
31.25
62.5
>125
>125
125
>62.5
15.63
31.25
15.63
31.25
62.5
0.11
CK1d
CK2e
CTf
1.96
15.63
7.81
>125
125
1.96
3.91
3.91
15.63
15.63
0.24
0.49
0.06
0.11
0.11
0.11
15.63
15.63
31.25
31.25
0.98
1.96
NTo
15.63
31.25
62.5
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>62.5
62.5
CGg
CLh
31.25
31.25
15.63
31.25
31.25
>62.5
31.25
>62.5
>62.5
>62.5
15.63
62.5
TBi
125
AFj
15.63
32.25
7.81
ACk
TMl
MGm
62.5
15.63
15.63
31.25
62.5
7.81
125
125
125
15.63
15.63
7.81
62.5
31.25
62.5
31.25
62.5
15.63
31.25
15.63
NT
a
b
c
d
Candida albicans ATCC44859, C. albicans ATCC90028, C. parapsilosis ATCC22019, C.
e
f
g
h
krusei ATCC 6258, C. krusei E28, C. tropicalis 156, C. glabrata 20/I, C. lusitaniae 2446/I,
i
j
k
l
Trichosporon beigelii 1188, Aspergillus fumigatus 231, Absidia corymbifera 272, Trichophyton
m
n
mentagrophytes 445, Microsporum gypseum, defin ed as 80% in h ibition of th e con trol growth ,
o
n ot tested.
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1168
Balšánek et al.:
TABLE II
Cytostatic activity (cell growth in h ibition – IC50, µm ol/l) of com poun ds 20b, 20c, 20e an d 20f
Com poun d
L1210
HL-60
HeLa S3
CCRF-CEM
20b
20c
20e
20f
NAa
4.6
6.3
6.0
NA
2.6
2.7
3.2
NA
8.4
NA
NA
NA
2.3
3.6
3.2
a
Not active at relevan t con cen tration s.
Th e com poun ds m an ifested cytostatic effect especially in th e leukem ic
lin es HL-60 an d CCRF-CEM; on ly com poun d 20c sh owed a rath er low ac-
tivity in th e HeLa S3 cells. Notably, th e cytostatic activity does n ot parallel
th e an tifun gal action sin ce th e an tifun gally in active ben zylsulfan yl deriva-
tive 20f possessed a cytostatic effect. It was th us in terestin g to speculate
wh ich part of th e m olecule was respon sible for th e cytostatic activity. In
order to sh ed m ore ligh t on th is issue, we prepared a series of 1-ph en yl-
5-(substituted ben zylsulfan yl)tetrazoles 21a–21f by alkylation of com poun d
6 with variously substituted ben zyl h alides (H, 3-OMe, 4-OMe, 4-Cl, 4-F,
4-NO2). All com poun ds were prepared un der ph ase tran sfer catalysis as de-
scribed in Sch em e 4, with th e exception of 4-n itroben zyl com poun d 21f,
th e preparation of wh ich was un successful un der th ese con dition s. In stead,
form ation of th e sodium salt of 6 with MeONa an d subsequen t SN reaction
with 4-n itroben zyl ch loride furn ish ed th is com poun d in a satisfactory yield
of 40%. Th e overview of th e 1,5-disubstituted tetrazoles is given in Ch art 2.
2
CHART 2
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Cytostatic Tetrazole–Butenolide Conjugates
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Screen in g of cytostatic activity of tetrazoles 21 again st th e above cell lin es
sh owed a m in im um , if an y, growth in h ibition at 10 µm ol/l. Th ese results
ren dered furth er IC50 determ in ation poin tless as th e values would h ave
been well above th is con cen tration . Hen ce, th e presen ce of th e buten olide
rin g in 20c, 20e an d 20f is an absolute n ecessity for th eir cytostatic effect,
even th ough its role m ust h ave been com bin ed with th at of sulfur (cf. in -
activity of 20b).
CONCLUSION
In sum m ary, we h ave n ot on ly exten ded th e previously publish ed series of
5-alkyl-3-h eteroarylfuran on es with a series of n ovel com poun ds possessin g
in terestin g biological properties, but also explored th e preparation of
1,5-disubstituted tetrazoles by cross couplin g reaction s. Notably, we con -
firm ed th at th e substitution of th e furan on e rin g in th e α-position with
stron gly electron -with drawin g h eteroaryl m oieties leads to com poun ds
with lim ited to low stability. Th is obstacle, h owever, can be circum ven ted
by in sertin g a suitable lin ker between both h eterocyclic rin gs. Wh ile th e
an tifun gal activity of th e com poun ds again st yeasts was rath er low, th e
MIC values again st A. corymbifera an d A. fumigatus ren der com poun d 20c
in terestin g for furth er developm en t, sin ce both th e alkylsulfan yl an d th e
eth yl groups at C5 of th e furan on e can be furth er varied. Cytostatic activity
requires th e presen ce of both th e furan on e rin g an d alkylsulfan yl group,
but th e structure–cytostatic activity relation sh ips are very likely differen t
from th e an tifun gal activity profile. Given th e low IC50 values again st
HL-60 an d CCRF-CEM lin es, furth er developm en t by varyin g th e RS group
in th e tetrazole rin g an d th e alkyl at C5 of th e furan on e m igh t also brin g
useful results.
EXPERIMENTAL
Gen eral Experim en tal Procedures
THF was distilled from ben zoph en on e ketyl an d diisopropylam in e from CaH2. Substituted
ph en ylacetic acids were obtain ed from Sigm a–Aldrich an d used as received. All an h ydrous
reaction s were perform ed in flam e-dried Sch len k tubes un der Ar atm osph ere. An alytical
th in -layer ch rom atograph y (TLC) was con ducted on Merck TLC plates (silica gel 60 F254, alu-
m in um back). Silica gel 60 (230–400 m esh ) for colum n ch rom atograph y was purch ased from
Merck. Melting points were determ ined on a Kofler block and are uncorrected. 1H and 13C NMR
spectra were recorded at am bien t tem perature on a Varian Mercury-Vx BB 300 spectrom eter
operatin g at 300 MHz for 1H. Ch em ical sh ifts are given as δ values in ppm an d are in directly
referen ced to tetram eth ylsilan e (TMS) via th e solven t sign al. Couplin g con stan ts (J) are
Collect. Czech. Chem. Commun. 2009, Vol. 74, Nos. 7–8, pp. 1161–1178

1170
Balšánek et al.:
given in Hz. All assign m en ts were m ade on th e basis of gCOSY, gHSQC an d gHMBC experi-
m en ts. Mixtures of isom ers are referred to as A an d B. IR spectra (νm ax, cm –1) were recorded
on a Nicolet Im pact 400 spectroph otom eter. Low-resolution m ass spectra were m easured on
a Magn um Fin n igan Mat apparatus.
