Design and synthesis of novel diphenacoum-derived, conformation-restricted vitamin K 2,3-epoxide reductase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.03.005

Two novel diphenacoum-derived analogues 5 and 6 are designed, synthesized and tested as potential vitamin K 2,3-epoxide reductase (VKOR) inhibitors. The inhibition studies indicated that 5 is a potent VKOR inhibitor, which confirmed that the replacement of the tetrahydronaphthalene on diphenacoum to a chroman functionality does not have a major impact on inhibition potency. The conformation-restricted compound 6 is a moderate inhibitor which may serve as a lead compound for further study of the mode of action of coumarin-type anticoagulants at the molecular level.

Full text of DOI:10.1016/j.bmcl.2005.03.005

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2665–2668
Design and synthesis of novel diphenacoum-derived,
conformation-restricted vitamin K 2,3-epoxide reductase inhibitors
Ding-Uei Chen, Pei-Yu Kuo and Ding-Yah Yang*
Department of Chemistry and Life Science Research Center, Tunghai University, 181, Taichung-Kang Road, Sec. 3,
Taichung 407, Taiwan, ROC
Received 8 July 2004; revised 25 February 2005; accepted 1 March 2005
Available online 11 April 2005
Abstract—Two novel diphenacoum-derived analogues 5 and 6 are designed, synthesized and tested as potential vitamin K 2,3-epox-
ide reductase (VKOR) inhibitors. The inhibition studies indicated that 5 is a potent VKOR inhibitor, which confirmed that the
replacement of the tetrahydronaphthalene on diphenacoum to a chroman functionality does not have a major impact on inhibition
potency. The conformation-restricted compound 6 is a moderate inhibitor which may serve as a lead compound for further study of
the mode of action of coumarin-type anticoagulants at the molecular level.
Ó 2005 Elsevier Ltd. All rights reserved.
Vitamin K 2,3-epoxide reductase (VKOR, E.C. 1.1.4.2)1
is a key enzyme involved in the vitamin K cycle,2,3 the
cyclic interconversion of vitamin K metabolites. It cata-
lyzes the conversion of vitamin K 2,3-epoxide to vitamin
K hydroquinone, as shown in Figure 1. Vitamin K
hydroquinone functions as an essential cofactor for vita-
min K-dependent carboxylases,4 which convert a limited
number of glutamic acid residues in targeted proteins to
c-carboxyglumate acid (Gla) residues. These Gla resi-
dues enable the proteins to bind Ca+2, as part of the
blood coagulation cascade.5 Concomitant with c-car-
boxylation, the hydroquinone cofactor is converted to
the metabolite vitamin K 2,3-epoxide, which is reduced
back to the hydroquinone by VKOR. Thus, inhibition
of the activity of VKOR, with its central position in
regulating biosynthesis of biologically active vitamin
K-dependent proteins,6 can prevent the formation of vita-
min K and subsequently reduced vitamin K. The lack of
carboxylase cofactor will then produce non-functional
vitamin K-dependent coagulation factors, which are
the basis for clinical drugs for control of thromboembo-
lic disease.7 It is well-documented that coumarin-type
anticoagulants target the vitamin K cycle by inhibition
of VKOR activity, which prevents reduction of vitamin
K, but their mode of action at the molecular level re-
mains unclear.8,9 For example, warfarin has long been
used as a therapeutic drug for anti-coagulation therapy
in humans as well as a poison in rodent pest control.
Unfortunately, this sixth most-prescribed cardiovascu-
lar drug in North America suffers from high interna-
tional normalized ratios (varying up to 120-fold),
which requires the treated patients to be monitored con-
stantly.10 Furthermore, the finding of hereditary warfa-
rin resistance in rats represented a major setback in
O
OH
VKOR
O
3
3
O
OH
Figure 1. Reaction catalyzed by vitamin K 2,3-epoxide reductase.
Keywords: VKOR inhibitor; Conformation restricted.
*
Corresponding author. Tel.: +886 4 2359 7613; fax: +886 4 2359 0426; e-mail: yang@mail.thu.edu.tw
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.005

