Triazolophthalazines: Easily Accessible Compounds with Potent Antitubercular Activity

Article abstract of DOI:10.1002/cmdc.201600085

Tuberculosis (TB) remains one of the major causes of death worldwide, in particular because of the emergence of multidrug-resistant TB. Herein we explored the potential of an alternative class of molecules as anti-TB agents. Thus, a series of novel 3-substituted triazolophthalazines was quickly and easily prepared from commercial hydralazine hydrochloride as starting material and were further evaluated for their antimycobacterial activities and cytotoxicities. Four of the synthesized compounds were found to effectively inhibit the Mycobacterium tuberculosis (M.tb) H₃₇Rv strain with minimum inhibitory concentration (MIC) values <10 μg mL^-1, whereas no compounds displayed cytotoxicity against HCT116 human cell lines (IC₅₀>100 μm). More remarkably, the most potent compounds proved to be active to a similar extent against various multidrug-resistant M.tb strains, thus uncovering a mode of action distinct from that of standard antitubercular agents. Overall, their ease of preparation, combined with their attractive antimycobacterial activities, make such triazolophthalazine-based derivatives promising leads for further development. Old trick, new drugs: A series of triazolophthalazines were synthesized and evaluated as potential antitubercular agents. A number of these easy-to-prepare compounds proved to be highly potent inhibitors of Mycobacterium tuberculosis (M.tb) and exhibited similar potency against multidrug-resistant M.tb strains.

Full text of DOI:10.1002/cmdc.201600085

DOI: 10.1002/cmdc.201600085
Full Papers
Triazolophthalazines: Easily Accessible Compounds with
Potent Antitubercular Activity
Damien Veau,[a, b] Serhii Krykun,[a] Giorgia Mori,[c] Beatrice S. Orena,[c] Maria R. Pasca,[c]
CØline Frongia,[b] ValØrie Lobjois,[b] Stefan Chassaing,*[a, b] Christian Lherbet,*[a] and
Michel Baltas*[a]
Tuberculosis (TB) remains one of the major causes of death
worldwide, in particular because of the emergence of multi-
drug-resistant TB. Herein we explored the potential of an alter-
native class of molecules as anti-TB agents. Thus, a series of
novel 3-substituted triazolophthalazines was quickly and easily
prepared from commercial hydralazine hydrochloride as start-
ing material and were further evaluated for their antimycobac-
terial activities and cytotoxicities. Four of the synthesized com-
pounds were found to effectively inhibit the Mycobacterium tu-
berculosis (M.tb) H37Rv strain with minimum inhibitory concen-
tration (MIC) values <10 mgmLÀ1, whereas no compounds dis-
played cytotoxicity against HCT116 human cell lines (IC50 >
100 mm). More remarkably, the most potent compounds
proved to be active to a similar extent against various multi-
drug-resistant M.tb strains, thus uncovering a mode of action
distinct from that of standard antitubercular agents. Overall,
their ease of preparation, combined with their attractive anti-
mycobacterial activities, make such triazolophthalazine-based
derivatives promising leads for further development.
Introduction
Mycobacterium tuberculosis (M.tb), the causative agent of tuber-
culosis (TB), represents an enduring, deadly infectious disease
for all mankind. According to the World Health Organization
(WHO), one-third of the world’s population is infected, current-
ly making TB the second leading cause of mortality world-
wide.[1] It is estimated that there were about 9 million cases of
TB and 1.5 million deaths in 2014. In recent years, the signifi-
cance of the disease has increased dramatically, as TB is also
the major cause of death among patients co-infected with HIV.
In a concerted effort to control and eradicate TB, an impressive
range of anti-TB drugs has been discovered.[2] Nevertheless,
treatment still remains lengthy and, most importantly, new
drug-resistant strains have emerged. In fact, nearly half a million
new cases of multidrug- and extremely drug-resistant TB
appear every year. In addition, one of the most potent TB
drugs, rifampicin, inactivates several antiviral drugs, making it
difficult to use for the treatment of TB–HIV co-infected patients
(Figure 1).[3] Due to an urgent need for the development of
novel drugs, the TB drug pipeline is filled with several new
therapeutic compounds for the first time in decades, many of
them in phase II or III clinical trials (Figure 1).[4] Whatever the
eventual outcome of these drugs currently in the TB pipeline,
continuous efforts should still be made in order to find new
compounds active against novel essential targets or com-
pounds with different modes of action against known targets.
Cinnamic acid and derivatives also represent a relevant class
of compounds having a century-old history as anti-TB agents.[5]
First reported to be used alone as early as 1894,[6] the structur-
[a] Dr. D. Veau, S. Krykun, Dr. S. Chassaing, Dr. C. Lherbet, Dr. M. Baltas
Laboratoire de Synth›se et Physicochimie de MolØcules d’IntØrÞt Biologique
(SPCMIB), CNRS-UMR5068, UniversitØ Paul Sabatier-Toulouse III,
118 Route de Narbonne, 31062 Toulouse Cedex 9 (France)
E-mail: christian.lherbet@itav.fr
baltas@chimie.ups-tlse.fr
[b] Dr. D. Veau, C. Frongia, Dr. V. Lobjois, Dr. S. Chassaing
ITAV, UniversitØ de Toulouse, CNRS, UPS,
1 place Pierre Potier, 31106 Toulouse Cedex 1 (France)
E-mail: stefan.chassaing@itav.fr
[c] Dr. G. Mori, Dr. B. S. Orena, Dr. M. R. Pasca
Dipartimento di Biologica e Biotecnologie ’Lazzaro Spallanzani’,
via Ferrata 1, 27100 Pavia (Italy)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600085.
Figure 1. Examples of anti-TB drugs: the commonly used rifampicin and iso-
niazid, as well as some drugs currently in the TB pipeline.
ChemMedChem 2016, 11, 1078 – 1089
1078
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
ally simple trans-cinnamic acid was later demonstrated to exert
a synergistic effect in combination with known anti-TB drugs,
such as rifampicin and isoniazid (INH)[7] (Figure 1). Also remark-
able is that the anti-TB profile of rifampicin could be improved
by replacing the methyl group of its piperazinyl moiety with
Synthesis of C0 series derivatives
We began with the synthesis of unmodified triazolophthalazine
2, which was obtained by condensation of hydralazine hydro-
chloride 1 with trimethyl orthoformate (Scheme 2). The reac-
tion was carried out either in pure trimethyl orthoformate or
with trimethyl orthoformate in dichloromethane as solvent in
a
cinnamyl group (i.e., cinnamyl-rifampicin, depicted in
Figure 1).[8]
In that context, and due to our interest in bioactive com-
pounds bearing the cinnamic scaffold,[5,9] we recently de-
scribed the synthesis of derivatives exhibiting promising anti-
TB activities.[10] Amongst all derivatives investigated so far, 4-
alkoxy cinnamoyl derivatives featuring both a styryl and triazo-
lophthalazine moiety were found to be the most promising
(Scheme 1).[11] Encouraged by these preliminary results, we
Scheme 2. Synthesis of C0 series derivatives. Reagents and conditions: a) 1
(1.0 equiv), HC(OMe)3 (1.0 equiv), Et3N (1.6 equiv), CH2Cl2, RT!408C, 15 h;
b) 1 (1.0 equiv), Br2 (excess), AcOH, reflux, 5 h; c) 1 (1.0 equiv), RN=C=S
(1.1 equiv), EDC (1.1 equiv), Na2CO3 (3 equiv), DMF, 808C, 4.5 h.
the presence of triethylamine to yield compound 2 in 75%
yield. Triazolophthalazine 2 was then selectively brominated at
position 3 with N-bromosuccinimide in acetic acid to furnish 3-
bromotriazolophthalazine 3 in 53% yield (Scheme 2). Product
Scheme 1. Triazolophthalazine derivatives as potential M.tb inhibitors: from
previously reported 3-styrylated triazolophthalazines[10] to the derivatives in-
vestigated in this study.
1
3 was easily identified by H NMR by disappearance of the cor-
report herein the synthesis of novel 3-substituted triazoloph-
thalazine derivatives, along with their biological evaluation as
anti-TB agents (Scheme 1). This will allow us to determine
whether the triazolophthalazine core could be regarded as
a novel and relevant pharmacophore in the TB context.
Indeed, although triazolophthalazines have been reported sev-
eral times for exhibiting other bioactivities,[9b,12] to the best of
our knowledge, they have received no attention as potential
anti-TB agents.[13] Our initial results with 35 3-substituted tria-
zolophthalazine derivatives are presented below.
responding singlet at 9 ppm.
Attempts to synthesize 3-amino derivatives 4a–c first started
from 3, but the expected nucleophilic aromatic substitution of
the bromine atom with amines proved unsuccessful in metha-
nol under either conventional or microwave heating
(Scheme 2). The starting compound 3 was fully recovered in
pure form, thus indicating that the bromine atom of 3 was
much less labile than previously postulated.[14] An alternative
approach, taking advantage of the fact that isothiocyanates
are known building blocks for the production of heterocycles,
was then investigated for the elaboration of 3-amino deriva-
tives 4a–c. In that respect, we took advantage of a well-estab-
lished cyclization/desulfurization process but, rather than using
DCC as originally reported,[15] we opted for EDC as the activat-
ing agent. Under these slightly modified conditions, 3-amino
derivatives 4a–c were obtained in good yields after purifica-
tion by flash chromatography.
Results and Discussion
Chemistry
As outlined in Scheme 1, we envisioned the preparation of
three series of triazolophthalazine derivatives (i.e., C0–2; with
reference to the number of carbon atoms between position 3
of the triazolophthalazine nucleus and the terminal moiety, X
Scheme 1). Of note, the synthesis of all derivatives was envis-
aged in a few steps using commercially available hydralazine
hydrochloride 1 as the starting material, regardless of the
series.
Synthesis of C1 series derivatives
In that series, we first prepared hydroxymethylated triazoloph-
thalazine 5 by reaction of triazolophthalazine 2 with an aque-
ous solution of formaldehyde in a small amount of dioxane, as
previously described for another triazolo system (Scheme 3).[16]
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1079
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
c using HOBt and EDC as coupling reagents in the
presence of N,N-diisopropylethylamine (DIPEA).
Beginning from cinnamic derivatives
As mentioned in the introduction (Scheme 1), we
previously reported the synthesis of 3-styrylated tria-
zolophthalazines, starting from trans-4-hydroxycin-
namic acid (i.e., trans-4-coumaric acid).[10] To further
evaluate the anti-TB potential of these derivatives,
we aimed toward building a small, focused library of
styryltriazolophthalazines. The reported four-step se-
quence nevertheless suffered from some drawbacks,
such as the number of steps, the need for long reac-
tion times, and its convergent approach (Scheme 5,
route a). According to route b (Scheme 5), we thus
Scheme 3. Synthesis of C1 series derivatives. Reagents and conditions: a) 1 (1.0 equiv),
HC(OMe)3 (1.0 equiv), Et3N (1.6 equiv), CH2Cl2, RT!408C, 15 h, 75%; b) 2, 37% aqueous
formaldehyde (excess), dioxane, 15 h, 81%; c) 1 (1.0 equiv), Et3N (8.0 equiv), CH2Cl2, RT,
0.5 h, then CHCl2CO2H (excess), 60!110 8C, 2.5 h; d) 6, morpholine (excess), 1008C, 5 h,
then 25% aqueous HCl, RT, 1 h.
