Umbelliferone aminoalkyl derivatives, a new class of squalene-hopene cyclase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2004.06.010

The synthesis is described of several aminoalkyl derivatives of coumarin, obtained in good yields under microwave or high-intensity ultrasound irradiation. These compounds proved uniformly active as inhibitors of squalene-hopene cyclase (SHC) from Alicycl

Full text of DOI:10.1016/j.ejmech.2004.06.010

European Journal of Medicinal Chemistry 39 (2004) 917–924
www.elsevier.com/locate/ejmech
Original article
Umbelliferone aminoalkyl derivatives,
a new class of squalene-hopene cyclase inhibitors
Giancarlo Cravotto a,*, Gianni Balliano a, Silvia Tagliapietra a,
Giovanni Palmisano b, Andrea Penoni b
a Dip. to di Scienza e Tecnologia del Farmaco, Università di Torino, via Giuria 9, 10125 Turin, Italy
b Dip. to di Scienze Chimiche e Ambientali; Università dell’Insubria, Via Valleggio 11, 22100 Como, Italy
Received 16 April 2004; accepted 29 June 2004
Available online 09 September 2004
Abstract
The synthesis is described of several aminoalkyl derivatives of coumarin, obtained in good yields under microwave or high-intensity
ultrasound irradiation. These compounds proved uniformly active as inhibitors of squalene-hopene cyclase (SHC) from Alicyclobacillus
acidocaldarius. Their design stemmed from our recent finding that the umbelliferone nucleus acquires inhibitory properties towards SHC
when functionalized with a suitable chain such as the x-epoxyfarnesyl group. Under our experimental conditions the most active ones, such
as 7-(4′-allylmethylamino-but-2-ynyloxy)chromen-2-one (IC50 0.75 mM), approached the potency of anticholesteremic drug Ro 48-8071
(IC50 0.35 mM), an effective inhibitor of both squalene- and oxidosqualene-cyclases (OSC). Tests are in progress to determine their efficacy
on different eukaryotic OSCs.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Coumarin derivatives; Squalene-hopene cyclase; Oxidosqualene cyclase; Microwave; High-intensity ultrasound
1. Introduction
sign is not feasible for the development of novel inhibitors.
However, the elucidation of the crystal structure of squalene-
hopene cyclase (SHC) [22], the functional analogue of OSCs
in bacteria, opened the road to understanding the catalytic
mechanism of OSCs and provided a good model for the
preliminary testing of potential inhibitors. Experiments of
co-crystallization of SHC with squalene-like [23] and other
molecules [24,25], in conjunction with homology-modelling
studies [26], have shed light on the structural requirements
for a molecule to fit the active site of these enzymes. One
such required feature is a tertiary amino group linked to a
hydrophobic moiety. Crystallographic studies have esta-
blished that the positive charge of the amine directly interacts
with the aspartate residue of the conserved DDTA motif at
the active site of SHC [23–25]. Recently, after investigating a
group of meroterpenoids that proved efficient inhibitors of
SHC [27], we proceeded to simplify the most active structu-
res (1,2, Fig. 1), gathering useful clues to improve molecular
design. We later found [28] that 2,4-dihydroxyacetophenone
also provides a convenient starting skeleton: compounds 3
and 4 tested as moderately active. The present work elabora-
tes further on the umbelliferone nucleus (7-
One of the most intensively studied enzymes of sterol
biosynthesis, oxidosqualene cyclase (OSC), catalyzes the
conversion of an acyclic compound, 2,3-oxidosqualene, to
the first cyclic intermediate along the pathway. This is lanos-
terol in all non-photosynthetic organisms, cycloartenol in
plants [1]. Several mammalian OSCs have been isolated,
characterized, cloned and sequenced [2–9]; in the same spate
hundreds of OSC inhibitors have been designed and tested
[10–14] as potential anti-fungal [15] or cholesterol-lowering
drugs [16]. Recently, a novel series of orally active inhibitors
have been tested on human liver microsomal OSC, and their
effectiveness as cholesterol-lowering drugs has been evalua-
ted in hyperlipidemic hamsters [17]. OSC inhibitors are also
under investigation as potential antifungal [18] and anti-
trypanosomal agents [19–21].
As no detailed crystal structure has been published yet for
eukaryotic OSCs, a straightforward structure-based drug de-
* Corresponding author. Tel.: +39-11-670-7687.
E-mail address: giancarlo.cravotto@unito.it (G. Cravotto).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.06.010

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G. Cravotto et al. / European Journal of Medicinal Chemistry 39 (2004) 917–924
suitable substrate for the Mannich reaction of terminal C(sp)
with putative methyliminium species originating from the
condensation of formaldehyde with secondary amines such
as N,N,N′-trimethylethylenediamine, pyrrolidine, diethy-
lamine and allylmethylamine (14–17, Scheme 2). Our previ-
ous protocol [31] was much improved working in tightly
sealed pressure-resistant tubes and irradiating with MW.
Table 1 shows the isolated yields for both methods.