An tifun gal Activity
In vitro an tifun gal activities of th e prepared com poun ds an d ketocon azole (Jan ssen –Cilag)
were evaluated on a pan el of four ATCC (Candida albicans ATCC 44859, C. albicans ATCC
90028, C. parapsilosis ATCC 22019, C. krusei ATCC 6258) an d n in e clin ical isolates of yeasts
(C. krusei E28, C. tropicalis 156, C. glabrata 20/I, C. lusitaniae 2446/I, Trichosporon beigelii
1188) an d filam en tous fun gi (Aspergillus fumigatus 231, Absidia corymbifera 272, Trichophyton
mentagrophytes 445, Microsporum gypseum) from th e collection of fun gal strain s deposited at
th e Departm en t of Biological an d Medical Scien ces, Faculty of Ph arm acy, Ch arles Un iversity,
Hradec Králové, Czech Republic. Th ree of th e above ATCC strain s (C. albicans ATCC 90028,
C. parapsilosis ATCC 22019, C. krusei ATCC 6258) also served as th e quality con trol strain s.
All th e isolates were m ain tain ed on Sabouraud dextrose agar prior to bein g tested.
Min im um in h ibitory con cen tration s (MICs) were determ in ed accordin g to th e m icro-
dilution form at of th e NCCLS M27-A guidelin es18. Dim eth yl sulfoxide (100%) served as a
diluen t for all com poun ds; th eir fin al con cen tration did n ot exceed 2%. RPMI 1640
(Sevaph arm a, Prague), a m edium supplem en ted with L-glutam in e an d buffered with 0.165
M
m orph olin e-1-propan esulfon ic acid (Serva) to pH 7.0 by 10 M NaOH was used as th e test
m edium . Th e wells of m icrodilution tray con tain ed 100 µl of th e RPMI 1640 m edium
with two-fold serial dilution s of th e com poun ds (1000–0.24 µm ol/l for n ew com poun ds) an d
100 µl of in oculum suspen sion . Fun gal in oculum in RPMI 1640 was prepared to give a fin al
con cen tration of 5 × 103 ± 0.2 cfu m l–1. Th e trays were in cubated at 35 °C an d MICs were
read visually for filam en tous fun gi an d ph otom etrically for yeasts as an absorban ce at
540 n m after 24 an d 48 h . Th e MIC values for th e derm atoph ytic strain (T. mentagrophytes
an d M. gypseum) were determ in ed after 72 an d 120 h . Th e MICs were defin ed as 80% in h ibi-
tion of th e con trol growth . MICs were determ in ed twice an d in duplicate. Th e deviation s
from th e usually obtain ed values given in Table I were n o h igh er th an th e n earest con cen -
tration value up an d down th e dilution scale.
Cytostatic Activity Assays
In h ibition of th e cell growth was estim ated in m ouse lym ph ocytic leukem ia L1210 cells
(ATCC CCL 219), CCRF-CEM T lym ph oblastoid cells (h um an acute lym ph oblastic leukem ia,
ATCC CCL 119), h um an prom yelocytic leukem ia HL-60 cells (ATCC CCL 240) an d h um an
cervix carcin om a HeLa S3 cells (ATCC CCL 2.2)19. L1210 cells, CCRF-CEM cells an d HL-60
cells were cultivated in RPMI 1640 m edium supplem en ted with calf foetal serum usin g
24-well tissue culture plates. Th e en dpoin t of th e cell growth was 72 h followin g th e drug
addition . HeLa S3 cells were seeded to 24-well dish es in RPMI 1640 HEPES m odification with
foetal calf serum . 48 h followin g th e drug addition th e cultivation was stopped an d th e cell
growth was evaluated. In parallel, cell viability was quan tified usin g MTT stan dard spectro-
ph otom etric assay20. Th e in h ibitory poten cy of th e com poun ds tested was expressed as IC50
values.
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Cytostatic Tetrazole–Butenolide Conjugates
1171
5-Eth yl-3-iodo-2,5-dih ydrofuran -2-on e (9)
A solution of 7 (0.030 g, 0.11 m m ol) in dry DMF (0.5 m l) was added to a stirred suspen sion
of [Pd2(dba)3]·CHCl3 (3 m g, 0.003 m m ol), Ph 3As (7 m g, 0.023 m m ol) an d CuI (3 m g,
0.016 m m ol) in dry DMF (3 m l) un der Ar. Th e resultan t m ixture was h eated up to 50 °C an d
a solution of 8 (0.040 g, 0.10 m m ol) in dry DMF (0.5 m l) was added dropwise. Th e reaction
m ixture was stirred at 50 °C for 6 h , th en saturated aqueous potassium fluoride solution
(1 m l) was added an d th e resultan t suspen sion was stirred at room tem perature for 30 m in .
Eth yl acetate (20 m l) was added, th e m ixture was filtered an d th e filtrate wash ed with a satu-
rated sodium ch loride solution (20 m l). Organ ic layer was dried (Na2SO4), solven ts were re-
m oved un der reduced pressure an d th e m ixture was purified by colum n ch rom atograph y
(gradien t elution , h exan e–eth yl acetate 95:5–7:3) to afford com poun d 9 (colorless oil, 8 m g,
13
yield 33%) an d 10
(wh ite crystals, 6 m g, yield 38%). For 9: 1H NMR (300 MHz, CDCl3):
7.72 (1 H, d, J = 1.7, H4), 5.00–4.92 (1 H, m , H5), 1.90–1.65 (2 H, m , -CH2-), 1.00 (3 H, t, J =
7.4, -CH3). 13C NMR (75 MHz, CDCl3): 170.06, 160.97, 86.33, 85.39, 26.24, 8.85. IR (CDCl3)
ν
m ax: 2974, 2938, 1763, 1595, 1463, 1332, 1279. LRMS: 239 (M• + + H, 100), 221 (4), 209
(10), 111 (7), 83 (4), 57 (18), 53 (7).
