Synthesis, bioevaluation and structural study of substituted phthalazin-1(2H)-ones acting as antifungal agents

Article abstract of DOI:10.3390/molecules18033479

Twenty-five polysubstituted phthalazinone derivatives were synthesized and tested for their antifungal activity against a panel of pathogenic and clinically important yeasts and filamentous fungi. Among them, the compound 4-(4-chlorobenzyl)-2- methylphthalazin-1(2H)-one (5) exhibited a remarkable antifungal activity against standardised strains of dermatophytes and Cryptococcus neoformans, as well as against some clinical isolates. A physicochemical study performed on compound 5 revealed its conformational and electronic characteristics, providing us with useful data for the future design of novel related antifungal analogues.

Full text of DOI:10.3390/molecules18033479

Molecules 2013, 18, 3479-3501; doi:10.3390/molecules18033479
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Bioevaluation and Structural Study of Substituted
Phthalazin-1(2H)-ones Acting as Antifungal Agents
Marcos Derita 1, Esther del Olmo 2,*, Bianca Barboza 2, Ana Esther García-Cadenas 2,
José Luis López-Pérez 2, Sebastián Andújar 3,4, Daniel Enriz 3,4, Susana Zacchino 1,* and
Arturo San Feliciano 2
1
Pharmacognosy Department, Faculty of Biochemical and Pharmaceutical Sciences,
National University of Rosario, Suipacha 531, 2000 Rosario, Argentine;
E-Mail: mgderita@hotmail.com (M.D.)
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, CIETUS-IBSAL,
University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain;
E-Mails: bianca.barboza@gmail.com (B.B.); anaester@usal.es (A.E.G.-C.);
lopez@usal.es (J.L.L.-P.); asf@usal.es (A.S.F.)
3
Faculty of Chemistry, Biochemistry and Pharmacy, National University of San Luis (UNSL),
Chacabuco 917, 5700 San Luis, Argentine; E-Mails: saanduja@unsl.edu.ar (S.A.);
denriz@unsl.edu.ar (D.E.)
4
IMIBIO-CONICET, Universidad Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina
* Authors to whom correspondence should be addressed; E-Mails: olmo@usal.es (E.D.O.);
szaabgil@citynet.net.ar (S.Z.); Tel.: +34-923-294-528 (E.D.O.) Fax: +34-923-294-515 (E.D.O.).
Received: 14 January 2013; in revised form: 31 January 2013 / Accepted: 14 March 2013 /
Published: 18 March 2013
Abstract: Twenty-five polysubstituted phthalazinone derivatives were synthesized and
tested for their antifungal activity against a panel of pathogenic and clinically important
yeasts and filamentous fungi. Among them, the compound 4-(4-chlorobenzyl)-2-
methylphthalazin-1(2H)-one (5) exhibited a remarkable antifungal activity against
standardised strains of dermatophytes and Cryptococcus neoformans, as well as against
some clinical isolates. A physicochemical study performed on compound 5 revealed its
conformational and electronic characteristics, providing us with useful data for the future
design of novel related antifungal analogues.

Molecules 2013, 18
3480
Keywords: benzylphthalazinones; antifungal; Cryptococcus neoformans; dermatophytes
structure–activity relationships; conformational study
1. Introduction
Fungal infections have emerged as a major cause of morbidity and often of mortality in
immunocompromised and debilitated patients over the past decades. A matter of concern in the
treatment of fungal infections is the limited number of efficacious antifungal drugs available [1,2].
Many of the currently available drugs are toxic, produce recurrence or lead to the development of
resistance, due in part to the prolonged periods of drug administration needed [3]. Although a new
generation of triazoles, polyenes in lipidic formulations and echinocandins have been introduced, and
several combination therapies have been configured as therapeutic alternatives during the last decade,
fungal infections remain difficult to eradicate [3]. There is, therefore, a clear need of discovering new
structures with antifungal properties, that could lead to the development of new useful agents for the
management of fungal infections.
In the course of our on-going screening program for new and selective antifungal compounds, we
have previously reported several series of antifungal compounds obtained from natural and synthetic
sources [4–9]. Considering that some phthalazine derivatives, including some polybrominated
compounds [10], 4-benzyl substituted ones [11] and others [12–15] have been evaluated for their
antimicrobial and particularly antifungal activities against yeasts (Candida and Cryptococcus strains)
and Aspergillus spp., we have prepared a series of twenty five differently substituted phthalazin-1-ones
to evaluate their antifungal activities against a panel of representative clinically important fungal
species. Then, taking into account the antifungal results, conformational and electronic studies on the
most interesting compound of the series were carried out.
2. Results and Discussion
2.1. Chemistry
A first group of phthalazinones 1–13 (Scheme 1) was synthesized from the intermediate
4-benzalphthalides B1–B7 by treatment with either hydrazine or methyl hydrazine. Previously, the
benzalphthalides were prepared in usually good though variable yields (90–45%) by high temperature
condensation of phthalic anhydride with mono-, di- or tri-substituted phenylacetic acids, in the
presence of toluene and potassium carbonate following a reported procedure [16], with a slight
variation. The condensation of benzalphthalides B1 to B4 and B6 with hydrazine hydrate at 80 °C
during 6–8 h yielded the phthalazinones 1–4 and 12 respectively, while the reaction of
benzalphthalides B1 to B5 and B7 with methylhydrazine under the same conditions gave
phthalazinones 5–9 and 13, respectively.
According to our preliminary evaluation results of this first group of phthalazinones, which will be
described below, the presence of the 4-chlorobenzyl substituent at position C-4 was considered as the
most relevant feature for the antifungal activity. Consequently, such a moiety was maintained in the

Molecules 2013, 18
3481
compounds synthesized later. Similarly, phthalazinones without a methyl group at the N-2 position
failed to show any noticeable antifungal activity (MIC values > 250 g/mL), whereas the
N2-methylated analogues displayed from fair to good inhibition results. In the continuation of the
research, the change of the methyl group at position N-2 of the phthalazinone for ethyl or allyl groups
led to compounds 10 and 11, respectively. These compounds were synthesized through direct
alkylation of the phthalazinone 1 with the corresponding alkyl or alkenyl bromide. Once evaluated, the
N2-ethyl derivative was less active and less potent than the N-methyl analogue, and the N-allyl derivative
resulted practically inactive. These observations influenced the criteria applied further in this research.
Scheme 1. Synthesis and structures of benzalphthalides B1–B7 and phthalazinones 1–13.
Ar
R2
Ar
O
3
4
R3
R4
Ar-CH2-COOH
H2N-NH-R1
Ar =
N
N
O
O
1
1
K2CO3, toluene
215-230 ºC
R1
O
B
O
O
Ar = Ph R1
R2
R3
Cl
CH3S
R4
Ar
B1 4ClPh
B2 4CH3SPh
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
B3 3,4(OCH2O)Ph
B4 3,4(CH3O)2Ph
B5 3,4,5(CH3O)3Ph
B6 1-naphthyl
H
-OCH2O-
CH3O CH3O
H
CH3
CH3
CH3
H
H
Cl
CH3S
B7 2-naphthyl
-OCH2O-
CH3 CH3O CH3O
Ar = Naphthyl
CH3 CH3O CH3O CH3O
12 1-naphthyl (R1 = H)
13 2-naphthyl (R1 =CH3)
R1-Br
10 C2H5
11 Allyl
H
H
Cl
Cl
H
H
1
Thus, the next step was focused to the introduction of structural modifications on the aromatic ring
of the starting phthalic anhydride, while retaining the 4-chlorobenzyl fragment at C-4 and the methyl
group at N-2. The modifications of the phthalazine system included the introduction of substituents
with electron donating (Me), withdrawing (Cl) and with extended resonance (NO2) properties. The
preparation of phthalazinones 14–25 was carried out by the procedures represented in Scheme 2. In
several cases, a microwave (MW)-based procedure (method B) applied to improve reaction times and
yields, also led to cleaner reaction products. The intermediate benzalphthalides B8–B16 were
previously prepared by the procedure mentioned above. The benzalphthalides monosubstituted on the
phthalazine system B8 to B11 and B13 were obtained as 1:1 mixtures of regioisomers with the
substituent indistinctly attached at positions C-5 or C-6 of the benzalphthalide. The benzalphthalide
B12 was obtained by sodium borohydride reduction of the mixed anhydride intermediate obtained by
treatment of B11 with ethyl chloroformate in THF at low temperature (−15 °C), in the presence of
triethylamine (TEA).
Phthalazinones 14–17 and 19–22 were obtained in good yields by treatment of the corresponding
benzalphthalides with methyl hydrazine at 80 °C, during 6–8 h. The phthalazinone 23 was obtained

Molecules 2013, 18
3482
from phthalazinone 17 after treatment with diazomethane. The phthalazinone-aldehyde 24 was
obtained from the 6(7)-hydroxymethylphthalazinone 18 under Swern oxidation conditions. Finally, the
treatment of aldehyde 24 with hydroxylamine under reflux in ethanol yielded the phthalazinone 25 in
good yield. Phthalazinones 20–22 were obtained by irradiation in a domestic multimode microwave
(MW) apparatus. Equimolar amounts of benzalphthalides B14–B16 and methylhydrazine were mixed
with SiO2 (10 mol) and irradiated at 350 W during 1–6 min, the mixture was percolated with ethyl
acetate and the crude purified by column chromatography to provide the desired phthalazinones 20–22
in 60–70% yield. It is interesting to note the advantages of the MW-based procedure that led to cleaner
reactions products in these cases and have previously served to prepare different phthalazine
derivatives [17]. Indeed, when method A was applied to the dichlorinated benzalphthalides B14–B16
more complex reaction mixtures were obtained, in which, apart from the expected phthalazinones 20–22,
in lowered yields, several compounds (not reported here) derived from chlorine substitution by
methylhydrazinyl groups were also found.
Scheme 2. Synthesis and structures of benzalphthalides B8–B16 and phthalazinones 14–25.
Ar
Ar
O
4'
Cl
4
5
4
Ar =
H2N-NH-CH3
Method A or B
Ar-CH2-COOH
R1
3'
R1
N
N
O
O
1
1'
R1
7
R2
K2CO3 toluene
6
CH3
1
O
O
B
O
Method A: K2CO3, / toluene
Method B: MW / SiO2
B
R1
R2 Comp. Method
6(7)
B8
B9
B10
CH3
CH3
CH3
H
2-Cl
3-Cl
H
14
15
16
17
18
19
20
21
22
A
A
A
B
ii)
B11 COOH
B12 CH2OH
17
18
R16(7) = -COOCH3 (23)
i
iii)
iv)
R16(7) = -CHO (24)
R16(7) = -C=NOH (25)
H
B13
B14
B15
B16
NO2
H
B
B
B
B
i) a: EtOOCCl, Et3N / THF, -15 ºC; b: NaBH4 / H2O.
ii) CH2N2 /Et2O; iii) Swern oxidation.
iv) NH2OH.HCl / EtOH reflux.
6,7Cl2
H
6,7Cl2 2-Cl
6,7Cl2 3-Cl
2.2. Antifungal Activity
The phthalazinone derivatives included in this research were tested in the range from 250 to 0.98 μg/mL
against a panel of clinically important fungi including yeasts, hyalohyphomycetes and dermatophytes
with the microbroth dilution method according to the CLSI guidelines [18,19]. Results against yeasts
showed that none of the compounds inhibited the yeasts Candida albicans, Saccharomyces cerevisiae
or the Aspergillus species filamentous fungi A. niger, A. fumigatus or A. flavus, with the exception of
compound 5 that inhibited the standardized strain Cryptococcus neoformans ATCC 32264. In contrast,
ten out of the twenty five phthalazinones tested (compounds 5–10, 14–16, 21) showed good to
moderate activities against the dermatophytes Microsporum canis (M.c.), Microsporum gypseum
(M.g.), Trichophyton mentagrophytes (T.m.)., Trichophyton rubrum (T.r.) and Epidermophyton
floccosum (E.f.), being also compound 5 the most active substance (Tables 1 and 2).

