5-(5,6-Dichloro-2-indolyl)-2-methoxy-2,4-pentadienamides: Novel and selective inhibitors of the vacuolar H+-ATPase of osteoclasts with bone antiresorptive activity

Article abstract of DOI:10.1021/jm9800144

The vacuolar H⁺-ATPase (V-ATPase), located on the ruffled border of the osteoclast, is a proton pump which is responsible for secreting the massive amounts of protons that are required for the bone resorption process. With the aim to identify n

Full text of DOI:10.1021/jm9800144

1568
J . Med. Chem. 1998, 41, 1568-1573
5-(5,6-Dich lor o-2-in d olyl)-2-m eth oxy-2,4-p en ta d ien a m id es: Novel a n d Selective
In h ibitor s of th e Va cu ola r H+-ATP a se of Osteocla sts w ith Bon e An tir esor p tive
Activity
Stefania Gagliardi,§ Guy Nadler,† Emanuela Consolandi,§ Carlo Parini,§ Marcel Morvan,† Marie-No¨elle Legave,†
Pietro Belfiore,‡ Andrea Zocchetti,‡ Geoffrey D. Clarke,‡ Ian J ames, Ponnal Nambi,| Maxine Gowen, and
Carlo Farina*,§
Departments of Chemistry and Biology, SmithKline Beecham S.p.A., Via Zambeletti, 20021 Baranzate, Milano, Italy,
Department of Medicinal Chemistry, SmithKline Beecham Research Unit, B.P. 58, 35762 St-Gregoire Cedex, France, and
Departments of Bone and Cartilage Biology and Renal Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland
Road, King of Prussia, Pennsylvania 19406-0939
Received J anuary 9, 1998
The vacuolar H+-ATPase (V-ATPase), located on the ruffled border of the osteoclast, is a proton
pump which is responsible for secreting the massive amounts of protons that are required for
the bone resorption process. With the aim to identify new agents which are able to prevent
the excessive bone resorption associated with osteoporosis, we have designed a novel class of
potent and selective inhibitors of the osteoclast proton pump, starting from the structure of
the specific V-ATPase inhibitor bafilomycin A1. Compounds 3a -d potently inhibited the
V-ATPase in chicken osteoclast membranes (IC50 ) 60-180 nM) and were able to prevent bone
resorption by human osteoclasts in vitro at low-nanomolar concentrations. Notably, the EC50
of compound 3c in this assay was 45-fold lower than the concentration required for half-maximal
inhibition of the V-ATPase from human kidney cortex. These results support the validity of
the osteoclast proton pump as a useful molecular target to produce novel inhibitors of bone
resorption, potentially useful as antiosteporotic agents.
Postmenopausal osteoporosis, the most common and
widespread metabolic bone disease, is characterized by
a net loss of bone mass which results from an imbalance
of the bone-remodeling process, with bone resorption
exceeding bone formation: bone resorption is a multi-
step process which ultimately requires secretion of
hydrochloric acid by osteoclasts.1 The lowering of pH
in the sealed microcompartment which underlies osteo-
clasts at their site of attachment to the bone surface is
necessary both to dissolve the bone mineral and to
provide the acidic environment required by collagenases
to degrade the bone matrix.2 Protons are extruded by
vacuolar-type H+-ATPases3 (V-ATPases) that are present
in large numbers on the ruffled border of the osteoclast,
while the chloride counterions diffuse passively through
a chloride channel.4
ATPases. This leads to systemic alteration of cellular
physiology and, ultimately, to death.
Proton-translocating V-ATPases9 are located in most
intracellular organelles of both constitutive and special-
ized secretory pathways of eukaryotic cells and play an
important role in membrane trafficking and protein
sorting as well as in protein degradation.10-12 In
kidney, plasma membrane V-ATPases participate in
urinary acidification.13
It is still controversial whether the osteoclast plasma
membrane possesses a unique proton pump isoform:
sequence differences between the chicken osteoclast
enzyme and V-ATPases in other cell types have been
reported,14-16 as well as a peculiar sensitivity of chicken
osteoclast V-ATPase to vanadate and nitrate.17 The
latter findings, however, have not been confirmed by
other investigators using either chicken18 or mam-
malian19,20 osteoclasts.
The osteoclast V-ATPase is, therefore, a novel poten-
tial target for reducing osteoclast activity. The mac-
rolide antibiotic bafilomycin A1 is a very potent and
specific inhibitor of V-ATPases5 which does not affect
significantly other important ATPases such as the H+/
K+-ATPase6 found in gastric parietal cells and which is
responsible for maintaining gastric acidity. Indeed,
bafilomycin A1 is able to inhibit bone resorption both
in vitro7 and in vivo;8 however, since it is not selective
for any particular type of V-ATPases, its administration
to animals causes inhibition of all the essential V-
To verify if different types of V-ATPases can be
distinguished pharmacologically, we have performed a
series of chemical modifications of the specific V-ATPase
inhibitor bafilomycin A1. As reported in a previous
paper,21 some key features required for biological activ-
ity have been identified, and these include the vinylic
methoxy at position 2, the dienic systems, a free hydroxy
group at position 7, and a portion of the side chain. Very
interestingly, a slight differential effect of some bafilo-
mycin derivatives on V-ATPase-driven proton transport
in membrane vesicles from chicken osteoclasts and
bovine chromaffin granules was observed suggesting
that selective modulation of different V-ATPases may
be possible.
§ Department of Chemistry.
‡ Department of Biology.
† Department of Medicinal Chemistry.
Department of Bone and Cartilage Biology.
| Department of Renal Pharmacology.
S0022-2623(98)00014-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/21/1998

Inhibitors of the V-ATPase of Osteoclasts
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1569
Ta ble 1. ATPase Inhibition of the Vacuolar ATPase from
Different Enzyme Sources and in Vitro Inhibition of Bone
Resorption by Isolated Human Osteoclastoma Cells
Ch a r t 1
Encouraged by these preliminary results, we have
continued our search for new analogues endowed with
a higher degree of selectivity for the osteoclast enzyme.
The inhibition of bafilomycin-sensitive ATPase activity
was measured in membrane vesicle preparations from
chicken osteoclasts (cOc) from the medullary bone of
calcium-starved egg-laying hens.22 Our strategy for
identifying selective inhibitors entailed the comparison
of the potency of the new derivatives in cOc against their
potency on V-ATPases from different origins. For this
purpose the enzyme from chicken adrenal glands (cAG)23
was used since this V-ATPase is philogenetically related
to the cOc proton pump. Potency in human tissues was
preliminarily assessed using membranes from human
kidney cortex (hK) since a high concentration of proton
secretory V-ATPases is present in the renal intercalated
cells where they regulate urinary acidification and
bicarbonate resorption.24
Since chemical modifications of bafilomycin are lim-
ited by its high complexity and low chemical stability,
a novel series of simplified derivatives was designed
which contained the essential features for V-ATPase-
inhibitory activity of bafilomycin, i.e., a vinylic methoxy
group at position 2 of the 5-(2-indolyl)-2,4-pentadienoyl
system in which the indole NH can possibly mimic the
necessary hydroxy group at position 7 of bafilomycin A1.
Thus, as indicated in Chart 1, methyl (2Z,4E)-5-(2-
indolyl)-2-methoxy-2,4-pentadienoate (1) was prepared
which was a moderate inhibitor of the chicken osteoclast
V-ATPase (IC50 ) 30 µM). Optimization of the aromatic
substitution led to the identification of lipophilic and
electron-withdrawing substituents at positions 5 and 6
of the indole ring as necessary requirements for high
potency. Conversely, introduction of any type of sub-
stituent at other positions of the indole ring, i.e., 1, 3,
4, or 7, was deleterious for activity. Thus, the 5,6-
dichloro derivative 2 showed a 15-fold increase in
potency, with an IC50 of 1.9 µM in the cOc ATPase assay.
A further increase of potency could be obtained by
converting the carbomethoxy group of 2 into the corre-
sponding carboxamides bearing a strongly basic nitro-
gen group separated by a three-carbon atom spacer from
the amidic nitrogen.
distance and the relative orientation of the two nitrogen
atoms were fixed, were even more potent (80 and 100
nM, respectively) with a similar degree of selectivity.
The 2-pyrimidylpiperazine derivative 3d , maintaining
the linear amidic chain of 3a but with a lipophilic basic
head, was the most potent compound in the V-ATPase
assay with an IC50 of 60 nM (Table 1).
The synthesis for the preparation of compounds 3a -d
is described in Scheme 1. Briefly, nitration of 3,4-
dichlorotoluene25 followed by reaction with diethyl
oxalate in the presence of potassium ethoxide and
subsequent reductive cyclization gave ethyl 5,6-di-
chlorindole-2-carboxylate (7). This intermediate was
reduced with LiAlH4, oxidized with MnO2, and homolo-
gated via a Horner-Emmons reaction to give ethyl (E)-
3-(5,6-dichloro-2-indolyl)-2-propenoate (9). This com-
pound was reduced with DIBAH and oxidized with
MnO2 to afford (E)-3-(5,6-dichloro-2-indolyl)-2-prope-
naldehyde (10). Wittig reaction of 10 with the phos-
phonium salt 1126 gave the dichloroindole ester 2 that
was subsequently hydrolyzed and condensed with the
appropriate amine affording derivatives 3a -d .
The bone antiresorptive activity of these novel inhibi-
tors was functionally evaluated in vitro using isolated
human osteoclasts obtained from giant cell tumors of
bone.27 Both the number of excavations (pits/bone slice)
and the generation of type I collagen telopeptides
(ELISA)28 produced after 48-h incubation were mea-
sured. As shown in Table 1, compounds 3a -d were
potent inhibitors of bone resorption by human osteo-
clasts with potencies that were comparable to their
ability to inhibit chicken osteoclast V-ATPase, although
all compounds were slightly more potent in the human
system. In the ELISA assay, the most potent com-
pounds were 3d ,c, with IC50’s of 10 and 30 nM,
respectively, and a selectivity of about 40-fold if com-
pared with the potency data from human kidney V-
ATPase assay. Selectivity is only indicative since
potency on different human enzymes should be com-
pared in the same assay. Unfortunately, however, it
has not yet been possible to produce a viable preparation
Initially, (2Z,4E)-5-(5,6-dichloro-2-indolyl)-N-[3-(di-
ethylamino)propyl]-2-methoxy-2,4-pentadienamide (3a )
was obtained which displayed a submicromolar potency
with an IC50 of 180 nM in cOc and a 15-fold selectivity
against the vacuolar enzyme in chicken adrenal glands.
Conformationally constrained analogues 3b,c, where the

1570 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Gagliardi et al.
Sch em e 1. Preparation of (2Z,4E)-5-(5,6-Dichloroindol-2-yl)-2-methoxypentadienamides 3a -d a
a
Reagents: (a) nitric acid/sulfuric acid (95%); (b) diethyl oxalate, potassium ethoxide (59%); (c) Fe/AcOH reduction (92%); (d) LiAlH4
in THF (80%); (e) MnO2 in Et2O (85%); (f) triethylphosphonoacetate, NaH in THF (89%); (g) DIBAH (100%); (h) MnO2 in EtOAc (92%);
(i) DBU in THF (89%); (l) NaOH (aq.) in MeOH/THF (97%); (m) R-NH2, HOBt/N′-[3-(dimethylamino)propyl]-N-ethylcarbodiimide
hydrochloride (56-80%).
brane proteins (about 70 µg of protein/mL) were used in the
of human osteoclast membrane vesicles containing a
bafilomycin-sensitive ATPase of suitable purity.
In conclusion a novel class of compounds endowed
with potent and selective osteoclast V-ATPase inhibitory
activity has been identified and characterized in in vitro
models of bone resorption. The results obtained clearly
support the validity of the proton pump located on the
ruffled border of the osteoclast as a useful molecular
target to produce novel inhibitors of bone resorption
which have potential as antiosteoporotic agents.
A more detailed description of our investigations
within this novel chemical class will be reported in
subsequent manuscripts, along with our studies aimed
at further optimizing the potency, therapeutic efficacy,
and selectivity of these new osteoclast V-ATPase inhibi-
tors.
ATPase assay.
Ba filom ycin -Sen sitive ATP a se Assa y. The reaction
medium contained 0.2 M sucrose, 50 mM KCl, 10 mM Hepes-
Tris, pH 7.4 (cAG and hKc) or pH 8 (cOc), 1 mM CDTA, 1 mM
ATP, 0.1 mM ammonium molybdate, 5 µM valinomycin, and
5 µM nigericin. The reaction was started by addition of 5 mM
MgSO4. After 30 min of incubation at 37 °C, the release of
phosphate was measured using a colorimetric assay (malachite
green) as described in the literature.29
P r ep a r a tion of Hu m a n Osteocla sts. Tissue from human
osteoclastoma tumor was used for the preparation of osteo-
clasts.27 The tissue was chopped into small pieces and placed
into a sterile 50-mL centrifuge tube. The pieces were disag-
gregated by incubation at 37 °C for 1 h in serum-free RPMI-
1640 medium, supplemented with 3 mg/mL (w/v) type I
collagenase. A cell suspension was obtained by gentle homog-
enization of the remaining tissue with a plunger from a 20-
mL syringe. The cell suspension was washed twice in medium
supplemented with 10% fetal calf serum (FCS). The cells were
frozen in cryotubes at a concentration of 5 million/mL in liquid
N2.
Exp er im en ta l Section
Bioch em istr y. P r ep a r a tion of Ch ick en Osteocla st
Mem br a n es (cOc). Bone microsomes were isolated from
medullary bones of calcium-starved egg-laying hens as de-
scribed previously.22 The crude preparation was collected as
100 000gmax fraction and resuspended in 10 mM Hepes buffer,
pH 7.4, 0.2 M sucrose, 50 mM KCl, 1 mM EGTA, and 2 mM
DTT. This fraction was further purified using a three-step
Hu m a n Osteocla st Bon e Resor p tion Assa y (HOcA).
The osteoclasts were negatively selected using Dynabeads Pan
Human HLAII (supplied by Dynal) and settled on bone slices
placed in a 96-well multiplate (4000 cells/well). After 30 min
the bone slices were transferred into a 24-well multiplate (4
bone slices/well). After 1 h, different concentrations of the test
compound were added (8 bone slices/each concentration). The
bone slices were incubated for 48 h in D-MEM culture medium
and then fixed by using 2% glutaraldehyde in 0.2 M cacodylate
buffer (15 min at 4 °C). After the buffer was removed and
the sample washed three times with distilled water, TRAP
(tartrate-resistant acid phosphatase) staining solution30 was
added and incubated for 60 min at 37 °C. The bone slices were
rinsed twice with distilled water, dried, and mounted on a
glass slide using bio-adhesive tape. After the number of
osteoclasts was read, the fixed-stained cells were removed by
5-min incubation in 5% bleach. The slices were washed, dried,
and sputter-coated with gold. Cellular activity was quanti-
tated by counting the number of pits formed during the
resorption using a light microscope, and the results were
expressed as number of pits/bone slice. The osteoclast activity
was further evaluated using the experimental procedure
15%, 30%, 45% (w/w) sucrose gradient spun at 280 000gmax
.
An amount of membrane proteins (20-40 µg/mL) was used in
the ATPase assay.
P r ep a r a t ion of Mem b r a n es fr om Ch ick en Ad r en a l
Gla n d s (cAG). The whole adrenal glands were used. Crude
microsomal membranes collected as 12 000gmax fraction were
resuspended in 0.3 M sucrose, 10 mM Hepes-Tris, 5 mM
EGTA, 1 mM DTT, 1 mM ATPNa2, 2 µg/mL pepstatin A, and
2 µg/mL leupeptin buffered at pH 7.5. This fraction was
further purified by a two-step 35%, 50% (w/w) sucrose gradient
and processed as previously described.23 An amount of mem-
brane proteins (20-40 µg of protein/mL) was used in the
ATPase assay.
P r ep a r a tion of Mem br a n es fr om Hu m a n Kid n ey Cor -
tex (h Kc). Brush border membranes were obtained from the
cortex of human kidney frozen immediately after surgery,
according to the method reported for bovine kidney.24 Mem-

Inhibitors of the V-ATPase of Osteoclasts
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1571
give 5,6-dichloroindole-2-carboxaldehyde (8) (84 g, 85%), mp
described in the literature27 utilizing in vitro ELISA kit28 for
quantification of type I collagen fragments released into the
bone cell culture supernatants during bone resorption.
205-208 °C. IR (Nujol): 3250, 1660 cm-1 1H NMR (DMSO-
.
d6): δ 12.26 (s, 1H); 9.89 (s, 1H); 8.09 (s, 1H); 7.66 (s, 1H);
7.41 (s, 1H). MS (ESI NEG): 212. Anal. (C9H5NOCl2) C, H,
N, Cl.
Ch em istr y. Melting points were determined with a Bu¨chi
530 hot stage apparatus and are uncorrected. Proton NMR
spectra were recorded on a Bruker ARX 300 spectrometer at
303 K unless otherwise indicated. Chemical shifts were
recorded in parts per million (δ units) downfield from tetram-
ethylsilane (TMS). IR spectra were recorded in Nujol mull or
in KBr with a Perkin-Elmer 1420 spectrometer. Mass spectra
were obtained on a Finnigan MAT TSQ-700 spectrometer.
Negative or positive electrospray (ESI NEG or ESI POS,
respectively) was recorded with the following conditions:
solvent methanol, spray 4.5 kV, skimmer (60 V, capillary
temperature 220 °C. Experimental conditions for electronic
impact (EI) were source 180 °C, 70 eV, 200 µA. Silica gel used
for flash column chromatography was Kiesegel 60 (230-400
mesh; E. Merk AG, Darmstadt, Germany). Solvent evapora-
tion was performed at reduced pressure, and oily products
were dried at 0.1 mbar for 16 h. 3,4-Dichlorotoluene (4) was
supplied by Acros and Florisil by Fluka. Activated manganese-
(IV) oxide (MnO2; <5 µm, ca. 85%) was supplied by Aldrich
Chemical and used without further activation. Data of the
combustion elemental analyses were within 0.4% of theoretical
values.
Eth yl (E)-3-(5,6-Dich lor o-2-in d olyl)-2-p r op en oa te (9).
To a solution of triethylphosphonoacetate (32.9 g, 147 mmol)
in THF (150 mL) was added a 60% oil dispersion of NaH (5.95
g, 149 mmol) portionwise in 30 min under nitrogen maintain-
ing the temperature between 0 and 5 °C. A solution of 5,6-
dichloroindole-2-carboxaldehyde (8) (29 g, 135.5 mmol) in THF
(200 mL) was added dropwise maintaining the internal tem-
perature at about 20 °C. The solvent was evaporated under
reduced pressure, and the residue was treated with H2O (200
mL) and EtOAc (500 mL). The organic layer was washed with
brine, dried over Na2SO4, and evaporated to dryness. The
residue was triturated with hexane, filtered, and dried under
vacuum to afford ethyl (E)-3-(5,6-dichloro-2-indolyl)propenoate
(9) (34.5 g, 89.6%) as a light-brown powder, mp 188-190 °C.
IR (Nujol): 3300, 1690, 1610 cm-1
.
1H NMR (DMSO-d6): δ
11.89 (s, 1H); 7.76 (d, 1H); 7.60 (s, 1H); 6.92 (s, 1H); 6.59 (d,
1H); 4.20 (q, 2H); 1.27 (t, 3H). MS (EI): 283 (M+), 237, 211,
174. Anal. (C13H11NO2Cl2) C, H, N, Cl.
(E)-3-(5,6-Dich lor o-2-in d olyl)-2-p r op en a ld eh yd e (10).
To a solution of ethyl (E)-3-(5,6-dichloro-2-indolyl)-2-prope-
noate (9) (34.5 g, 121 mmol) in dry THF (500 mL) was added
DIBAH (1 M solution in hexane, 242 mL, 2.4 mequiv) dropwise
under nitrogen maintaining the temperature below -20 °C.
Stirring was continued for 1 h; then the reaction was quenched
with 10% H2SO4 (400 mL) and a saturated solution of potas-
sium sodium tartrate tetrahydrate (400 mL). The organic
layer was separated, and the aqueous phase was extracted
with Et2O (200 mL). The combined organic phases were dried
over MgSO4, filtered, and evaporated to dryness to yield 29 g
of (E)-3-(5,6-dichloro-2-indolyl)-2-propen-1-ol (100%). 1H NMR
(DMSO-d6): δ 11.51 (s, 1H); 7.70 (s, 1H); 7.48 (s, 1H); 6.60 (d,
1H); 6.45 (m, 1H); 6.41 (s, 1H); 4.98 (t, 1H); 4.15 (t, 2H). Crude
(E)-3-(5,6-dichloro-2-indolyl)-2-propen-1-ol (29 g, 119.8 mmol)
was suspended in EtOAc (800 mL), and activated MnO2 (50
g, 574 mmol) was added. The mixture was stirred at room
temperature for 2 days and then filtered on a Celite pad,
washing with EtOAc (400 mL). The combined organic phases
were evaporated to give (E)-3-(5,6-dichloro-2-indolyl)-2-prope-
naldehyde (10) (26.7 g, 92.8%) as a brown powder, mp 216-
Eth yl 2-Oxo-3-(2-n itr o-4,5-d ich lor op h en yl)p r op a n oa te
(6). To a suspension of potassium (24.5 g, 0.63 ga) in
anhydrous Et2O (245 mL) was added a solution of EtOH (158
mL) and anhydrous Et2O (126 mL) dropwise under nitrogen
during 4 h. The resulting solution was diluted with Et2O (600
mL), and then diethyl oxalate (85.5 mL, 0.63 mol) was added
dropwise in about 30 min. A solution of 525 (130 g, 0.63 mol)
dissolved in anhydrous Et2O (225 mL) was added dropwise to
the resulting yellow mixture in 1 h. Stirring was continued
for an additional 3 h, and the dark-brown mixture was allowed
to settle at room temperature for 2 days. The potassium salt
was filtered, washed with anhydrous Et2O (200 mL), and dried
to give 210 g of a dark-brown powder. The solid was
suspended in a mixture of water (200 mL) and EtOAc (400
mL) and then acidified with 10% HCl. The organic layer was
washed with a saturated solution of NaHCO3 and brine, dried
over Na2SO4, filtered, and evaporated to give ethyl 2-oxo-3-
(2-nitro-4,5-dichlorophenyl)propanoate (6) (115.1 g, 59.7%) as
a light-brown solid, mp 104-107 °C. IR (Nujol): 3400, 1750,
1710 cm-1
.
1H NMR (CDCl3): δ 8.41 (s, 1H); 8.32 (s, 1H); 6.89
218 °C. IR (Nujol): 3210, 1685, 1620-1610 cm-1 1H NMR
.
(DMSO-d6): δ 11.95 (s br, 1H); 9.65 (d, 1H); 7.89 (s, 1H); 7.71
(d, 1H); 7.61 (s, 1H); 7.00 (s, 1H); 6.78 (dd, 1H). MS (ESI
NEG): 238 (M - H-), 210. Anal. (C11H7NOCl2) C, H, N, Cl.
(s, 2H); 4.41 (q, 2H); 1.41 (t, 3H). MS (ESI NEG): 304 (M -
H). Anal. (C11H9NO5Cl2) C, H, N, Cl.
Eth yl 5,6-Dich lor oin d ole-2-ca r boxyla te (7). A mixture
of ethyl 2-oxo-3-(2-nitro-4,5-dichlorophenyl)propanoate (6) (20
g, 65 mmol) and iron powder (32 g, 0.57 ga) in EtOH (125 mL)
and glacial acetic acid (125 mL) was refluxed for 2 h. After
cooling, the resulting mixture was evaporated under vacuum.
THF (200 mL) was added to the solid residue, and the
suspension was filtered on Florisil (100 g) eluting with a large
amount of THF (1000 mL). The solvent was evaporated
obtaining ethyl 5,6-dichloroindole-2-carboxylate (7) (15.5 g,
92%) as a light-brown powder, mp 216-218 °C. IR (Nujol):
Meth yl 2-Meth oxy-2-(tr ip h en ylp h osp h on iu m )a ceta te
Br om id e (11). A mixture of methyl methoxyacetate (88.2 g,
847 mmol), N-bromosuccinimide (151 g, 847 mmol), benzoyl
peroxide (375 mg), and 420 mL of CCl4 was refluxed overnight
(WARNING: the reaction mixture must be warmed slowly and
carefully because the reaction is very vigorous). The mixture
was filtered and the solvent concentrated to dryness. The
residue was distilled under vacuum (40-45 °C/0.1 mbar) to
afford 134 g (86.4%) of methyl 2-bromo-2-methoxyacetate,
which was dissolved in 1 L of Et2O, added dropwise to a
solution of triphenylphosphine (192 g, 732 mmol) in Et2O (500
mL), and stirred at room temperature overnight. The pre-
cipitate was collected by filtration, washed with Et2O, and
dried in vacuo to give methyl 2-methoxy-2-(triphenylphospho-
nium)acetate bromide (11) (289 g, 88.6%) as a white powder,
3310, 1695 cm-1
.
1H NMR (CDCl3): δ 9.01 (s, 1H); 7.78 (s,
1H); 7.14 (s, 1H); 4.43 (q, 2H); 1.43 (t, 3H). MS (ESI POS):
258 (MH+). Anal. (C11H9NO2Cl2) C, H, N, Cl.
5,6-Dich lor oin d ole-2-ca r boxa ld eh yd e (8). To an ice-cold
1 M solution of LiAlH4 in anhydrous THF (1.6 L, 1 M) under
Ar was added ethyl 5,6-dichloroindole-2-carboxylate (7) (150
g, 581 mmol), dissolved in anhydrous THF (1 L), dropwise
under stirring, keeping the temperature below 5 °C. After
stirring for 2 h at room temperature, the reaction was
quenched by successive addition of water (75 mL), 15% NaOH
(75 mL), and water (150 mL). The organic phase was dried
over MgSO4, filtered, and concentrated under vacuum afford-
ing (5,6-dichloro-2-indolyl)methanol (100 g, 80%) which was
suspended in 3 L of Et2O, treated with MnO2 (200 g, 2.3 mol),
and stirred at room temperature for 2 days. The suspension
was filtered on a Celite pad washing with Et2O and warm
acetone. The combined organic phases were concentrated to
mp 143-145 °C. IR (Nujol): 2740, 2620, 1720 cm-1 1H NMR
.