2-[1-(4-Meth oxyph en yl)-1H-tetrazol-5-yl]acetic Acid (11b)
Butyllith ium (1.6 M solution in h exan es, 10.35 m l, 16.56 m m ol) was added to a stirred solu-
tion of 1-(4-m eth oxyph en yl)-5-m eth yl-1H-tetrazole (3.000 g, 15.77 m m ol) in dry THF (40 m l)
at 0 °C un der Ar. Th e resultan t solution was stirred at 50 °C for 30 m in , th en dry ice (100 g)
was added, an d th e m ixture was allowed to warm up to room tem perature. Th en 5% aqueous
sodium h ydroxide solution (50 m l) was added, an d th e reaction m ixture wash ed with eth yl
acetate (50 m l). Th e aqueous layer was acidified with aqueous h ydroch loric acid to pH ~ 2,
th e resultan t solution wash ed th ree tim es with eth yl acetate (50 m l), th e organ ic layer dried
(Na2SO4) an d th e solven ts were rem oved un der reduced pressure. Yield 70%. Brown ish oil.
1H NMR (300 MHz, CDCl3): 7.40–7.35 (2 H, m , AA′BB′), 7.06–7.02 (2 H, m , AA′BB′), 4.03
(2 H, s, -CH2-), 3.86 (3 H, s, -OCH3). 13C NMR (75 MHz, CDCl3): 177.48, 161.28, 149.44,
126.59, 125.56, 115.09, 55.67, 29.52. IR (CDCl3), νm ax: 3012, 2938, 2841, 1716, 1610, 1514,
1408, 1291, 1258.
Preparation of Com poun ds 12a an d 12b. Gen eral Procedure
Dowex 50® (2.4 g) was added to a stirred solution of 11 (12.00 m m ol) in m eth an ol (50 m l)
at 0 °C un der Ar. Th e resultan t m ixture was stirred at room tem perature for 20 h , th e ion
exch an ger filtered off, wash ed with eth yl acetate (50 m l), an d th e solven ts rem oved un der
reduced pressure. Th e resultan t colorless oil was dissolved in dry THF (5 m l) an d added to
a stirred solution of dry diisopropylam in e (12.60 m m ol) an d butyllith ium (1.6 M solution in
h exan es, 13.20 m m ol) in dry THF (60 m l) at –60 °C un der Ar. After 30-m in stirrin g, allyl
brom ide (12.6 m m ol) was added, an d th e reaction m ixture allowed to gradually warm to
room tem perature over 2 h . Th e m ixture was th en diluted with eth yl acetate (100 m l),
wash ed with a saturated am m on ium ch loride solution (50 m l), th e organ ic layer was dried
(Na2SO4) an d th e solven ts rem oved un der reduced pressure. Th e crude product was purified
by colum n ch rom atograph y (h exan e–dieth yl eth er 8:2) to afford com poun d 12.
Methyl 2-(1-phenyl-1H-tetrazol-5-yl)pent-4-enoate (12a): Yield 43%. Colorless oil. 1H NMR
(300 MHz, CDCl3): 7.62–7.56 (3 H, m , Ar), 7.45–7.40 (2 H, m , Ar), 5.71–5.53 (1 H, m , H4),
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1172
Balšánek et al.:
5.09–4.94 (2 H, m , H5), 3.97 (1 H, dd, J1 = 9.1, J2 = 6.3, H2), 3.66 (3 H, s, -CH3), 3.04–2.82
(2 H, m , H3). 13C NMR (75 MHz, CDCl3): 168.84, 152.73, 133.28, 132.94, 130.87, 129.95,
125.60, 118.93, 52.95, 40.91, 34.39. IR (CDCl3), νm ax: 2956, 1747, 1598, 1497, 1437, 1268.
LRMS: 258 (M• +, 1), 239 (2), 201 (30), 171 (17), 158 (4), 144 (17), 131 (100), 117 (25), 104
(7), 91 (19), 77 (56), 64 (15), 51 (34).
Methyl 2-[1-(4-methoxyphenyl)-1H-tetrazol-5-yl]pent-4-enoate (12b): Yield 59%. Colorless oil.
1H NMR (300 MHz, CDCl3): 7.36–7.30 (2 H, m , AA′BB′), 7.07–7.03 (2 H, m , AA′BB′),
5.71–5.52 (1 H, m , H4), 5.09–4.96 (2 H, m , H5), 3.93 (1 H, dd, J1 = 9.3, J2 = 6.3, H2), 3.87
(3 H, s, Ar-OCH3), 3.67 (3 H, s, -CH3), 3.01–2.82 (2 H, m , H3). 13C NMR (75 MHz, CDCl3):
168.93, 161.24, 153.00, 133.02, 126.18, 125.78, 118.86, 114.97, 55.65, 52.94, 41.00, 34.34.
IR (CDCl3), νm ax: 3084, 2984, 2955, 2841, 1743, 1609, 1518, 1444, 1303, 1258. LRMS: 289
(M• + – H, 84), 259 (41), 245 (10), 228 (13), 219 (100), 201 (71), 186 (36), 174 (68), 161 (16),
149 (82), 133 (25), 121 (53), 106 (25), 92 (28), 78 (40), 63 (38), 53 (39).
5-Meth yl-3-(1-ph en yl-1H-tetrazol-5-yl)tetrah ydrofuran -2-on e (13a)
Ester 12a (1.175 g, 4.55 m m ol) was dissolved in a m ixture of m eth an ol–water 3:1 (40 m l)
an d lith ium h ydroxide (0.267 g, 5.92 m m ol) was added. Th e reaction m ixture was stirred at
room tem perature for 20 h , m eth an ol was rem oved un der reduced pressure, an d th e resul-
tan t solution was diluted with water (15 m l) an d acidified with aqueous h ydroch loric acid
to pH ~ 3. Th e m ixture was wash ed th ree tim es with eth yl acetate (50 m l), th e organ ic layer
was dried (Na2SO4) an d th e solven t rem oved un der reduced pressure. Th e product was
cooled down to 0 °C an d con cen trated sulfuric acid (5 m l) was added dropwise un der con -
stan t stirrin g. Th e reaction m ixture was stirred at 0 °C for 20 m in , th en cautiously poured
in to a saturated sodium ch loride solution (50 m l) an d pH of th e m ixture was adjusted with
a saturated aqueous sodium carbon ate solution to ~8. Th e m ixture was th en wash ed with
eth yl acetate (50 m l), th e organ ic layer dried (Na2SO4) an d th e solven t rem oved un der re-
duced pressure. Th e crude product was purified by colum n ch rom atograph y (h exan e–dieth yl
eth er 7:3) to afford lacton e 13a as a m ixture of two diastereom ers. Yield 62%. Wh ite crys-
tals, m .p. 111–114 °C. 1H NMR (300 MHz, CDCl3): A: 7.74–7.68 (2 H, m , Ar), 7.67–7.54 (3 H,
m , Ar), 5.29–5.16 (1 H, m , H5), 4.11 (1 H, dd, J1 = 9.2, J2 = 3.4, H3), 3.09–2.97 (1 H, m ,
H4A), 2.38–2.26 (1 H, m , H4B), 1.48 (3 H, d, J = 6.3, -CH3); B: 7.74–7.68 (2 H, m , Ar),
7.67–7.54 (3 H, m , Ar), 4.75–4.61 (1 H, m , H5), 4.15 (1 H, t, J = 10.4, H3), 2.84–2.71 (2 H,
m , H4), 1.56 (3 H, d, J = 6.0, -CH3). 13C NMR (75 MHz, CDCl3): A: 172.30, 151.68, 132.92,
130.84, 129.93, 125.34, 77.20, 36.33, 35.63, 20.91; B: 171.79, 151.18, 132.99, 130.88,
129.90, 125.41, 76.51, 38.02, 36.12, 20.62. IR (CDCl3), νm ax: 2984, 2933, 1773, 1598, 1500,
1457, 1387, 1349. LRMS: 245 (M• + + H, 23), 228 (3), 216 (14), 207 (8), 197 (9), 185 (11), 171
(56), 157 (25), 145 (37), 130 (100), 117 (20), 103 (17), 91 (54), 77 (62), 64 (33), 51 (53).