Product 5 was isolated in good yield, and its structure was fur-
ther confirmed by its X-ray crystallography.
Oxidation of 5 to aldehyde and/or to its acid derivative was
then attempted but without success. Upon exposure of 5 to
strong oxidizers (such as KMnO4 or CrO3[17]), only compound 2
was obtained, due to easy decarboxylation of the resulting
acid (Scheme 2).[18] We also tried to use milder oxidation condi-
tions, such as the IBX/oxone system[19] in acetonitrile/water,
but still without success. Other oxidation methods that selec-
tively lead to the formation of aldehyde were then examined.
Reactions using IBX or Dess–Martin periodinane did not afford
selective oxidation and delivered a complex mixture of prod-
ucts. Similar results were observed using manganese(IV) oxide
as the oxidant in dichloromethane. Taking into account these
Scheme 4. Synthesis of C2 series derivatives from diethyl malonate. Reagents
and conditions: a) 1 (1.0 equiv), Et3N (8.0 equiv), CH2Cl2, RT, 0.5 h, then
CH2(CO2Et)2 (excess), 110 8C, 5 h; b) 8 (1.0 equiv), NaBH4 (excess), EtOH, 808C,
5 h; c) 8, 5m aqueous NaOH/EtOH (1:2), 10 h; d) 10 (1.0 equiv), HOBt
(1.1 equiv), EDC (1.1 equiv), DMF, 0.5 h, then RNH2 (1.1 equiv), DIPEA
(2.1 equiv), 10 h.
difficulties for oxidizing 5, we opted for an alternative two-step
approach involving 3-dichloromethylated triazolophthalazine 6
as an intermediate. The reaction of 1 in boiling dichloroacetic
acid for 2 h allowed easy formation of 6, which was isolated in
good yield (80%). Treatment of 6 with two equivalents of mor-
pholine, followed by hydrolysis (25% hydrochloric acid), afford-
ed gem-diol 7 in its hydrochloride form with 52% yield. It is
noteworthy that 7 was predominant in methanol or DMSO in
its gem-diol form, whereas the aldehyde form was observed in
chloroform. This result was confirmed by NMR spectroscopy,
mass spectrometry, and X-ray analysis.
Synthesis of C2 series derivatives
Beginning from diethyl malonate
The synthesis of compounds 8–11 was based on the conden-
sation of 1 with diethyl malonate and further modifications of
the resulting compound 8 (Scheme 4). The condensation was
carried out in an excess of diethyl malonate with subsequent
purification using short-path vacuum distillation to afford ethyl
ester 8 in 86% yield. The reduction and saponification of 8
proved efficient under standard conditions, furnishing primary
alcohol 9 and carboxylic acid 10, respectively, in excellent
yields. Acid 10 could be readily converted into amides 11 a–
Scheme 5. Retrosynthetic analyses of alkoxylated triazolophthalazine deriva-
tives 13: route a: previously reported four-step and convergent route;
route b: two-step and divergent route proposed herein.
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1080
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
envisioned that alkoxylated derivatives of type 13 could be
prepared in a more straightforward and divergent manner
through O-alkylation of phenolic precursor 12b, whose synthe-
sis was envisaged in one pot from 1 and trans-4-coumaric acid
as condensation partners.
densation process and were thus able to prepare derivatives
12c–j in modest to satisfactory yields, ranging from 40 to
83%. As an additional derivative, we prepared trihydroxylated
compound 14 through demethylation of 12j using standard
conditions (excess BBr3 in dichloromethane at À788C).
Using a similar approach, we also planned the synthesis of
another C2 derivative, 3-ethynyltriazolophthalazine 15
(Scheme 7), but our attempts to obtain this compound directly
through microwave-mediated condensation of 1 and 3-trime-
thylsilyl propiolic acid failed. A very complex mixture of prod-
ucts was obtained, thus revealing the instability of
the propiolic acid under our microwave conditions.
Slight modification of the conditions nevertheless
enabled the synthesis of 15. Construction of the tria-
zolophthalazine scaffold indeed proved effective
when performed in two steps rather than in a one-
pot manner. Trimethylsilyl propiolic acid was first
coupled with 1 under milder temperature conditions
to afford acyclic compound 16, which was isolated
and then cyclized to silylated intermediate 17 under
microwave irradiation in almost quantitative yield.
Final deprotection of 17 under standard conditions
(i.e., K2CO3 in MeOH) furnished desired derivative 15
in 31% overall yield over three steps.
Accordingly, we first focused our attention on finding ade-
quate conditions for the one-pot synthesis of trans-styryltriazo-
lophthalazines (Scheme 6). Using 1 and cinnamic acid as
model partners, we examined the effect of microwave irradia-
tion on the condensation process, furnishing 12a as the de-
With phenolic derivative 12b in hand, this opened
a route to access previously mentioned derivatives
13 (Scheme 5, route b). In our hands, the phenolic
functionality of 12b proved easy to alkylate under
standard reaction conditions, thus allowing the
preparation of six additional alkoxylated derivatives,
13a–f, with yields ranging from 46 to 85%
(Scheme 8). Of note, the alkylating agents used were
chosen based on their chain lengths as well as their
unsaturation/branching profiles.
Finally, because stereochemical configurations of
double bonds were demonstrated several times to
Scheme 6. One-pot synthesis of C2 series derivatives from cinnamic derivatives. Reagents
and conditions: a) reactions run with cinnamic acid (1.0 equiv), 1 (1.5 equiv), HOBt
(1.1 equiv), EDC (1.1 equiv), Et3N (4 equiv), CH3CN, microwave 1028C, 1 h, unless other-
wise stated; b) reaction run under conventional heating at 958C in a sealed tube;
c) DIPEA was used instead of Et3N; d) reaction run with 12k (1.0 equiv), BBr3 (9 equiv),
CH2Cl2, À788C, 48 h.
have crucial effects on biological activities,[20] we also
envisioned preparation of some cis-styryltriazoloph-
thalazines from their trans isomers. In the TB con-
text, an illustrative example is given by cinnamic
acid itself, the cis isomer of which was recently
sired triazolophthalazine adduct. To our delight, microwaving
was found to highly activate the condensation, with complete
conversion achieved in only 1 h under microwave irradiation,
while 48 h was required under conventional heating. With mi-
crowave irradiation in acetonitrile as solvent and in the pres-
ence of triethylamine as base, derivative 12a was isolated in
pure form and good yield (83%). The same condensation con-
ditions proved further successful for preparing the expected
phenolic derivative, 12b. In that case, the time needed for
complete conversion was similar (1 h), and 12b was obtained
in 69% yield after a simple filtration–recrystallization sequence.
Due to their efficiency, we decided to further exploit these re-
action conditions for the synthesis of other triazolophthalazine
derivatives (Scheme 6). We submitted various commercial cin-
namic acids (dihydrogenated and/or 4-substituted) to the con-
Scheme 7. Synthesis of 3-ethynylated derivative 15. Reagents and conditions:
a) 3-(trimethylsilyl)propynoic acid (1.0 equiv), 1 (1.5 equiv), HOBt (1.1 equiv),
EDC (1.1 equiv), Et3N (4 equiv), CH3CN, microwave 1028C, 1 h; b) 3-(trime-
thylsilyl)propynoic acid (1.0 equiv), 1 (1.5 equiv), HOBt (1.1 equiv), EDC
(1.1 equiv), Et3N (4 equiv), CH3CN, 08C!RT, 3 h; c) 16, CH3CN, microwave
1028C, 1 h; d) 17 (1.0 equiv), K2CO3 (3.2 equiv), MeOH, 12 h, 31% (steps b–d).
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1081
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
method. Minimum inhibitory concentrations (MICs)
are reported in Table 1 using INH and ethambutol
(ETH) as controls. For clarity, compounds are separat-
ed in the table according to the series to which they
belong (i.e., C0–2).
Compounds of the C0 and C1 series (Schemes 2
and 3) all proved to be poor inhibitors, with 4a (MIC
33 mm) and 7 (MIC 43 mm) being the most active
(Table 1). More gratifyingly, some compounds of the
C2 series displayed good to excellent activities
against the M.tb H37Rv strain. Within the amide
series (compounds 11 a–d, Scheme 4), inhibitory ac-
tivity appeared to be closely related to the chain
length. Indeed the longer the R chain, the higher the
potency (i.e., MIC: 11 a >11 b >11 c). Of note, a simi-
lar trend was found in diverse triazole series that we
recently developed.[24–26]
Scheme 8. Synthesis of alkoxylated trans-styryltriazolophthalazine derivatives 13. Re-
agents and conditions: a) 12b (1.0 equiv), RÀBr (1.2–1.5 equiv), KI (1.5 equiv), K2CO3
(1.5 equiv), DMF, RT!608C, 12 h.
Table 1. Inhibitory activity of triazolophthalazines against the M.tb H37Rv
strain.
found to be 120-fold more active against M.tb than the trans
isomer.[21] We then selected five trans derivatives and submit-
ted them to photoisomerization conditions, similar to condi-
tions reported elsewhere for promoting the trans–cis isomeri-
zation of stilbenes (Scheme 9).[22] While photoirradiation of
12a, 12e, and 13a promoted expected trans–cis conversion in
a clean manner,[23] the formation of complex mixtures of com-
pounds was observed in the cases of 13c and 13e. Good to
excellent conversions were attained after 50 h irradiation in
acetonitrile, and the resulting mixture of diastereomers was
purified by column chromatography to give 18a–c.
Compd
MIC [mgmLÀ1 (mm)][a]
C0 compounds
Compd
MIC [mgmLÀ1 (mm)][a]
C2 compounds
2
3
4a
4b
4c
>10 (>59)
>10 (>40)
10 (33)
12e
12 f
12g
12h
12i
4 (11.4)
>4 (>12.6)
>4 (>13.9)
>4 (>12.6)
>4 (>11.5)
>5 (>14.4)
6 (19.0)
>4 (>10.5)
1 (2.8)
>4 (>11.2)
0.5 (1.2)
>4 (>9.3)
>5 (>15.6)
2.25 (12.9)
>4 (>14.7)
8 (22.8)
>10 (>31)
0.05 (0.36)
1 (4.90)
>10 (>36)
>10 (>44)
C1 compounds
>10 (>50)
>10 (>40)
10 (43)
C2 compounds
>10 (>39)
>10 (>40)
>10 (>44)
>10 (>33)
10 (32)
12j
5
6
7
13a
13b
13c
13d
13e
13 f
14
8
9
10
11 a
11 b
11 c
12a
12b
12c
12d
15
18a
18b
18c
INH[b]
ETH[b]
2.5 (7)
>4 (>14.7)
>4 (>13.9)
>4 (>14.7)
>4 (>13.8)
[a] Values are the meanÆ0.025 mgLÀ1 (n=3). [b] INH (isoniazid) and ETH
(ethambutol) were used as controls.