Molecules 2013, 18
3483
Table 1. Antifungal activity (MIC values, g/mL) of phthalazinones 1–13 against dermatophytes.
Comp.
E.f.
M.c.
M.g.
T.r.
T.m.
1
2
i
i
i
i
i
i
i
i
i
i
3
i
i
i
i
i
i
i
i
4
i
i
25
50
i
5
6.25
250
100
125
i
6.25
100
125
125
i
25
12.5
125
100
i
6
100
7
i
8
i
62.5
125
50
i
9
i
100
50
i
10
11
12
13
AmB
Terb
50
i
62.5
i
50
i
i
i
i
i
i
i
i
i
i
i
0.075
0.04
0.50
0.04
0.125
0.04
0.075
0.01
0.075
0.025
i: Compound considered inactive (MIC > 250 g/mL); AmB: Amphotericin B; Terb: Terbinafine;
E f. = Epidermophyton floccosum; M.c. = Microsporum canis; M.g. = M. gypseum;
T.r. = Trichophyton rubrum; T.m.= T. mentagrophytes.
Table 2. Antifungal activity (MIC values, g/mL) of phthalazinones 14–25 against dermatophytes.
Comp.
5
M.c.
M.g.
T.r.
T.m.
6.25
25
12.5
25
14
i
125
100
50
15
i
250
50
100
16
i
i
100
100
17
i
i
i
i
18
i
i
i
i
19
i
i
i
i
20
i
i
i
i
21
i
i
i
i
22
i
i
i
i
i
i
i
23
i
i
24
i
i
i
25
i
125
0.125
0.04
100
0.075
0.01
100
0.075
0.025
AmB
Terb
0.50
0.04
i: Compounds considered inactive (MIC > 250 g/mL); AmB: Amphotericin B; Terb: Terbinafine;
E.f. = Epidermophyton floccosum; M.c. = Microsporum canis; M.g. = M. gypseum;
§
T.r. = Trichophyton rubrum; T.m.= T. mentagrophytes. Compounds with only one substituent at
position 6 (7), actually contain 1:1 mixtures of both regioisomers.

Molecules 2013, 18
3484
2.2.1. Analysis of the Activity against Dermatophytes
Table 1 summarizes the results of the antifungal activity found for phthalazinones 1–13, all of
which possess no substituent at the fused benzene ring of the phthalazine system. As it can be seen,
the phthalazinone derivatives 1–4 and 12, without substitution at N-2, were inactive (MIC values >
250 g/mL). The comparison between those 2-methyl compounds 1, 5, 10 and 11, easily led us to
define the Me group as the best substituent at N-2, within those compounds tested. However, it is
noteworthy that the N-Me substitution is not by itself sufficient for phthalazinones to display
antifungal activity, since a change of the substituent at C-4 (benzyl to 2-naphthylmethyl), led to
compound 13 which is devoid of antifungal activity. At this respect, another fact that can be observed
when comparing the results related to the absence or presence of a 4-chlorobenzyl substituent at C-4,
that seems to be determinant for the activity and is present in the two most potent compounds of this
group, 5 and 10. Accordingly, the concurrence of both substituents, Me on N-2, and Cl at the p-position
of benzyl group, would be the structural features that combine for the antifungal properties of compound 5.
Other substituents (MeS-, -OCH2O-, MeO-) on the benzyl side chain along with N-Me, provide the
antifungal phthalazinones 6–9, which showed just moderate activity. In addition, the comparison of
antifungal potencies of compounds 1 vs. 5, 2 vs. 6, 3 vs. 7 and 4 vs. 8 showed that the different
substituents at the p-position of the benzyl moiety need to be accompanied by an N-Me group to show
antifungal activity.
The interesting antifungal activities of compound 5 led us to prepare the analogues 14–25, all of
them containing both a Me substituent at N-2 and the 4-chlorobenzyl fragment at the C-4 position.
These compounds were evaluated against the complete panel of fungi, though only positive results are
included in Table 2.
Activity results in Table 2 show that the introduction of a methyl substituent at C-6(7) (compounds
14–16) rendered compounds with 2–10 times lower antifungal activity than compound 5 and a narrower
spectrum of action. Interestingly enough, the change of the Me on C-6(7) to a variety of
electron-withdrawing groups as COOH, COOMe, CH2OH, NO2 or CHO (compounds 17–19 and 23–24),
or even the introduction of two chlorine substituents on C-6 and C-7 (compounds 20–22) led to
inactive compounds. However, compound 25 with a hydroxylimino function at positions C-6(7), and
the 4-chlorobenzyl group at C-4, showed moderate activity against three dermatophyte strains.
We note also that the addition of an extra chlorine substituent at either position 2' or 3' of the benzyl
fragment attached to C-4 of the phthalazinone system in compounds 15, 16, 21 and 22 did not produce
significant changes in the antifungal activity in comparison with their respective monosubstituted
4-ClBn analogues 14 and 20.
2.2.2. Analysis of the Activity against Yeasts
Results against yeasts showed that compound 5 was the only one that showed antifungal activity in
at least one yeast (C. neoformans) of the panel with a value of MIC = 12.5 µg/mL. C. neoformans
remains as an important life-threatening complication for immunocompromised hosts, particularly for
patients who have undergone transplantation of solid organs. The seriousness of this pathogenic yeast
has increased in the last decade, because of the appearance of fluconazole-resistant Cryptococcus

Molecules 2013, 18
3485
strains. Consequently, new compounds acting against this fungus are highly desirable [20,21].
Therefore, we decided to test compound 5 against an extended panel of C. neoformans clinical isolates
provided by the Malbrán Institute (MI, Buenos Aires, Argentina). The results are shown in Table 3.
For the sake of comparison the MIC and Minimum Fungicidal Concentration (MFC), values found
against an ATCC standardized strain of C. neoformans are included. MIC values were determined
against this new panel by using three endpoints: MIC100, MIC80 and MIC50 (the minimum
concentration of compounds that inhibit 100, 80 and 50% of fungal growth, respectively). The
application of less stringent endpoints such as MIC80 and MIC50 has been shown to represent the
in vitro activity of compounds more consistently [22] and many times provides a better correlation
with other measurements of antifungal activity [23]. The evaluation of the MFC for compound 5 was
accomplished by sub-culturing a sample of culture medium from MIC tubes showing no growth, onto
drug-free agar plates.
Table 3. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration
(MFC) values of phthalazinone 5 against clinical isolates of Cryptococcus neoformans.
Phthalazinone 5
AmB
MIC100
0.25
Itz
Vcz
Voucher
specimen
Strain
MIC100 MIC80
MIC50
3.9
MFC
15.6
7.8
MIC100
0.15
MIC100
<0.015
<0.015
<0.015
<0.015
<0.015
<0.015
<0.015
0.03
C. neoformans
C. neoformans
C. neoformans
C. neoformans
C. neoformans
C. neoformans
C. neoformans
C. neoformans
C. neoformans
ATCC 32264
IM 983040
IM 972724
IM 042074
IM 983036
IM 000319
IM 972751
IM 031631
IM 031633
7.8
3.9
7.8
3.9
3.9
7.8
i
3.9
0.13
<0.015
0.25
3.9
3.9
15.6
31.3
i
0.06
15.6
i
3.9
0.25
<0.015
<0.015
<0.015
<0.015
<0.015
0.25
i
0.25
125
62.5
62.5
15.6
62.5
62.5
31.3
7.8
31.3
31.3
15.6
7.8
250
250
125
31.3
0.13
0.25
0.25
0.13
0.25
MIC100, MIC80 and MIC50: concentration of compound 5 (g/mL) that inhibits 100, 80 or 50% the control growth
respectively. ATCC: Voucher specimen from American Type Culture Collection (Manassas, Virginia, USA);
IM: specimens from the Malbrán Institute (Buenos Aires, Argentina). AmB = Amphotericin B; Itz = Itraconazole;
Vcz = Voriconazole; i: MIC ≥ 250 µg/mL.
Results in Table 3 showed that 5 was fungicidal rather than fungistatic against seven out of the eight
clinical isolates. It displayed strong antifungal activity (MIC50 and MIC80 between 3.9 and 15.6 µg/mL)
against five out of the eight clinical isolates tested, and showed lower but still significant activity
against the rest of the isolates. Although MIC values of the reference drugs amphotericin B,
itraconazole and voriconazole against Cryptococcus neoformans are considerably lower than those
displayed by compound 5, it is worth to take into account that five or six MIC100, MIC80 or MIC50
values found for this compound against the nine fungal strains tested (Table 3), were lower than
20 µg/mL, which is indicative of a high antifungal potency.
2.3. Conformational and Electronic Study of Compound 5
With the purpose of obtaining a better structural information, and aiming to facilitate future design
of better drugs in this field, we conducted a computer-assisted conformational and electronic study on

Molecules 2013, 18
3486
compound 5 focused on its spatial orientations and electronic distribution. Compound 5 looks like a
simple conformational problem with mainly two torsional angles (θ1 and θ2, Figure 1). For the sake of
clarity, we have given the names A, B and C to the three rings of the whole molecule.
Figure 1. Phthalazinone 5 with definition of rings and main torsional angles.
Cl
C
1
2
N
A
B
N
CH3
O
In a preliminary and exploratory step, the conformational study of this molecule was carried out
from a double scan of θ1 vs. θ2 using semiempirical PM6 calculations. To obtain such a surface we
rotated the torsional angle θ1 vs. θ2 each 20°. PM6 calculations predict that the conformationally
allowed space for compound 5 is somewhat restricted. In this surface, we observed four
conformational allowed zones; however, we noted that this compound possesses at least four
equivalent conformers. The surface also suggests that the planar conformations possessing θ2 ≈ 180°
display very high energies. Although the semi-empirical calculations can define broad conformational
features, one should employ a more accurate method, such as DFT calculations to ensure that the
molecular flexibility and relative stability of the conformers are correct. Thus, we performed
B3LYP/6-31G(d,p) optimizations in order to confirm the preliminary results obtained from PM6
calculations. DFT optimizations confirm the semiempirical calculations giving four energetically
equivalent conformations for this molecule. These preferred form displayed half-extended
conformations. The conformational analysis of compound 5 requires, at this point, the evaluation of the
flexibility, i.e., the energy determination of the transitional barrier between the predicted conformers.
This is of crucial importance because, if the barriers are low, during a molecular recognition, this
compound could be converted, with a low energy cost, to the preferred form. Energy profiles of
compound 5 obtained from B3LYP/6-31G(d,p) calculations are given in Figure 2(A and B), which
show the influence of ring orientations on the potential energy of the rotamers. To understand the
significance of the rotation barrier, it is important to look not just to the magnitude of the energy
barriers, but also to the complete behaviour energy vs rotation angle. Figure 2(A) shows that
B3LYP/6-31G(d,p) calculations predict two conformations for θ1, those with θ1 near to 130° and
330°. We obtained barriers of about 2.5 Kcal mol−1 for the conformational interconversion at DFT
level, indicating a significant molecular flexibility for this rotation.