3-[1-(4-Meth oxyph en yl)-1H-tetrazol-5-yl]-5-m eth yltetrah ydrofuran -2-on e (13b)
Ester 12b (1.156 g, 5.41 m m ol) was dissolved in a m ixture of m eth an ol–water 3:1 (40 m l)
an d lith ium h ydroxide (0.318 g, 7.03 m m ol) was added. Th e reaction m ixture was stirred at
50 °C for 11 h , m eth an ol was rem oved un der reduced pressure, th e resultan t solution di-
luted with water (15 m l) an d acidified with aqueous h ydroch loric acid to pH ~ 3. Th e m ix-
ture was wash ed th ree tim es with eth yl acetate (50 m l), th e organ ic layer was dried (Na2SO4)
an d th e solven t rem oved un der reduced pressure. A 30% solution of h ydrobrom ic acid in
AcOH (10 m l) was slowly added to th e product un der Ar at room tem perature, an d th e reac-
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Cytostatic Tetrazole–Butenolide Conjugates
1173
tion m ixture was stirred for 6 h . Th e rem ain in g reagen t was rem oved un der reduced pres-
sure, th e crude product was dissolved in m eth an ol (15 m l) an d sodium carbon ate (1.232 g,
11.63 m m ol) was added. After 45 m in of stirrin g at room tem perature, th e m ixture was di-
luted with eth yl acetate (50 m l), wash ed with a saturated aqueous sodium ch loride solution
(50 m l), th e organ ic layer was dried (Na2SO4) an d th e solven ts were rem oved un der reduced
pressure. Th e crude product was purified by colum n ch rom atograph y (h exan e–dieth yl eth er
7:3) to afford lacton e 13b as a m ixture of two diastereom ers. Yield 30%. Colorless oil.
1H NMR (300 MHz, CDCl3): A: 7.67–7.50 (2 H, m , AA′BB′), 7.12–6.98 (3 H, m , Ar), 5.31–5.15
(1 H, m , H5), 4.12 (1 H, dd, J1 = 9.0, J2 = 3.5, H3), 3.88 (3 H, s, -OCH3), 3.09–2.96 (1 H, m ,
H4A), 2.36–2.21 (1 H, m , H4B), 1.48 (3 H, d, J = 6.3, -CH3); B: 7.67–7.50 (2 H, m , Ar),
7.12–6.98 (3 H, m , Ar), 4.72–4.62 (1 H, m , H5), 4.14 (1 H, t, J = 10.6, H3), 3.88 (3 H, s,
-OCH3), 2.80–2.69 (2 H, m , H4), 1.54 (3 H, d, J = 6.0, -CH3). 13C NMR (75 MHz, CDCl3):
A: 172.00, 151.32, 127.11, 122.94, 115.01, 77.19, 55.71, 36.21, 35.50, 20.83; B: 171.69,
160.69, 151.11, 127.03, 122.94, 115.16, 76.50, 55.71, 37.91, 35.97, 20.54.
3,4-Epoxy-5-m eth yl-3-(1-ph en yl-1H-tetrazol-5-yl)tetrah ydrofuran -2-on e (15)
A solution of dry diisopropylam in e (0.30 m l, 2.15 m m ol) an d butyllith ium (1.6 M solution
in h exan es, 2.26 m m ol) in dry THF (5 m l) was stirred at 0 °C un der Ar for 10 m in . Th e LDA
solution was th en cooled to –60 °C, an d a solution of lacton e 13a (0.500 g, 2.05 m m ol) in
dry THF (2 m l) was added dropwise. After 30-m in stirrin g at –60 °C, a solution of ph en yl-
selan yl ch loride (0.589 g, 3.08 m m ol) in dry THF (5 m l) was added, an d th e resultan t m ix-
ture was allowed to warm to room tem perature over 2 h . Th e m ixture was th en diluted with
eth yl acetate (50 m l), wash ed with a saturated am m on ium ch loride solution (50 m l), th e or-
gan ic layer was dried (Na2SO4) an d th e solven ts were rem oved un der reduced pressure. Th e
crude product was purified by colum n ch rom atograph y (h exan e–dieth yl eth er 8:2) to afford
ph en ylselan yl derivative 14a, wh ich was im m ediately used in th e n ext step.
Lactone 14a (0.267 g, 0.67 m m ol) was dissolved in a m ixture of ethyl acetate–THF (2:1, 3 m l),
th e reaction m ixture was cooled down to 0 °C, an d sodium h ydrogen carbon ate (0.150 g) an d
a 30% aqueous h ydrogen peroxide solution (1 m l) were added. Th e resultan t m ixture was
stirred at room tem perature for 1 h , th en diluted with eth yl acetate (50 m l), an d wash ed
with a saturated sodium ch loride solution (50 m l). Th e organ ic layer was dried (Na2SO4) an d
th e solven ts rem oved un der reduced pressure. Th e crude product was purified by colum n
ch rom atograph y (h exan e–dieth yl eth er 7:3) to afford lacton e 15. Yield 5%. Colorless oil. 1H
NMR (300 MHz, CDCl3): 7.70–7.65 (2 H, m , Ar), 7.63–7.58 (3 H, m , Ar), 4.81 (1 H, q, J = 6.9,
H5), 4.57 (1 H, s, H4), 1.56 (3 H, d, J = 6.9, CH3). 13C NMR (75 MHz, CDCl3): 166.06,
145.14, 133.39, 130.91, 129.78, 124.38, 75.77, 64.84, 52.24, 17.84.
