2-Acylhydrazino-5-arylpyrrole derivatives: Synthesis and antifungal activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2008.08.003

The synthesis and antifungal activity of 2-acylhydrazino-5-arylpyrroles 21-62 are described. Pyrrole derivatives 21-62 were evaluated for their antifungal activity towards Candida albicans ATCC 10231 and three Candida non-albicans isolated from clinical s

Full text of DOI:10.1016/j.ejmech.2008.08.003

European Journal of Medicinal Chemistry 44 (2009) 1288–1295
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-Acylhydrazino-5-arylpyrrole derivatives: Synthesis and antifungal activity
evaluation
,
b
a
a
b
Valentina Onnis a , Alessandro De Logu , Maria T. Cocco , Roberta Fadda , Rita Meleddu ,
*
Cenzo Congiu a
a
`
Dipartimento di Tossicologia, Sezione di Chimica Farmaceutica, Universita degli Studi di Cagliari, via Ospedale 72, Cagliari I-09124, Italy
Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia Medica, Universita degli Studi di Cagliari, viale Sant’Ignazio da Laconi 38, Cagliari I-09124, Italy
b
`
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2008
Received in revised form 14 July 2008
Accepted 13 August 2008
Available online 22 August 2008
The synthesis and antifungal activity of 2-acylhydrazino-5-arylpyrroles 21–62 are described. Pyrrole
derivatives 21–62 were evaluated for their antifungal activity towards Candida albicans ATCC 10231 and
three Candida non-albicans isolated from clinical specimens. Most of them showed very good antifungal
activities against Candidae, having MIC values in the 0.39–3.12 mg/mL range and enhanced inhibition
potency as compared to that of fluconazole. In addition, some of the most active compounds were tested
for cytotoxic activities against breast (MCF-7), lung (H-460), and central nervous system (SF-268) human
cancer cell lines with the NCI anticancer drug screen. The activity of pyrroles described in this paper,
along with the low toxicity, shows promise for the future development of non-toxic new antimycotic
agents. The relationship between functional group variation and biological activity of the evaluated
compounds is also discussed.
Keywords:
2-Acylhydrazino-5-arylpyrroles
Candidae
Antifungal activity
Cytotoxic activity
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
antifungal agents is still unacceptably high [10,13–15]. Currently,
a small number of agents are available, and all have some draw-
The incidence of fungal infections has increased significantly in
the past two decades mainly due to the growing number of
immunocompromised patients, such as cancer patients, patients
who have undergone organ transplantation, and patients with
AIDS, and the frequent use of cytotoxic and/or antibacterial drugs
[1–6]. Clinically, candidiasis and aspergillosis account for between
80% and 90% of systemic fungal infections in immunocompromised
patients. Candida species have been found to be the fourth most
prevalent group of pathogens and have been isolated from 8% of
patients with nosocomial bloodstream infections [7]. They are also
identified as critical pathogens in infections of wounds and other
body fluids [8]. The change in mortality rate associated with
disseminated candidiasis has been insignificant even after treat-
ment with effective antifungal drugs [9]. Until recently, ampho-
tericin B was the standard therapy for many fungal infections, but
a high frequency of renal toxicity has limited its use [10,11]. In
recent times, new antifungal agents, such as azoles (fluconazole
and voriconazole), and echinocandins (caspofungin), have
increased the ability to treat fungal infections [10,12]. However, all
of them have a narrow spectrum of activity, the development of
high resistance has been documented, especially, for fluconazole,
and the mortality due to fungal infections even with the novel
backs regarding their spectrum, toxicity, tissue distribution, central
nervous system (CNS) penetration, and high cost [13–17]. In view of
the increasing resistance to existing azole antifungals [18,19] and
the lack of sufficient chemical diversity in the existing classes of
antifungals, the need for new antifungals remains high. As part of
our ongoing development of efficient protocols for the preparation
of biologically active heterocycles from common intermediates and
keeping in view the biological activity of the pyrrole derivatives, we
have published the synthesis and preliminary evaluation of in vitro
antifungal activity of a series of 2-acetyl (or ethoxycarbonyl)-
hydrazinopyrrole derivatives [20]. Our results indicated that some
of the derivatives displayed appreciable fungistatic effect, espe-
cially against a strain of Candida albicans isolated from clinical
specimens, making this class of compounds suitable for further
development as potential antimycotic agents. Therefore we present
the design, synthesis, and biological evaluation of a novel group of
potent antifungal pyrroles derived from the substances described
in our previous study.
2. Results and discussion
2.1. Synthesis
The synthetic approach to obtain 2-acylhydrazino-5-arylpyrrole
derivatives 21–62 followed the reactions shown in Scheme 1. The
* Corresponding author. Tel.: þ39 0706758632; fax: þ39 0706758612.
E-mail address: vonnis@unica.it (V. Onnis).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.08.003

V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
1289
X
described in our previous papers [20–23]. Intermediates 4–20 were
converted to pyrroles 21–62 (Tables 1 and 2) by treatment with the
appropriate a-bromoketone in 1:1 molar ratio, in the presence of
sodium hydrogencarbonate, in anhydrous MeCN solution. All the
newly synthesized compounds gave corrected analytical data. The
IR and NMR spectral data were consistent with the assigned
structure.
X
H
N
(i)
R
H2N
N
H
H2N
OE t
O
4- 10: X = COOEt
11-14: X = COOMe
15-20: X = CN
1: X = COOEt
2: X = COOMe
3: X = CN
2.2. Antifungal activity
(ii)
X
Compd.
R
The antifungal properties of compounds 21–62 were evaluated
by the broth microdilution method according to the NCCLS refer-
ence document M27-A [24] against a panel of four Candidae: C.
albicans ATCC 10231, and Candida glabrata, Candida parapsilosis and
Candida krusei isolated from clinical specimens. Fluconazole and
amphotericin B were used as positive controls.
Me
OEt
i-Pr
n-Pr
4-OMeBz
Et
MeO(CH2)2
MeOCH2
4, 11, 15
5, 13, 18
6, 16
7
8
9, 12, 17
10, 14, 20
19
H
N
R
Ar
N
H
N
H
O
21-62
2.3. Discussion
The antifungal effect of compounds 21–43 is reported in Table 1.
From the analysis of data we can note that compounds 24, 25, 27,
28, 31, 32, and 40–43 display minimum inhibitory concentration
Scheme 1. Synthetic pathway to pyrroles 21–62. Reagents and conditions: (i)
RCONHNH2, EtOH, r.t.; (ii) ArCOCH2Br, NaHCO3, MeCN, reflux.
(MIC) values against C. albicans in the 3.12–6.25 mg/mL range.
