Novel benzoyl nitrogen mustard derivatives of pyrazole analogues of distamycin A: Synthesis and antileukemic activity

Article abstract of DOI:10.1016/S0968-0896(98)00205-3

The design and synthesis of novel benzoic acid mustard (BAM) derivatives of distamycin A bearing one or more pyrazole rings replacing the pyrrole rings of the latter are described. In vitro and in vivo activities against L1210 leukemia are reported and discussed. Some of these compounds show an activity profile comparable to tallimustine 1. All the compounds bearing the pyrazole ring close to the BAM moiety show reduced cytotoxicity in comparison to derivatives characterized by the BAM linked to a pyrrole: the same effect has not been observed when occurring at the amidine terminus of the oligopeptidic frame. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00205-3

Bioorganic & Medicinal Chemistry 7 (1999) 251±262
Novel Benzoyl Nitrogen Mustard Derivatives of Pyrazole
Analogues of Distamycin A: Synthesis and Antileukemic Activity
Pier Giovanni Baraldi,a,* Paolo Cozzi,b Cristina Geroni,b Nicola Mongelli,b
Romeo Romagnolia and Giampiero Spallutoa
a
Á
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, 44100 Ferrara, Italy
bPharmacia and Upjohn, Oncology, Preclinical Research, Milano, Italy
Received 23 June 1998; accepted 4 September 1998
AbstractÐThe design and synthesis of novel benzoic acid mustard (BAM) derivatives of distamycin A bearing one or more pyrazole
rings replacing the pyrrole rings of the latter are described. In vitro and in vivo activities against L1210 leukemia are reported and
discussed. Some of these compounds show an activity pro®le comparable to tallimustine 1. All the compounds bearing the pyrazole
ring close to the BAM moiety show reduced cytotoxicity in comparison to derivatives characterized by the BAM linked to a pyr-
role: the same e€ect has not been observed when occurring at the amidine terminus of the oligopeptidic frame. # 1999 Elsevier
Science Ltd. All rights reserved.
Introduction
analogue 7, we have also substituted the alkyl amidine
side chain at the C-terminus either with a dimethylami-
nopropylamino (compound 8) or a p-[bis(2-chloroethyl)
amino] phenylamino moiety (compound 9). Finally, we
have also investigated two four-ring homologues 10 and
11 of the compound 3, both obtained by insertion of a
pyrrole and pyrazole ring, respectively, on the N-temi-
nus of the tripeptidic frame.
Tallimustine 1 (FCE 24517),1 a distamycin A derivative
in which the formyl group has been replaced by a ben-
zoyl nitrogen mustard moiety (BAM), is a potent cyto-
toxic agent which exhibits a broad spectrum of anti-
tumor activity in a series of experimental tumor mod-
els.2 This compound retains the AT preference of dis-
tamycin A and appears to possess a high preference for
alkylation of 30-adenine-N3 atom located in the
sequence 50-TTTTGA-30.3 The potent anti-tumor activ-
ity of tallimustine may be due to its ability to inhibit the
binding of not well characterized protein factor(s)
recognizing this sequence of the DNA.4
Chemistry
Compounds 2±4 were prepared as previously reported.5
For the preparation of the compounds 5 and 6 two dif-
ferent convergent approaches were employed. Starting
from the known amino-amidine 12,8 condensation
with the acid 136 by 1-ethyl-3[3-(dimethylamino)propyl]-
carbodiimide hydrochloride (EDC) as coupling agent
a€orded the dipeptide 14 in 89% yield. Catalytic
hydrogenation of 14, followed by condensation with
the mustard derivatives 17 and 18 in presence of
EDC, gave the tripeptides 5 and 6 in 73 and 78% yield,
respectively (Scheme 1).
We have recently published5 the synthesis of three talli-
mustine analogues 2±4 which contain either one or two
pyrazoles instead of pyrroles and the encouraging
activity of these compounds prompted us to complete
this series of derivatives, considering other pyrrole±pyr-
azole combinations 5±7 in order to verify a possible
relationship between the number of pyrazoles, their
position and cytotoxicity. Since polypyrrole compounds
are susceptible to oxidative breakdown,6 all these pyr-
azolo tallimustine-like derivatives were prepared as
potentially more stable DNA minor groove binders
aimed to improve the relative instability of the poly-
pyrrolic frame.7 Moreover, in the case of tripyrazole
The syntheses of the acids 17 and 18 were performed by
coupling methyl 1-methyl-4-aminopyrrole-2-carboxylate9
and methyl 1-methyl-3-aminopyrazole-5-carboxylate,6
respectively, with p-[bis-(2-chloroethyl)amino]benzoyl
chloride,10,11 and subsequent alkaline hydrolysis of
esters 15 and 16, in 76 and 78% yield, respectively.
Key words: Anti-tumor compounds; DNA, leukemia; substituent
e€ects.
*Corresponding author. Tel: +39-(0)532-291293; fax: +39-(0)532-
291296; e-mail: pgb@ifeuniv.unife.it
In the second approach, the BAM moiety was intro-
duced at the last step, after the preparation of tripep-
tidic frame, as described in Scheme 2. The unstable
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00205-3

252
P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
Chart 1.
amino-nitrile intermediate 1912 was condensed with the
acid 13 to a€ord 20 in 87% yield, which after a reduc-
tion±condensation process with the acids 2112 and 22 led
to the corresponding derivatives 23 and 24 in 71% and
73% yield, respectively. Treatment of 23 and 24 under
Pinner reaction conditions13 (HCl in anhydrous ethanol
and then ammonia), a€orded the corresponding nitro
amidines 25 and 26 in 93 and 89% yield, respectively.
The subsequent catalytic reduction of the nitro group
gave the relative stable amine intermediates, which
without isolation or puri®cation, were directly coupled
with 4-[bis(2-chloroethyl)amino]benzoyl chloride to give
5 and 6 in 58 and 68% yield, respectively.
synthesis of 7, in which the amidino group was intro-
duced at an early stage and that proved to be unsa-
tisfactory owing to the high sensitivity and instability of
the amino-amidines 34 and 35. The compound 34 was
obtained after catalytic reduction of the nitro-amidine
32 prepared by Pinner reaction from nitrile 29.
Attempted condensation of 34 and the acid 18 with
EDC did not supply the desire product.
In the second attempt, the nitrogen mustard was teth-
ered to the pyrazole tripeptide 35, which was prepared
by catalytic hydrogenation of the nitro-amidine 33
obtained submitting to Pinner reaction the nitro-nitrile
31. Latter compound was prepared by condensation
between the known compound 306 and the acid 13 in
83% yield. Condensation of 35 and p-[bis-(2-chloro-
ethyl)amino]benzoyl chloride gave only a mixture of
undetectable products, therefore we resolved to introduce
the amidine side chain at the last step (Scheme 5).
Starting from the already synthesized ester 36,6 catalytic
removal of the benzyloxycarbonyl (Cbz or Z) protecting
group and subsequent condensation with 18 provided
the tripeptide 38 in 75% yield. In order to e€ect con-
densation with the b-aminopropionamidine moiety
(prepared as already reported14) the ester 38 was con-
verted quantitatively into the carboxylic acid 39 by
For the syntheses of compounds 3, 10, and 11, the
nitrile 27 was employed as starting material. The treat-
ment of 27 under Pinner reaction conditions gave the
corresponding nitro-amidine 28 in 93% yield, which
after a reduction±condensation process in the presence
of EDC with the pyrrole and pyrazole derivatives 17
and 18, led to the tetrapeptides 10 and 11, respectively,
with 53 and 46% yield, respectively (Scheme 3).
We have employed three di€erent routes for the syn-
thesis of the pyrazole-tallimustine analogue 7. In the
Scheme 4 were reported two initial approaches to the

P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
253
Scheme 1.
Scheme 2.

254
P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
alkaline hydrolysis. Latter compound was subsequently
coupled by the corresponding N-hydroxysuccinimide
ester, with preformed b-aminopropionamidine dihydro-
bromide to give desired compound 7 in 93.5% yield.
The same strategy was followed for the synthesis of C-
terminus-modi®ed analogues, in which a dimethylami-
nopropylamino (compound 8) or a p-[bis-(2-chloro-
ethyl)amino]anilino (compound 9) moiety replaced the
parent amidine group (Scheme 5): these compounds
were obtained in 68 and 66% yield, respectively.
cytotoxicity. In fact, the introduction of pyrrole ring
between the benzoic acid mustard and the N-terminal
pyrrole of 3, to give the homologue 10, yielded an
increase of 10-fold cytotoxicity with respect to the par-
ent compound 3 (225.2 and 20.10 ng/mL for 3 and 10,
respectively). Moreover, this increase in the number of
pyrrole units led to an increase of in vivo potency, but
failed to improve anti-leukemic activity. In fact, at their
optimal doses (O.D.) (12.5 and 6.25 mg/Kg for 3 and 10,
respectively) the less potent compound 3 was more
e€ective than the pyrrole homologue 10 producing an
increase of the median survival time (T/C% 213 versus
167 for 3 and 10, respectively). On the contrary, the
pyrazole homologue 11 showed threefold less potent
antiproliferative activity than the parent compound 3
(225.2 and 608.3 ng/mL for 3 and 11, respectively).
Results and Discussion
All the synthesized compounds (5±11) were assayed in
vitro against L1210 murine leukemia cell lines, L1210
cells resistant to tallimustine (L1210/tallimustine) and
to doxorubicin (L1210/DX) (obtained from NCI,
Bethesda, MD). Only some representative compounds
were tested in vivo against L1210 murine leukemia and
M5076 murine reticulosarcoma as solid tumor model.
All the results for the compounds tested both in vivo
and in vitro against L1210 murine leukemia were given
in Tables 1 and 2.
The tripyrazole analogue 7 resulted to be about 20-fold
less cytotoxic in vitro than the tripyrrole counterpart
tallimustine (1325 versus 50.3 ng/mL), and substantially
devoid of antileukemic activity in vivo (O.D.= 12.5 mg/
Kg, T/C%=100 versus O.D.=6.25 mg/Kg, T/C%=
125).
The cytotoxicities and the anti-leukemic activities of all
synthesized compounds were evaluated as previously
described and compared with that of tallimustine.15
In the design of the compound 8, an analogue of 7 that
contains a dimethylaminopropyl group (extimated pKa
9.3) at the C-terminus instead of strongly basic propio-
namidino moiety (extimated pKa 12), this function
should be protonated at physiological pH of 7.4 and
thus it would be attracted to DNA as the amidine moiety.
For the compound 3, the increased number of pyrrole
units in the oligopeptidic frame led to an increase of
Scheme 3.

P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
255
Scheme 4.
Scheme 5.

256
P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
Table 1 In vivo and in vitro activity against L1210 murine leukemia
Kg, T/C%=125 for tallimustine). The derivatives 2
and 3 exhibited also signi®cant activity in vivo against
M 5076 murine reticulosarcoma, although they were
less potent than the tallimustine (O.D=1.56 mg/Kg,
T/C%=150 for tallimustine 1, O.D=6.25 mg/Kg,
T/C%=225 for 2; O.D=12.5 mg/Kg, T/C%=185 for
3). These data reveal that 2 was the best compound
of this series, possessing a very high activity both
against L1210 murine leukemia and M 5076 solid tumor
model.
Compound
in vitro L1210
in vivo L1210
%c T/C
a
IC50
(ng/mL)
O.D.b
(mg/Kg)
1
2
3
4
5
6
7
8
50.3
35.00
225.20
1398.00
78.60
1986.80
1325.00
3000.00
50000
20.10
608.30
6.25
6.25
12.50
12.50
n.dd
n.d
12.50
n.d
n.d
125
213
213
218
n.d
n.d
100
n.d
n.d
167
n.d
The compounds 4 and 6, in which one or two pyrrole
units near the BAM moiety were replaced by corre-
sponding pyrazole rings, respectively, were substantially
inactive (IC50 ng/mL; 1398 and 1986.8, respectively).
9
10
11
6.25
n.d
aIC50=50% inhibitory concentration represents the mean from dose±
response curves of at least three experiments.
The anti-leukemic activity of derivatives of this series
was strictly dependent from the position of the pyrazole
ring, and appeared lower when pyrazole was close to
alkylating moiety.
bO.D.=optimal dose; optimal non toxic dose <LD10
.
c%T/C=median survival time of treated versus untreated miceÂ100.
dn.d.=not determined.
The cytoxicities of synthesized compounds on resistant
leukaemic cell lines were reported in the Table 2. The
results obtained showed that only 10 and 11 were inac-
tive (resistance index (R.I.) from 180 to 60), whereas the
other compounds presented low R.I. (from 30 to 1). It
may be noted that, while the compound 10 showed a
potent cytostatic activity against L1210 leukemia (IC50
20.1 ng/mL), it was completely inactive against L1210/
Dx and L1210/tallimustine (IC50 2465.8 and 2550.7 ng/
mL, respectively).
Table 2 In vitro activity of compounds 1±11 against L1210 leukemia
cells, L1210 resistants to the doxorubicin (DX) and tallimustine
Compound
L1210
L1210/DX
L1210/tallimustine
IC50
(ng/mL)
IC50
(ng/mL)
R.I.
IC50
(ng/mL)
R.I
1
2
3
4
5
6
7
8
50.3
35.00
225.20
1398.00
78.60
1986.80
1325.00 11100.00
3000.00 14500.00
638.80
915.00
6894.20
7980.00
533.97
28.02
26.14
30.6
5.71
6.79
1.64
8.38
4.83
1
528.80
1015.00
3089.50
2658.00
n.d
21.7
29
13.72
1.9
n.d
n.d
4.36
1.48
1
3262.50
n.d
5800.00
4450.00
50000
2550.70 127
175.5
Conclusions
9
10
11
50000
20.10
608.30 36585.5
50000
2465.80 122.6
In this series of pyrrole±pyrazole tallimustine analogue
2±7, we have found that the pyrrole position was critical
for the activity. The presence of this heterocycle nearby
benzoyl nitrogen mustard moiety was necessary for
activity both in vitro and in vivo. This was con®rmed
for the compound 3, where increasing the number of
pyrrole rings by the introduction of a pyrrole nearby to
the alkylating moiety gave the corresponding derivative
10 which was much more active than the parent com-
pound 3. On the contrary, the opposite e€ect was
obtained with the incorporation of a pyrazole ring, as in
compound 11, which caused a decrease of L1210 activ-
ity in comparison 3 and 10.
60.14 106752.5
IC50=50% inhibitory concentration represents the mean from dose±
response curves of at least three experiments. R.I (resistance
index)=ratio between IC50 values on resistant cells and sensitive cells.
Nevertheless, this derivative 8 was twofold less active
than the corresponding compound 7 bearing the ami-
dino moiety (IC50 ng/mL, 1325 versus 3000). Introduc-
tion of a second benzoyl nitrogen mustard moiety, as
for the compound 9, in place of the charged amidine
end group on the C-terminus proved to be completely
ine€ective (IC50 ng/mL, 1325 for 7 versus 5000 for 9).
Experimental
Materials and methods
Among the hybrids pyrrole±pyrazole tripeptidics syn-
thesized, the two tallimustine isosters 2 and 5, in which
the pyrrole near the amidine terminus and the central
pyrrole were replaced by corresponding pyrazole rings
respectively, showed cytotoxicity L1210 equivalent to
tallimustine (IC50 ng/mL; 35 and 78.6 ng/mL versus
50.3 ng/mL, respectively). Compound 3, in which two
pyrrole units near the amidine terminus was replaced
with the same number of pyrazole rings, was less active
than tallimustine (IC50 ng/mL, 225 versus 50.3) but as
compound 2 showed superior antileukemic activity in
vivo (O.D.=6.25 mg/Kg, T/C%=213 for 2 and O.D.=
12.5 mg/Kg, T/C%=213 for 3 versus O.D.=6.25 mg/
All reactions were carried out under argon atmosphere,
unless otherwise described. Standard syringe techniques
were applied for tranferring anhydrous solvents. Reac-
tion courses and product mixtures were routinely mon-
itored by TLC on silica gel (precoated F254 Merk plates)
1
and visualized with aqueous KMnO4. H NMR spectra
were obtained in DMSO solutions with a Bruker AC
200 spectrometer. Chemical shifts (d) are given in ppm
up®eld from tetramethylsilane. Melting points (mp)
were determined on a Buchi±Tottoli apparatus and are
1
uncorrected. All products reported showed H NMR

P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
257
spectra in agreement with the assigned structures. Ele-
mental analyses were conducted by the Mycroanalytical
Laboratory of the Chemistry Department of the Uni-
versity of Ferrara. All compounds obtained commercially
were used without further puri®cation. Organic solu-
tions were dried over anhydrous MgSO4. Methanol was
distilled from magnesium turnings, dioxane was distilled
from calcium hydride and anhydrous DMF was distilled
from calcium chloride and stored over molecular sieves
(3 A). In high-pressure hydrogenation experiments, a
Parr shaker on a high-pressure autoclave was used.
16 was obtained as a yellow powder. Yield 1.49 g
(3.72 mmol, 72%); mp 155±157 ꢀC; H NMR (DMSO-
1
d6) d 3.66 (d, J=6.2 Hz, 4H), 3.77 (d, J=6.2 Hz, 4H),
3.88 (s, 3H), 4.02 (s, 3H), 6.67 (d, J=9 Hz, 2H), 7.40 (s,
1H), 7.78 (d, J=9 Hz, 2H), 8.82 (s, 1H); anal. calcd for
C17H20N4O3Cl2: C, 51.14; H, 5.05; N, 14.03; Cl, 17.76.
Found: C, 51.05; H, 4.97; N, 13.89; Cl, 17.68.
1-Methyl-4-[4-bis-(2-chloroethyl)aminophenylamido]-pyrrole-
2-carboxylic acid (17). To a well-stirred solution of 15
(300 mg, 0.75 mmol) in 3 mL of dioxane was added 2 N
aqueous KOH (0.75 mL) and the resulting mixture stir-
red at room temperature for 2 h. The clear solution was
evaporated to remove dioxane, diluted with water
(5 mL), cooled on an icewater bath and acidi®ed with
10% HCl to pH 2. The suspension was extracted with
EtOAc (2Â10 mL) and the organic layers were com-
bined, dried (Na2SO4), and concentrated. The resulting
residue was precipitated from EtOAc/hexane to give the
3-[1-Methyl-4-(1-methyl-3-benzyloxycarbonylaminopyrazole-
5-carboxamido)-pyrrole-2-carboxamido] propioniamidine
hydrochloride (14). To a solution of amino-amidine 12
(706 mg, 2.5 mmol) in anhydrous DMF (3 mL), cooled
to 0 ꢀC, were added triethylamine (TEA) (0.35 mL,
2.5 mmol), the acid 13 (675 mg, 0.25 mmol) and then
EDC (960 mg, 5 mmol). The reaction mixture was
slowly warmed to room temperature and allowed to stir
for 18 h. After this time, the solution was acidi®ed with
20% HCl at pH 2 and DMF was removed under
reduced pressure The resulting solid puri®ed by ¯ash
column chromatography with CH2Cl2/EtOH/H2O (10/
10/0.8, v/v) a€orded a yellow oil which was crystallized
from methanol/ethyl ether to give 14 as a white solid.
product 17 as
a brown powder. Yield 219 mg
(0.57 mmol, 76%); mp 185±187 ꢀC; H NMR (DMSO-
d6) d 3.34 (m, 8H), 3.78 (s, 3H), 6.63 (s, 1H), 6.80 (d,
J=6.8 Hz, 2H), 7.28 (s, 1H), 7.79 (d, J=6.8 Hz, 2H),
9.98 (s, 1H), 12.3 (bs, 1H); anal. calcd for
C17H19N3O3Cl2: C, 53.14; H, 4.98; N, 10.94; Cl, 18.4.
Found: C, 52.6; H, 4.1; N, 10.1; Cl, 17.9.
1
Yield 1.12 g (2.22 mmol, 89%); mp 213 ꢀC; H NMR
1
(DMSO-d6) d 2.63 (t, J=6 Hz, 2H), 3.50 (m, 2H), 3.82
(s, 3H), 3.97 (s, 3H), 5.16 (s, 2H), 6.96 (s, 1H), 7.11 (s,
2H), 7.25 (s, 1H), 7.39 (m, 5H), 8.33 (t, J=6 Hz, 1H),
8.77 (s, 2H), 9.02 (s, 2H), 10.24 (s, 1H), 10.43 (s, 1H);
Anal calcd for C22H27N8O4Cl: C, 52.54; H, 5.41; N,
22.28; Cl, 7.05. Found: C, 52.48; H, 5.32; N, 22.20; Cl,
6.98.
1-Methyl-3-[4-bis-(2-chloroethyl)aminophenylamido]-pyr-
azole-5-carboxylic acid (18). Following the same proce-
dure reported for the synthesis of 17, starting from 16
(500 mg, 1.25 mmol) and 2 N aqueous KOH (1 mL,
2 equiv), after workup the acid 18 was obtained as
brown solid. Yield 404 mg (1.05 mmol, 84%); mp 233±
235 ꢀC; H NMR (DMSO-d6) d 2.50 (m, 4H), 3.78 (m,
1
4H), 4.03 (s, 3H), 6.82 (d, J=9 Hz, 2H), 7.12 (s, 1H),
7.92 (d, J=9 Hz, 2H), 10.66 (s, 1H), 13.25 (bs, 1H);
anal. calcd for C16H18N4O3Cl2: C, 49.9; H, 4.71; N,
14.5; Cl, 18.4. Found: C, 49; H, 4.1; N, 13.7; Cl, 17.7.
Methyl 1-methyl-4-[4-bis-(2-chloroethyl)aminophenylamido]-
pyrrole-2-carboxylate (15). To a solution of p-N,N-
bis(2-chloroethyl)aminobenzoyl
chloride
(700 mg,
2.5 mmol) in anhydrous dioxane (5 mL) was added
dropwise to a mixture of methyl 1-methyl-4-amino-
pyrrole-2-carboxylate9 (385 mg, 2.5 mmol) and triethyl-
amine (TEA) (0.35 mL, 2.5 mmol) in anhydrous
dioxane (10 mL) cooled to 0 ꢀC. The reaction mixture
was allowed to stir at room temperature overnight and
then concentrated under reduced pressure to a brown
solid, which was dissolved in EtOAc and washed with
water (2Â10 mL). The organic layer was dried
(Na2SO4), concentrated, and the resulting residue pre-
cipitated from EtOAc/petroleum ether to give 15 as a
®ne brown powder. Yield 736 mg (1.87 mmol, 74%); mp
3-[1-Methyl-4-(1-methyl-3-benzyloxycarbonylaminopyrazole-
5-carboxamido)-pyrrole-2-carboxamido]propionitrile (20).
To a solution of amino-nitrile 19 (384 mg, 2 mmol) and
acid 13 (522 mg, 2 mmol) in DMF (5 mL) at 0 ꢀC was
added EDC (786 mg, 4 mmol). The reaction mixture was
allowed to warm to room temperature and then stirred
for 24 h. The solvent was removed under reduced pres-
sure, the residue taken up in EtOAc (10 mL) and then
washed once with 5% aq HCl. The organic layer was
dried on Na2SO4 and solvent removed under reduced
pressure. The product puri®ed by crystallization
(EtOAc/petroleum ether) a€orded 20 as a yellow pow-
der. Yield 760 mg (1.74 mmol, 87%); mp 232 ꢀC; 1H
NMR (DMSO-d6) d 2.68 (t, J=6.2 Hz, 2H), 3.58 (d,
J=6.4 Hz, 2H), 3.81 (s, 3H), 4.01 (s, 3H), 5.22 (s, 2H),
6.97 (s, 1H), 7.10 (s, 2H), 7.23 (s, 1H), 7.39 (m, 5H), 8.38
(t, J=6 Hz, 1H), 10.33 (s, 1H), 10.52 (s, 1H); anal. calcd
for C22H23N7O4: C, 58.79; H, 5.16; N, 21.81. Found: C,
58.67; H, 5.07; N, 21.74.
175 ꢀC; H NMR (DMSO-d6) d 3.34 (m, 8H), 3.78 (s,
1
3H), 4.10 (s, 3H), 6.8 (d, J=6.8 Hz, 2H), 6.88 (s, 1H),
7.46 (s, 1H), 7.79 (d, J=6.4 Hz, 2H), 9.98 (s, 1H); anal.
calcd for C18H21N3O3Cl2: C, 54.28; H, 5.31; N, 10.55;
Cl, 17.8. Found: C, 54.16; H, 5.12; N, 10.41; Cl, 17.68.
Methyl 1-methyl-3-[4-bis-(2-chloroethyl)aminophenylamido]-
pyrazole-5-carboxylate (16). Following the same proce-
dure reported for the synthesis of 15, starting from
p-N,N-bis(2-chloroethyl)aminobenzoyl chloride (1.6 g,
5.17 mmol), methyl 1-methyl-3-aminopyrazole 5-car-
boxylate6 (802 mg, 5.17 mmol), and anhydrous triethyl-
amine (0.85 mL, 5.17 mmol), after workup compound
1-Methyl-3-nitropyrazole-5-carboxylic acid (22). Follow-
ing the same procedure reported for the synthesis of 17,
starting from methyl 1-methyl-3-nitropyrazole-5-car-
boxylate16 (600 mg, 3.24 mmol) and 2 N aqueous KOH

258
P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
(3.5 mL, 6.48 mmol), after workup the compound 22
was obtained as a yellow powder. Yield 537 mg
(3.14 mmol, 97%); mp 170±172 C; H NMR (DMSO-
d6) d 4.27 (s, 3H), 7.38 (s, 1H); anal. calcd for
C5H5N3O4: C, 35.1; H, 2.95; N, 24.56. Found: C, 35.01;
H, 2.88; N, 24.48.
7.32 (bs, 2H), 7.56 (s, 1H), 7.74 (s, 1H), 7.86 (s, 1H),
8.21 (bs, 1H), 8.78 (bs, 2H), 8.98 (bs, 2H), 10.98 (s, 1H),
11.16 (s, 1H); anal. calcd for C20H25N10O5Cl: C, 46.11;
H, 4.84; N, 26.89; Cl, 6.81. Found: C, 46.02; H, 4.73; N,
26.80; Cl, 6.73.
ꢀ
1
3-[1-Methyl-4-[1-methyl-3-(1-methyl-3-nitropyrazole-5-
carboxamido)pyrazole-5-carboxamido]pyrrole-2-carbox-
amido]propionamidine hydrochloride (26). According to
the procedure reported for the Pinner reaction on the
compound 23, starting from 24 (940 mg, 2 mmol), the
amidine 26 was obtained in 89% yield (932 mg): mp
232 C; H NMR (DMSO-d6) d 2.61 (t, J=6 Hz, 2H),
3.44 (t, J=6 Hz, 2H), 4.02 (s, 3H), 4.08 (s, 3H), 4.24 (s,
3H), 7.24 (s, 1H), 7.31 (s, 1H), 7.63 (s, 1H), 7.97 (s, 1H),
8.84 (bs, 1H), 8.86 (bs, 2H), 9.04 (bs, 2H), 11.32 (s, 1H),
11.58 (s, 1H); anal. calcd for C19H24N11O5Cl: C, 43.72;
H, 4.63; N, 29.52; Cl, 6.79. Found: C, 43.64; H, 4.55;
N,29.45; Cl, 6.68.
3-[1-Methyl-4-[1-methyl-3-(1-methyl-4-nitropyrrole-2-car-
boxamido)pyrazole-5-carboxamido]pyrrole-2-carboxamido]-
propionitrile (23). A solution of 20 (868 mg, 2 mmol) in
15 mL of a mixture MeOH/dioxane (2/1, v/v) was
hydrogenated over 100 mg of 10% Pd/C at 50 psi for
3 h. The catalyst was removed by ®ltration, the ®ltrate
was concentrated to give a green oil which was used
without puri®cation for the next step. This crude amine
was dissolved in anhydrous DMF (7 mL) and at this
solution were added 21 (340 mg, 2 mmol), HOBt
(270 mg, 2 mmol), and EDC (384 mg, 2 mmol). The
reaction mixture was stirred at room temperature for
12 h, and the evaporation of the DMF under reduced
pressure gave a brown solid which was suspended in a
mixture EtOAc (10 mL) and 10% aq HCl. This suspen-
sion was stirred for 2 h and then ®ltered, rinsed with
water, to give 23 as a pale-yellow solid. Yield 663 mg,
ꢀ
1
3-[1-Methyl-4-[1-methyl-4-[1-methyl-4-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrrole-2-carboxamido]pyrazole-5-
carboxamido]pyrrole-2-carboxamido]
propionamidine
hydrochloride (5). Procedure A. A solution of 14
(500 mg, 1 mmol) in 15 mL of a mixture methanol/diox-
ane (1/1, v/v) and 5% aq HCl (6 drops) was hydro-
genated at 50 psi over 10% Pd/C using a Parr
hydrogenator for 4 h. The catalyst was removed by ®l-
tration, the yellow ®ltrate concentrated, and the residue
used immediately in the following step. The above
amine dissolved in 5 mL of anhydrous DMF was treated
at 0 ꢀC with N-ethyldiisopropylamine (DIPEA or
Hunig's base) (192 mL, 1 mmol) and after 5 min, with 17
(385 mg, 1 mmol) and then EDC (384 mg, 2 mmol). The
reaction mixture was stirred for 18 h at room tempera-
ture. The solvent was evaporated to dryness in vacuo,
and the resulting residue was puri®ed by ¯ash column
chromatography with CH2Cl2/MeOH (4/1, v/v) to give
5 as a brown solid. Yield 536 mg, 0.73 mmol, 73%; mp
71%; mp 211±213 ꢀC; H NMR (DMSO-d6) d 2.62 (t,
1
J=6 Hz, 2H), 3.41 (t, J=6 Hz, 2H), 3.85 (s, 3H), 4.03 (s,
3H), 4.12 (s, 3H), 7.24 (s, 1H), 7.29 (s, 1H), 7.46 (s, 1H),
7.72 (s, 1H), 7.89 (s, 1H), 8.15 (bs, 1H), 10.92 (s, 1H),
11.28 (s, 1H); anal. calcd for C20H21N9O5: C, 51.39; H,
4.53; N, 26.97. Found: C, 51.29; H, 4.48; N, 26.89.
3-[1-Methyl-4-[1-methyl-3-(1-methyl-3-nitropyrazole-5-
carboxamido)pyrazole-5-carboxamido]pyrrole-2-carbox-
amido]propionitrile (24). Starting from 20 (651,
1.5 mmol), 22 (391 mg, 1.5 mmol), HOBt (405 mg,
3 mmol), and EDC (576 mg, 3 mmol), the reaction was
carried out via a similar procedure as described above
for 23 to a€ord 24 as a white solid. Yield 512 mg, 73%;
mp 255±256 ꢀC; 1H NMR (DMSO-d6) d 2.62 (t,
J=6 Hz, 2H), 3.41 (t, J=6 Hz, 2H), 3.85 (s, 3H), 4.03 (s,
3H), 4.12 (s, 3H), 7.29 (s, 1H), 7.46 (s, 1H), 7.72 (s, 1H),
7.89 (s, 1H), 8.15 (bs, 1H), 8.56 (s, 2H), 8.99 (s, 2H),
10.92 (s, 1H), 11.28 (s, 1H); anal. calcd for
C19H20N10O5: C, 48.72; H, 4.3; N, 29.9. Found: C,
48.67; H, 4.25; N, 29.79.
254±256 ꢀC; H NMR (DMSO-d6) d 2.56 (m, 2H), 3.32
1
(m, 2H), 3.49 (m, 8H), 3.79 (s, 3H), 3.82 (s, 3H), 4.04 (s,
3H), 6.83 (d, J=8.8 Hz, 2H), 6.98 (s, 1H), 7.15 (s, 1H),
7.24 (s, 1H), 7.42 (s, 2H), 7.84 (d, J=8.8 Hz, 2H), 8.29
(bs, 1H), 8.51 (s, 2H), 8.94 (s, 2H), 10.07 (s, 1H), 10.44
(s, 1H), 10.64 (s, 1H); anal. calcd for C31H38N11O4Cl3:
C, 50.65; H, 5.21; N, 20.96; Cl, 14.47. Found: C, 50.58;
H, 5.11; N, 20.88; Cl, 14.40.
3-[1-Methyl-4-[1-methyl-3-(1-methyl-4-nitropyrrole-2-car-
boxamido)pyrazole-5-carboxamido]pyrrole-2-carboxamido]-
propionamidine hydrochloride (25). To a cooled suspen-
sion (0 ꢀC) of 23 (467 mg, 1 mmol) in absolute ethanol
(200 mL) was bubbled with anhydrous HCl for 20 min.
The reaction mixture was stirred at 0 ꢀC for an addi-
tional hour and at room temperature for 10 min.
Removal of the solvent under reduced pressure gave a
solid residue, which was redissolved in absolute ethanol
(150 mL). The mixture was cooled (0 ꢀC) and anhydrous
ammonia was bubbled inside for 10 min. After 24 h at
room temperature, the solvent was removed in vacuo to
give a white solid, which was washed with ethyl ether
(twice), ®ltered, and dried under high vacuum at 40 ꢀC
to give amidine 25. Yield 519 mg, 93%; mp 287±289 ꢀC;
1H NMR (DMSO-d6) d 2.53 (t, J=6 Hz, 2H), 3.44 (t,
J=6 Hz, 2H), 3.82 (s, 3H), 4.00 (s, 3H), 4.08 (s, 3H),
Procedure B. The compound 25 (260 mg, 0.5 mmol) was
submitted to hydrogenation following the same proce-
dure reported for 14. After the workup, the amine was
used immediately in the following step. The above
amine was dissolved in 5 mL of a mixture dioxane/water
(4/1, v/v) was treated at 0 ꢀC with NaHCO3 (168 mg,
2 mmol) and p-N,N-bis(2-chloroethyl)aminobenzoyl
chloride (140 mg, 0.5 mmol). The reaction mixture was
stirred for 3 h at room temperature. After TLC analysis
indicated that all of the starting material had dis-
appeared, the reaction was acidi®ed to pH 5 with 5% aq
HCl and concentrated under reduced pressure. The
resulting residue was puri®ed by ¯ash column chroma-
tography with CH2Cl2/MeOH (4/1, v/v) to give 5 as a
brown solid. Yield 213 mg, 0.29 mmol, 58%.

P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
259
3-[1-Methyl-4-[1-methyl-3-[1-methyl-3-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrazole-5-carboxamido]pyrazole-5-
for the synthesis of 5, starting from 28 (230 mg,
0.44 mmol), NaHCO3 (130 mg, 1.32 mmol) and p-N,N-
carboxamido]pyrrole-2-carboxamido]
propionamidine
bis(2-chloroethyl)aminobenzoyl
chloride
(308 mg,
hydrochloride (6). According to the procedure A repor-
ted for the synthesis of 5, starting from 14 (400 mg,
0.78 mmol), Hunig's base (133 mL, 0.78 mmol), 18
(300 mg, 0.78 mmol), and EDC (300 mg, 1.56 mmol),
after workup the resulting residue was puri®ed by ¯ash
column chromatography with CH2Cl2/MeOH (5/1, v/v)
to give 6 as a yellow solid. Yield 390 mg, 0.53 mmol,
1.1 mmol), after workup and ¯ash column chromatog-
raphy with CH2Cl2/MeOH (40/10, v/v), 3 was obtained
as a white solid. Yield 216 mg, 0.295 mmol, 67%; mp
108±110 ꢀC; H NMR (DMSO-d6) d 2,63 (m, 2H), 3.54
1
(m, 2H), 3.79 (m, 8H), 3.87 (s, 3H), 4.02 (s, 3H), 4.05 (s,
3H), 6.83 (d, J=9 Hz, 2H), 7.16 (s, 1H), 7.31 (s, 1H),
7.41 (s, 1H), 7.54 (s, 1H), 7.86 (d, J=9 Hz, 2H), 8.65
(bs, 2H), 8.82 (m, 1H), 9.02 (bs, 2H), 10.07 (s, 1H),
10.55 (s, 1H), 11.15 (s, 1H); anal. calcd for
C30H37N12O4Cl3: C, 48.95; H, 5.07; N, 22.83; Cl, 14.45.
Found: C, 48.87; H, 4.98; N, 22.75; Cl, 14.38.
68%; mp 278±280 ꢀC; H NMR (DMSO-d6) d 2.63 (m,
1
2H), 3.50 (m, 2H), 3.79 (s, 3H), 3.83 (s, 3H), 4.07 (s,
3H), 4.43 (m, 8H), 6.82 (d, J=8.6 Hz, 2H), 6.99 (s, 1H),
7.26 (s, 1H), 7.45 (s, 1H), 7.57 (s, 2H), 7.93 (d,
J=8.8 Hz, 2H), 8.32 (bs, 1H), 8.66 (s, 2H), 9.00 (s, 2H),
10.48 (s, 1H), 10.64 (s, 1H), 11.19 (s, 1H); anal. calcd for
C30H38N12O4Cl3: C, 48.89; H, 5.2; N, 22.8; Cl, 14.43.
Found: C, 48.78; H, 5.11; N, 22.72; Cl, 14.31.
3-[1-Methyl-3-[1-methyl-3-[1-methyl-4-[1-methyl-4-[4-bis-
(2-chloroethyl)aminobenzenamido-pyrrole-2-carboxamido]-
pyrrole-2-carboxamido]pyrazole-5-carboxamido]pyrazole-
5-carboxamido] propionamidine hydrochloride (10).
Following the procedure A reported for the synthesis
of 5, starting from the nitro-amidine 28 (300 mg,
0.516 mmol), DIPEA (88 mL, 0.516 mmol), 17 (270 mg,
0.69 mmol) and then EDC (240 mg, 1.2 mmol), after
workup and ¯ash column chromatography with CH2Cl2/
MeOH (50/10, v/v), 10 was obtained as a white solid.
Yield 234 mg, 0.273 mmol, 53%; mp 257±260 ꢀC; 1H
NMR (DMSO-d6) d 2.3±2.6 (m, 4H), 3.4±3.5 (m, 8H),
3.79 (s, 3H), 3.81 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 6.83
(d, J=7 Hz, 2H), 6.96 (s, 1H), 7.07 (s, 1H), 7.09 (s, 1H),
7.18 (s, 1H), 7.24 (s, 1H), 7.30 (s, 1H), 7.84 (d, J=7 Hz,
2H), 8.24 (m, 1H), 8.52 (s, 2H), 8.94 (s, 2H), 10.01
(s, 1H), 10.03 (s, 1H), 10.55 (s, 1H), 11.15 (s, 1H);
anal. calcd for C36H43N14O5Cl3: C, 50.39; H, 5.05; N,
22.85; Cl, 12.39. Found: C, 50.28; H, 4.96; N, 22.78;
Cl, 12.29.
According to the procedure B reported for the synthesis
of 5, starting from 26 (390 mg, 0.75 mmol), NaHCO3
(252 mg, 3 mmol) and p-N,N-bis (2-chloroethyl)amino-
benzoyl chloride (210 mg, 0.75 mmol), after workup
and ¯ash column chromatography with CH2Cl2/MeOH
(4/1, v/v), 6 was obtained as a brown solid. Yield
375 mg, 0.51 mmol, 68%.
3-[1-Methyl-3-[1-methyl-3-(1-methyl-4-nitropyrrole-2-
carboxamido)pyrazole-5-carboxamido]pyrazole-5-carbox-
amido] propionitrile (27). Following the same procedure
reported for the synthesis of 23, starting from 1-methyl-
3-(1-methyl-3-aminopyrazole-5-carboxamido)-pyrazole-
5-carboxamidopropionitrile 306 (1.26 g, 4 mmol), 1-methyl-
3-nitropyrrole-5-carboxylic acid (680 mg, 4 mmol),
HOBt (540 mg, 4 mmol) and EDC (800 mg, 4 mmol)
dissolved in DMF (20 mL), after the workup the prod-
uct 27 was obtained as a white solid. Yield 1.34 g,
1.3 mmol, 72%; mp 288±290 ꢀC; 1H NMR (DMSO-d6) d
2.78 (t, J=6.2 Hz, 2H), 3.45 (t, J=6.2 Hz, 2H), 3.97 (s,
3H), 4.03 (s, 3H), 4.07 (s, 3H), 7.34 (s, 1H), 7.58 (s, 1H),
7.81 (s, 1H), 8.21 (s, 1H), 8.95 (bs, 1H), 10.98 (s, 1H),
11.22 (s, 1H); anal. calcd for C19H20N10O5: C, 48.72; H,
4.3; N, 29.9. Found: C, 48.61; H, 4.19; N, 29.82.
3-[1-Methyl-3-[1-methyl-3-[1-methyl-4-[1-methyl-4-[4-bis-
(2-chloroethyl)aminobenzenamido]-pyrrole-2-carboxamido]-
pyrrole-2-carboxamido]pyrazole-5-carboxamido]pyrazole-
5-carboxamido] propionamidine hydrochloride (11).
Following the procedure A reported for the synthesis
of 5, starting from the nitro-amidine 28 (500 mg,
0.86 mmol), DIPEA (0.2 mL), 18 (415 mg, 1.06 mmol)
and EDC (370 mg, 1.84 mmol), after workup and ¯ash
column chromatography with CH2Cl2/MeOH (40/20,
v/v), 11 was obtained as a white solid: Yield 317 mg,
3-[1-Methyl-3-[1-methyl-3-(1-methyl-4-nitropyrrole-2-
carboxamido)pyrazole-5-carboxamido]pyrazole-5-carbox-
amido] propioniamidine hydrochloride (28). Following
the same procedure reported for the synthesis of 25,
starting from the compound 27 (500 mg, 1.07 mmol),
after workup the amidine 28 was obtained as a green
solid. Yield: 519 mg, 0.92 mmol, 93%; mp 290±291 ꢀC;
1H NMR (DMSO-d6) d 2.50 (t, J=6.1 Hz, 2H), 3.38 (t,
J=6.2 Hz, 2H), 3.87 (s, 3H), 4.01 (s, 3H), 4.05 (s, 3H),
7.40 (bs, 2H), 7.56 (s, 1H), 7.80 (s, 1H), 7.95 (s, 1H),
8.24 (bs, 1H), 8.88 (bs, 2H), 9.16 (bs, 2H), 10.98 (s, 1H),
11.16 (s, 1H); anal. calcd for C19H24N11O5Cl: C, 43.72;
H, 4.63; N, 29.52; Cl, 6.79. Found: C, 43.67; H, 4.58; N,
29.44; Cl, 6.72.
0.37 mmol, 46%; mp 220±223 ꢀC; H NMR (DMSO-d6)
1
d 2.63 (t, J=6 Hz, 4H), 3.55 (m, 8H), 3.63 (t, J=6 Hz,
4H), 3.79 (s, 3H), 3.88 (s, 3H), 4.02 (s, 3H), 4.07 (s, 3H),
6.82 (d, J=9 Hz, 2H), 7.17 (s, 1H), 7.31 (s, 1H), 7.42 (s,
1H), 7.47 (s, 1H), 7.54 (s, 1H), 7.94 (d, J=8.8 Hz, 2H),
8.66 (bs, 2H), 8.81 (m, 1H), 9.03 (b, 2H), 10.30 (s, 1H),
10.60 (s, 1H), 10.66 (s, 1H), 11.41 (s, 1H); anal. calcd for
C35H42N15O5Cl3: C, 48.93; H, 4.93; N, 24.45; Cl, 12.38.
Found: C, 48.86; H, 4.87; N, 24.33; Cl, 12.29.
3-[1-Methyl-4-[1-methyl-4-[1-methyl-3-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrazole-5-carboxamido]pyrrole-2-
carboxamido]pyrrole-2-carboxamido]
propionamidine
3-[1-Methyl-3-[1-methyl-3-[1-methyl-4-[4-bis(2-chloroethyl)-
aminophenylamido)]pyrrole - 2 - carboxamido]pyrazole - 5-
carboxamido]pyrazole-5-carboxamido] propionamidine
hydrochloride (3). Following the procedure B reported
hydrochloride (4). Following the procedure A reported
for the synthesis of 5, starting from 1-methyl-4-(1-
methyl-4-aminopyrrole-2-carboxamido)-pyrrole-2-carbox-
amidopropionamidine hydrochloride (496 mg, 1.36 mmol),

260
P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
87%), mp 176±178 ꢀC. H NMR (DMSO-d6): d 2.79 (t,
1
DIPEA (0.23 mL, 1.36 mmol), 18 (520 mg, 1.36 mmol)
and then EDC (522 mg, 2.72 mmol), after workup the
residue was dissolved in a small volume of methanol
and then ethyl ether was added to precipitate the crude
product as a brown solid. The procedure was repeated
®ve times to give 4 as a yellow solid. Yield 700 mg,
J=6.2 Hz, 2H), 3.51 (m, 2H), 4.03 (s, 3H), 4.22 (s, 3H),
7.34 (s, 1H), 7.97 (s, 1H), 8.68 (bs, 4H), 9.01 (m, 1H),
11.51 (s, 1H); anal. calcd for C13H18N9O4Cl: C, 39.09;
H, 4.54; N, 31.53; Cl, 8.87. Found: C, 38.99; H, 4.47; N,
31.46; Cl, 8.81.
0.952 mmol, 70%; mp 210±215 ꢀC; H NMR (DMSO-
1
d6) d 2.49 (m, 2H), 2.61 (m, 2H), 3.38 (m, 4H), 3,49 (m,
4H), 3.61 (s, 3H), 3.66 (s, 3H), 4.06 (s, 3H), 6.82 (d,
J=9 Hz, 2H), 6.96 (s, 1H), 7.10 (s, 1H), 7.20 (s, 1H),
7.32 (s, 1H), 7.47 (s, 1H), 7.95 (d, J=9.1 Hz, 2H), 8.24
(bt, 1H), 8.63 (bs, 2H), 8.99 (bs, 2H), 10.00 (s, 1H),
10.07 (s, 1H), 10.49 (s, 1H); anal. calcd for
C31H38N11O4Cl3: C, 50.65; H, 5.21; N, 20.96; Cl, 14.47.
Found: C, 50.58; H, 5.11; N, 20.87; Cl, 14.39.
Attempted formation of 3-[1-Methyl-3-[1-methyl-3-[1-
methyl-3-[4-bis(2-chloroethyl)aminophenylamido]-pyrazole-
5-carboxamido]pyrazole-5-carboxamido]pyrazole-5-carbox-
amido] propionamidine hydrochloride (7). Following the
procedure A reported for the synthesis of 5, starting
from the nitro-amidine 32 (200 mg, 0.5 mmol), DIPEA
(86 mL, 0.5 mmol), acid 18 (160 mg, 0.61 mmol) and
EDC (192 mg, 1 mmol) after work-up, no signi®cant
products were detectable on TLC.
3-[1-Methyl-3-(1-methyl-3-nitropyrazole-5-carboxamido)-
pyrazole-5-carboxamido] propionitrile (29). The nitroacid
13 (350 mg, 2.24 mmol) was re¯uxed in 4 mL of thionyl
chloride for 1 h. The reaction mixture was concentrated
in vacuo and the resulting residue was co-evaporated
with toluene. The resulting acid chloride was used
without further puri®cation.
3-[1-Methyl-3-[1-methyl-3-(1-methyl-3-nitropyrazole-5-
carboxamido)pyrazole-5-carboxamido]pyrazole-5-carbox-
amido] propionamidine hydrochloride (33). According to
the general procedure reported for the preparation of
25, starting from 31 (470 mg, 1 mmol), after work-up,
the nitroamidine 33 was obtained as a yellow solid.
Yield 500 mg (0.83 mmol, 83%), mp 265±267 ꢀC. 1H
NMR (DMSO-d6): d 2.72 (t, J=6.2 Hz, 2H), 3.54 (m,
2H), 4.01 (s, 3H), 4.07 (s, 3H), 4.12 (s, 3H), 7.13 (s, 1H),
7.31 (s, 1H), 7.59 (s, 1H), 8.73 (m, 1H), 8.82 (s, 2H), 9.15
(s, 2H), 11.17 (s, 1H), 11.3 (s, 1H); anal. calcd for
C18H23N12O5Cl: C, 41.35; H, 4.43; N, 32.14; Cl, 6.78.
Found: C, 41.27; H, 4.35; N, 32.07; Cl, 6.71.
To a cooled solution of 1-methyl-3-aminopyrazole-5-
carboxamidopropionitrile6 (295 mg, 1.53 mmol) and
NaHCO3 (252 mg, 3 mmol) in a mixture water/dioxane
(10 mL, 1/4, v/v), the above acid chloride in dioxane
(5 mL) was added dropwise. After 2 h at 0 ꢀC, the reac-
tion mixture was allowed to rise to room temperature
and stirred for 18 h. Solvent was evaporated, the solid
dissolved in EtOAc (15 mL) and washed successively
with 5% HCl (5 mL), saturated aq NaHCO3 (5 mL) and
brine. The organic layer was dried (Na2SO4), evap-
orated under reduced pressure and the residue puri®ed
by cristallization (EtOAc/petroleum ether) give 29 as a
brown solid. Yield 439 mg, 1.27 mmol, 81%; mp 254 ꢀC;
1H NMR (DMSO-d6) d 2.77 (t, J=5.6 Hz, 2H), 3.44 (m,
2H), 4.04 (s, 3H), 4.23 (s, 3H), 7.36 (s, 1H), 7.96 (s, 1H),
8.97 (m, 1H), 11.48 (s, 1H); anal. calcd for C13H14N8O4:
C, 49.09; H, 4.07; N, 32.36. Found: C, 49.00; H, 3.98; N,
32.25.
Attempted formation of 3-[1-Methyl-3-[1-methyl-3-[1-
methyl-3-[4-bis(2-chloroethyl)aminophenylamido]-pyrazole-
5-carboxamido]pyrazole-5-carboxamido]pyrazole-5-carbox-
amido] propionamidine hydrochloride (7). Using the
procedure B reported for the synthesis of 5, starting
from the nitro-amidine 33 (522 mg, 1 mmol), NaHCO3
(168 mg, 2 mmol) and p-[bis-(2-chloroethyl)amino]-
benzoyl chloride (270 mg, 1 mmol), the reaction was
stirred for 30 min at 0 ꢀC and then at room temperature:
no signi®cant products were detectable on TLC during
reaction progress.
3-[1-Methyl-3-[1-methyl-3-(1-methyl-3-nitropyrazole-5-
carboxamido)pyrazole-5-carboxamido]pyrazole-5-carbox-
amido] propionitrile (31). Following the same procedure
reported for the synthesis of 20, starting from 306 (2 g,
6.4 mmol), 13 (1 g, 6.4 mmol) and EDC (1.7 g,
8.32 mmol), after work-up, the product 31 was obtained
as a white solid. Yield 2.37 g (5.06 mmol, 79%), mp
Methyl 3-[1-Methyl-3-[1-methyl-3-[1-methyl-3-[4-bis(2-
chloroethyl)aminophenylamido]-pyrazole-5-carboxamido]-
pyrazole-5-carboxamido]pyrazole-5-carboxylate (38). Using
the same procedure reported for the synthesis of 23,
starting from 36 (385 mg, 1 mmol), the acid 18 (462 mg,
1.2 mmol), HOBt (135 mg, 1 mmol) and EDC (192 mg,
1 mmol) after usual work-up and ¯ash column chro-
matography with EtOAc, 38 was obtained as a white
solid. Yield 485 mg (0.75 mmol, 75%), mp 256 C. H
NMR (DMSO-d6): d 3,79 (m, 8H), 3.85 (s, 3H), 4.02 (s,
3H), 4.03 (s, 3H), 4.04 (s, 3H), 6.82 (d, J=8.8 Hz, 2H),
7.16 (s, 1H), 7.57 (s, 1H), 7.60 (s, 1H), 7.93 (d, J=8.6 Hz,
2H), 10.64 (s, 1H), 11.15 (s, 1H), 11.28 (s, 1H); anal. calcd
for C27H30N10O5Cl2: C, 50.24; H, 4.68; N, 21.7; Cl, 10.98;
Found: C, 50.17; H, 4.61; N, 21.62; Cl, 10.89.
173±175 ꢀC. H NMR (DMSO-d6): d 2.76 (t, J=6 Hz,
1
ꢀ
1
2H), 3.47 (m, 2H), 4.03 (s, 3H), 4.08 (s, 3H), 4.23 (s,
3H), 7.33 (s, 1H), 7.64 (s, 1H), 7.96 (s, 1H), 8.95 (m,
1H), 11.26 (s, 1H), 11.41 (s, 1H); anal. calcd for
C18H19N11O5: C, 46.06; H, 4.08; N, 32.82; Found: C,
45.98; H, 4.00; N, 32.73.
3-[1-Methyl-3-(1-methyl-3-nitropyrazole-5-carboxamido)-
pyrazole-5-carboxamido] propionamidine hydrochloride
(32). According to the general procedure reported for
the preparation of 25, starting from 29 (600 mg,
1.73 mmol), after work-up, the nitroamidine 32 was
obtained as a green solid. Yield 600 mg (1.5 mmol,
3-[1-Methyl-3-[1-methyl-3-[1-methyl-3-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrazole-5-carboxamido]pyrazole-5-
carboxamido]pyrazole-5-carboxylic acid (39). Using the
same procedure reported for the synthesis of 17, starting

P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
261
from 38 (129 mg, 0.2 mmol) and 2N aqueous KOH
(0.25 mL), after work-up the compound 39 was
obtained as a white solid. Yield 126 mg (0.2 mmol,
[1-Methyl-3-[1-methyl-3-[1-methyl-3-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrazole-5-carboxamido]pyrazole-5-
carboxamido]pyrazole-5-carboxamido] 4-bis(2-chloroethyl)-
aminoanilino (9). To a solution of 4-[bis(2-chloro-
ethyl)amino]anilino hydrochloride17 (88.4 mg, 0.1 mmol)
and NaHCO3 (25 mg, 0.3 mmol) in 6 mL of a mixture
dioxane/water (3/1, v/v) cooled to 0 ꢀC, was added
dropwise the solution of 40 (73.2 mg, 0.1 mmol) in DMF
(3 mL). The reaction mixture was stirred at rt for 18 h
and concentrated in vacuo. The residue was puri®ed by
¯ash column chromatography with EtOAc to give 9 as a
white solid. Yield 147 mg, 0.198 mmol, 66.5%; mp 255±
258 C; H NMR (DMSO-d6) d 3.79 (m, 8H), 4.05 (s,
3H), 4.07 (s, 3H), 4.09 (s, 3H), 4.12 (m, 8H), 7.49 (s,
1H), 7.57 (s, 1H), 7.58 (d, J=8.6 Hz, 2H), 7.60 (s, 1H),
7.96 (d, J=8.4 Hz, 2H), 10.1 (s, 1H), 10.7 (s, 1H), 11.2
(s, 1H), 11.3 (s, 1H); anal. calcd for C36H40N12O4Cl4: C,
51.07; H, 4.76; N, 19.85; Cl, 16.75. Found: C, 50.96; H,
4.68; N, 19.76; Cl, 16.68.
100%), mp 280 ꢀC. H NMR (DMSO-d6): d 3,79 (m,
1
8H), 4.02 (s, 3H), 4.03 (s, 3H), 4.06 (s, 3H), 6.82 (d,
J=8.4 Hz, 2H), 7.04 (s, 1H)), 7.57 (s, 1H), 7.58 (s, 1H),
7.93 (d, J=9.1 Hz, 2H), 10.6 (s, 1H), 11.11 (s, 1H),
11.13 (s, 1H); anal. calcd for C26H28N10O5Cl2: C, 49.45;
H, 4.47; N, 22.18; Cl, 11.23. Found: C, 49.37; H, 4.41;
N, 22.09; Cl, 11.17.
N-Succinimidyl-[1-Methyl-3-[1-methyl-3-[1-methyl-3-[4-
bis(2-chloroethyl)aminophenylamido]-pyrazole-5-carbox-
amido]pyrazole-5-carboxamido]pyrazole-5-carboxylate (40).
To a mixture of 39 (640 mg, 1 mmol) and N-hydroxy-
succinimide (200 mg, 1.46 mmol) in 10 mL of anhy-
drous DMF cooled to 0 ꢀC was added DCC (400 mg,
1.8 mmol) in a one portion and the stirring was con-
tinued for 24 h at rt. The dicyclohexylurea formed was
®ltered o€ and washed with cold DMF. The ®ltered was
concentrated to small volume (3 mL) and this solution,
containing the active ester, was used for the next step
without puri®cation.
ꢀ
1
Biological testing in vitro. All the tested hybrid com-
pounds were dissolved in DMSO at 1 mg/mL immedi-
ately before use and diluted in medium prior to adding
cells. The murine lymphocytic leukemia cells L1210,
L1210/Dx and L1210/tallimustine were grown in vitro
as a stationary suspension culture in RPMI 1640 med-
ium (GIBCO) supplemented with 10% FCS (Flow,
Irvine, UK), 2 mM l-glutamine (GIBCO), 10 mM b-
mercaptoethanol, 100 U/mL penicillin and 100 mg/mL
streptomycin.
3-[1-Methyl-3-[1-methyl-3-[1-methyl-3-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrazole-5-carboxamido]pyrazole-5-
carboxamido]pyrazole-5-carboxamido] propionamidine
hydrochloride (7). To a solution of p-aminopropion-
amide dihydrobromide (74.6 mg, 0.3 mmol) and
NaHCO3 (25 mg, 0.3 mmol) in 6 mL of a mixture diox-
ane/water (3/1, v/v) cooled to 0 ꢀC, was added dropwise
the solution of 40 (73.2 mg, 0.1 mmol) in DMF (3 mL).
The reaction mixture was stirred at rt for 18 h, acidi®ed
to pH 5 with 5% aqueous HCl and concentrated in
vacuo. The residue was puri®ed by ¯ash column chro-
matography with MeOH/CH2Cl2 (1/2, v/v) to give 7 as
a white solid. Yield 70 mg (0.093 mmol, 93.5%), mp
To determine survival after drug exposure, exponen-
tially growing L1210 cells were continously exposed to
various concentrations of drugs for 48 h. The anti-
proliferative activity of the drugs was evaluated by
counting surviving cells in a model ZBI Coulter Counter
(Coulter Electronics, Hialeah, FL). Results were
expressed as IC50 (dose causing 50% inhibition of cell
growth in treated cultures relative to untreated con-
trols). All experiments were repeated at least twice. For
each drug concentration, duplicate cultures were used.
Vehicle or solvent controls were run with each experi-
ment.
177±180 ꢀC; H NMR (CD3OD) d 2.57 (m, 2H), 3.53
1
(m, 2H), 3.79 (m, 8H), 4.02 (s, 3H), 4.06 (s, 3H), 4.08 (s,
3H), 6.81 (d, J=8.6 Hz, 2H), 7.31 (s, 1H), 7.57 (s, 1H),
7.59 (s, 1H), 7.93 (d, J=8.4 Hz, 2H); anal. calcd for
C29H36N13O4Cl3: C, 47.33; H, 4.93; N, 24.76; Cl, 14.27.
Found: C, 47.20; H, 4.81; N, 24.68; Cl, 14.15.
3-[1-Methyl-3-[1-methyl-3-[1-methyl-3-[4-bis(2-chloroethyl)-
aminophenylamido]-pyrazole-5-carboxamido]pyrazole-5-
carboxamido]pyrazole-5-carboxamido]dimethylaminopro-
pane (8). To an ice-cooled solution of 40 (73.2 mg,
0.1 mmol) in 5 mL of anhydrous DMF was added N,N-
dimethylaminopropylamine (40 mL, 0.3 mmol 0.1 mmol)
in DMF (3 mL) and the mixture was stirred at rt for
18 h. Concentration of the solution provided a residue
which was puri®ed by ¯ash column chromatography
with MeOH/CH2Cl2 (2/1, v/v) to give 8 as a yellow
solid. Yield 42 mg (0.058 mmol, 58%), mp 220±222 ꢀC;
1H NMR (DMSO-d6) d 1.67 (t, J=7 Hz, 2H), 2.38 (m,
2H), 3.15 (s, 3H), 3.17 (s, 3H), 3.26 (m, 2H), 3.85 (m,
8H), 4.07 (s, 3H), 4.10 (s, 3H), 4.13 (s, 3H), 6.82 (d,
J=9 Hz, 2H), 7.28 (s, 1H), 7.57 (s, 1H), 7.59 (s, 1H),
7.95 (d, J=8.8 Hz, 2H), 8.65 (t, J=6.4 Hz, 1H), 10.64 (s,
1H), 11.13 (s, 1H), 11.18 (s, 1H); anal. calcd for
C31H40N12O4Cl2: C, 52.03; H, 5.63; N, 23.49; Cl, 9.91.
Found: C, 51.94; H, 5.54; N, 23.38; Cl, 9.80.
Biological testing in vivo. Inbred DBA/2 and C57BL/
6N, ®rst generation hybrid (C57BL/6No  DBA/2No)
B6D2F1 adult female mice were used for the evaluation
of the antitumor activity. Mice were 2±3 months of age
and weighed 20±22 g at the time of tumor implantation.
All mice were supplied by Charles River Italia (Calco,
Como, Italy). Animal health was monitored by ser-
ological testing; the animals were free of infectious
pathogens, including mouse hepatitis virus, Sendai virus
and Mycoplasma pulmonis, during the course of experi-
mentation. L1210 murine leukemia (originally obtained
from the National Cancer Institute, Frederick, MD)
was maintained by continuous i.p. passage (106 cells/
mouse) in syngenic DBA/2N mice.
All drug solutions were prepared immediately before
use and given intravenously (i.v.) in a volume of 10 mL/
Kg of body weight. The vehicle used in preparation of
solutions consisted of 10% Tween 80 and 90% saline.

262
P. G. Baraldi et al. / Bioorg. Med. Chem. 7 (1999) 251±262
L1210 leukemia (105 cells/mouse, CDF1 mice) was
implanted i.v. The solid tumor M5076 was inoculated
i.v. (106 cells/mouse) in CDF1 mice. A dose response was
determined in all experiments. Toxicity was evaluated
on the basis of the gross autopsy ®ndings and the weight
loss, mainly in terms of reduction of spleen and liver size.
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Acknowledgements
We wish to thank Pharmacia & Upjhon for the ®nancial
support of this work.
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