Umbelliferone 5 was O-alkylated with 6-bromohexanol
[32], 8-bromooctanol and 1,6-dibromohexane by refluxing in
acetone in the presence of potassium carbonate [33]. The
primary alcoholic groups were successively oxidised to alde-
hyde by stirring at room temperature with pyridinium chlo-
rochromate in CH2Cl2. A longer alkyl chain (C10) was in-
serted by Mitsunobu dehydroalkylation of umbelliferone
with 10-undecen-1-ol [29], then exploiting the C=C double
bond as a latent carbonyl function. Oxidative demolition of
the terminal olefin with osmium tetroxide/N-methyl-
morpholine N-oxide and potassium periodate afforded the
10-formyl derivative [34]. Products easily underwent reduc-
tive amination under HIU. This reaction was carried out in a
sonochemical reactor developed in the authors’ laboratory
[35] using methanol–water 7:3 containing 3% CH3COOH
and excess amine (benzylamine, piperidine, morpholine,
dimethylamine or allylmethylamine) followed by treatment
with NaBH3CN (Scheme 3). After about 1 h sonication,
amino derivatives 18–25 were isolated in good yields (70–
85%).
Products 19, 20 and 24 were obtained in comparable
yields but after much longer reaction times (36–48 h) by
nucleophilic substitution of 7-(6-bromohexyloxy)coumarin
(7) [36] prepared by O-alkylation of umbelliferone with
1,6-dibromohexane.
Although we had previously found that derivatives of
umbelliferone were generally more active than those of
4-hydroxycoumarin bearing the same chain [27], we decided
to determine the effect of introducing an amino group into
Fig.
1
.
SHC inhibitors derived from umbelliferone and 2,4-
dihydroxyacetophenone: farnesyferol C (1) (IC50 7.0 µM), umbelliprenine-
10′,11′-monoepoxyde (2) (IC50 2.5 µM), secoammoresinol (3) (IC50
30 µM), and 4-(10-undecenyl) resacetophenone (4) (IC50 48 µM).
hydroxycoumarin) by the insertion of alkyl chains bearing a
terminal amino group; this appears in fact to be most prom-
ising feature for OSC inhibitory activity.
2. Chemistry
O-alkylation of umbelliferone (5) was achieved by treat-
ment with alkyl bromides and base or, more efficiently, by the
Mitsunobu reaction (alcohol, DIAD, TPP, THF) carried out
under sonochemical conditions as previously described [29].
High-intensity ultrasound (HIU) considerably reduced reac-
tion times and gave uniformly good yields (Scheme 1).
Among the strategies we explored to insert the amino
function in the side chain, the most efficient ones were
microwave (MW)-promoted Mannich aminomethylation of
a terminal alkyne, reductive amination under HIU and classic
nucleophilic substitution with alkyl bromides. O-propar-
gylation of 5 afforded the well-studied compound 6 [30], a
Scheme 2. MW-promoted Mannich aminomethylations of O-propargyl
umbelliferone.
Table 1
Yield % of Mannich aminomethylations
Compound
MW (90 °C)
Heating under reflux
(15 min), yield % (9 h), yield %
14
15
16
17
81
92
96
94
34
54
53
60
Scheme 1
tions of the side chain.
. Conditions for umbelliferone alkylation and further modifica-

G. Cravotto et al. / European Journal of Medicinal Chemistry 39 (2004) 917–924
919
4. Result and discussion
As described in paragraph 2, several aminoalkyl deriva-
tives of coumarin were prepared in good yields by MW- and
HIU-promoted reactions. Umbelliferone derivatives proved
uniformly effective on SHC from Alicyclobacillus acidocal-
darius. Their design stemmed from our recent finding that
the umbelliferone nucleus acquired inhibitory properties to-
wards SHC when functionalized with a suitable side chain
such as the x-epoxyfarnesyl group [27]. The tip of the prenyl
moiety plays here a crucial role by mimicking the portion of
the squalene molecule that interacts with the substrate-
protonating groups of the enzyme [37], while the coumarin
moiety interacts with the aromatic residues at the active site
that are involved in stabilizing the high-energy intermediates.
As the literature abundantly documents the importance of an
amino group in prenyl- and non prenyl-type inhibitors of
squalene and oxidosqualene cyclases, we replaced the ep-
oxyfarnesyl arm with a 6–10 C chain bearing a tertiary amino
group.
Scheme 3
derivatives.
. Sonochemical reductive amination of umbelliferone formyl
The majority of derivatives tested in the present work
proved to be active with IC50 values of 2–7 µM. The type of
amino group, the length and carbon unsaturation of the chain
influenced activity only slightly. Most active was 17 (IC50
0.75 µM), in which the rigid acetylenic arm bears an allylm-
ethylamino function. Under our experimental conditions the
activity of 17 approached that of the anticholesteremic drug
Ro 48-8071 (IC50 0.35 µM), an effective inhibitor of both
squalene- and oxidosqualene cyclases [16,17,25] that has a
very different structure but also contains the same amino
group.
Scheme 4. Synthesis of 3-aminoacyl derivatives of 4-hydroxycoumarin.
one of the latter. The allylmethylamino derivative of
4-hydroxycoumarin (28) was prepared in three steps: acyla-
tion with 10-undecenoyl chloride, oxidative demolition of
the terminal double bond and reductive amination under HIU
(Scheme 4).
5. Conclusion
3. Biology
In summary, we showed that umbelliferone aminoalkyl
derivatives are a new promising class of SHC inhibitors, and
pinpointed the structural details of the aminoalkyl group that
are critical for molecular recognition. These compounds are
easier to prepare and much more stable than monoepoxyfar-
nesyl derivatives previously studied by us [27]. Tests are in
progress to determine their efficacy on different eukaryotic
OSCs.
Inhibition tests carried out on SHC from Alicyclobacillus
acidocaldarius at concentrations up to 100 µM showed that,
excepting the adduct of umbelliferone with the N,N,N′-
trimethylethylenediamine (14) and the 3-aminoacyl deriva-
tive of 4-hydroxycoumarin (28), all compounds listed in
Table 2 had good and comparable inhibitory activities. Most
active were 17 (IC50 0.75 µM) and 25 (IC50 2 µM), that
shared a methylallylamino group.