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D.-U. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2665–2668
O
mals. In addition, the commercial preparation of
superwarfarins like diphenacoum is laborious and time
consuming. Although an improved procedure has been
recently reported,13 large scale production of diphena-
coum is still currently unavailable. Thus, developing
an easily prepared, potentially less toxic VKOR inhibi-
tor is highly desired. Here, we report the rational design,
efficient synthesis, and in vitro evaluation of a diphena-
coum derivative and a conformation-restricted dioxabi-
cycle as potential VKOR inhibitors.
OH
O
OH
O
O
O
coumatetralyl
warfarin
Br
Previous studies14 have demonstrated that the 4-
hydroxycoumarin moiety and the right hand phenyl part
of superwarfarins are absolutely required for inhibitor
potency. Yet the upper right hand moiety has minimal
effect and the incorporation of new functional groups
will not alter its biological activity dramatically. Hence,
we envisioned replacing the tetrahydronaphthalene moi-
ety of diphenacoum with a chroman functionality. The
resulting potential VKOR inhibitor presumably will of-
fer benefits of easy preparation, higher water solubility,
and lower toxicity when compared with diphenacoum.
In addition, it will be more susceptible to further chemi-
cal modification, that is, cyclization to give a conforma-
tion-restricted molecule, which can reduce the original
four possible stereoisomers to two enantiomers. Biologi-
cal evaluation of these analogues may shed light on the
mode of action of coumarin-type VKOR inhibitors.
OH
O
OH
OH
O
O
O
difenacoum
bromadialone
Figure 2. Structures of warfarin and superwarfarins.
rodent control. While the second generation anticoagu-
lants, superwarfarins11 (coumatetralyl, bromadiolone,
and diphenacoum, depicted in Fig. 2), have so far been
quite successful in fighting resistant rodents, their ex-
treme toxicity is a danger to humans12 and other ani-
O
OH
O
O
O
H3PO4
40% KOH
+
H
EtOH, reflux
MeOH, reflux
OH
R
R
O
2. R=Ph
2a. R=H
3. R=Ph
3a. R=H
1. R=Ph
1a. R=H
R
NaBH4
MeOH
r t
R
OH
4-hydroxycoumarin
OH
O
O
O
p-TsOH,CH2ClCH2Cl
reflux
R
O
4. R=H
4a. R=Ph
5. R=Ph
5a. R=H
4-hydroxycoumarin
AlCl3
130~140 oC
R
AlCl3
130~140 o
C
O
O
O
O
6. R=Ph
6a. R=H
Scheme 1. Synthesis of compounds 5, 5a, 6, and 6a.

D.-U. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2665–2668
2667
The preparation of the proposed compound 5 is
depicted in Scheme 1. The synthesis started with a
base-catalyzed aldol condensation of 20-hydroxyace-
tophenone and 40-phenylbenzaldehyde 1 under reflux
conditions in methanol for 3 h to give the a,b-unsatu-
rated ketone 2 with a 75% yield.15 Ketone 2 was then
cyclized by refluxing with phosphoric acid in ethanol
for 2 days to yield 2-biphenylchroman-4-one 3 with a
70% yield.15 Further reduction of the ketone 3 (or the
mixtures of 2 and 3) with sodium borohydride in meth-
anol under room temperature for 30 min gave the corre-
inhibition result is quite encouraging if we consider that
the 4-hydroxyl group on the coumarin moiety, which is
crucial for potent VKOR inhibition is not available in
compound 6. Since the importance of hydrogen bonding
in the enzyme–inhibitor interaction is well-recognized,
the inhibition activity of 6 may be attributed, partially
at least, to its novel rigid bicyclic skeleton. Therefore,
future preparation of 4-hydroxycoumarin conserved as
well as conformation-restricted diphenacoum analogues
not only can facilitate the development of potent VKOR
inhibitors but also can serve as a lead compound for fur-
ther study of the mode of action of coumarin-type anti-
coagulants at the molecular level.
sponding alcohol
4
quantitatively. The final
condensation of 4 with 4-hydroxycoumarin in 1,2-
dichloroethane in the presence of a catalytic amount
of p-toluenesulfonic acid under reflux conditions for
6 h afforded the target compound 5 with a 78% yield.
This concise synthetic route is highly attractive since
no column chromatography is needed. When compound
5 was further heated with aluminum chloride at 130–
140 °C for 30 min, a cyclized compound 6 formed as ex-
pected. Alternatively, this dioxabicyclic derivative 6 can
be prepared directly from condensation of alcohol 4
with 4-hydroxycoumarin in AlCl3.16 Numerous attempts
to obtain a satisfactory crystal structure of 6 proved fu-
tile, presumably due to the extended biphenyl moiety in
6 preventing proper crystal formation. Nevertheless, the
structure of 6 was indirectly confirmed by X-ray crystal-
lographic analysis of 6a,17 as depicted in Figure 3, which
was prepared by the exact same procedure as 6 except
benzaldehyde 1a was used as the starting material.
In conclusion, we have rationally designed, chemically
synthesized, and characterized two novel diphena-
coum-derived, conformation-restricted analogues. Bio-
logical evaluation demonstrated that 5 is a potent and
6 is a moderate VKOR inhibitor. Further design and
synthesis of a rigid diphenacoum-derived analogue,
which retains the 4-hydroxycoumarin functionality is
currently underway.
Acknowledgements
The financial assistance provided by National Science
Council of Republic of China is gratefully
acknowledged.
The inhibition studies of 5 and 6 with partially purified
VKOR18 from beef liver indicated that compound 5
remains a potent VKOR inhibitor with IC50 value of
0.04 lM, which is 2.5-fold more potent than warfarin.19
This result confirmed that the replacement of the tetra-
hydronaphthalene on diphenacoum with a chroman
functionality does not have a major impact on inhibition
potency. Although further toxicity tests are needed, the
four-step, highly efficient synthesis of 5 from relatively
inexpensive starting materials and reagents makes it
suitable for mass production on an industrial scale.
The conformation-restricted compound 6, on the other
hand, was a moderate VKOR inhibitor with IC50 value
of 1 lM, which is 10-fold less potent than warfarin. This
References and notes
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16. For preparation of 5 and 6 from 4: To a solution of 2-
biphenyl-4-yl-chroman-4-ol 4 (0.2 g, 0.66 mmol) and 4-
hydroxycoumarin (0.1 g, 0.62 mmol) in 1,2-dichloroethane
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Figure 3. X-ray crystal structure of compound 6a.

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D.-U. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2665–2668
solvent was concentrated in vacuo. This dark red mixture
over MgSO4, filtered, and concentrated. The crude final
product was purified by column chromatography
(EtOAc–hexanes = 1.5:8.5) to give a white solid 6-biphen-
yl-6,12-methano-6H,12H,13H-[1]benzopyrano[4,3-d][1,3]-
benzodioxocin-13-one 6 in a 62% yield. Mp 211–212 °C.
1H NMR (CDCl3, 400 MHz): d 7.90–6.96 (m, 17H, Ar
HÕs), 4.41 (t, J = 2.8 Hz, 1H), 2.51, 2.45 (ABdq, J = 13.6,
2.8 Hz, 1H each); 13C NMR (CDCl3, 100 MHz): d 163.0,
161.2, 155.2, 152.6, 141.1, 140.6, 139.1, 132.1, 130.4, 128.7,
128.6, 127.3, 127.1, 127.0, 126.5, 124.0, 123.2, 122.1, 118.9,
118.8, 116.4, 116.1, 107.0, 75.4, 34.7, 32.7, 29.7. IR (KBr) m
1715, 1231, 761 cmꢀ1. HRMS (FAB) calcd for C30H20O4
(MH+), 445.1449, found 445.1450. Anal. Calcd for
C30H20O4: C, 81.07; H, 4.54; O, 14.40. Found: C, 80.78;
H, 4.53; O, 14.51.
was poured into water and basified with dilute sodium
hydride solution. The solution was then extracted with
ethyl acetate twice. The combined organic extracts were
dried over MgSO4, filtered, and concentrated. The crude
final product was purified by column chromatography
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yl-hydroxy-30,40-dihydro-20H-[3,40]bichromenyl-2-one 5 in
a
78% yield. Mp 225–226 °C. 1H NMR (CDCl3,
300 MHz): d 7.76–7.00 (m, 17H, Ar HÕs), 5.18 (t,
J = 6.3 Hz, 1H), 4.59 (t, J = 4.2 Hz, 1H), 2.49 (q,
J = 1.5 Hz, 2H); 13C NMR (CDCl3, 75 MHz): d 161.6,
158.2, 152.4, 151.3, 142.4, 140.4, 138.6, 132.0, 128.9, 128.4,
128.2, 127.7, 127.4, 127.2, 126.2, 125.2, 124.1, 122.8, 122.1,
116.7, 116.4, 115.1, 106.1, 100.3, 32.9, 27.2. IR (KBr) m
3393, 1673, 1612, 1222, 758 cmꢀ1. HRMS (EI) calcd for
C30H22O4 (M+), 446.1518, found 446.1511. Anal. Calcd
for C30H22O4: C, 80.70; H, 4.97; O, 14.33. Found: C,
80.30; H, 5.23; O, 14.23. To a mixture of 2-biphenyl-4-yl-
chroman-4-ol 4 (0.2 g, 0.66 mmol) and 4-hydroxycouma-
rin (0.1 g, 0.62 mmol) was added a catalytic amount of
aluminum chloride. This mixture was then gently heated
to 130–140 °C. After completion of the reaction within
30 min (monitored by TLC), the stiff solid was allowed to
stand 15 min at room temperature. This mixture was
poured into water and basified with dilute sodium hydride
solution. The solution was then extracted with ethyl
acetate twice. The combined organic extracts were dried
17. Crystallographic data (excluding structure factors) for 6a
has been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number
CCDC-236132. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12, Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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