In sharp contrast, a wide disparity in MIC values was ob-
served for the C2 series of cinnamic derivatives (Schemes 6 and
8). The substituent at the para position of the aryl group was
clearly found to be crucial for inhibitory activity. Amongst all of
the cinnamic-derived compounds, compound 12e, bearing
a bromine atom, and compounds 13c and e, bearing either an
isopentenyl or a geranyl side chain, proved to be the best in-
hibitors with regard to their MIC values.[27] Compounds 13d
and f, corresponding to saturated analogues 13c and e, were
found to be inactive, thus revealing the key role played by un-
saturation on inhibitory potential. With regard to the C2 unit
linking the triazolophthalazine and the aromatic groups, nei-
ther its oxidation state nor its stereochemistry appeared to be
Scheme 9. Synthesis of cis-styryltriazolophthalazine derivatives 18 from their
trans isomers. Reagents and conditions: a) reactions run with trans derivative
at 0.015m in CH3CN, hn, 50 h; b) the formation of a complex mixture of pho-
toproducts was observed.
Biology
Antimycobacterial activities
The newly synthesized compounds were evaluated for their in-
hibitory activity against M.tb (H37Rv strain) using a dilution
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1082
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
critical for the targeted anti-TB activity. Indeed, shifting from
the C=C to its reduced CÀC form (12c vs. 12a and 12d vs.
12b) or from the E to its diastereomeric Z forms (18a vs. 12a,
18b vs. 12e, and 18c vs. 13a) did not cause any profitable
effect. It is also noteworthy that all of the prepared phenolic
derivatives, from monophenolic 12b, i, and j to catechol- and
pyrogallol-based derivatives 12h and 14, unfortunately proved
ineffective as anti-TB agents. Finally, and to our delight, com-
pound 15, the shortest C2 derivative in our series, showed
good anti-TB activity with a MIC value of ~10 mm.
Conclusions
A series of 35 3-substituted triazolophthalazine derivatives was
prepared from a single commercially available precursor (hy-
dralazine hydrochloride) using straightforward synthetic se-
quences (one- to three-step syntheses). These easy-to-prepare
derivatives did not manifest cytotoxicity toward HCT116
human cells, and among these, four compounds (11 c, 12e,
13e, and 15) exhibited attractive inhibitory activities against
M.tb H37Rv strain. More significantly, these compounds present-
ed similar activities against multidrug-resistant M.tb strains,
thus revealing an alternative mode of action for these triazo-
lophthalazine-based compounds compared with standard anti-
TB drugs. Further work is now underway to tackle the identifi-
cation of the protein targets of this class of potent anti-TB
compounds. The synthesis of other triazolophthalazine-based
heterocyclic systems will be envisaged as soon as the target is
clearly identified.
Antimycobacterial activities against M.tb-resistant strains and
cytotoxicities
To further explore the potential of our compounds, a number
of active triazolophthalazine derivatives were screened against
drug-resistant strains and cytotoxicity against a human colon
cancer cell line (HCT116) as a model cell line (Table 2). Al-
though most of the synthesized compounds were tested for
Table 2. Inhibitory activity against M.tb-resistant strains and cytotoxic ac-
tivity of selected triazolophthalazines.
Experimental Section
Chemistry
Compd
MIC [mgmLÀ1 (mm)][a,b]
IC50 [mm][c]
General: All starting materials were purchased from commercial
sources and were used as received. Anhydrous solvents were fresh-
ly distilled before use or were obtained from the M. Braun solvent
purification system (MB-SPS-800). The reactions were monitored by
thin-layer chromatography carried out on silica plates (silica gel 60
F254, Merck) using a UV light for visualization. Column chromatogra-
phies were performed 1) on silica gel 60 (0.040–0.063 mm, Merck)
using mixtures of ethyl acetate (or diethyl ether) and cyclohexane
as eluents, or 2) with a puriFlash 430 system using puriFlash col-
umns from Interchim. Evaporation of solvents was conducted
under reduced pressure at temperatures below 308C unless other-
wise noted. Melting points (mp) were measured with a Stuart
SMP30 apparatus in open capillary tubes or with a Mettler Toledo
MP50 melting point system and are uncorrected. IR spectra were
obtained from the Service Commun de Spectroscopie Infrarouge
et Raman of the Plateforme Technique, Institut de Chimie de Tou-
louse. Values are reported in cmÀ1. 1H and 13C NMR spectra were re-
corded on a Bruker Avance 300 spectrometer at 300 and 75 MHz,
respectively. Chemical shifts (d) and coupling constants (J) are
given in ppm and Hz, respectively. Chemical shifts are reported rel-
ative to residual solvent as an internal standard (CDCl3: 7.26 ppm
CI1
CI2
CI3
2
nd[d]
nd[d]
nd[d]
4 (11.4)
nd[d]
nd[d]
0.5 (1.2)
2.25 (12.9)
>0.2 (1.5)
nd[d]
nd[d]
nd[d]
4 (11.4)
nd[d]
nd[d]
0.5 (1.2)
2.25 (12.9)
>0.2 (1.5)
nd[d]
nd[d]
nd[d]
4 (11.4)
nd[d]
nd[d]
0.5 (1.2)
2.25 (12.9)
>0.2 (1.5)
>100
>100
>100
>100
>100
53
>100
>100
nd[d]
12b
12c
12e
12i
13d
13e
15
INH[e]
[a] Drug resistance profiles of M.tb clinical isolates (CI): CI1: resistant to
streptomycin, INH, rifampicin, and ETH; CI2: resistant to streptomycin,
INH, rifampicin, ETH, pyrazinamide, ethionamide, and capreomicin; CI3:
resistant to streptomycin, INH, rifampicin, ETH, pyrazinamide, and ethio-
namide. [b] Values are the meanÆ0.025 mgLÀ1 (n=3). [c] Cytotoxicity IC50
values in the HCT116 human colon cancer cell line. [d] Not determined.
[e] INH (isoniazid) was used as a control.
cytotoxicity, only a selection of data, characteristic of the gen-
eral cytotoxic profiles we observed for triazolophthalazine de-
rivatives, is given in Table 2. With the exception of compound
13d, which exhibited moderate cytotoxicity toward HCT116
(i.e., IC50 =53 mm), no tested triazolophthalazine derivatives
demonstrated cytotoxicity against this human cell line (i.e.,
IC50 >100 mm).
In light of their relevant inhibitory efficiencies against the
M.tb H37Rv strain, compounds 12e, 13e, and 15 were selected
and further evaluated for their inhibitory potential against
three multidrug-resistant M.tb clinical isolates (i.e., CI1–3 in
Table 2). The results showed that the level of efficiency of each
compound was preserved regardless of the nature of the
strain, thus constituting another valuable asset over standard
anti-TB drugs, such as INH and ETH.
1
1
for H and 77.0 ppm for 13C; CD3OD: 3.31 ppm for H and 49.0 ppm
for 13C; [D6]DMSO: 2.50 ppm for 1H NMR and 39.5 ppm for
1
13C NMR). H multiplicities are designated by the following abbrevi-
ations: s=singlet, d=doublet, t=triplet, m=multiplet, b=broad.
Carbon multiplicities were determined by DEPT135 or Jmod experi-
ments. Electrospray (ESI) and desorption chemical ionization (DCI)
low-/high-resolution mass spectra were obtained from the Service
Commun de Spectroscopie de Masse of the Plateforme Technique,
Institut de Chimie de Toulouse. Crystallographic data for com-
pounds 5, 7, and 8[28] were collected on a Bruker-AXS SMART
APEX II diffractometer (8) and a Bruker-AXS Quazar APEX II diffrac-
tometer using a 30 W air-cooled microfocus source (ImS) with fo-
cusing multilayer optics (5 and 7) at a temperature of 193(2) K,
with MoKa radiation (l: 0.71073 Š) using F and W scans. The data
were integrated with SAINT,[29] and an empirical absorption correc-
tion with SADABS[30] was applied. The structures were solved by
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1083
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
direct methods, using SHELXS-97, and refined using the least-
squares method on F2.[31]
HRMS (ESI+) m/z [M+H]+ calcd for C16H14N5: 276.1249, found:
276.1239.
N-Octyl[1,2,4]triazolo[3,4-a]phthalazin-3-amine (4c): Compound
4c was obtained as a yellow powder (yield: 120 mg, 81%): mp:
109–1108C; 1H NMR (300 MHz, CDCl3): d=8.48 (d, J=7.9 Hz, 1H),
8.35 (s, 1H), 7.82 (td, J=7.1 Hz, 1.2 Hz, 1H), 7.78 (d, J=7.1 Hz, 1H),
7.67 (td, J=7.1 Hz, 1.1 Hz, 1H), 4.74 (t, J=5.8 Hz, 1H), 3.65–3.58
(m, 2H), 1.79–1.69 (m, 2H), 1.43–1.21 (m, 10H), 0.86 ppm (t, J=
6.4 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=150.9, 145.8, 139.5, 133.7,
129.8, 128.0, 124.2, 122.74, 122.69, 43.4, 31.8, 29.8, 29.3, 29.2, 26.8,
22.6, 14.1 ppm; FTIR-ATR (neat): n˜ =3378, 3265, 3047, 3014, 2924,
2855, 1607, 1581, 1535, 1377 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd
for C17H24N5: 298.2032, found: 298.2026.
Synthesis of [1,2,4]triazolo[3,4-a]phthalazine (2): 1-Hydrazinyl-
phthalazine hydrochloride 1 (500 mg, 2.5 mmol) and Et3N (0.55 mL,
4 mmol) in dry CH2Cl2 (10 mL) were stirred for 30 min at room tem-
perature, then trimethyl orthoformiate (0.43 mL, 4 mmol) was
added. The resulting mixture was stirred for 15 h at 408C, filtered,
and recrystallized from CH2Cl2/MeOH (12:1) to furnish the title
compound 2 in pure form as a white solid (yield: 320 mg, 75%):
1
mp: 1858C; H NMR (300 MHz, CDCl3): d=9.00 (s, 1H), 8.61 (s, 1H),
8.59 (m, 1H), 7.94–7.91 (m, 2H), 7.78 ppm (td, J=7.1 Hz, 1.1 Hz,
1H); 13C NMR (75 MHz, [D6]DMSO): d=150.8, 149.6, 142.5, 135.1,
132.5, 129.8, 123.8, 123.2, 122.1 ppm; FTIR-ATR (neat): n˜ =3100,
3070, 2990, 2960, 1610, 1520, 1450, 1318 cmÀ1; HRMS (ESI+) m/z
[M+H]+ calcd for C9H7N4: 171.0671, found: 171.0665.
Synthesis of [1,2,4]triazolo[3,4-a]phthalazin-3-ylmethanol (5): Tri-
azolophthalazine 2 (200 mg, 1 mmol) was heated at 1008C in a mix-
ture of 37% aqueous formaldehyde solution (5 mL) and dioxane
(2 mL). After complete conversion of the starting material (TLC
monitoring), the reaction mixture was evaporated under reduced
pressure to furnish title compound 5 in pure form as a white solid
(yield: 210 mg, 81%): mp: 244–2468C; 1H NMR (300 MHz,
[D6]DMSO) d=9.23 (s, 1H), 8.60 (d, J=7.9 Hz, 1H), 8.29 (d, J=
7.8 Hz, 1H), 8.12 (td, J=7.6 Hz, 1.1 Hz, 1H), 8.01 (td, J=7.5 Hz,
1.1 Hz, 1H), 6.60 (bs, 1H), 4.99 ppm (s, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=150.3, 149.2, 142.0, 134.7, 132.0, 129.3, 123.4, 122.7,
121.7, 52.6 ppm; FTIR-ATR (neat): n˜ =3264 (OH), 3042, 2843, 2580,
2551, 1639, 1593, 1460, 1218 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd
for C10H9N4O: 201.0776, found: 201.0781.