required 3-(2-acylhydrazino)-3-aminopropenoates 4–14 and 3-(2-
acylhydrazino)-3-aminopropenenitriles 15–20 were prepared by
treatment of imidates 1–3 with acylhydrazines using the method
Furthermore MIC values of compounds 24, 25, 27, 28, 29, and 40–42
against C. glabrata are 2- to 4-fold better than that of the reference
drug fluconazole. The same 24, 25, 28, 32, as well as 40 showed
Table 1
Antifungal activity of pyrroles 21–43a
COOEt
H
N
R
Ar
N
H
N
H
O
21-43
Compound Ar
R
c log Pb MIC (
m
g/mL)
MFCc
(mg/mL)
C. albicans ATCC 10231 C. glabrata C. parapsilosis C. krusei C. albicans ATCC 10231 C. glabrata C. parapsilosis C. krusei
21
Ph
4-ClPh
4-MePh
4-OMePh Me
Ph
4-ClPh
4-MePh
4-OMePh OEt
Ph
4-ClPh
4-MePh
4-OMePh i-Pr
Ph
4-ClPh
4-MePh
4-OMePh n-Pr
Me
Me
Me
1.02
1.58
1.51
0.9
1.95
2.51
2.43
1.82
2.24
2.8
2.73
2.12
2.09
2.65
2.58
1.97
2.61
3.3
12.5
>100
>100
3.12
3.12
25
12.5
>100
>100
6.25
6.25
100
50
12.5
50
50
25
100
12.5
12.5
50
100
12.5
>100
>100
25
12.5
>100
>100
3.12
3.12
>100
6.25
3.12
>100
>100
12.5
12.5
12.5
>100
>100
12.5
>100
>100
>100
12.5
12.5
6.25
>100
>100
>100
12.5
12.5
>100
25
25
12.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
12.5
12.5
25
100
1.56
>100
100
25
50
50
50
>100
25
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
AmBd
FLCe
50
50
OEt
OEt
OEt
6.25
12.5
100
6.25
25
>100
25
12.5
12.5
>100
25
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
25
>100
>100
100
>100
>100
100
>100
>100
>100
>100
100
>100
>100
>100
25
6.25
3.12
3.12
6.25
6.25
i-Pr
i-Pr
i-Pr
>100
>100
6.25
6.25
12.5
>100
>100
12.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6.25
6.25
6.25
12.5
0.78
12.5
6.25
12.5
n-Pr
n-Pr
n-Pr
100
>100
25
>100
>100
>100
50
50
>100
100
12.5
25
12.5
50
0.78
25
12.5
Ph
4-OMeBz
>100
>100
>100
50
>100
100
6.25
12.5
6.25
25
4-ClPh
4-MePh
Ph
4-OMePh Et
Ph
4-OMePh MeO(CH2)2 0.85
4-OMeBz
4-OMeBz
Et
3.23
1.68
1.55
3.12
3.12
3.12
6.25
0.39
0.78
25
25
100
1.56
>100
100
50
100
1.56
50
MeO(CH2)2 0.98
6.25
0.78
>100
0.78
0.39
a
Antifungal activity was determined with the microbroth dilution assay following the NCCLS guidelines.
Calculated using ChemDraw Ultra software version 8.0.3.
Defined as the minimum concentration of compound which resulted in the killing of >99% of the original inoculum.
Amphotericin B.
Fluconazole.
b
c
d
e

1290
V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
Table 2
Antifungal activity of pyrroles 44–62a
X
H
N
R
Ar
N
H
N
H
O
44-62
Compound
X
Ar
Ph
R
c log Pb MIC (
m
g/mL)
MFCc
(mg/mL)
C. albicans ATCC 10231 C. glabrata C. parapsilosis C. krusei C. albicans ATCC 10231 C. glabrata C. parapsilosis C. krusei
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
Me
0.9
3.12
1.56
3.12
0.39
3.12
3.12
3.12
6.25
6.25
6.25
3.12
3.12
3.12
3.12
3.12
3.12
3.12
6.25
6.25
0.39
0.78
6.25
3.12
3.12
0.78
3.12
3.12
3.12
0.78
25
6.25
25
6.25
6.25
6.25
6.25
6.25
3.12
6.25
3.12
3.12
3.12
1.56
1.56
0.78
50
3.12
25
6.25
6.25
6.25
3.12
3.12
6.25
6.25
6.25
3.12
3.12
3.12
6.25
6.25
6.25
6.25
6.25
3.12
6.25
3.12
6.25
6.25
12.5
12.5
12.5
12.5
>100
6.25
50
50
50
12.5
12.5
25
12.5
25
12.5
12.5
50
25
25
1.56
>100
12.5
6.25
12.5
12.5
>100
12.5
100
6.25
12.5
25
6.25
3.12
12.5
12.5
>100
6.25
50
25
12.5
12.5
6.25
12.5
6.25
25
6.25
25
>100
25
4-OMePh Me
Ph Et
4-OMePh Et
4-OMePh i-Pr
Ph
4-OMePh OEt
Ph
4-OMePh MeOCH2
Ph
0.77
1.55
1.43
1.99
1.82
1.70
0.56
0.43
50
OEt
3.12
12.5
25
MeOCH2
25
MeO(CH2)2 0.85
3.25
6.25
6.25
3.12
6.25
6.25
3.12
12.5
6.25
6.25
0.78
12.5
4-OMePh MeO(CH2)2 0.73
12.5
25
6.25
COOMe Ph
COOMe 4-OMePh Me
COOMe Ph Et
COOMe 4-OMePh Et
COOMe Ph
COOMe 4-OMePh OEt
Me
0.69
0.56
1.34
1.21
1.61
1.48
12.5
3.12
12.5
6.25
6.25
50
12.5
6.25
0.78
25
12.5
12.5
12.5
>100
100
12.5
1.56
>100
OEt
25
12.5
6.25
0.78
0.39
COOMe Ph
COOMe 4-OMePh MeO(CH2)2 0.51
MeO(CH2)2 0.64
12.5
6.25
0.78
62
AmBd
FLCe
25
1.56
50
>100
a
Antifungal activity was determined with the microbroth dilution assay following the NCCLS guidelines.
Calculated using ChemDraw Ultra software version 8.0.3.
Defined as the minimum concentration of compound which resulted in the killing of >99% of the original inoculum.
Amphotericin B.
Fluconazole.
b
c
d
e
a 2-fold inhibition potency against C. krusei as compared to fluco-
nazole. Also it is interesting to note that pyrroles 21, 24, 25, 27, 28,
and 43 exhibit fungicidal effect against C. albicans ATCC 10231 at
concentrations of the corresponding MICs.
The results of antifungal activity of pyrroles 44–62 are displayed
in Table 2. Pyrroles 44–62 show a general enhancement in anti-
fungal activity with respect to compounds 21–43. MIC values of
these compounds are in the 0.39–6.25 and 0.78–50
mg/mL range
As regards structure–activity relationships (SARs), we found
that activity of this series of compounds was dependent on the
substituent present at 5-position of the pyrrole ring. These
compounds with a methoxy substituent in the para position or with
an unsubstituted phenyl showed, in general, a more potent anti-
fungal activity than other compounds synthesized. Replacement of
the methoxy group with chlorine or methyl resulted in decreased
or impaired activity. Furthermore the presence at 2-position of the
pyrrole ring of butyrylhydrazino, isobutyrylhydrazino, or (4-
methoxyphenyl)acetylhydrazino moieties led to compounds
endowed with poor activity. With the mechanism of the antifungal
effect being unknown, we assumed that while a certain degree of
lipophilicity is necessary for the compound to penetrate the fungal
cell wall, the presence of a hydrophilic moiety can play a most vital
role in binding to its biological target molecule. As matter of fact,
the active compounds are characterized by c log P values minor
than 2.4. In view of the results obtained and bearing in mind that
the desired biological activity is supported by the presence of
a phenyl ring or 4-methoxyphenyl in 5-position, we shifted
attention to the substitution at C(3) of the pyrrole ring. We settled
on to determine the effect of replacing the COOEt moiety with CN or
COOMe groups. Thus, we designed a new series of pyrroles
(compounds 44–62) bearing CN or COOMe groups at C-3 of pyrrole
ring as well as a 2-acylhydrazino moiety that give to the molecule
c log P values minor than 2.
against C. albicans ATCC 10231, and the three Candida non-albicans
isolated from clinical specimens, respectively. Compound 47
showed the highest activity of the whole series. In particular, the
analysis of pyrrole 47 MIC data reveals a 2- to 32-fold inhibition
potency against C. albicans, C. glabrata and C. krusei as compared to
fluconazole. On the contrary the same compound displays anti-
fungal activity minor than fluconazole against C. parapsilosis.
Furthermore 47 is endowed of the same antifungal potency of
amphotericin B against C. albicans ATCC 10231 and against clinically
isolated Candida species. Compound 47 was shown to be fungicidal
against all the strains tested at concentrations 8 doubling dilutions
greater than the corresponding MICs (MCF 6.25–12.5 mg/mL).
Replacing the 3-COOEt (pyrrole 41) or 3-COOMe (pyrrole 58) with
a 3-cyano group, to produce pyrrole 47, led to an approximate 8- to
32-fold increase in activity. Replacement of the ethyl-
carbonylhydrazino moiety of pyrrole 47 by an ethoxycarbonylhy-
drazino (compound 50), methoxymethylcarbonylhydrazino
(compound 52) or methoxyethylcarbonylhydrazino (compound 54)
resulted in reduced activity (4- to 32-fold).