3-[1-(4-Meth oxyph en yl)-5-m eth yl-1H-tetrazol-5-yl]-2,5-dih ydrofuran -2-on e (16)
A solution of dry diisopropylam in e (0.22 m l, 1.57 m m ol) an d butyllith ium (1.6 M solution
in h exan es, 1.65 m m ol) in dry THF (5 m l) was stirred at 0 °C un der Ar for 10 m in , th en
cooled down to –60 °C an d a solution of lacton e 13b (0.411 g, 1.50 m m ol) in dry THF
(2 m l) was added dropwise. After 30 m in of stirrin g at –60 °C, a solution of ph en ylselan yl
ch loride (0.430 g, 2.24 m m ol) in dry THF (5 m l) was added an d th e resultan t m ixture was
allowed to warm to room tem perature over 2 h . Th e m ixture was th en diluted with eth yl
acetate (50 m l), wash ed with a saturated am m on ium ch loride solution (50 m l), th e organ ic
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1174
Balšánek et al.:
layer was dried (Na2SO4) an d th e solven ts were rem oved un der reduced pressure. Th e crude
product was purified by colum n ch rom atograph y (h exan e–eth yl acetate 95:5) to afford
ph en ylselan yl derivative 14b, wh ich was im m ediately used in th e n ext step.
Lacton e 14b (0.286 g, 0.67 m m ol) was dissolved in a m ixture of m eth an ol–THF–water
(5:5:1, 15 m l), an d sodium h ydrogen carbon ate (0.068 g, 0.80 m m ol) an d sodium periodate
(0.330 g, 1.54 m m ol) were added. Th e resultan t m ixture was stirred at room tem perature for
2.5 h , th en diluted with water (30 m l), an d wash ed with eth yl acetate (50 m l). Th e organ ic
layer was dried (Na2SO4) an d th e solven ts were rem oved un der reduced pressure. Th e crude
product was purified by colum n ch rom atograph y (h exan e–eth yl acetate 9:1) to afford
lacton e 16. Yield 19%. Colorless oil (wh en fresh ly prepared). 1H NMR (300 MHz, CDCl3):
7.58–7.51 (3 H, m , AA′BB′ + H4), 7.12–7.01 (2 H, m , AA′BB′), 5.22 (1 H, qd, J1 = 6.9, J2 = 1.6,
H5), 3.88 (3 H, s, -OCH3), 1.55 (3 H, d, J = 6.9, -CH3).
5-Eth yl-3-(pyrim idin -5-yl)-2,5-dih ydrofuran -2-on e (18)
A solution of 5-iodopyrim idin e (0.227 g, 1.10 m m ol) in dry DMF (1 m l) was added to a
stirred suspen sion of [Pd2(dba)3]·CHCl3 (21 m g, 0.02 m m ol), Ph 3As (49 m g, 0.16 m m ol) an d
CuI (15 m g, 0.08 m m ol) in dry DMF (4 m l) un der Ar. Th e resultan t m ixture was h eated up
to 50 °C, an d a solution of 8 (0.401 g, 1.00 m m ol) in dry DMF (1 m l) was added dropwise.
Th e reaction m ixture was stirred at 50 °C for 6 h , a saturated aqueous potassium fluoride so-
lution (1 m l) was th en added, an d th e resultan t suspen sion was stirred at room tem perature
for 30 m in . Eth yl acetate (50 m l) was added, th e m ixture was filtered, an d th e filtrate
wash ed with a saturated sodium ch loride solution (50 m l). Th e organ ic layer was dried
(Na2SO4), th e solven ts were rem oved un der reduced pressure, an d th e crude product was
purified by colum n ch rom atograph y (gradien t elution , h exan e–eth yl acetate 95:5–7:3) to af-
ford lacton e 18. Yield 80%. Colorless oil, quickly decom posin g. 1H NMR (300 MHz, CDCl3):
9.22 (1 H, s, Ar2), 9.20 (2 H, s, Ar4,6), 7.78 (1 H, d, J = 1.9, H4), 5.15–5.07 (1 H, m , H5),
2.01–1.75 (2 H, m , -CH2-), 1.08 (3 H, t, J = 7.4, -CH3). 13C NMR (75 MHz, CDCl3): 170.58,
158.79, 154.96, 150.13, 127.03, 123.95, 82.57, 26.57, 9.22.
1-(4-Iodoph en yl)-1H-tetrazole (19b)
Tetrazole 19a (0.250 g, 0.82 m m ol) was dissolved in acetic acid (3.5 m l), a 30% aqueous
h ydrogen peroxide solution (7.5 m l) was added, an d th e resultan t m ixture h eated un der re-
flux for 1.5 h . After coolin g down to room tem perature, sodium carbon ate was slowly added
(to pH ~ 8), an d th e m ixture wash ed with eth yl acetate (60 m l). Th e organ ic layer was dried
(Na2SO4), th e solven ts rem oved un der reduced pressure, an d th e crude product purified by
colum n ch rom atograph y (h exan e–eth yl acetate 8:2) to afford tetrazole 19b . Yield 63%.
Wh ite crystals, m .p. 189–191 °C. 1H NMR (300 MHz, CDCl3): 10.10 (1 H, s, Het), 8.04–7.98
(1 H, m , AA′BB′), 7.76–7.69 (1 H, m , AA′BB′). 13C NMR (75 MHz, CDCl3): 142.47, 139.01,
133.67, 123.18, 95.97. IR (KBr), νm ax: 3455, 3132, 1503, 1495, 1385, 1212, 996, 819. LRMS:
271 (M• + – H, 1), 244 (100), 234 (3), 207 (3), 191 (2), 165 (2), 149 (4), 127 (20), 117 (35), 90
(66), 74 (11), 63 (47), 50 (24).
Preparation of Com poun ds 19c–19f. Gen eral Procedure
Tetrazole 19a (0.800 g, 2.63 m m ol) was dissolved in cycloh exan e (20 m l), an d a 10 M aqueous
sodium h ydroxide solution (10 m l), an d alkyl h alide (2.63 m m ol) an d tetrabutylam m on ium
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Cytostatic Tetrazole–Butenolide Conjugates
1175
iodide (0.100 g, 0.27 m m ol) were added. Th e reaction m ixture was h eated un der reflux with
rapid stirrin g for 30 m in . After coolin g down to room tem perature, th e resultan t m ixture
was filtered an d th e filtrate wash ed with water (30 m l). Th e organ ic layer was dried
(Na2SO4), th e solven ts were rem oved un der reduced pressure an d th e crude product purified
by recrystallization from eth an ol.