Synthesis of 3-bromo[1,2,4]triazolo[3,4-a]phthalazine (3): An
excess of bromine (2 mL) was added to a solution of compound 2
(200 mg, 1 mmol) in pure acetic acid (5 mL). After stirring at reflux
for 5 h, the mixture was cooled to room temperature, quenched
with aqueous NaHCO3 until pH~10 was reached, and then stirred
at room temperature for an additional 8 h. The resulting precipi-
tate was filtered to furnish the title compound 3 in pure form as
1
a white solid (yield: 160 mg, 53%): mp: 2028C; H NMR (300 MHz,
CD3OD): d=9.01 (s, 1H), 8.57 (bd, J=7.9 Hz, 1H), 8.21 (bd, J=
7.9 Hz, 1H), 8.21 (td, J=7.9, 1.2 Hz, 1H), 7.98 ppm (td, J=7.9,
1.2 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=149.3, 144.2, 134.6,
131.5, 129.2, 125.9, 123.2, 122.2, 121.8 ppm; FTIR-ATR (neat): n˜ =
3050, 3023, 2981, 1526, 1458, 1414, 1208 cmÀ1; HRMS (ESI+) m/z
[M+H]+ calcd for C9H6BrN4: 248.9796/250.9755, found: 248.9788/
250.9764.
Synthesis of 3-(dichloromethyl)[1,2,4]triazolo[3,4-a]phthalazine
(6): 1-Hydrazinylphthalazine hydrochloride 1 (100 mg, 0.5 mmol)
and Et3N (0.55 mL, 4 mmol) in dry CH2Cl2 (10 mL) were stirred for
30 min at room temperature, then dichloroacetic acid (10 mL) was
carefully added. After stirring for an additional 30 min at 608C until
complete evaporation of CH2Cl2, the reaction mixture was heated
at 110 8C for 2 h. After cooling to room temperature, the mixture
was diluted with H20 (40 mL), and the resulting precipitate was fil-
tered and finally recrystallized from MeOH to furnish title com-
pound 6 in pure form as a yellow powder (yield: 120 mg, 80%):
General procedure for the synthesis of 3-amino[1,2,4]triazo-
lo[3,4-a]phthalazines 4a–c: A DMF (10 mL) mixture of 1-hydrazi-
nylphthalazine hydrochloride
1 (100 mg, 0.5 mmol, 1 equiv),
Na2CO3 (150 mg, 1.5 mmol, 3 equiv), and the corresponding iso-
thiocyanate (0.55 mmol, 1.1 equiv) was stirred at 808C for 30 min.
EDC·HCl (110 mg, 0.55 mmol, 1.1 equiv) was then added, and the
resulting mixture was stirred for additional 4 h at 808C. After cool-
ing to room temperature and adding water (20 mL), the resulting
mixture was extracted with EtOAc (3”20 mL), and the combined
EtOAc layers were dried over Na2SO4, filtered, and evaporated. Pu-
rification of the residue by flash chromatography, eluting with
EtOAc/MeOH (9:1), gave title compounds 4a–c in pure forms.
1
mp: 2258C; H NMR (300 MHz, [D6]DMSO): d=9.27 (s, 1H), 8.57 (d,
J=7.9 Hz, 1H), 8.28 (d, J=7.6 Hz, 1H), 8.12 (td, J=7.6 Hz, 1.2 Hz,
1H), 8.09 (s, 1H), 8.01 ppm (td, J=7.6 Hz, 1.2 Hz, 1H); 13C NMR
(75 MHz, [D6]DMSO): d=149.3, 147.1, 143.8, 134.8, 131.9, 129.3,
123.2, 122.4, 122.2, 60.1 ppm; FTIR-ATR (neat): n˜ =3057, 3018,
1526, 1456, 1216 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd for C10H7
Cl2N4: 253.0048, found: 253.0037.
N-Phenyl[1,2,4]triazolo[3,4-a]phthalazin-3-amine
(4a):
Com-
pound 4a was obtained as a yellow powder (yield: 110 mg, 81%):
mp: 219–2208C; 1H NMR (300 MHz, CDCl3): d=8.53 (d, J=8.1 Hz,
1H), 8.44 (s, 1H), 7.89–7.77 (m, 4H), 7.73–7.68 (m, 1H), 7.40–7.35
(m, 2H), 7.7 (brs, 1H), 7.06–7.00 ppm (m, 1H); 13C NMR (75 MHz,
CDCl3): d=146.7, 146.3, 139.2, 138.9, 133.9, 130.3, 129.2, 128.2,
123.9, 122.9, 121.9, 117.0 ppm; FTIR-ATR (neat): n˜ =3454, 3275,
3057, 2919, 1616, 1577, 1452, 1246 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C15H12N5: 262.1093, found: 262.1092.
Synthesis of [1,2,4]triazolo[3,4-a]phthalazine-3-ylmethanediol
(7): A solution of phthalazine 6 (100 mg, 0.4 mmol) in morpholine
(5 mL) was heated for 5 h at 1008C. The resulting red solution was
then cooled to room temperature, concentrated to dryness, and
treated with a 25% HCl solution (5 mL). After stirring for 1 h, the
resulting precipitate was filtered and finally recrystallized from
MeOH to furnish title compound 7 in pure form as an off-white
powder (yield: 50 mg, 52%): mp: 228–2308C; 1H NMR (300 MHz,
[D6]DMSO): d=9.25 (s, 1H), 8.56 (d, J=7.9 Hz, 1H), 8.27 (d, J=7.9,
1.1 Hz, 1H), 8.11 (td, J=7.6, 0.9 Hz, 1H), 8.07 (s, 1H), 8.00 ppm (d,
J=7.6 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=149.4, 147.1,
143.9, 134.9, 132.0, 129.4, 123.2, 122.5, 121.2, 60.1 ppm; HRMS
(ESI+) m/z [M+H]+ calcd for C10H7N4O: 199.0620, found: 199.0621.
N-Benzyl[1,2,4]triazolo[3,4-a]phthalazin-3-amine
(4b):
Com-
pound 4b was obtained as a yellow powder (yield: 110 mg, 82%):
mp: 1148C; 1H NMR (300 MHz, CDCl3): d=8.49 (d, J=7.9 Hz, 1H),
8.33 (s, 1H), 7.83 (td, J=8.3 Hz, 1.1 Hz, 1H), 7.78 (d, J=7.6 Hz, 1H),
7.67 (td, J=8.1 Hz, 0.9 Hz, 1H), 7.48–7.45 (m, 2H), 7.37–7.27 (m,
3H), 5.08 (t, J=5.7 Hz, 1H), 4.81 ppm (d, J=6.0 Hz, 2H); 13C NMR
(75 MHz, CDCl3): d=150.7, 145.9, 139.7, 138.4, 133.7, 129.9, 128.6,
128.1, 128.0, 127.6, 124.1, 122.72, 122.67, 47.3 ppm; FTIR-ATR
Synthesis of ethyl [1,2,4]triazolo[3,4-a]phthalazin-3-ylacetate (8):
1-Hydrazinylphthalazine hydrochloride 1 (100 mg, 0.5 mmol) and
Et3N (0.55 mL, 4 mmol) in dry CH2Cl2 (10 mL) were stirred for
30 min at room temperature, then diethyl malonate (10 mL) was
(neat): n˜ =3391, 3255, 3084, 3030, 2866, 1608, 1538, 1356 cmÀ1
;
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1084
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
carefully added. After heating for 5 h at 110 8C, the reaction mix-
ture was evaporated and further distilled under vacuum. Purifica-
tion of the residue by flash chromatography, eluting with CH2Cl2/
MeOH (9:1), gave title compound 8 in pure form as a yellow
powder (yield: 110 mg, 86%): mp: 1358C; 1H NMR (300 MHz,
CDCl3): d=8.63–8.60 (m, 1H), 8.60 (s, 1H), 7.95–7.89 (m, 2H), 7.78
(td, J=7.3 Hz, 1.0 Hz, 1H), 4.30 (s, 2H), 4.19 (q, J=7.2 Hz, 2H),
1.23 ppm (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=167.6,
147.6, 145.1, 142.9, 134.0, 130.8, 128.0, 123.4, 123.1, 123.0, 61.6,
30.8, 14.1 ppm; FTIR-ATR (neat): n˜ =3446, 3024, 2984, 2880, 1731,
1527, 1458, 1323, 1200 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd for
C13H13N4O2: 257.1039, found: 257.1039.
N-Benzyl-2-([1,2,4]triazolo[3,4-a]phthlazin-3-yl)acetamide (11b):
Compound 11 b was obtained as a white powder (yield: 92 mg,
72%): mp: 1948C; H NMR (300 MHz, CDCl3): d=8.64 (s, 1H), 8.58
(d, J=7.5 Hz, 1H), 7.97–7.79 (m, 4H), 7.30–7.24 (m, 5H), 4.50 (d, J=
5.8 Hz, 2H), 4.27 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=165.8,
147.9, 146.2, 143.0, 137.9, 134.2, 131.1, 128.6, 128.2, 127.7, 127.4,
123.22, 123.16, 123.1, 43.8, 31.8 ppm; FTIR-ATR (neat): n˜ =3203,
3025, 2966, 2901, 1646, 1560, 1461, 1272 cmÀ1; HRMS (ESI+) m/z
[M+H]+ calcd for C18H16N5O: 318.1355, found: 318.1357.
1
N-Nonyl-2-([1,2,4]triazolo[3,4-a]phthalazin-3-yl)acetamide (11c):
Compound 11 c was obtained as a yellow powder (yield: 120 mg,
1
85%): mp: 1738C; H NMR (300 MHz, CDCl3): d=8.65 (s, 1H), 8.63
(d, J=8.0 Hz, 1H), 7.99–7.93 (m, 2H), 7.82 (td, J=7.5 Hz, 1.1 Hz,
1H), 7.46 (bs, 1H), 4.22 (s, 2H), 3.32–3.25 (m, 2H), 1.54–1.49 (m,
2H), 1.31–1.22 (m, 12H), 0.85 ppm (t, J=7.0 Hz, 3H); 13C NMR
(75 MHz, CDCl3): d=165.7, 148.0, 146.5, 143.04, 134.2, 131.2, 128.3,
123.4, 123.26, 123.24, 40.0, 31.93, 31.89, 29.5, 29.4, 29.32, 29.29,
27.0, 22.7, 14.2 ppm; FTIR-ATR (neat): n˜ =3263, 3081, 3030, 2921,
2853, 2394, 1664, 1569, 1457, 1230 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C20H28N5O: 354.2294, found: 354.2290.
Synthesis of 2-(5,6-dihydro[1,2,4]triazolo[3,4-a]phthalazin-3-yle-
thanol (9): An EtOH (10 mL) solution of phthalazine 8 (100 mg,
0.4 mmol) and NaBH4 (300 mg) was heated for 5 h at 808C. After
cooling to room temperature, the resulting mixture was extracted
with EtOAc (3”20 mL), and the combined EtOAc layers were dried
over Na2SO4, filtered, and evaporated, giving title compound 9 in
pure form as a white solid (yield: 90 mg, 95%): mp: 2008C;
1H NMR (300 MHz, CD3OD): d=7.98–7.96 (m, 1H), 7.54–7.40 (m,
3H), 4.33 (s, 2H), 3.95 (t, J=6.7 Hz, 2H), 3.09 ppm (t, J=6.7 Hz,
2H); 13C NMR (75 MHz, CD3OD): d=152.7, 149.9, 134.9, 132.1,
129.4, 127.2, 124.6, 123.4, 60.3, 50.2, 28.5 ppm. FTIR-ATR (neat): n˜ =
3464, 3268, 2923, 2328, 1524, 1462, 1367 cmÀ1; HRMS (ESI+) m/z
[M+H]+ calcd for C11H13N4O: 217.1089, found: 217.1084.