In addition to the antifungal evaluation, the most active
compounds 45, 47, 50, and 56 were evaluated by the National
Cancer Institute (NCI, Bethesda, MD) for cytotoxic activities against
breast (MCF-7), lung (H-460), and central nervous system (SF-268)
human cancer cell lines [25], in order to determine the selectivity of
pyrroles towards fungi. Results showed (Table 3) that all the tested

V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
1291
Table 3
NH). IR (Nujol) 3417 (NH), 3234 (NH), 3059 (NH), 1716 (C]O), 1657,
1607 cmꢀ1. Anal. Calcd for C9H17N3O3: C, 50.22; H, 7.96; N, 19.52.
Found: C, 50.18; H, 7.98; N, 19.54.
Cytotoxic activitya of compounds 45, 47, 50 and 56
Compound
Growth inhibition percentageb at 10ꢀ5 M concentration
MCF-7
H-460
SF-268
4.1.2.2. Ethyl 3-amino-3-(2-propanoylhydrazino)-2-propenoate (9).
45
47
50
56
0
0
0
31
0
0
2.1
0
Yield 86%. Mp 119–120 ꢁC (benzene). 1H NMR (CDCl3):
d 1.05 (t, 3H,
4.6
0.9
0
J ¼ 7.6 Hz, CH3),1.18 (t, 3H, J ¼ 7.3 Hz, CH3), 4.10 (m, 4H, CH2), 5.57 (s,
0
1H, CH), 6.21 (s, 2H, NH2), 10.22, 10.39 (s, 2H, NH). IR (Nujol) 3400
(NH), 3210 (NH), 3047 (NH), 1742 (C]O), 1653, 1618, 1595 cmꢀ1
.
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen
(see Refs. [25–27] for details).
Anal. Calcd for C8H15N3O3: C, 47.75; H, 7.51; N, 20.88. Found: C,
47.71; H, 7.49; N, 20.92.
b
Calculated with respect to untreated cells.
4.1.2.3. Ethyl 3-amino-3-(2-(3-methoxypropanoyl)hydrazino)-2-pro-
pyrroles displayed poor or no cytotoxic activity at 10ꢀ5 M concen-
tration. Thus compounds 45, 47, 50, and 56 showed high selectivity
towards Candidae.
penoate (10). Yield 82%. Mp 64–65 ꢁC (n-hexane). 1H NMR (CDCl3):
d
1.20 (t, 3H, J ¼ 7.3 Hz, CH3), 2.43 (m, 2H, CH2), 3.63 (m, 2H, CH2),
4.13 (q, 2H, J ¼ 7.3 Hz, CH2), 5.61 (s, 1H, CH), 5.97 (s, 2H, NH2), 10.02,
10.42 (s, 2H, NH). IR (Nujol) 3432 (NH), 3180 (NH),1723 (C]O),1687
(C]O),1600,1582 cmꢀ1. Anal. Calcd for C9H17N3O4: C, 46.74; H, 7.41;
N, 18.17. Found: C, 46.71; H, 7.43; N, 18.13.
3. Conclusion
We synthesized a novel series of 2-acylhydrazinopyrrole deriv-
atives. Most of the synthesized compounds exhibited potent anti-
fungal activity against C. albicans ATCC 10231 and clinically isolated
Candida species. These novel compounds may serve as lead for the
development of drugs with different structural feature from those
of the currently utilized antifungal agents. The structure–activity
relationships discussed above can provide useful information for
further design and synthesis of compounds with optimized bioac-
tivity profile. Additional studies to optimize the structural feature as
well as to explore the mechanism of action of this class of
compounds is planned to start in our group in near future.
4.1.2.4. Methyl 3-(2-acetylhydrazino)-3-amino-2-propenoate (11).
Yield 90%. Mp 149–150 ꢁC (toluene). 1H NMR (DMSO-d6):
d 1.89 (s,
3H, CH3), 3.60 (s, 3H, CH3), 5.06 (s, 1H, CH), 6.12 (s, 2H, NH2), 9.32
(s, 2H, NH). IR (Nujol) 3498 (NH), 3414 (NH), 3184 (NH), 1724
(C]O), 1684 (C]O), 1614 cmꢀ1. Anal. Calcd for C6H11N3O3: C,
41.61; H, 6.40; N, 24.27. Found: C, 41.67; H, 6.39; N, 24.24.
4.1.2.5. Methyl 3-amino-3-(2-propanoylhydrazino)-2-propenoate
(12). Yield 88%. Mp 139–140 ꢁC (2-PrOH). 1H NMR (DMSO-d6):
d
0.94 (t, 3H, J ¼ 5.3 Hz, CH3), 2.04 (q, 2H, J ¼ 5.3 Hz, CH2), 3.56 (s,
3H, CH3), 5.07 (s, 1H, CH), 6.09 (s, 2H, NH2), 9.24 (s, 2H, NH). IR
(Nujol) 3420 (NH), 3178 (NH), 1727 (C]O), 1682 (C]O), 1657,
1624 cmꢀ1. Anal. Calcd for C7H13N3O3: C, 44.91; H, 7.00; N, 22.45.
Found: C, 44.95; H, 7.02; N, 22.42.
4. Experimental protocols
4.1. Synthesis
4.1.1. Materials and general methods
4.1.2.6. Methyl 3-amino-3-(2-ethoxycarbonylhydrazino)-2-propenoate
Melting points were determined on a Stuart Scientific Melting
point SMP1 and are uncorrected. Proton NMR spectra were recorded
on a Varian Unity 300 spectrometer. The chemical shift are reported
(13). Yield 94%. Mp 137–138 ꢁC (2-PrOH). 1H NMR (DMSO-d6):
d 1.10
(t, 3H, J ¼ 6.9 Hz, CH3), 3.56 (s, 3H, CH3), 4.00 (q, 2H, J ¼ 6.9 Hz, CH2),
5.04 (s, 1H, CH), 5.99 (s, 2H, NH2), 8.88, 9.21 (s, 2H, NH). IR (Nujol)
3416 (NH), 3232 (NH), 3043 (NH), 1720 (C]O), 1694 (C]O), 1639,
1584 cmꢀ1. Anal. Calcd for C7H13N3O4: C, 41.38; H, 6.45; N, 20.68.
Found: C, 41.35; H, 6.44; N, 20.72.
in parts per million (d, ppm) downfield from tetramethylsilane
(TMS), which was used as internal standard. Infrared spectra were
obtained with a Bruker Vector 22 spectrophotometer. Elemental
analyses were carried out with a Carlo Erba model 1106 Elemental
Analyzer and the values found were within 0.4% of theoretical
values. Ethyl 3-(2-acetylhydrazino)-3-amino-2-propenoate (4) [20],
ethyl 3-amino-3-(2-ethoxycarbonylhydrazino)-2-propenoate (5)
[20], ethyl 3-amino-3-(2-isobutyrylhydrazino)-2-propenoate (6)
[21], ethyl 3-amino-3-(2-(4-methoxyphenyl)acetyl)hydrazino-2-
propenoate (8) [22], 3-amino-3-(2-acetylhydrazino)-2-propeneni-
trile (15) [23], 3-amino-3-(2-isobutyrylhydrazino)-2-propenitrile
(16) [23] and pyrroles 21–28 [20] were obtained with previously
described procedures.
4.1.2.7. Methyl 3-amino-3-(2-(3-methoxypropanoyl)hydrazino)-2-pro-
penoate (14). Yield 84%. Mp 65–66 ꢁC (n-hexane). 1H NMR (CDCl3):
d
2.25 (m, 2H, CH2), 3.06 (s, 3H, CH3), 3.45 (s, 3H, CH3), 3.54 (m, 2H,
CH2), 5.47 (s, 1H, CH), 5.90 (s, 2H, NH2), 9.91, 10.02 (s, 2H, NH). IR
(Nujol) 3410 (NH), 3320 (NH), 3186 (NH), 1741 (C]O), 1722,
1648 cmꢀ1. Anal. Calcd for C8H15N3O4: C, 44.23; H, 6.96; N, 19.34.