Table 2
6. Experimental protocols
Inhibitory activities on SHC for compounds 14–28 in comparison with Ro
48-8071 (Hoffman-La Roche)
6.1. General methods
Compound
IC50 (µM) a
Compound
IC50 (µM) a
14
15
16
17
18
19
20
na a
5
21
22
23
24
25
28
7
Anhydrous conditions were achieved (when indicated) by
flame-drying flasks and other equipment. Reactions were
monitored by TLC on Alugram Sil—Macherey-Nagel F254
(0.25 mm) plates, which were visualized by UV inspection
and stained with a 5% KMnO4 solution or by heating after a
spray of 5% H2SO4 in ethanol. Macherey-Nagel silica gel
was used for column chromatography (CC). A Waters mi-
5–7
5
5
0.75
8
4–5
2
8
100
0.35
6
Ro 48-8071
a Values are means of three experiments (na = not active at 100 µM).

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G. Cravotto et al. / European Journal of Medicinal Chemistry 39 (2004) 917–924
croPorasil column 7.8-300 was used for semipreparative
HPLC, a Gilson 133 refractive index refractometer serving
for peak detection. Melting points were obtained on a Büchi
SMP-20 apparatus and are uncorrected. 1H NMR (300 MHz)
spectra were recorded on a Bruker 300 Advance spectrom-
eter at 25 °C. CIMS were performed on a Finnigan-MAT
TSQ70 with isobutane as reactant gas. Unless otherwise
noted commercially available reagents and solvents were
used without further purification. Immediately prior to use
CH2Cl2 and THF were distilled under N2 from P4O10 and
sodium-benzophenone ketyl, respectively.
The sonochemical apparatus used in the present work was
designed for stringent reaction conditions. The transducer,
consisting of two high-efficiency pre-stressed piezoelectric
rings, is lodged in a Delrin® housing that can be cooled by a
flow of refrigerated oil. Moreover, the whole probe system
(comprising transducer, booster and immersion horn) is re-
frigerated by an oil forced-circulation circuit connected to an
oil–freon heat exchanger; this is cooled in turn by a refrigera-
tor unit. Frequency can be tuned between 17 and 45 kHz and
power output can be varied up to 200 W/cm2. To achieve
optimal acoustic efficiency the reactor can rotate eccentri-
cally around the horn and the probe can be made to move
alternatively up and down by a predetermined excursion at a
chosen speed. Transducer resonance is maintained by a true
motional feedback network. Electrical and acoustic param-
eters are monitored. Reactions were carried out in a Teflon®
tube (1 mm thick) inserted in a Delrin® reactor that was
thermostatted by four Peltier modules.
6.2.2. Mitsunobu reaction under HIU
A solution in anhydrous THF (35 ml) containing triph-
enylphosphine (2 mmol), the alcohol (2 mmol) and umbellif-
erone (2 mmol), was sonicated (18.0 kHz, 100 W/cm2) at
10 °C under an argon atmosphere. Diisopropyl azodicar-
boxylate (DIAD) (2 mmol) was added dropwise over 5 min.
The orange-red colour of DIAD immediately disappeared
and a weakly exothermic reaction occurred. The mixture was
sonicated for 35–55 min at 20 °C. When the reaction was
complete, as indicated by TLC (eluent: hexane/EtOAc, 9:1),
the mixture was evaporated to dryness. The residue was
diluted with hexane–ether 3:1 v/v, filtered through a thin pad
of Celite® to remove the precipitate of triphenylphosphine
oxide and concentrated under reduced pressure. Finally the
product was purified by silica gel CC. The reaction was
carried out under nitrogen in a Teflon tube inserted in the
thermostatted reactor.
6.2.3. 7-(6-Bromohexyloxy)chromen-2-one (7)]
White powder; m.p. 45 °C. IR (KBr) 3422, 1722, 1624,
1400, 1293, 1236, 829 cm–1; 1H NMR (CDCl3) d = 7.64 (1H,
d, J = 9.5 Hz, H-4), 7.37 (1H, d, J = 8.4 Hz, H-5), 6.85–6.81
(2H, m, H-6, H-8), 6.25 (1H, d, J = 9.46 Hz, H-3), 4.03 (2H,
t, J = 6.34 Hz, H-1′), 3.44 (2H, t, J = 6.72 Hz, H-6′),
1.90–1.52 (8H, m, CH2). CIMS: 325 (M + H)+. Rf = 0.50
(hexane/EtOAc, 1:1).
6.2.4. 7-(6′-hydroxyhexyloxy)chromen-2-one (8)]
White powder; m.p. 49 °C. IR (KBr) 3450, 1720, 1620,
For microwave-promoted reactions a modified version of
a domestic oven (Candy MSA 20M) was used. The oven was
placed in a protective cage. CAUTION: reaction vessels must
be allowed to cool down below the boiling point of the
solvent before they are removed from the oven and opened.
Analyses for C, H, N were within 0.3% of the theoretical
value.
1
1540, 1295, 1232, 1120 cm–1; H NMR (CDCl3) d = 7.64
(1H, d, J = 9.5 Hz, H-4), 7.36 (1H, d, J = 8.4 Hz, H-5),
6.85–6.79 (2H, m, H-6, H-8), 6.24 (1H, d, J = 9.5 Hz, H-3),
4.02 (2H, t, J = 6.5 Hz, H-1′), 3.67 (2H, t, J = 6.5 Hz, H-6′),
1.83 (2H, m, H-2′) 1.60 (2H, m, H-5′) 1.55–1.44 (4H, m, H-3′
and 4′). CIMS: 263 (M + H)+. Rf = 0.46 (hexane/EtOAc, 1:1).