General procedure for the synthesis of 3-styrylated derivatives
12a–k
and
14:
1-Hydrazinylphthalazine
hydrochloride
1
(1.5 equiv), HOBt (1.1 equiv), EDC·HCl (1.5 equiv), and Et3N
(4 equiv) were successively added to a CH3CN solution of the re-
quired (dehydro)cinnamic acid (1.0 equiv) in a 5 mL microwave re-
actor. After microwave irradiation for 40 to 60 min (~1008C,
260 W), the resulting mixture was cooled to room temperature and
evaporated under reduced pressure. The thus-obtained crude ma-
terials were diluted with CH2Cl2, and the resulting organic layer
was washed with saturated aqueous NH4Cl solution, water, and
brine, dried over Na2SO4, filtered, and evaporated. Purification of
the residue by flash chromatography, eluting with the appropriate
eluent, gave title compounds 12a–k and 14 in pure forms.
Synthesis of [1,2,4]triazolo[3,4-a]phthalazin-3-ylacetic acid (10):
An EtOH (10 mL) solution of phthalazine 8 (100 mg, 0.4 mmol) and
NaOH (5m, 5 mL) was stirred at room temperature for 10 h. The re-
action mixture was then acidified with concentrated HCl to pH~4,
and the resulting precipitate was filtered, giving title compound
10 in pure form as a white solid (yield: 85 mg, 93%): mp: 1758C;
1H NMR (300 MHz, [D6]DMSO); d=12.93 (s, 1H), 9.10 (s, 1H), 8.51
(d, J=7.9 Hz, 1H), 8.23 (d, J=7.7 Hz, 1H), 8.08 (td, J=7.7 Hz,
0.9 Hz, 1H), 7.95 (td, J=7.8 Hz, 0.9 Hz, 1H), 4.24 ppm (s, 2H);
13C NMR (75 MHz, [D6]DMSO): d=169.3, 148.3, 145.6, 142.2, 134.4,
131.1, 129.2, 122.8, 122.5, 122.1, 30.5 ppm; FTIR-ATR (neat): n˜ =
E-3-Styryl-2-([1,2,4]triazolo[3,4-a]phthalazine (12a): Compound
12a was obtained as a yellow powder (yield: 83%): mp: 1968C;
1H NMR (300 MHz, CDCl3): d=8.76 (d, J=8.0 Hz, 1H), 8.72 (s, 1H),
8.20 (d, J=16.6 Hz, 1H), 8.02–7.96 (m, 2H), 7.85 (td, J=7.5 Hz,
1.1 Hz, 1H), 7.69–7.67 (m, 2H), 7.52 (d, J=16.6 Hz, 1H), 7.45–
7.34 ppm (m, 3H); 13C NMR (75 MHz, CDCl3): d=148.5, 147.4, 142.7,
135.90, 135.89, 133.9, 130.7, 129.0, 128.7, 128.0, 127.2, 123.3, 123.1,
122.9, 110.5 ppm; FTIR-ATR (neat): n˜ =3041, 3012, 2983, 2958,
2886, 2360, 2317, 2157, 1980, 1640, 1627, 1518, 1504, 1468, 1450,
1392 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd for C17H13N4: 273.1140,
found: 273.1141.
3070, 3038, 2979, 2771, 2434, 1712, 1530, 1469, 1272, 1159 cmÀ1
;
HRMS (ESI+) m/z [M+H]+ calcd for C11H9N4O2: 229.0726, found:
229.0729.
General procedure for the synthesis of amides 11 a–c: A DMF
(10 mL) mixture of phthalazine 10 (100 mg, 0.4 mmol, 1.0 equiv),
EDC·HCl (92 mg, 1.1 equiv), and HOBt (64 mg, 1.1 equiv) was stirred
for 30 min at room temperature. DIPEA (120 mg, 2.1 equiv) and the
corresponding amine (1.1 equiv) was then added to the mixture.
After stirring for 10 h at room temperature, the resulting mixture
was diluted with water (20 mL) and extracted with EtOAc (3”
20 mL). The combined EtOAc layers were dried over Na2SO4, fil-
tered, and evaporated. Purification of the residue by flash chroma-
tography, eluting with CH2Cl2/MeOH (9:1), gave title compounds
11 a–c in pure forms.
E-4-(2-([1,2,4]Triazolo[3,4-a]phthalazin-3-yl)vinyl)phenol
(12b):
Compound 12b was obtained as a yellow powder (yield: 69%):
mp> 2508C; 1H NMR (300 MHz, [D6]DMSO): d=9.34 (s, 1H), 8.57
(bd, J=7.7 Hz, 1H), 8.33 (bd, J=7.5 Hz, 1H), 8.12 (td, J=7.5 Hz,
1.1 Hz, 1H), 8.06–8.00 (m, 2H), 7.67–7.64 (m, 2H), 7.35 (d, J=
16.6 Hz, 1H), 6.88–6.85 ppm (m, 2H); 13C NMR (75 MHz, 5% [D1]TFA
in [D6]DMSO): d=159.2, 148.3, 148.2, 143.9, 142.6, 136.4, 134.3,
131.2, 131.1, 130.9, 129.3, 128.9, 127.7, 126.6, 125.8, 124.3, 123.0,
122.7, 122.1, 117.1, 115.6 ppm; FTIR-ATR (neat): n˜ =3325, 3063,
3022, 2959, 2888, 2766, 2695, 2359, 2332, 1734, 1607, 1590, 1517,
1500, 1454, 1391, 1360, 1289 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd
for C17H13N4O: 289.1089, found: 289.1089.
N-Phenyl-2-([1,2,4]triazolo[3,4-a]phthalazin-3-yl)acetamide (11a):
Compound 11 a was obtained as a white powder (yield: 75 mg,
61%): mp: 2268C; 1H NMR (300 MHz, [D6]DMSO): d=9.71 (s, 1H),
8.70 (s, 1H), 8.66 (d, J=7.7 Hz, 1H), 8.02–7.96 (m, 2H), 7.85 (td, J=
7.5 Hz, 1.1 Hz, 1H), 7.62–7.59 (m, 2H), 7.32–7.27 (m, 2H), 7.11–7.06
(m, 1H), 4.43 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=163.7,
154.3, 148.2, 137.7, 134.4, 131.3, 128.9, 128.3, 124.5, 123.31, 123.27,
123.25, 120.1, 115.0, 32.8 ppm; FTIR-ATR (neat): n˜ =3298, 3054,
3031, 2924, 1661, 1527, 1447, 1233 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C17H14N5O: 304.1198, found: 304.1207.
E-3-Phenethyl-[1,2,4]triazolo[3,4-a]phthalazine (12c): Compound
12c was obtained as a yellow powder (yield: 56%): mp: 1458C;
1H NMR (300 MHz, CDCl3): d=8.65 (dd, J=8.0, 0.6 Hz, 1H), 8.59 (s,
1H), 7.97–7.90 (m, 2H), 7.80 (td, J=7.5 Hz, 1.1 Hz, 1H), 7.32–7.19
(m, 5H), 3.56–3.50 (m, 2H), 3.30–3.24 ppm (m, 2H); 13C NMR
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1085
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
(75 MHz, CDCl3): d=150.6, 147.3, 142.6, 140.5, 133.9, 130.7, 128.5,
128.4, 128.0, 126.3, 123.5, 123.2, 123.0, 33.0, 26.3 ppm; FTIR-ATR
(neat): n˜ =3485, 3432, 3242, 3054, 3025, 2929, 2356, 2329, 1625,
1603, 1526, 1497, 1354, 1318 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd
for C17H15N4: 275.1297, found: 275.1294.
7.4, 1.1 Hz, 1H), 7.35 (d, J=16.5 Hz, 1H), 6.89 (s, 2H), 3.97 ppm (s,
6H); 13C NMR (75 MHz, CDCl3): d=148.8, 147.4, 147.3, 142.8, 136.2,
136.1, 134.1, 130.7, 128.0, 127.6, 123.7, 123.3, 123.0, 108.8, 104.2,
56.4 ppm; FTIR-ATR (neat): n˜ =3090, 3025, 2986, 2932, 2851, 2831,
1639, 1598, 1517, 1453, 1328, 1260, 1218, 1121 cmÀ1; HRMS (ESI+)
m/z [M+H]+ calcd for C19H17N4O3: 349.1301, found: 349.1303.
E-4-[1,2,4]Triazolo[3,4-a]phthalazin-3-yl)phenol (12d): Compound
12d was obtained as a yellow powder (yield: 47%): mp: 2358C;
1H NMR (300 MHz, 5% [D1]TFA in CD3CN): d=9.25 (s, 1H), 8.59–
8.56 (m, 1H), 8.37–8.34 (m, 1H), 8.29–8.17 (m, 2H), 7.11–7.08 (m,
2H), 6.75–6.72 (m, 2H), 3.61 (t, J=7.5 Hz, 2H), 3.18 ppm (t, J=
7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=153.9, 151.7, 151.1, 141.4,
136.3, 135.1, 130.5, 129.5, 124.8, 124.2, 116.6, 115.7, 112.8, 31.1,
25.7 ppm; FTIR-ATR (neat): n˜ =3317, 3032, 2792, 2587, 1736, 1605,
1583, 1532, 1461, 1372, 1280, 1225 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C17H15N4O: 291.1246, found: 291.1247.
E-3-(2-(3,4,5-Trimethoxyphenyl)ethenyl[1,2,4]triazolo[3,4-
a]phthalazine (12j): Compound 12j was obtained as a yellow
powder (yield: 71%): mp: 2008C; 1H NMR (300 MHz, CDCl3): d=
8.64 (s, 1H), 8.61 (bd, J=7.5 Hz, 1H), 8.01 (d, J=16.5 Hz, 1H), 7.93–
7.88 (m, 2H), 7.76 (td, J=7.5, 1.1 Hz, 1H), 7.33 (d, J=16.5 Hz, 1H),
6.82 (s, 2H), 3.91 (s, 6H), 3.87 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=153.4, 148.4, 147.4, 142.8, 139.0, 135.7, 134.0, 131.6,
130.7, 128.0, 123.4, 123.1, 122.9, 110.1, 104.2, 60.9, 56.1 ppm; FTIR-
ATR (neat): n˜ =3264, 3065, 3032, 3003, 2968, 2939, 2837, 1643,
1585, 1524, 1511, 1450, 1333, 1251, 1123 cmÀ1; HRMS (ESI+) m/z
[M+H]+ calcd for C20H19N4O3: 363.1457, found: 363.1472.