Found: C, 44.29; H, 6.97; N, 19.30.
4.1.2.8. 3-Amino-3-(2-propanoylhydrazino)-2-propenenitrile (17).
Yield 98%. Mp 154–155 ꢁC (toluene). 1H NMR (DMSO-d6):
d 0.93 (t,
4.1.2. General procedure for the synthesis of compounds 4–20
A solution of ethyl 3-amino-3-ethoxypropenoate (1), methyl 3-
amino-3-ethoxypropenoate (2), or 3-amino-3-ethoxypropeneni-
trile (3) (0.01 mol) and the appropriate acylhydrazine (0.01 mol) in
dry EtOH (50 mL) was heated at 70 ꢁC for 5 min and then allowed to
stand overnight at r.t. The formed precipitate was filtered off,
washed with diethyl ether and recrystallized from the solvent
indicated.
3H, J ¼ 7.7 Hz, CH3), 2.37 (q, 2H, J ¼ 7.7 Hz, CH2), 5.39 (s, 1H, CH),
6.30 (s, 2H, NH2), 9.42, 9.98 (s, 2H, NH). IR (Nujol) 3451 (NH), 3408
(NH), 3205 (NH), 2257 (CN), 1612, 1599 cmꢀ1. Anal. Calcd for
C6H10N4O: C, 46.74; H, 6.54; N, 36.34. Found: C, 46.77; H, 6.56; N,
36.36.
4.1.2.9. 3-Amino-3-(2-ethoxycarbonylhydrazino)-2-
propenenitrile (18). Yield 97%. Mp 169–170 ꢁC (MeCN). 1H NMR
(DMSO-d6):
d
1.11 (t, 3H, J ¼ 6.9 Hz, CH3), 3.99 (q, 2H, J ¼ 6.9 Hz,
4.1.2.1. Ethyl 3-amino-3-(2-butyrylhydrazino)-2-propenoate (7). Yield
CH2), 5.35 (s, 1H, CH), 6.21 (s, 2H, NH2), 9.01, 9.62 (s, 2H, NH). IR
(Nujol) 3451 (NH), 3408 (NH), 3205 (NH), 2257 (CN), 1612,
1599 cmꢀ1. Anal. Calcd for C6H10N4O2: C, 42.35; H, 5.92; N, 32.92.
Found: C, 42.40; H, 5.90; N, 32.90.
86%. Mp 177–180 ꢁC (cyclohexane). 1H NMR (DMSO-d6):
d 0.81 (t, 3H,
J ¼ 5.1, CH3), 1.57 (m, 2H, CH2), 2.29 (m, 2H, CH2), 4.03 (q, 2H,
J ¼ 5.1 Hz, CH2), 5.04 (s, 1H, CH), 6.09 (s, 2H, NH2), 9.24, 10.02 (s, 2H,

1292
V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
4.1.2.10. 3-Amino-3-(2-(2-methoxyacetyl)hydrazino)-2-propeneni-
6.48 (s, 1H, H-4), 7.08–7.53 (m, 5H, Ph), 7.82, 9.80 (s, 2H, NH), 10.73
(s, 1H, NH). IR (Nujol) 3320 (NH), 3269 (NH), 1666 (C]O), 1611,
1597 cmꢀ1. Anal. Calcd for C17H21N3O3: C, 64.74; H, 6.71; N, 13.32.
Found: C, 64.77; H, 6.70; N, 13.36.
trile (19). Yield 86%. Mp 159–160 ꢁC (MeCN). 1H NMR (DMSO-d6):
d
3.29 (s, 3H, CH3), 4.13 (s, 2H, CH2), 5.24 (s, 1H, CH), 6.33 (s, 2H,
NH2), 9.36, 10.00 (s, 2H, NH). IR (Nujol) 3451 (NH), 3408 (NH), 3205
(NH), 2257 (CN), 1612, 1599 cmꢀ1. Anal. Calcd for C6H10N4O2: C,
42.35; H, 5.92; N, 32.92. Found: C, 42.40; H, 5.90; N, 32.90.
4.1.3.6. Ethyl 5-(4-chlorophenyl)-2-(2-butyrylhydrazinyl)-
1H-pyrrole-3-carboxylate (34). Yield 76%. Mp 149–150 ꢁC (MeCN).
4.1.2.11. 3-Amino-3-(2-(3-methoxypropanoyl)hydrazino)-2-propeni-
1H NMR (DMSO-d6):
d
0.88 (t, 3H, J ¼ 6.9 Hz, CH3), 1.22 (t, 3H,
trile (20). Yield 87%. Mp 124–125 ꢁC (n-hexane). 1H NMR (DMSO-
J ¼ 7.3 Hz, CH3), 1.56 (m, 2H, CH2), 2.20 (m, 2H, CH2), 4.13 (q, 2H,
J ¼ 6.9 Hz, CH2), 6.20 (s, 1H, H-4), 7.28 (d, 2H, J ¼ 8.0 Hz, Ph), 7.58 (d,
2H, J ¼ 8.0 Hz, Ph), 7.83, 9.63 (s, 2H, NH), 10.67 (s, 1H, NH). IR (Nujol)
3374 (NH), 3337 (NH), 3299 (NH), 1684 (C]O), 1656, 1616,
1595 cmꢀ1. Anal. Calcd for C17H20ClN3O3: C, 58.37; H, 5.76; N, 12.01.
Found: C, 58.34; H, 5.75; N, 12.03.
d6):
d
2.26 (t, 2H, J ¼ 6.5 Hz, CH2), 3.40 (s, 3H, CH3), 3.67 (t, 2H,
J ¼ 6.5 Hz, CH2), 5.16 (s, 1H, CH), 6.31 (s, 2H, NH2), 9.42, 9.73 (s, 2H,
NH). IR (Nujol) 3410 (NH), 3320 (NH), 3186 (NH), 1741 (C]O), 1722,
1648 cmꢀ1. Anal. Calcd for C7H12N4O2: C, 45.64; H, 6.57; N, 30.42.
Found: C, 45.69; H, 6.56; N, 30.46.
4.1.3. General procedure for the synthesis of pyrrole derivatives
(21–62)
4.1.3.7. Ethyl 5-(4-methylphenyl)-2-(2-butyrylhydrazinyl)-
1H-pyrrole-3-carboxylate (35). Yield 82%. Mp 194–195 ꢁC (MeCN).
A mixture of compounds 4–20 (10 mmol), NaHCO3 (0.92 g,
11 mmol), and the appropriate phenacyl bromide (10 mmol) in dry
MeCN (10 mL) was refluxed for 20 min and then stirred at room
temperature for 2 h. The formed precipitate was filtered off and
purified by crystallization from the adequate solvent to give the
pyrrole derivatives 21–62.
1H NMR (DMSO-d6):
d
0.85 (t, 3H, J ¼ 7.7 Hz, CH3), 1.21 (t, 3H,
J ¼ 7.3 Hz, CH3), 1.52 (m, 2H, CH2), 2.13 (m, 2H, CH2), 2.22 (s, 3H,
CH3), 4.30 (q, 2H, J ¼ 7.3 Hz, CH2), 6.41 (s, 1H, H-4), 7.10 (d, 2H,
J ¼ 7.7 Hz, Ph, H-3 and H-5), 7.60 (d, 2H, J ¼ 7.7 Hz, Ph, H-2 and H-6),
7.80, 9.59 (s, 2H, NH), 10.69 (s, 1H, NH). IR (Nujol) 3324 (NH), 1659
(C]O), 1602 cmꢀ1. Anal. Calcd for C18H23N3O3: C, 65.63; H, 7.04; N,
12.76. Found: C, 65.68; H, 7.05; N, 12.74.
4.1.3.1. Ethyl 2-(2-isobutyrylhydrazinyl)-5-phenyl-1H-pyrrole-3-
carboxylate (29). Yield 83%. Mp 168–170 ꢁC (MeCN). 1H NMR
4.1.3.8. Ethyl 5-(4-methoxyphenyl)-2-(2-butyrylhydrazinyl)-
(DMSO-d6):
d
1.00 (d, 6H, J ¼ 6.9 Hz, CH3), 1.20 (t, 3H, J ¼ 7.2 Hz,
1H-pyrrole-3-carboxylate (36). Yield 92%. Mp 180–182 ꢁC (MeCN).