6.2.5. 7-(8′-hydroxyoctyloxy)chromen-2-one (9)]
6.2. General procedures for O-alkylation of umbelliferone
White powder; m.p. 101 °C. IR (KBr) 3450, 1738, 1646,
1473, 1410, 1298, 838 cm–1; 1H NMR (CDCl3) d = 7.58 (1H,
d, J = 9.5 Hz, H-4), 7.29 (1H, d, J = 8.4 Hz, H-5), 6.77–6.69
(2H, m, H-6, H-8), 6.16 (1H, d, J = 9.5 Hz, H-3), 3.57 (2H, t,
J = 6.5 Hz, H-8′), 3.32 (2H, t, J = 6.4 Hz, H-1′), 1.70 (2H, m,
H-2′) 1.50 (2H, m, H-7′), 1.50–1.25 (8H, m, CH2). CIMS:
291 (M + H)+. Rf = 0.55 (hexane/EtOAc, 1:1).
6.2.1. Conventional protocol
In a 50 ml two-necked, round-bottomed flask equipped
with a magnetic stirrer, a condenser and a nitrogen inlet,
umbelliferone (891 mg, 5.5 mmol), K2CO3 (1.52 g,
11.0 mmol) and 33 ml of anhydrous acetone were added. The
stirred mixture was heated under reflux for 2 h under nitrogen
atmosphere, then allowed to cool down to room temperature
before the alkylbromide or bromoalcohol (5.5 mmol) was
added dropwise. The resulting mixture was heated under
reflux for another 7 h. Eluents used for TLC were
hexane/EtOAc 1:1 or 7:3.
Work-up: the reacted mixture was quenched with water
(20 ml), extracted with ethyl acetate (50 ml) and washed with
brine (20 ml). After drying (MgSO4) and removal of the
solvent, the residue was purified by silica gel CC
(hexane/EtOAc, 9:1) to afford the corresponding
7-alkyloxycoumarin in high yield.
6.3. General procedure for the oxidation of primary
hydroxyls in the side chain
In a 100 ml round-bottomed flask containing the alcohol
solution (2 mmol in 40 ml CH2Cl2), pyridinium chlorochro-
mate (PCC, 4 mmol) and Celite® (0.2 g) were added. After
2 h stirring at room temperature the reaction was monitored
by TLC (eluent: hexane/EtOAc, 9:1). The residue was fil-
tered through a pad of Celite® and concentrated under
vacuum. Products were purified by silica gel CC.

G. Cravotto et al. / European Journal of Medicinal Chemistry 39 (2004) 917–924
921
6.3.1. 7-(6′-oxohexyloxy)chromen-2-one (10)]
screw cap, the following were placed: dioxane (10 ml),
paraformaldehyde (4.0 mmol), copper(II) acetate (5 mg) and
the amine (1.5 mmol). The mixture was irradiated with mi-
crowave (200 W) for 5 min. Temperature rose to 80 °C and a
violet color developed. After cooling down to 25 °C,
7-propargyloxycoumarin (300 mg, 1.5 mmol) dissolved in
dioxane (5 ml) was added and the mixture was irradiated
further (200 W) for 10 min. The reaction was monitored by
TLC using CHCl3/CH3OH (9:1) as eluent (starting material
Rf = 0.8, products Rf all below 0.6). The reacted mixture was
quenched with water (20 ml), extracted with ethyl acetate
(50 ml) and washed with brine (20 ml). After drying
(MgSO4) and removal of the solvent, the residue was purified
Gum; IR (liquid film) 1732, 1614, 1556, 1506, 1350,
1
1230, and 1122 cm–1; H NMR (CDCl3) d = 9.79 (1H, s,
CHO), 7.64 (1H, d, J = 9.5 Hz, H-4), 7.37 (1H, d, J = 8.4 Hz,
H-5), 6.84–6.79 (2H, m, H-6, H-8), 6.25 (1H, d, J = 9.5 Hz,
H-3), 4.02 (2H, t, J = 6.5 Hz, H-1′), 2.50 (2H, td, J = 7.2,
1.6 Hz, H-5′), 1.87–1.80 (2H, m, H-2′), 1.75–1.68 (2H, m,
H-4′), 1.58–1.50 (2H, m, H-3′). CIMS: 261 (M + H)+; Rf =
0.60 (hexane/EtOAc, 1:1).
6.3.2. 7-(8′-Oxooctyloxy)chromen-2-one (11)]
White powder, m.p. 53 °C. IR (KBr) 1732, 1618, 1402,
1290, 1242, 1132, and 845 cm–1; 1H NMR (CDCl3) d = 9.77
(1H, s, CHO), 7.63 (1H, d, J = 9.5 Hz, H-4), 7.36 (1H, d, J =
8.4 Hz, H-5), 6.77–6.69 (2H, m, H-6, H-8), 6.24 (1H, d, J =
9.5 Hz, H-3), 4.01 (2H, t, J = 6.5 Hz, H-1′), 2.44 (2H, td, J =
7.2 Hz, J = 1.6 Hz, H-7′), 1.82 (2H, m, H-2′) 1.65 (2H, m,
H-6′), 1.48–1.37 (6H, m, H-3′-5′). CIMS: 289 (M + H)+. Rf =
0.51 (hexane/EtOAc, 1:1).
by silica gel column chromatography (CHCl3
→
CHCl3/MeOH, 9:1) to afford the corresponding Mannich
adduct.