E-3-(4-Bromostyryl)-[1,2,4]triazolo[3,4-a]phthalazine (12e): Com-
pound 12e was obtained as a white powder (yield: 78%): mp:
1
2428C; H NMR (300 MHz, CDCl3): d=8.73–8.70 (m, 2H), 8.10 (d, J=
General procedure for the synthesis of 3-styrylated derivatives
13a–f: K2CO3 (1.5 equiv) and KI (1.5 equiv) were added to a DMF
solution of 12b. After stirring the suspension at room temperature
for 15 min, the corresponding alkylating agent (1.2 equiv) was
added. After heating overnight at 608C, the mixture was cooled to
room temperature and filtered. The resulting filtrate was evaporat-
ed and diluted with EtOAc, and the organic layer was then washed
with saturated aqueous NH4Cl solution, water, and brine, dried
over Na2SO4, filtered, and evaporated. Purification of the residue by
flash chromatography, eluting with the appropriate eluent, gave
title compounds 13a–f.
16.6 Hz, 1H), 8.01–7.96 (m, 2H), 7.84 (td, J=7.5 Hz, 1.1 Hz, 1H),
7.57–7.48 ppm (m, 5H); 13C NMR (75 MHz, CDCl3): d=148.4, 147.6,
147.5, 142.9, 135.0, 134.7, 134.2, 132.0, 130.9, 128.7, 128.1, 123.5,
123.4, 123.1, 111.3 ppm; FTIR-ATR (neat): n˜ =3063, 3020, 2925,
1762, 1725, 1615, 1532, 1450, 1389, 1268, 1232 cmÀ1; HRMS (ESI+)
m/z [M+H]+ calcd for C17H12N479Br: 351.0245, found: 351.0235.
E-3-(4-Nitrostyryl)-[1,2,4]triazolo[3,4-a]phthalazine (12 f): Com-
pound 12 f was obtained as a yellow powder (yield: 62%): mp:
1908C; 1H NMR (300 MHz, 5% [D1]TFA in CDCl3): d=9.16 (s, 1H),
8.78 (bd, J=7.8 Hz, 1H), 8.43–8.23 (m, 6H), 7.90–7.87 (m, 2H),
7.67 ppm (d, J=16.7 Hz, 1H); 13C NMR (75 MHz, 5% [D1]TFA in
CDCl3): d=150.7, 148.9, 146.9, 141.8, 141.1, 140.0, 136.7, 135.3,
129.5, 128.9, 125.1, 124.5, 124.4, 119.0, 109.6 ppm; FTIR-ATR (neat):
n˜ =3041, 2913, 1750, 1732, 1501, 1464, 1425, 1333, 1264,
E-3-(4-Ethoxystyryl)-[1,2,4]triazolo[3,4-a]phthalazine (13a): Com-
pound 13a was obtained as a yellow powder (yield: 46%): mp:
1
1958C; H NMR (300 MHz, CDCl3): d=8.69 (bd, J=7.9 Hz, 1H), 8.66
(s, 1H), 8.10 (d, J=16.6 Hz, 1H), 7.98–7.93 (m, 2H), 7.81 (td, J=
7.5 Hz, 1.1 Hz, 1H), 7.62–7.58 (m, 2H), 7.37 (d, J=16.6 Hz, 1H),
6.96–6.91 (m, 2H), 4.08 (q, J=6.0 Hz, 2H), 1.44 ppm (t, J=6.0 Hz,
3H); 13C NMR (75 MHz, CDCl3): d=159.8, 148.9, 147.2, 142.6, 135.5,
133.9, 130.6, 128.6, 127.9, 123.6, 123.1, 122.9, 114.7, 108.2, 63.5,
14.7 ppm; FTIR-ATR (neat): n˜ =3060, 3027, 2974, 2849, 1623, 1604,
1515, 1452, 1392, 1347, 1301, 1250, 1217 cmÀ1; MS (ESI+) m/z [M+
H]+ 317.14.
1207 cmÀ1
;
HRMS (ESI+) m/z [M+H]+ calcd for C17H12N5O2:
318.0991, found: 318.1001.
E-4-(2-[1,2,4]Triazolo[3,4-a]phthalazin-3-yl)vinyl)benzene-1,2-diol
(12g): Compound 12g was obtained as a yellow powder (yield:
40%): mp: 1968C; 1H NMR (300 MHz, [D6]DMSO): d=9.13 (s, 1H),
8.53 (bd, J=7.9 Hz, 1H), 8.24 (bd, J=7.5 Hz, 1H), 8.07 (bt, J=
7.5 Hz, 1H), 7.95–7.81 (m, 2H), 7.23–7.03 (m, 3H), 6.80 ppm (d, J=
8.0 Hz, 1H); 13C NMR (75 MHz, 5% [D1]TFA in [D6]DMSO): d=148.5,
148.1, 147.3, 145.7, 142.2, 135.7, 134.5, 131.1, 129.1, 127.1, 123.1,
122.7, 122.2, 120.0, 115.9, 113.7, 106.9 ppm; FTIR-ATR (neat): n˜ =
3437, 3318, 3020, 2602, 1618, 1601, 1520, 1445, 1350, 1304, 1268,
E-3-(4-Benzyloxystyryl)-[1,2,4]triazolo[3,4-a]phthalazine
(13b):
Compound 13b was obtained as a yellow powder (yield: 60%):
1
mp: 2108C; H NMR (300 MHz, CDCl3): d=8.69 (bd, J=7.7 Hz, 1H),
8.67 (s, 1H), 8.10 (d, J=16.5 Hz, 1H), 7.98–7.92 (m, 2H), 7.80 (td,
J=7.5 Hz, 1.0 Hz, 1H), 7.63–7.59 (m, 2H), 7.47–7.32 (m, 6H), 7.04–
6.99 (m, 2H), 5.12 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=159.6,
148.9, 147.3, 142.7, 136.6, 135.4, 133.9, 130.6, 129.1, 128.7, 128.6,
128.0, 127.9, 127.4, 123.7, 123.2, 122.9, 115.2, 108.6, 70.0 ppm;
FTIR-ATR (neat): n˜ =3324, 3048, 2953, 2853, 2360, 2333, 1638, 1604,
1574, 1520, 1460, 1389, 1346, 1210 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C24H19N4O: 379.1559, found: 379.1555.
1236 cmÀ1
;
HRMS (ESI+) m/z [M+H]+ calcd for C17H13N4O2:
305.1039, found: 305.1036.
E-4-(2-[1,2,4]Triazolo[3,4-a]phthalazin-3-yl)vinyl)-2-methoxyphe-
nol (12h): Compound 12h was obtained as a yellow powder
1
(yield: 58%): mp: 2408C; H NMR (300 MHz, 5% [D1]TFA in CDCl3)
d=9.05 (s, 1H), 8.67 (bd, J=7.6 Hz, 1H), 8.32 (d, J=16.5 Hz, 1H),
8.24–8.13 (m, 3H), 7.34–7.27 (m, 2H), 7.20 (d, J=1.7 Hz, 1H), 7.01
(d, J=8.2 Hz, 1H), 4.00 ppm (s, 3H); 13C NMR (75 MHz, 5% [D1]TFA
in CDCl3): d=152.0, 148.9, 147.4, 147.2, 146.4, 142.1, 136.3, 134.8,
129.6, 126.8, 124.4, 124.3, 124.1, 120.1, 115.1, 109.9, 100.8,
56.0 ppm; FTIR-ATR (neat): n˜ =3084, 2359, 2332, 1636, 1594, 1526,
1498, 1468, 1452, 1423, 1381, 1303, 1286, 1244 cmÀ1; HRMS (ESI+)
m/z [M+H]+ calcd for C18H15N4O2: 319.1195, found: 319.1199.
E-3-(4-((3-Methylbut-2-en-1-yl)oxy)styryl)-[1,2,4]triazolo[3,4-
a]phthalazine (13c): Compound 13c was obtained as a yellow
powder (yield: 66%): mp: 1708C; 1H NMR (300 MHz, CDCl3): d=
8.69 (bd, J=8.1 Hz, 1H), 8.67 (s, 1H), 8.10 (d, J=16.6 Hz, 1H), 7.98–
7.93 (m, 2H), 7.80 (td, J=7.5 Hz, 1.0 Hz, 1H), 7.62–7.59 (m, 2H),
7.38 (d, J=16.6 Hz, 1H), 6.97–6.94 (m, 2H), 5.54–5.49 (m, 1H), 4.56
(d, J=6.7 Hz, 2H), 1.77 (s, 3H), 1.62 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=159.7, 149.0, 147.2, 142.7, 138.4, 135.6, 133.9, 130.6,
128.7, 128.6, 128.0, 123.6, 123.1, 123.0, 119.4, 114.9, 108.3, 64.8,
25.8, 18.2 ppm; FTIR-ATR (neat): n˜ =3035, 3023, 2961, 2877, 1609,
E-4-(2-[1,2,4]Triazolo[3,4-a]phthalazin-3-yl)vinyl)-2,6-dimethoxy-
phenol (12i): Compound 12i was obtained as a yellow powder
(yield: 60%): mp: 2368C; 1H NMR (300 MHz, CDCl3): d=8.70–8.68
(m, 2H), 8.07 (d, J=16.5 Hz, 1H), 7.99–7.93 (m, 2H), 7.82 (td, J=
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1086
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
1602, 1573, 1511, 1449, 1346, 1305, 1258, 1244 cmÀ1; MS (ESI+) m/z
[M+H]+ 357.17.
1.1 equiv) and EDC·HCl (148 mg, 1.1 equiv) at 08C. The mixture was
stirred for 30 min at 08C and then stirred for additional 2 h at
room temperature. The resulting precipitate was filtered, washed
with saturated aqueous NH4Cl solution (10 mL), water (2”10 mL)
and Et2O (3”10 mL), thus giving the crude product 16, which was
used without further purification in the next step: 1H NMR
(300 MHz, [D6]DMSO): d=11.51 (bs, 1H), 10.45 (bs, 1H), 8.20–7.91
(m, 2H), 7.77–7.60 (m, 3H), 0.26 ppm (s, 9H); 13C NMR (75 MHz,
[D6]DMSO): d=152.4, 151.9, 145.5, 136.4, 135.1, 129.2, 127.8, 124.2,
96.5, 94.7, 59.8, À0.9 ppm; MS (DCI+) m/z [M+H]+ 285.1. After mi-
crowave irradiation for 60 min (~1008C, 260 W) of a CH3CN solu-
tion (3 mL) of crude 16 (10 mg, 0.035 mmol), the resulting mixture
was cooled to room temperature and evaporated under reduced
pressure. The thus-obtained crude product was diluted with EtOAc
(20 mL), and the resulting organic layer was washed with water
(2”10 mL) and brine (2”10 mL), dried over Na2SO4, filtered, and
evaporated. The resulting crude product 17 was directly submitted
to deprotection conditions. Compound 17 and K2CO3 (15 mg,
3.2 equiv) were suspended in MeOH, and this mixture was stirred
for 12 h at room temperature. The mixture was then diluted with
EtOAc (20 mL), and the resulting organic layer was washed with sa-
turated aqueous NH4Cl solution (2”10 mL), water (2”10 mL), brine
(2”10 mL), dried over Na2SO4, filtered, and evaporated. Purification
of the residue by flash chromatography, eluting with EtOAc/petro-
leum ether (1:1), gave title compound 15 in pure form (6 mg, 31%
E-3-(4-Isopentyloxy)styryl)-[1,2,4]triazolo[3,4-a]phthalazine
(13d): Compound 13d was obtained as a yellow powder (yield:
1
85%): mp 1618C; H NMR (300 MHz, CDCl3): d=8.69 (bd, J=8.0 Hz,
1H), 8.66 (s, 1H), 8.10 (d, J=16.6 Hz, 1H), 7.98–7.93 (m, 2H), 7.80
(td, J=7.5 Hz, 1.1 Hz, 1H), 7.62–7.58 (m, 2H), 7.37 (d, J=16.6 Hz,
1H), 6.96–6.91 (m, 2H), 4.04 (t, J=6.6 Hz, 2H), 1.90–1.81 (m, 1H),
1.78 (q, J=6.9 Hz, 2H), 0.98 ppm (d, J=6.6 Hz, 6H); 13C NMR
(75 MHz, CDCl3): d=160.0, 148.9, 147.2, 142.6, 135.5, 133.9, 130.5,
128.6, 127.9, 123.6, 123.1, 122.9, 114.7, 108.2, 66.4, 37.9, 25.0,
22.5 ppm; FTIR-ATR (neat): n˜ =3072, 3031, 2957, 2872, 2359, 1743,
1631, 1601, 1570, 1515, 1455, 1387, 1310, 1252 cmÀ1; HRMS (ESI+)
m/z [M+H]+ calcd for C22H23N4O: 359.1872, found: 359.1864.