CH3), 2.45 (m, 1H, CH), 4.18 (q, 2H, J ¼ 7.2 Hz, CH2), 6.53 (s, 1H, H-4),
7.25–7.60 (m, 5H, Ph), 7.87, 9.63 (s, 2H, NH), 10.81 (s, 1H, NH). IR
(Nujol) 3311 (NH), 3278 (NH), 1677 (C]O), 1657, 1607 cmꢀ1. Anal.
Calcd for C17H21N3O3: C, 64.74; H, 6.71; N, 13.32. Found: C, 64.77; H,
6.70; N, 13.36.
1H NMR (DMSO-d6):
d
0.86 (t, 3H, J ¼ 7.3 Hz, CH3), 1.21 (t, 3H,
J ¼ 6.9 Hz, CH3), 1.52 (m, 2H, CH2), 2.13 (m, 2H, CH2), 3.79 (s, 3H,
CH3), 4.13 (q, 2H, J ¼ 6.9 Hz, CH2), 6.33 (s, 1H, H-4), 6.85 (d, 2H,
J ¼ 8.8 Hz, Ph, H-3 and H-5), 7.45 (d, 2H, J ¼ 8.8 Hz, Ph, H-2 and H-6),
7.77, 9.58 (s, 2H, NH), 10.65 (s, 1H, NH). IR (Nujol) 3268 (NH), 1669
(C]O), 1633, 1596 cmꢀ1. Anal. Calcd for C18H23N3O4: C, 62.59; H,
6.71; N, 12.17. Found: C, 62.64; H, 6.70; N, 12.14.
4.1.3.2. Ethyl 5-(4-chlorophenyl)-2-(2-isobutyrylhydrazinyl)-
1H-pyrrole-3-carboxylate (30). Yield 70%. Mp 173–174 ꢁC (MeCN).
1H NMR (DMSO-d6):
d
1.03 (d, 6H, J ¼ 6.9 Hz, CH3), 1.21 (t, 3H,
4.1.3.9. Ethyl 2-(2-(2-(4-methoxyphenyl)acetyl)hydrazinyl)-5-
phenyl-1H-pyrrole-3-carboxylate (37). Yield 86%. Mp 144–145 ꢁC
J ¼ 7.3 Hz, CH3), 2.42 (m, 1H, CH), 4.13 (q, 2H, J ¼ 7.3 Hz, CH2), 6.55
(s, 1H, H-4), 7.30 (d, 2H, J ¼ 8.1 Hz, Ph), 7.56 (d, 2H, J ¼ 8.1 Hz, Ph),
7.83, 9.62 (s, 2H, NH), 10.82 (s, 1H, NH). IR (Nujol) 3311 (NH), 3278
(NH), 1677 (C]O), 1657, 1607 cmꢀ1. Anal. Calcd for C17H20ClN3O3: C,
58.37; H, 5.76; N, 12.01. Found: C, 58.32; H, 5.77; N, 12.04.
(MeCN). 1H NMR (DMSO-d6):
d
1.20 (t, 3H, J ¼ 6.9 Hz, CH3), 3.42 (s,
2H, CH2), 3.67 (s, 3H, CH3), 4.13 (q, 2H, J ¼ 6.9 Hz, CH2), 6.49 (s, 1H,
H-4), 6.82 (d, 2H, J ¼ 7.7 Hz, Ph, H-3 and H-5), 7.10–7.28 (m, 5H, Ph),
7.52 (d, 2H, J ¼ 7.7 Hz, Ph, H-2 and H-6), 7.85, 9.88 (s, 2H, NH), 10.76
(s, 1H, NH). IR (Nujol) 3444 (NH), 3291 (NH), 1670 (C]O), 1608,
1593 cmꢀ1. Anal. Calcd for C22H23N3O4: C, 67.16; H, 5.89; N, 10.68.
Found: C, 67.21; H, 5.90; N, 10.66.
4.1.3.3. Ethyl 5-(4-methylphenyl)-2-(2-isobutyrylhydrazinyl)-
1H-pyrrole-3-carboxylate (31). Yield 80%. Mp 190–192 ꢁC (MeCN).
1H NMR (DMSO-d6):
d
1.03 (d, 6H, J ¼ 6.5 Hz, CH3), 1.21 (t, 3H,
J ¼ 7.7 Hz, CH3), 2.23 (s, 3H, CH3), 2.41 (m, 1H, CH), 4.11 (q, 2H,
J ¼ 7.7 Hz, CH2), 6.42 (s, 1H, H-4), 7.08 (d, 2H, J ¼ 7.7 Hz, Ph), 7.42 (d,
2H, J ¼ 7.7 Hz, Ph), 7.80, 9.59 (s, 2H, NH), 10.72 (s, 1H, NH). IR (Nujol)
4.1.3.10. Ethyl 5-(4-chlorophenyl)-2-(2-(2-(4-
methoxyphenyl)acetyl)hydrazinyl)-1H-pyrrole-3-carboxylate
(38). Yield 39%. Mp 179–180 ꢁC (MeCN). 1H NMR (DMSO-d6):
d 1.21
3350 (NH), 3312 (NH), 3208 (NH), 1745 (C]O), 1657, 1599 cmꢀ1
.
(t, 3H, J ¼ 7.0 Hz, CH3), 3.42 (s, 2H, CH2), 3.67 (s, 3H, CH3), 4.13 (q,
2H, J ¼ 7.0 Hz, CH2), 6.45 (s, 1H, H-4), 6.84 (d, 2H, J ¼ 8.0 Hz, 4-
OMePh), 7.09 (d, 2H, J ¼ 7.8 Hz, 4-OMePh), 7.25 (d, 2H, J ¼ 8.0 Hz, 4-
ClPh), 7.44 (d, 2H, J ¼ 7.8 Hz, 4-ClPh), 7.86, 9.83 (s, 2H, NH), 10.85 (s,
Anal. Calcd for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76. Found: C,
65.67; H, 7.02; N, 12.79.
4.1.3.4. Ethyl 5-(4-methoxyphenyl)-2-(2-isobutyrylhydrazinyl)-1H-
pyrrole-3-carboxylate (32). Yield 62%. Mp 187–188 ꢁC (MeCN). 1H
1H, NH). IR (Nujol) 3380 (NH), 3233 (NH), 1669 (C]O), 1611 cmꢀ1
.
Anal. Calcd for C22H22ClN3O4: C, 61.75; H, 5.18; N, 9.82. Found: C,
61.69; H, 5.19; N, 9.80.
NMR (DMSO-d6):
d
1.04 (d, 6H, J ¼ 6.9 Hz, CH3), 1.22 (t, 3H,
J ¼ 6.9 Hz, CH3), 2.40 (m, 1H, CH), 3.71 (s, 3H, CH3), 4.13 (q, 2H,
J ¼ 6.9 Hz, CH3), 6.34 (s, 1H, H-4), 6.86 (d, 2H, J ¼ 8.8 Hz, Ph), 7.46 (d,
2H, J ¼ 8.8 Hz, Ph), 7.79, 9.59 (s, 2H, NH), 10.70 (s, 1H, NH). IR (Nujol)
4.1.3.11. Ethyl 5-(4-methylphenyl)-2-(2-(2-(4-
methoxyphenyl)acetyl)hydrazinyl)-1H-pyrrole-3-carboxylate
3286 (NH), 1686 (C]O), 1650, 1596 cmꢀ1
.
Anal. Calcd for
(39). Yield 66%. Mp 169–170 ꢁC (MeCN). 1H NMR (DMSO-d6):
d 1.20
C18H23N3O4: C, 62.59; H, 6.71; N, 12.17. Found: C, 64.57; H, 6.70; N,
12.20.
(t, 3H, J ¼ 6.9 Hz, CH3), 2.23 (s, 3H, CH3), 3.41 (s, 2H, CH2), 3.67 (s,
3H, CH3), 4.11 (q, 2H, J ¼ 6.9 Hz, CH2), 6.42 (s, 1H, H-4), 6.82 (d, 2H,
J ¼ 8.1 Hz, 4-OMePh), 7.10 (d, 2H, J ¼ 7.7 Hz, 4-OMePh), 7.20 (d, 2H,
J ¼ 8.1 Hz, 4-MePh), 7.42 (d, 2H, J ¼ 7.7 Hz, 4-MePh), 7.83, 9.87 (s,
2H, NH), 10.71 (s, 1H, NH). IR (Nujol) 3444 (NH), 3286 (NH), 1669
(C]O), 1597 cmꢀ1. Anal. Calcd for C23H25N3O4: C, 67.80; H, 6.18; N,
10.31. Found: C, 67.77; H, 6.20; N, 10.28.
4.1.3.5. Ethyl 2-(2-butyrylhydrazinyl)-5-phenyl-1H-pyrrole-3-
carboxylate (33). Yield 71%. Mp 194–195 ꢁC (MeCN). 1H NMR
(DMSO-d6):
d
0.86 (t, 3H, J ¼ 7.3 Hz, CH3),1.22 (t, 3H, J ¼ 7.3 Hz, CH3),
1.54 (m, 2H, CH2), 2.13 (m, 2H, CH2), 4.13 (q, 2H, J ¼ 7.3 Hz, CH2),

V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
1293
4.1.3.12. Ethyl
carboxylate (40). Yield 27%. Mp 159–160 ꢁC (MeCN). 1H NMR
(DMSO-d6):
2.17 (q, 2H, J ¼ 7.3 Hz, CH2), 4.13 (q, 2H, J ¼ 7.3 Hz, CH2), 6.49 (s, 1H,
H-4), 7.08–7.54 (m, 5H, Ph), 7.80, 9.61 (s, 2H, NH), 10.75 (s, 1H, NH).
IR (Nujol) 3319 (NH), 1661 (C]O), 1597 cmꢀ1. Anal. Calcd for
C16H19N3O3: C, 63.77; H, 6.36; N, 13.94. Found: C, 63.70; H, 6.37; N,
13.96.
5-phenyl-2-(2-propanoylhydrazinyl)-1H-pyrrole-3-
CH3), 6.32 (s, 1H, H-4), 6.86 (d, 2H, J ¼ 7.0 Hz, Ph, H-3 and H-5), 7.42
(d, 2H, J ¼ 7.0 Hz, Ph, H-2 and H-6), 7.96, 9.76 (s, 2H, NH), 10.96 (s,
1H, NH). IR (Nujol) 3332 (NH), 3321 (NH), 2201 (CN), 1735 (C]O),
1708, 1617 cmꢀ1. Anal. Calcd for C15H16N4O2: C, 63.37; H, 5.67; N,
19.71. Found: C, 63.42; H, 5.66; N, 19.74.
d
1.00 (t, 3H, J ¼ 7.3 Hz, CH3),1.22 (t, 3H, J ¼ 7.3 Hz, CH3),
4.1.3.20. 5-(4-Methoxyphenyl)-2-(2-isobutyrylhydrazinyl)-1H-
pyrrole-3-carbonitrile (48). Yield 24%. Mp 208–210 ꢁC (MeCN). 1H
NMR (DMSO-d6):
d
0.98 (d, 6H, J ¼ 6.9 Hz, CH3), 2.39 (m, 1H, CH),
4.1.3.13. Ethyl 5-(4-methoxyphenyl)-2-(2-propanoylhydrazinyl)-1H-
pyrrole-3-carboxylate (41). Yield 35%. Mp 187–188 ꢁC (MeCN). 1H
3.68 (s, 3H, CH3), 6.54 (s, 1H, H-4), 7.18 (d, 2H, J ¼ 7.8 Hz, Ph, H-3
and H-5), 7.50 (d, 2H, J ¼ 7.8 Hz, Ph, H-2 and H-6), 7.94, 9.86 (s,
2H, NH), 11.09 (s, 1H, NH). IR (Nujol) 3259 (NH), 2204 (CN), 1696
(CO), 1672, 1599 cmꢀ1. Anal. Calcd for C16H18N4O2: C, 64.41; H,
6.08; N, 18.78. Found: C, 64.47; H, 6.07; N, 18.76.
NMR (DMSO-d6):
d
0.99 (t, 3H, J ¼ 7.3 Hz, CH3),1.21 (t, 3H, J ¼ 6.9 Hz,
CH3), 2.15 (q, 2H, J ¼ 7.3 Hz, CH2), 3.60 (s, 3H, CH3), 4.12 (q, 2H,
J ¼ 6.9 Hz, CH2), 6.32 (s, 1H, H-4), 6.85 (d, 2H, J ¼ 8.6 Hz, Ph, H-3 and
H-5), 7.49 (d, 2H, J ¼ 8.6 Hz, Ph, H-2 and H-6), 7.75, 9.59 (s, 2H, NH),
10.66 (s, 1H, NH). IR (Nujol) 3519 (NH), 3320 (NH), 1668 (C]O),
1642,1604,1585 cmꢀ1. Anal. Calcd for C17H21N3O4: C, 61.62; H, 6.39;
N, 12.68. Found: C, 61.67; H, 6.38; N, 12.65.
4.1.3.21. 2-(2-Ethoxycarbonylhydrazinyl)-5-phenyl-1H-pyrrole-3-
carbonitrile (49). Yield 34%. Mp 204–205 ꢁC (MeCN). 1H NMR
(DMSO-d6):
d
1.16 (t, 3H, J ¼ 6.9 Hz, CH3), 4.03 (q, 2H, J ¼ 6.9 Hz,
CH2), 6.51 (s, 1H, H-4), 7.05–7.53 (m, 5H, Ph), 8.13, 9.17 (s, 2H, NH),
11.09 (s, 1H, NH). IR (Nujol) 3286 (NH), 2200 (CN), 1699 (C]O),
1675, 1612 cmꢀ1. Anal. Calcd for C14H14N4O2: C, 62.21; H, 5.22; N,
20.73. Found: C, 62.16; H, 5.20; N, 20.78.
4.1.3.14. Ethyl 2-(2-(3-methoxypropanoyl)hydrazinyl)-5-phenyl-1H-
pyrrole-3-carboxylate (42). Yield 22%. Mp 141–142 ꢁC (MeCN). 1H
NMR (DMSO-d6):
d
1.23 (t, 3H, J ¼ 6.9 Hz, CH3), 2.41 (m, 2H, CH2),
3.21 (s, 3H, CH3), 3.54 (m, 2H, CH2), 4.13 (q, 2H, J ¼ 6.9 Hz, CH2), 6.50
(s,1H, H-4), 7.09–7.50 (m, 5H, Ph), 7.85, 9.75 (s, 2H, NH),10.64 (s,1H,
NH). IR (Nujol) 3274 (NH), 3257 (NH), 1672 (C]O), 1641, 1605,
1592 cmꢀ1. Anal. Calcd for C17H21N3O4: C, 61.62; H, 6.39; N, 12.68.
Found: C, 61.66; H, 6.38; N, 12.72.
4.1.3.22. 2-(2-Ethoxycarbonylhydrazinyl)-5-(4-methoxyphenyl)-1H-
pyrrole-3-carbonitrile (50). Yield 38%. Mp 205–206 ꢁC (MeCN). 1H
NMR (DMSO-d6):
d
1.00 (t, 3H, J ¼ 7.7 Hz, CH3), 3.86 (s, 3H, CH3),
4.02 (q, 2H, J ¼ 7.7 Hz, CH2), 6.32 (s, 1H, H-4), 6.86 (d, 2H, J ¼ 8.4 Hz,
Ph, H-3 and H-5), 7.41 (d, 2H, J ¼ 8.4 Hz, Ph, H-2 and H-6), 7.94, 9.86
(s, 2H, NH), 10.95 (s, 1H, NH). IR (Nujol) 3301, 2198 (CN), 1695
(C]O), 1673, 1619 cmꢀ1. Anal. Calcd for C15H16N4O3: C, 59.99; H,
5.37; N, 18.66. Found: C, 60.06; H, 5.38; N, 18.63.
4.1.3.15. Ethyl 5-(4-methoxyphenyl)-2-(2-(3-methoxypropanoyl)
hydrazinyl)-1H-pyrrole-3-carboxylate (43). Yield 68%. Mp 157–
158 ꢁC (MeCN). 1H NMR (DMSO-d6):
d
1.21 (t, 3H, J ¼ 7.0 Hz, CH3),
2.39 (m, 2H, CH2), 3.19 (s, 3H, CH3), 2.54 (m, 2H, CH2), 3.70 (s, 3H,
CH3), 4.12 (q, 2H, J ¼ 7.0 Hz, CH2), 6.33 (s, 1H, H-4), 6.85 (d, 2H,
J ¼ 7.3 Hz, Ph, H-3 and H-5), 7.44 (d, 2H, J ¼ 7.3 Hz, Ph, H-2 and H-
6), 7.79, 9.72 (s, 2H, NH), 10.54 (s, 1H, NH). IR (Nujol) 3281 (NH),
4.1.3.23. 2-(2-(2-Methoxyacetyl)hydrazinyl)-5-phenyl-1H-pyrrole-3-
carbonitrile (51). Yield 24%. Mp 181–182 ꢁC (MeCN). 1H NMR
(DMSO-d6):
d 2.40 (s, 2H, CH2), 3.90 (s, 3H, CH3), 6.50 (s, 1H, H-4),
3261 (NH), 1674 (C]O), 1639, 1596 cmꢀ1
.
Anal. Calcd for
7.07–7.51 (m, 5H, Ph), 8.16, 9.87 (s, 2H, NH), 11.07 (s, 1H, NH). IR
(Nujol) 3296 (NH), 3232 (NH), 2204 (CN), 1702 (C]O), 1678, 1613,
1595 cmꢀ1. Anal. Calcd for C14H14N4O2: C, 62.21; H, 5.22; N, 20.73.
Found: C, 62.27; H, 5.23; N, 20.68.
C18H23N3O5: C, 59.82; H, 6.41; N, 11.63. Found: C, 59.77; H, 6.40; N,
11.66.
4.1.3.16. 2-(2-Acetylhydrazinyl)-5-phenyl-1H-pyrrole-3-carbonitrile
(44). Yield 98%. Mp 198–200 ꢁC (MeCN). 1H NMR (DMSO-d6):
d
1.83
4.1.3.24. 2-(2-(2-Methoxyacetyl)hydrazinyl)-5-(4-methoxyphenyl)-
(s, 3H, CH3), 6.50 (s,1H, H-4), 7.07–7.50 (m, 5H, Ph), 8.09, 9.81 (s, 2H,
NH), 11.05 (s, 1H, NH). IR (Nujol) 3364 (NH), 3260 (NH), 2215 (CN),
1679 (C]O), 1618 cmꢀ1. Anal. Calcd for C13H12N4O: C, 64.99; H,
5.03; N, 23.32. Found: C, 65.03; H, 5.02; N, 23.36.
1H-pyrrole-3-carbonitrile (52). Yield 35%. Mp 189–190 ꢁC (MeCN).
1H NMR (DMSO-d6):
d 2.48 (s, 2H, CH2), 3.36 (s, 3H, CH3), 3.70 (s, 3H,
CH3), 6.33 (s, 1H, H-4), 6.86 (d, 2H, J ¼ 8.4 Hz, Ph, H-3 and H-5), 7.41
(d, 2H, J ¼ 8.4 Hz, Ph, H-2 and H-6), 8.07, 9.85 (s, 2H, NH), 10.97 (s,
1H, NH). IR (Nujol) 3312 (NH), 2201 (CN), 1682 (C]O), 1663,
1619 cmꢀ1. Anal. Calcd for C15H16N4O3: C, 59.99; H, 5.37; N, 18.66.
Found: C, 59.92; H, 5.38; N, 18.70.
4.1.3.17. 2-(2-Acetylhydrazinyl)-5-(4-methoxyphenyl)-1H-pyrrole-3-
carbonitrile (45). Yield 40%. Mp 194–195 ꢁC (MeCN). 1H NMR
(DMSO-d6):
d 1.83 (s, 3H, CH3), 3.80 (s, 3H, CH3), 6.32 (s, 1H, H-4),
6.33 (d, 2H, J ¼ 8.4 Hz, Ph, H-3 and H-5), 7.41 (d, 2H, J ¼ 8.4 Hz, Ph,
H-2 and H-6), 7.98, 9.78 (s, 2H, NH), 10.93 (s, 1H, NH). IR (Nujol)
3279 (NH), 2197 (CN), 1697 (C]O), 1673, 1602 cmꢀ1. Anal. Calcd for
C14H14N4O2: C, 62.21; H, 5.22; N, 20.73. Found: C, 62.27; H, 5.21; N,
20.77.
4.1.3.25. 2-(2-(3-Methoxypropanoyl)hydrazinyl)-5-phenyl-1H-
pyrrole-3-carbonitrile (53). Yield 29%. Mp 187–188 ꢁC (MeCN). 1H
NMR (DMSO-d6):
d
2.38 (t, 2H, J ¼ 6.1 Hz, CH2), 3.17 (s, 3H, CH3),
3.54 (t, 2H, J ¼ 6.1 Hz, CH2), 6.50 (s, 1H, H-4), 7.10–7.48 (m, 5H, Ph),
8.17, 9.87 (s, 2H, NH), 11.01 (s, 1H, NH). IR (Nujol) 3298 (NH), 2205
(CN), 1696 (C]O), 1672, 1611 cmꢀ1. Anal. Calcd for C15H16N4O2: C,
63.37; H, 5.67; N, 19.71. Found: C, 63.41; H, 5.69; N, 19.68.
4.1.3.18. 5-Phenyl-2-(2-propanoylhydrazinyl)-1H-pyrrole-3-carbon-
itrile (46). Yield 27%. Mp 189–190 ꢁC (MeCN). 1H NMR (DMSO-d6):
d
1.33 (t, 3H, J ¼ 5.1 Hz, CH3), 4.03 (q, 2H, J ¼ 5.1 Hz, CH2), 6.50 (s, 1H,
4.1.3.26. 5-(4-Methoxyphenyl)-2-(2-(3-methoxypropanoyl)hy-
H-4), 7.10–7.50 (m, 5H, Ph), 8.11, 9.17 (s, 2H, NH),11.08 (s,1H, NH). IR
(Nujol) 3280 (NH), 2205 (CN), 1731 (C]O), 1707, 1613 cmꢀ1. Anal.
Calcd for C14H14N4O: C, 66.13; H, 5.55; N, 22.03. Found: C, 66.17; H,
5.56; N, 22.06.
drazinyl)-1H-pyrrole-3-carbonitrile (54). Yield 37%. Mp 174–175 ꢁC
(MeCN). 1H NMR (DMSO-d6):
d
2.38 (t, 2H, J ¼ 6.6 Hz, CH2), 3.18 (s,
3H, CH3), 3.54 (t, 2H, J ¼ 6.6 Hz, CH2), 3.70 (s, 3H, CH3), 6.32 (s, 1H,
H-4), 6.86 (d, 2H, J ¼ 8.6 Hz, Ph, H-3 and H-5), 7.41 (d, 2H, J ¼ 8.6 Hz,
Ph, H-2 and H-6), 8.07, 9.84 (s, 2H, NH), 10.89 (s, 1H, NH). IR (Nujol)
4.1.3.19. 5-(4-Methoxyphenyl)-2-(2-propanoylhydrazinyl)-1H-
3278 (NH), 1668 (C]O), 1644, 1596 cmꢀ1
. Anal. Calcd for
pyrrole-3-carbonitrile (47). Yield 39%. Mp 179–180 ꢁC (MeCN). 1H
C16H18N4O3: C, 61.13; H, 5.77; N, 17.82. Found: C, 61.17; H, 5.76; N,
17.86.