6.4.2. Heating under reflux
In a 50 mL two-necked, round-bottomed flask equipped
with a magnetic stirrer and a condenser, the following were
placed: dioxane (10 mL), paraformaldehyde (4.0 mmol),
copper (II) acetate (5 mg) and the amine (1.5 mmol). The
stirred mixture was heated at 60 °C for 1 h, during which time
a violet color developed. Then 7-(propargyloxy)coumarin
(300 mg, 1.5 mmol) in dioxane (5 ml) was added, and heating
continued at 90 °C for 8 h.
6.3.3. 7-(10-undecenyloxy)chromen-2-one (12)
White powder; m.p. 85 °C. IR (KBr) 1726, 1624, 1134,
1
837 cm–1; H NMR (CDCl3) d = 7.60 (1H, d, J = 9.5 Hz,
H-4), 7.35 (1H, d, J = 8.4 Hz, H-5), 6.80 (2H, m, H-6, H-8),
6.25 (1H, d, J = 9.5 Hz, H-3), 3.95 (2H, t, J = 6.50 Hz, H-1),
2.75 (2H, t, J = 4.90 Hz, H-11′), 2.45 (1H, m, H-10′), 1.80
(2H, m, H-2′), 1.65 (2H, m, H-9′), 1.50–1.20 (12H, m, CH2).
CIMS: 315 (M + H)+; Rf = 0.45 (hexane/EtOAc, 4:1).
6.4.3. 7-[4′-(N,N,N′-trimethylethylendiamino)-but-2-yny-
loxy]chromen-2-one (14)
6.3.4. Oxidative demolition of 12
Yellow oil; IR (liquid film) 1721, 1607, 1566, 1283, 1231,
In a 50 mL round-bottomed flask equipped with a mag-
netic stirrer 628 mg of 12 (2.0 mmol), water/acetone, 1:1
mixture (15 ml), 0.2 M OsO4 in toluene (100 µl,
0.025 eq/mol) and N-methylmorpholine-N-oxide (232 mg)
were added. After 1 h stirring at room temperature, NaIO4
(422 mg, 1.97 mmol) was added portionwise and the mixture
was stirred for another 3 h, monitoring the reaction course by
TLC (eluant: hexane/EtOAc, 7:3). The reacted mixture was
quenched with water (20 ml), extracted with CH2Cl2 (40 ml),
and washed with brine (20 ml). After drying (MgSO4) and
removal of the solvent, the residue was purified by silica gel
CC yielding 405 mg of 13 (yield 63%).
1
1123, 1086 cm–1; H NMR (CDCl3) d = 7.61 (1H, d, J =
9.5 Hz, H-4), 7.38 (1H, d, J = 8.7 Hz, H-5), 6.96–6.87 (2H,
m, H-6, H-8), 6.24 (1H, d, J = 9.5 Hz, H-3), 4.79 (2H, s,
H-1′), 3.78 (2H, s, H-4′), 2.71 (4H, s, H-1″, 2″), 2.37 (6H, s,
2 × CH3), 2.26 (3H, s, CH3). CIMS: 315 (M + H)+; Rf = 0.19
(CHCl3/MeOH, 9:1).
6.4.4. 7-[4′-(N-pyrrolidyn)-but-2-ynyloxy]chromen-2-one
(15)
White powder; m.p. 66 °C; IR (KBr) 1725, 1616, 1277,
1232, 1123, 999, and 831 cm–1; 1H NMR (CDCl3) d = 7.62
(1H, d, J = 9.5 Hz, H-4), 7.36 (1H, d, J = 8.3 Hz, H-5), 6.88
(2H, m, H-6, H-8), 6.23 (1H, d, J = 9.5 Hz, H-3), 4.76 (2H, s,
H-1′), 3.43 (2H, s, H-4′), 2.56 (4H, t, J = 5.4 Hz, H-1″, 4″),
1.75 (4H, m, H-2″, 3″). CIMS: 284 (M + H)+; Rf = 0.51
(CHCl3/MeOH, 85:15).
6.3.5. 7-(10′-Oxodecyloxy)chromen-2-one (13)
White powder. m.p. 55 °C. IR (KBr) 3343, 2980, 2853,
1736, 1616, 1128, and 833 cm–1; 1H NMR (CDCl3) d = 9.78
(1H, s, CHO), 7.64 (1H, d, J = 9.46 Hz, H-4), 7.37 (1H, d, J =
8.4 Hz, H-5), 6.85–6.81 (2H, m, H-6, H-8), 6.25 (1H, d, J =
9.46 Hz, H-3), 4.02 (2H, t, J = 6.47 Hz, H-1′), 2.44 (2H, t, J =
7.27 Hz, H-10’), 1.82 (2H, m, H-9′) 1.66–1.27 (12H, m,
CH2). CIMS: 317 (M + H)+. Rf = 0.22 (hexane/EtOAc, 7:3).
6.4.5. 7-[4′-(N-diethylamino)-but-2-ynyloxy]chromen-2-
one (16)
Gum; IR (KBr) 1744, 1730, 1618, 1607, 1275, 1207,
1120, 1007, and 839 cm–1; 1H NMR (CDCl3) d = 7.33 (1H, d,
J = 8.3 Hz, H-5), 6.59 (1H, d, J = 9.5 Hz, H-4), 6.83 (2H, m,
H-6, H-8), 6.18 (1H, d, J = 9.5 Hz, H-3), 4.71 (2H, s, H-1′),
3.37 (2H, s, H-4′), 2.41 (4H, q, J = 7.2 Hz, CH2N), 0.95 (6H,
t, J = 7.2 Hz, CH3). CIMS: 286 (M + H)+; Rf = 0.52
(CHCl3/MeOH, 4:1).
