Design, synthesis, and biological evaluation of hybrid molecules containing α-methylene-γ-butyrolactones and polypyrrole minor groove binders

Article abstract of DOI:10.1021/jm031104y

We have synthesized and evaluated a series of hybrids of polypyrrole minor groove binders structurally related to the natural antitumor agent distamycin A, and α-methylene-γ-butyrolactones with methyl, phenyl, and 4-substituted phenyl groups at the lacton

Full text of DOI:10.1021/jm031104y

J . Med. Chem. 2004, 47, 2877-2886
2877
Design , Syn th esis, a n d Biologica l Eva lu a tion of Hybr id Molecu les Con ta in in g
r-Meth ylen e-γ-bu tyr ola cton es a n d P olyp yr r ole Min or Gr oove Bin d er s
Pier Giovanni Baraldi,*,† Maria del Carmen Nunez,† Mojgan Aghazadeh Tabrizi,† Erik De Clercq,‡
J an Balzarini,‡ J aime Bermejo,§ Francisco Este´vez,# and Romeo Romagnoli†
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, 44100 Ferrara, Italy, Laboratory of Virology and
Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium, Instituto de Productos
Naturales y Agrobiolog´ıa del CSICsInstituto de Bioorga´nica “Antonio Gonza´lez”, Avenida Astrof´ısico Francisco Sa´nchez 2,
38206 La Laguna, Tenerife, Spain, and Departamento de Bioqu´ımica, Facultad de Medicina, Universidad de Las Palmas de
Gran Canaria, Avenida S. Cristo´bal, 35016 Las Palmas de Gran Canaria, Spain
Received November 17, 2003
We have synthesized and evaluated a series of hybrids of polypyrrole minor groove binders
structurally related to the natural antitumor agent distamycin A, and R-methylene-γ-
butyrolactones with methyl, phenyl, and 4-substituted phenyl groups at the lactone C(γ)
position, denoted 5-17, for in vitro cytotoxic activity against a variety of cancer cell lines. The
apoptotic and cytotoxic activities against several tumor cell lines are reported and discussed
in terms of their structural differences in relation to both the number of N-methylpyrrole rings
and the type of the alkylating unit tethered to the oligopeptidic frame. It may be noted that in
general, and especially for 11, 12, and 17, the cytotoxicity of the hybrids was much greater
than that of the R-methylene-γ-butyrolactone units 24a -g alone. Using the human leukemia
cell line HL-60, we have tested the effects of a selected series of compounds on programmed
cell death (apoptosis). The results clearly indicate that 11, 12, and 17, but not 9, are able to
induce apoptosis as demonstrated from (i) identification of nuclear changes associated with
apoptosis using fluorescence microscopy and (ii) by DNA laddering on agarose gel electro-
phoresis. Compound 12 was the most potent, especially after a short incubation period. It
induced extensive hydrolysis of poly ADP-ribose polymerase (PARP), considered to be a hallmark
of apoptosis, which plays a critical role in chromatin architecture and DNA metabolism.
In tr od u ction
tional structure in a wide range of natural products,11,12
particularly cytotoxic sesquiterpene lactones such as
methylenolactocin 2,13 protolichesterinic acid 3,14 and
vernolepin 4.15 It was soon determined that the struc-
tural requirement for the biological activities is mainly
associated with the exocyclic, conjugated double bond
(the OdCdCdCH2 moiety), which acts as an alkylating
agent in a Michael-type reaction with biological cellular
nucleophiles or sulfydryl-containing enzymes.16 Because
of the interesting biological activities and their unique
structural features, R-methylene-γ-butyrolactones present
an important scientific theme.17
Many natural and synthetic anticancer agents with
the ability to interact with DNA have been discovered,
but most have little sequence specificity and often
exhibit severe toxicity to normal tissues.1 For these
reasons, DNA minor groove binders have been an
attractive source of novel antitumor agents.2 The in-
creasing interest in this group of compounds stems from
their ability to interact in a sequence-selective fashion
at quite long DNA binding sites, suggesting the pos-
sibility of targeting specific DNA sequences within the
genome. Distamycin A 1 is a naturally occurring anti-
biotic characterized by the presence of an oligopeptidic
pyrrolecarbamoyl frame ending with an amidino moi-
ety,3,4 which binds to the DNA minor groove, preferen-
tially to the AT-rich sequence, and in a reversible
manner.5-7 Distamycin A 1 can be used as a DNA
sequence selective vector for both alkylating and non-
alkylating agents leading to a substantial increase in
cytotoxicity against cancer cell lines in comparison to
that of distamycin alone.8-10
R-Methylene-γ-butyrolactone derivatives have at-
tracted much attention over the years because the
R-methylene-γ-butyrolactone ring is an important func-
* To whom correspondence should be addressed. Phone: 39-(0)532-
291293. Fax: 39-(0)532-291296. E-mail: pgb@ifeuniv.unife.it.
† Universita` di Ferrara.
‡ Rega Institute for Medical Research.
§ Instituto de Productos Naturales.
#
Universidad de Las Palmas de Gran Canaria.
10.1021/jm031104y CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/09/2004

2878 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Baraldi et al.
Here, we report on the preparation and biological
evaluation of a novel series of conjugates, 5-17, that
have two moieties in their structure. One is a distamy-
cin-like moiety, to acquire DNA minor groove binding
activity, which is joined by a pyrazole, which acts as a
rigid linker, to the R-methylidene-γ-butyrolactone resi-
dues 24a -g to acquire alkylation with biological nu-
cleophiles. In fact, via the hydroxy and carbonyl func-
tions present in the 3- and 5-position, respectively, on
the pyrazolic moiety, it was possible to join the R-me-
thylidene-γ-butyrolactone residues 24a -g and the ami-
nopolypyrrolic amides 25-28.
We have synthesized three different series of com-
pounds. In the first series of hybrids 5, 9, and 10, an
R-methylidene-γ-methyl-γ-butyrolactone residue joined
by a methylene residue to a pyrazole moiety (derivative
24a ) was linked to an oligopyrrole consisting of a
varying number of pyrrole amide units (from one to
three). For the derivative characterized by the presence
of deformyl distamycin A as the oligopyrrole carrier
(compound 5), the effect of the replacement of the
C-terminal amidino moiety by a more basic and ioniz-
able guanidino group (derivative 16) was also evaluated.
In the second series of derivatives 6-8, we have
evaluated the effects in terms of cytotoxic activity by
varying the length of the methylene chain, from three
to seven methylene units, between the R-methylidene-
γ-methyl-γ-butyrolactone residue and the pyrazole moi-
ety (compounds 24b-d , respectively).
In an attempt to better understand the effect of
structural modifications on optimal cytotoxic activity,
we have finally synthesized analogues of 24a , in which
the C(γ)-methyl of the lactone ring was replaced by a
more lipophilic moiety, such as a phenyl (compound 24e)
or a para-substituted phenyl (compounds 24f and 24g).
For the latter derivatives, the effects of the presence of
both electron-withdrawing (Cl, compound 24f) and
electron-donating (Ph, compound 24g) substituents on
the γ-phenyl moiety of the lactone skeleton were deter-
mined. The derivative 24e has been used for the
synthesis of the third novel series of hybrids 11, 12, 13
containing, respectively, three, two, and one pyrrole
amide units, while compounds 24f and 24g were linked
to the N-terminal pyrrole residue of distamycin A to
afford distamycin conjugates 14 and 15, respectively.
By the synthesis of 17, we have evaluated the biological
effects due to the substitution in compound 11 of the
C-terminal amidine moiety with a guanidine function.
The synthesis of these hybrid compounds 5-17 re-
quired the preparation of pyrazole R-methylidene-γ-
butyrolactone bearing a carboxylic acid moiety to pro-
Sch em e 1a
Ch em istr y
The synthetic route followed for the synthesis of
hybrid compounds 5-17 is outlined in Scheme 1. The
key step was the assembly between pyrazole carboxylic
acids 24a -g bearing the R-methylidene-γ-methyl/aryl-
γ-butyrolactone as the alkylating moiety and the ami-
nopolypyrroleamide moieties 25-28.18 The condensation
of the acylating agents 24a -g with amines 25-28 was
performed using an excess (2 equiv) of 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide
hydrochloride
(EDCl) as the coupling agent, in dry DMF as solvent,
in the presence of Hunig’s base at room temperature
and with identical reaction times (18 h). Compounds
5-17 were prepared in acceptable yields, after purifica-
tion by silica gel flash-chromatography.
a
Reagents: (a) EDCl, Hunig’s base, DMF.