NMR (DMSO-d6): d 1.00 (m, 3H, CH3), 2.13 (m, 2H, CH2), 3.70 (s, 3H,

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V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
4.1.3.27. Methyl 2-(2-acetylhydrazinyl)-5-phenyl-1H-pyrrole-3-
carboxylate (55). Yield 34%. Mp 174–175 ꢁC (MeCN). 1H NMR
3.53 (m, 2H, CH2), 3.64 (s, 3H, CH3), 3.70 (s, 3H, CH3), 6.34 (s, 1H, H-4),
6.85 (d, 2H, J ¼ 6.5 Hz, Ph, H-3 and H-5), 7.44 (d, 2H, J ¼ 6.5 Hz, Ph, H-2
and H-6), 7.80, 9.72 (s, 2H, NH), 10.54 (s, 1H, NH). IR (Nujol) 3278 (NH),
1668 (C]O),1644,1596 cmꢀ1. Anal. Calcd for C17H21N3O5: C, 58.78; H,
6.09; N, 12.10. Found: C, 58.72; H, 6.07; N, 12.12.
(DMSO-d6):
d 1.87 (s, 3H, CH3), 3.64 (s, 3H, CH3), 6.50 (s, 1H, H-4),
7.08–7.53 (m, 5H, Ph), 7.81, 9.67 (s, 2H, NH), 10.74 (s, 1H, NH). IR
(Nujol) 3224 (NH), 1686 (C]O), 1647, 1611 cmꢀ1. Anal. Calcd for
C14H15N3O3: C, 61.53; H, 5.53; N, 15.38. Found: C, 61.49; H, 5.54; N,
15.36.
4.2. Antifungal susceptibility assays
4.1.3.28. Methyl 2-(2-acetylhydrazinyl)-5-(4-methoxyphenyl)-1H-
pyrrole-3-carboxylate (56). Yield 31%. Mp 179–180 ꢁC (MeCN). 1H
MICs were determined by the broth microdilution method
according to the NCCLS reference document M27-A [24]. RPMI
1640 medium (Sigma Chemicals) without NaHCO3, supple-
NMR (DMSO-d6):
d 1.86 (s, 3H, CH3), 3.63 (s, 3H, CH3), 3.70 (s, 3H,
CH3), 6.33 (s, 1H, H-4), 6.85 (d, 2H, J ¼ 8.8 Hz, Ph, H-3 and H-5), 7.46
(d, 2H, J ¼ 8.8 Hz, Ph, H-2 and H-6), 7.76, 9.65 (s, 2H, NH), 10.65 (s,
1H, NH). IR (Nujol) 3327 (NH), 3340 (NH), 1686 (C]O), 1642,
1603 cmꢀ1. Anal. Calcd for C15H17N3O4: C, 59.40; H, 5.65; N, 13.85.
Found: C, 59.35; H, 5.66; N, 13.89.
mented with L-glutamine (Gibco, Invitrogen) and buffered with
0.165 M morpholinepropanesulfonic acid (MOPS, Sigma Chem-
icals) at pH 7.0 was used as test medium. Twofold dilutions of
the drugs with concentrations ranging between 0.008 and
200 mg/L were obtained in RPMI 1640 and dispensed into the
wells of plastic microdilution trays. Starting inoculum suspen-
sions were obtained by the spectrophotometric method of
inoculum preparation, adjusted to 106 CFU/mL and then diluted
4.1.3.29. Methyl 5-phenyl-2-(2-propanoylhydrazinyl)-1H-pyrrole-3-
carboxylate (57). Yield 34%. Mp 149–150 ꢁC (MeCN). 1H NMR
in test medium to 2 ꢂ104 cells/mL. A 100-
m
l yeast inoculum was
(DMSO-d6):
d
1.00 (t, 3H, J ¼ 7.7 Hz, CH3), 2.16 (q, 2H, J ¼ 7.7 Hz,
CH2), 3.64 (s, 3H, CH3), 6.50 (s, 1H, H-4), 7.05–7.53 (m, 5H, Ph),
7.61, 9.61 (s, 2H, NH), 10.74 (s, 1H, NH). IR (Nujol) 3311 (NH),
added to each well of the microdilution trays to obtain final
concentrations of the drugs ranging between 0.004 and 100 mg/
L and final inocula of 104 cells/mL. The inoculated plates were
incubated overnight at 35 ꢁC in a humid atmosphere. After
agitation, plates were visually read with the aid of a reading
mirror and spectrophotometrically with an automatic plate
reader (Sunrise Tecan, Gro¨dig/Salzburg, Austria) set at 450 nm.
MICs were determined either visually or by spectrophotometric
evaluation showed excellent agreement.
3272 (NH), 1685 (C]O), 1647, 1609 cmꢀ1
. Anal. Calcd for
C15H17N3O3: C, 62.71; H, 5.96; N, 14.63. Found: C, 62.77; H, 5.95;
N, 14.60.
4.1.3.30. Methyl 5-(4-methoxyphenyl)-2-(2-propanoylhydrazinyl)-
1H-pyrrole-3-carboxylate (58). Yield 44%. Mp 185–186 ꢁC (MeCN).
1H NMR (DMSO-d6):
d
1.00 (t, 3H, J ¼ 8.4 Hz, CH3), 2.16 (q, 2H,
J ¼ 8.4 Hz, CH2), 3.64 (s, 3H, CH3), 3.70 (s, 3H, CH3), 6.34 (s, 1H, H-4),
6.85 (d, 2H, J ¼ 8.1 Hz, Ph, H-3 and H-5), 7.46 (d, 2H, J ¼ 8.1 Hz, Ph,
H-2 and H-6), 7.76, 9.59 (s, 2H, NH), 10.66 (s, 1H, NH). IR (Nujol)
3518 (NH), 3307 (NH), 1675 (C]O), 1643, 1601 cmꢀ1. Anal. Calcd for
C16H19N3O4: C, 60.56; H, 6.03; N, 13.24. Found: C, 60.63; H, 6.02; N,
13.26.
After the MIC determination, a 20-ml sample from each well was
seeded in plates of Sabouraud Dextrose agar. Plates were incubated
for 72 h at 35 ꢁC. The minimum fungicidal concentration (MFC) was
defined as the minimum concentration of compound which
resulted in the growth of less than two colonies representing the
killing of >99% of the original inoculum.
4.1.3.31. Methyl 2-(2-ethoxycarbonylhydrazinyl)-5-phenyl-1H-
pyrrole-3-carboxylate (59). Yield 21%. Mp 151–152 ꢁC (MeCN). 1H
Acknowledgements
NMR (DMSO-d6):
d
1.16 (t, 3H, J ¼ 6.8 Hz, CH3), 3.64 (s, 3H, CH3),
We thank the Antitumor Evaluation Branch of the National
Cancer Institute for performing cytotoxicity evaluations.
4.03 (q, 2H, J ¼ 6.8 Hz, CH2), 6.51 (s, 1H, H-4), 7.07–7.49 (m, 5H, Ph),
7.84, 9.02 (s, 2H, NH), 10.88 (s, 1H, NH). IR (Nujol) 3554 (NH), 3309
(NH), 1703 (C]O), 1650 (C]O), 1614 cmꢀ1
. Anal. Calcd for
References
C15H17N3O4: C, 59.40; H, 5.65; N, 13.85. Found: C, 59.46; H, 5.64; N,
13.86.
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1.14 (t, 3H, J ¼ 6.8 Hz, CH3),
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(s, 1H, H-4), 6.85 (d, 2H, J ¼ 8.1 Hz, Ph, H-3 and H-5), 7.52 (d, 2H,
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1H NMR (DMSO-d6):
d 2.40 (m, 2H, CH2), 3.20 (s, 3H, CH3), 3.54 (m,
2H, CH2), 3.65 (s, 3H, CH3), 6.50 (s, 1H, H-4), 7.06–7.53 (m, 5H, Ph),
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(MeCN). 1H NMR (DMSO-d6):
d 2.40 (m, 2H, CH2), 3.19 (s, 3H, CH3),

V. Onnis et al. / European Journal of Medicinal Chemistry 44 (2009) 1288–1295
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