New retinoid derivatives as back-ups of Adarotene

Article abstract of DOI:10.1016/j.bmc.2012.01.042

Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carbamate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in tumor models.

Full text of DOI:10.1016/j.bmc.2012.01.042

Bioorganic & Medicinal Chemistry 20 (2012) 2405–2415
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New retinoid derivatives as back-ups of Adarotene
Giuseppe Giannini a, , Tiziana Brunetti a, Gianfranco Battistuzzi a, Domenico Alloatti a,
⇑
Gianandrea Quattrociocchi a, Maria Grazia Cima a, Lucio Merlini b, Sabrina Dallavalle b,
Raffaella Cincinelli b, Raffaella Nannei b, Loredana Vesci a, Federica Bucci a, Rosanna Foderà a,
Mario Berardino Guglielmi a, Claudio Pisano a, Walter Cabri a
a Sigma-Tau Industrie Farmaceutiche Riunite S.p.a, via Pontina Km 30.400, 00040 Pomezia, Italy
b DISMA—Dipartimento di Scienze Molecolari Agroalimentari, Università di Milano, via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl
group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination
of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carba-
mate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at
different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovar-
ian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in
tumor models.
Received 22 December 2011
Accepted 26 January 2012
Available online 6 February 2012
Keywords:
Adarotene derivatives
Atypical retinoids
Prodrugs
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
studies by Pellicciari and co-workers showed that the novel nucle-
ar receptor small heterodimer partner (SHP) is involved in the
Adamantyl-substituted retinoid-related molecules are a unique
class of compounds that have been found to induce apoptosis in a
large number of tumor types, many of which display resistance to
classic retinoids. Adarotene (1)1 belongs to this so-called class of
atypical retinoids.2
induction of apoptosis by Adarotene and similar compounds.4,5
Adarotene was selected by Sigma-Tau for clinical development
as a ‘chemotherapy enhancer’ in solid tumors. In pre-clinical mod-
els of haematological as well as solid tumors, it induced a DNA
damage response and affected the modulation of cancer cell sur-
vival pathways.6 When used in combination with other anticancer
agents, it showed a significant synergistic effect in most of the
combinations and models tested.7
However, despite the high activity, the pharmacokinetic proper-
ties of 1 were not satisfactory and its bioavailability was compro-
mised by premature metabolism. In fact, the presence of a
phenolic group allowed a rapid O-glucuronidation as a major
mechanism of elimination of the drug, favouring a fast excretion
of its glucuronide metabolite in the urines.
O
OH
HO
1
In recent years efforts have been made to increase the plasma
concentration of the leads generated in the drug screening facili-
ties. In some cases the prodrug approach may be feasible, enabling
transient masking of functional groups so that the degree of first
pass metabolism is reduced. The so-called prodrugs have a longer
physiological lifetime, eventually reverting to the therapeutic form
by hydrolyzing in the acidic environment of the stomach or the
alkaline conditions in the intestine.8
It represents a new first-in-class potent proapoptotic and
cytodifferentiating agent. Whereas it was initially designed as a
selective activator of RARs b and c, it has been found to inhibit cell
growth and to induce apoptosis in a large number of tumor types,
many of which display resistance to classic retinoids, using a
RAR-receptor independent mechanism.3 Furthermore, recent
The purpose of this investigation was to improve the drug-like
properties of our lead compound 1. Herein a group of compounds
are reported, some of them are Adarotene bio-reversible
derivatives, whereas some others could be considered as new
⇑
Corresponding author.
E-mail address: giuseppe.giannini@sigma-tau.it (G. Giannini).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.042

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G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
compounds, with a biological profile overlapping with or different
from that of the parent drug.
prodrugs are often used as soft alkyl derivatives of active drugs to
overcome bioavailability problems.10 It was obtained by reaction of
AOCOM iodide with tert-butylester 5 under phase-transfer condi-
tions. The free acid 3e was isolated after treatment with montmo-
rillonite KSF in acetonitrile (Scheme 2).
The second group of derivatives included a series of esters
(6a–i). Esterification of therapeutically active agents to provide
prodrugs with improved properties has become a familiar strategy
for the circumvention of adverse physicochemical limitations. It is
of course essential for the success of this strategy that the ester
progenitor be capable of delivering the parent drug at a practical
rate in vivo. Ester derivatives of 1 with different chains were syn-
thesized (6a–i) as described in Scheme 3.
Compound 6a was prepared by exhaustive acetylation of 1 with
acetyl chloride and subsequent hydrolysis of the resulting anhy-
dride in THF/H2O. For compounds 6b,c the coupling step was per-
formed with bromobutanoyl chloride, followed by nucleophilic
displacement with morpholine or N-methylpiperazine, respec-
tively. Conjugates of Adarotene with undecanoic acid and linoleic
acid (6f–g) were also synthesized. The choice of fatty acids was
based on the results reported by Sauer et al.,11 who demonstrated
that tumor cells selectively take up certain kinds of natural fatty
acids from the arterial blood, presumably for use as biochemical
precursors and energy source. Bradley et al.12 synthesized an ester
between DHA (cis-4,7,10,13,16,19-docosahexaenoic acid) and
paclitaxel and showed that the derivative had a greatly extended
Several compounds were synthesized and their stability was
evaluated at different pH (1.2–6.8–7.4). The cytotoxic activity
in vitro on NCI-H460 non-small cell lung carcinoma and A2780
ovarian tumor cell lines was also tested. A potential back-up of
Adarotene was selected to be evaluated in in vivo tumor models.
2. Chemistry
With the overall objective of improving the pharmacokinetics
properties of Adarotene, a series of its bio-reversible derivatives
was studied. According to chemical structures, these can be
grouped into three classes: ether (3a–e), ester (6a–h) and carba-
mate (7a–i) derivatives. Ethers 3a–c were prepared by alkylation
of Adarotene methyl ester, followed by basic hydrolysis, as de-
picted in Scheme 1.
An imidomethyl derivative of 1 was also prepared as a ‘soft’ al-
kyl phenolic ether. The hydrolysis of imidomethyl prodrugs of phe-
nols has been extensively studied by Sloan and co-workers.9 These
types of prodrugs rely on simple chemical hydrolysis rather than
enzymatic hydrolysis to revert to the parent drug. With this pur-
pose, compound 3d was obtained by coupling the acid-labile
tert-butyl ester of 1 (5) with a hydroxymethylimide using Mitsun-
obu chemistry. Finally, an alkyloxycarbonyloxymethyl (AOCOM)
ether of Adarotene (3e) was synthesized, considering that AOCOM
O
O
OH
OMe
a, b
HO
RO
2
3a-c
CH2CH2N
O
3a R =
3b R =
3cR =
CH2 CN
MEM
Scheme 1. Reagents and conditions: (a) for 3a–b: R-X, K2CO3, DMF, rt to 60 °C, 4–12 h; for 3c: NaH, MEMCl, DMF, 20 °C, overnight; (b) LiOHꢀH2O, THF–H2O 1:1, rt, overnight.
Br
COOtBu
b, c
a
HO
HO
4
5
COOH
d, e
COOH
O
N
O
3d
O
O
O
O
3e
O
Scheme 2. Reagents and conditions: (a) tert-Butyl acrylate, Pd(OAc)2, TEA, tri-o-tolylphosphine, 110 °C, 1 h; (b) N-hydroxymethylphthalimide, K2CO3, DIAD, acetone, N2, rt;
overnight; (c) TFA–CH2Cl2 1:1, 0 °C, 10 min; (d) ICH2OCOOC3H7, K2CO3, CH2Cl2/H2O, Bu4Nþ ꢀ HSO4ꢁ, rt, overnight; (e) Montmorillonite KSF, CH3CN, reflux, 2 h.

G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
2407
COOH
COOH
a,b
CH3COO
HO
6a
1
c,d,e
COOH
O
6i
O
(CH2)3
(CH2)3
N
N
O
N
6b R=
6c R =
COOtBu
HO
6d R =
6e R =
NH3*Cl-
5
-CH2-O-(CH2)2-O-(CH2)2-O-CH2-COOH
f,g
6f R =
6g R =
6h R =
(CH2)9-CH3
COOH
-(CH2)7-CH=CH-CH2-CH=CH-(CH2)4-CH3
O
O
TFA-
R
NH3+
6b-h
Scheme 3. Reagents and conditions: (a) acetyl chloride, DIPEA, DCM, rt, 2 h; (b) THF–H2O 1:1, rt, 4 h; (c) TBDPSCl, morpholine, DMF, rt, 20 min; (d) cyclopropane–COCl,
pyridine, 50 °C, 30 min; (e) TBAF, THF, ꢁ78 °C, 30 min; (f) for 6b–c: bromobutanoyl chloride, DIPEA, DCM, rt, 1 h, then morpholine or N-methylpiperazine, DMF, 50 °C,
overnight; for 6d–g: RCOOH, PyBOP, DMF, rt, 2–5 h; for 6h: cyclopropyl(NHBoc)COOH, PyBOP, DIPEA, rt, overnight; (g) for 6d–e DCM–TFA 8:2, rt; for 6b,c and 6f HCl 4 M in
dioxane, rt; for 6g–h TFA–CH2Cl2 1:1, 0 °C, 30 min.
half-life and a significantly higher therapeutic index than free pac-
litaxel in mice bearing tumors.
designed di-substituted carbamates susceptible to chemical con-
version, by intramolecular cyclization reaction, in addition to enzy-
molysis. Namely, compounds bearing a nucleophile on their
promoiety were prepared. In this way, ideally, generation of the ac-
tive drug is not dependent upon the host environment but rather
solely upon the rate of a predictable, intramolecular cyclization–
elimination reaction.15
All the carbamates (7a–i) were prepared following the general
procedure depicted in Scheme 4. The tert-butyl ester of Adarotene
(5) was converted to the corresponding carbamates using either a
phenol/p-nitrophenyl chloroformate/amine or a phenol/isocyanate
approach. The final removal of the protecting group was performed
with CH2Cl2/TFA or HCl in dioxane.
Conversion of ester prodrugs to drugs depends upon chemical
or enzymatic hydrolysis of the ester bond. Generally, ester bonds
are easily hydrolyzed by carboxylate esterase, which exists abun-
dantly in the serum. According to a recent paper by McCarthy
and co-workers,13 the stability of a prodrug can be maximized by
employing cyclopropanecarboxylic acid esters, presumably be-
cause of hyperconjugative stabilization. On the basis of these re-
sults esters 6h and 6i were synthesized following the pathway
depicted in Scheme 3. Adarotene was converted into the corre-
sponding tert-butyldiphenylsilylester and then treated with cyclo-
propanecarbonyl chloride at 50 °C. Hydrolysis of the ester with
TBAF afforded the desired Adarotene derivative 6i. Compound 6h
was obtained following a similar strategy. tert-Butyl ester 5 was
treated with N-Boc protected 1-aminocyclopropylcarboxylic acid
in the presence of PyBOP and DIPEA. Hydrolysis with TFA–CH2Cl2
1:1 at 0 °C gave 6h as a trifluoroacetate.
3. Results and discussion
3.1. Chemical stability
The third class of prodrugs included a series of carbamates. Car-
bamate esters are one of the most popular types of prodrugs, with
examples reported for duocarmycin, camptothecin, entacapone
and 3-PPP. A carbamoyl bond is more stable than an ester bond
to hydrolysis by carboxylate esterase and carbamates are fre-
quently employed to improve solubility, absorption and bioavail-
ability and to extend the duration of action of the parent drug.14
Basic carbamates were synthesized as examples of cyclization-
activated prodrugs. It is well known that carbamates are converted
to alcohols by carboxylesterases, P450 oxidation and/or intramo-
lecular cyclization reaction. To increase bioconversion, we
Chemical stability of Adarotene derivatives was ascertained at
37 °C at the stomach pH (1.2), intestine pH (6.8) and blood pH
(7.4). Due to the low water solubility of the compounds, stability
experiments were performed in aqueous buffer containing 5%
DMSO and 5% Tween 80. The stability results are reported (Table
1a, S.I.) as percentage of recovery, as determined by HPLC and UPLC
measurements (see Section 5). All compounds appeared unchanged
after 12 h at pH 1.2. Compounds 6b–d, 6h and 7b were partially
hydrolysed at pH 6.8 and 7.4, with the lowest percentage of recovery
for 6d and 7b (12% and 36% resp.) at pH 7.4. All the partially hydro-
lysed compounds contain a basic nitrogen in the side chain.

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G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
Scheme 4. Reagents and conditions: (a) TEA, isopropyl isocyanate, DCM, rt, 5 d; (b) for 7b–i p-nitrophenylchloroformate, DIPEA, DCM or DCM/DMF, 0 °C to rt, 1–5 h; then RH,
rt to 55 °C, overnight; (d) for 7c DCM–TFA 8:2, rt; for 7b and 7d–i HCl 4 M in dioxane, rt.
3.2. Esterase stability
Table 1
Antiproliferative effect of Adarotene derivatives on NCI-H460 non-small cell lung and
In order to evaluate the influence of the esterase activity on the
half-life of the prodrugs, selected compounds, that turned out to be
stable at various pH values (3c, 3e, 6h, 6i, 7b, and 7i), were treated
with murine plasma, and, for comparison, with murine plasma pre-
treated with PMSF (phenylmethyl sulfonyl fluoride), a known
inhibitor of esterase. Results are reported in Table 2a, S.I. Whereas
the MEM ether 3c was stable up to 6 h, the cyclopropyl ester 6i was
immediately deprotected, even in presence of the inhibitor. The
same behaviour was shown within 1 h by the piperidinopiperidyl
carbamate 7i. The compounds 3e and 7i were deprotected up to
95% within 30 min, clearly only by action of the esterase, which
was inhibited by PMSF. The aminocyclopropyl ester 6h showed a
similar behaviour, however starting to be deprotected even in
the presence of PMSF after 30 min.
A431 epidermoid carcinoma cells
Entry
IC50 SD (lM)
NCI-H460
A431
Adarotene (1)
0.2 0.01
>10
0.85 0.06
4.2 0.04
8.4 1.6
0.32 0.01
0.26 0.008
0.22 0.009
0.24 0.01
0.51 0.003
0.17 0.007
1.54 0.08
7.8 0.7
0.12 0.01
n.e.
n.e.
n.e.
n.e.
3a
3b
3c
3d
3e
6a
6b
6c
6d
6e
6f
n.e.
0.53 0.03
0.34 0.02
0.41 0.04
0.41 0.03
0.33 0.01
n.e.
n.e.
0.40 0.02
n.e.
6g
6h
6i
0.10 0.003
0.85 0.03
3.3. Growth inhibition assay
7a
7b
7c
7d
7e
7f
7g
7h
7i
5
0.1
0.24 0.008
0.1
n.e.
0.32 0.03
n.e.
The inhibitory effect of Adarotene derivatives was assessed on
NCI-H460 non-small cell lung carcinoma and A431 epidermoid
carcinoma (Table 1).
Among ethers 3, only compounds 3b and 3e showed an antipro-
liferative activity comparable to Adarotene: 3e likely acts as a real
prodrug whereas 3b can be considered active on its own. All the es-
ters 6 showed a high activity, except for the two highly lipophilic
compounds 6f and 6g. On the contrary the carbamates were in
general less active than Adarotene, the only ones comparable being
7b and 7f.
5
3.0 0.1
2.6 0.08
0.92 0.05
1.39 0.04
3.5 0.2
>10
n.e.
3.4 0.3
1.4 0.1
n.e.
n.e.
>10
Tumor cells were treated for 24 h followed by 48 h of recovery.
Cell survival was evaluated by sulphorodamine B test.
n.e. = not evaluated.

G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
2409
Table 2
while against A431 (Table 3), 6b (iv) showed more potent activity
than the parent drug.
Antitumor activity of 6b delivered intravenously in comparison with Adarotene
administered by oral route (qdx3/wx2w) and iv against NCI-H460 NSCLC sc
xenografted in CD1 nude mice
In conclusion, Adarotene derivatives are active in vivo only if
endowed with groups that release the parent compound rapidly.
Moreover, when these groups led to an increase in solubility,
the corresponding prodrugs could be administered iv as well as
orally.
The obtained results confirm that prodrug derivatives may con-
stitute a feasible platform for improving pharmacokinetics of 1 and
that widely varying hydrolysis rates in aqueous solutions may be
achieved depending on the selected pro-moiety.
Compound
Dose mg/kg
Route
BWL%
TVI% +22
Vehicle
0
25
13
18
iv
po
iv
iv
0
9
8
/
Adarotene (1)
Adarotene (1)
6b
⁄34
⁄37
⁄37
10
Vehicle was 10% DMSO, 9% HPBCD, 14.4% PEG1000 in water. ⁄P <0.05 vs vehicle-
treated group (Mann–Whitney).
Table 3
5. Experimental
Antitumor activity of 6b delivered intravenously (qdx3/wx2w) in comparison with
Adarotene administered by oral route and other derivatives (ip and po) against A431
epidermoid ca. sc xenografted in CD1 nude mice
5.1. General chemical methods
Compound
Dose mg/Kg
Route
BWL%
TVI% +17
All reagents and solvents were of reagent grade or were purified
by standard methods before use. Column chromatography was car-
ried out on flash Silica Gel (Merck 230–400 mesh). TLC analysis
was conducted on silica gel plates (Merck 60F254). NMR spectra
were recorded at 300 MHz with a Bruker instrument. All final com-
pounds were analyzed by HPLC and their purity turned out to be
>97%. The HPLC analyses were performed on a Waters Alliance
2695 pump unit using a photo diode array detector Waters 2996.
Chemical shifts (d values) and coupling constants (J values) are gi-
ven in ppm and Hz, respectively.
Vehicle
Adarotene (1)
6b
0
25
37
10
35
35
37
37
40
40
ip
po
iv
iv
ip
po
ip
po
ip
0
12
18
0
6
3
0
0
0
4
/
⁄36
⁄⁄⁄59
⁄34
1
7e
7f
7i
0
21
0
26
22
po
Vehicle was 10% DMSO, 9% HPBCD, 14.4% PEG1000 in water. ⁄P <0.05, ⁄⁄⁄P <0.001 vs
vehicle-treated group; P <0.05 vs Adarotene-treated group (Mann–Whitney test).
5.1.1. (E)-3-[30-Adamantan-1-yl-40-(2-morpholin-4-yl-ethoxy)-
biphenyl-4-yl]-acrylic acid hydrochloride (3a)
3.4. In vivo studies
A solution of Adarotene methyl ester (2) (250 mg, 0.64 mmol)
and K2CO3 (265 mg, 1.92 mmol) in 2.5 mL of DMF was stirred at
room temperature for 30 min, then 4-(2-chloroethyl)morpholine
hydrochloride (155 mg, 0.83 mmol) was added. After having stir-
red for 12 h at 60 °C, the solution was poured into water and ex-
tracted with ethyl acetate. The organic phase was washed with
water, saturated aqueous sodium bicarbonate, water and brine,
then dried and evaporated. The residue was purified by flash
chromatography (hexane/ethyl acetate 50:50) to afford 138 mg of
(E)-3-[30-adamantan-1-yl-40-(2-morpholin-4-yl-ethoxy)-biphenyl-
4-yl]-acrylic acid methyl ester. Yield 43%. 1H NMR (300 MHz,
CDCl3) d 7.73 (d, 1H, J = 15.6 Hz), 7.68–7.32 (m, 6H), 6.95 (d, 1H,
J = 8.5 Hz), 6.49 (d, 1H, J = 15.6 Hz), 4.22–4.19 (m, 2H), 3.89–3.63
(m, 7H), 2.95–2.81 (m, 2H), 2.69–2.48 (m, 4H), 2.30–2.02 (m,
9H), 1.89–1.70 (m, 6H).
The above compound (123 mg, 0.24 mmol) was dissolved in a
solution of 50 mg (1.2 mmol) of LiOHꢀH2O in 10 mL of THF/H2O
1:1 and the mixture was stirred overnight at room temperature
in the dark. THF was evaporated and the aqueous phase was
acidified with 1 N HCl. The solid formed was filtered and dried
to give 103 mg of the desired compound. Yield 83%. 1H NMR
(300 MHz, DMSO) d 11.40 (br s, 1H), 7.81–7.46 (m, 7H), 7.15
(d, 1H, J = 8.3 Hz), 6.56 (d, 1H, J = 15.8 Hz), 4.58–4.41 (m, 2H),
4.09–3.92 (m, 2H), 3.90–3.75 (m, 2H), 3.70–30.48 (m, 4H),
2.18–2.00 (m, 9H), 1.82–1.63 (m, 6H). HRMS ESI-MS (ESI+) m/
z = 524.2 [M+H]+.
On the basis of the in vitro data, compound 6b was chosen for
the in vivo investigation. It was delivered intravenously at
18 mg/10 mL/kg against NCI-H460 NSCLC sc xenografted in CD1
nude mice in comparison with Adarotene administered intrave-
nously at 13 mg/10 mL/kg and by oral route at 25 mg/10 mL/kg
according to the schedule qdx3/wx2w. Compound 6b and Adaro-
tene (po and iv) were administered at the maximum tolerated dose
(BWL 10% with no lethal toxicity) and showed both a comparable
significant antitumor activity (TVI were 37% and 34%, P <0.05 vs
vehicle-treated group, Mann–Whitney test) (Table 2).
In another experiment 6b was again evaluated for its efficacy on
A431 epidermoid carcinoma xenografted in CD1 nude mice. Com-
pound 6b at 37 mg/10 mL/kg iv (qdx3/wx2w) significantly inhib-
ited the tumor growth of 59%, whereas Adarotene delivered at
25 mg/kg po with the same schedule exhibited a lower antitumor
effect with a TVI of 36%. Due to their poor solubility 7e, 7f and
7i, derivatives were evaluated both intraperitoneally and by oral
route. As reported, these compounds did not show any antitumor
effect (Table 3).
4. Conclusion
Adarotene (1) represents a new first-in-class potent proapop-
totic and cytodifferentiating agent. Aiming at the circumvention
of the first pass metabolism, we designed and synthesized a series
of potentially bio-reversible derivatives of 1. From the results so far
produced, Adarotene derivatives with a stable bond, that is, carba-
mates, showed, as expected, no (7e) or poor activity (7f), while the
less stable carbamate (7i), appeared active both po and iv. When
the bond was labile, as in ester derivatives, for example, 6b, the
activity was equivalent or higher than for the parent compound.
Compound 6b, when administered iv, against NCI-H460 (Table
2), showed an activity similar to that of Adarotene (iv and po),
5.1.2. (E)-3-(30-Adamantan-1-yl-40-cyanomethoxy-biphenyl-4-
yl)-acrylic acid (3b)
To
0.64 mmol) in 2 mL of DMF, K2CO3 (166 mg, 1.2 mmol), KI
(53 mg, 0.32 mmol) and bromoacetonitrile (49 L, 0.7 mmol) were
a solution of Adarotene methyl ester (2) (250 mg,
l
added. The mixture was stirred at room temperature for 4 h under
nitrogen, then it was poured into water and extracted with ethyl
acetate. The organic phase was washed with water, saturated

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G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
aqueous sodium bicarbonate, water and brine, then dried and
evaporated. The crude product was purified by flash chromatogra-
phy (hexane/ethyl acetate 80:20) to give 100 mg of (E)-3-(30-ada-
mantan-1-yl-40-cyanomethoxy-biphenyl-4-yl)-acrylic acid methyl
ester. Yield 37%. 1H NMR (300 MHz, DMSO-d6) d 7.88–7.42 (m,
7H), 7.20 (d, 1H, J = 8.5 Hz), 6.78 (d, 1H, J = 16 Hz), 5.28 (s, 2H),
3.71 (s, 3H), 2.21–1.99 (m, 9H), 1.81–1.67 (m, 6H).
The above ester (50 mg, 0.12 mmol) was dissolved in a solution
of 24 mg (0.58 mmol) of LiOHꢀH2O in 5 mL of THF/H2O 1:1 and the
mixture was stirred overnight at room temperature in the dark.
THF was evaporated and the aqueous phase was acidified with
1 N HCl. The solid formed was filtered and dried to give 40 mg of
the title compound. Yield 81%. 1H NMR (300 MHz, DMSO-d6) d
7.82–7.39 (m, 7H), 6.98 (d, 1H, J = 8.5 Hz), 6.56 (d, 1H, J = 16 Hz),
4.52 (s, 2H), 2.20–2.00 (m, 9H), 1.82–1.68 (m, 6H). HRMS ESI-MS
(ESI+) m/z = 436.2 [M+Na]+.
5.1.5. (E)-3-[30-Adamantan-1-yl-40-(1,3-dioxo-1,3-
dihydroisoindol-2-yloxy)-biphenyl-4-yl] acrylic acid (3d)
Equimolar amounts (0.464 mmol) of N-hydroxymethylphthali-
mide, 5, potassium carbonate, DIAD and sodium iodide were added
to 0.928 mL of dry acetone and stirred in the dark, under nitrogen,
overnight. Acetone was evaporated, the residue was dissolved in
ethyl acetate and washed with water. The organic phase was dried,
filtered and the solvent evaporated. The crude was purified by flash
chromatography (hexane/ethyl acetate 85:15) to give 150 mg of
(E)-3-[30-adamantan-1-yl-40-(1,3-dioxo-1,3-dihydroisoindol-2-
yloxy)-biphenyl-4-yl] acrylic acid tert-butyl ester as a yellow
solid. Yield 55%. 1H NMR (300 MHz, DMSO-d6) d 8.08–7.90
(m, 4H), 7.82–7.62 (m, 4H), 7.60–7.53 (m, 2H), 7.44 (s, 1H);
7.36 (d, J = 8.23 Hz, 1H), 6.56 (d, J = 16 Hz, 1H), 5.70 (s, 2H),
2.08–1.88 (m, 9H), 1.70–1.52 (m, 6H), 1.51 (s, 9H).
The above tert-butyl ester (110 mg, 0.186 mmol) was dissolved
in dry methylene chloride (1.86 mL) and this solution was treated
with trifluoroacetic acid (1.86 mL) at 0 °C under stirring. After 10
minutes at 0 °C the solvent was evaporated and the residue was
rinsed with hexane to obtain 97 mg of the title compound as a
white solid (3d). Yield 98%. 1H NMR (300 MHz, DMSO-d6) d 8.08–
7.90 (m, 4H), 7.87–7.64 (m, 4H), 7.64–7.55 (m, 2H), 7.44 (s, 1H),
7.36 (d, J = 8.05 Hz, 1H), 6.54 (d, J = 16 Hz, 1H), 5.70 (s, 2H), 2.08–
1.86 (m, 9H), 1.71–1.50 (m, 6H). HRMS ESI-MS (ESI+) m/z = 556.2
[M+Na]+.
5.1.3. (E)-3-[30-Adamantan-1-yl-40-(2-methoxyethoxymethoxy)-
biphenyl-4-yl]-acrylic acid (3c)
To an ice-cooled suspension of sodium hydride (60% in mineral
oil, 14.8 mg, 0.371 mmol) in 1.3 mL of dry DMF, Adarotene methyl
ester (2) (120 mg, 0.309 mmol) was slowly added while maintain-
ing the temperature at 0–5 °C. The resulting red solution was
stirred at room temperature for 30 min, then MEMCl (42 lL,
0.371 mmol) was added. After having stirred overnight at 20 °C,
iced water was added and the mixture was extracted several times
with ethyl acetate. The combined organic phases were dried over
Na2SO4, filtered and evaporated. The resulting crude product was
purified by flash chromatography (hexane/acetone 85:15) to give
123 mg of (E)-3-[30-adamantan-1-yl-40-(2-methoxyethoxyme-
thoxy)-biphenyl-4-yl]-acrylic acid methyl ester as a white solid.
Yield 84%. 1H NMR (300 MHz, acetone-d6) d 7.82–7.69 (m, 5H),
7.60–7.48 (m, 2H), 7.26 (d, J = 8.5 Hz, 1H), 6.60 (d, J = 15.7 Hz,
1H), 5.43 (s, 2H), 3.92–3.84 (m, 2H), 3.76 (s, 3H), 3.64–3.52 (m,
2H), 2.81 (s, 3H), 2.20 (s, 6H), 2.10 (s, 3H), 1.81(s, 6H).
5.1.6. (E)-3-(30-Adamantan-1-yl-40-
propoxycarbonyloxymethoxy- biphenyl-4-yl) acrylic acid (3e)
A mixture of 5 (250 mg, 0.581 mmol) and K2CO3 (241 mg,
1.74 mmol) in water (2.86 mL) was allowed to stir 30 min before
adding tetrabutylammonium hydrogen sulphate (197 mg,
0.581 mmol) and 1.43 mL of methylene chloride. After 10 min, a
solution of iodomethylpropyl carbonate (184 mg, 1.39 mmol) in
1.43 mL of methylene chloride was added in portions to the reac-
tion mixture. The resulting biphasic system was stirred at room
temperature overnight. The phases were separated and the water
layer was extracted with methylene chloride. The organic phases
were combined and concentrated to give an oily residue. This
was purified by flash chromatography (hexane/ethyl acetate
88:12) to give 37 mg of (E)-3-(30-adamantan-1-yl-40-propoxycar-
bonyloxymethoxy biphenyl-4-yl) acrylic acid tert-butyl ester as a
yellow oil. Yield 37%, 1H NMR (300 MHz, acetone-d6) d 7.81–7.54
(m, 7H), 7.28 (d, J = 8.6 Hz, 1H), 6.50 (d, J = 16 Hz, 1H), 6.00 (s,
2H), 4.20–4.10 (m, 2H), 2.20 (s, 6H), 2.12–2.02 (m, 3H), 1.85 (s,
6H), 1.75–1.60 (m, 2H), 1.53 (s, 9H), 1.01–0.90 (m, 3H).
A mixture of the above tert-butyl ester (40 mg, 0.0732 mmol)
and montmorillonite KSF (15 mg) in acetonitrile (1 mL) was stirred
at reflux for 2 h. The reaction mixture was filtered and washed
with Et2O. After evaporation of the solvent the crude was purified
by flash chromatography (hexane/ethyl acetate 40:60) to give
10 mg of the title compound (3e). Yield 28%. 1H NMR (300 MHz,
acetone-d6) d 7.84–7.68 (m, 5H), 7.65–7.54 (m, 2H), 7.28 (d,
J = 8.6 Hz, 1H), 6.56 (d, J = 16 Hz, 1H), 6.00 (s, 2H), 4.20–4.10 (m,
2H), 2.20 (s, 9H), 1.83 (s, 6H), 1.78–1.62 (m, 2H), 1.00–0.90 (m,
3H). HRMS ESI-MS (ESI+) m/z = 513.2 [M+Na]+.
The above obtained (E)-3-[30-adamantan-1-yl-40-(2-methoxy-
ethoxymethoxy)-biphenyl-4-yl]-acrylic acid methyl ester (56 mg,
0.117 mmol) was added to
a
solution of LiOHꢀH2O (25 mg,
0.585 mmol) in 4.82 mL of a mixture THF/H2O 1:1 and stirred at
room temperature, in the dark, overnight. After the evaporation
of THF, the aqueous phase was cooled with an ice-bath and acidi-
fied with HCl 2 N. The white precipitate was filtered and dried to
give 55 mg of title compound (3c). Yield 100%. 1H NMR
(300 MHz, DMSO-d6) d 7.78–7.63 (m, 4H), 7.59 (d, J = 16.0 Hz,
1H), 7.52 (d, J = 8.1 Hz, 1H), 7.43 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H),
6.55 (d, J = 16 Hz, 1H), 5.36 (s, 2H), 3.82–3.73 (m, 2H), 3.57 -3.44
(m, 2H), 3.25 (s, 3H), 2.14–2.08 (m, 9H), 1.75 (s, 6H). HRMS ESI-
MS (ESI+) m/z = 485.2 [M+Na]+.
5.1.4. (E)-3-(30-Adamantan-1-yl-40-hydroxybiphenyl-4-yl)-
acrylic acid tert-butyl ester (5)
A round flask containing a mixture of 3-adamantan-1-yl-40-bro-
mobiphenyl-4-ol (4) (2.0 g, 5.22 mmol), tert-butyl acrylate
(1.21 mL, 8.35 mmol), Pd(OAc)2 (11.7 mg, 0.0522 mmol) and tri-
o-tolylphosphine in 2.42 mL of Et3N was immersed in an oil bath
and kept at 110 °C for 1 h. Iced water was then added, followed
by 1 N HCl. The aqueous phase was extracted repeatedly with ethyl
acetate, dried over Na2SO4, filtered and evaporated. The crude
product was purified by flash chromatography (hexane/ethyl ace-
tate 85:15) to give 2.025 g of the title compound as a white solid.
Yield 90%. 1H NMR (300 MHz, CDCl3) d 7.65 (d, J = 15.5 Hz, 1H),
7.58–7.52 (m, 4H), 7.49 (d, J = 2.2 Hz, 1H), 7.34 (dd, J = 8.25 Hz,
2.2 Hz, 1H), 6.76 (d, J = 8.25 Hz, 1H), 6.40 (d, J = 15.5 Hz, 1H), 5.08
(br s, 1H), 2.20 (s, 6H), 2.15 (s, 3H), 1.8 (s, 6H), 1.57 (s, 9H).
5.1.7. Acetic acid 3-adamantan-1-yl-40-((E)-2-carboxy-vinyl)-
biphenyl-4-yl ester (6a)
Acetyl chloride (74
(E)-3-(30-adamantan-1-yl-40-hydroxybiphenyl-4-yl)acrylic acid
(156 mg, 0.42 mmol), DIPEA (290 L, 1.68 mmol) in DCM (5 mL).
lL, 1.05 mmol) was added to a solution of
l
The reaction mixture was allowed to stir for 2 h at room tempera-
ture, then diluted with CH2Cl2 and washed several times with
water. The organic phase was concentrated under reduced

G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
2411
pressure and the crude product was subsequent hydrolysed in
THF/water 1:1. After complete conversion (4 h), DMF and brine
were added and the organic phase was separated, dried over
Na2SO4, filtered and concentrated. The white solid obtained was
washed with Et2O and dried to give 145 mg of the title compound
(6a). Yield 80%, 1H NMR (300 MHz, DMSO-d6): 12.40 (br s, 1H);
7.52–7.80 (M, 7H); 7.10 (d, J = 8.8 Hz, 1H); 6.56 (d, J = 15.8 Hz,
1H, CH); 2.30 (s, 3H); 1.95–2.10 (m, 10H); 1.78 (m, 6H,). ESI-MS
m/z = 417.0 [M+H]+.
5.1.10. (S)-2-Amino-3-methyl-butyric acid 3-adamantan-1-yl-
40-((E)-2-carboxy-vinyl)-biphenyl-4-yl ester hydrochloride (6d)
To a solution of BOC-Val-OH (24 mg, 0.11 mmol) in DMF (1 mL)
were added PyBOP (57 mg, 0.11 mmol), DIPEA (65 mL, 0.5 mmol)
and the reaction mixture, monitored by TLC, was stirred until com-
plete activation of the acid. 5 was then added (43 mg, 0.10 mmol)
and the reaction mixture was stirred for 5 h at 0 °C. After standard
work-up, the crude product was purified by flash column chroma-
tography (hexane/ethyl acetate 90:10) to get the desired product.
Yield 45%.
5.1.8. 4-Morpholin-4-yl-butyric acid 3-adamantan-1-yl-40-((E)-
2-carboxy-vinyl)-biphenyl-4-yl ester hydrochloride (6b)
The above tert-butyl ester was dissolved at room temperature in
a DCM/TFA (8:2) mixture and stirred until complete cleavage. The
reaction mixture was then concentrated under reduced pressure
and diluted with DCM. The latter procedure was repeated twice
to get the crude desired product. The latter was then dissolved in
DMSO and then freeze-dried to obtain the desired compound
(6d). Yield 83%; 1H NMR (300 MHz, DMSO-d6) 12.40 (br s, 1H);
8.99 (br s, 2H); 7.76 (d, J = 8.5 Hz, 2H); 7.70 (d, J = 8.5 Hz, 2H);
7.614 (m, 3H); 7.25 (d, J = 8.5 Hz, 1H); 6.57 (d, J = 15.9 Hz, 1H);
4.2 (d, J = 3.1 Hz, 1H); 2.53 (m, 10H); 2.05 (s, 5H); 1.97 (m, 1 H);
1.15 (d, J = 7.0 Hz, 3H); 1.12 (d, J = 7.0 Hz, 3H). ESI-MS: m/
z = 474.2 [M+H]+.
To a solution of 5 (100 mg, 0.23 mmol) and DIPEA (80
0.46 mmol) in anhydrous DCM (4 mL) was added dropwise at
0 °C 4-bromobutyryl chloride (53 L, 0.46 mmol). The reaction
lL,
l
mixture was stirred for 1 h at room temperature. The reaction mix-
ture was diluted with DCM and washed with H2O. The organic
phase was dried over Na2SO4 and the solvent was removed under
reduced pressure. The crude material thus obtained was used in
the next step without any further purification.
Morpholine (140 lL, 1.61 mmol) was added to a suspension of
the bromo derivative in DMF (3 mL) and the reaction mixture
was stirred at 50 °C for 12 h. Solvent were removed under reduced
pressure, and the residue was purified by flash chromatography
(hexane/ethyl acetate 40:60) to get the expected product. Yield
50%.
The above tert-butyl ester (68 mg, 0.116 mmol) was dissolved
in dioxane (3 mL) and then this solution was treated with HCl
4 M in dioxane (3 mL) at room temperature. The reaction mixture
was stirred until complete conversion of the starting material to
the corresponding carboxylic derivative. The white solid precipi-
tate was filtered, washed with hexane and dried to give 66 mg of
the title (6b) as a white solid. Yield 99%, 1H NMR (300 MHz,
DMSO-d6) 12.40 (br s, 1H); 7.76 (d, J = 7.7 Hz, 2H); 7.70 (d,
J = 7.7 Hz, 2H); 7.65 (d, J = 15.7 Hz, 1H); 7.56 (m, 2H); 7.15 (d,
J = 9.1 Hz, 1H); 6.56 (d, J = 15.7 Hz, 1H); 3.94 (br d, 2H); 3.75 (br
t, 2H); 3.44 (br m, 2H); 3.18–3.04 (br m, 6H); 2.80 (t, 2H); 2.03
(m, 9H); 1.74 (br s, 6H). ESI-MS m/z = 530.3 [M+H]+.
5.1.11. (E)-3-(30-Adamantan-1-yl-40-{2-[2-(2-carboxymethoxy-
ethoxy)-ethoxy]-acetoxy}-biphenyl-4-yl)-acrylic acid (6e)
To
0.93 mmol) in DMF (3 mL) were added PyBOP (531 mg,
1.02 mmol), DIPEA (832 L, 4.6 mmol) and the reaction mixture,
a solution of 3,6,9-trioxaundecanedioic acid (206 mg,
l
monitored by TLC, was stirred until complete activation of the acid.
Compound 5 (100 mg, 0.23 mmol) was then added and the reac-
tion mixture was stirred for 2.5 h at room temperature. The reac-
tion mixture was diluted with AcOEt and washed with HCl 0.5 N
and brine. The organic phase was dried with Na2SO4, filtered and
concentrated under reduced pressure; the crude product was puri-
fied by flash column chromatography (DCM/MeOH 90:10) to get
the desired product. Yield 72%.
The above tert-butyl ester was dissolved at room temperature in
a DCM/TFA (8:2) mixture and stirred until complete deprotection.
The reaction mixture was then concentrated under reduced pres-
sure; the crude product was washed with hexane and dried under
vacuum to obtain the desired compound (6e). Yield 98%; 1H NMR
(300 MHz, DMSO-d6) 12.40 (br s, 1H); 7.76 (d, J = 8.4 Hz, 2H);
7.70 (d, J = 8.4 Hz, 2H); 7.62 (d, J = 16.0 Hz, 1H); 7.56 (m, 2H);
7.18 (d, J = 8.7 Hz, 1H); 6.56 (d, J = 16.0 Hz, 1H); 4.49 (s, 2H); 4 (s,
2H); 3.71 (m, 2H); 3.57 (m, 6H); 2.02(m, 9H); 1.73 (m, 6H). ESI-
MS m/z = 601.3 [M+Na]+.
5.1.9. 4-(4-Methyl-piperazin-1-yl)-butyric acid 3-adamantan-1-
yl-40-((E)-2-carboxy-vinyl)-biphenyl-4-yl ester dihydrochloride
(6c)
To a solution of 5 (94 mg, 0.22 mmol) and DIPEA (77
0.44 mmol) in anhydrous DCM (4 mL) was added dropwise at
0 °C 4-bromobutyryl chloride (100 L, 0.88 mmol). The reaction
lL,
l
mixture was stirred for 1 h at room temperature. The crude mate-
rial thus obtained was used in the next step without any further
purification.
5.1.12. Undecanoic acid 3-adamantan-1-yl-40-((E)-2-carboxy-
vinyl)-biphenyl-4-yl ester (6f)
N-Methyl piperazine (171 lL, 1.54 mmol) was added to a sus-
pension of the bromo derivative in DMF (3 ml) and the reaction
mixture was stirred at 50 °C for 12 h. Solvent was removed under
reduced pressure, and the residue was purified by flash chromatog-
raphy (DCM/MeOH 90:10) to allow to give product protected as
tert-butyl ester. Yield 26%.
The above tert-butyl ester (34 mg, 0.057 mmol) was dissolved
in dioxane (1 mL) and then this solution was treated with HCl
4 M in dioxane (2 mL) at room temperature. The reaction mixture
was stirred until complete conversion of the starting material to
the corresponding carboxylic derivative. The white precipitate
was filtered, purified by HPLC preparative (water/acetonitrile
30:70) and dried to give 16 mg of the title compound (6c). Yield
46%, 1H NMR (300 MHz, DMSO-d6) 12.40 (br s, 1H); 7.76 (d,
J = 8.5 Hz, 2H); 7.69 (d, J = 8.5 Hz, 2H); 7.62 (d, J = 15.8 Hz, 1H);
7.56 (m, 2H); 7.10 (d, J = 8.8 Hz, 1H); 6.56 (d, J = 15.8 Hz, 1H);
2.8–3 (br m, 4H); 2.6–2.8 (m, 6H); 2.4 (m, 2H); 2.1 (s, 3H); 2.02
(br s, 9H); 1.9 (m, 2H); 1.78 (br m, 6H). ESI-MS m/z = 543.4 [M+H]+.
To a solution of undecanoic acid (133 mg, 0.71 mmol) in DMF
(1 mL) were added PyBOP (407 mg, 0.78 mmol), DIPEA (619 lL,
3.55 mmol) and the reaction mixture was stirred for 30 min. Com-
pound 5 was then added (275 mg, 0.64 mmol) and the reaction
mixture was stirred for 13 h at room temperature. After standard
work-up, the crude product was purified by flash column chroma-
tography (hexane/ethyl acetate 90:10) to get the desired product.
Yield 63%.
The above tert-butyl ester was dissolved at room temperature in
0.5 mL dioxane (0.5 mL). HCl 4 M in dioxane (2 mL) was added and
the reaction mixture was stirred until complete conversion of the
starting material to the corresponding carboxylic derivative. The
reaction mixture was concentrated under reduced pressure, the so-
lid obtained was dissolved in DCM and concentrated (two times)
and then washed with hexane and dried under vacuum to give
110 mg of the desired product as a white solid (6f). Yield 71%, 1H
NMR (300 MHz, DMSO-d6) 12.40 (br s, 1H); 7.75 (d, J = 8.5 Hz,

2412
G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
2H); 7.68 (d, J = 8.5 Hz, 2H); 7.62 (d, J = 16.0 Hz, 1H); 7.54 (m, 1H);
7.54 (s, 1H); 7.07 (d, J = 9.0 Hz, 1H); 6.55 (d, J = 16.0 Hz, 1H); 2.64
(t, J = 7.2 Hz, 2H); 2.04 (br s, 3H); 1.99 (br s, 6H); 1.72 (br s, 6H);
1.68 (m, 2H); 1.40–1.20 (br m + br s, 14H); 0.84 (t, J = 7.1 Hz).
ESI-MS m/z = 543.3 [M+H]+.
(E)-Octadeca-9,12-dienoic acid 3-adamantan-1-yl-40-(2-tert-
butoxycarbonylvinyl)-biphenyl-4-yl ester (110 mg, 0.159 mmol)
was dissolved in dry methylene chloride (1.59 mL) and treated
with TFA (1.59 mL) at 0 °C under stirring. After 30 min at 0 °C the
solvent was evaporated to give 100 mg of title compound (6g) as
a white solid. Yield 99%. 1H NMR (300 MHz, DMSO-d6) d 7.84–
7.68 (m, 4H), 7.62 (d, J = 15.0 Hz, 1H), 7.58–7.50 (m, 2H), 7.10 (d,
J = 8.5 Hz, 1H), 6.58 (d, J = 16.0 Hz, 1H), 5.40–5.22 (m, 4H), 2.76
(t, J = 2.25 Hz, 2H), 2.70 (t, J = 2.65 Hz, 2H), 2.10–1.90 (m, 13H),
1.80–1.67 (m, 8H), 1.45–1.16 (m, 14H), 0.86 (t, J = 3.0 Hz, 3H).
HRMS ESI-MS (ESI+) m/z = 659.4 [M+Na]+.
5.1.13. (E)-Cyclopropanecarboxylic acid 3-adamantan-1-yl-40-
(2-carboxyvinyl)-biphenyl-4-yl ester (6i)
Morpholine (1.3 equiv) was added to a suspension of (E)-3-(30-
adamantan-1-yl-40-hydroxybiphenyl-4-yl) acrylic acid (200 mg,
0.534 mmol) in DMF (3 mL). TBDPSCl (1.1 equiv) was added via sir-
inge upon rigorous stirring. The reaction mixture was allowed to
stir for 20 min under nitrogen, at room temperature, then diluted
with CH2Cl2 and washed several times with water. The organic
layer was evaporated and the crude was purified by flash
chromatography (hexane/ethyl acetate 85:15) to give 237 mg of
(E)-3-(30-adamantan-1-yl-40-hydroxybiphenyl-4-yl) acrylic acid
tert-butyldiphenylsilyl ester as a yellow solid. Yield 72%. 1H NMR
(300 MHz, DMSO-d6) d 9.59 (s, 1H), 7.87–7.61 (m, 9H), 7.56–7.36
(m, 8H), 6.88 (d, J = 8.6 Hz, 1H), 6.75 (d, J = 16.0 Hz, 1H), 2.27–2.0
(m, 9H), 1.74 (s, 6H), 1.09 (s, 9H).
5.1.15. (E)-1-[3-Adamantan-1-yl-40-(2-carboxyvinyl)-biphenyl-
4-yloxycarbonyl]-cyclopropyl ammoniun trifluoroacetate (6h)
To a solution of 5 (2.5 g, 5.81 mmol) in dry DMF (28.8 mL) Py-
BOP (3.32 g, 6.39 mmol), 1-tert-butoxycarbonylaminocyclopro-
pane-carboxylic acid (1.29 g, 6.39 mmol) and DIPEA (5.06 mL,
29.05 mmol) were sequentially added. The reaction was stirred
overnight at room temperature under nitrogen. The mixture was
then cooled at 0 °C and 0.1 N HCl was added. The white solid
formed was filtered and crystallized from Et2O to give 2.56 g of
1-tert-butoxycarbonyl aminocyclopropanecarboxylic acid 3-ada-
mantan-1-yl-40-(2-tert-butoxycarbonylvinyl)-biphenyl-4-yl ester.
Yield 72%. 1H NMR (300 MHz, DMSO-d6) d 7.82–7.66 (m, 5H),
7.63–7.53 (m, 2H), 6.96 (d, J = 8.25 Hz, 1H), 6.58 (d, J = 15.2 Hz,
1H), 2.09–2.0 (m, 9H), 1.86–1.67 (m, 6H), 1.60–1.45 (m, 11H),
1.39 (s, 9H); 1.30–1.20 (m, 3H).
The above tert-butyl ester (135 mg, 0.220 mmol) was dissolved
in dry DCM (2.2 mL) and then this solution was treated with TFA
(2.2 mL) at 0 °C under stirring. After 30 min at 0 °C the solvent
was evaporated to give 126 mg of the title compound (6h) as a
white solid. Yield 99%. 1H NMR (300 MHz, DMSO-d6) d 8.96 (br s,
3H), 7.84–7.72 (m, 4H), 7.69–7.57 (m, 3H), 7.09 (d, J = 8.21 Hz,
1H), 6.58 (d, J = 15.0 Hz, 1H), 2.16–2.04 (m, 4H), 2.0–1.89 (m,
6H), 1.88–1.79 (m, 8H), 1.67–1.56 (m, 2H). HRMS ESI-MS (ESI+)
m/z = 594.2 [M+Na]+.
Cyclopropanecarbonyl chloride (29
lL, 0.318 mmol) was added
to
a
solution of the above obtained derivative (130 mg,
0.212 mmol) in pyridine (1 mL). The mixture was heated at 50 °C
for 30 min, then diluted with water and extracted several times
with ethyl acetate. The combined organic phases were washed
with 1 N hydrochloric acid, dried over Na2SO4 and evaporated to
dryness in vacuo. The desired cyclopropanecarboxylic acid (E)-3-
adamantan-1-yl-40-(2-tert-butyldiphenylsilyloxyvinyl)-biphenyl-
4-yl ester was obtained after purification on silica gel (ethyl
acetate/hexane 10:90) as a white solid (61 mg). Yield 42%. 1H
NMR (300 MHz, CDCl3) d 7.80–7.72 (m, 5H), 7.61–7.52 (m, 5H),
7.50–7.30 (m, 7H), 7.08 (d, J = 8.6 Hz, 1H), 6.55 (d, J = 16 Hz, 1H),
2.11 (s, 9H), 1.98–1.91 (m, 1H), 1.76 (s, 6H), 1.26–1.20 (m, 2H),
1.15 (s, 9H), 1.08–1.00 (m, 2H).
To a solution of cyclopropanecarboxylic acid (E)-3-adamantan-
1-yl-40-(2-tert-butyldiphenylsilyloxyvinyl)-biphenyl-4-yl
ester
(30 mg, 0.044 mmol) in dry THF (2 mL) at ꢁ78 °C, under nitrogen,
was added a solution of TBAF (1 M, 0.221 mL) in THF. The reaction
mixture was stirred for 30 min at ꢁ78 °C and then a saturated solu-
tion of NH4Cl was added. THF was evaporated and the residue was
taken up with water. The white solid precipitate was filtered,
washed with Et2O and dried to give 12 mg of the title compound
(6i). Yield 62%. 1H NMR (300 MHz, DMSO-d6) d 7.82–7.68 (m,
4H), 7.65 (d, J = 16.0 Hz, 1H), 7.59–7.51 (m, 2H), 7.09 (d,
J = 8.6 Hz, 1H), 6.57 (d, J = 16.0 Hz, 1H), 2.10–1.96 (m, 10H), 1.76
(s, 6H), 1.16–1.01 (m, 4H). HRMS ESI-MS (ESI+) m/z = 465.2
[M+Na]+.
5.1.16. (E)-3-(30-Adamantan-1-yl-40-isopropylcarbamoyloxy-
biphenyl-4-yl)-acrylic acid (7a)
Isopropyl isocyanate (327
tion of (E)-3-(30-adamantan-1-yl-40-hydroxybiphenyl-4-yl) acrylic
acid (200 mg, 0.534 mmol) and NEt3 (314 L, 2.44 mmol) in anhy-
lL, 3.33 mmol) was added to a solu-
l
drous DCM (8 mL). The reaction mixture was stirred for 5 days at
room temperature. The reaction mixture was diluted with DCM
and washed with H2O. The organic phase was dried over Na2SO4
and the solvent was removed under reduced pressure. The desired
compound was obtained without any further purification as a
white powder (7a). Yield 68%, 1H NMR (300 MHz, DMSO-d6)
12.40 (br s, 1H); 7.7 (br d, H); 7.74 (d, J = 8.2 Hz, 2H); 7.67 (d,
J = 8.2 Hz, 2H); 7.60 (d, J = 15.9 Hz, 1H); 7.51 (m, 2H); 7.04 (d,
J = 8.9 Hz, 1H); 6.58 (d, J = 15.9 Hz, 1H); 3.70 (m,1H); 2.02 (br s,
9H); 1.74 (m, 6H); 1.14 (s, 3H); 1.12 (s, 3H). ESI-MS m/z = 458.1
[MꢁH]ꢁ.
5.1.14. (E)-Octadeca-9,12-dienoic acid 3-adamantan-1-yl-40-(2-
carboxyvinyl)-biphenyl-4-yl ester (6g)
To a solution of 5 (200 mg, 0.464 mmol) and a few crystals of
DMAP in 2.19 mL of dry pyridine were added 208 mg (0.696 mmol)
of linoleoyl chloride. After stirring overnight at room temperature
the reaction mixture was poured into iced water and the aqueous
layer was extracted twice with ethyl acetate. The combined organ-
ic layers were washed twice with 1 N HCl, water and dried over
Na2SO4. Flash chromatography of the reaction mixture (hexane/
ethyl acetate 95:5) gave 210 mg of (E)-octadeca-9,12-dienoic acid
3-adamantan-1-yl-40-(2-tert-butoxycarbonylvinyl)-biphenyl-4-yl
ester as a colourless oil. Yield 65%, 1H-MNMR (300 MHz, DMSO-d6)
d 7.81–7.65 (m, 4H), 7.64–7.53 (m, 3H), 7.10 (d, J = 8.5 Hz, 1H), 6.56
(d, J = 16.0 Hz, 1H), 5.43–5.24 (m, 4H), 2.73 (t, J = 2.25 Hz, 2H), 2.67
(t, J = 3.75 Hz, 2H), 2.10–1.93 (m, 13H), 1.80–1.60 (m, 8H), 1.50 (s,
9H), 1.43–1.21 (m, 14H), 0.85 (t, J = 3.0 Hz, 3H).
5.1.17. (E)-3-[30-Adamantan-1-yl-40-(2-methylamino-
ethylcarbamoyloxy)-biphenyl-4-yl]-acrylic acid hydrochloride
(7b)
To a solution of 5 (104 mg, 0.24 mmol) and DIPEA (252 lL,
1.45 mmol) in anhydrous DCM (5 mL) was added dropwise at
0 °C para-nitrophenylchloroformate (174 mg, 0.58 mmol). The
reaction mixture was stirred for 2 h at room temperature. The
crude material thus obtained was used in the next step without
any further purification.
N-Boc-N-methylethylenediamine (87
lL, 0.49 mmol) was then
added and the reaction mixture was stirred at 55 °C for 14 h. The

G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
2413
reaction mixture was diluted with DCM and washed with H2O. The
organic phase was dried over Na2SO4 and the solvent was removed
under reduced pressure. The resulting residue was purified by flash
chromatography (hexane/ethyl acetate 80:20) to get the desired
intermediate. Yield 65%.
J = 16.1 Hz, 1H); 3.76 (d, J = 5.0 Hz, 2H); 2.04 (s, 9H); 1.74 (m,
6H). ESI-MS: m/z = 476.0 [M+H]+.
5.1.20. (E)-3-[30-Adamantan-1-yl-40-(4-amino-
butylcarbamoyloxy)-biphenyl-4-yl]-acrylic acid Hydrochloride
(7e)
The above tert-butyl ester was dissolved in dioxane (1 mL) and
then this solution was treated with HCl 4 M in dioxane (2 mL) at
room temperature. The reaction mixture was stirred until com-
plete conversion of the starting material to the corresponding car-
boxylic derivative that precipitates as a white solid. The white solid
was filtered, washed with Et2O and dried to give the title com-
pound (7b). Yield 94%, 1H NMR (300 MHz, DMSO-d6) 12.40 (br s,
1H); 8.90 (br s, 1H); 8.11 (t, J = 5.7 Hz 1H); 7.75 (d, J = 8.3 Hz,
2H); 7.68 (d, J = 8.3 Hz, 2H); 7.62 (d, J =16.0 Hz, 1H); 7.53 (dd,
J = 8.1 Hz, 1.9 Hz, 1H); 7.51 (s, 1H); 7.17 (d, J = 8.1 Hz, 1H); 6.55
(d, J = 16.0 Hz, 1H); 3.42 (q, J = 6.1 Hz, 2H); 3.03 (t, J = 6.4 Hz, 2H);
2.59 (s, 3H); 2.02 (br s, 9H); 1.74 (br t, 6H). ESI-MS m/z = 475.1
[M+H]+.
To a solution of 5 (100 mg, 0.23 mmol) and DIPEA (202 lL,
1.16 mmol) in anhydrous DCM (6 mL) was added dropwise at
0 °C para-nitrophenylchloroformate (141 mg, 0.7 mmol). The reac-
tion mixture was stirred for 2 h at room temperature. The crude
material thus obtained was used in the next step without any fur-
ther purification.
N-Boc-1,4-diaminobutane (175 mg, 0.92 mmol) and DIPEA
(126 lL, 0.70 mmol) were then added and the reaction mixture
was stirred at 55 °C for 12 h. The reaction mixture was diluted with
DCM and washed with HCL 0.01 N and H2O. The organic phase was
dried over Na2SO4 and the solvent was removed under reduced
pressure. The resulting residue was purified by flash chromatogra-
phy (hexane/ethyl acetate 60:40) to get the desired intermediate.
Yield 60%.
The above tert-butyl ester was dissolved in dioxane (0.5 mL) at
0 °C and then this solution was treated with HCl 4 M in dioxane
(4 mL). The ice-bath was removed and the reaction mixture was
stirred until complete conversion of the starting material to the
corresponding carboxylic derivative that precipitates as a white
powder. The white solid precipitate was filtered, washed with
Et2O and dried to give 40 mg of the title compound (7e). Yield
66%, 1H NMR (300 MHz, DMSO-d6) 12.40 (br s, 1H); 7.4–8 (br m,
8H); 7.05 (m, 1H); 6.55 (m, 1H); 3.1 (m, 2H); 2.8 (m, 2H); 2.02
(s, 9H); 1.6–1.8 (br s, 6H); 1.4–1.6 (br s, 4H). EI-MS m/z = 489.4
[M+H]+.
5.1.18. 4-[3-Aadamantan-1-yl-40-((E)-2-carboxy-vinyl)-
biphenyl-4-yloxycarbonylamino]-piperidine-1-carboxylic acid
benzyl ester (7c)
Benzyl 4-isocyanatotetrahydro-1(2H)-pyridinecarboxylate
(120 mg, 0.46 mmol) was added to a solution of 5 (100 mg,
0.23 mmol) and NEt3 (64 lL, 0.46 mmol) in anhydrous DCM
(4 mL). The reaction mixture was stirred for 2 days at room
temperature. Yield 37%.
The above tert-butyl ester (60 mg, 0.087 mmol) was dissolved at
room temperature in a DCM/TFA (8:2 v/v, 2.4 mL) and stirred until
complete cleavage. After complete deprotection, mixture was con-
centrated under reduced pressure, then diluted with DCM and con-
centrated to remove acid excess. Mixture was dried with high
vacuum to give 38 mg of pure product. (7c). Yield 69%, 1H NMR
(300 MHz, DMSO-d6) 12.40 (br s, 1H); 7.92 (d, 1H); 7.74 (d,
J = 8.2 Hz, 2H); 7.68 (d, J = 8.2 Hz, 2H); 7.61 (d, J = 15.9 Hz, 1H);
7.5 (m, 2H); 7.32–7.42 (m, 5H); 7.03 (d, J = 8.9 Hz, 1H); 6.54 (d,
J = 15.9 Hz, 1H); 5.07 (s, 2H); 3.95 (dd, 2H); 3.6 (m, 1H); 2.9–3.1
(m, 2H); 2.02 (br s, 9H); 1.9–1.8 (dd, 2H); 1.73 (m, 6H); 1.42 (m,
2H). ESI-MS m/z = 633.3 [MꢁH]ꢁ.
5.1.21. (E)-3-[30-Adamantan-1-yl-40-(2-morpholin-4-yl-ethyl-
carbamoyloxy)-biphenyl-4-yl]-acrylic acid hydrochloride (7f)
To a solution of 5 (100 mg, 0.23 mmol) and DIPEA (240 lL,
1.38 mmol) in anhydrous DCM (6 mL) was added dropwise at
0 °C p-nitrophenylchloroformate (115 mg, 0.57 mmol). The reac-
tion mixture was stirred for 2 h at room temperature. The crude
material thus obtained was used in the next step without any fur-
ther purification.
5.1.19. (E)-3-(30-Adamantan-1-yl-40-
4-(2-Aminoethyl)-morpholine (120 lL, 0.92 mmol) was then
carboxymethylcarbamoyloxy-biphenyl-4-yl)-acrylic acid (7d)
added and the reaction mixture was stirred at 55 °C for 12 h. The
reaction mixture was diluted with DCM and washed with H2O.
The organic phase was dried over Na2SO4 and the solvent was re-
moved under reduced pressure. The resulting residue was purified
by flash chromatography (DCM/MeOH 98:2) to get the desired
intermediate. Yield 56%.
The above tert-butyl ester (76 mg, 0.13 mmol) was dissolved in
dioxane (1 mL) and then this solution was treated with HCl 4 M in
dioxane (3 mL) at room temperature. The reaction mixture was
stirred until complete conversion of the starting material to the
corresponding carboxylic derivative. The white solid precipitate
was filtered, washed with DCM and dried to give 48 mg of the title
compound (7f). Yield 81%, 1H NMR (300 MHz, DMSO-d6): 12.40 (br
s, 1H); 8.2 (m, 1H); 7.75 (d, J = 8 Hz, 2H); 7.68 (d, J = 8 Hz, 2H); 7.61
(d, J = 16.1 Hz, 1H); 7.53 (m, 2H); 7.15 (d, J = 7.86 Hz, 1H); 6.55 (d,
J = 16.1 Hz, 1H); 3.95 (m, 2H); 3.81 (t, 2H); 3.53 (m, 4H); 3.1–3.3
(m, 4H); 2.02 (br s, 9H); 1.74 (m, 6H). ESI-MS m/z = 531.4 [M+H]+.
To a solution of 5 (120 mg, 0.28 mmol) and DIPEA (269 lL,
2.23 mmol) in dry DCM (7 mL) was added dropwise at 0 °C para-
nitrophenylchloroformate (174 mg, 0.83 mmol). The reaction mix-
ture was stirred for 2 h at room temperature. The crude material
thus obtained was used in the next step without any further
purification.
GlyO-tert-butyl HCl (188 mg, 1.12 mmol) was then added and
the reaction mixture was stirred at 55 °C for 12 h. The reaction
mixture was diluted with DCM and washed with H2O. The organic
phase was dried over Na2SO4 and the solvent was removed under
reduced pressure. The resulting residue was purified by flash chro-
matography (hexane/ethyl acetate 80:20) to get 100 mg
(0.17 mmol) of the desired intermediate. Yield 61%.
The above tert-butyl ester was dissolved in dioxane (2.2 mL)
and then this solution was treated with HCl 4 M in dioxane
(2.2 mL) at room temperature. The reaction mixture was stirred
until complete conversion of the starting material to the corre-
sponding carboxylic derivative. The white solid precipitate was fil-
tered, washed with Et2O and dried to give 12 mg (0.025 mmol;
15%) of the title compound (7d). Yield 62%, 1H NMR (300 MHz,
DMSO-d6) 12.50 (br s, 1H); 8.15 (t, J = 6.1 Hz 1H); 7.75 (d,
J = 8.5 Hz, 2H); 7.68 (d, J = 8.5 Hz, 2H); 7.62 (d, J = 16.1 Hz, 1H);
7.54 (m, 1H); 7.50 (s, 1H); 7.02 (d, J = 8.4 Hz, 1H); 6.54 (d,
5.1.22. (E)-3-(30-Adamantan-1-yl-40-undecyl-carbamoyloxy-
biphenyl-4-yl)-acrylic acid (7g)
To a solution of 5 (158 mg, 0.43 mmol) and DIPEA (299 lL,
1.72 mmol) in anhydrous DCM/DMF mixture (7:3 v/v; 2.8 mL)
was added dropwise at 0 °C p-nitrophenylchloroformate (174 mg,
0.86 mmol). The reaction mixture was stirred for 2 h at room

2414
G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
temperature. The crude material thus obtained was used in the
next step without any further purification.
room temperature. The reaction mixture was stirred until com-
plete conversion of the starting material to the corresponding car-
boxylic derivative as white powder. The white solid precipitate
was filtered, washed with Et2O and dried to give 70 mg
(0.12 mmol) of the title compound (7i). Yield 54%, 1H NMR
(300 MHz, DMSO-d6) 12.40 (br s, 1H); 10.57 (br s, 1H); 7.77 (d,
J = 8.4 Hz, 2H7.71 (d, J = 8.4 Hz, 2H); 7.64 (d, J = 15.9 Hz, 1H); 7.55
(m, 2H); 7.10 (d, J = 9.0 Hz, 1H); 6.58 (d, J = 15.9 Hz, 1H); 4.29 (m,
2H); 3.39 (m, 2H); 2.94 (m, 4H); 2.24 (m, 2H); 2.05 (m, 9H); 1.81
(m, 14H). ESI-MS m/z = 569.4 [M+H]+.
Undecylamine (370 lL, 1.72 mmol) was then added and the
reaction mixture was stirred at room temperature overnight. The
reaction mixture was concentrated under reduced pressure and
the resulting residue was purified by flash chromatography (hex-
ane/ethyl acetate 80:20) to get the desired intermediate. Yield 89%.
The above tert-butyl ester was dissolved in dioxane (1 mL) and
then this solution was treated with HCl 4 M in dioxane (2 mL) at
room temperature. The reaction mixture was stirred until com-
plete conversion of the starting material to the corresponding car-
boxylic derivative. The reaction mixture was concentrated under
reduced pressure, the solid obtained was dissolved in DCM and
concentrated (two times) and then washed with hexane and dried
under vacuum to give 110 mg of the title compound (7f). Yield 81%,
1H NMR (300 MHz, DMSO-d6) 12.40 (br s, 1H); 7.82 (t, J = 5.8 Hz
1H); 7.75 (d, J = 8.5 Hz, 2H); 7.67 (d, J = 8.5 Hz, 2H); 7.61 (d,
J = 15.8 Hz, 1H); 7.50 (m, 1H); 7.49 (s, 1H); 7.01 (d, J = 8.9 Hz,
1H); 6.54 (d, J = 15.8 Hz, 1H); 3.08 (q, J = 6.4 Hz, 2H); 2.02 (br s,
9H); 1.74 (br t, 6H); 1.44 (m, 2H); 1.40–1.23 (m, 16H); 0.83 (t,
J = 6.9 Hz, 3H). ESI-MS m/z = 572.5 [M+H]+.
5.2. Chromatographic analysis to assess chemical stability
For chromatographic analysis a Waters ACQUITY UPLC system
equipped with a computer and EMPOWER 2 software, a binary sol-
vent manager, a sample manager and a photodiode array spectro-
photometer detector PDA were used. A Waters BEH C18 1.7 lm
(2.1 ꢂ 50 mm) reverse-phase analytical column was used. A gen-
eral gradient was used at room temperature with the following sol-
vent system: solvent A = Water + 0.1% TFA; solvent B = acetonitrile;
starting at 60% A:40% B, at t = 8 min 5% A:95% B, at t = 9 min 5%
A:95% B, at t = 9.5 min 60% A:40% B, and ending 11 min. A flow rate
set at 0.5 mL/min. Absorbance was monitored using a photodiode
array detector from 190 to 450 nm.
5.1.23. (E)-3-{30-Adamantan-1-yl-40-[(S)-1-(carboxymethyl-
carbamoyl)-3-methyl-butylcarbamoyloxy]-biphenyl-4-yl}-
acrylic acid (7h)
To a solution of 5 (43 mg, 0.1 mmol) and DIPEA (108
lL,
5.3. Growth inhibition assay
0.6 mmol) in anhydrous DCM (1.5 mL) was added dropwise at
0 °C p-nitrophenylchloroformate (50 mg, 0.25 mmol). The reaction
mixture was stirred for 2 h at room temperature. The crude mate-
rial thus obtained was used in the next step without any further
purification.tert-BuOGlyLeuNH2 (112 mg, 0.4 mmol) was then
added and the reaction mixture was stirred at room temperature
for 16 h. The desired intermediate was obtained after purification
by silica gel chromatography with a gradient hexane/ethyl acetate
80:20 to 70:30. Yield 71%.
The above tert-butyl ester was dissolved in dioxane (2.2 mL)
and then this solution was treated with HCl 4 M in dioxane
(2.2 mL) at room temperature. The reaction mixture was stirred
until complete conversion of the starting material to the corre-
sponding carboxylic derivative as an oil. The white solid precipitate
was filtered, washed with Et2O and dried to give 30 mg
(0.051 mmol) of the title compound (7f). Yield 51%, 1H NMR
(300 MHz, DMSO-d6) 8.29 (d, J = 8.6 Hz, 1H); 7.64 (s, 4H); 7.45
(br s, 3H); 7.40 (d, J = 16.2 Hz, 1H); 7.05 (d, J = 8.6 Hz, 1H); 6.52
(d, J = 16.2 Hz, 1H); 4.03 (m, 2H); 3.44 (m, 1H); 2.02 (m, 10H);
1.73 (m, 8H); 0.89 (d, J = 6.4 Hz, 3H); 0.83 (d, J = 6.4 Hz, 3H). ESI-
MS: m/z = 587.3 [MꢁH]ꢁ.
The inhibitory effect of Adarotene derivatives on NCI-H460 non-
small cell lung carcinoma and A431 epidermoid carcinoma growth
was assessed after 24 h of drug exposure followed by 48 h of recov-
ery. Cells in the logarithmic phase of growth were seeded in octu-
plicate into 96-well plates. Twenty-four hours after seeding, the
drugs were added to the complete medium RPMI-1640. Twenty-
four hours later the drugs were removed and growth inhibition
was assessed after 48 h by sulphorodamine B test.16 IC50 was de-
fined as the drug concentration causing a 50% reduction of cell sur-
vival compared with that of untreated control and was evaluated
by ALLFIT program.17
5.4. Assessment of the in vivo cytotoxicity tumor growth
An in vivo experiment was carried out using 5–6 week-old fe-
male CD1 nude mice (Harlan, Italy). The mice were kept in laminar
flow rooms at constant temperature and humidity. They had free
access to food and water. Experimental protocol was approved
by the Ethics Committee for Animal Experimentation of Sigma-
Tau, according to the United Kingdom Coordinating Committee
on Cancer Research Guidelines.18 The human tumor cell line used
is a large cell lung carcinoma, known to be highly resistant to che-
motherapy. NCI-H460 NSCLC cells (3 ꢂ 106) or A431 epidermoid
carcinoma cells (5 ꢂ 106) from in vitro cultures were s.c. injected
into the right flank of mice. Each experimental group included
eight mice. Tumors were implanted on day 0. Drug treatment
started 3 days upon tumor injection when tumor lesions were
around 50 mm3 and tumor growth was followed by biweekly mea-
surements of tumor diameters with a Vernier caliper. TV3 was cal-
culated according to the following formula: TV (mm3) = d2 ꢂ D ꢂ 2,
where d and D were the shortest diameter and the longest diame-
ter, respectively.
5.1.24. [1,40]Bipiperidinyl-10-carboxylic acid 3-adamantan-1-yl-
40-((E)-2-carboxy-vinyl)-biphenyl-4-yl ester hydrochloride (7i)
To a solution of 5 (100 mg, 0.23 mmol) and DIPEA (240 lL,
1.38 mmol) in anhydrous DCM (6 mL) was added dropwise at
0 °C p-nitrophenylchloroformate (115 mg, 0.57 mmol). The reac-
tion mixture was stirred for 2 h at room temperature. The crude
material thus obtained was used in the next step without any fur-
ther purification.
4-Piperidinopiperidine (155 mg, 0.92 mmol) was then added
and the reaction mixture was stirred at room temperature for
12 h. The reaction mixture was diluted with DCM and washed with
H2O. The organic phase was dried over Na2SO4 and the solvent was
removed under reduced pressure. The resulting residue was puri-
fied by flash chromatography (DCM/MeOH 96:4) to get the desired
intermediate. Yield 46%.
Drug efficacy was assessed as TVI% that was 100 ꢁ (mean TV
treated/mean TV control ꢂ 100). Toxic effects of drug treatment
were assessed as BWL% that was 100 ꢁ (mean body weight day
x/mean body weight day 1 ꢂ 100), where day 1 was the first day
of treatment and day x was any day thereafter. The highest (max-
imum) BWL is reported in Table 2. Mice were weighed every day
The above tert-butyl ester was dissolved in dioxane (1 mL) and
then this solution was treated with HCl 4 M in dioxane (2 mL) at

G. Giannini et al. / Bioorg. Med. Chem. 20 (2012) 2405–2415
2415
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.042.
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Differ. 2004, 11, 280; (d) Kadara, H.; Schroeder, C. P.; Lotan, D.; Pisano, C.; Lotan,