(DMSO-d6): δ 7.95 (m, 3H); 7.85-7.75 (m, 12H); 6.90 (d, 1H);
3.62 (s, 3H); 3.59 (s, 3H). MS (ESI POS): 365 (M+), 277, 262,
103. Anal. (C22H22BrO3P) C, H, Br, P.
Meth yl (2Z,4E)-5-(5,6-Dich lor o-2-in d olyl)-2-m eth oxy-
2,4-p en ta d ien oa te (2). To a solution of (E)-3-(5,6-dichloro-
2-indolyl)-2-propenaldehyde (10) (11.5 g, 47.9 mmol) dissolved
in 200 mL of THF were added methyl 2-methoxy-2-(tri-
phenylphosphonium)acetate bromide (11) (23.5 g, 52.7 mmol)
and DBU (7.84 mL, 1.1 equiv). The mixture was refluxed for
1.5 h. The solvent was removed under reduced pressure, and

1572 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Gagliardi et al.
the crude compound was purified by flash chromatography
(hexane/EtOAc, 7/3). After trituration with iPr2O, methyl
(2Z,4E )-5-(5,6-dich lor o-2-in dolyl)-2-m et h oxy-2,4-pen t a -
dienoate (2) (14 g, 89.6%) was obtained as a yellow powder,
IR (Nujol): 3358, 3298, 1664, 1620 cm-1
.
1H NMR (DMSO-
d6): δ 11.70 (s br, 1H); 7.82 (d, 1H); 7.73 (s, 1H); 7.51 (s, 1H);
7.18 (dd, 1H); 6.82 (d, 1H); 6.58 (d, 1H); 6.58 (s, 1H); 4.23-
4.10 (m, 1H); 3.70 (s, 3H); 1.60 (dd, 2H); 1.18 (dd, 2H); 1.17 (s,
mp 202-204 °C. IR (Nujol): 3400, 1700, 1610 cm-1
.
1H NMR
6H); 1.00 (s, 6H). MS (ESI POS): 450 (MH+). Anal. (C14H11
NO3Cl2) C, H, N, Cl.
-
(DMSO-d6): δ 11.82 (s, 1H); 7.77 (s, 1H); 7.52 (s, 1H); 7.21
(dd, 1H); 7.00 (d, 1H); 6.89 (d, 1H); 6.62 (s, 1H); 3.76 (s, 1H);
(2Z,4E)-exo-5-(5,6-Dich lor o-2-in d olyl)-2-m eth oxy-N-[8-
m et h yl-8-a za b icyclo[3.2.2]oct -3-yl]-2,4-p en t a d ien a m id e
(3c): prepared according to the general procedure by using
3-amino-8-methyl-8-azabicyclo[3.2.1]octane,31 to give a yellow
powder that was converted into the hydrochloride salt (56%),
mp >250 °C. IR (Nujol): 3610, 3404, 3296, 3150, 2722, 1658,
3.73 (s, 1H). MS (ESI NEG): 324 (M - H). Anal. (C15H13
NO3Cl2) C, H, N, Cl.
-
(2Z,4E)-5-(5,6-Dich lor o-2-in d olyl)-2-m eth oxy-2,4-p en ta -
d ien oic Acid . A suspension of methyl (2Z,4E)-5-(5,6-dichloro-
2-indolyl)-2-methoxy-2,4-pentadienoate (2) (7.5 g, 23 mmol)
and 20% NaOH (10 mL, 50 mmol) in MeOH (20 mL) and THF
(40 mL) was heated at 50 °C for 1 h. After cooling to room
temperature, the organic solvent was evaporated. The residue
was acidified with 20% HCl, and the precipitate was collected
by filtration, washed with water, and dried at 60 °C under
vacuum to obtain (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-
2,4-pentadienoic acid (7 g, 97.5%) as a yellow powder, mp 249-
1612 cm-1 1H NMR (DMSO-d6): δ 11.80 (s, 1H); 9.88 (s br,
.
1H); 8.12 (s br, 1H); 7.75 (s, 1H); 7.51 (s, 1H); 7.19 (dd, 1H);
6.86 (d, 1H); 6.61 (d br, 1H); 6.58 (s, 1H); 4.20-4.05 (m, 1H);
3.90-3.80 (m, 2H); 3.71 (s, 3H); 2.70-2.60 (m, 3H); 2.29-2.11
(m, 2H); 2.05-190 (m, 6H). MS (EI): 433 (M+), 402, 262, 235,
188, 124, 96, 82. Anal. (C14H11NO3Cl2‚HCl) C, H, N, Cl.
(2Z,4E)-5-(5,6-Dich lor o-2-in d olyl)-2-m eth oxy-N-[3-[4-(2-
p y r im id in y l)p ip e r a zin -1-y l]p r o p y l]-2,4-p e n t a d ie n -
a m id e (3d ): was prepared according to the general procedure
by using the 3-[4-(2-pyrimidinyl)piperazin-1-yl]propylamine to
give a yellow powder (75%), mp 227-228 °C. IR (Nujol): 3380,
250 °C. IR (Nujol): 3390, 1675, 1610 cm-1 1H NMR (DMSO-
.
d6): δ 11.80 (s, 1H); 7.76 (s, 1H); 7.52 (s, 1H); 7.19 (dd, 1H);
6.95 (s, 1H); 6.84 (d, 1H); 6.60 (s, 1H); 3.75 (s, 3H); 3.37 (s,
1H). MS (ESI NEG): 310 (M - H). Anal. (C14H11NO3Cl2) C,
H, N, Cl.
3212, 1642, 1606 cm-1 1H NMR (DMSO-d6): δ 11.72 (s, 1H);
.
8.34 (d, 2H); 8.25 (t br, 1H); 7.73 (s, 1H); 7.50 (s, 1H); 7.18
(dd, 1H); 6.84 (d, 1H); 6.66 (d, 1H); 6.60 (dd, 1H); 6.58 (s, 1H);
3.72 (m, 7H); 3.22 (dt, 2H); 2.40 (m, 4H); 2.35 (t, 2H); 1.72-
3-[4-(2-P yr im idin yl)piper azin -1-yl]pr opylam in e. A mix-
ture of 4-(2-pyrimidinyl)piperazine (1.2 g, 7.3 mmol), CH3CN
(15 mL), K2CO3 (3 g), and 3-bromo-1-[(tert-butoxycarbonyl)-
amino]propane (1.9 g, 8 mmol) was refluxed for 4 h. After
cooling, the suspension was filtered, washed with CH3CN (10
mL), and evaporated at reduced pressure. The residue was
1.62 (m, 2H). MS (ESI POS): 515 (MH+). Anal. (C25H28
Cl2N6O2) C, H, N, Cl.
-
i
triturated with Pr2O (10 mL) and filtered to give 1.02 g (43%)
Ack n ow led gm en t. The authors gratefully acknowl-
edge A. Cerri and R. Mena for NMR and MS spectra
recording and interpretation and Professor L. Merlini
(University of Milan) for valuable discussions and
helpful advice.
of 3-[4-(2-pyrimidinyl)piperazin-1-yl]-1-[(tert-butoxycarbonyl)-
amino]propane as a white powder, mp 99-100 °C. IR (Nu-
jol): 3340, 1695 cm-1
. A solution of 3-[4-(2-pyrimidinyl)-
piperazin-1-yl]-1-[(tert-butoxycarbonyl)amino]propane (4 g, 12.4
mmol) in CH2Cl2 (20 mL) was treated dropwise with TFA (20
mL) at 10 °C. The mixture was allowed to reach room
temperature and stirred for 1 h. The solvent was removed at
reduced pressure, and the residue was dissolved in water (10
mL), treated with solid K2CO3, extracted with EtOAc (50 mL),
washed with brine (20 mL), and dried over Na2SO4. The
solvent was evaporated to dryness to give the 3-[4-(2-pyrim-
idinyl)piperazin-1-yl]propylamine (2.3 g, 83%) as an oil. IR
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(film): 3370, 1681 cm-1
.
1H NMR (CDCl3): δ 8.30 (d, 2H);
6.46 (dd, 1H); 3.81 (m, 4H); 2.86 (t, 2H); 2.70 (s br, 2H); 2.50
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(2Z,4E)-5-(5,6-Dich lor o-2-in d olyl)-N-[3-(d ieth yla m in o)-
p r op yl]-2-m eth oxy-2,4-p en ta d ien a m id e (3a ): prepared ac-
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.
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