E-3-(4-((E-3,7-Dimethylocta-2,6-dien-1-yl)oxy)styryl)-[1,2,4]triazo-
lo[3,4-a]phthalazine (13e): Compound 13e was obtained as
a yellow powder (yield: 56%): mp: 1508C; 1H NMR (300 MHz,
CDCl3): d=8.69 (bd, J=7.9 Hz, 1H), 8.66 (s, 1H), 8.10 (d, J=
16.6 Hz, 1H), 7.98–7.92 (m, 2H), 7.80 (td, J=7.5 Hz, 1.2 Hz, 1H),
7.61–7.59 (m, 2H), 7.37 (d, J=16.6 Hz, 1H), 6.97–6.93 (m, 2H),
5.52–5.48 (m, 1H), 5.12–5.09 (m, 1H), 4.59 (d, J=6.5 Hz, 2H), 2.15–
2.11 (m, 4H), 1.76 (s, 3H), 1.68 (s, 3H), 1.61 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=159.7, 148.9, 147.2, 142.6, 141.4, 135.5, 133.9,
131.8, 130.6, 128.7, 128.6, 127.9, 123.7, 123.6, 123.1, 123.0, 119.2,
115.0, 108.3, 64.9, 39.5, 26.2, 25.7, 17.7, 16.7 ppm; FTIR-ATR (neat):
n˜ =3239, 3063, 2965, 2926, 2362, 2338, 1711, 1633, 1603, 1514,
1452, 1375, 1348, 1243 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd for
C27H29N4O: 425.2341 found: 425.2344.
1
in 3 steps): mp: 2248C; H NMR (300 MHz, CDCl3): d=8.76 (s, 1H),
8.73 (bd, J=7.7 Hz, 1H), 8.05–7.98 (m, 2H), 7.88 (td, J=7.3 Hz,
1.2 Hz, 1H), 3.80 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=150.0,
142.3, 135.4, 133.0, 128.8, 124.2, 123.8, 121.5, 113.1, 89.5, 66.8 ppm;
FTIR-ATR (neat): n˜ =3626, 3344, 3179, 3079, 3046, 2536, 2121, 1968,
1673, 1640, 1623, 1553, 1521, 1470, 1446, 1378, 1314, 1264,
1233 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd for C11H7N4: 195.0671,
found: 195.0668.
E-3-(4-(3,7-Dimethyloctyl)oxy)styryl)-[1,2,4]triazolo[3,4-a]phthal-
azine (13 f): Compound 13 f was obtained as a yellow powder
(yield: 82%): mp: 1248C; 1H NMR (300 MHz, CDCl3): d=8.75 (bd,
J=7.7 Hz, 1H), 8.71 (s, 1H), 8.18 (d, J=16.5 Hz, 1H), 8.01–7.95 (m,
2H), 7.84 (td, J=7.5 Hz, 1.1 Hz, 1H), 7.63–7.59 (m, 2H), 7.37 (d, J=
16.5 Hz, 1H), 6.95–6.92 (m, 2H), 4.07–4.02 (m, 2H), 1.89–1.20 (m,
10H), 0.96 (d, J=6.4 Hz, 3H), 0.88 ppm (d, J=6.6 Hz, 6H); 13C NMR
(75 MHz, CDCl3): d=160.0, 148.9, 147.3, 142.6, 135.8, 133.9, 130.7,
128.7, 128.5, 128.0, 123.6, 123.2, 123.0, 114.8, 108.1, 66.4, 39.2, 37.3,
36.1, 29.8, 27.9, 24.6, 22.7, 22.6, 19.6 ppm; FTIR-ATR (neat): n˜ =
3060, 3027, 2951, 2918, 2836, 1636, 1602, 1575, 1522, 1468, 1449,
1383, 1363, 1348, 1295, 1242, 1225, 1210 cmÀ1; HRMS (ESI+) m/z
[M+H]+ calcd for C27H33N4O: 429.2654 found: 429.2657.
General procedure for the photoisomerization of (E) to (Z) deriv-
atives 18a–c: A 0.02m CH3CN solution of E-styrylated triazoloph-
thalazine 12a, 12e, or 13a was irradiated by a 350 W halogen
lamp for 50 h. A solution of K2CO3 (1.5 equiv) and KI (1.5 equiv)
was added. After cooling to room temperature, the mixture was
evaporated and the residue was purified by flash chromatography,
eluting with the appropriate eluent, to furnish title compounds
18a–c in pure forms.
Z-3-Styryl-2-([1,2,4]triazolo[3,4-a]phthalazine (18a): Compound
18a was obtained as a yellow oil (yield: 75%): H NMR (300 MHz,
Synthesis of E-4-(2-[1,2,4]triazolo[3,4-a]phthalazin-3-yl)vinyl)ben-
zene-1,2,3-triol (14): BBr3 (1m in CH2Cl2, 3 mL, 9 equiv) was added
dropwise to a suspension of 12j (120 mg, 1.0 equiv) at À788C. The
mixture was allowed to warm to room temperature, and after stir-
ring for 2 days, the mixture was quenched with water (10 mL) and
treated 30 min later with CH2Cl2 (30 mL). After evaporation of the
aqueous layer, title compound 14 was precipitated from CH2Cl2 as
a brown powder in 82% yield (90 mg): mp: 2998C; 1H NMR
(300 MHz, [D6]DMSO); d=9.16 (s, 1H), 8.52 (d, J=7.8 Hz, 1H), 8.25
(d, J=7.7 Hz, 1H), 8.08 (t, J=7.4 Hz, 1H), 7.95 (t, J=7.8 Hz, 1H),
7.75 (d, J=16.4 Hz, 1H), 7.14 (d, J=16.4 Hz, 1H), 6.68 ppm (s, 2H);
13C NMR (75 MHz, [D6]DMSO); d=148.7, 148.0, 146.3, 142.2, 136.2,
135.3, 134.5, 131.3, 129.2, 126.0, 123.2, 122.6, 122.2, 106.7,
106.5 ppm; FTIR-ATR (neat): n˜ =3093, 3060, 3030, 2931, 2851, 1631,
1605, 1535, 1462, 1321, 1228, 1185 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C17H13N4O3: 321.0988, found: 321.0988.
1
CDCl3): d=8.75 (bd, J=8.0 Hz, 1H), 8.63 (s, 1H), 8.01–7.93 (m, 4H),
7.83 (td, J=7.5 Hz, 1.1 Hz, 1H), 7.39–7.33 (m, 3H), 7.12 (d, J=
13.0 Hz, 1H), 6.92 ppm (d, J=16.6 Hz, 1H); 13C NMR (75 MHz,
CDCl3): d=147.6, 147.3, 142.1, 137.9, 135.5, 134.0, 130.9, 130.0,
128.9, 128.2, 128.0, 123.5, 123.4, 123.1, 110.0 ppm; FTIR-ATR (neat):
n˜ =3041, 3012, 2983, 2958, 2886, 2360, 2317, 2157, 1980, 1640,
1627, 1518, 1504, 1468, 1450, 1392 cmÀ1; HRMS (ESI+) m/z [M+H]+
calcd for C17H13N4: 273.1140, found: 273.1141.
Z-3-(4-bromostyryl)-[1,2,4]triazolo[3,4-a]phthalazine (18b): Com-
pound 18b was obtained as a colorless oil (yield: 90%): 1H NMR
(300 MHz, 5% [D1]TFA in CDCl3): d=8.70 (bd, J=7.8 Hz, 1H), 8.64
(s, 1H), 8.00–7.93 (m, 4H), 7.84 (td, J=7.5 Hz, 1.0 Hz, 1H), 7.51–7.48
(m, 2H), 7.01 (d, J=13.1 Hz, 1H), 6.96 ppm (d, J=13.1 Hz, 1H);
13C NMR (75 MHz, CDCl3): d=147.6, 147.3, 142.1, 136.4, 134.3,
134.2, 131.7, 131.3, 131.1, 128.1, 123.5, 123.24, 123.20, 123.1,
110.3 ppm; FTIR-ATR (neat): n˜ =3063, 3020, 2925, 1762, 1725, 1615,
1532, 1450, 1389, 1268, 1232 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd
for C17H12N479Br: 351.0185, found: 351.0235.
Synthesis of 3-ethynyl-[1,2,4]triazolo[3,4-a]phthalazine (15): Et3N
(0.4 mL, 4 equiv) was added to a CH2Cl2 solution (3 mL) of 3-(trime-
thylsilyl)propynoic acid (100 mg, 0.7 mmol, 1 equiv), 1-hydrazi-
nylphthalazine hydrochloride 1 (207 mg, 1.5 equiv), HOBt (104 mg,
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1087
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Z-3-(4-ethoxystyryl)-[1,2,4]triazolo[3,4-a]phthalazine (18c): Com-
pound 18c was obtained as a yellow oil (yield: 55%): 1H NMR
(300 MHz, CDCl3): d=8.72 (bd, J=7.9 Hz, 1H), 8.62 (s, 1H), 8.11–
8.08 (m, 2H), 7.99–7.92 (m, 2H), 7.84–7.79 (m, 1H), 7.01 (d, J=
13.1 Hz, 1H), 6.90–6.87 (m, 2H), 6.79 (d, J=13.1 Hz, 1H), 4.06 (q,
J=7.0 Hz, 2H), 1.41 ppm (t, J=7.0 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=159.6, 148.0, 147.2, 142.0, 137.4, 134.0, 132.0, 130.9,
128.2, 128.0, 123.5, 123.4, 123.2, 114.1, 107.1, 63.4, 14.8 ppm; FTIR-
ATR (neat): n˜ =3060, 3027, 2974, 2849, 1623, 1604, 1515, 1452,
1392, 1347, 1301, 1250, 1217 cmÀ1; HRMS (ESI+) m/z [M+H]+ calcd
for C19H17N4O: 317.1389, found: 317.1398.
versitØ Paul Sabatier for a PhD fellowship. S.K. was supported by
the French Embassy in Kiev (Ukraine), and investigations were
performed within the framework of the Groupement Franco-Uk-
rainien en Chimie MolØculaire (GDRI), funded by the CNRS.
Keywords: antimycobacterial activity · cytotoxicity · organic
synthesis · triazolophthalazines · tuberculosis
[1] Global Tuberculosis Control: WHO Report 2015, World Health Organiza-
tion, Geneva (www.who.int).