Butyrolactones and Polypyrrole Binders
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2879
Sch em e 2a
Ta ble 1. In Vitro Activity of Hybrids 5-17 and Alkylating
Units 24a ,e-g against the Proliferation of Murine Leukemia
(L1210), Murine Mammary Carcinoma (FM3A), and Human
T-Lymphoblast (Molt/4 and CEM) Cellsa
IC50 (µM)
compd
L1210/0
FM3A/0
Molt4/C8
CEM/0
5
6
7
8
14.7 ( 4.9 49.0 ( 3.7
14.7 ( 2.5
254 ( 11
106 ( 13
71.1 ( 2.2
6.8 ( 0.3
15.9 ( 1.2
255 ( 22
74.3 ( 3.8
35.6 ( 5.6
9.6 ( 4.7
195 ( 12
109 ( 10
243 ( 21
92 ( 16
26.7 ( 1.1 63.3 ( 6.7
8.02 ( 2.9 27.6 ( 11
24.3 ( 4.0 32.4 ( 6.1
9
10
11
12
13
14
15
16
17
24a
24e
24f
24g
22.3 ( 10.1 26.3 ( 2.0
4.2 ( 0.5
4.1 ( 0.1
17.5 ( 1.2
18.5 ( 1.3
5.2 ( 0.1
4.6 ( 0.4
15.8 ( 13
79.1 ( 31
65.3 ( 8.0
12.3 ( 7.7
10.1 ( 7.1
746 ( 21
189 ( 58
9.7 ( 4.2
5.7 ( 1.1
18.9 ( 3.2
86.3 ( 3.6
95.0 ( 0
15.9 ( 2.7
10.0 ( 1.1
402 ( 143
174 ( 36
57.9 ( 8.3
18.5 ( 4.7
113 ( 26
12.5 ( 5.8 36.3 ( 20
25.2 ( 2.4 64.7 ( 29
33.2 ( 1.1 21.8 ( 9.2
9.6 ( 4.5
5.1 ( 1.4
522 ( 26
107 ( 24
13.0 ( 0
42.5 ( 23
20.5 ( 4.8
755 ( 12
189 ( 46
99.2 ( 44.1 46.9 ( 5.5
14.8 ( 0.5 42.0 ( 2.5
9.4 ( 1.5
64.6 ( 15
distamycin A (1) 133 ( 18
150 ( 29
a
IC50 is the compound concentration required to inhibit tumor
cell proliferation by 50%. Data are expressed as the mean ( SE
from the dose-response curves of at least three independent
experiments.
24g, respectively), where compound 24f bearing the 4′-
Cl-substituted phenyl moiety exhibited in most cases a
lower potency than its 4′-Ph counterpart 24g. The good
antiproliferative activity of the latter compound implies
that a lipophilic and bulky substituent at the benzene
increases the cytostatic potency. Otherwise, tethering
24f and 24g to deformyl distamycin A 27 to give the
corresponding hydrids 14 and 15, respectively, proved
to be less cytotoxic than the alkylating unit alone with
respect to all cell lines, with the exception of the murine
mammary carcinoma (FM3A) cells.
a
Reagents: (a) 19a -g, NaH, DMF; (b) LiOH THF/MeOH, H2O;
(c) (CH3)3CBr, K2CO3, BTEAC, DMF; (d) CH2dC(CH2Br)CO2Et,
Zn, THF; (e) TFA, CH2Cl2.
vide a point of attachment for the appropriate aminopoly-
pyrroleamide moieties. O-alkylation of the methyl ester
of 5-hydroxy-2-methyl-2H-pyrazole-3-carboxylic acid 1819
with the appropriate ketone 19a -g20 using sodium
hydride (NaH, 50% in mineral oil) in dry DMF afforded
the corresponding derivatives 20a -g, which were then
transformed into the corresponding tert-butyl esters
22a -g after mild alkaline hydrolysis of the methyl ester
derivatives 20a -g with LiOH, to give the corresponding
carboxylic acids 21a -g, and subsequent reaction with
tert-butyl bromide in K2CO3.21 The intermediates 22a -g
were then cyclized by the reaction with ethyl 2-(bro-
momethyl)acrylate22 and zinc powder in dry THF (Re-
formatsky-type condensation) to form the γ-lactones
23a -g. Subsequent treatment with trifluoroacetic acid
(TFA) in dichloromethane, at room temperature, af-
forded the carboxylic acids 24a -g (Scheme 2).
It can be seen in Table 1 that tethering the lactones
24a -e to the DNA binders 25-28 (to afford the hybrids
5-13, 16, and 17) resulted, with only a few exceptions,
in improved cytotoxic potency against most of the tumor
cell lines compared to the alkylating units alone.
Compounds 11, 12, and 17, which possess the phenyl
at the γ-position of the R-methylene-γ-butyrolactone
moiety (24e) as the alkylating moiety, were found to
have both the best and broadest antiproliferative activ-
ity. As previously reported, the lower inhibitory potency
of 14 and 15 with respect to 11 implies that both an
electron-donating substituent (Ph, compound 15) and
an electron-withdrawing substituent (Cl, compound 14)
at the benzene moiety of the lactone reduced their
antiproliferative activity.
Resu lts a n d Discu ssion
An titu m or Activity. In Table 1 we have reported
the in vitro cytostatic activity of hybrid compounds 5-17
and R-methylene γ-substituted γ-butyrolactone pyrazole
derivatives 24a and 24e-g against a panel of tumor
cell lines, using distamycin A as reference compound.
Several compounds demonstrated profound growth-
inhibitory effects on the proliferation of murine leuke-
mia (L1210), murine mammary carcinoma (FM3A), and
human T-lymphoblastoid (Molt/4 and CEM) cells.
In the series of lactones 24a and 24e-g, compound
24e, possessing a phenyl substituent at the γ-position
of the lactone, was more active against all cell lines than
its methyl counterpart 24a . However, a significant
increase in cytostatic activity was observed when the
γ-phenyl group of 24e was replaced by a 4′-chloro or a
4′-phenyl-substituted phenyl group (compounds 24f and
In compounds 5-8, we set out to investigate the
influence of the length (n) of the polymethylene chain
between the lactone ring and the pyrazole moiety on
the cytotostatic activity. With an increase in the number
of methylene units from one to three (compounds 5 and
6, respectively), the cytostatic activity dramatically
decreased. When the methylene linker length was
increased from three (compound 6) to five (compound
7) methylene units, the in vitro antiproliferative activity
increased at least 2-fold but proved to be lower than that
with n ) 1 (derivative 5). Compound 8 with the longest
linker length in the series (n ) 7) was more active with
respect to the derivatives with a propyl and pentyl chain

2880 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Baraldi et al.
(compounds 6 and 7, respectively) and comparable to
the compound (5) with the shortest spacer (n ) 1).
A correlation between cytostatic activity and the
length of the polypyrrolic frame was observed for the
compounds bearing 24a and 24e as alkylating moieties.
In the series of derivatives 5, 9, and 10, which possess
an R-methylene-γ-methyl-γ-butyrolactone moiety, the
derivative 9 with two N-methylpyrrolic units was almost
2-fold more cytostatic than the corresponding pyrrole
homologue 5. The derivative 5 showed an activity
comparable to that of compound 10 characterized by the
presence of a single pyrrole ring in its structure. The
same behavior was not observed for the hybrids 11-
13, characterized by the presence of an R-methylene-γ-
phenyl-γ-butyrolactone moiety as the alkylating unit.
Compounds 11 and 12, which possess three and two
pyrrolic rings, respectively, showed a similarly pro-
nunced antiproliferative activity and proved to be 3-fold
higher than that observed for the monopyrrolic com-
pound 13. Of compounds 11-13, the hybrids 11 and 12
exhibited the highest potency across the panel of tumor
cell lines, especially against L1210, Molt/4, and CEM
cells, with IC50 values ranging between 4 and 10 µM.
F igu r e 1. Effects of compounds 9, 11, 12, and 17 on induction
of apoptosis in HL-60 cells. Cells were incubated with no drug
(C, control) or with 10-30 µM of the indicated compounds for
24 h. After treatment they were stained with bisbenzimide
trihydrochloride, and apoptotic cells were quantitated by
fluorescence microscopy (A). The results of a representative
experiment are shown, and each point represents the average
( SE of triplicate determinations. Also shown are photomi-
crographs of representative fields of cells treated with 10 µM
of the indicated products and stained as above (B).
Taken together, for all tumor cell lines, compounds
possessing the same number of pyrrole rings and the
alkylating unit 24e appeared to be more cytostatic than
those containing 24a as the alkylating agent.
The nature of the substituent at the C(γ)-position of
the lactone moiety had a great effect on the antiprolif-
erative activity of compounds having the same oligopep-
tide frame. From a comparison of the cytostatic activity
of the compounds with a different alkylating unit (24a
and 24e) and with the same number of pyrrolic rings,
the derivatives 5, 9, and 10 characterized by the
presence of three, two, and one pyrrole units, respec-
tively, and with the aliphatic methyl substituent at the
γ-position of the lactone were less active than their
γ-phenyl counterparts 11, 12, 13, respectively. Other-
wise, the data show that the two series of derivatives
5, 9, 10 and 11-13 retain a higher antiproliferative
activity when compared to the lactones 24a and 24e
alone. Modification of the C-terminal amidine group in
compounds 5 and 11, with a guanidino moiety to furnish
the corresponding derivatives 16 and 17, substantially
maintained the cytostatic activity.
F igu r e 2. Analysis of DNA fragmentation induced by com-
pounds 9, 11, 12, and 17 in HL-60 cells. Cells were incubated
in the absence (control) or presence of 30 µM of the indicated
compounds for 8 h. Low molecular weight DNA was isolated,
electrophoresed on 2% agarose gel containing ethidium bro-
mide, and visualized under UV light: M, size marker; bp, base
pairs.
less effective. Figure 1B shows the morphological changes
associated with the cells in response to 10 µM of the
compounds and their comparison with those from cells
incubated with the vehicle alone.
We also examined whether fragmentation of DNA,
which is considered as the end point of the apoptotic
pathway, is activated in response to compounds 9, 11,
12, and 17. As demonstrated in Figure 2, DNA frag-
ments formed by intranucleosomal hydrolysis of chro-
matin was observed after 8 h of treatment with 30 µM
of each compound. In agreement with the results
obtained in Figure 1, only 9 did not induce DNA
laddering, as revealed by agarose gel electrophoresis.