6.4. General procedure for Mannich aminomethylation
6.4.1. Microwave-assisted (N.B. always work behind
a shield)
In a 50 ml Carius-type pyrex tube (heavy-wall borosilicate
glass, ACE Glassware) stoppered with a pressure-resistant

922
G. Cravotto et al. / European Journal of Medicinal Chemistry 39 (2004) 917–924
6.4.6. 7-(4′-Allylmethylamino-but-2-ynyloxy)chromen-2-
one (17)
6.5.4. 7-(Morpholinyl-N-octyloxy)chromen-2-one (21)
Yellow oil; IR (liquid film) 1732, 1614, 1352, 1280, 1230,
1121, and 837 cm–1; 1H NMR (CDCl3) d = 7.61 (1H, d, J =
9.5 Hz, H-4), 7.34 (1H, d, J = 8.4 Hz, H-5), 6.81–6.75 (2H,
m, H-6, H-8), 6.20 (1H, d, J = 9.5 Hz, H-3), 3.97 (2H, t, J =
6.5 Hz, H-1′), 3.72 (4H, m, H-3″, H-5″), 2.52 (2H, t, J =
7.6 Hz, H-2″, H-6″), 2.39 (2H, m, H-8′), 1.75 (2H, m, H-2′),
1.52–1.31 (10H, m, CH2). CIMS: 360 (M + H)+; Rf = 0.13
(hexane/EtOAc, 1:9).
Yellow oil; IR (liquid film) 1721, 1618, 1279, 1107, and
812 cm–1; 1H NMR (CDCl3) d = 7.63 (1Η, d, J = 9.5 Hz,
H-4), 7.37 (1H, d, J = 8.3 Hz, H-5), 6.90 (1H, s, H-8), 6.88
(1H, overlap, H-6), 6.24 (1H, d, J = 9.5 Hz, H-3), 5.76 (1H,
m, H-7′), 5.11 (2H, m, H-8′), 4.77 (2H, s, H-1′), 3.33 (2H, s,
H-4′), 2.97 (2H, d, J = 6.6 Hz, H-6′), 2.24 (3H, s, CH3).
CIMS: 284 (M + H)+; Rf = 0.58 (CHCl3/MeOH, 1:1).
6.5.5. 7-[8′-(Dimethylamino-N-octyloxy)]chromen-2-one
(22)
6.5. General procedure for reductive amination
White powder; m.p. 50 °C; IR (KBr) 1732, 1614, 1470,
1363, 1124, and 854 cm–1; 1H NMR (CDCl3) d = 7.59 (1H, d,
J = 9.5 Hz, H-4), 7.32 (1H, d, J = 8.4 Hz, H-5), 6.80–6.73
(2H, m, H-6, H-8), 6.18 (1H, d, J = 9.5 Hz, H-3), 3.95 (2H, t,
J = 6.5 Hz, H-1′), 2.63 (2H, t, J = 7.8 Hz, H-8′), 2.49 (6H, s,
H-1″, H-2″), 1.75 (2H, m, H-2′), 1.59 (2H, m, H-7′), 1.40–
1.20 (8H, m, CH2). CIMS: 318 (M + H)+; Rf = 0.53
(CHCl3/MeOH, 4:1).
Reactions were performed in a 1-mm thick Teflon® tube
inserted in a Delrin® reactor thermostatted by two Peltier
modules. 7-(formylalkyloxy)coumarin (0.4 mmol) and an
excess of the amine (1.5 mmol) dissolved in methanol–water
(7:3) containing 3% CH3COOH (20 ml) were sonicated
(18.1 kHz, 80 W/cm2) for 15 min at 30 °C. Then NaBH3CN
(0.4 mmol) was added portionwise and the mixture was
sonicated for an additional 45 min. TLC: eluent
hexane/EtOAc, 1:1. Work-up: the mixture was diluted with
water (20 ml), extracted with CH2Cl2 (30 ml) and washed
with brine (20 ml). After drying (MgSO4) and removal of the
solvent the residue was purified by silica gel column chroma-
tography (hexane/EtOAc, 7:3 → 1:1) to afford the corre-
sponding amino derivatives.
6.5.6. 7-[10′-(Dimethylamino-N-decyloxy)]chromen-2-one
(23)
White powder; m.p. 57 °C; IR (KBr) 1732, 1614, 1470,
1364, 1126, and 854 cm–1; 1H NMR (CDCl3) d = 7.59 (1H, d,
J = 9.5 Hz, H-4), 7.32 (1H, d, J = 8.4 Hz, H-5), 6.80–6.73
(2H, m, H-6, H-8), 6.18 (1H, d, J = 9.5 Hz, H-3), 3.95 (2H, t,
J = 6.5 Hz, H-1′), 2.64 (2H, t, J = 7.8 Hz, H-10′), 2.50 (6H, s,
H-1″, H-2″), 1.75 (2H, m, H-2′), 1.61 (2H, m, H-9′), 1.46–
1.20 (12H, m, CH2). CIMS: 318 (M + H)+; Rf = 0.56
(CHCl3/MeOH, 4:1).
6.5.1. 7-[6′-(benzylamino-hexyloxy)]chromen-2-one (18)
Yellow oil; IR (liquid film) 3300, 1732, 1614, 1271, 1230,
1122, 835, and 698 cm–1; 1H NMR (CDCl3) d = 8.11 (1H, d,
J = 9.5 Hz, H-4), 7.82 (5H, m, Ph), 7.80–7.71 (1H, m, H-5),
7.34–7.27 (2H, m, H-6, H-8), 6.71 (1H, d, J = 9.5 Hz, H-3),
4.49 (2H, t, J = 6.5 Hz, H-1′), 4.29 (2H, s, CH2-Ph), 3.15 (2H,
t, J = 6.9 Hz, H-6′), 2.31 (2H, m, H-2′), 2.08–1.87 (6H, m,
H-3′, H-4′, H-5′). CIMS: 352 (M + H)+; Rf = 0.45
(CHCl3/MeOH, 9:1).
6.6. Amination of 7 with N-methylallylamine
In a 50 ml round-bottomed flask 7 (300 mg, 0.92 mmol),
DMA (15 ml) and N-methylallylamine (177 µl, 1.85 mmol)
were added. The reaction mixture was stirred 24 h at 45 °C
and monitored by TLC (CHCl3/MeOH, 9:1). CC (eluent
CHCl3/MeOH, 19:1) yielded 210 mg of 24 (yield 72%).
6.5.2. 7-(piperidinyl-N-hexyloxy)chromen-2-one (19)
Yellow oil; IR (liquid film) 1732, 1614, 1350, 1280, 1230,
1122, and 835 cm–1; 1H NMR (CDCl3) d = 8.10 (1H, d, J =
9.5 Hz, H-4), 7.63 (1H, d, J = 8.4 Hz, H-5), 6.84–6.79 (2H,
m, H-6, H-8), 6.23 (1H, d, J = 9.5 Hz, H-3), 4.02 (2H, t, J =
6.4 Hz, H-1′), 2.55 (4H, br s, H-2″, H-6″), 2.31 (2H, t, J =
7.6 Hz, H-6′), 1.81 (2H, m, H-2′), 1.63–1.36 (12H, m, CH2),
CIMS: 330 (M + H)+; Rf = 0.24 (CHCl3/MeOH, 9:1).
6.6.1. 7-[6-(Allylmethylamino)-hexyloxy]-chromen-2-one
(24)
White powder. m.p. 145 °C. IR (KBr) 1728, 1626, 1473,
1400, 1298, and 843 cm–1; 1H-NMR (CDCl3) d = 7.64 (1H,
d, J = 9.5 Hz, H-4), 7.37 (1H, d, J = 8.4 Hz, H-5), 6.85–6.81
(2H, m, H-6, H-8), 6.25 (1H, d, J = 9.46 Hz, H-3), 6.19–6.07
(1H, m, H-2″), 5.55–5.46 (2H, m, H-3″), 4.02 (2H, td, J =
2.22, 3.88 Hz, H-1′), 3.56 (2H, d, J = 6.64 Hz, H-1″), 2.91
(2H, t, J = 7.79 Hz, H-6′), 2.67 (3H, s, CH3), 1.93–1.46 (8H,
m, CH2). CIMS: 316 (M + H)+. Rf = 0.29 (CHCl3/MeOH,
9:1).
6.5.3. 7-(Morpholinyl-N-hexyloxy)chromen-2-one (20)
Yellow oil; IR (liquid film) 1732, 1614, 1350, 1280, 1242,
1120, and 835 cm–1; 1H NMR (CDCl3) d = 7.54 (1H, d, J =
9.5 Hz, H-4), 7.25 (1H, d, J = 8.4 Hz, H-5), 6.73–6.63 (2H,
m, H-6, H-8), 6.11 (1H, d, J = 9.5 Hz, H-3), 3.90 (2H, t, J =
6.5 Hz, H-1′), 3.60 (4H, t, J = 4.4 Hz, H-3″, H-5″), 2.24 (2H,
t, J = 7.4 Hz, H-6′), 2.33 (4H, s, H-2″, H-6″),1.71 (2H, m,
H-2′), 1.48–1.09 (6H, m, CH2). CIMS: 332 (M + H)+; Rf =
0.40 (CHCl3/MeOH, 9:1).
6.6.2. 7-[10-(Allylmethylamino)-decyloxy]-chromen-2-one
(25)
White powder; m.p. 99 °C. IR (KBr) 3450, 1724, 1622,
1300, 1134, and 851 cm–1; 1H NMR (CDCl3) d = 7.64 (1H, d,

G. Cravotto et al. / European Journal of Medicinal Chemistry 39 (2004) 917–924
923
J = 9.5 Hz, H-4), 7.37 (1H, d, J = 8.4 Hz, H-5), 6.85–6.81
(2H, m, H-6, H-8), 6.25 (1H, d, J = 9.46 Hz, H-3), 6.18–6.05
(1H, m, H-13′), 5.59–5.49 (2H, m, H-14′), 4.01 (2H, t, J =
12.9 Hz, H-1′), 3.63 (2H, d, J = 6.99 Hz, H-12′), 2.98–2.93
(2H, m, H-10′), 2.73 (3H, s, H-11′), 1.83–1.32 (16H, m,
CH2). CIMS: 372 (M + H)+; Rf = 0.68 (CHCl3/MeOH, 1:1).
7.76 Hz, H-5), 7.41 (1H, t, J = 6.88 Hz, H-7), 7.28–7.18 (2H,
m, H-6, H-8), 6.04–5.91(1H, m, H-2″), 5.42 (2H, m, H-3″),
3.45 (2H, d, J = 6.91 Hz, H-1″), 2.79 (2H, t, J = 10.60 Hz,
H-2’), 2.59 (3H, s, CH3), 1.65-1.18 (16H, m, CH2). CIMS:
386 (M + H)+. Rf = 0.40 (CHCl3/MeOH, 9:1).
6.7. SHC inhibition tests
6.6.3. 3-(10′-Undecenoyl)chroman-2,4-dione (26)
In a 50 ml two-necked round-bottomed flask equipped
with a magnetic stirrer and a nitrogen inlet, 4-hydroxy-
coumarin (5.0 g, 30.8 mmol) and a catalytic amount of
piperidine were dissolved in anhydrous pyridine (35 ml). The
solution was placed under stirring, cooled down to 0 °C and
10-undecenoyl chloride was added dropwise. The mixture
was stirred 8 h under nitrogen at 40 °C and the reaction
monitored by TLC. Work-up: the reacted mixture was diluted
with EtOAc and washed with 5% HCl and with NaHCO3,
dried over Na2SO4, filtered and evaporated to dryness. CC
(eluent hexane/EtOAc, 19:1) yielded 7.0 g of 26 (21.3 mmol,
70%).
White powder, m.p. 98 C. IR (KBr) 1717, 1607, 1547,
1225, 980, and 901 cm–1. 1H NMR (CDCl3) d = 8.08 (1H, d,
J = 7.90 Hz, H-5), 7.73 (1H, td, J = 7.14 Hz, J′ = 1.39 Hz,
H-7), 7.38–7.28 (2H, m, H-6, H-8), 5.90–5.76 (1H, m,
H-10′), 5.03–4.93 (2H, m, H-11′), 3.21 (2H, t, J = 7.45 Hz,
H-2′), 1.83–1.30 (14H, m, CH2). CIMS: 329 (M + H)+. Rf =
0,4 (hexane/EtOAc 4:1).
[14C]Squalene, the substrate of squalene-hopene cyclase,
was obtained by incubating 1 µCi of [14C]mevalonolactone
with S10 supernatant of a pig liver homogenate (25 mg of
protein) following the method of Popják [38], in the presence
of oxidosqualene cyclase inhibitor U-18666A. Under these
conditions the bulk of radioactivity in the unsaponifiable
extract was shared between squalene and oxidosqualene,
which could be easily separated by TLC [39].
E. coli cells expressing the SHC gene were provided by
Prof. G. Schulz (Universität Freiburg, Germany). For en-
zyme preparation we modified the procedure of Ochs et al.
[40]. Briefly, a colony was suspended in 8 mL of Luria broth
containing 30 mg ampicillin per liter. After incubation for
15 h at 37 °C (pH 7.0), the culture was diluted to 100 ml with
the same medium. After growing to mid-log phase (OD578
0.6–0.8), the cells were induced for 5–7 h with 1 mM
isopropylthio-b-galactoside, after which they were harvested
and resuspended in 200 mM Tris buffer pH 8.0 containing
10 mM b-mercaptoethanol and 5 mg/ml lysozyme. The sus-
pension was frozen at—80 °C for 2 h, then the cells were
thawed at room temperature and lysed. The lysate was cen-
trifuged at 40 000 × g for 60 min and membrane-bound
proteins were solubilized by adding 1% Triton X-100.
The enzymatic activity of SHC was assayed as previously
reported [41].
6.6.4. 3-(10′-oxodecanoyl)chroman-2,4-dione (27)
In a 50 ml round-bottomed flask 26 (350 mg, 1.07 mmol),
30 ml of H2O/acetone 1:1, 54 µl (0.02 eq/mol) of 0.2 M OsO4
in toluene and N-methylmorpholine-N-oxide (125 mg,
1 eq/mol, 1.067 mmol) were placed and the mixture was
magnetically stirred for a few minutes before adding 502 mg
(2.2 eq/mol, 2.34 mmol) of NaIO4 in small portions over an
interval of 40 min at 24–26 °C. The stirring was continued for
3 h and the reaction was monitored by TLC on silica gel
plates, eluent hexane/EtOAc, 7:3.
IC50 values (the concentration of inhibitor that reduced by
50% the enzymatic conversion of squalene to hopene) were
determined at 10 µM substrate concentration.
Acknowledgements
Work up: the reacted mixture was diluted with H2O,
poured into a separatory funnel and extracted with CH2Cl2.
The organic phase was washed with saturated NaCl solution,
dried with Na2SO4, filtered and evaporated under vacuum.
Products were purified by CC, eluent petrol ether/EtOAc,
9:1. 102 mg of product were obtained (yield: 30%).
White powder. m.p. 119 °C. IR (KBr) 1717, 1609, 1549,
1225, 982, 768 and cm–1; 1H NMR (CDCl3) d = 9.78 (1H, s,
CHO), 8.08 (1H, d, J = 7.90 Hz, H-5), 7.73 (1H, td, J = 1.39,
7.14 Hz, H-7), 7.38–7.28 (2H, m, H-6, H-8), 3.21 (2H, t, J =
7.45 Hz, H-2′), 2.44 (2H, td, J = 5.98, 1.40 Hz, H-9’),
1.77–1.27 (12H, m, CH2). CIMS: 331 (M + H)+. Rf = 0.52
(hexane/EtOAc, 7:3).
The authors are very grateful to Prof. G. Schulz (Universität
Freiburg, Germany) for providing cultures of E. coli express-
ing squalene-hopene cyclase gene, to Dr. H. Dehmlow
(Hoffmann-La Roche, Basel, Switzerland) for a generous
gift of Ro 048871 and to the University of Turin for the
financial support.
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