Molecules 2013, 18
3487
Figure 2. Potential Energy Curves (PECs) obtained for torsional the angles θ1 and θ2 φ
1
of
compound 5. The curves were calculated at B3LYP/6-31G(d,p) level of theory.
(A)
(B)
In turn, Figure 2B shows the rotational behaviour obtained for the torsional angle θ2. In this case,
conformations near to 0.0°, 120.0° and 240° are the preferred forms, whereas the planar form
possessing θ2 near to 180° is a markedly disfavoured conformation due to the steric hindrance. For this
torsion, the barrier for the interconversions is somewhat higher (3.8 Kcal/mol) than that obtained for
θ1. From these results, we can conclude that the molecular flexibility of this compound is significant
but moderate.
Once obtained the energetically preferred form of compound 5, then we performed an electronic
analysis using molecular electrostatic potentials (MEPs). Figure 3 shows the MEPs obtained for the
preferred conformation of compound 5. The MEP map of this molecule exhibited three clear minima,
one deep red zone located in the proximity of the carbonyl group (V(r) of about –0.045 el/au3), a second
minimum in the vicinity of the N atom (orange zone, V(r) of about −0.025 el/au3). Near to the ring C
we observed a relatively extended hydrophobic zone (yellow and green area with V(r) ranging from
−0.02 to 0.008 el/au3). This third minimum correspond to ring C and from the V(r) values obtained for
this zone it is evident that the presence of a chlorine substituent at p-position of ring C polarizes this
ring. We consider that, despite its symmetrical nature, this aromatic ring could make a specific
contribution to the binding via its particular aromatic ring orientation. Thus, considering our
experimental results, it appears that the presence of a chlorine substituent at the p-position at ring C
could be important for attaining such an interaction. In this sense, the stereoelectronic changes induced
by the presence of an additional chlorine atom, the common feature of many synthetic antifungal
drugs, at the ortho or meta positions, could be the reason of the decreased activity found for the
dichlorobenzyl derivatives 15, 16, 21 and 22 in comparison with 5.
Predictions of ADME, absorption and distribution parameters and the calculated physicochemical
properties (log S = −4.4, clog P = 3.7 ) for compound 5 and its analogues, are within the typical ranges
desired for a drug, as well as the fulfillment of Lipinski's rule permit us to consider this substance as a
good lead compound for antifungal activity.

Molecules 2013, 18
Figure 3. Electrostatic potential-encoded electron density surface obtained for compound 5.
3488
The surface was generated with GAUSSIAN 03 using a B3LYP/6-311++G(d,p) single point
calculation. The colouring represents electrostatic potential with red indicating the strongest
attraction to a positive point charge and blue indicating the strongest repulsion. The electrostatic
potential is the energy of interaction of the positive point charge with the nuclei and electrons of a
molecule. It provides a representative measure of overall molecular charge distribution. The
colour-coding is shown on the left.
3. Experimental
3.1. Chemistry
Melting points (mp) were determined in a Büchi apparatus in open capillaries and were uncorrected.
All commercial chemicals were used as purchased and solvents purified by the standard procedures
prior to use [24]. Thin-layer chromatography was performed on Merck 60 silica gel GF-254 precoated
plates and the identification was done with UV light and colorization with 10% phosphomolybdic acid
or ninhydrin spray followed by heating. Flash column chromatography was performed on Merck 60
silica gel (0.063–0.2 mesh). Infrared spectra were recorded using neat samples, without solvent or
KBr, on a FT-IR spectrometer Nicolet Impact 410 model. NMR spectra were recorded on Bruker AC
200 (200 MHz) and Bruker DRX 400 (400 MHz) instruments. Chemical shifts (δ) are expressed in
parts per million (ppm) relative to the residual solvent peak: CDCl3 7.26 ppm/77.0 ppm and coupling
constants (J) are reported in Hertz (Hz). High-resolution mass spectra (HRMS) were recorded on a
QSTAR XL mass spectrometer, by electron spray ionisation (ESI-MS) technique (5 kV).
3.1.1. General Procedure for the Synthesis of Benzalphthalides B1–B16
Phthalic anhydride (2.2 mmol), the corresponding phenylacetic (naphthylacetic) acid (2.7 mmol),
sodium acetate (0.26 mmol) and toluene (5 mL) were placed in a round-bottom flask to which a
Dean-Stark separator was adapted. The mixtures were maintained at 210–245 °C under nitrogen and

Molecules 2013, 18
3489
with magnetic stirring for 9–33 h. After cooling, the reaction mixtures were dissolved with ethyl
acetate and washed with aqueous Na2CO3 (sat.), brine and water, dried over Na2SO4 and concentrated
under reduced pressure to give the crude reaction products. Solid products were purified by
crystallization and oily products chromatographed over silica gel; yields ranged from 40–95%. All the
benzalphthalides were obtained as the Z isomer, and the configuration was confirmed through
NOE-difference and/or 2D-ROESY experiments.
(Z)-3-(4-Chlorobenzylidene)isobenzofuran-1-one (B1). Yield 75%. Yellow crystals; mp 172–174 °C;
IR (KBr), max: 2919, 1796, 1656, 1450, 1366, 1270, 1078, 969, 850, 825, 758, 606 cm−1. 1H-NMR 
6.30 (s, 1H, H-8), 7.30 (d, J = 8.8 Hz, 2H, H-3'+ H-5'), 7.53 (d, J = 7.8 Hz, 1H, H-4), 7.54 (m,1H,
H-6), 7.68 (m, 1H, H-5), 7.70 (d, J = 8.8 Hz, 2H, H-2'+ H-6'), 7.87 (d, J = 7.8 Hz, 1H, H-7) ppm.
13C-NMR : 105.7 (C-8), 119.9 (C-4), 123.3 (C-7a), 125.6 (C-7), 129.0 (C-3' + C-5'), 130.0 (C-6),
131.3 (C-2' + C-6'), 131.6 (C-4'), 134.2 (C-1'), 134.7 (C-5), 140.3 (C-3a), 144.9 (C-3), 166.9 (C-1)
ppm. ESI-MS: m/z 257.0291 [M+H]+; Anal. Calcd for C15H9ClO2: C, 70.19; H, 3.53. Found: C, 70.20;
H, 3.49.
3.1.2. General Procedure for the Synthesis of Phthalazinones 1–4 and 12
Benzalphthalides B (1 mol) were mixed with an excess of hydrazine hydrate (4 mL), and few drops
of toluene, and the mixture maintained at 70–80 °C under stirring for 3–12 h. After cooling reaction
mixtures were extracted with ethyl acetate and washed with water, dried over Na2SO4 and concentrated
under reduced pressure to give crude products that were purified by flash chromatography on silica gel
and/or crystallisation.
4-(4-Chlorobenzyl)phthalazin-1(2H)-one (1). Yield 77%. Colourless oil. IR (NaCl), max: 3159, 2902,
1
1664, 1609, 1488, 1258, 815, 798, 684 cm−1. H-NMR : 4.28 (s, 2H, H-9), 7.21 (d, J = 8.8 Hz, 2H,
H-3' + H-5'), 7.27 (d, J = 8.8 Hz, 2H, H-2' + H-6'), 7.75 (m, 3H, H-5 + H-6 + H-7), 8.47 (dd, J = 7.5,
2.5 Hz, 1H, H-8), 11.74 (br s, 1H, NH) ppm. 13C-NMR  38.2 (C-9), 125.2 (C-7), 127.1 (C-8), 128.3
(C-8a), 128.9 (C-4a + C-3' + C-5'), 129.9 (C-2' + C-6'), 131.5 (C-5); 132.7 (C-1'), 133.6 (C-6), 136.0
(C-4'), 146.0 (C-4), 160.6 (C-1) ppm. ESI-MS: m/z 271.0560 [M+H]+; Anal. Calcd for C15H11ClN2O:
C, 66.55; H, 4.10; N, 10.35. Found: C, 66.49; H, 4.11; N, 10.30.
4-(4-Methylsulfanylbenzyl)phthalazin-1(2H)-one (2). Yield 50%. Colourless oil. IR (NaCl), max: 3188,
2920, 1657, 1492, 1260, 1017, 966, 793, 770 cm−1. 1H-NMR : 2.43 (s, 3H, SCH3), 4.26 (s, 2H, H-9),
7.18 (d, J = 8.0 Hz, 2H, H-3' + H-5'), 7.26 (d, J = 8.0 Hz, 2H, H-2' + H-6'), 7.73 (m, 3H, H-5 + H-6 + H-7),
8.46 (m, 1H, H-8), 11.48 (br s, 1H, NH) ppm. 13C-NMR : 15.7 (SCH3), 38.3 (C-9), 125.2 (C-7), 126.9
(C-8 + C-3' + C-5'), 128.2 (C-8a), 128.9 (C-2' + C-6'), 129.7 (C-4a), 131.3 (C-5); 133.4 (C-6), 134.4 (C-1'),
136.7 (C-4'), 146.2 (C-4), 160.8 (C-1) ppm. ESI-MS: m/z 283.0827 [M+H]+; Anal. Calcd for
C16H14N2OS: C, 68.06; H, 5.00; N, 9.92. Found: C, 68.01; H, 4.96; N, 9.93.
4-(3,4-Methylenedioxybenzyl)phthalazin-1(2H)-one (3). Yield 100%. Colourless oil. IR (NaCl), max
:
1
3,216, 2916, 2852, 1661, 1496, 1248, 925, 860, 764 cm−1. H-NMR : 4.20 (s, 2H, H-9), 5.90 (br s,
2H, OCH2O), 6.73 (br s, 1H, H-2'), 6.74 (br s, 2H, H-5' + H-6'), 7.74 (m, 3H, H-5 + H-6 + H-7), 8.47