5-(Ethylsulfanyl)-1-(4-iodophenyl)-1H-tetrazole (19c): R–X = eth yl brom ide. Yield 63%. Wh ite
crystals, m .p. 78–79 °C. 1H NMR (300 MHz, CDCl3): 7.94–7.86 (2 H, m , AA′BB′), 7.39–7.31 (2 H,
m , AA′BB′), 3.42 (2 H, q, J = 7.4, -SCH2-), 1.50 (3 H, t, J = 7.4, -CH3). 13C NMR (75 MHz,
CDCl3): 154.22, 138.97, 133.36, 125.26, 95.63, 27.88, 14.54. IR (KBr), νm ax: 3432, 3088,
2958, 2928, 1489, 1384, 1246, 1056, 1005, 829. LRMS: 316 (1), 244 (100), 207 (2), 127 (9),
117 (18), 90 (33), 74 (5), 63 (24), 50 (12).
1-(4-Iodophenyl)-5-isopropylsulfanyl-1H-tetrazole (19d ): R–X = isopropyl iodide. Yield 63%.
Wh ite crystals, m .p. 112–113 °C. 1H NMR (300 MHz, CDCl3): 7.93–7.85 (2 H, m , AA′BB′),
7.37–7.29 (2 H, m , AA′BB′), 4.22–4.07 (1 H, m , -SCH), 1.51 (6 H, d, J = 6.9, -CH3). 13C NMR
(75 MHz, CDCl3): 153.98, 138.89, 133.37, 125.40, 95.61, 39.98, 23.22. IR (KBr), νm ax: 2965,
1492, 1385, 1240, 1059, 1006, 829. LRMS: 347 (M• + + H, 1), 318 (100), 276 (10), 249 (42),
219 (1), 191 (2), 150 (5), 127 (4), 116 (8), 108 (2), 90 (13), 76 (5), 63 (23), 50 (10).
5-(Butylsulfanyl)-1-(4-iodophenyl)-1H-tetrazole (19e): R–X = butyl brom ide. Yield 75%.
Wh ite crystals, m .p. 110–111 °C. 1H NMR (300 MHz, CDCl3): 7.93–7.87 (2 H, m , AA′BB′),
7.38–7.32 (2 H, m , AA′BB′), 3.41 (2 H, t, J = 7.3, -SCH2-), 1.87–1.74 (2 H, m , -CH2-),
1.54–1.41 (2 H, m , -CH2-), 0.95 (3 H, t, J = 7.3, -CH3). 13C NMR (75 MHz, CDCl3): 154.46,
138.96, 133.39, 125.28, 95.61, 33.17, 31.01, 21.76, 13.48. IR (KBr), νm ax: 2961, 2931, 1491,
1423, 1398, 1379, 1247, 1006, 827. LRMS: 361 (M• + + H, 1), 244 (100), 234 (2), 207 (2), 127
(6), 117 (12), 90 (20), 75 (3), 63 (20), 50 (8).
5-(Benzylsulfanyl)-1-(4-iodophenyl)-1H-tetrazole (19f): R–X = ben zyl ch loride. Yield 69%.
Wh ite crystals, m .p. 115–116 °C. 1H NMR (300 MHz, CDCl3): 7.89–7.83 (2 H, m , AA′BB′),
7.45–7.38 (2 H, m , Ar), 7.36–7.25 (5 H, m , AA′BB′ + Ar), 4.63 (2 H, s, -CH2-). 13C NMR
(75 MHz, CDCl3): 153.78, 138.91, 135.01, 133.21, 129.20, 128.84, 128.25, 125.21, 95.68,
37.74. IR (KBr), νm ax: 3059, 1495, 1402, 1382, 1248, 1060, 1005, 828, 699. LRMS: 244 (100),
234 (1), 207 (1), 127 (9), 117 (17), 90 (33), 74 (4), 63 (22), 50 (12).
Preparation of Com poun ds 20b–20f. Gen eral procedure
A solution of an aryl iodide 19 (1.10 m m ol) in dry DMF (1 m l) was added to a stirred sus-
pen sion of [Pd2(dba)3]·CHCl3 (21 m g, 0.02 m m ol), Ph 3As (49 m g, 0.16 m m ol) an d CuI (15 m g,
0.08 m m ol) in dry DMF (4 m l) un der Ar. Th e resultan t m ixture was h eated up to 50 °C an d
a solution of 8 (0.401 g, 1.00 m m ol) in dry DMF (1 m l) was added dropwise. Th e reaction
m ixture was stirred at 50 °C for 6 h , a saturated aqueous potassium fluoride solution (1 m l)
was added, an d th e resultan t suspen sion stirred at room tem perature for 30 m in . Eth yl
acetate (50 m l) was added, th e m ixture was filtered an d th e filtrate was wash ed with a satu-
rated sodium ch loride solution (50 m l). Th e organ ic layer was dried (Na2SO4), th e solven ts
were rem oved un der reduced pressure, an d th e crude product purified by colum n ch rom ato-
graph y (gradien t elution , h exan e–eth yl acetate 99:1–8:2) to afford lacton es 20b–20f.
5-Ethyl-3-[4-(1H-tetrazol-1-yl)phenyl]-2,5-dihydrofuran-2-one (20b): Yield 79%. Wh ite crys-
tals, m .p. 158–160 °C. 1H NMR (300 MHz, (CD3)2CO): 9.79 (1 H, s, Het), 8.26–8.20 (2 H, m ,
AA′BB′), 8.19 (1 H, d, J = 1.7, H4), 8.04–7.96 (2 H, m , AA′BB′), 5.20–5.13 (1 H, m , H5),
1.98–1.72 (2 H, m , -CH2-), 1.04 (3 H, t, J = 7.4, -CH3). 13C NMR (75 MHz, (CD3)2CO):
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Balšánek et al.:
171.80, 151.94, 142.37, 135.24, 132.27, 129.99, 129.37, 121.93, 82.46, 27.08, 9.31. IR (KBr),
ν
m ax: 3448, 3135, 2975, 1736, 1522, 1461, 1209, 1148, 1131, 975, 838, 534. LRMS: 255
(M• + – H, 6), 221 (6), 207 (23), 193 (7), 172 (16), 157 (6), 144 (22), 131 (100), 117 (20), 104
(12), 91 (35), 77 (78), 65 (16), 51 (34). For C13H12N4O2 calculated: 60.93% C, 4.72% H, 21.86% N;
foun d: 60.63% C, 4.92% H, 21.71% N.
5-Ethyl-3-{4-[5-(ethylsulfanyl)-1H-tetrazol-1-yl]phenyl}-2,5-dihydrofuran-2-one (20c): Yield
80%. Wh ite crystals, m .p. 64–66 °C. 1H NMR (300 MHz, CDCl3): 8.10–8.05 (2 H, m , AA′BB′),
7.71 (1 H, d, J = 1.9, H4), 7.68–7.62 (2 H, m , AA′BB′), 5.11–5.02 (1 H, m , H5), 3.42 (2 H, q,
J = 7.4, -SCH2-), 2.01–1.76 (2 H, m , -CH2-), 1.49 (3 H, t, J = 7.4, -SCH2CH3), 1.08 (3 H, t, J =
7.4, -CH3). 13C NMR (75 MHz, CDCl3): 171.18, 154.29, 149.42, 134.08, 131.19, 130.34,
128.39, 123.78, 81.71, 27.82, 26.61, 14.52, 9.21. IR (KBr), νm ax: 2927, 1750, 1636, 1515,
1385, 1120, 968, 845. LRMS: 315 (M• + – H, 1), 291 (13), 269 (100), 229 (4), 208 (13), 167
(8), 155 (17), 138 (4), 119 (8), 91 (2), 69 (3), 57 (23). For C15H16N4O2S calculated: 56.94% C,
5.10% H, 17.71% N, 10.14% S; foun d: 57.38% C, 5.41% H, 17.33% N, 9.80% S.
5-Ethyl-3-{4-[5-(isopropylsulfanyl)-1H-tetrazol-1-yl]phenyl}-2,5-dihydrofuran-2-one (20d ): Yield
98%. Wh ite crystals, m .p. 122–123 °C. 1H NMR (300 MHz, CDCl3): 8.10–8.02 (2 H, m ,
AA′BB′), 7.71 (1 H, d, J = 1.7, H4), 7.66–7.58 (2 H, m , AA′BB′), 5.10–5.03 (1 H, m , H5),
4.21–4.05 (1 H, m , -SCH), 2.04–1.72 (2 H, m , -CH2-), 1.50 (6 H, d, J = 6.9, -CH3), 1.07 (3 H,
t, J = 7.4, -CH3). 13C NMR (75 MHz, CDCl3): 171.19, 154.04, 149.50, 134.06, 131.17, 130.27,
128.31, 123.92, 81.70, 39.87, 26.56, 23.17, 9.17. IR (CDCl3), νm ax: 2973, 2930, 1758, 1607,
1517, 1462, 1390, 1338. LRMS: 330 (M• +, 7), 327 (16), 315 (13), 281 (8), 267 (4), 255 (5),
245 (22), 219 (10), 202 (18), 193 (6), 160 (8), 149 (14), 135 (18), 127 (24), 119 (10), 91 (56),
70 (46), 61 (100). For C16H18N4O2S calculated: 58.16% C, 5.49% H, 16.96% N, 9.71% S;
foun d: 58.51% C, 5.81% H, 16.68% N, 9.40% S.
3-{4-[5-(Butylsulfanyl)-1H-tetrazol-1-yl]phenyl}-5-ethyl-2,5-dihydrofuran-2-one (20e): Yield
90%. Wh ite crystals, m .p. 62–64 °C. 1H NMR (300 MHz, CDCl3): 8.10–8.04 (2 H, m , AA′BB′),
7.71 (1 H, d, J = 1.7, H4), 7.67–7.62 (2 H, m , AA′BB′), 5.10–5.03 (1 H, m , H5), 3.40 (2 H, t,
J = 7.3, -SCH2-), 1.99–1.73 (4 H, m , -CH2-), 1.54–1.39 (2 H, m , -CH2-), 1.08 (3 H, t, J = 7.4,
-CH3), 0.94 (3 H, t, J = 7.3, -CH3). 13C NMR (75 MHz, CDCl3): 171.19, 154.53, 149.44,
134.09, 131.18, 130.32, 128.38, 123.78, 81.71, 33.10, 30.97, 26.59, 21.72, 13.45, 9.19. IR
(KBr), νm ax: 3087, 2956, 2931, 2873, 1747, 1515, 1381, 1340, 1121, 837. LRMS: 344 (M• +, 1),
281 (2), 246 (10), 207 (2), 181 (11), 147 (2), 121 (3), 91 (100), 77 (5), 65 (17), 51 (7). For
C
17H20N4O2S calculated: 59.28% C, 5.85% H, 16.27% N, 9.29% S; foun d: 59.60% C, 6.10% H,
15.96% N, 8.98% S.
3-{4-[5-(Benzylsulfanyl)-1H-tetrazol-1-yl]phenyl}-5-ethyl-2,5-dihydrofuran-2-one (20f): Yield
77%. Wh ite crystals, m .p. 119–121 °C. 1H NMR (300 MHz, CDCl3): 8.07–8.01 (2 H, m ,
AA′BB′), 7.70 (1 H, d, J = 1.6, H4), 7.60–7.54 (2 H, m , AA′BB′), 7.44–7.38 (2 H, m , Ar),
7.35–7.26 (3 H, m , Ar), 5.08–5.02 (1 H, m , H5), 4.61 (2 H, s, -SCH2-), 2.01–1.72 (2 H, m ,
-CH2-), 1.06 (3 H, t, J = 7.4, -CH3). 13C NMR (75 MHz, CDCl3): 171.13, 153.83, 149.62,
134.99, 133.81, 131.20, 130.08, 129.12, 128.73, 128.30, 128.12, 123.67, 81.67, 37.59, 26.48,
9.12. IR (CDCl3), νm ax: 2974, 2939, 2881, 1755, 1606, 1516, 1455, 1387, 1338. LRMS: 377
(M• + – H, 1), 246 (13), 181 (13), 121 (3), 91 (100), 77 (5), 65 (17), 51 (6). For C20H18N4O2S
calculated: 63.47% C, 4.79% H, 14.80% N, 8.47% S; found: 63.14% C, 4.89% H, 14.70% N,
8.28% S.
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Cytostatic Tetrazole–Butenolide Conjugates
1177
Preparation of Com poun ds 21a–21e. Gen eral Procedure
Tetrazole 6 (1.000 g, 5.61 m m ol) was dissolved in toluen e (20 m l), an d a 1 M aqueous so-
dium h ydroxide solution (20 m l), an d alkyl h alide R–X (15.00 m m ol) an d tetrabutyl-
am m on ium brom ide (0.160 g, 0.31 m m ol) were added. Th e reaction m ixture was h eated
un der reflux with rapid stirrin g for 3 h . After coolin g to room tem perature, th e layers were
separated an d th e organ ic layer was wash ed with water (3 × 30 m l), dried (Na2SO4) an d th e
solven t was rem oved un der reduced pressure. Th e crude product was purified by colum n
ch rom atograph y (h exan e–eth yl acetate 9:1).
5-(Benzylsulfanyl)-1-phenyl-1H-tetrazole (21a)21: R–X = ben zyl brom ide. Yield 84%. Wh ite
crystals, m .p. 68–69 °C. 1H NMR (300 MHz, CDCl3): 7.56–7.50 (5 H, m , H2, H3, H4, H5,
H6), 7.47–7.40 (2 H, m , H2′, H6′), 7.38–7.27 (3 H, m , H3′, H4′, H5′), 4.62 (2 H, s, CH2).
5-[(3-Methoxybenzyl)sulfanyl]-1-phenyl-1H-tetrazole (21b): R–X = 3-m eth oxyben zyl brom ide.
Yield 74%. Wh ite crystals, m .p. 79–81 °C. 1H NMR (300 MHz, CDCl3): 7.56–7.49 (5 H, m ,
H2, H3, H4, H5, H6), 7.22 (1 H, d, J = 8.0, H5′), 7.02–6.93 (2 H, m , H2′, H6′), 6.83 (1 H, dd,
J = 8.0, J = 2.5, H4′), 4.67 (2 H, s, CH2), 3.78 (s, 3 H, OCH3). 13C NMR (75 MHz, CDCl3):
159.8, 153.9, 136.6, 133.5, 130.1, 129.8, 129.7, 123.8, 121.5, 114.6, 113.8, 55.2, 37.6.
5-[(4-Methoxybenzyl)sulfanyl]-1-phenyl-1H-tetrazole (21c)22: R–X = 4-m eth oxyben zyl ch lo-
ride. Yield 55%. Wh ite crystals, m .p. 51–53 °C. 1H NMR (300 MHz, CDCl3): 7.55–7.50
(5 H, m , H2, H3, H4, H5, H6), 7.38–7.31 (2 H, m , AA′BB′, H3′, H5′), 6.87–6.82 (2 H, m ,
AA′BB′, H2′, H6′), 4.59 (2 H, s, CH2), 3.79 (3 H, s, OCH3). 13C NMR (75 MHz, CDCl3): 159.5,
154.0, 133.6, 130.5, 130.0, 129.7, 127.0, 123.8, 114.2, 55.3, 37.3.
5-[(4-Chlorobenzyl)sulfanyl]-1-phenyl-1H-tetrazole (21d )22: R–X = 4-ch loroben zyl ch loride.
Yield 25%. Wh ite crystals, m .p. 98–101 °C. 1H NMR (300 MHz, CDCl3): 7.55–7.50 (5 H, m ,
H2, H3, H4, H5, H6), 7.40–7.34 (2 H, m , AA′BB′, H3′, H5′), 7.31–7.26 (2 H, m , AA′BB′, H2′,
H6′), 4.58 (2 H, s, CH2). 13C NMR (75 MHz, CDCl3): 153.5, 134.1, 133.9, 133.5, 130.6, 130.2,
129.8, 129.0, 123.7, 36.7.
5-[(4-Fluorobenzyl)sulfanyl]-1-phenyl-1H-tetrazole (21e)22: R–X = 4-fluoroben zyl ch loride.
Yield 94%. Wh ite crystals, m .p. 87–90 °C. 1H NMR (300 MHz, CDCl3): 7.55–7.50 (5 H, m ,
H2, H3, H4, H5, H6), 7.45–7.37 (2 H, m , H3′, H5′), 7.05–6.96 (2 H, m , H2′, H6′), 4.59 (2 H, s,
CH2). 13C NMR (75 MHz, CDCl3): 162.5 (d, J = 247.4), 153.7, 133.5, 131.1, 131.0 (d, J = 8.3),
130.2, 129.8, 123.8, 115.7 (d, J = 21.8), 36.8.
5-[(4-Nitroben zyl)sulfan yl]-1-ph en yl-1H-tetrazole (21f)21
Tetrazole 6 (1.000 g, 5.61 m m ol) an d fresh ly prepared sodium m eth oxide (7.30 m m ol) were
dissolved in dry DMF (6 m l) an d, after 10-m in stirrin g at room tem perature, 4-n itroben zyl
ch loride (1.440 g, 8.41 m m ol) was added. Th e reaction m ixture was stirred at 100 °C for
48 h , th en cautiously poured in to water (30 m l), an d th e resultan t m ixture was wash ed with
eth yl acetate (50 m l). Th e organ ic layer was wash ed with a 5% aqueous h ydroch loric acid
solution (30 m l), water (30 m l) an d a 5% aqueous sodium h ydrogen carbon ate solution
(30 m l). Th e organ ic layer was th en dried (Na2SO4), th e solven ts rem oved un der reduced
pressure, an d th e crude product purified by colum n ch rom atograph y (h exan e–eth yl acetate
75:25) to afford tetrazole 21f. Yield 40%. Yellowish crystals, m .p. 151–153 °C. 1H NMR
(300 MHz, CDCl3): 8.21–8.14 (2 H, m , AA′BB′, H3′, H5′), 7.69–7.61 (2 H, m , AA′BB′, H2′,
H6′), 7.58–7.48 (5 H, m , H2, H3, H4, H5, H6), 4.67 (2 H, s, CH2). 13C NMR (75 MHz,
CDCl3): 153.0, 147.6, 143.1, 133.3, 130.4, 130.2, 129.9, 123.9, 123.7, 36.3.
Collect. Czech. Chem. Commun. 2009, Vol. 74, Nos. 7–8, pp. 1161–1178

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Balšánek et al.:
This work was supported by the Centre for New Antivirals and Antineoplastics funded by the
Ministry of Education, Youth and Sports of the Czech Republic (1M0508) and by the Czech Science
Foundation (project No. 203/07/1302). It is also a part of the research project MSM0021620822 of
the Ministry of Education, Youth and Sports of the Czech Republic.
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