We further performed an immunoblot assay to exam-
ine whether the compounds induced poly ADP-ribose
polymerase (PARP) cleavage. This protein plays an
important role in chromatin architecture and DNA
metabolism.23 It is also considered to be a hallmark of
apoptosis and a recognized caspase substrate whose
degradation from 116 kDa to an 85 kDa fragment is
indicative of activation of the apoptotic process. In dose-
response experiments, PARP cleavage was detected in
12-treated cells after 4 h of exposure at a concentration
as low as 10 µM and increased in a dose-dependent
manner (Figure 3). On the other hand, PARP cleavage
In d u ction of Ap op tosis by Com p ou n d s 9, 11, 12,
a n d 17. To determine whether a selected series of the
most active compounds 9, 11, 12, and 17 induced
apoptosis, we incubated HL-60 cells with 10 or 30 µM
of these agents for 24 h. Compounds 11, 12, and 17 were
able to induce apoptosis, judging by the appearance of
typical morphological changes that included chromatin
condensation, its compaction along the periphery of the
nucleus, and nuclear segmentation into three or more
chromatin fragments visualized by fluorescence micros-
copy. As shown in Figure 1A, the percentage of apoptotic
cells increased in a dose-dependent manner, with the
exception of derivative 9. During this period, 11 and 12
caused an increase in the percentage of apoptotic cells
to the same extent, approximately 16% with 10 µM and
about 26% with 30 µM. These values are significantly
higher than those obtained with control cells (7.4 ( 0.1%
of apoptotic cells). Compound 17 also induced apoptosis
at both concentrations tested, although it was slightly

Butyrolactones and Polypyrrole Binders
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2881
30 min and 1 h at room temperature. To this solution was
added the appropriate ω-halo ketone (1-4 mmol) dissolved in
dry DMF (2 mL), and the resulting mixture continued to stir
at room temperature for 18 h and then concentrated in vacuo.
The resulting residue was dissolved in EtOAc (10 mL) and
washed with water (2 × 5 mL). The recombined organic phases
were dried on Na2SO4 and concentrated under vacuum, and
the crude product was purified by flash chromatography
(EtOAc/petroleum ether).
Meth yl Ester of 2-Meth yl-5-(2-oxop r op oxy)-2H-p yr a -
zole-3-ca r boxylic Acid (20a ). Following the general proce-
dure A, starting from 18 (5.8 g, 37 mmol) and 1-chloropropan-
2-one 19a (13.6 g, 11.7 mL, 147 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
2:8), compound 20a was obtained as a white solid (4.1 g, 52%
yield), mp 56-57 °C. 1H NMR (CDCl3) δ: 2.22 (s, 3H), 3.85 (s,
3H), 3.99 (s, 3H), 4.70 (s, 2H), 6.23 (s, 1H).
F igu r e 3. Western blot analysis of poly ADP-ribose poly-
merase (PARP) cleavage. Cells were incubated in the presence
of 10 µM (top) or 30 µM (bottom) of the indicated compounds
9, 11, 12, and 17 for 4 h, and cell lysates were subjected to
SDS-PAGE. The PARP level was determined by immunob-
lotting with a monoclonal antibody that also recognizes the
85 kDa fragment.
Meth yl Ester of 2-Meth yl-5-(4-oxop en tyloxy)-2H-p yr a -
zole-3-ca r boxylic Acid (20b). Following the general proce-
dure A, starting from 18 (781 mg, 5 mmol) and 5-iodopentan-
2-one 19b (4.24 g, 20 mmol), after workup and purification by
flash chromatography (EtOAc/petroleum ether 1:9), compound
20b was obtained as a white solid (650 mg, 54% yield), mp
49-51 °C. 1H NMR (CDCl3) δ: 2.06 (m, 2H), 2.17 (s, 3H), 2.64
(t, J ) 7.2 Hz, 2H), 3.66 (s, 3H), 4.03 (s, 3H), 4.11 (t, J ) 6.1
Hz, 2H), 6.17 (s, 1H).
was minimal in cells exposed to 11 and 17, although
the percentage of apoptotic cells was comparable (see
Figure 1A). These results indicate that PARP cleavage
was involved in the apoptosic process induced by 12 but
was minimally, if at all, involved in apoptosis induced
by 11 and 17 within the time frame of the study.
Con clu sion s
Meth yl Ester of 2-Meth yl-5-(6-oxoh ep tyloxy)-2H-p yr a -
zole-3-ca r boxylic Acid (20c). Following the general proce-
dure A, starting from 18 (520 mg, 3.3 mmol) and 7-bromohep-
tan-2-one 19c (1.95 g, 10 mmol), after workup and purification
by flash chromatography (EtOAc/petroleum ether 2:8), com-
pound 20c was obtained as a white solid (894 mg, 95% yield),
The present work demonstrated not only the molec-
ular design and the chemical synthesis of novel R-me-
thylene-γ-butyrolactones-lexitropsin hybrids but also
their apoptotic profiles and cytostatic activities. Many
of the compounds prepared in this study were shown to
demonstrate a significant antiproliferative activity to-
ward four different tumor cell lines. We found that
compounds 11, 12, and 17 activate apoptosis on HL-60
cells. Treatment of the tumor cells with these com-
pounds induced morphological changes and DNA frag-
mentation characteristic of apoptotic cell death. We have
also demonstrated that only 12 induces PARP hydroly-
sis, which is considered a marker of apoptosis. PARP is
an important nuclear chromatin-associated enzyme that
plays an important role in mediating the normal cellular
response to DNA damage, and its cleavage implicates
activation of caspase(s).
1
mp 54-56 °C. H NMR (CDCl3) δ: 1.65 (m, 6H), 2.14 (s, 3H),
2.45 (t, J ) 7.4 Hz, 2H), 3.86 (s, 3H), 4.03 (s, 3H), 4.08 (t, J )
6.5 Hz, 2H), 6.16 (s, 1H).
Meth yl Ester of 2-Meth yl-5-(8-oxon on yloxy)-2H-p yr a -
zole-3-ca r boxylic Acid (20d ). Following the general proce-
dure A, starting from 18 (99 mg, 0.63 mmol) and 9-bromononan-
2-one 19d (420 mg, 1.9 mmol), after workup and purification
by flash chromatography (EtOAc/petroleum ether 2:8), com-
pound 20d was obtained as a white solid (187 mg, 95% yield),
mp 55-57 °C. 1H NMR (CDCl3) δ: 1.34 (m, 10H), 2.13 (s, 3H),
2.42 (t, J ) 7.2 Hz, 2H), 3.66 (s, 3H), 4.03 (s, 3H), 4.08 (t, J )
6.7 Hz, 2H), 6.17 (s, 1H).
Meth yl Ester of 2-Meth yl-5-(2-oxo-2-p h en yleth oxy)-2H-
p yr a zole-3-ca r boxylic Acid (20e). Following the general
procedure A, starting from 18 (4 g, 25 mmol) and 2-bromo-1-
phenylethanone 19e (5.47 g, 27.5 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
2:8), compound 20e was obtained as a white solid (6.85 g, 70%
Exp er im en ta l Section
Ch em istr y. Ma ter ia ls a n d Meth od s. 1H NMR spectra
were recorded on a Bruker AC 200 spectrometer. Chemical
shifts (δ) are given in ppm upfield from tetramethylsilane as
the internal standard, and the spectra were recorded in
appropriate deuterated solvents indicated in the procedure.
Melting points (mp) were determined on a Buchi-Tottoli
apparatus and are uncorrected. All products reported showed
1H NMR spectra in agreement with the assigned structures.
Elemental analyses were conducted by the Microanalytical
Laboratory of the Chemistry Department of the University of
Ferrara. All reactions were carried out under an inert atmo-
sphere of dry nitrogen, unless otherwise described. Standard
syringe techniques were applied for transferring dry solvents.
Reaction courses and product mixtures were routinely moni-
tored by TLC on silica gel (precoated F254 Merck plates) and
visualized with aqueous KMnO4. Flash chromatography was
performed using 230-400 mesh silica gel and the solvent
system indicated in the procedure. All commercially available
compounds were used without further purification. Organic
solutions were dried over anhydrous MgSO4. Dioxane was
distilled from calcium hydride, and dry DMF was distilled from
calcium chloride and stored over molecular sieves (3 Å).
Gen er a l P r oced u r e A for t h e Syn t h esis of 20a -g.
Compound 18 (156 mg, 1 mmol), NaH (50% in mineral oil, 1
mmol, 48 mg), and dry DMF (5 mL) were stirred at 0 °C for
1
yield), mp 102-105 °C. H NMR (CDCl3) δ: 3.85 (s, 3H), 3.98
(s, 3H), 5.54 (s, 2H), 6.37 (s, 1H), 7.60 (m, 2H), 7.68 (t, J ) 7.6
Hz, 1H), 7.98 (d, J ) 7.6 Hz, 2H).
Met h yl E st er of 5-[2-(4-Ch lor op h en yl)-2-oxoet h oxy]-
2-m eth yl-2H-p yr a zole-3-ca r boxylic Acid (20f). Following
the general procedure A, starting from 18 (1 g, 6.4 mmol) and
2-bromo-1-(4-chlorophenyl)ethanone 19f (2.68 g, 19 mmol),
after workup and purification by flash chromatography (EtOAc/
petroleum ether 2:8), compound 20f was obtained as a yellow
1
solid (1.97 g, 95% yield), mp 118-120 °C. H NMR (CDCl3) δ:
3.86 (s, 3H), 3.98 (s, 3H), 5.41 (s, 2H), 6.28 (s, 1H), 7.45 (d, J
) 7.6 Hz, 2H), 7.95 (d, J ) 7.6 Hz, 2H).
Met h yl E st er of 5-(2-Bip h en yl-4-yl-2-oxoet h oxy)-2-
m eth yl-2H-p yr a zole-3-ca r boxylic Acid (20g). Following the
general procedure A, starting from 18 (510 mg, 3.26 mmol)
and 1-biphenyl-4-yl-2-bromoethanone 19g (1.23 g, 4.5 mmol),
after workup and purification by flash chromatography (EtOAc/
petroleum ether 2:8), compound 20f was obtained as a yellow
1
solid (750 mg, 71% yield), mp 168-170 °C. H NMR (CDCl3)
δ: 3.66 (s, 3H), 3.99 (s, 3H), 5.49 (s, 2H), 6.31 (s, 1H), 7.45 (m,
3H), 7.62 (m, 2H), 7.69 (d, J ) 7.8 Hz, 2H), 8.04 (d, J ) 7.8
Hz, 2H).
Gen er a l P r oced u r e B for t h e Syn t h esis of 21a -g.
Lithium hydroxide monohydrate (46 mg, 1.1 mmol, 1.1 equiv)

2882 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Baraldi et al.
was added to a solution of 20a -g (1 mmol) in 6 mL of THF/
MeOH/H2O (4:1:1) at room temperature. The reaction mixture
was stirred for 1 h and then concentrated in vacuo. The crude
residue was dissolved in water (5 mL), and the solution was
acidified with 10% aqueous HCl and extracted with EtOAc (3
× 5 mL). The combined organic extracts were washed with
brine (5 mL), dried over Na2SO4, and concentrated in vacuo.
The products obtained were used without any purification for
the next reaction.
2-Met h yl-5-(2-oxop r op oxy)-2H -p yr a zole-3-ca r b oxylic
Acid (21a ). Following the general procedure B, starting from
20a (4.03 g, 19 mmol), after workup compound 21a was
obtained as a yellow oil (3.7 g, 97% yield). 1H NMR (CDCl3) δ:
2.25 (s, 3H), 4.02 (s, 3H), 4.74 (s, 2H), 6.36 (s, 1H), 7.7 (bs,
1H).
2-Meth yl-5-(4-oxop en tyloxy)-2H-p yr a zole-3-ca r boxyl-
ic Acid (21b). Following the general procedure B, starting
from 20b (240 mg, 1 mmol), after workup compound 21b was
obtained as a colorless oil (220 mg, 97% yield). 1H NMR
(CDCl3) δ: 2.05 (m, 2H), 2.18 (s, 3H), 2.65 (t, J ) 7.2 Hz, 2H),
4.04 (s, 3H), 4.12 (t, J ) 6.2 Hz, 2H), 6.30 (s, 1H), 10.3 (bs,
1H).
1H NMR (CDCl3) δ: 1.56 (s, 9H), 2.05 (m, 2H), 2.16 (s, 3H),
2.63 (t, J ) 7.2 Hz, 2H), 4.02 (s, 3H), 4.09 (t, J ) 6.2 Hz, 2H),
6.09 (s, 1H).
ter t-Bu tyl Ester of 2-Meth yl-5-(6-oxoh ep tyloxy)-2H-
p yr a zole-3-ca r boxylic Acid (22c). Following the general
procedure C, starting from 21c (620 mg, 2.4 mmol), after
workup 22c was obtained as a brown oil (500 mg, 67% yield).
1H NMR (CDCl3) δ: 1.63 (m, 6H), 1.56 (s, 9H), 2.14 (s, 3H),
2.45 (t, J ) 7.2 Hz, 2H), 4.03 (s, 3H), 4.07 (t, J ) 6.4 Hz, 2H),
6.10 (s, 1H).
ter t-Bu t yl E st er of 2-Met h yl-5-(8-oxon on yloxy)-2H -
p yr a zole-3-ca r boxylic Acid (22d ). Following the general
procedure C, starting from 21d (840 mg, 2.97 mmol), after
workup 22d was obtained as a brown oil (940 mg, 94% yield).
1H NMR (CDCl3) δ: 1.35 (m, 10H), 1.55 (s, 9H), 2.13 (s, 3H),
2.42 (t, J ) 7.2 Hz, 2H), 3.99 (s, 3H), 4.06 (t, J ) 6.4 Hz, 2H),
6.09 (s, 1H).
ter t-Bu tyl Ester of 2-Meth yl-5-(2-oxo-2-p h en yleth oxy)-
2H-p yr a zole-3-ca r boxylic Acid (22e). Following the general
procedure C, starting from 21e (1 g, 3.8 mmol), after workup
1
22e was obtained as a brown oil (1.13 g, 94% yield). H NMR
(CDCl3) δ: 1.54 (s, 9H), 3.94 (s, 3H), 5.44 (s, 2H), 6.33 (s, 1H),
7.59 (m, 2H), 7.68 (t, J ) 7.2 Hz, 1H), 8.00 (d, J ) 7.2 Hz,
2H).
ter t-Bu tyl Ester of 5-[2-(4-Ch lor op h en yl)-2-oxoeth oxy]-
2-m eth yl-2H-p yr a zole-3-ca r boxylic Acid (22f). Following
the general procedure C, starting from 21f (1.53 g, 5.2 mmol),
after workup compound 22f was obtained as a brown oil (1.60
g, 89% yield). 1H NMR (CDCl3) δ: 1.56 (s, 9H), 3.95 (s, 3H),
5.40 (s, 2H), 6.22 (s, 1H), 7.49 (d, J ) 8.0 Hz, 2H), 7.95 (d, J
) 8.00 Hz, 2H).
2-Meth yl-5-(6-oxoh ep tyloxy)-2H-p yr a zole-3-ca r boxyl-
ic Acid (21c). Following the general procedure B, starting
from 20c (894 mg, 1 mmol), after workup compound 21c was
obtained as an oil (620 mg, 74% yield). 1H NMR (CDCl3) δ:
1.65 (m, 6H), 2.14 (s, 3H), 2.45 (t, J ) 7.4 Hz, 2H), 4.03 (s,
3H), 4.11 (t, J ) 6.5 Hz, 2H), 6.16 (s, 1H), 8.5 (bs, 1H).
2-Met h yl-5-(8-oxon on yloxy)-2H -p yr a zole-3-ca r b oxyl-
ic Acid (21d ). Following the general procedure B, starting
from 20d (990 mg, 3.3 mmol), after workup compound 21d
1
was obtained as a colorless oil (860 mg, 92% yield). H NMR
ter t-Bu tyl Ester of 5-(2-Bip h en yl-4-yl-2-oxoeth oxy)-2-
m eth yl-2H-p yr a zole-3-ca r boxylic Acid (22g). Following the
general procedure C, starting from 21g (504 mg, 1.5 mmol),
after workup 22g was obtained as a yellow oil (410 mg, 78%
(CDCl3) δ: 1.32 (m, 10H), 2.14 (s, 3H), 2.43 (t, J ) 7.6 Hz,
2H), 4.05 (s, 3H), 4.12 (t, J ) 6.6 Hz, 2H), 6.29 (s, 1H), 8.3 (bs,
1H).
1
2-Meth yl-5-(2-oxo-2-p h en yleth oxy)-2H-p yr a zole-3-ca r -
boxylic Acid (21e). Following the general procedure B,
starting from 20e (4.79 g, 17.5 mmol), after workup compound
21e was obtained as a white solid (4.33 g, 96% yield), mp 136-
yield). H NMR (CDCl3) δ: 1.56 (s, 9H), 3.97 (s, 3H), 5.49 (s,
2H), 6.25 (s, 1H), 7.45 (m, 3H), 7.62 (m, 2H), 7.74 (d, J ) 8.2
Hz, 2H), 8.07 (d, J ) 8.4 Hz, 2H).
Gen er a l P r oced u r e D for th e Syn th esis of 23a -g. To a
solution of 22a -g (1 mmol) in dry THF (7 mL), activated Zn
powder (261 mg, 4 mmol), hydroquinone (2 mg), and ethyl
2-(bromomethyl)acrylate (503 mg, 0.26 mL, 2.6 mmol) were
added. The mixture was refluxed under nitrogen for 24 h. After
cooling, the reaction mixture was filtered on Celite and the
filtrate was diluted with CH2Cl2 (15 mL). The organic solution
washed with 1 N HCl solution (5 mL) and water (5 mL), dried
(Na2SO4), and then evaporated gave a residual solid, which
was purified by column chromatography on silica gel (EtOAc/
petroleum ether).
ter t-Bu tyl Ester of 2-Meth yl-5-(2-m eth yl-4-m eth ylen e-
5-oxotetr a h yd r ofu r a n -2-ylm eth oxy)-2H-p yr a zole-3-ca r -
boxylic Acid (23a ). Following the general procedure D,
starting from 22a (400 mg, 1.6 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
2:8), compound 23a was obtained as a colorless oil (240 mg,
50% yield). 1H NMR (CDCl3) δ: 1.51 (s, 3H), 1.55 (s, 9H), 2.74
(dt, J ) 17.2 and 2.6 Hz, 1H), 3.11 (dt, J ) 17.2 and 2.6 Hz,
1H), 3.99 (s, 3H), 4.13 (s, 2H), 5.63 (t, J ) 2.4 Hz, 1H), 6.10 (s,
1H), 6.25 (t, J ) 2.8 Hz, 1H).
ter t-Bu tyl Ester of 2-Meth yl-5-[3-(2-m eth yl-4-m eth yl-
en e-5-oxotetr a h yd r ofu r a n -2-yl)p r op oxy]-2H-p yr a zole-3-
ca r boxylic Acid (23b). Following the general procedure D,
starting from 22b (800 mg, 3 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
3:7), compound 23b was obtained as a colorless oil (852 mg,
81% yield). 1H NMR (CDCl3) δ: 1.43 (s, 3H), 1.56 (s, 9H), 1.84
(m, 4H), 2.60 (dt, J ) 17.2 and 2.4 Hz, 1H), 3.12 (dt, J ) 17.2
and 2.4 Hz, 1H), 3.99 (s, 3H), 4.14 (t, J ) 7.4 Hz, 2H), 5.63 (t,
J ) 2.0 Hz, 1H), 6.09 (s, 1H), 6.24 (t, J ) 2.8 Hz, 1H).
ter t-Bu tyl Ester of 2-Meth yl-5-[5-(2-m eth yl-4-m eth yl-
en e-5-oxotetr a h yd r ofu r a n -2-yl)p en tyloxy]-2H-p yr a zole-
3-ca r boxylic Acid (23c). Following the general procedure D,
starting from 22c (500 mg, 1.6 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
1
138 °C. H NMR (CDCl3) δ: 3.96 (s, 3H), 5.51 (s, 2H), 6.25 (s,
1H), 7.53 (m, 2H), 7.65 (m, 2H), 7.99 (m, 1H), 10.8 (bs, 1H).
5-[2-(4-Ch lor op h en yl)-2-oxoeth oxy]-2-m eth yl-2H-p yr a -
zole-3-ca r boxylic Acid (21f). Following the general proce-
dure B, starting from 20f (1.80 g, 5.80 mmol), after workup
compound 21f was obtained as a white solid (1.58 g, 92% yield),
mp 147-150 °C. 1H NMR (CDCl3) δ: 3.87 (s, 3H), 5.55 (s, 2H),
6.27 (s, 1H), 7.65 (d, J ) 7.4 Hz, 2H), 8.01 (d, J ) 7.4 Hz, 2H),
10.8 (bs, 1H).
5-(2-Bip h en yl-4-yl-2-oxoeth oxy)-2-m eth yl-2H-p yr a zole-
3-ca r boxylic Acid (21g). Following the general procedure B,
starting from 20g (702 mg, 2 mmol), after workup compound
21g was obtained as a white solid (504 mg, 75% yield), mp
208-210 °C. 1H NMR (CDCl3) δ: 3.88 (s, 3H), 5.60 (s, 2H),
6.30 (s, 1H), 7.52 (m, 3H), 7.78 (d, J ) 7.4 Hz, 2H), 7.88 (d, J
) 7.4 Hz, 2H), 8.09 (d, J ) 8.2 Hz, 2H), 10.7 (bs, 1H).
Gen er a l P r oced u r e C for t h e Syn t h esis of 22a -g.
Compounds 21a -g (1 mmol) dissolved in a solution of BTEAC
(228 mg, 1 mmol) in dry DMF (10 mL) was treated with K2-
CO3 (3.59 g, 26 mmol) and tert-butyl bromide (6.57 g, 5.4 mL,
48 mmol). After 24 h at 55 °C, the mixture was poured into
cold water (10 mL) and extracted with EtOAc (3 × 15 mL).
The recombined organic phases were dried (Na2SO4), and after
concentration in vacuo, the crude product (22a -g) was used
without purification for the next step.
ter t-Bu t yl E st er of 2-Met h yl-5-(2-oxop r op oxy)-2H -
p yr a zole-3-ca r boxylic Acid (22a ). Following the general
procedure C, starting from 21a (1.23 g, 6 mmol), after workup
22a was obtained as a brown oil (400 mg, 27% yield). 1H NMR
(CDCl3) δ: 1.56 (s, 9H), 2.23 (s, 3H), 3.97 (s, 3H), 4.70 (s, 2H),
6.16 (s, 1H).
ter t-Bu tyl Ester of 2-Meth yl-5-(4-oxop en tyloxy)-2H-
p yr a zole-3-ca r boxylic Acid (22b). Following the general
procedure C, starting from 21b (724 mg, 3.2 mmol), after
workup 22b was obtained as an orange oil (850 mg, 94% yield).

Butyrolactones and Polypyrrole Binders
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2883
3/7), compound 23c was obtained as a colorless oil (460 mg,
76% yield). H NMR (CDCl3) δ: 1.33 (t, J ) 2.8 Hz, 2H), 1.40
(s, 3H), 1.56 (s, 9H), 1.76 (m, 6H), 2.72 (dt, J ) 17.2 and 2.6
Hz, 1H), 3.23 (dt, J ) 17.2 and 2.6 Hz, 1H), 4.04 (s, 3H), 4.10
(t, J ) 7.4 Hz, 2H), 5.61 (t, J ) 2.2 Hz, 1H), 6.10 (s, 1H), 6.23
(t, J ) 2.6 Hz, 1H).
23c (460 mg, 1.2 mmol), after workup, compound 24c was
obtained as a colorless oil (228 mg, 59% yield). 1H NMR
(CDCl3) δ: 1.26 (t, J ) 7.01 Hz, 2H), 1.40 (s, 3H), 1.74 (m,
6H), 2.72 (dt, J ) 17.2 and 2.4 Hz, 1H), 3.02 (dt, J ) 17.2 and
2.4 Hz, 1H), 4.05 (s, 3H), 4.13 (t, J ) 7.43 Hz, 2H), 5.62 (t, J
) 2.33 Hz, 1H), 6.24 (t, J ) 2.63 Hz, 1H), 6.29 (s, 1H), 8.4 (bs,
1H).
1
ter t-Bu tyl Ester of 2-Meth yl-5-[7-(2-m eth yl-4-m eth yl-
en e-5-oxotetr a h yd r ofu r a n -2-yl)h ep tyloxy]-2H-p yr a zole-
3-ca r boxylic Acid (23d ). Following the general procedure D,
starting from 22d (950 mg, 2.8 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
2:8), compound 23d was obtained as a colorless oil (790 mg,
2-Meth yl-5-[7-(2-m eth yl-4-m eth ylen e-5-oxotetr a h yd r o-
fu r an -2-yl)h eptyloxy]-2H-pyr azole-3-car boxylic Acid (24d).
Following the general procedure E, starting from compound
23d (406 mg, 1 mmol), after workup, compound 24d was
obtained as a colorless oil (350 mg, 77% yield). 1H NMR
(CDCl3) δ: 1.17 (t, J ) 4.6 Hz, 2H), 1.39 (s, 3H), 1.71 (m, 10H),
2.72 (dt, J ) 17.0 and 2.6 Hz, 1H), 3.12 (dt, J ) 17.0 and 2.6
Hz, 1H), 4.05 (s, 3H), 4.12 (t, J ) 2.4 Hz, 2H), 5.62 (t, J ) 2.4
Hz, 1H), 6.25 (t, J ) 2.6 Hz, 1H), 6.30 (s, 1H), 8.5 (bs, 1H).
2-Meth yl-5-(4-m eth ylen e-5-oxo-2-p h en yltetr a h yd r ofu -
r a n -2-ylm et h oxy)-2H-p yr a zole-3-ca r b oxylic Acid (24e).
Following the general procedure E, starting from compound
23e (2.17 g, 5.6 mmol), after workup, compound 24e was
obtained as a colorless oil (1.71 g, 95% yield). 1H NMR (CDCl3)
δ: 3.19 (dt, J ) 17.2 and 2.4 Hz, 1H), 3.63 (dt, J ) 17.2 and
2.4 Hz, 1H), 3.86 (s, 3H), 4.38 (s, 3H), 5.66 (t, J ) 2.4 Hz, 1H),
6.11 (s, 1H), 6.24 (t, J ) 2.8 Hz, 1H), 7.42 (m, 4H), 8.4 (bs,
1H).
5-[2-(4-Ch lor op h en yl)-4-m eth ylen e-5-oxotetr a h yd r ofu -
r an -2-ylm eth oxy]-2-m eth yl-2H-pyr azole-3-car boxylic Acid
(24f). Following the general procedure E, starting from
compound 23f (280 mg, 0.67 mmol), after workup, compound
24f was obtained as a colorless oil (242 mg, 95% yield). 1H
NMR (CDCl3) δ: 3.10 (dt, J ) 16.8 and 2.4 Hz, 1H), 3.61 (dt,
J ) 16.8 and 2.4 Hz, 1H), 3.95 (s, 3H), 4.28 (s, 2H), 5.69 (t, J
) 2.4 Hz, 1H), 6.10 (s, 1H), 6.31 (t, J ) 2.8 Hz, 1H), 7.38 (m,
4H), 8.7 (bs, 1H).
5-(2-Bip h en yl-4-yl-4-m eth ylen e-5-oxotetr a h yd r ofu r a n -
2-ylm et h oxy)-2-m et h yl-2H -p yr a zole-3-ca r b oxylic Acid
(24g). Following the general procedure E, starting from
compound 23g (400 mg, 0.87 mmol), after workup, compound
24g was obtained as a white solid (351 mg, 95% yield), mp
101-103 °C. 1H NMR (CDCl3) δ: 3.25 (dt, J ) 17.0 and 2.4
Hz, 1H), 3.61 (dt, J ) 17.0 and 2.4 Hz, 1H), 4.01 (s, 3H), 4.40
(s, 2H), 5.67 (t, J ) 2.4 Hz 1H), 6.10 (s, 1H), 6.28 (t, J ) 2.8
Hz, 1H), 7.51 (m, 9H), 8.6 (bs, 1H).
Gen er a l P r oced u r e F for th e Cou p lin g of P yr a zole
Ca r boxylic Acid (24a -g) w ith P yr r ole Oligom er s (25-
28). To a stirred 0.4 M solution of the pyrrole oligomer 25-28
in anhydrous DMF under argon atmosphere Hu¨nig’s base (1
equiv) was added at 0 °C. After 5 min, the acids 24a -g (1.1
equiv) followed by EDCl (2 equiv) were added. The resulting
mixture was stirred for 18 h as it warmed to room tempera-
ture, acidified with 10% HCl to pH 3, and then evaporated to
dryness in a vacuum. The resulting residue was purified by
column chromatography (CH2Cl2/CH3OH 8:2 v/v) and recrys-
tallized (CH3OH/diethyl ether) to give 5-17.
1
70% yield). H NMR (CDCl3) δ: 1.01 (t, J ) 4.6 Hz, 2H), 1.39
(s, 3H), 1.56 (s, 9H), 1.66 (m, 10H), 2.71 (dt, J ) 17.2 and 2.4
Hz, 1H), 3.21 (dt, J ) 17.2 and 2.4 Hz, 1H), 3.99 (s, 3H), 4.10
(t, J ) 6.5 Hz, 2H), 5.61 (t, J ) 2.4 Hz, 1H), 6.10 (s, 1H), 6.22
(t, J ) 2.6 Hz, 1H).
ter t-Bu tyl Ester of 2-Meth yl-5-(4-m eth ylen e-5-oxo-2-
p h en yltetr a h yd r ofu r a n -2-ylm eth oxy)-2H-p yr a zole-3-ca r -
boxylic Acid (23e). Following the general procedure D,
starting from 22e (2.26 g, 7.0 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
3/7), compound 23e was obtained as a colorless oil (2.69 g, 80%
yield). 1H NMR (CDCl3) δ: 1.54 (s, 9H), 3.20 (dt, J ) 17.2 and
2.6 Hz, 1H), 3.66 (dt, J ) 17.2 and 2.6 Hz, 1H), 3.97 (s, 3H),
4.33 (s, 3H), 5.64 (t, J ) 2.4 Hz, 1H), 6.08 (s, 1H), 6.26 (t, J )
2.8 Hz, 1H), 7.40 (m, 4H).
ter t-Bu tyl Ester of 5-[2-(4-Ch lor op h en yl)-4-m eth ylen e-
5-oxot et r a h yd r ofu r a n -2-ylm et h oxy]-2-m et h yl-2H -p yr a -
zole-3-ca r boxylic Acid (23f). Following the general proce-
dure D, starting from 22f (410 mg, 1.2 mmol), after workup
and purification by flash chromatography (EtOAc/petroleum
ether 2:8), compound 23f was obtained as a colorless oil (280
mg, 60% yield). 1H NMR (CDCl3) δ: 1.54 (s, 9H), 3.12 (dt, J )
17.4 and 2.4 Hz, 1H), 3.56 (dt, J ) 17.4 and 2.4 Hz, 1H), 3.97
(s, 3H), 4.30 (s, 2H), 5.66 (t, J ) 2.40 Hz, 1H), 6.07 (s, 1H),
6.28 (t, J ) 2.78 Hz, 1H), 7.40 (m, 4H).
ter t-Bu tyl Ester of 5-(2-Bip h en yl-4-yl-4-m eth ylen e-5-
oxotetr ah ydr ofu r an -2-ylm eth oxy)-2-m eth yl-2H-pyr azole-
3-ca r boxylic Acid (23g). Following the general procedure D,
starting from 22g (410 mg, 1 mmol), after workup and
purification by flash chromatography (EtOAc/petroleum ether
3:7), compound 23g was obtained as a colorless oil (400 mg,
71% yield). 1H NMR (CDCl3) δ: 1.55 (s, 9H), 3.21 (dt, J ) 16.8
and 2.6 Hz, 1H), 3.62 (dt, J ) 16.8 and 2.6 Hz, 1H), 3.98 (s,
3H), 4.37 (s, 2H), 5.66 (t, J ) 2.4 Hz, 1H), 6.10 (s, 1H), 6.28 (t,
J ) 2.8 Hz, 1H), 7.51 (m, 9H).
Gen er a l P r oced u r e E for th e Syn th esis of 24a -g. To a
solution of the tert-butyl esters 23a -g (1 mmol) in CH2Cl2 (5
mL) was added CF3COOH (1 mL), and the mixture was stirred
at room temperature for 3 h. The solvent was removed under
reduced pressure and triturated with petroleum ether (5 mL)
to furnish 23a -g, which were collected by filtration.
2-Meth yl-5-(2-m eth yl-4-m eth ylen e-5-oxotetr a h yd r ofu -
r a n -2-ylm eth oxy)-2H-p yr a zole-3-ca r boxylic Acid (24a ).
Following the general procedure E, starting from compound
23a (240 mg, 0.7 mmol), after workup, compound 24a was
obtained as a light-yellow powder (210 mg, 95% yield), mp
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-(2-
m eth yl-4-m eth ylen e-5-oxotetr ah ydr ofu r an -2-ylm eth oxy)-
1H -p yr a zole -5-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]-
p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]p r op ion -
a m id in e Hyd r och lor id e (5). Yield, 33%; white solid; mp
220-221 °C. 1H NMR (DMSO) δ: 1.45 (s, 3H), 2.65 (t, J ) 6.3
Hz, 2H), 2.94 (dt, J ) 17.6 and 2.4 Hz, 1H), 3.22 (dt, J ) 17.6
and 2.4 Hz, 1H), 3.43 (m, 2H), 3.81 (s, 3H), 3.85 (s, 3H), 3.87
(s, 3H), 3.95 (s, 3H), 4.17 (s, 2H), 5.74 (t, J ) 2.32 Hz, 1H),
6.05 (t, J ) 2.77 Hz, 1H), 6.42 (s, 1H), 6.97 (s, 1H), 7.07 (s,
1H), 7.08 (s, 1H), 7.19 (s, 1H), 7.25 (s, 1H), 7.30 (s, 1H), 8.25
(t, J ) 7.2 Hz, 1H), 8.62 (bs, 2H), 8.99 (bs, 2H), 9.95 (s, 1H),
10.02 (s, 1H), 10.32 (s, 1H). Anal. (C33H40ClN11O7) C, H, Cl,
N.
1
131-134 °C. H NMR (DMSO) δ: 1.42 (s, 3H), 2.81 (dt, J )
17 and 2.4 Hz, 1H), 3.08 (dt, J ) 17 and 2.4 Hz, 1H), 3.91 (s,
3H), 4.14 (s, 2H), 5.73 (t, J ) 2.0 Hz, 1H), 6.04 (t, J ) 2.8 Hz,
1H), 6.22 (s, 1H), 13.2 (bs, 1H).
2-Meth yl-5-[3-(2-m eth yl-4-m eth ylen e-5-oxotetr a h yd r o-
fu r a n -2-yl)pr opoxy]-2H-p yr a zole-3-ca r boxylic Acid (24b).
Following the general procedure E, starting from compound
23b (858 mg, 2.5 mmol), after workup, compound 24b was
obtained as a colorless oil (720 mg, 95% yield). 1H NMR
(CDCl3) δ: 1.43 (s, 3H), 1.86 (m, 4H), 2.76 (dt, J ) 16.8 and
2.4 Hz, 1H), 3.00 (dt, J ) 16.8 and 2.4 Hz, 1H), 4.04 (s, 3H),
4.14 (t, J ) 7.21 Hz, 2H), 5.64 (t, J ) 2.04 Hz, 1H), 6.24 (t, J
) 2.85 Hz, 1H), 6.29 (s, 1H), 8.7 (bs, 1H).
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-[3-
(2-m e t h y l-4-m e t h y le n e -5-o x o t e t r a h y d r o fu r a n -2-y l)-
p r op oxy]-1H-p yr a zole-5-ca r boxa m id o]p yr r ole-2-ca r box-
a m id o]p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]-
p r op ion a m id in e Hyd r och lor id e (6). Yield, 36%; yellow
2-Meth yl-5-[5-(2-m eth yl-4-m eth ylen e-5-oxotetr a h yd r o-
fu r an -2-yl)pen tyloxy]-2H-pyr azole-3-car boxylic Acid (24c).
Following the general procedure E, starting from compound
1
solid; mp 146-149 °C. H NMR (DMSO) δ: 1.36 (s, 3H), 1.74

2884 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Baraldi et al.
(m, 4H), 2.51 (t, J ) 6.3 Hz, 2H), 2.80 (dt, J ) 17.0 and 2.6
Hz, 1H), 2.92 (dt, J ) 17.0 and 2.6 Hz, 1H), 3.49 (m, 2H), 3.61
(s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 3.94 (s, 3H), 4.07 (s, 2H),
5.73 (t, J ) 2.4 Hz, 1H), 6.05 (t, J ) 2.8, 1H), 6.37 (s, 1H),
6.96 (s, 1H), 7.06 (s, 1H), 7.08 (s, 1H), 7.18 (s, 1H), 7.24 (s,
1H), 7.28 (s, 1H), 8.22 (t, J ) 7.2 Hz, 1H), 8.58 (bs, 2H), 8.92
(12). Yield, 49%; white solid; mp 132-134 °C. 1H NMR
(DMSO) δ: 2.62 (t, J ) 2.6 Hz, 2H), 3.11 (dt, J ) 17.4 and 2.5
Hz, 1H), 3.49 (m, 2H), 3.52 (dt, J ) 17.4 and 2.5 Hz, 1H), 3.81
(s, 3H), 3.85 (s, 3H), 3.93 (s, 3H), 4.36 (d, J ) 11 Hz, 1H), 4.48
(d, J ) 11 Hz, 1H), 5.79 (t, J ) 2.2 Hz, 1H), 6.10 (t, J ) 2.7
Hz, 1H), 6.37 (s, 1H), 6.94 (s, 1H), 7.03 (s, 1H), 7.18 (s, 1H),
7.26 (s, 1H), 7.49 (m, 5H), 8.22 (t, J ) 7.2 Hz, 1H), 8.54 (bs,
(bs, 2H), 9.94 (s, 1H), 10.00 (s, 1H), 10.27 (s, 1H). Anal. (C35H44
ClN11O7) C, H, Cl, N.
-
2H), 8.91 (bs, 2H), 9.97 (s, 1H), 10.23 (s, 1H). Anal. (C32H36
ClN9O6) C, H, Cl, N.
-
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-[5-
(2-m e t h y l-4-m e t h y le n e -5-o x o t e t r a h y d r o fu r a n -2-y l)-
pen tyloxy]-1H-pyr azole-5-car boxam ido]pyr r ole-2-car box-
a m id o]p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]-
p r op ion a m id in e Hyd r och lor id e (7). Yield, 29%; yellow
3-[1-Me t h yl-4-[1-m e t h yl-3-(2-p h e n yl-4-m e t h yle n e -5-
oxotetr ah ydr ofu r an -2-ylm eth oxy)-1H-pyr azole-5-car box-
a m id o]p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r o-
ch lor id e (13). Yield, 45%; white solid; mp 114-117 °C. 1H
NMR (DMSO) δ: 2.58 (t, J ) 2.6 Hz, 2H), 3.11 (dt, J ) 17.4
and 2.5 Hz, 1H), 3.49 (m, 2H), 3.64 (dt, J ) 17.4 and 2.5 Hz,
1H), 3.81 (s, 3H), 3.91 (s, 3H), 4.35 (d, J ) 11 Hz, 1H), 4.47 (d,
J ) 11 Hz, 1H), 5.78 (t, J ) 2.2 Hz, 1H), 6.10 (t, J ) 2.7 Hz,
1H), 6.37 (s, 1H), 6.92 (s, 1H), 7.20 (s,1H), 7.48 (m, 5H), 8.27
(t, J ) 7.2, 1H), 8.56 (bs, 2H), 8.91 (bs, 2H), 10.20 (s, 1H). Anal.
(C26H30ClN7O5) C, H, Cl, N.
1
solid; mp 112-114 °C. H NMR (DMSO) δ: 1.25 (t, J ) 2.89
Hz, 2H), 1.33 (s, 3H), 1.73 (m, 6H), 2.49 (t, J ) 6.3 Hz, 2H),
2.77 (m, 2H), 3.46 (m, 2H), 3.81 (s, 3H), 3.87 (s, 3H), 3.94 (s,
3H), 4.02 (s, 3H), 4.08 (s, 2H), 5.71 (t, J ) 2.3 Hz, 1H), 6.03 (t,
J ) 2.8, 1H), 6.45 (s, 1H), 6.95 (s, 1H), 7.07 (s, 1H), 7.11 (s,
1H), 7.21 (s, 1H), 7.25 (s, 1H), 7.30 (s, 1H), 8.27 (t, J ) 7.2 Hz,
1H), 8.74 (bs, 2H), 9.05 (bs, 2H), 9.96 (s, 1H), 10.03 (s, 1H),
10.37 (s, 1H). Anal. (C37H48ClN11O7) C, H, Cl, N.
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-[2-
(4-ch lor op h en yl)-4-m et h ylen e-5-oxot et r a h yd r ofu r a n -2-
ylm et h oxy]-1H -p yr a zole-5-ca r b oxa m id o]p yr r ole-2-ca r -
b oxa m id o]p yr r ole -2-ca r b oxa m id o]p yr r ole -2-ca r b ox-
a m id o]p r op ion a m id in e Hyd r och lor id e (14). Yield, 32%;
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-[7-
(2-m e t h y l-4-m e t h y le n e -5-o x o t e t r a h y d r o fu r a n -2-y l)-
h eptyloxy]-2H-pyr azole-5-car boxam ido]pyr r ole-2-car box-
a m id o]p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]-
p r op ion a m id in e Hyd r och lor id e (8). Yield, 40%; yellow
1
white solid; mp 215-218 °C. H NMR (DMSO) δ: 2.61 (t, J )
1
solid; mp 132-136 °C. H NMR (DMSO) δ: 1.20 (t, J ) 4.61
6.1 Hz, 2H), 2.71 (dt, J ) 17.0 and 2.4 Hz, 1H), 2.93 (dt, J )
17.0 and 2.4 Hz, 1H), 3.43 (m, 2H), 3.81 (s, 3H), 3.84 (s, 3H),
3. 86 (s, 3H), 3.93 (s, 3H), 4.37 (d, J ) 11 Hz, 1H), 4.42 (d, J
) 11 Hz, 1H), 5.80 (t, J ) 2.2 Hz, 1H), 6.11 (t, J ) 2.7 Hz,
1H), 6.40 (s, 1H), 6.96 (s, 1H), 7.06 (s, 1H), 7.19 (s, 1H), 7.25
(s, 1H), 7.28 (s, 1H), 7.50 (s, 1H), 7.52 (m, 4H), 8.24 (t, J ) 7.2
Hz, 1H), 8.62 (bs, 2H), 8.99 (bs, 2H), 9.94 (s, 1H), 10.01 (s,
1H), 10.29 (s, 1H). Anal. (C38H41Cl2N11O7) C, H, Cl, N.
Hz, 2H), 1.32 (s, 3H), 1.68 (m, 10H), 2.63 (t, J ) 6.1 Hz, 2H),
2.71 (dt, J ) 16.8 and 2.4 Hz, 1H), 2.82 (dt, J ) 16.8 and 2.4
Hz, 1H), 3.49 (m, 2H), 3.72 (s, 3H), 3.75 (s, 3H), 3.81 (s, 3H),
3.94 (s, 3H), 4.17 (s, 2H), 5.70 (t, J ) 2.4 Hz, 1H), 6.03 (t, J )
2.4 Hz, 1H), 6.37 (s, 1H), 6.96 (s, 1H), 7.07 (s, 1H), 7.09 (s,
1H), 7.19 (s, 1H), 7.25 (s, 1H), 7.29 (s, 1H), 8.23 (t, J ) 7.2 Hz,
1H), 8.61 (bs, 2H), 8.92 (bs, 2H), 9.94 (s, 1H), 10.00 (s, 1H),
10.27 (s, 1H). Anal. (C39H52ClN11O7) C, H, Cl, N.
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-[2-
b ip h en yl-4-yl-4-m et h ylen e-5-oxot et r a h yd r ofu r a n -2-yl
m eth oxy]-1H-p yr a zole-5-ca r boxa m id o]p yr r ole-2-ca r box-
a m id o]p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]-
p r op ion a m id in e Hyd r och lor id e (15). Yield, 38%; white
solid; mp 227-230 °C. 1H NMR (DMSO) δ: 2.89 (t, J ) 6.1
Hz, 2H), 3.04 (dt, J ) 17.4 and 2.4 Hz, 1H), 3.48 (m, 2H), 3.64
(dt, J ) 17.4 and 2.4 Hz, 1H), 3.81 (s, 3H), 3.84 (s, 3H), 3. 86
(s, 3H), 3.93 (s, 3H), 4.14 (d, J ) 11 Hz, 1H), 4.47 (d, J ) 11
Hz, 1H), 5.80 (t, J ) 2.24 Hz, 1H), 6.20 (t, J ) 2.7 Hz, 1H),
6.43 (s, 1H), 6.96 (s, 1H), 7.06 (s, 1H), 7.24 (s, 1H), 7.27 (s,
1H), 7.44 (s, 1H), 7.48 (s, 1H), 7.56 (m, 9H), 8.26 (t, J ) 7.2
Hz, 1H), 8.60 (bs, 2H), 8.98 (bs, 2H), 9.94 (s, 1H), 10.01 (s,
1H), 10.29 (s, 1H). Anal. (C44H46ClN11O7) C, H, Cl, N.
3-[1-Me t h yl-4-[1-m e t h yl-4-[1-m e t h yl-3-(2-m e t h yl-4-
m e t h y le n e -5-o x o t e t r a h y d r o fu r a n -2-y lm e t h o x y )-1H -
p y r a z o le -5-c a r b o x a m i d o ]p y r r o le -2-c a r b o x a m i d o ]-
p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e
(9). Yield, 43%; white solid; mp 154-156 °C. 1H NMR (DMSO)
δ: 1.44 (s, 3H), 2.64 (t, J ) 6.1 Hz, 2H), 2.82 (dt, J ) 17.4 and
2.4 Hz, 1H), 3.05 (dt, J ) 17.4 and 2.4 Hz, 1H), 3.48 (m, 2H),
3.81 (s, 3H), 3.85 (s, 3H), 3.94 (s, 3H), 4.16 (s, 2H), 5.73 (t, J
) 2.2 Hz, 1H), 6.05 (t, J ) 2.6 Hz, 1H), 6.43 (s, 1H), 6.94 (s,
1H), 7.06 (s, 1H), 7.19 (s, 1H), 7.28 (s, 1H), 8.25 (t, J ) 7.2 Hz,
1H), 8.66 (bs, 2H), 9.00 (bs, 2H), 9.99 (s, 1H), 10.32 (s, 1H).
Anal. (C27H34ClN9O6) C, H, Cl, N.
3-[1-Meth yl-4-[1-m eth yl-3-(2-m eth yl-4-m eth ylen e-5-ox-
otetr a h yd r ofu r a n -2-ylm eth oxy)-1H-p yr a zole-5-ca r boxa -
m id o]p yr r ole-2-ca r b oxa m id o]p r op ion a m id in e H yd r o-
ch lor id e (10). Yield, 48%; white solid; mp 112-114 °C. 1H
NMR (DMSO) δ: 1.44 (s, 3H), 2.62 (t, J ) 2.6 Hz, 2H), 3.17
(dt, J ) 17.4 and 2.4 Hz, 1H), 3.48 (m, 2H), 3.62 (dt, J ) 17.4
and 2.4 Hz, 1H), 3.81 (s, 3H), 3.93 (s, 3H), 4.16 (s, 2H), 5.73 (t,
J ) 2.2 Hz, 1H), 6.05 (t, J ) 2.7 Hz, 1H), 6.43 (s, 1H), 6.95 (s,
1H), 7.24 (s, 1H), 8.33 (t, J ) 7.2 Hz, 1H), 8.70 (bs, 2H), 9.04
(bs, 2H), 10.33 (s, 1H). Anal. (C21H28ClN7O5) C, H, Cl, N.
3-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-(2-
ph en yl-4-m eth ylen e-5-oxotetr ah ydr ofu r an -2-ylm eth oxy)-
1H -p yr a zole -5-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]-
p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]p r op ion -
a m id in e Hyd r och lor id e (11). Yield, 33%; white solid; mp
225-228 °C. 1H NMR (DMSO) δ: 2.61 (t, J ) 6.1 Hz, 2H),
2.93 (m, 2H), 3.49 (m, 2H), 3.81 (s, 3H), 3.84 (s, 3H), 3.86 (s,
3H), 3.93 (s, 3H), 4.36 (d, J ) 11 Hz, 1H), 4.48 (d, J ) 11 Hz,
1H), 5.76 (t, J ) 2.2 Hz, 1H), 6.10 (t, J ) 2.7 Hz 1H), 6.40 (s,
1H), 6.96 (s, 1H), 7.06 (s, 1H), 7.19 (s, 1H), 7.25 (s, 1H), 7.28
(s, 1H), 7.38 (s, 1H), 7.49 (m, 5H), 8.24 (t, J ) 7.2, 1H), 8.58
(bs, 2H), 8.97 (bs, 2H), 9.94 (s, 1H), 10.02 (s, 1H), 10.27 (s,
1H). Anal. (C38H42ClN11O7) C, H, Cl, N.
2-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-(2-
m eth yl-4-m eth ylen e-5-oxotetr ah ydr ofu r an -2-ylm eth oxy)-
1H -p yr a zole -5-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]-
p yr r ole -2-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]e t h yl
Gu a n id in e Hyd r och lor id e (16). Yield, 47%; yellow solid; mp
1
128-132 °C. H NMR (DMSO) δ: 1.45 (s, 3H), 2.82 (dt, J )
17.6 and 2.5 Hz, 1H), 3.02 (dt, J ) 17.6 and 2.5 Hz, 1H), 3.15
(m, 2H), 3.43 (m, 2H), 3.82 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H),
3.95 (s, 3H), 4.17 (s, 2H), 5.76 (t, J ) 2.3 Hz, 1H), 6.12 (t, J )
2.8 Hz, 1H), 6.42 (s, 1H), 6.96 (s, 1H), 7.07 (s, 1H), 7.12 (s,
1H), 7.15 (bs, 2H), 7.20 (s, 1H), 7.25 (s, 1H), 7.29 (s, 1H), 7.38
(bs, 2H), 7.54 (bs, 1H), 8.25 (t, J ) 7.2 Hz, 1H), 9.96 (s, 1H),
10.02 (s, 1H), 10.32 (s, 1H). Anal. (C33H41ClN12O7) C, H, Cl,
N.
2-[1-Met h yl-4-[1-m et h yl-4-[1-m et h yl-4-[1-m et h yl-3-(2-
ph en yl-4-m eth ylen e-5-oxotetr ah ydr ofu r an -2-ylm eth oxy)-
1H -p yr a zole -5-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]-
p yr r ole-2-ca r b oxa m id o]p yr r ole-2-ca r b oxa m id o]et h yl-
gu a n id in e Hyd r och lor id e (17). Yield, 51%; white solid; mp
1
161-162 °C. H NMR (DMSO) δ: 3.09 (dt, J ) 17.0 and 2.4
Hz, 1H), 3.15 (m, 2H), 3.55 (m, 2H), 3.64 (dt, J ) 17.0 and 2.4
Hz, 1H), 3.82 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 3.93 (s, 3H),
4.36 (d, J ) 11 Hz, 1H), 4.48 (d, J ) 11 Hz, 1H), 5.79 (t, J )
2.2 Hz, 1H), 6.10 (t, J ) 2.7 Hz 1H), 6.41 (s, 1H), 6.96 (s, 1H),
7.07 (s, 1H), 7.15 (bs, 2H), 7.20 (s, 1H), 7.25 (s, 1H), 7.28 (s,
1H), 7.38 (s, 1H), 7.41 (bs, 2H), 7.45 (m, 5H), 7.52 (bs, 1H),
3-[1-Me t h yl-4-[1-m e t h yl-4-[1-m e t h yl-3-(2-p h e n yl-4-
m e t h y le n e -5-o x o t e t r a h y d r o fu r a n -2-y lm e t h o x y )-1H -
p y r a z o le -5-c a r b o x a m i d o ]p y r r o le -2-c a r b o x a m i d o ]-
p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e

Butyrolactones and Polypyrrole Binders
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2885
8.15 (t, J ) 7.2, 1H), 9.96 (s, 1H), 10.02 (s, 1H), 10.28 (s, 1H).
blocked with 5% nonfat milk in Tris-buffered saline containing
0.1% Tween-20 for 1 h, followed by overnight incubation with
anti-PARP monoclonal antibody (BD PharMingen; 1:1000
dilution in TBST supplemented with 3% nonfat milk). After
being washed and incubated with anti-mouse antibody con-
jugated to horseradish peroxidase (HRP) (Amersham Phar-
macia Biotech), the antigen-antibody complexes were visu-
alized by enhanced chemiluminescence (ECL, Amersham
Pharmacia Biotech) using the manufacturer’s protocol.
Anal. (C38H43ClN12O7) C, H, Cl, N.
Cytosta tic Assa ys. Murine leukemia L1210, murine mam-
mary carcinoma FM3A, human T-lymphocyte Molt 4/ C8, and
CEM cells were suspended at 300000-500000 cells/mL of
culture medium, and an amount of 100 µL of these cell
suspensions was added to 200 µL microtiter plate wells
containing 100 µL of an appropriate dilution of the test
compounds. After 2 days (L1210 and FM3A) or 3 days (Molt
4/ C8 and CEM) of incubation at 37 °C, the cell number was
determined using a Coulter counter. The 50% cytostatic
concentration (IC50) was defined as the compound concentra-
tion required to inhibit cell proliferation by 50%.
Ack n ow led gm en t. We thank Pharmacia & Upjohn
for providing distamycin A. Funding for this study was
provided by the Fonds voor Wetenschappelijk Onder-
zoek (FWO-Vlaanderen krediet Grant No. G. 0/04.98)
and the Geconcerteerde Onderzoeksacties (Contract No.
2000/12) to J . Balzarini and E. De Clercq. We thank
Lizette van Berckelaer, Anita Van Lierde, and Frieda
De Meyer for their excellent technical assistance. This
research was supported in part by FEDER Grant No.
1FD1997-1831 (J .B.B.). We thank J ose´ Este´vez (Hos-
pital Universitario Insular de Gran Canaria) for his
collaboration in the Western blot assays. This work was
also supported in part by Grant No. 156/1999 from the
Direccio´n General de Universidades e Investigacio´n,
Spain.
Biologica l Assa ys for t h e Ap op t ot ic St u d ies. Cell
Cu ltu r e. The human promyelocytic leukemia HL-60 cell line
established by Gallagher et al.24 was used in this study. Cells
were cultured in a suspension in RPMI-1640 medium (Invit-
rogen) supplemented with 10% (v/v) heat-inactivated fetal
bovine serum, penicillin (100 units mL-1), and streptomycin
(100 µg mL-1) in a humidified atmosphere of 95% air and 5%
CO2 at 37 °C. Cells were maintained at a density of <1 × 106
cells/mL. Compounds were dissolved in dimethyl sulfoxide
(DMSO) and stored as stock solutions at -20 °C. Further
dilutions were made in culture medium immediately prior to
use. In all experiments, the final concentration of DMSO did
not exceed 1% (v/v), a concentration that was nontoxic to the
host cells. Cell viability was determined using the trypan blue
exclusion test. To ensure an exponential growth, cells were
resuspended in fresh medium 24 h before each treatment.
Qu a n tita tive F lu or escen ce Micr oscop y. Cells (∼1 × 106)
were fixed in 3% paraformaldehyde for 10 min at room
temperature and then stained with 10 µg/mL bisbenzimide
trihydrochloride (Hoechst 33258) for 30 min at 37 °C before
fluorescence microscopy analysis. Apoptotic cells were identi-
fied by their fragmented chromatin.25 Stained nuclei with
condensed chromatin (supercondensed chromatin at the nuclear
periphery) or nuclei that were fragmented into multiple
smaller dense bodies were considered as apoptotic. Nuclei with
uncondensed and dispersed chromatin were considered as
nonapoptotic. A minimum of 500 cells were counted for each
sample, and each experiment was done in triplicate.
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