Molecules 2013, 18
3490
13
(dd, J = 8.7, 2.0 Hz, 1H, H-8), 10.68 (br s, 1H, NH) ppm. C-NMR : 38.5 (C-9), 101.1 (OCH2O),
108.5 (C-5'), 108.9 (C-2'), 121.5 (C-6'), 125.4 (C-7), 127.0 (C-8), 128.3 (C-8a), 129.8 (C-4a), 131.3
(C-1'), 131.5 (C-5); 133.6 (C-6), 146.5 (C-4 + C-3'), 148.0 (C-4'), 160.2 (C-1). ESI-MS: m/z 281.0848
[M+H]+; Anal. Calcd for C16H12N2O3: C, 68.56; H, 4.32; N, 9.99. Found: C, 68.49; H, 4.26; N, 9.87.
4-(3,4-Dimethoxybenzyl)phthalazin-1(2H)-one (4). Yield 92%. Colourless oil. IR (NaCl), max: 3294,
2919, 1651, 1514, 1352, 1259, 1029, 860, 783, 730 cm−1. 1H-NMR : 3.82 (s, 6H, 2 × OCH3); 4.26 (s,
2H, H-9), 6.73 (br s, 1H, H-2'), 6.77 (d, J = 8.0 Hz, 1H, H-5'), 7.74 (m, 3H, H-5 + H-6 + H-7), 7.82
13
(dd, J = 8.0, 1.2 Hz, 1H, H-6'), 8.47 (m, 1H, H-8), 11.50 (bs, 1H, NH) ppm. C-NMR  38.5 (C-9),
55.8 (2 × OCH3), 111.2 (C-5'), 115.5 (C-2'), 120.4 (C-6'), 125.4 (C-7), 126.9 (C-8), 128.2 (C-8a),
129.8 (C-4a), 130.1 (C-1'), 131.2 (C-5), 133.3 (C-6), 146.5 (C-4), 147.9 (C-4'), 149.1 (C-3'), 161.0
(C-1) ppm. ESI-MS: m/z 297.1161 [M+H]+; Anal. Calcd for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45.
Found: C, 68.88; H, 5.39; N, 9.46.
1-Naphthylmethylphthalazin-1-one (12). Yield 98%. Oil. IR (NaCl), max: 3417, 2919, 1653, 1595,
1470, 1023, 870, 787 cm−1. 1H-NMR : 4.18 (s, 2H, H-9), 7.12 (d, J = 7.0 Hz, 1H, H-4'), 7.53 (m, 1H,
H-5'), 7.55 (m, 1H, H-9'), 7.57 (m, 1H, H-8'), 7.74 (m, 3H, H-5 + H-6 + H-7), 7.75 (m, 1H, H-10'),
7.76 (m, 1H, H-6'), 8.18 (dd, J = 8.2, 1.8 Hz, 1H, H-7'), 8.44 (dd, J = 8.4, 2.0 Hz, 1H, H-8), 11.00 (br
13
s, 1H, NH) ppm. C-NMR : 35.3 (C-9), 123.1 (C-7'), 125.1 (C-7), 125.5 (C-5'), 126.1 (C-8'), 126.4
(C-9'), 127.2 (C-8 + C-4'), 127.7 (C-6'), 128.5 (C-8a), 128.9 (C-10'),129.8 (C-4a), 131.3 (C-5), 131.9
(C-2'), 133.3 (C-1' + C-3'), 133.4 (C-6), 146.3 (C-4), 160.7 (C-1) ppm. ESI-MS: m/z 287.1106
[M+H]+; Anal. Calcd for C19H14N2O: C, 79.70; H, 4.93; N, 9.78. Found: C, 79.71; H, 4.89; N, 9.72.
3.1.3. General Procedure for the Synthesis of Phthalazinones 5–9 and 14–16.
Benzalphthalides B (1 mol) were mixed with an excess of methylhydrazine (4 mL), the mixtures
maintained at 70–80 °C under stirring for 4–11 h. After cooling reaction mixtures were extracted with
ethyl acetate and washed with water, dried over Na2SO4 and concentrated under reduced pressure to
give crude products that were purified by flash chromatography on silica gel. In the case of compounds
14–16 the starting benzalphthalides were 1:1 mixtures or regioisomers with the substituent at positions
C-5 and C-6 and correspondingly yielded mixtures of 6(7)-substituted phthalazinones in the
same proportion.
4-(4-Chlorobenzyl)-2-methylphthalazin-1(2H)-one (5). Yield 75%, oil. IR (NaCl): max 3068, 2920,
1
1650, 1587, 1489, 1262, 1093, 815, 797, 749, 700 cm−1. H-NMR : 3.87 (s, 3H, CH3), 4.25 (s, 2H,
H-9), 7.20 (d, J = 8.8 Hz, 2H, H-3' + H-5'), 7.26 (d, J = 8.8 Hz, 2H, H-2' + H-6'), 7.70 (m, 3H, H-5 + H-6
13
+ H-7), 8.42 (m, 1H, H-8) ppm. C-NMR : 38.3 (C-9), 39.4 (CH3), 125.0 (C-7), 127.2 (C-8), 128.2
(C-8a), 128.9 (C-3' + C-5'), 129.2 (C-4a), 129.8 (C-2' + C-6'), 131.3 (C-5); 132.8 (C-1' + C-6), 136.4
(C-4'), 144.5 (C-4), 159.6 (C-1) ppm. ESI-MS: m/z 285.0716 [M+H]+; Anal. Calcd for C16H13ClN2O:
C, 67.49; H, 4.60; N, 9.84. Found: C, 67.39; H, 4.52; N, 9.81.
2-Methyl-4-(4-methylsulfanylbenzyl)phthalazin-1(2H)-one (6). Yield 80%, Colourless oil. IR (NaCl):
1
max 2921, 2852, 1651, 1585, 1492, 1435, 1257, 1080, 810, 795, 775 cm−1. H-NMR : 2.43 (s, 3H,

Molecules 2013, 18
3491
SCH3), 3.86 (s, 3H, NCH3), 4.23 (s, 2H, H-9), 7.17 (d, J = 8.8 Hz, 2H, H-3' + H-5'), 7.24 (d, J = 8.8 Hz,
2H, H-2' + H-6'), 7.67 (m, 3H, H-5 + H-6 + H-7), 8.43 (dd, J = 6.0, 2.9 Hz 1H, H-8) ppm. 13C-NMR :
15.9 (SCH3), 38.4 (C-9), 39.4 (NCH3),125.2 (C-7), 127.0 (C-8 + C-3' + C-5'), 128.2 (C-8a), 128.9 (C-2' +
C-6'), 129.3 (C-4a), 131.2 (C-5); 132.8 (C-6), 134.8 (C-1'), 136.8 (C-4'), 143.9 (C-4), 159.6 (C-1) ppm.
ESI-MS: m/z 297.0983 [M+H]+; Anal. Calcd for C H16N2OS: C, 68.89; H, 5.44; N, 9.45. Found: C,
17
68.81; H, 5.43; N, 9.39; S, 10.76.
2-Methyl-4-(3,4-methylenedioxybenzyl)phthalazin-1(2H)-one (7). Yield 65%, Colourless oil. IR
(NaCl): max2924, 2854, 1651, 1586, 1490, 1245, 1037, 925, 742, 698 cm−1. 1H-NMR : 3.89 (s, 3H,
CH3), 4.19 (s, 2H, H-9), 5.90 (s, 2H, OCH2O), 6.70 (d, J = 8.0 Hz, 1H, H-5'), 6.71 (br s, 1H, H-2'),
13
6.74 (d, J = 8.0 Hz, 1H, H-6'), 7.69 (m, 3H, H-5 + H-6 + H-7), 8.41 (m, 1H, H-8) ppm. C-NMR :
38.6 (C-9), 39.4 (CH3), 100.9 (OCH2O), 108.3 (C-5'), 108.7 (C-2'), 121.3 (C-6'), 125.1 (C-7), 127.0
(C-8), 128.2 (C-8a), 129.2 (C-4a), 131.6 (C-1'), 131.1 (C-5); 132.6 (C-6), 145.1 (C-4), 146.3 (C-3'),
147.9 (C-4'), 159.6 (C-1) ppm. ESI-MS: m/z 295.1004 [M+H]+; Anal. Calcd for C17H14N2O3: C, 69.38;
H, 4.79; N, 9.52. Found: C, 69.31; H, 4.77; N, 9.53.
4-(3,4-Dimethoxybenzyl)-2-methylphthalazin-1(2H)-one (8). Yield 93%, Colourless oil. IR (NaCl):
1
max 2926, 1652, 1515, 1453, 1260, 1236, 1029, 791, 744 cm−1. H-NMR : 3.83 (s, 6H, 2 × OCH3),
3.89 (s, 3H, CH3), 4.24 (s, 2H, H-9), 6.77 (d, J = 7.0 Hz, 1H, H-5'), 6.78 (s, 1H, H-2'), 6.79 (d, J = 7.0 Hz,
1H, H-6'), 7.69 (m, 3H, H-5 + H-6 + H-7), 8.44 (m, 1H, H-8) ppm. 13C-NMR : 38.6 (C-9), 39.4
(CH3), 55.9 (2 × OCH3), 111.3 (C-5'), 111.6 (C-2'), 120.5 (C-6'), 125.2 (C-7), 127.0 (C-8), 128.2 (C-8a),
128.4 (C-4a), 130.4 (C-1'), 131.2 (C-5), 132.7 (C-6), 145.2 (C-4), 147.9 (C-4'), 149.1 (C-3'), 159.6 (C-1)
ppm. ESI-MS: m/z 311, 1317 [M+H]+; Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03. Found:
C, 69.59; H, 5.81; N, 9.04.
4-(3,4,5-Trimethoxybenzyl)-2-methylphthalazin-1(2H)-one (9). Yield 70%, oil. IR (NaCl): max: 2937,
2837, 1651, 1587, 1330, 804, 776, 743 cm−1. 1H-NMR 3.77 (s, 9H, 3 × OCH3), 3.89 (s, 3H, CH3), 4.21
(s, 2H, H-9), 6.44 (s, 2H, H-2' + H-6'), 7.68 (m, 3H, H-5 + H-6 + H-7), 8.42 (m, 1H, H-8) ppm.
13C-NMR : 39.2 (C-9), 39.4 (CH3), 56.1 (2 × OCH3), 60.8 (OCH3), 105.4 (C-2' + C-6'), 125.2 (C-7),
127.0 (C-8), 128.1 (C-8a), 129.4 (C-4a), 131.3 (C-5), 132.8 (C-6), 136.8 (C-1'), 145.0 (C-4), 153.4
(C-3' + C-4' + C-5'), 159.6 (C-1) ppm. ESI-MS: m/z 341.1423 [M+H]+; Anal. Calcd for C19H20N2O4:
C, 67.05; H, 5.92; N, 8.23. Found: C, 67.01; H, 5.88; N, 8.24.
2-Methyl-4-(naphthalen-2-ylmethyl)phthalazin-1(2H)-one (13). Yield 99%, oil. IR (NaCl): max 3052,
2926, 1651, 1584, 1257, 1033, 806, 785, 740, 691 cm−1. 1H-NMR : 3.91 (CH3) 4.39 (s, 2H, H-9), 7.38
(m, 1H, H-5), 7.40 (m, 1H, H-7), 7.52 (m, 1H, H-6), 7.60 (m, 3H, H-7' + H-8' + H-9'), 7.65 (m, 1H,
H-6'), 7.75 (m, 1H, H-10'), 7.77 (br s, 1H, H-2'), 7.78 (m, 1H, H-5'), 8.46 (m, 1H, H-8) ppm. 13C-NMR
: 39.5 (C-9), 39.8 (CH3), 125.6 (C-7), 126.1 (C-10'), 126.6 (C-7'), 127.0 (C-9'), 127.3 (C-8), 127.4
(C-6'), 128.0 (C-8'), 128.1 (C-8a), 128.5 (C-5'), 128.8 (C-2'), 129.7 (C-4a + C-5), 132.7 (C-1' + C-6),
133.9 (C-3'), 135.9 (C-4'), 145.3 (C-4), 160.0 (C-1) ppm. ESI-MS: m/z 301.1263 [M+H]+; Anal. Calcd
for C20H16N2O: C, 79.98; H, 5.37; N, 9.33. Found: C, 79.91; H, 5.39; N, 9.30.

Molecules 2013, 18
3492
4-(4-Chlorobenzyl)-2,6(7)-dimethylphthalazin-1(2H)-one (14). Yield 93%, oil. IR (NaCl): max2922,
1651, 1618, 1490, 1091, 1015, 838 cm−1. ESI-MS: m/z 299.0873 [M+H]+; Anal. Calcd for
C17H15ClN2O: C, 68.34; H, 5.06; N, 9.38. Found: C, 68.19; H, 4.95; N, 9.40.
1
4-(4-Chlorobenzyl)-2,6-dimethylphthalazin-1(2H)-one (14a). H-NMR : 2.42 (s, 3H, CH3), 3.84 (s,
3H, NCH3), 4.21 (s, 2H, H-9), 7.24–7.22 (m, 4H, H-2' + H-6' and H-3' + H-5'), 7.40 (br s, 1H, H-5),
13
7.52 (d, J = 8.4, 1H, H-7), 8.30 (d, J = 8.4, 1H, H-8) ppm. C-NMR : 21.8 (CH3), 37.7 (C-9), 39.1
(NCH3), 124.3 (C-5), 126.7 (C-8), 128.5 (C-3'+ C-5'), 128.6 (C-8a), 128.9 (C-4a), 129.5 (C-2' + C-6'),
132.2 (C-4'), 132.4 (C-7), 136.2 (C-1'), 144.1 (C-6), 143.3 (C-4), 159.2 (C-1) ppm.
1
4-(4-Chlorobenzyl)-2,7-dimethylphthalazin-1(2H)-one (14b). H-NMR : 2.45 (s, 3H, CH3), 3.85 (s,
3H, NCH3), 4.20 (s, 2H, H-9), 7.24–7.22 (m, 4H, H-2' + H-6' and H-3' + H-5'), 7.45 (d, J = 8.4 Hz, 2H,
H-5 + H-6), 8.30 (br s, 1H, H-8) ppm. 13C-NMR : 21.4 (CH3), 37.7 (C-9), 39.1 (NCH3), 124.7 (C-5),
126.4 (C-8), 126.6 (C-8a), 127.7 (C-4a), 128.5 (C-3'+ C-5'), 129.5 (C-2' + C-6'), 132.2 (C-4'), 136.2
(C-1'), 138.3 (C-6), 141.8 (C-7), 143.3 (C-4), 159.2 (C-1).
4-(2,4-Dichlorobenzyl)-2,6(7)-dimethylphthalazin-1(2H)-one (15). Yield 94%, oil. IR (NaCl):
max2921, 1653, 1618, 1472, 1347, 1048, 860, 837 cm−1. ESI-MS: m/z 333, 0483 [M+H]+; Anal.
Calcd for C17H14Cl2N2O: C, 61.28; H, 4.23; N, 8.41. Found: C, 61.30; H, 4.11; N, 8.30.
1
4-(2,4-Dichlorobenzyl)-2,6-dimethylphthalazin-1(2H)-one (15a). H-NMR : 2.46 (s, 3H, CH3), 3.81
(s, 3H, NCH3), 4.29 (s, 2H, H-9), 7.00 (d, J = 8.4 Hz, H-6'), 7.05 (dd, J = 8.4, 1.8 Hz, H-5'), 7.38 (d,
J = 1.8 Hz, H-3'), 7.48 (s, 1H, H-5), 7.51 (d, J = 8.0 Hz, 1H, H-7), 8.32 (d, J = 8.0 Hz, 1H, H-8) ppm.
13C-NMR : 21.9 (CH3), 34.9 (C-9), 39.1 (NCH3), 124.0 (C-5), 126.7 (C-8), 127.0 (C-5'), 127.8
(C-8a), 129.1 (C-4a + C-6'), 130.7 (C-3'), 132.7 (C-7), 132.9 (C-2'), 134.1 (C-1' + C-4'), 143.1 (C-4),
143.6 (C-6), 159.3 (C-1) ppm.
1
4-(2,4-Dichlorobenzyl)-2,7-dimethylphthalazin-1(2H)-one (15b). H-NMR : 2.49 (s, 3H, CH3), 3.82
(s, 3H, NCH3), 4.29 (s, 2H, H-9), 6.97 (d, J = 8.4 Hz, H-6'), 7.10 (dd, J = 8.4, 1.8 Hz, H-5'), 7.38 (d,
J = 1.8 Hz, H-3'), 7.38 (d, 1H, J = 7.7 Hz, H-6), 7.50 (d, 1H, J = 7.7 Hz, H-5), 8.32 (br s, 1H, H-8)
ppm. 13C-NMR : 21.6 (CH3), 34.9 (C-9), 39.1 (NCH3), 124.4 (C-5), 126.6 (C-8), 127.0 (C-5'), 125.6
(C-8a), 129.1 (C-4a + C-6'), 130.7 (C-3'), 142.1 (C-7), 132.9 (C-2'), 134.1 (C-1' + C-4'), 143.4 (C-4),
134.1 (C-6), 159.3 (C-1) ppm.
4-(3,4-Dichlorobenzyl)-2,6(7)-dimethylphthalazin-1(2H)-one (16). Yield 86%, oil. IR (NaCl):

max2921, 1651, 1618, 1470, 1347, 1031, 823 cm−1. ESI-MS: m/z 333.0483 [M+H]+; Anal. Calcd for
C17H14Cl2N2O2: C, 61.28; H, 4.23; N, 8.41. Found: C, 61.17; H, 4.12; N, 8.49.
1
4-(3,4-Dichlorobenzyl)-2,6-dimethylphthalazin-1(2H)-one (16a). H-NMR : 2.45 (s, 3H, CH3), 3.84
(s, 3H, NCH3), 4.20 (s, 2H, H-9), 7.10 (dd, J = 8.6, 2.0 Hz, H-6'), 7.33 (d, J = 8.6 Hz, H-5'), 7.35 (d,
J = 2.0 Hz, H-2'), 7.39 (s, 1H, H-5), 7.51 (d, J = 8.0 Hz, 1H, H-7), 8.32 (d, J = 9.0 Hz, 1H, H-8) ppm.
13C-NMR : 22.0 (CH3), 37.6 (C-9), 39.2 (NCH3), 124.3 (C-5), 125.8 (C-8a), 127.1 (C-8), 128.0
(C-4a + 5'), 130.2 (C-2'), 130.4 (C-6'), 130.7 (C-3'), 132.5 (C-4'), 132.8 (C-7), 138.1 (C-1'), 143.5 (C-4),
143.6 (C-6), 159.4 (C-1) ppm.

Molecules 2013, 18
3493
1
4-(3,4-Dichlorobenzyl)-2,7-dimethylphthalazin-1(2H)-one (16b). H-NMR : 2.48 (s, 3H, CH3), 3.86
(s, 3H, NCH3), 4.20 (s, 2H, H-9), 7.11 (dd, J = 8.0, 2.0 Hz, H-6'), 7.32 (d, J = 8.0, 1.8 Hz, H-5'), 7.35
(d, J = 2.0 Hz, H-2'), 7.48 (dd, 1H, J = 7.7,1.5 Hz, H-6), 7.50 (d, 1H, J = 7.7 Hz, H-5), 8.23 (br s, 1H,
13
H-8) ppm. C-NMR : 21.6 (CH3), 37.8 (C-9), 39.2 (NCH3), 124.6 (C-5), 125.8 (C-8a), 126.8 (C-8),
128.0 (C-5'), 129.1 (C-4a), 130.4 (C-6'), 130.7 (C-3'), 130.2 (C-2'), 132.5 (C-4'), 134.1 (C-6), 138.1
(C-1'), 142.2 (C-7), 143.8 (C-4), 159.4 (C-1) ppm.
3.1.4. Procedure for the Synthesis of Compounds 10 and 11
A mixture of phthalazinone 1 (0.20 mmol), ethyl bromide or allyl bromide (0.22 mmol), potassium
carbonate (33 mg) and acetonitrile (5 mL) were maintained under reflux for 25 h. Solvent was
removed under vacuum and the crude mixture dissolved in ethyl acetate, washed with water, dried over
Na2SO4 and concentrated under reduced pressure to give crude products that were purified by flash
chromatography on silica gel.
4-(4-Chlorobenzyl)-2-ethylphthalazin-1(2H)-one (10). Yield 89%, Colourless oil. IR (NaCl):
1

max2930, 1650, 1585, 1350, 1262, 1090, 830, 798, 691 cm−1. H-NMR : 1.43 (t, J = 7.3 Hz, 3H,
CH3), 4.26 (s, 2H, H-9), 4.33 (q, J = 7.3 Hz, 2H, CH2), 7.18 (d. J = 8.5 Hz, H-3' + H-5'), 7.26 (d, J =
13
8.5 Hz, 2H, H-2' + H-6'), 7.66 (m, 3H, H-5 + H-6 + H-7), 8.45 (m, 1H, H-8) ppm. C-NMR : 13.6
(CH3), 38.3 (C-9), 46.2 (CH2), 124.9 (C-7), 127.3 (C-8), 128.4 (C-8a), 128.8 (C-3' + C-5'), 129.0 (C-4a),
129.7 (C-2' + C-6'), 131.2 (C-5); 132.7 (C-1'), 132.8 (C-6), 136.5 (C-4'), 144.6 (C-4), 159.0 (C-1) ppm.
ESI-MS: m/z 299.0873 [M+H]+; Anal. Calcd for C17H15ClN2O: C, 68.34; H, 5.06; N, 9.38. Found: C,
68.27; H, 5.04; N, 9.30.
2-Allyl-4-(4-Chlorobenzyl)phthalazin-1(2H)-one (11). Yield 73%, oil. IR (NaCl): max3073, 2930,
1
1655, 1586, 1490, 1092, 810, 796 cm−1. H-NMR : 4.25 (s, 2H, H-9), 4.85 (m, 2H, CH2), 5.20/5.27
(m, 2H, =CH2), 6.06 (m, 1H, CH=), 7.18 (d. J = 8.2 Hz, H-3' + H-5'), 7.22 (d, J = 8.2 Hz, 2H, H-2' +
13
H-6'), 7.67 (m, 3H, H-5 + H-6 + H-7), 8.42 (m, 1H, H-8) ppm. C-NMR : 38.2 (C-9), 53.4 (CH2),
117.8 (=CH2), 124.8 (C-7), 127.3 (C-8), 128.3 (C-8a), 128.7 (C-3' + C-5'), 129.1 (C-4a), 129.6 (C-2' +
C-6'), 130.1 (C-1'), 131.2 (C-5); 132.5 (CH=), 132.8 (C-6), 136.2 (C-4'), 144.8 (C-4), 158.9 (C-1) ppm.
ESI-MS: m/z 311.0873 [M+H]+; Anal. Calcd for C18H15ClN2O: C, 69.57; H, 4.86; N, 9.01. Found: C,
69.48; H, 4.80; N, 9.02.
3.1.5. General Procedure for the Synthesis of Phthalazinones 17–22
A solution of the corresponding benzalphthalide B (1 mol), methylhydrazine (3 mL) in
dichloromethane (6 mL) was absorbed in silica gel (10:1 respecting the benzalphthalide). The solvent
was removed under vacuum and the mixture MW irradiated (350 W) for 1-6 minutes. Then, 3 drops of
water were added and stirred for 20 min at room temperature. Ethyl acetate was added to the mixture
and the silica gel filtered out. The solvent was removed under vacuum and the crude mixture purified
by flash chromatography on silica gel. Phthalazinones 17–19 were obtained as 1:1 mixtures of
regioisomers at the 6/7 positions.

Molecules 2013, 18
3494
4-(4-Chlorobenzyl)-6(7)-hydroxycarbonyl-2-methylphthalazin-1(2H)-one (17). Yield 90%, oil. IR
(NaCl): max3430–2715, 1720, 1645, 1614, 1352, 1088, 803, 720 cm−1. ESI-MS: m/z 330.0611
[M+H]+; Anal. Calcd for C17H13ClN2O3: C, 62.11; H, 3.99; N, 8.52. Found: C, 62.15; H, 3.90; N, 8.50.
4-(4-Chlorobenzyl)-6-hydroxycarbonyl-2-methylphthalazin-1(2H)-one (17a). 1H-NMR (CD3OD +
CDCl3) : 3.88 (s, 3H, NCH3), 4.29 (s, 2H, H-9), 7.18–7.30 (m, 4H, H-2' + H-6' and H-3'+ H-5'), 8.45
(br s, 1H, H-5), 8.33 (d, J = 8.4 Hz, 1H, H-7), 7.73 (d, J = 8.4 Hz, 1H, H-8) ppm. 13C-NMR (CD3OD +
CDCl3) : 38.0 (C-9), 39.4 (NCH3), 125.3 (C-8), 127.7 (C-8a), 128.7 (C-3' + H-5'), 128.9 (C-5), 129.7
(C-2' + H-6'), 130.2 (C-4a), 131.6 (C-4'), 132.6 (C-6), 133.4 (C-7), 135.7 (C-1'), 144.8 (C-4), 159.5
(C-1), 166.7 (COOH) ppm.
4-(4-Chlorobenzyl)-7-hydroxycarbonyl-2-methylphthalazin-1(2H)-one (17b). 1H-NMR (CD3OD +
CDCl3) : 3.88 (s, 3H, NCH3), 4.31 (s, 2H, H-9), 7.18–7.30 (m, H-2' + H-6' and H-3' + H-5'), 8.33 (d,
13
J = 8.0 Hz, 1H, H-5), 8.49 (d, J = 8.0 Hz, 1H, H-6), 9.09 (s, 1H, H-8) ppm. C-NMR (CD3OD +
CDCl3) : 37.8 (C-9), 39.4 (NCH3), 127.0 (C-8), 127.2 (C-5), 127.7 (C-8a), 128.7 (C-3' + H-5'), 129.7
(C-2' + H-6'), 130.3 (C-4a), 131.6 (C-6), 131.7 (C-4'), 135.7 (C-1'), 145.5 (C-4), 134.8 (C-7), 159.2
(C-1), 166.5 (COOH) ppm.
4-(4-Chlorobenzyl)-6(7)-hydroxymethyl-2-methylphthalazin-1(2H)-one (18). Yield 91%, Colourless
oil. IR (NaCl): max3306, 1632, 1617, 1582, 1356, 1060, 844, 821 cm−1. ESI-MS: m/z 313.0611
[M+H]+; Anal. Calcd for C17H15ClN2O3: C, 64.87; H, 4.80; N, 8.90. Found: C, 64.76; H, 4.70; N, 8.87.
1
4-(4-Chlorobenzyl)-6-hydroxymethyl-2-methylphthalazin-1(2H)-one (18a). H-NMR  3.84 (s, 3H,
NCH3), 4.22 (s, 2H, H-9), 4.80 (s, 2H, CH2OH), 7.16–7.22 (m, 4H, H-2'+ H-6'and H-3' + H-5'), 7.60
(d, J = 8.0 Hz, 1H, H-7), 7.66 (s, 1H, H-5), 8.31 (d, J = 8.0 Hz, 1H, H-8) ppm. 13C-NMR : 38.1 (C-9),
39.5 (NCH3), 64.3 (CH2), 122.1 (C-5), 127.2 (C-8a), 127.3 (C-8), 128.9 (C-7 + C-3' + H-5'), 129.2
(C-4a), 129.8 (C-2' + H-6'), 132.6 (C-4'), 136.3 (C-1'), 144.8 (C-4), 146.6 (C-6), 159.6 (C-1) ppm.
1
4-(4-Chlorobenzyl)-7-hydroxymethyl-2-methylphthalazin-1(2H)-one (18b). H-NMR : 3.84 (s, 3H,
NCH3), 4.23 (s, 2H, H-9), 4.82 (s, 2H, CH2OH), 7.16–7.22 (m, 4H, H-2' + H-6' and H-3' + H-5'), 7.64
13
(d, J = 8.4 Hz, 1H, H-5), 7.72 (dd, J = 8.4, 1.5 Hz, 1H, H-6), 8.36 (br s, 1H, H-8) ppm. C-NMR :
38.3 (C-9), 39.5 (NCH3), 64.3 (CH2), 124.5 (C-5), 125.3 (C-8), 127.2 (C-8a), 128.3 (C-4a), 128.9 (C-3' +
H-5'), 129.8 (C-2' + H-6'), 131.5 (C-6), 132.6 (C-4'), 136.3 (C-1), 144.6 (C-4), 145.2 (C-7), 159.6 (C-1) ppm.
4-(4-Chlorobenzyl)-6(7)-nitro-2-methylphthalazin-1(2H)-one (19). Yield 53%, yellowish oil. IR
(NaCl): max2918, 1662, 1618, 1531, 1344, 1090, 794 cm−1. ESI-MS: m/z 329.0567 [M+H]+; Anal.
Calcd for C16H12ClN3O3: C, 58.28; H, 3.67; N, 12.74. Found: C, 58.18; H, 3.72; N, 12.50.
1
4-(4-Chlorobenzyl)-6-nitro-2-methylphthalazin-1(2H)-one (19a). H-NMR (400 MHz) : 3.82 (s, 3H,
CH3), 4.23 (s, 2H, H-9), 7.08–7.18 (m, 4H, H-3' + H-5' + H-2' + H-6'), 8.47 (s, 1H, H-5), 7.72 (d,
13
J = 8.7 Hz, 1H, H-7), 8.53 (d, J = 8.7Hz, 1H, H-8) ppm. C-NMR (100 MHz) : 38.5 (C-9), 39.8
(CH3), 123.3 (C-8), 125.1 (C-5); 129.0 (C-8a), 129.3 (C-3' + C-5'), 129.8 (C-7 + C-2' + C-6'), 131.9
(C-4'), 132.8 (C-4a), 135.3 (C-1'), 143.4 (C-4), 149.0 (C-6), 158.4 (C-1) ppm.

Molecules 2013, 18
3495
1
4-(4-Chlorobenzyl)-7-nitro-2-methylphthalazin-1(2H)-one (19b). H-NMR (400 MHz) : 3.82 (s, 3H,
CH3), 4.23 (s, 2H, H-9), 7.08–7.18 (m, 4H, H-3' + H-5' + H-2'+ H-6'), 8.37 (d, J = 8.4 Hz, 1H, H-6),
8.49 (d, J = 8.4 Hz, 1H, H-5), 9.18 (s, 1H, H-8) ppm. 13C-NMR (100 MHz) : 38.3 (C-9), 39.8 (CH3),
120.7 (C-8), 126.9 (C-6), 129.0 (C-8a), 129.3 (C-3' + C-5'), 129.8 (C-5 + C-2' + C-6'), 131.9 (C-4'),
133.8 (C-4a), 135.5 (C-1'), 143.1 (C-4), 150.2 (C-7), 158.1 (C-1) ppm.
6,7-Dichloro-4-(4-chlorobenzyl)-phthalazin-1(2H)-one (20). Yield 64%, Colourless oil. IR (NaCl):
1

max2943, 1652, 1490, 1090, 1015, 804, 732 cm−1. H-NMR : 3.85 (s, 3H, CH3), 4.20 (s, 2H, H-9),
7.18 (d, J = 8.4, 2H, H-2' + H-6'), 7.28 (d, J = 8.4 Hz, 2H, H-3' + H-5'), 7.71 (s, 1H, H-5), 8.49 (s, 1H,
13
H-8) ppm. C-NMR : 38.1 (C-9), 39.6 (CH3), 126.7 (C-8), 127.5 (C-8a), 128.4 (C-1'), 129.1 (C-5 +
C-3' + C-5'), 129.7 (C-2' + C-6'), 133.0 (C-4'), 135.5 (C-7), 136.5 (C-4a), 138.0 (C-6), 143.0 (C-4),
157.9 (C-1) ppm. ESI-MS: m/z 352.9937 [M+H]+; Anal. Calcd for C16H11Cl3N2O: C, 54.34; H, 3.14;
N, 7.92. Found: C, 54.40; H, 3.07; N, 7.79.
6,7-Dichloro-4-(2,4-dichlorobenzyl)-phthalazin-1(2H)-one (21). Yield 86%, oil. IR (NaCl): max2923,
1
1660, 1581, 1471, 1128, 1101, 850 cm−1. H-NMR (400 MHz) 3.80 (s, 3H, CH3), 4.20 (s, 2H, H-9),
7.03 (d, J = 8.0 Hz, 1H, H-6'), 7.15 (dd, J = 8.0, 1.7 Hz, 1H, H-5'), 7.47 (d, J = 1.7 Hz, 1H, H-3'), 7.76
13
(s, 1H, H-5), 8.53 (s, 1H, H-8) ppm. C-NMR : 35.0 (C-9), 39.5 (CH3), 126.3 (C-8), 127.3 (C-8a),
127.4 (C-5'), 128.4 (C-1'), 129.1 (C-5), 129.5 (C-3'), 131.0 (C-6'), 133.3 (C-2'), 133.6 (C-4'), 134.3 (C-7),
136.6 (C-4a), 138.2 (C-6), 142.0 (C-4), 158.0 (C-1) ppm. ESI-MS: m/z 386.9547 [M+H]+; Anal. Calcd
for C16H10Cl4N2O: C, 49.52; H, 2.60; N, 7.22. Found: C, 49.43; H, 2.71; N, 7.14.
6,7-Dichloro-4-(3,4-dichlorobenzyl)-phthalazin-1(2H)-one (22). Yield 89%, oil. IR (NaCl): max 2921,
1
1651, 1618, 1470, 1347, 1031, 823 cm−1. H-NMR : 3.81 (s, 3H, CH3), 4.25 (s, 2H, H-9), 7.09 (d,
J = 8.6 Hz, 1H, H-6'), 7.10 (d, J = 8.6 Hz, 1H, H-5'), 7.40 (br s, 1H, H-2'), 7.80 (s, 1H, H-5), 8.49 (s,
1H, H-8) ppm. 13C-NMR : 37.8 (C-9), 39.6 (CH3), 126.1 (C-8), 127.3 (C-8a), 128.1 (C-5'), 129.0 (C-5),
130.8 (C-6'), 131.9 (C-2'), 132.3 (C-3'), 133.5 (C-4'), 133.9 (C-7), 136.3 (C-4a), 137.5 (C-1'), 138.3 (C-6),
143.1 (C-4), 158.2 (C-1) ppm. ESI-MS: m/z 386.9547 [M+H]+; Anal. Calcd for C16H10Cl4N2O: C,
49.52; H, 2.60; N, 7.22. Found: C, 49.61; H, 2.53; Cl, 36.57; N, 7.17.
3.1.6. Synthesis of Phthalazinone Carboxymethyl ester 23
The phthalazinone 17 (20 mg, 0,06 mmoles) was treated with a saturated solution diazomethane in
ether (2 mL), and maintaind in darkness at room temperature overnight. The solvent was removed to
give 22 mg (99%) of the ester 23, as a regioisomeric mixture.
4-(4-Chlorobenzyl)-6(7)-methoxycarbonyl-2-methylphthalazin-1(2H)-one (23). Oil. IR (NaCl): max
2928, 1704, 1652, 1614, 1490, 1347, 1090, 1015, 845 cm−1. ESI-MS: m/z 343.0771 [M+H]+; Anal.
Calcd for C18H15ClN2O3: C, 63.07; H, 4.41; N, 8.17. Found: C, 62.97; H, 4.51; N, 8.22.
1
4-(4-Chlorobenzyl)-6-methoxycarbonyl-2-methylphthalazin-1(2H)-one (23a). H-NMR : 3.88 (s, 3H,
CH3), 3.97 (s, 3H, OCH3), 4.28 (s, 2H, H-9), 7.19 (d, J = 8.8 Hz, 2H, H-3' + H-5'), 7.27 (d, J = 8.8 Hz,
2H, H-2' + H-6'), 7.70 (d, J = 8.8 Hz, 1H, H-7), 8.29 (d, J = 8.8 Hz, 1H, H-8), 8.40 (s, 1H, H-5) ppm.
13C-NMR : 38.2 (C-9), 39.6 (CH3), 52.7 (OCH3), 125.4 (C-5); 128.2 (C-8a), 129.0 (C-8 + C-3' + C-5'),

Molecules 2013, 18
3496
129.8 (C-2' + C-6'), 131.3 (C-7), 132.0 (C-4'), 133.0 (C-4a), 133.1 (C-6), 136.0 (C-1'), 144.1 (C-4),
159.1 (C-1), 165.6 (COO) ppm.
1
4-(4-Chlorobenzyl)-7-methoxycarbonyl-2-methylphthalazin-1(2H)-one (23b). H-NMR : 3.88 (s, 3H,
CH3), 3.97 (s, 3H, OCH3), 4.30 (s, 2H, H-9), 7.19 (d, J = 8.8 Hz, 2H, H-3' + H-5'), 7.27 (d, J = 8.8 Hz,
2H, H-2' + H-6'), 8.29 (d, J = 8.4 Hz, 1H, H-6), 8.51 (d, J = 8.4 Hz, 1H, H-5), 9.03 (s, 1H, H-8).
13C-NMR : 38.4 (C-9), 39.6 (CH3), 52.7 (OCH3), 126.9 (C-5); 127.8 (C-8), 128.2 (C-8a), 129.0
(C-3' + C-5'), 129.8 (C-2' + C-6'), 132.0 (C-4'), 132.6 (C-4a), 132.9 (C-6), 134.0 (C-7), 136.0 (C-1'),
144.8 (C-4), 159.1 (C-1), 165.6 (COO).
3.1.7. Synthesis of the Phthalazinone Aldehyde 24
To a three-neck round-bottom flask filled with dichloromethane (15 mL) and a stirring bar, two
compensated pressure addition funnels were adapted. Air was removed, the system filled with Ar and
taken to −55 °C, then a solution of 2M oxallyl chloride in dichloromethane (1.10 mL, 2.20 mmol) was
added. Five min later a mixture of dimethylsulfoxide (0.4 mL, 4.44 mmol) in dichloromethane (2.3 mL)
was added dropwise. After 5 min a solution of phthalazinone 18 (230 mg, 0.73 mmol) in
dichloromethane (6.5 mL) was added slowly. The mixture was maintained with stirring for 30 min at
−55 °C. Then, triethylamine (1.0 mL, 7.20 mmol) was added and the mixture taken to 0 °C for 60 min.
Then, water (5 mL) was added to the mixture, which was transferred to a separatory funnel, where it
was washed with aqueous solutions of 2N HCl, NaHCO3 (saturated) and NaCl to pH = 7. The organic
layer was dried over Na2SO4, concentrated under reduced pressure to give a crude mixture, that was
purified by flash chromatography on silica gel in CH2Cl2/AcOEt (9:1) to provide 138 mg (61%) of
aldehyde 24.
4-(4-Chlorobenzyl)-6(7)-formyl-2-methylphthalazin-1(2H)-one (24). Oil. IR (NaCl): max 2928, 1704,
1652, 1614, 1490, 1347, 1090, 1015, 845 cm−1. ESI-MS: m/z 313.0666 [M+H]+; Anal. Calcd. for
C17H13ClN2O2: C, 65.29; H, 4.19; N, 8.96. Found: C, 65.31; H, 4.12; N, 8.83.
4-(4-Chlorobenzyl)-6-formyl-2-methylphthalazin-1(2H)-one (24a). 1H-NMR : 3.89 (s, 3H, CH3), 4.32
(s, 2H, H-9), 7.19 (d, J = 8.8 Hz, 2H, H-3' + H-5'), 7.29 (d, J = 8.8 Hz, 2H, H-2' + H-6'), 8.17 (s, 1H,
H-5), 8.18 (d, J = 8.8 Hz, 1H, H-7), 8.61 (d, J = 8.8 Hz, 1H, H-8), 10.10 (s, 1H, CHO) ppm. 13C-NMR
: 38.3 (C-9), 39.7 (CH3), 127.2 (C-5); 128.8 (C-8a), 128.6 (C-8), 129.1 (C-3' + C-5'), 129.8 (C-2 '+ C-6'),
130.7 (C-7), 131.9 (C-4a), 133.0 (C-4'), 135.8 (C-1'), 138.9 (C-6), 144.8 (C-4), 158.8 (C-1), 190.8
(CHO) ppm.
4-(4-Chlorobenzyl)-7-formyl-2-methylphthalazin-1(2H)-one (24b). 1H-NMR : 3.90 (s, 3H, CH3), 4.29
(s, 2H, H-9), 7.19 (d, J = 8.8 Hz, 2H, H-3'+ H-5'), 7.29 (d, J = 8.8 Hz, 2H, H-2'+ H-6'), 7.77 (d, J = 8.4 Hz,
13
1H, H-6), 8.17 (d, J = 8.4 Hz, 1H, H-5), 8.90 (br s, 1H, H-8), 10.17 (s, 1H, CHO) ppm. C-NMR :
38.3 (C-9), 39.7 (CH3), 126.1 (C-5); 128.8 (C-8a), 129.1 (C-3'+C-5'), 129.6 (C-8), 129.8 (C-2' +
C-6'), 131.3 (C-6), 133.0 (C-4'), 135.8 (C-1'), 137.8 (C-4a), 138.9 (C-7), 144.1 (C-4), 159.0 (C-1),
190.7 (CHO) ppm.

Molecules 2013, 18
3497
3.1.8. Synthesis of the 6(7)hydroxylimino-phthalazinone 25
To a solution of 24 (100 mg, 0.32 mmol) in ethanol (5 mL), dry pyridine (83 μL, 1.03 mmol) and
hydroxylamine clorhydrate (25 mg, 0.35 mmol) were added. The mixture was refluxed under stirring
for 2 hours. Solvents were removed under vacuum and the mixture dissolved in ethyl acetate. The
organic layer was washed with solutions of 2N HCl and NaCl to pH = 7, dried over Na2SO4, and taken
do dryness to give 95 mg (92%) of the regioisomers 25.
4-(4-Chlorobenzyl)-6(7)-hydroxylimino-2-methylphthalazin-1(2H)-one (25). Oil. IR (NaCl): max3441,
2927, 1632, 1579, 1111, 995, 796, 674 cm−1. ESI-MS: m/z 328.0775 [M+H]+; Anal. Calcd for
C17H14ClN3O2: C, 62.30; H, 4.31; N, 12.82. Found: C, 62.35; H, 4.38; N, 12.83.
4-(4-Chlorobenzyl)-6-hydroxylimino-2-methylphthalazin-1(2H)-one (25a). 1H-NMR (400 MHz,
DMSO-d6) : 3.72 (s, 3H, CH3), 4.29 (s, 2H, H-9), 7.33-7.34 (m, 4H, H-2' + H-6' and H-3' + H-5'),
7.90 (d, J = 8.5 Hz, 1H, H-8), 8.04 (d, J = 8.5 Hz, 1H, H-7), 8.33 (s, 1H, HC=N), 8.41 (s, 1H, H-5)
13
ppm. C-NMR (100 MHz) : 36.8 (C-9), 39.1 (CH3), 124.3 (C-5); 126.2 (C-8), 127.8 (C-4a), 128.5
(C-3' + C-5'), 128.7 (C-8a), 130.0 (C-7), 130.3 (C-2' + C-6'), 131.2 (C-4'), 136.2 (C-6), 144.3 (C-4),
137.0 (C-1'), 147.1 (HC=N) 158.2 (C-1), ppm.
4-(4-Chlorobenzyl)-7-hydroxylimino-2-methylphthalazin-1(2H)-one (25b). 1H-NMR (400 MHz,
MDSO-d6) : 3.71 (s, 3H, CH3), 4.29 (s, 2H, H-9), 7.33-7.34 (m, 4H, H-2' + H-6' and H-3' + H-5'),
8.04 (d, J = 8.4 Hz, 1H, H-6), 8.33 (s, 1H, HC=N), 8.50 (d, J = 8.4 Hz, 1H, H-5), 8.60 (d, J = 8.5 Hz,
1H, H-8 ppm. 13C-NMR (100 MHz) : 38.8 (C-9), 39.1 (CH3), 123.9 (C-5); 127.6 (C-8), 127.9 (C-8a),
128.0 (C-4a), 128.5 (C-3' + C-5'), 130.3 (C-6 + C-2'+C-6'), 131.2 (C-4'), 137.0 (C-7 + C-1'), 144.3
(C-4), 147.1 (HC=N) 158.2 (C-1) ppm.
3.2. Antifungal Evaluation
3.2.1. Microorganisms And Media
For the antifungal evaluation, standardized strains from the American Type Culture Collection
(ATCC), Manassas, Virginia, USA, and Culture Collection of the Reference Center of Mycology
(CCC), Faculty of Biochemical and Pharmacuetical Sciences, Suipacha 531-(2000)-Rosario, Argentina
were used in a first instance of screening: C. albicans ATCC 10231, S. cerevisiae ATCC 9763,
C. neoformans ATCC 32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029,
T. rubrum CCC 110, T. mentagrophytes ATCC 9972, M. gypseum CCC 115, M. canis CCC 113 and
E. floccosum CCC 112.
Active compounds were tested against clinical isolates from the Malbrán Institute [(MI), Av. Velez
Sarsfield 563. Buenos Aires)]. The isolates included eight strains of C. neoformans. The voucher
specimen numbers are presented in Table 3. Strains were grown on Sabouraud-chloramphenicol agar
slants for 48 h at 30 °C, maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid, Hampshire,
UK) and sub-cultured every 15 d to prevent pleomorphic transformations. Inocula of cell or spore
suspensions were obtained according to reported procedures and adjusted to 1-5 x103 cells/spores with
colony forming units (CFU) per mL [18,19].

Molecules 2013, 18
3498
3.2.2. Antifungal Susceptibility Testing
Minimum Inhibitory Concentration (MIC) of each compound was determined by using broth
microdilution techniques according to the guidelines of the Clinical and Laboratory Standards Institute
(CLSI, formerly National Committee for Clinical Laboratory Standards, NCCLS) for yeasts (M27-A3)
and for filamentous fungi (M 38 A2) [18,19].
MIC values were determined in RPMI-1640 (Sigma, St. Louis, MO, USA) buffered to pH 7.0 with
MOPS. Microtiter trays were incubated at 35 °C for yeasts and at 28–30 °C for the rest of fungi in a
moist, dark chamber, and MICs were visually recorded at 48 h for yeasts, and at a time according to
the control fungus growth, for the rest of fungi.
For the assay, stock solutions of pure compounds were two-fold diluted with RPMI from
250–0.98 g/mL (final volume = 100 L) and a final DMSO concentration ≤ 1%. A volume of 100 L
of inoculum suspension was added to each well with the exception of the sterility control where sterile
water was added to the well instead. Terbinafine, amphotericin B, voriconazole and itraconazole, were
used as positive controls.
Endpoints were defined as the lowest concentration of drug resulting in total inhibition (MIC100) of
visual growth compared to the growth in the control wells containing no antifungal. MIC80 and MIC50
were defined as the lowest concentration of a compound that induced 80% or 50% reduction of the
growth control respectively (culture media with the microorganism but without the addition of any
compound) and was determined spectrophotometrically with the aid of a VERSA Max microplate
reader (Molecular Devices, Sunnyvale, CA, USA).
3.3. Computational Methods
All calculations were carried out using the Gaussian 03 program [25]. The search for low-energy
conformations on the potential energy surface for compound 5 was carried out by first using semi-
empirical PM6 calculations. Subsequently, DFT (B3LYP/6-31G (d,p)) calculations were used in the
geometry optimisation jobs. Minima were characterized through harmonic frequency analysis.
Correlations effects were included using Density Functional Theory (DFT) with the Becke-3-Lee-
Yang-parr (RB3LYP) [26] functional and 6-31++G(d,p) basis set for all complexes obtained at the
lower level of computation. During the DFT calculations, the RHF/6-31G geometries were kept fixed.
Potential energy curves (PEC) have been obtained via one-dimensional (1D)-scans using DFT
(B3LYP/6-31G (d,p)) calculations. In these curves the energy has been calculated at 30 ° intervals of
the dihedral angles.
The electronic study of the compounds was carried out by using molecular electrostatic potentials.
MEPs have been shown to provide reliable information, both on the interaction sites of the molecules
with point charges and on the comparative reactivities of these sites [27]. These MEPs were calculated
using B3LYP/6-311++G(d,p) single point calculations from the MOLEKEL program [28].
4. Conclusions
In summary, we have described here a group of 2-methylphthalazin-1(2H)-one derivatives acting as
antifungal agents. Among them, the compound 4-(4-chlorobenzyl)-2-methylphthalazin-1(2H)-one (5)

Molecules 2013, 18
3499
exhibited remarkable antifungal activity against dermatophytes and against C. neoformans
standardized strains, as well as against a number of clinical isolates. Complementarily, we have carried
out a structural molecular and electronic study on compound 5 to reveal the conformational and
electronic characteristics of this compound. Predictions of ADME, absorption and distribution
parameters and the calculated physicochemical properties (log S = −4.4, clog P = 3.7) for compound 5
and its analogues, are within the typical ranges desired for a drug, as well as the fulfillment of
Lipinski's rule permit us to consider this substance as a good lead compound for antifungal activity.
All these aspects serve to justify future research on new series of phthalazinones focused on the
structural optimization that could lead to a substantial improvement of potency and antifungal activity
spectrum. Such research must be complemented with in vivo toxicity and efficacy evaluations and the
elucidation of the mechanism of action.
Acknowledgments
This work is a collaborative research performed under the auspices of “Programa Iberoamericano
de Ciencia y Tecnología para el Desarrollo - CYTED X.7”, and supported by grants from Spanish
ISCIII (RD06/0021/0022 to A.S.F.), from Argentineana Agencia Nacional de Promoción Científica y
Tecnológica (ANPCyT PICT 0608 to S.Z. and PICT to D.E). B.B. and A.E.G-C thank to the EC-ALFA
Program and the Spanish MEC their respective PhD fellowships. R.D.E. is member of the Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET-Argentina) staff.
References
1. Mathew, B.; Nath, M. Recent approaches to antifungal therapy for invasive mycoses.
ChemMedChem 2009, 4, 310–323.
2. Monk, B.; Goffeau, A. Outwitting multidrug resistance to antifungals. Science 2008, 321, 367–369.
3. Chen, S.; Playford, E.; Sorrell, T. Antifungal therapy in invasive fungal infections. Curr. Opin.
Pharmacol. 2010, 10, 522–530.
4. Olivella, M.; Rodríguez, A.M.; Zacchino, S.A.; Somlai, C.; Penke, B.; Farkas, V.; Perczel, A.;
Enriz, R.D. New antifungal peptides. Synthesis, bioassays and initial structure prediction by CD
spectroscopy. Bioorg. Med. Chem. Lett. 2010, 20, 4808–4811.
5. Yunes, R.; Leal, P.; Mascarello, A.; Derita, M.; Zuljan, F.; Nunes, R.; Zacchino, S. Relation
between lipophilicity of alkyl gallates and antifungal activity against yeasts and filamentous fungi.
Bioorg. Med. Chem. Lett. 2009, 19, 1793–1796.
6. Masman, M.; Rodríguez, A.; Raimondi, M.; Zacchino, S.; Luiten, P.; Somlai, C.; Kortvelyesi, T.;
Penke, B.; Enriz, R. Penetratin and derivatives acting as antifungal agents. Eur. J. Med. Chem.
2009, 44, 212–228.
7. Escalante, A.; Gattuso, M.; Pérez, P.; Zacchino, S. Evidence for the mechanism of action of the
antifungal phytolaccoside B from Phytolacca tetramera. J. Nat. Prod. 2008, 71, 1720–1725.
8. Kouznetsov, V.; Vargas, L.; Sortino, M.; Vasquez, Y.; Gupta, M.; Freile, M.; Enriz, R.;
Zacchino, S. Antifungal and cytotoxic activities of some N-substituted aniline derivatives bearing
an hetaryl fragment. Bioorg. Med. Chem. 2008, 16, 794–809.

Molecules 2013, 18
3500
9. Sortino, M.; Cechinel Filho, V.; Correa R.; Zacchino, S. N-phenyl and N-phenylalkyl-maleimides
acting against Candida spp.: time-to-kill, stability, interaction with maleamic acids. Bioorg. Med. Chem.
2008, 16, 560–568.
10. Mohamed, F.K. Synthesis, reactions and antimicrobial activity on some novel phthalazinones
derivatives. Der Chemica Sinica 2010, 1, 20–31.
11. Abd El-Wahab, A.H.; Mohamed, H.M.; El-Agrody, A.M.; El-Nassag, M.A.; Bedair, A.H.
Synthesis and Biological Screening of 4-Benzyl-2H-phthalazine derivatives. Pharmaceuticals
2011, 4, 1158–1170.
12. Sridhara, K.R.; Reddy, K.R.V.; Keshavayya, J.; Ambika, D.S.; Goud, S.; Peethambar, S.K.
Synthesis and antimicrobial studies of some new 3-isoxazoline substituted phthalazine
methylsulfonyl oxadiazoles. Eur. J. Chem. 2011, 8, 1022–1029.
13. Sridhara, A.M.; Reddy, K.R.V.; Keshavayya, J.; Ambika, D.M.S.; Gopinath, V. Bose, P.;
Goud, S.K.; Peethambar, S.K. Synthesis, Antimicrobial and Cytotoxicity Studies of Some Novel
Modified Strobilurin Derivatives. J. Braz. Chem. Soc. 2011, 22, 849–856.
14. Sridhara, A.M.; Reddy, K.R.V.; Keshavayya, J.; Goud, P.S.K.; Somashekar, B.C.; Bose, P.;
Peethambar, S.K.; Gaddam, S.K. Synthesis and antimicrobial activity of 2-substituted [4-(1,3,4-
oxadiazol-2-yl methyl)] phthalazin-1(2H)-one derivatives. Eur. J. Med. Chem. 2010, 45, 4983–4989.
15. Ryu, C.K.; Kim, D.H.; Lee, I.K.; Kim, S.J. The evaluation of in vitro antifungal activities of
6-[(N-4-fluorophenyl)amino]-7-chloro-5,8-quinolinedione. Yakhak Hoeji 1996, 40, 90–94.
16. Zamilpa, A.; Herrera-Ruiz, M.; del Olmo, E.; López-Pérez, J.L.; Anxiolytic effects of
benzalphthalides. Bioorg. Med. Chem. Lett. 2005, 15, 3483–3486.
17. Nascimento-Júnior, N.M.; Kümmerle, A.E.; Barreiro, E.J.; Fraga, C.A.M. MAOS and Medicinal
Chemistry: Some Important Examples from the Last Years. Molecules 2011, 16, 9274–9297.
18. Clinical and Laboratory Standards Institute (CLSI). Document M27-A3, 3rd ed.; NCCLS: Wayne,
PA, USA, 2008; Volume 28, pp. 1–25.
19. Clinical and Laboratory Standards Institute (CLSI). Document M38-A2, 2nd ed.; NCCLS:
Wayne, PA, USA, 2008; Volume 28, pp. 1–35.
20. Singh, N. Treatment of opportunistic mycoses: how long is long enough? Lancet Infect. Dis.
2003, 3, 703–707.
21. Polak, A. The past, Present and future of antimycotic combination therapy. Mycoses 1999, 42,
355–370.
22. Insuasty, B.; Gutiérrez, A.; Quiroga, J.; Abonia, R.; Nogueras, M.; Cobo, J.; Svetaz, L.;
Raimondi, M.; Zacchino, S. Fungicide activity of 5-(4-chlorobenzylidene)-(Z)-2-dimethylamino-
1,3-thiazol-4-one against Cryptococcus neoformans. Arch. Pharm. Chem. Life Sci. 2010, 343, 48–53.
23. Ernst, E.; Roling, E.; Petzold, R.; Keele, D.; Klepser, M. In vitro activity of micafungin (FK-463)
against Candida spp: Microdilution, time-kill, and postantifungal-effect studies. Antimicrob.
Agents Chemother. 2002, 46, 3846–3853.
24. Armarego, W.L.F.; Perrin, D.D. Purification of Laboratory Chemicals, 4th ed.; Pergamon Press:
Oxford, UK, 2000.
25. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.;
Montgomery, J.A., Jr.; Vreven, T.; Kudin, K.N.; Burant, J.C.; et al. GAUSSIAN 03, Revision B.01;
Gaussian, Inc.: Wallingford, CT, USA, 2004.

Molecules 2013, 18
3501
26. Lee, C.; Yang, W.; Parr, R. Development of the Colle-Salvetti correlation energy formula into a
functional of the electron density. Phys. Rev. B 1988, 37, 785–789.
27. Polilzer, P.; Truhlar, D.G. Chemical Applications of Atomic and Molecular Electrostatic
Potentials; Plenum Publishing: New York, NY, USA, 1981; p. 34.
28. Flükiger, P.; Lüthi, H.P.; Portmann, S.; Weber, J. MOLEKEL 4.0; Swiss Center for Scientific
Computing: Manno, Switzerland, 2000.
Sample Availability: Samples of the compounds 1–25 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).