[2] M. Stehr, A. A. Elamin, M. Singh, Curr. Top. Med. Chem. 2014, 14, 110–
129.
Biology
[3] C. E. Barry III, H. I. Boshoff, V. Dartois, T. Dick, S. Ehrt, J. Flynn, D. Schnap-
pinger, R. J. Wilkinson, D. Young, Nat. Rev. Microbiol. 2009, 7, 845–855.
[4] The current drug pipeline can be monitored at: www.newtbdrugs.org/
pipeline.php; see also: A. Zumla, P. Nahid, S. T. Cole, Nat. Rev. Drug Dis-
covery 2013, 12, 388–404.
[5] a) P. De, D. Veau, F. Bedos-Belval, S. Chassaing, M. Baltas in “Understand-
ing Tuberculosis—New Approaches to Fighting Against Drug Resist-
ance”, Mycobacterium Tuberculosis/Book 2, InTech Publisher, 2012,
pp. 337–362; b) P. De, K. De, D. Veau, F. Bedos-Belval, S. Chassaing, M.
Baltas, Expert Opin. Ther. Pat. 2012, 22, 155–168; c) P. De, F. Bedos-
Belval, C. Vanucci-BacquØ, M. Baltas, Curr. Org. Chem. 2012, 16, 747–
768.
Growth conditions of M.tb strains and determination of mini-
mum inhibitory concentrations (MICs): M.tb H37Rv was used as
the reference strain, and clinical isolates were grown at 378C in
Middlebrook 7H9 broth (Difco), supplemented with 0.05%
Tween 80, or on solid Middlebrook 7H11 medium (Difco) supple-
mented with oleic acid–albumin–dextrose–catalase (OADC). MIC
values for the new compounds were determined by means of the
micro-broth dilution method. Dilutions of M.tb culture (~105–
106 cfumLÀ1) were streaked onto 7H11 solid medium containing
a range of drug concentrations (0.25 mgmLÀ1 to 40 mgmLÀ1). Plates
were incubated at 378C for about 21 days, and the growth was vis-
ually evaluated. The lowest drug dilution at which visible growth
failed to occur was taken as the MIC value. Results were expressed
as the average of at least three independent determinations.
[6] J. P. Warbasse, Ann. Surg. 1894, 19, 102–111.
[7] N. Rastogi, K. S. Goh, L. Horgen, W. W. Barrow, FEMS Immunol. Med. Mi-
crobiol. 1998, 21, 149–157.
[8] a) V. M. Reddy, G. Nadadhur, D. Daneluzzi, V. Dimova, P. R. J. Gangadhar-
am, Antimicrob. Agents Chemother. 1995, 39, 2320–2324; b) V. Velichka,
A. Ivana, T. Haruaki, S. Katsumasa, R. Venkata, G. Nadadhur, D. Donna, K.
Todor, D. Arvind, F. Yurii, Y. Ljudmila, A. Toumanov, Z. Zvetana, S. Chiaki,
Recent Pat. Anti-Infect. Drug Discovery 2010, 5, 76–90.
[9] a) C. Lapeyre, M. DelomØn›de, F. Bedos-Belval, H. Duran, A. N›gre-Sal-
vayre, M. Baltas, J. Med. Chem. 2005, 48, 8115–8124; b) P. De, M. Baltas,
D. Lamoral-Theys, C. Bruy›re, R. Kiss, F. Bedos-Belval, N. Saffon, Bioorg.
Med. Chem. 2010, 18, 2537–2548.
[10] a) G. K. Yoya, F. Bedos-Belval, P. Constant, H. Duran, M. DaffØ, M. Baltas,
Bioorg. Med. Chem. Lett. 2009, 19, 341–343; b) P. De, G. K. Yoya, P. Con-
stant, F. Bedos-Belval, H. Duran, N. Saffon, M. DaffØ, M. Baltas, J. Med.
Chem. 2011, 54, 1449–1461.
[11] For clarity and simplicity, triazolophthalazine is used in the course of
this article instead of [1,2,4]triazolo[3,4-a]phthalazine, which is the
actual IUPAC name.
[12] For examples of triazolophthalazine derivatives of biological relevance,
see: a) H. M. F. Allah, R. Soliman, J. Heterocycl. Chem. 1987, 24, 667–671;
b) M. Kai, A. Noda, H. Noda, S. Goto, Chem. Pharm. Bull. 1988, 36, 3604–
3608; c) T. Seki, T. Takezaki, R. Ohuchi, H. Ohuyabu, T. Ishimori, K.
Yasuda, Chem. Pharm. Bull. 1994, 42, 1609–1616; d) X. Zhang, A. Amer,
X. Fan, J. Balzarini, J. Neyts, E. De Clercq, M. Prichard, E. Kern, P. F. Tor-
rence, Bioorg. Chem. 2007, 35, 221–232; e) M. N. Drwal, K. Agama,
L. P. G. Wakelin, Y. Pommier, R. Griffith, PLoS ONE 2007, 6, e25150; f) O.
Fedorov, H. Lingard, C. Wells, O. P. Monteiro, S. Picaud, T. Keates, C.
Yapp, M. Philpott, S. J. Martin, I. Felletar, B. D. Marsden, P. Filippakopou-
los, S. Müller, S. Knapp, P. E. Brennan, J. Med. Chem. 2014, 57, 462–476.
[13] Within the context of TB, it is nevertheless worth noting that [1,2,4]tria-
zolo[3,4-a]phthalazine itself is the end product of the hydralazine trans-
formation of M. tuberculosis N-acetyl transferase, an enzyme that active-
ly participates in the increased resistance of the bacilli against INH. For
details, see: A. M. Abuhammad, E. D. Lowe, E. Fullam, M. Noble, E. F.
Garman, E. Sim, Protein Cell 2010, 1, 384–392.
M.tb clinical isolates and drug susceptibility testing:[26] Three
M.tb multidrug-resistant isolates were collected at the Sondalo Di-
vision of the Valtellina and Valchiavenna, Italy, hospital authority in
2012. Their resistance profile is shown in Table 2. All clinical isolates
were grown in BACTEC MGIT 960 and Lowenstein–Jensen slants.
Drug susceptibility testing for all first-line antitubercular drugs was
performed with the BACTEC MGIT 960 System (BectoneDickinson
Diagnostic Systems, Sparks, Maryland) for isoniazid (0.1 mgmLÀ1
0.4 mgmLÀ1), rifampicin (1 mgmLÀ1), streptomycin (1 mgmLÀ1
;
;
4 mgmLÀ1),
ethambutol
(5 mgmLÀ1),
and
pyrazinamide
(100 mgmLÀ1), in accordance with the manufacturer’s instructions.
MIC value determination for second-line drugs (cycloserine,
50 mgmLÀ1; amikacin, 1–5 mgmLÀ1; ciprofloxacin, 2 mgmLÀ1; ethio-
namide, 5 or 10 mgmLÀ1; para-aminosalicylic acid, 4 or 8 mgmLÀ1
;
ofloxacin, 10 mgmLÀ1) was also performed using the MGIT 960
System.
Cytotoxicity: HCT116 human colon cancer cells (ATCC) were cul-
tured in DMEM supplemented with 10% fetal calf serum. For cyto-
toxicity evaluation, 3000 cells were seeded per well in 96-well
plates and, 24 h later, were treated with test compounds at con-
centrations ranging from 50 nm to 100 mm (eight replicates for
each). After 4 days of treatment, cells were incubated with the cell
proliferation reagent WST-1 (Roche) for 3–4 h, and the absorbance
was then measured with a scanning multiwell spectrophotometer.
The measured absorbance directly correlates to the number of
viable cells. Data were analyzed using Excel (Microsoft) and Prism
(GraphPad) software.
[14] K. T. Potts, C. Lovelette, J. Org. Chem. 1969, 34, 3221–3230.
[15] A.-M. M. E. Omar, M. G. Kasem, I. M. Laabota, J. Bourdais, J. Heterocycl.
Chem. 1981, 18, 499–501.
Acknowledgements
[16] K. Wang, W. Xu, W. Zhang, M. Mo, Y. Wang, J. Shen, Bioorg. Med. Chem.
Lett. 2013, 23, 2897–2901.
[17] H. Zimmer, R. Glaser, J. Kokosa, D. A. Garteiz, E. V. Hess, A. Litwin, J. Med.
Chem. 1975, 18, 1031–1033.
We gratefully acknowledge the Centre National de la Recherche
Scientifique (CNRS) and the UniversitØ Paul Sabatier for financial
support. D.V. thanks the French Ministry of Research and the Uni-
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1088
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[18] Notably, the corresponding acid has already been described in the liter-
ature as unstable. For example, see Ref. [14].
[25] C. Menendez, A. Chollet, F. Rodriguez, C. Inard, M. R. Pasca, C. Lherbet,
M. Baltas, Eur. J. Med. Chem. 2012, 52, 275–283.
[26] C. Menendez, F. Rodriguez, A. L. de Jesus Lopes Ribeiro, F. Zara, C. Fron-
gia, V. Lobjois, N. Saffon, M. R. Pasca, C. Lherbet, M. Baltas, Eur. J. Med.
Chem. 2013, 69, 167–173.
[27] Notably, compounds 13c and 13e were previously reported by us in
Ref. [10b].
[28] CCDC 1448411 (5), 1448412 (7), and 1448413 (8) contain the supple-
mentary crystallographic data for this paper. These data are provided
free of charge by The Cambridge Crystallographic Data Centre.
[29] SAINT-NT, Bruker-AXS, Madison, WI (USA), 2000.
[19] A. P. Thottumkara, M. S. Bowsher, T. K. Vinod, Org. Lett. 2005, 7, 2933–
2936.
[20] For examples with bioactive stilbenes, see: a) M. Cushman, D. Nagarath-
nam, D. Gopal, A. K. Chakraborti, C. M. Lin, E. Hamel, J. Med. Chem.
1991, 34, 2579–2588; b) J. A. Woods, J. A. Hadfield, G. R. Pettit, B. W.
Fox, A. T. McGown, Br. J. Cancer 1995, 71, 705–711; c) C. Rius, M. Abu-
Taha, C. Hermenegildo, L. Piqueras, J. M. Cerda-Nicolas, A. C. Issekutz, L.
EstaÇ, J. Cortijo, E. J. Morcillo, F. Orallo, M. J. Sanz, J. Immunol. 2010,
185, 3718–3727.
[30] SADABS, Program for Data Correction, Bruker-AXS, Madison, WI (USA).
[31] G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112–122.
[21] Y.-L. Chen, S.-T. Huang, F.-M. Sun, Y.-L. Chiang, C.-J. Chiang, C.-M. Tsai, C.-
J. Weng, Eur. J. Pharm. Sci. 2011, 43, 188–194.
[22] D. H. Waldeck, Chem. Rev. 1991, 91, 415–436.
[23] As revealed when monitoring the photoconversion by H NMR and TLC.
1
Received: February 7, 2016
Revised: March 13, 2016
[24] C. Menendez, S. Gau, C. Lherbet, F. Rodriguez, C. Inard, M. R. Pasca, M.
Baltas, Eur. J. Med. Chem. 2011, 46, 5524–5531.
Published online on April 21, 2016
ChemMedChem 2016, 11, 1078 – 1089
www.chemmedchem.org
1089
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim