Synthesis of photoactivable inhibitors of osteoclast vacuolar ATPase

Article abstract of DOI:10.1016/S0968-0896(03)00106-8

Amides of (2Z,4E)-5-[(5,6-dichloroindol-2-yl)]-2-methoxy-N-[3-[4-[3-(carboxymethoxy) phenyl)] piperazin-1-yl]propyl]-2,4-pentadienamide (1) and of 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienoic acid (2) are strong inhibitors of the vacuolar ATPase located on the plasma membrane of osteoclasts. In order to understand which V-ATPase subunit is involved in the interaction with these novel inhibitors, analogues containing a photoactivable group and an iodine atom were designed. A series of alcohols or amines containing the photoactivable trifluoroaziridinophenyl or benzophenone moiety and an iodine atom were linked to the above acids via an ester or amide group. These compounds could be thereafter used as a radioactive photoprobe to label the protein. Whereas the compounds containing the photoactivable groups maintained good inhibitory activity, the introduction of the bulky iodine atom was generally detrimental, decreasing potency significantly. Better results were obtained by linking 3-(4-aminopiperidinomethyl)-3′-iodobenzophenone to 3-ethoxy-4-(2-(5,6-dichlorobenzimidazolyl))benzoic acid to give the corresponding amide 27, that inhibited vacuolar ATP-ase with a IC₅₀=140 nM. The feasibility of introducing a radioactive ¹²⁵I atom was ascertained by exchanging the iodine with a tributylstannyl group, that was again substituted by iodine.

Full text of DOI:10.1016/S0968-0896(03)00106-8

Bioorganic & Medicinal Chemistry 11 (2003) 2247–2254
Synthesis of Photoactivable Inhibitors of Osteoclast
Vacuolar ATPase
Barbara Biasotti,a Sabrina Dallavalle,a Lucio Merlini,a,* Carlo Farina,b
Stefania Gagliardi,b,* Carlo Parinib and Pietro Belfiorec
a
`
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, 20133 Milan, Italy
bNiKem Research Srl, Via Zambeletti 25, 20021 Baranzate di Bollate (Mi), Italy
cGlaxoSmithKline, MuscoloSkeletal Disease, 709 Swedeland Road, King of Prussia PA 19406, USA
Received 30 October 2002; accepted 7 February 2003
Abstract—Amides of (2Z,4E)-5-[(5,6-dichloroindol-2-yl)]-2-methoxy-N-[3-[4-[3-(carboxymethoxy)phenyl)] piperazin-1-yl]propyl]-
2,4-pentadienamide (1) and of 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienoic acid (2) are strong inhibitors of the vacuolar
ATPase located on the plasma membrane of osteoclasts. In order to understand which V-ATPase subunit is involved in the inter-
action with these novel inhibitors, analogues containing a photoactivable group and an iodine atom were designed. A series of
alcohols or amines containing the photoactivable trifluoroaziridinophenyl or benzophenone moiety and an iodine atom were linked
to the above acids via an ester or amide group. These compounds could be thereafter used as a radioactive photoprobe to label the
protein. Whereas the compounds containing the photoactivable groups maintained good inhibitory activity, the introduction of the
bulky iodine atom was generally detrimental, decreasing potency significantly. Better results were obtained by linking 3-(4-amino-
piperidinomethyl)-30-iodobenzophenone to 3-ethoxy-4-(2-(5,6-dichlorobenzimidazolyl))benzoic acid to give the corresponding
amide 27, that inhibited vacuolar ATP-ase with a IC50=140 nM. The feasibility of introducing a radioactive 125I atom was ascer-
tained by exchanging the iodine with a tributylstannyl group, that was again substituted by iodine.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Inhibitors of the osteoclast proton pump may therefore
represent useful agents able to prevent the excessive
bone resorption associated with osteoporosis.4 Among
them, natural macrolide antibiotics such as bafilomycins
and concanamycins are potent but completely unselec-
tive inhibitors of V-ATPases,5 ruling out any possible
therapeutic utility of these compounds. It has been
reported that chemical modifications of the backbone of
bafilomycin A1 may be able to differentiate potency and
selectivity against different vacuolar-type ATPases6 as
assessed by measuring the effects of novel bafilomycin
analogues on the V-ATPase-driven proton transport in
membrane vesicles from different tissues.
Osteoporosis is a widespread metabolic bone disease,
particularly among postmenopausal women, charac-
terised by low bone mass and microarchitectural dete-
rioration of bone with consequent bone fragility and an
increased risk of vertebral, wrist and hip fractures. It
results from an imbalance of the bone remodeling process,
with bone resorption exceeding bone formation. Bone
resorption is a multistep process which ultimately requires
secretion of hydrochloric acid1 from osteoclasts into the
resorption compartment. Protons are extruded by vacuo-
lar-type ATPases2 (V-ATPases) that are present in large
number on the ruffled border of the osteoclasts, while the
chloride counter-ions diffuse passively through a chlor-
ide channel to dissipate the membrane potential gener-
ated in the resorption lacuna by proton transport.3
7
Extensive SAR studies on bafilomycin A1 led to the
identification of the key structural requirements for
biological activity and allowed the design and synthesis
of novel indole derivatives as simpler and more easily
accessible inhibitors of the osteoclast V-ATPase.8
*Corresponding authors. Tel.: +39-025-031-6812; fax: +39-025-031-
6801; e-mail: lucio.merlini@unimi.it (L. Merlini). Tel.: +39-0238062459;
fax: +39-0230862606; e-mail: stefania.gagliardi@nikemresearch.com
(S. Gagliardi).
SB-242784 (Fig. 1), the most representative compound
of this class, was able to inhibit bone resorption in vitro
in the human osteoclast (hOc) resorption assay9 (Table
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00106-8

2248
B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
groups, which in a first instance were inserted without
the cold iodine label. Among the photolabelled inhibi-
tors thus obtained, those retaining the most significant
activity at the molecular target were iodinated to verify
that the presence of this large substituent was not detri-
mental for V-ATPase inhibitory activity.
Figure 1. Structure of SB-242784.
Results
Table 1. In vitro activity of SB-242784 (IC50 nM)
As the active compounds were esters or amides of the
acids 1 and 2 (Fig. 2),8 amines or alcohols containing
the selected photophores were prepared, and coupled
with the appropriate acid. Acid 1 was obtained from 2 by
coupling with the appropriate amine. The esters and the
amides were prepared by a standard procedure which
requires coupling of the acid with the appropriate alco-
hol or amine in the presence of WSC and HOBt.
V-ATPase assaya
COc hOc
SB-242784 26.5ꢀ1.5 22ꢀ0.9
Human osteoclast resorption assay
Pits/bone slices
ELISA
13.5ꢀ2.0
3.4ꢀ1.9
acOc, chicken osteoclasts; hOc, human osteoclasts.
1) and to completely prevent the bone loss induced by
ovariectomy in rats after 6 months treatment at 10 mg/
kg/d po, thus suggesting a possible utility of selective
inhibition of the osteoclast V-ATPase as a novel
approach for the prevention of bone loss in humans.10
The synthesis of meta-trifluoromethyldiazirinoaniline 8
was performed according to Scheme 1 starting from
3-nitrotrifluoroacetophenone 318,19 following the general
procedure of Brunner and Semenza20 for the synthesis of
the trifluoromethyldiazirino group. A synthesis of 8, dif-
fering in some steps from ours, was recently reported by
Darrow et al.21 but without experimental details.
V-ATPases are complex, heteromultimeric enzymes,
composed of at least 20 different subunits. The osteo-
clast V-ATPase is characterized by the presence of a
specific isoform of the 116 kDa subunit (subunit a)
which is predominantly expressed in these multi-
nucleated cells.11 Together with the 16 kDa proteolipid
(c subunit) and some accessory subunits, subunit a
makes up the V0 portion of the V-ATPase, which con-
tains the proton channel. It has been shown that both
bafilomycin and SB-242784 do inhibit V-ATPase activity
by binding to the V0 subcomplex.12 However, whilst
bafilomycin and concanamycin were shown, very
recently, to bind to the ubiquitous 16 kDa (c) subunit,13,14
it is not known whether SB-242784 and related inhibitors
bind the same subunit c or do indeed interact with the
osteoclast-specific subunit a, thus providing a mechan-
istic rationale for osteoclast selectivity of these inhibitors.
The other ligands used to obtain products reported in
Table 2 were prepared according to the literature or
following standard methods.
Table 2 reports the value of the inhibition of V-ATPase
(IC50, nM) of some derivatives of acid 1, and of their
analogues containing the trifluoroazirino or benzophe-
none photoactive group, or this group and an iodine
atom. The amide 8 from aniline exhibited an acceptable
IC50 of 340 nM and the introduction of the tri-
fluoroazirinyl group did not change the activity sub-
stantially (9, IC50=380 nM). Therefore the effect of the
presence of the necessary, but bulky iodine atom was
assessed by testing the ester 10. The potency of this
compound, unfortunately, decreased significantly to an
IC50 of 1200 nM. The possibility that such an effect was
due to the replacement of the amide group by an ester, was
excluded by comparing the activity of the corresponding
amide 11, which showed an even higher IC50. Again the
possible effect of the benzylamino versus the anilino group
was excluded by comparing the potencies of the amides
with benzylamine (12) and with m-iodoaniline (13). The
resulting conclusion was that the introduction of a bulky
iodine atom in the photolabelling group was detrimental
to the activity. The introduction of the benzophenone
photophoric group instead of the trifluoroazirino led to a
compound (14) with unsufficient potency (IC50=800 nM)
even if iodine was not present. Consequently, the insertion
of the halogen on this group was not attempted.
To try to answer this question, we started a chemical
approach to obtain novel analogues of SB-242784 and/
or analogues bearing photoactivable groups, a suitable
radiolabel and, at the same time, maintaining a sig-
nificant inhibitory potency against osteoclast V-
ATPase. The availability of such compounds, would
then allow photoaffinity labelling experiments to iden-
tify the primary binding proteins to which they bind. In
a second level of identification, the tagged protein target
can be fragmented into short peptides to localize the
binding region within the biopolymer.15
This approach entailed the preparation of the com-
pounds with a ‘cold’ label, assessment of their biological
activity and, finally, optimization of the conditions for
the synthesis of the ‘hot’ molecules. Among the possible
photoreactive groups that have been mostly used
because of their reliable and reproducible high-efficiency
labelling of target proteins, such as tetrafluoro-
phenylazide, trifluoromethylphenyldiazirine and benzo-
phenone photophores,16,17 we selected the two latter
Our attention then shifted toward the amides of the
shorter acid 2 with 4-aminopiperidine (Table 3). Amide
15 exhibited an acceptable IC50, but introduction of the
photophore and of iodine (16) strongly decreased the
activity. A similar, but less marked, effect was shown by
iodobenzophenone-derived amides (18–19), where a

B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
2249
Figure 2. Structure of acids 1 and 2.
ꢁ
.
Scheme 1. (a) NH2OH HCl, Py, EtOH, 80 C; (b) TsCl, DMAP, Et3N, CH2Cl2, rt; (c) liq NH3, Et2O/CH2Cl2; (d) I2, Et3N, EtOH, rt; (e) Na2S2O4,
Bu4NBr, NaOH, THF.
7–10-fold increase of the IC50 with respect to 17 was
observed, depending on the position of the iodine atom.
In this case, the activity (140 nM) was high enough, and
compound 27 was chosen for the photoaffinity test
(Table 4).
Compounds possessing the dienic chain, such as SB-
242784, undergo photo-induced isomerisation of the
double bonds when they are in solution. This liability led
us to evaluate the possibility to replace the dienic chain
with a phenyl ring. Potency was increased when a meth-
oxy group was inserted in the position close to the indole
ring. The replacement of the indole ring by benzimidazole
improved water solubility while retaining the same inhi-
bitory potency. Finally substitution of the methoxy by an
ethoxy group gave the most potent inhibitor (25, Scheme
2), with an IC50 in hO c ATPase assay of 8 nM2.2 O n the
basis of these data it was decided to produce photoaffinity
derivatives also in this new class of ATPase inhibitors.
Amide 26 maintained a reasonable potency and therefore
an iodinated benzoylbenzylpiperidine (27) was synthesised.
In order to allow the introduction of radioactive 125I,
compound 27 had to be stannylated, and the tributyl-
stannyl group exchanged again with iodine. The reac-
tion of 27 with hexabutylditin to give 28, followed by
iodination with chloramine-T and sodium iodide23 pro-
ceeded satisfactorily (Scheme 3), so that this procedure
can be used for the preparation of the radioactive deri-
vative.
In conclusion, we have obtained a iodinated strong
inhibitor of vacuolar ATPase containing a photo-
activable benzophenone group, that, once labelled with
125I, can be used as a photoprobe for the photoaffinity
labelling of this enzyme.

2250
B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
Experimental
Table 2. In vitro activity (V-ATPase inhibition in human osteoclasts)
of derivatives of compound 1
All reagents and solvents were reagent grade or were
purified by standard methods before use. Melting points
were determined in open capillaries on a Buchi melting
point apparatus and are uncorrected. Column chroma-
tography was carried 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, in CDCl3, if not
otherwise stated. Chemical shifts (d values) and cou-
pling constants (J values) are given in ppm and Hz,
respectively. Mass spectra were recorded at an ionizing
voltage of 70 eV on a Finnigan TQ70 spectrometer. The
relative intensities of mass spectrum peaks are listed in
parentheses. Solvents were routinely distilled prior to
use; anhydrous tetrahydrofuran (THF) and ether (Et2O)
were obtained by distillation from sodium-benzophe-
none ketyl; dry methylene chloride was obtained by
distillation from phosphorus pentoxide. All reactions
requiring anhydrous conditions were performed under a
positive nitrogen flow, and all glassware was oven-dried
and/or flame dried.
Compd
R
Mp (ꢁC)
Yield (%)
IC50 (nM)
1
–OH
–OEt
240–242
184–185
Ref 8
Ref 8
84
45
8
9
100–105
90–92
27
28
340
380
10
113–116
30
1200
(2Z,4E)-5-[(5,6-Dichloroindol-2-yl)]-2-methoxy-N-[3-[4-
[3-(carboxymethoxy)phenyl)] piperazin-1-yl]propyl]-2,4-
pentadienamide hydrochloride (1). (a) 4-(3-Hydroxy-
phenyl)piperazine (9 g, 50.5 mmol) was dissolved in
CH3CN and Et3N (18 mL, 130 mmol) and 3-bromo-N-
t-butoxycarbonylpropylamine (18 g, 75.6 mmol) were
added. The solution was refluxed for 5 h. The organic
solvent was evaporated to dryness and the residue was
re-dissolved in EtOAc, washed with water, brine and
dried over Na2SO4. After solvent evaporation an oil was
obtained (17 g) that was used in the next step without
further purification.
11
12
80–85
48
61
2400
120
130–133
13
14
216–220
34
63
490
800
The crude compound was dissolved in DMF (170 mL)
and solid K2CO3 (51 g) was added. The suspension was
stirred at room temperature and ethyl bromoacetate
(6.21 mL, 56 mmol) was added dropwise. Stirring was
Scheme 2. (a) NaNO2, HCl, CuCN–NaCN; (b) SOCl2; (c) o-NH2C6H2Cl2NH2, Et3N, then P2O5; (d) NaOH; (e) SOCl2, then 4-aminote-
tramethylpiperidine.

B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
2251
Table 3. In vitro activity (V-ATPase assay in human osteoclasts) of derivatives of compound 2
Compd
R
Mp (ꢁC)
255–258
Yield (%)
71
IC50 (nM)
93
15
16
17
18
19
235–240
241–249
218–220
184–188
28
10
37
60
> >1000
100
1000
710
Scheme 3. (a) (Sn(Bu)3)2, Pd(PPh3)4, Et3N; (b) chloramine-T, NaI.
continued for 24 h. Et2O(200 mL) was added and the
suspension was filtered and evaporated to dryness. The
residue was re-dissolved in EtOAc and washed with
water, brine and dried over Na2SO4. The residue was
triturated with hexane and filtered obtaining 12.4 g of 3-[4
-[3-(ethoxycarbonylmethoxy)phenyl)]piperazin-1-yl]-N-t-
butoxycarbonylpropylamine, mp 93–97 ꢁC. The protec-
tive group was removed dissolving the compound in
CH2Cl2 (50 mL) and adding TFA (25 mL). After stirring
3 h at room temperature, the solvent was evaporated to
dryness obtaining 14 g of 3-[4-[3-(ethoxycarbonyl-
methoxy)phenyl)]piperazin-1-yl]propylamine as a tri-
fluoroacetate salt, NMR d=7.15 (dd, J=7.4, 7.4 Hz,
1H, 5ArH); 6.90 (s br, 3H, NH+3 ); 6.57 (dd, J=7.4, 1.2
Hz, 1H, 4(6) ArH); 6.49 (dd, J=1.2, 1.2 Hz, 1H, 2ArH);
6.38 (dd, J=7.4, 1.2 Hz, 1H, 6(4) ArH); 4.59 (s, 2H,
OCH2CO); 4.27 (q, J=7.2 Hz, 2H, COCH2); 3.21–3.10
(m, 6H, ArN(CH2)2+CH2NH+3 ); 2.80–2.68 (m, 6H,
N(CH2)3); 1.92 (m, 2H, NCH2–CH2); 1.30 (t, J=7.2,
3H, CH3).
(b) Acid 28 (3.6 g, 11.5 mmol), dissolved in a mixture of
acetonitrile and THF (3:1), was coupled with 3-[4-[3-
(ethoxycarbonylmethoxy)phenyl)]piperazin-1-yl]propy-
lamine using HOBT (1.55 g) and WSC (2.21 g). The
reaction mixture was refluxed for 1 h. Solvent was evap-
orated to dryness and the residue was dissolved in
EtOAc and washed with water, NaHCO3 and brine.
The organic phase was dried over Na2SO4, filtered and
concentrated to give a solid that was triturated with
Et2O. The solid (5.1 g) was filtered and then re-dissolved
in THF. 20% NaOH was added (12 mL) and the reac-

2252
B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
Table 4. In vitro activity (V-ATPase assay on human osteoclasts) of
5,6-dichlorobenzimidazole derivatives
mixture stirred in a closed vessel 2 days at room tem-
perature, then cooled at ꢂ78 ꢁC, gaseous ammonia
bubbled for 5 min, left 2 days at room temperature.
Evaporation of ammonia, filtration and evaporation of
the solvent gave a red-orange oil (1.67 g), NMR d 2.4
(NH), 2.95 (NH), 7.63 (t, J=7, H-5), 7.97 (dd, J=7,1.5,
H-4), 8.30 (dd, J=7,1.5, H-6), 8.48 (s, H-2), that was
used without further purification.
Compd
R
IC50 (nM)
8
3-(3-Nitrophenyl)-3-trifluoromethyldiazirine (7). Diazir-
idine 6 (0.8 g, 3.43 mmol) in 10 mL of MeOH was treated
with 0.96 mL (6.86 mmol) of Et3N and with 0.87 g (3.43
mmol) of iodine, the mixture stirred in the dark for 40 min,
then ether (20 mL) was added and the mixture washed with
satd aq citric acid, satd aq sodium thiosulfate, and water,
dried and evaporated to give 0.64 g of crude 7. Chroma-
tography with hexane/CH2Cl2 4:1 gave 0.59 g (74%) of 7,
NMR d 7.5–7.7 (m, 2H Ar), 8.05 (s, 1H Ar), 8.3 (d, J=7,
1H Ar). Anal. calcd for C9H8F3N3O2: C, 43.73; H, 3.26, N,
17.00. Found: C, 43.87; H, 3.29; N, 17.18.
25
26
27
90
140
3-(3-Aminophenyl)-3-trifluoromethyldiazirine (8). Diazir-
ine 7 (101 mg, 0.44 mmol) in 6 mL of THF was added
with 3 mL of aq NaOH at pH 10, then with 229 mg
(1.32 mmol) of sodium dithionite and 14 mg (0.04
mmol) of tetrabutylammonium bromide, and heated in
an oil bath. After 15 min, when pH was 6 and tem-
perature 40 ꢁC, the mixture was added with further 229
mg of dithionite, refluxed 6.5 h, keeping the solution
basic by addition of 6 M NaOH every 30 min, and
adding dithionite (115 mg) every h. The mixture was
cooled, treated with NaHCO3 and extracted with ether.
Drying, evaporation and chromatography with hexane/
CH2Cl2 1:1 gave 41.6 mg (47%) of 8, mp 68–70 ꢁC,
NMR d 6.45 (s, H-2), 6.52 (d, J=7, H-4), 6.69 (d, J=7,
H-6), 7.17 (t, J=7, H-5), m/z 201(77), 173(76), 83(28),
49(100). Anal. calcd for C9H10F3N3: C, 49.77; H, 4.64,
N, 19.35. Found: C, 49.88; H, 4.73; N, 19.18..
tion was stirred at 50 ꢁC for 1 h. After evaporating to
dryness, 10% HCl was added and the solid obtained
was filtered, washing twice with water. Crude com-
pound was triturated with acetonitrile to give 3.7 g of 1,
mp 240–242 ꢁC, NMR (DMSO-d6 and TFA) d 11.81 (s
br, 1H); 10.36 (s br, 1H); 8.34 (t br, J=6.0 Hz, 1H); 7.74
(s, 1H); 7.53 (s, 1H); 7.20 (dd, J=15.5,11.6 Hz, 1H);
7.15 (dd, J=8.5, 8.5 Hz, 1H); 6.86 (d, J=15.5 Hz, 1H);
6.69 (d, J=11.6 Hz, 1H); 6.60 (dd, J=8.2, 1.9 Hz, 1H);
6.58 (d, J=1.6 Hz, 1H); 6.54 (dd, J=2.2, 1.9 Hz, 1H);
6.41 (dd, J=8.2, 2.2 Hz, 1H); 4.64 (s, 2H); 3.88–3.88 (m,
2H); 3.76 (s, 3H); 3.55 (m, 2H); 3.27 (m, 2H); 3.20–3.04
(m, 6H); 2.00–1.89 (m, 2H), m/z 587; 555; 351; 319; 262;
236. Anal. calcd for C30H34Cl2N4O5: C, 59.90; H, 5.70,
N, 9.31. Found: C, 59.78; H, 5.82; N, 9.21.
3-Nitrotrifluoroacetophenone oxime (4). Compound 3
(1.44 g, 6.57 mol) was dissolved in 8 mL of dry ethanol,
added with 8 mL of pyridine and 1.37 g (19.7 mmol) of
General method for the synthesis of esters or amides of
(1) and (2)
.
hydroxylamine HCl, and refluxed 2.5 h. Evaporation,
taking up with ether, washing with acid water (pH 5),
drying, evaporation and chromatography (hexane–
AcOEt 4:1) gave 2.58 g of 4,17 mp 48–51 ꢁC.
The acid (0.5 mmol) was suspended in 12 mL of
CH3CN–THF 2:1 in the dark and under nitrogen. WSC
(0.75 mmol) and HOBt (0.75 mmol) were added and the
mixture heated at 40 ꢁC. After 1–2 h, if the acid was still
detectable by TLC, WSC (0.35 mmol) and HOBt (0.35
mmol) were added. After 2 h, the appropriate alcohol or
amine (0.55 mmol) was added and the mixture refluxed
2–3 h. Then water and AcOEt were added, and the
mixture stirred overnight at room temperature. Addi-
tion of 10% NaOH, extraction with AcOEt, and chro-
matography afforded the products.
3-Nitro-(1-tosyloxyimino)trifluoroethylbenzene
(5).
Oxime 4 (2.46 g, 10.5 mmol) in 25 mL of CH2Cl2 was
cooled at 0 ꢁC, added with Et3N (2.2 mL, 15.8 mmol),
DMAP (0.64 g, 5.25 mmol) and tosyl chloride (2.2 g,
11.6 mmol), stirred 24 h at room temperature, evapo-
rated, taken up with AcOEt, washed with water and
brine, evaporated and the crude product crystallised
from CH2Cl2/hexane to give 3.06 g (75%) of 5 as a
white solid, mp 95–98 ꢁC, NMR d 2.50 (s, CH3), 7.40
(m, 2H, H-30+H-50), 7.65–7.78 (m, H-5 and H-6), 7.9
(m, H-20 and H-60), 8.22 (m, H-2), 8.40 (m, H-4). Anal.
calcd for C15H11F3N2O5S: C, 46.39; H, 2.86, N, 7.21.
Found: C, 46.54; H, 2.81; N, 7.28.
2-Ethoxy-4-aminobenzoic acid (20). A suspension of
methyl 2-ethoxy-4-acetamidobenzoate (50 g, 211 mmol)
in aqueous solution of NaOH (15% w/w, 200 mL) was
gently refluxed for 16 h. The resulting pale brown solu-
tion was allowed to cool to room temperature and then
further cooled in an ice water bath. Concentrated HCl
(37% w/w) was added until the solution reached pH 6.
The solid precipitated from the solution was filtered
under vacuum, dried at 50 ꢁC obtaining 38.3 g (yield
3-(3-Nitrophenyl)-3-trifluoromethyldiaziridine (6). To 3
mL of liquid ammonia at ꢂ78 ꢁC was added a suspen-
sion of 2.62 g (6.75 mmol) of 5 in 30 mL of CH2Cl2, the

B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
2253
100%) of 20, mp 152–154 ꢁC, NMR (DMSO-d6) d 1.33
(t, J=6.9 Hz, Me), 4.01 (q, J=6.9 Hz, CH2), 5.79 (br s,
2H exch. with D2O), 6.12 (dd, J=8.5, 2.2 Hz, ArH),
6.20 (d, J=2.2 Hz, ArH), 7.48 (d, J=8.5 Hz, ArH),
11.98 (br s, 1H exch. with D2O). ). Anal. calcd for
C9H11NO3: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.48;
H, 6.05; N, 7.65.
filtered, washed with water and dried under vacuum at
50 ꢁC to give 16.5 g of the title compound as a light
brown powder (yield 82.6%), mp >250 ꢁC, NMR
(DMSO-d6) d 1.45 (t, J=6.9 Hz, Me), 4.43 (q, J=6.9
Hz, CH2), 7.55 (dd, J=8.2, 0.3 Hz, ArH), 7.79 (d,
J=0.3 Hz, ArH), 7.91 (s, ArH benzimidazole), 8.36 (d,
J=8.2 Hz, ArH), 11.62 (s, 1H). Anal. calcd for
C16H11Cl2N3O: C, 57.85; H, 3.34, N, 12.65. Found: C,
57.87; H, 3.26; N, 12.58.
2-Ethoxy-4-cyanobenzoic acid (21). CuCN (12 g, 134
mmol) was suspended in 100 mL of distilled water.
NaCN (18.3 g, 373 mmol) was added with vigorous
stirring and the internal temperature was kept below
40 ꢁC until all the CuCN went into solution. A suspen-
sion of 2-ethoxy-4-aminobenzoic acid (20 g, 110 mmol),
in water (200 mL) and concentrated HCl (33 mL) was
stirred and cooled in an ice bath. When the temperature
reached 5 ꢁC, a solution of NaNO2 (9.7 g, 140 mmol) in
water (30 mL) was added dropwise at such a rate as to
maintain the temperature below 5 ꢁC. When all the
NaNO2 was added, the solution was slowly introduced
through an ice-cooled dropping funnel into the reactor
containing the NaCN/CuCN solution. A reaction took
place with the vigorous formation of N2. A few drops of
1-octanol were added to keep the foaming under con-
trol. Stirring was continued for 4 h. The resulting sus-
pension was then extracted with ethyl acetate (3ꢃ100
mL) and the organic phase dried over MgSO4 and eva-
porated under vacuum obtaining 15 g of the title com-
pound (yield 71.1%) as a light brown powder, mp 170–
172 ꢁC. NMR (DMSO-d6) d 1.60 (t, J=6.9 Hz, Me), 4.37
(q, J=6.9 Hz, CH2), 7.30 (d, J=0.9 Hz, ArH), 7.41 (dd,
J=8.2, 0.9 Hz, ArH), 8.27 (d, J=8.2 Hz, ArH), 10.42 (s
br, 1H exch. with D2O). ). Anal. calcd for C10H9NO3: C,
62.82; H, 4.74, N, 7.33. Found: C, 62.93; H, 4.82; N, 7.22.
5,6-Dichloro-2-(4-carboxy-2-ethoxyphenyl)benzimidazole
hydrochloride (24). A mixture of 5,6-dichloro-2-(4-
cyano-2-ethoxyphenyl)benzimidazole
hydrochloride
(16.3 g, 44.2 mmol) and 32% NaOH (40 mL) in ethanol
(100 mL) was refluxed for 8 h. After cooling to room
temperature the organic solvent was removed under
vacuum and the aqueous phase was acidified with
37% HCl, and stirred for 1 h. The solid was filtered,
washed with water (100 mL) and dried at 50 ꢁC
under vacuum to give 12 g as a light brown powder
(yield 70%), mp >250 ꢁC, NMR (DMSO-d6) d 8.70
(s br, 3H, exch. with D2O); 1.45 (t, J=6.9 Hz, Me),
4.44 (q, J=6.9 Hz, CH2), 7.69 (dd, J=8.2, 0.2 Hz,
ArH), 7.71 (d, J=0.2 Hz, ArH), 8.02 (s, ArH benzi-
midazole), 8.29 (d, J=8.2 Hz, ArH). Anal. calcd for
C16H13Cl3N2O3: C, 49.57; H, 3.38; N, 7.23. Found:
C, 49.77; H, 3.20; N, 7.38.
4-(5,6-Dichlorobenzimidazol-2-yl)-N-(2,2,6,6-tetrame-
thylpiperidin-4-yl)-3-ethoxybenzamide (25). A mixture of
5,6-dichloro-2-(4-carboxy-2-ethoxyphenyl)benzimidazole
hydrochloride (1 g, 2.58 mmol) and of thionyl chloride
(8 mL) in dichloromethane (20 mL) was refluxed for 3 h.
After cooling to room temperature the solvent was
removed under vacuum and the crude residue was used
in the following reaction without further purification.
The acid chloride was added portionwise to a solution
of 4-amino-2,2,6,6-tetramethylpiperidine (0.612 g, 3.87
mmol), triethylamine (5 mL, 36 mmol) in dichloro-
methane (50 mL). Stirring was continued at room tem-
perature for 2 h. The solvent was removed under
vacuum and the residue was suspended in water (50 mL)
and filtered. The solid was dried at 50 ꢁC under vacuum
and then crystallised with ethanol to give 0.6 g of the
title compꢁound as a light brown powder (yield 47.5%),
2-Ethoxy-4-cyanobenzoyl chloride (22). 2-Ethoxy-4-cya-
nobenzoic acid (10 g, 52.3 mmol) and thionyl chloride
(50 mL) were refluxed in CH2Cl2 (80 mL) for 5 h. Sol-
vent was removed under vacuum to leave 10.9 g of an
off white solid (52 mmol, yield 99%) that was used
without further purification.
5,6-Dichloro-2-(4-cyano-2-ethoxyphenyl)benzimidazole
hydrochloride (23). A solution of 2-ethoxy-4-cyano-
benzoyl chloride (10.9 g, 52 mmol) in dichloromethane
(109 mL) was added dropwise in 5 h to a solution of 4,5-
dichlorophenylenediamine (18.5 g, 104.5 mmol) and
triethylamine (53 g=72.6 mL, 523 mmol) in dichloro-
methane (550 mL). Stirring was continued for addi-
tional 2 h. The solvent was removed under vacuum and
the residue was triturated with water (100 mL), filtered
and dried at 50ꢁC under vacuum. The solid was sus-
pended in diethyl ether (200 mL), stirred for 1 h, filtered
and dried under vacuum to give 19 g of N-(2-amino-4,5-
dichlorophenyl)-2-ethoxy-4-cyanobenzamide (yield 51%),
mp 195–198 ꢁC. A suspension of N-(2-amino-4,5-
dichlorophenyl)-2-ethoxy-4-cyanobenzamide (19 g, 54.2
mmol) and P2O5 (19 g, 134 mmol) in xylene (380 mL)
was refluxed for 24 h. Additional P2O5 (9.5 g, 67 mmol)
was added and the mixture was refluxed for 48 h. Sol-
vent was removed under reduced pressure. The residue
was treated with 30% NaOH (80 mL) and water (100
mL) and then acidified with 37% HCl. The solid was
1
mp >280 C, H NMR (DMSO-d6) d 1.05 (s, 6H), 1.20
(dd, 2H), 1.05 (s, 6H), 1.20 (s, 6H), 1.50 (t, 3H), 1.73 (d,
2H), 4.35–4.22 (m, 1H), 4.40 (q, 2H), 7.59 (d, 1H), 7.64
(s, 1H), 7.90 (s, 2H), 8.30 (m, 2H), 12.20 (s br, 1H), m/z
488 (MH+); 473; 124. Anal. calcd for C25H30Cl2N4O2:
C, 61.35; H, 6.18; N, 11.45. Found: C, 61.22; H, 6.27; N,
11.58.
N-4-Phenylmethylpiperidinamide of 3-ethoxy-4-(5,6-di-
chlorobenzimidazol-2-yl)benzoic acid (26). Prepared fol-
lowing the general procedure described above, for
example, for 25. Obtained in 63% yield after tri-
turation with abs. EtOH. Mp 295 ꢁC, NMR d 1.48
(t, CH3), 1.54–1.69 (CH2), 1.80 (CH2), 2.03 (CH2),
2.84 (CH2), 3.48 (s, ArylCH2N), 3.87–3.82 (m,
CHNH), 4.28 (q, 2H, OCH2), 6.12 (d, NHCO),
7.2–8.0 (11H arom), 8.12 (s, 1H), 8.59 (d, 1H,
J=8), 11.63 (1H, NH). m/z 522 (MH+). Anal.

2254
B. Biasotti et al. / Bioorg. Med. Chem. 11 (2003) 2247–2254
calcd for C28H28Cl2N4O2: C, 64.25; H; 5.39; N,
10.70. Found: C, 64.42; H, 5.48; N, 10.58.
References and Notes
1. Baron, R.; Neff, L.; Louvard, D.; Courtoy, P. J. J. Cell
Biol. 1985, 101, 2210.
2. Blair, H. C.; Teitelbaum, S. L.; Ghiselli, R.; Gluck, S.
Science 1989, 245, 855.
3. Schlesinger, P. H.; Blair, H. C.; Teitelbaum, S. L.;
Edwards, J. C. J. Biol. Chem. 1997, 272, 18636.
4. Farina, C.; Gagliardi, S. Curr. Pharm. Des. 2002, 8, 2033.
5. Bowman, E. M.; Siebers, A.; Altendorf, K. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 7972.
N-4-(3-Iodobenzoyl)-phenylmethylpiperidinamide of 3-
ethoxy-4-(5,6-dichlorobenzimidazol-2-yl)benzoic acid (27).
Prepared following the general procedure described
above, for example, for 25. Obtained in 25% yield after
chromatography
with
CH2Cl2–MeOH–NH4OH
80:15:1, mp 137–139 ꢁC, NMR d 1.65 (t, CH3), 1.75–
1.85 (CH2), 2.1–2.3 (CH2), 2.2–2.35 (CH2), 2.9–3.1
(CH2), 3.64 (s, ArylCH2N), 4.0–4.15 (m, CHNH), 4.46
(q, 2H, OCH2), 6.12 (d, NHCO), 7.2–8.0 (11H arom),
8.12 (s, 1H), 8.59 (d, 1H, J=8 Hz), 10.7 (1H, NH) m/z
754(55), 753(29), 752(62), 751(15), 403(50), 82(100).
Anal. calcd for C35H31Cl2IN4O3: C, 55.79; H, 4.15; N,
7.44. Found: C, 55.92; H, 4.27; N, 7.53.
6. Gagliardi, S.; Rees, M.; Farina, C. Curr. Med. Chem. 1999,
6, 1197.
7. Gagliardi, S.; Gatti, P. A.; Belfiore, P.; Zocchetti, A.;
Clarke, G. D.; Farina, C. J. Med. Chem. 1998, 41, 1883.
8. Gagliardi, S.; Nadler, G.; Consolandi, E.; Parini, C.; Mor-
van, M.; Legave, M.-N.; Belfiore, P.; Zocchetti, A.; Clarke,
G. D.; James, I.; Nambi, P.; Gowen, M.; Farina, C. J. Med.
Chem. 1998, 41, 1568.
N-4-(3-Tributylstannylbenzoyl)-phenylmethylpiperidina-
mide of 3-ethoxy-4-(5,6-dichlorobenzimidazol-2-yl)ben-
zoic acid (28). Compound 27 (20 mg, 0.026 mmol)
was dissolved in 0.3 mL of THF, triethylamine (0.5
mL, 0.052 mmol), hexabutylditin (0.02 mL, 0.04
mmol) and tetrakis(triphenylphosphine)Pd (6 mg,
0.005 mmol) were added, and the mixture refluxed 12
h. Evaporation and chromatography with CH2Cl2/
MeOH 100:2 gave 10 mg of 28, NMR d 0.7–0.9 (9H,
3 CH3), 0.9–1.65 (20H), 1.95–2.05 (CH2), 2.1–2.25
(CH2), 2.75–2.90 (CH2), 3.53 (s, ArylCH2N), 3.90–
4.05 (m, 1H, CHNH), 4.33 (q, 2H, OCH2), 6.06 (d,
NHCO), 7.15–7.80 (12H arom), 8.51 (d, 1H, J=8
Hz), 10.65 (s, 1H, NH). Anal. calcd for
C47H58Cl2N4O3Sn: C, 61.59; H, 6.38; N, 6.11. Found:
C, 61.82; H, 6.17; N, 6.44.
9. Nadler, G.; Morvan, M.; Delimoge, I.; Belfiore, P.; Zoc-
chetti, A.; James, I.; Zembryki, D.; Lee-Rycakzewski, E.;
Parini, C.; Consolandi, E.; Gagliardi, S.; Farina, C. Bioorg.
Med. Chem. Lett. 1998, 8, 3621.
10. Visentin, L.; Dodds, R. A.; Valente, M.; Misiano, P.;
Bradbeer, J. N.; Oneta, S.; Liang, X.; Gowen, M.; Farina, C.
J. Clin. Invest. 2000, 106, 309.
11. Li, Y.-P.; Chen, W.; Liang, Y.; Li, E.; Stashenko, P. Nat.
Gen. 1999, 23, 447.
12. Mattsson, J. P.; Li, X.; Peng, S.-B.; Nilsson, F.; Andersen,
P.; Lundberg, L. G.; Stone, D. K.; Keeling, D. J. Eur. J. Bio-
chem. 2000, 267, 4115.
13. Drose, S.; Boddien, C.; Gassel, M.; Ingerhorst, G.; Zeeck,
A.; Altendorf, K. Biochemistry 2001, 40, 2816.
14. Huss, M.; Ingenhorst, G.; Konig, S.; Gassel, M.; Drose,
S.; Zeeck, A.; Altendorf, K.; Wieczorek, H. J. Biol. Chem.
2002, 277, 40544.
´
15. Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000,
18, 64.
Synthesis of 27 from 28. To a solution of 28 (10 mg,
0.011 mmol) in 10 mL of EtOH were added 1.1 mg of
NaI in 1.1 mL of 0.01 M NaOH, then HCl 0.05 N to pH
4–5, then chloramine-T (6.2 mg, 0.022 mmol) in 6.2 mL of
H2O. After 15 min, sodium metabisulfite (125 mg in 12.5
mL of H2O) was added, then, after 10 min, 30 mL of satd
aq NaHCO3, and the solution extracted with CH2Cl2.
Drying, evaporation and taking up with ether gave 8 mg
(75%) of 27. Anal. calcd for C35H31Cl2IN4O3: C, 55.79; H,
4.15; N, 7.44. Found: C, 55.97; H, 4.09; N, 7.33.
16. Fleming, S. A. Tetrahedron 1995, 51, 12479.
17. Weber, P. J. A.; Beck-Sickinger, A. G. J. Pept. Res. 1997,
49, 375.
18. Stewart, R.; Van der Linden, R. Can. J. Chem. 1960, 38,
399.
19. Nassal, M. Liebigs Ann. Chem. 1983, 1510.
20. Brunner, J.; Semenza, G. Biochemistry 1981, 20, 7174.
21. Darrow, J. W.; Hadac, E. M.; Miller, L. J.; Sugg, E. E.
Bioorg. Med. Chem. Lett. 1998, 8, 3127.
22. Farina, C.; Gagliardi, S.; Nadler, G.; Morvan, M.; Parini,
C.; Belfiore, P.; Visentin, L.; Gowen, M. Il Farmaco 2001, 56,
113.
23. John, C. S.; Lim, B. B.; Vilner, B. J.; Geyer, B. C.; Bowen,
W. D. J. Med. Chem. 1998, 41, 2445.
24. Boyd, M. R.; Farina, C.; Belfiore, P.; Gagliardi, S.;
Kim, J. W.; Hayakawa, Y.; Beutler, J. A.; Mckee, T. C.;
Bowman, B.; Bowman, E. J. J. Pharmacol. Exp. Ther.
2001, 297, 114.
ATPase inhibition activity. ATPase inhibition data were
obtained using chicken osteoclast membrane prepara-
tion or human osteoclastic tumor cell membrane pre-
paration following the procedure described in refs 7 or
24, respectively. All the data are the average of two
independent measurements.