Synthesis of jacaranone-derived nitrogenous cyclohexadienones and their antiproliferative and antiprotozoal activities

Article abstract of DOI:10.3390/molecules23112902

The cytotoxic and antiprotozoal activities of the phytoquinoide, jacaranone, and related compounds have been an ongoing topic in recent drug discovery. Starting from the natural product-derived cyclohexadienone scaffold, a series of nitrogen-containing derivatives were synthesized and subsequently evaluated for their antiproliferative and antiprotozoal activity. Anticancer potency was analyzed using different types of cancer cell lines: MDA-MB-231 breast cancer, CCRF-CEM leukemia, HCT-116 colon cancer, U251 glioblastoma, and, in addition, non-tumorigenic MRC-5 lung fibroblasts. Antiproliferative activities at micromolar concentrations could be shown. Antiprotozoal activity was assessed against Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STIB900. For all compounds, selectivity indices (SI) were calculated based on assessed cytotoxicity towards L6 cells. In addition, the structure-activity-relationships and physicochemical parameters of these compounds are discussed.

Full text of DOI:10.3390/molecules23112902

molecules
Article
Synthesis of Jacaranone-Derived Nitrogenous
Cyclohexadienones and Their Antiproliferative and
Antiprotozoal Activities
Armin Presser 1,
*
, Gunda Lainer 1, Nadine Kretschmer 2, Wolfgang Schuehly 2, Robert Saf 3,
Marcel Kaiser 4,5 and Marc-Manuel Kalt 1
1
Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, Schubertstrasse 1,
8010 Graz, Austria; gunda.lainer@edu.uni-graz.at (G.L.); marc.kalt@edu.uni-graz (M.-M.K.)
Institute of Pharmaceutical Sciences, Pharmacognosy, University of Graz, Universitaetsplatz 4,
2
8010 Graz, Austria; nadine.kretschmer@uni-graz.at (N.K.); wolfgang.schuehly@uni-graz.at (W.S.)
Institute for Chemistry and Technology of Materials (ICTM), Graz University of Technology,
3
Stremayrgasse 9, 8010 Graz, Austria; robert.saf@tugraz.at
Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland;
4
marcel.kaiser@swisstph.ch
University of Basel, Petersplatz 1, 4003 Basel, Switzerland
5
*
Correspondence: armin.presser@uni-graz.at; Tel.: +43-316-380-5369
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Claudio Santi and Luca Sancineto
Received: 26 September 2018; Accepted: 29 October 2018; Published: 7 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The cytotoxic and antiprotozoal activities of the phytoquinoide, jacaranone, and related
compounds have been an ongoing topic in recent drug discovery. Starting from the natural
product-derived cyclohexadienone scaffold, a series of nitrogen-containing derivatives were
synthesized and subsequently evaluated for their antiproliferative and antiprotozoal activity.
Anticancer potency was analyzed using different types of cancer cell lines: MDA-MB-231 breast cancer,
CCRF-CEM leukemia, HCT-116 colon cancer, U251 glioblastoma, and, in addition, non-tumorigenic
MRC-5 lung fibroblasts. Antiproliferative activities at micromolar concentrations could be shown.
Antiprotozoal activity was assessed against Plasmodium falciparum NF54 and Trypanosoma brucei
rhodesiense STIB900. For all compounds, selectivity indices (SI) were calculated based on assessed
cytotoxicity towards L6 cells. In addition, the structure-activity-relationships and physicochemical
parameters of these compounds are discussed.
Keywords: antiproliferative activity; antiprotozoal activity; green chemistry; natural products;
jacaranone
1. Introduction
Natural products (NPs) play vital roles in drug discovery. More than half of the drugs that
have been approved over the past 30 years are natural compounds or compounds based on these [
Approximately 68% of anti-infectives are classified as nature-derived or inspired, and 80% of all
anticancer compounds fall into this category [ ]. Examples of well-known drugs derived from
1].
2
natural products are paclitaxel or doxorubicin (anticancer), artemisinin (antimalarial), daptomycin
(antibacterial), and morphine (analgesic). The most striking feature of many natural products is their
structural diversity, which is still largely untapped. About 40% of the chemical scaffolds found in NPs
are still absent in today’s medicinal chemistry [
been described in a number of reviews and reports [2,4–10].
Jacaranone ( ) and its derivatives, phytoquinoids isolated from several Jacaranda and Senecio
species, exhibited promising pharmacophore qualities in previous investigations. The remarkable
3]. The importance of NPs in drug development has
1
Molecules 2018, 23, 2902; doi:10.3390/molecules23112902
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2902
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cytotoxic and antiprotozoal properties of jacaranone have been especially well-studied both in vitro
and in vivo 11 14]. Related nitrogenous NPs (e.g., verongiaquinol ( ) or melodamide A ( )) are
[
–
2
3
also valuable drug candidates that possess antiproliferative, antibiotic, antiviral, antiprotozoal,
and anti-inflammatory activities [15–20].
Herein, we report the design and synthesis of jacaranone-inspired N-containing cyclohexadienones
and our findings on the antiproliferative, antiplasmodial, and antitrypanosomal activities of these
NP-derived quinols (Figure 1).
Figure 1. Naturally occurring quinols with remarkable biological activity.
2. Results and Discussion
2.1. Chemistry
Imide derivatives are a valuable group of bioactive compounds. In spite of their wide applicability,
available procedures for their synthesis are limited [21]. During the course of our work on jacaranone
imides, we searched for an efficient method that we could use to construct the N-dienone scaffold.
During our initial attempts, we followed a Mitsunobu route [22] as summarized in Scheme 1
(method A). Starting from the commercially available compound, methyl 4-hydroxyphenyl acetate (
4),
the temporarily protected alcohol was obtained in two steps in almost quantitative yields.
5
The introduced thexyldimethylsilyl (TDS) group is superior in comparison with other commonly
used silyl protecting strategies because of its greater stability and ease of handling [23].
Subsequently, the intermediates 6a–c were prepared from TDS ether 5 under Mitsunobu conditions
and then treated with an excess amount of tetrabutylammonium fluoride (TBAF) [24] to afford the
unprotected imides 7a–c in moderate overall yields (58–71%).
Walker [25] reported that yields of the crucial Mitsunobu reaction could be increased by simply
altering the order in which the reagents were combined. Although we observed these instructions,
following the Mitsunobu route still did not allow us to produce a wide range of imide derivatives.
Therefore, we focused on tyramine (
material for our imide design. A previously described method [26] used
condensation with phthalic anhydride in refluxing acetic acid. Following this published protocol as
shown in Scheme 1, a series of cyclic imides (7a ) were synthesized in generally good to excellent
yields (method B). Furthermore, tyramine has an advantage in that it is significantly more reactive than
the formerly used alcohol , and the tedious process of protecting the phenolic group is not necessary.
8) as a commercially available and more suitable starting
8
for the chemoselective
–
j
5
Interestingly, the preparation of 7f and 7g failed using this procedure, perhaps due to its basicity (7f
)
or hydrolytic degradation (7g).
Recently, we have become interested in the development of synthetic methods using polyethylene
glycol (PEG) as a novel, environmentally and industrially friendly medium and promoter [27].
It is known that PEG can act as an excellent reaction medium for the synthesis of N-alkyl and
N-arylphthalimides [28]. For this reason, we investigated whether the substitution of the established
solvent acetic acid (method B) by the nontoxic, inexpensive, nonionic liquid PEG 400 (method C) was
a viable alternative procedure for the condensation of various anhydrides with 8.

Molecules 2018, 23, 2902
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OH
OTDS
OTDS
i
ii
H
N
O
O
O
O
OH
X
4
5
6a-c
iii
OH
OH
O
iv
O
v
O
O
HO
NH2
X
X
8
7a-j
13a-j
O
O
O
N
O
a X =
N
O
O
N
O
b
N
O
c
X =
X =
O
N
O
O
Cl
Cl
Cl
Cl
N
f X =
N
O
d X =
e X =
O
O
h X =
g X =
N
O
N
O
O
N
O
O
O
i X =
N
O
j X =
Scheme 1. Reagents and conditions: meth◦od A: (i) 1. TDSCl, DBU, RT, 1.5 h; 2. LiAlH4, THF, 0 ◦C,
2 h (98%); (ii) imide, PPh3, DIAD, THF, 0 C
CH2Cl2, RT, 1 h (7a: 80%, 7b: 85%, 7c: 82%); (iv) anhydride, AcOH, 120 C, 1.5 h (method B: 7a: 73%,
7b: 55%, 7c: 58%, 7d: 89%, 7e: 90%, 7f: 0%, 7g: 0%, 7h: 86%, 7i: 92%, 7j: 79%) or PEG 400, 140 C, 4 h
→
RT, 2.5–24 h (6a: 90%, 6b: 70%, 6c: 77%); (iii) TBAF,
◦
◦
(method C: 7a: 23%, 7b: 51%, 7c: 98%, 7d: 67%, 7e: 67%, 7f: 86%, 7g: 71%, 7h: 79%, 7i: 98%, 7j: 98%);
◦
(v) PhI(OAc)2, CH3CN/H2O (12:5), 0 C, 7 min (13a: 67%, 13b: 17%, 13c: 55%, 13d: 40%, 13e: 64%, 13f
:
19%, 13g: 18%, 13h: 79%, 13i: 88%, 13j: 49%.
Initially, the modified procedure provided only poor yields because PEG acted as a solubilizer.
This led to difficulties during the workup of the highly water-soluble imides. By referring to a
PEG-assisted solvent and catalyst-free synthesis of 3,4-dihydropyrimidinones [29], we significantly

Molecules 2018, 23, 2902
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decreased the amount of PEG used in our synthesis. In fact, the results obtained by modifying the
method demonstrated that we could considerably increase the product yield in most cases, including
the cases of the elusive imides, 7f and 7g.
Overall, upon comparing the yields achieved with methods A and B/C, respectively, we could
clearly demonstrate the great advantage of using tyramine (
8) as a starting material to prepare the cyclic
imides, 7a , as well as the potential of using PEG as an excellent reagent to promote organic reactions.
–j
Next, we turned our attention to the preparation of the jacaranone-derived amines. N-alkyl
amines can be obtained by the catalytic amination of alcohols [30] or by the reaction of the respective
amine with alkyl halides and an auxiliary base [31]. Heterocyclic N-imides have also been smoothly
converted into the corresponding amines with LiAlH4 in excellent yields [32,33]. This route was
initially adopted for the synthesis of amines 11a
–d
from the previous synthesized imides (Scheme 2,
method D).
◦
Scheme 2. Reagents and conditions: (i) Pyrrolidine, [Cp*IrCl2]2 (5 mol% Ir), NaHCO3, toluene, 110 C,
25 h (method E: 10b: 45%); (ii) PPh3, DDQ, TBAB, CH2Cl2, RT, 50 min (81%); (iii) secondary amine, NaI,
proton sponge®, CH3CN, 20 h (method F: 10a: 72%, 10b: 87%, 10c: 87%, 10d: 94%); (iv) LiOH, DMF,
RT, 3–17 h (11a: 84%, 11b: 93%, 11c: 100%, 11d: 82%); (v) ammonium formate, Pd(OH)2/C, MeOH,
◦
reflux, 16 h (100%); (vi) LiAlH4, THF, 0 C, 90 min (method D: 11a: 72%, 11b: 0%, 11c: 0%, 11d: 0%, 12
:
◦
88%); (vii) PhI(OAc)2, CH3CN/H2O/phosphate buffer (12:3:2), pH = 6.4, 0 C, 7 min (14a: 16%, 14b
0%, 14c: 28%, 14d: 0%, 14e: 0%); (viii) PhI(OAc)2, CH3CN/H2O (12:5), 0 C, 7 min (15: 65%).
:
◦

Molecules 2018, 23, 2902
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Watson et al. [32] showed that the reduction of the phthalimide functionality might be problematic
due to the comparatively facile oxidation of the emerging isoindoline to the respective isoindole.
Surprisingly, the conversion of phthalimide 7a with LiAlH4 gave essentially the isoindoline, 11a
in good yields, whereas the reduction to the amines, 11b , failed. In contrast, the partially reduced
derivative, 12, was exclusively obtained when 7h was treated with LiAlH4.
,
–d
As an alternative route to the desired jacaranone-derived amines, we examined the frequently
used iridium-catalyzed alkylation of alcohols [34] (method E). However, treatment of the primary
alcohol, 5, with pyrrolidine in the presence of [Cp*IrCl2]2 [35] delivered 10b in only moderate yields;
thus, this synthetic route was abandoned.
Finally, we applied the traditional amination of alkyl halogenides for the preparation of jacaranone
amines (method F). For this purpose, alcohol
(TBAB), PPh3, and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) [36] to the corresponding bromide
This intermediate was then coupled with the appropriate secondary amine in the presence of NaI and
proton sponge® [37] to obtain the amines, 10b
, in good to excellent yields. Only the N-alkylation
5 was first converted with tetrabutyl ammonium bromide
9.
–
d
of isoindoline (10a) led to slightly lower yields under these conditions. Deprotection of the obtained
amines, 10a–d, with LiOH in DMF [38] was successful, resulting in the desired derivatives, 11a–d.
Previous studies have demonstrated that isoindolines can be readily converted to
tetrahydroisoindoles by palladium hydroxide-catalyzed hydrogenation [39,40]. To broaden the range
of usable amines, we examined the reported procedure and observed a quantitative conversion of 11a
to the expected tetrahydroisoindole, 11e.
Finally, we investigated the oxidative dearomatization of the synthesized N-containing
phenols into the respective p-alkyl quinols. Such cyclic dienones exhibit not only promising
pharmacophores [3,41], but are also attractive intermediates for enantioselective natural product
synthesis [42
–
44]. Phenol dearomatization processes are generally mediated by hypervalent iodine
45]. The commonly used protocol using
reagents, and are well-documented in the literature [43
,
phenyliodine(III) diacetate (PIDA) in aqueous CH3CN [46] appeared to be most suitable for
our purpose.
The conversion of the compounds, 7a
cyclohexadienones, 13a and 15, in satisfactory yields, whereas the oxidation of all tertiary amines
11a ) failed. To improve the rate of conversion for these compounds, we examined the impact
–j and 12, with PIDA afforded the respective p-substituted
–
j
(
–e
of varying the pH values on the reaction outcome. The best results were obtained applying a
1 M phosphate buffer that adjusted the pH to 6.4 during the oxidation. This variation led to the
availability of a few additional tertiary jacaranone amines (14a,14c), but the yields still remained far
from being satisfactory.
Subsequently, the synthesized dienones were evaluated for their antiproliferative and
antiprotozoal activity, following the method described in the Experimental section.
2.2. Physicochemical Properties
Physicochemical parameters play crucial roles in the selection process of drug candidates for
product development. The properties of small molecules, especially those that are orally bioavailable,
are concentrated in a relatively narrow range of physicochemical space known as the “drug-like
space” [47]. An optimal lipophilicity range, along with low molecular weight and small polar
surface area, are major prerequisites that lead to good absorption of chemicals by the intestine
through passive diffusion [48,49]. For this reason, an assessment of drug-likeness was made,
and various physicochemical properties were calculated for all tested compounds (Table 1 and
Supplementary Materials).

Molecules 2018, 23, 2902
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All compounds had relatively low molecular weights within the range of 223–352 g
Their polar surface areas are low and well within the range where good central nervous system (CNS)
penetration is plausible [50 51]. The latter parameter is especially important to treat the CNS-persistent
·
mol−1
.
,
second stage of human African trypanosomiasis (HAT) [52]. All derivatives fulfill the Lipinski rule
of five [53] and the Veber rule [54]. Except for imides, 13c and 13g, the logP data of which lie slightly
outside the proposed region, all mentioned compounds also comply with the drug-likeness classifier
defined by Ghose et al. [55].
The adequate application of ligand efficiency (LE) metrics is of utmost relevance in guiding
lead discovery—and, more importantly, lead optimization—towards drug-like chemical space [56
The ligand efficiency metrics of our synthesized compounds (LE > ~0.3), lipophilic ligand efficiency
(LLE) (LLE > ~5), and lipophilicity-corrected ligand efficiency (LELP) ( 10 < LELP < 10) agree closely
,57].
−
with the values proposed for drug candidates [56] (see Supplementary Materials, Table S3).
In addition, all synthesized dienones were subjected to the BOILED-Egg analysis [58
,
59],
an improvement upon the well-known Egan egg model [60]. This tool utilizes the computed
lipophilicity and polarity of small drugs as input, allowing researchers to predict brain and intestinal
permeation efficacies. This model is frequently used in industrial and academic contexts for drug
discovery and development. All computed substances are within the thresholds of the model and,
therefore, are predicted to show good gastrointestinal absorption. Furthermore, compounds, 13d and
14a, also lie within the physicochemical space of molecules that have a high probability of permeating
the blood-brain barrier (BBB) (see Supplementary Materials, Figure S1).
2.3. Biological Evaluation
2.3.1. Antiproliferative Activity
All synthesized dienones (13a–j, 14a, 14c, and 15) were evaluated for their cytotoxic activity
against human (cancer) cell lines. To cover a range of different tumor entities, we used a panel
of cancer cell lines (CCRF-CEM leukemia, MDA-MB-231 breast cancer, HCT-116 colon cancer,
and U251 glioblastoma cells) as well as a non-tumorigenic human cell line (MRC-5 lung fibroblasts).
All derivatives were screened for their cytotoxicity at 5 µg/mL and 50 µg/mL to cover a broad
concentration range. Cells were exposed to the derivatives for 72 h. As can be seen in Figure 2,
the highest cytotoxicity was found for 13b and 13i. Both compounds reduced the metabolic activity
below 40% of the control at 5
all tested compounds displayed activities against leukemia and breast cancer cells lines, but the
overall cytotoxicity of compounds 13f 13g 13j, and 14a was negligible. Furthermore, the 50 g/mL
dilution corresponds to concentrations in the 140–215 M range, which is not considered to be active.
Interestingly, the morpholine derivative, 14c, displayed only minor cytotoxicity against most cell lines
at 5 g/mL, but subsequently exhibited the highest antitrypanosomal activity. No clear correlation
between cytotoxic and antiprotozoal effects could be drawn. Against human MRC-5 cells, 13f 13g 13j
and 14a did not show any cytotoxicity at 5 g/mL and only a weak activity at 50 g/mL. These results
µg/mL in all (13i) or almost all (13b) cell lines. At 50 µg/mL, almost
,
,
µ
µ
µ
,
,
,
µ
µ
essentially match those of the subsequent L6 cytotoxicity assay, which was performed during the
antiprotozoal screening. In that series, the highest IC50 values (and, therefore, the lowest cytotoxicity)
were found for compounds 13b, 13f, and 13g (for detailed results, see Supplementary Materials,
Table S4).

Molecules 2018, 23, 2902
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Figure 2. Results of the XTTa viability assay using leukemia (CCRF-CEM), breast cancer
(MDA-MB-231), colon cancer (HCT-116), and glioblastoma cells (U251) as well as non-tumorigenic
lung fibroblasts (MRC-5). Cells were treated with 5 g/mL ( ) or 50 g/mL ( ) of the derivatives
for 72 h. Afterwards, the metabolic activities of the cells were measured. Vinblastine (VBN) served
µ
A
µ
B
as the positive control (0.01
(0.5% DMSO) control cells (ctrl) (mean
2H-tetrazolium-5-carboxanilide).
µ
g/mL). The results are expressed as a percentage of vehicle-treated
a
±
s.e.m., n = 6). (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2.3.2. Antiprotozoal Activity
The synthesized dienones were also investigated for their activity against P. falciparum NF54 and
T. brucei rhodesiense STIB900 as well as for their cytotoxicity against L6 rat skeletal myoblasts (Table 1).
For each parasite, a selectivity index (SI = IC50(L6)/IC50(parasite)) was calculated. The TDR (Special
Program for Research and Training in Tropical Diseases, World Health Organization) criteria [61] were
adopted to interpret antiparasitic activity and selectivity.
All derivatives showed moderate (IC50 = 1–10
T. brucei rhodesiense, except the inactive pyridine-2,3-dicarboximide, 13f, and the morpholine-3,5-dione,
13g, with IC50 values >10 M. These results were surprising, especially in the case of compound
µM) or high (IC50 < 1 µM) activity towards
µ
13f, as basic, nitrogen-containing compounds often show favorable activities against protozoal
parasites. Interestingly, the corresponding morpholine derivative, 14c, showed, in contrast, the highest
antitrypanosomal activity (IC50 = 0.27
µM) of all the tested N-dienones. Compared to the
antitrypanosomal activity, the antiplasmodial effects of all tested compounds against P. falciparum
NF54 strains were rather weak. Notable in this series, unfortunately, was the lack of selectivity of most
compounds, as the compounds showed selectivity indices (SI) of <13.

Molecules 2018, 23, 2902
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Table 1. In vitro antiparasitic activity, host toxicity, and key physicochemical properties of tested compounds.
a
c
SI b
SI b
Cyt. L6 d
Compd.
P. falc.
T.b.rhod.
Chemical Structure
logP
tPSA (7.4)
IC50 (µM)
IC50 (µM)
IC50 (µM)
Chl.
Mel.
Pod.
0.002
0.004
0.007
5.08
13a
3.78
1.3
1.58
3.2
0.85
74.68
13b
13c
4.42
8.63
6.1
1.1
2.15
6.93
12.5
1.4
26.80
9.90
−0.18
−0.60
74.68
74.68
13d
13e
3.24
1.9
0.1
5.71
2.26
1.1
4.5
6.28
1.89
74.68
74.68
74.38
10.26
−0.38

Molecules 2018, 23, 2902
9 of 22
Table 1. Cont.
a
c
SI b
SI b
Cyt. L6 d
Compd.
P. falc.
T.b.rhod.
Chemical Structure
logP
tPSA (7.4)
IC50 (µM)
IC50 (µM)
IC50 (µM)
13f
13g
13h
28.04
5.5
44.75
3.4
153.48
77.54
2.96
−0.06
87.57
11.82
3.04
6.6
1.0
15.32
1.70
5.1
1.7
−1.13
83.91
74.68
0.81
13i
13j
1.28
3.93
2.7
2.0
0.80
0.97
4.4
8.3
3.52
8.04
0.66
0.55
74.68
74.68

Molecules 2018, 23, 2902
10 of 22
Table 1. Cont.
a
c
SI b
SI b
Cyt. L6 d
Compd.
P. falc.
T.b.rhod.
Chemical Structure
logP
tPSA (7.4)
IC50 (µM)
IC50 (µM)
IC50 (µM)
14a
14c
15
2.91
1.0
0.57
4.9
2.81
3.46
1.89
41.74
10.93
6.18
0.3
2.0
0.27
1.52
12.9
8.3
0.04
0.58
49.77
77.84
12.63
a
P. falciparum, strain NF54, erythrocytic stages; b SI is the ratio: IC50 in L6 cells/IC50 in each parasite; c T. brucei rhodesiense, strain STIB900 trypomastigote forms; d cytotoxicity L6 cells rat
skeletal myoblasts. Reference drugs: Chloroquine (chl., IC50 = 0.002 0.001), melarsoprol (mel., IC50 = 0.004 0.003), podophyllotoxin (pod., IC50 = 0.007 0.002). The IC50 value of each
±
±
±
reference drug is the mean from multiple measurements taken in parallel with the compounds of interest. IC50 values of the tested compounds are means of two to three independent
assays. The individual IC50 values within each assay varied <25%. The physical properties were predicted by using Marvin 18.10.0, ChemAxon (https://www.chemaxon.com).

Molecules 2018, 23, 2902
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2.3.3. Structure-Activity Relationships (SAR) of the Antiproliferative and Antiprotozoal Activity
The mechanism of action of compounds with quinoid structural elements is based on redox cycling
with excessive generation of reactive oxygen species (ROS) in the intracellular environment [62].
ROS play central roles in cell signaling and are able to activate the intrinsic pathway of cell
apoptosis [63]. It is expected that these processes are responsible for the cytotoxic action of quinoids on
microorganisms, as well as on tumor cells [64]. Due to the nature of the presented compounds, target
specific effects are few and far between. For instance, the antiproliferative effects of the phytoquinoid,
jacaranone, are caused by its interactions with the protein kinase B (AKT) and mitogen-activated
protein kinase (p38 MAPK) signaling pathways [13].
Nevertheless, a closer examination of the SARs of jacaranone-based nitrogenous cyclohexadienones
with regard to their antiproliferative activity (Figure 2) suggests that the most potent compounds, 13b
13e, and 13i, share an α,β-unsaturated imide as a core structural element (Figure 3).
,
Figure 3. Crucial scaffold for antiproliferative activity.
These compounds showed high activity levels against some (13b: CCRF-CEM, MDA-MB-231,
HCT-116; 13e: CCRF-CEM) or all (13i) tested cell lines at low concentrations. Compound 13b
also showed a comparably low cytotoxicity against L6 cells with an IC50 of 26.8
µM, hinting
that it has a target-specific inhibitory effect. In contrast, pyridine-2,3-dicarboximide, 13f, and the
morpholine-3,5-dione, 13g, exhibited neither cytotoxic nor antiprotozoal activity.
Unfortunately, our antiprotozoal assays revealed no specific effects. However, a certain rank
correlation between antiplasmodial activity and logP (rS =
on one side and antitrypanosomal activity and ASApol (rS = 0.870) on the other side was observed
(see also Supplementary Materials, Table S2).
−
0.772) or ASApho values (rS =
−
0.711)
3. Experimental Section
3.1. Chemicals and Instruments
Melting points were obtained on a digital melting point apparatus (Electrothermal IA 9200,
Staffordshire, UK). The NMR spectra were measured on a Unity Inova 400 MHz instrument (Varian,
Darmstadt, Germany) and a Avance III 300 MHz NMR Spectrometer (Bruker, Rheinstetten, Germany)
◦
at 25 C using 5 mm tubes. Chemical shifts were given in parts per million (ppm), the tetramethylsilane
(TMS) resonance (0.00 ppm) was used as an internal standard. Coupling constants (J) were reported in
1
1
hertz (Hz). H and 13C-resonances were assigned using 1H,1H, and 1H,13C correlation spectra. H and
13C resonances are numbered as given in the formulae (see Supplementary Materials).
High-resolution EI mass spectra (70 eV, source temperature 220 ◦C) were recorded on an
orthogonal TOF spectrometer (Waters GCT Premier, Milford, MA, USA) equipped with a direct
insertion (DI) probe. Typically, 0.2 µL of a solution of the sample (c = 0.1 mg/mL) were placed
in the glass cup used for DI, dried under atmospheric pressure, and transferred into the vacuum.
Mass spectra (50–800 Da; 1 spectrum/s; resolution appr. 7500 FWHM) were continuously acquired
while the sample was evaporated rapidly. ESI mass spectra were acquired on an Exactive Orbitrap mass
spectrometer equipped with a heated ESI II source (ThermoFisher Scientific, Inc., Bremen, Germany).
HPLC separations were performed on an Agilent HPLC instrument 1200 series (Santa Clara,
CA, USA) with quaternary pump, autosampler, autoinjector, column oven, and DAD detection.
A Eurospher C18 column (particle size 1.8
µm; 2.0
×
125 mm with guard cartridge) (Knauer, Berlin,

Molecules 2018, 23, 2902
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Germany) was used for analysis of the compounds at a flow rate of 150
µ
L/min and at a constant
◦
temperature of 25 C. The chromatographic method was performed with a gradient of acetonitrile (A)
in millipore water (B), both with each 0.1% HCOOH, from 10% to 90% A in B within 20 min, then to
100% A within 5 min, followed by returning to starting conditions within 1 min, and re-equilibration
for 8 min. 5.0 µL of sample dissolved in methanol were injected and detection was done at 205, 220,
and 254 nm. As assayed through HPLC-DAD analysis, all tested compounds possessed a purity higher
than 95%.
Materials: TLC was carried out on TLC plates (silica gel 60 F254 0.2 mm, 200
Darmstadt, Germany). TLCs were visualized by spraying with cerium(IV) sulfate/ammonium
molybdate and subsequent heating with a heat gun. The phosphate buffer (1 M, pH = 6.4) was prepared
×
200 mm) (Merck,
as follows: 7.1 g (0.05 mmol) Na2HPO4 and 6.9 g (0.05 mmol) NaH2PO4
×
H2O was dissolved in
H2O and diluted to 100 mL with the same solvent. The pH was controlled using a pH-meter and,
if necessary, adjusted to a pH of 6.4. Solvents were concentrated by rotary evaporation below 50 ◦C.
Purity and homogeneity of compounds were assessed by the TLC and HPLC methods.
The intermediates, 6a–c, were prepared via Mitsunobu reaction according to the literature [22], and
the subsequent deprotection step was accomplished with TBAF according to the literature [24]. Proton
sponge® (1,8-bis(dimethylamino)naphthalene) and other chemicals were purchased from Sigma-Aldrich
(Vienna, Austria). All reagents and chemicals were used without any further purification.
3.2. Synthesis
2-[4-(Thexyldimethylsilyloxy)phenyl]ethanol (5). To a stirred solution of methyl 4-hydroxyphenylacetate
4
(1.2 g, 7.2 mmol) in anhydrous CH2Cl2 (12 mL), 1.3 mL 1,8-diazabicyclo[5.4.0]undec-7-ene (8.7 mmol)
and 1.6 mL thexyldimethylsilyl chloride (8.0 mmol) were added successively at 0 ◦C. The mixture
was allowed to reach ambient temperature until TLC showed complete consumption of the starting
material (1.5 h). The reaction mixture was diluted with H2O and extracted three times with EtOAc.
The combined organic layers were dried over Na2SO4 and evaporated to dryness. The crude
product was purified by flash chromatography using cyclohexane (CH)/EtOAc (3:1) to obtain
2-[4-(thexyldimethylsilyloxy)phenyl]-acetate in quantitative yield as a colourless oil.
The residual oil was dissolved in 12 mL of anhydrous THF and then slowly treated with 7.2 mL
LiAlH4 (1 M solution in THF) at 0 ◦C. After the addition of LiAlH4 was completed, the reaction was
allowed to reach ambient temperature for 2 h. A 2 M aqueous solution of Na/K tartrate (40 mL) was
slowly added at 0 ◦C to quench the reaction and the mixture was stirred for 1 h. Then, the solution
was extracted three times with MTBE, the combined organic layers were dried over Na2SO4, and
concentrated in vacuo to yield 2.0 g (98%) of crude
5
as a clear, colourless oil, which was used without
7.08 (d, J = 8.5 Hz, 2H,
further purification. Rf = 0.27 (CH:EtOAc = 2:1); 1H-NMR (400 MHz, CDCl3)
δ
H-2/6), 6.78 (d, J = 8.5 Hz, 2H, H-3/5), 3.82 (t, J = 6.5 Hz, 2H, H-8), 2.80 (t, J = 6.5 Hz, 2H, H-7), 1.73
(hept, J = 6.9 Hz, 1H, CH-(CH3)2), 0.94 (d, J = 6.9 Hz, 6H, (CH3)2-CH), 0.94 (s, 6H, (CH3)2-C), 0.21
(s, 6H, (CH3)2-Si) ppm; 13C-NMR (100 MHz, CDCl3)
δ 154.1 (C-4), 130.8 (C-1), 129.9 (C-2/6), 120.2
(C-3/5), 63.8 (C-8), 38.4 (C-7), 34.1 (CH-(CH3)2), 25.0 (C-(CH3)2), 20.1 ((CH3)2-C), 18.6 ((CH3)2-CH),
−2.5 ((CH3)2-Si) ppm; HRMS (ESI) calcd. for C16H29O2Si [M + H]+ = 281.1937; Found: 281.1931.
3.2.1. General Procedure for the Synthesis of the Compounds, 7a–7j
AcOH-assisted condensation. A mixture of tyramine
8 (274 mg, 2.0 mmol) and the corresponding
anhydride (1.9 mmol) in glacial acetic acid (3 mL) were refluxed for 1.5 h. After cooling of the reaction
mixture to ambient temperature, cold H2O (10 mL) was added and the resultant precipitate was
filtered, washed several times with cold water, and dried under reduced pressure (7a
, 7e). When the
product did not precipitate from the solution (7b 7h ), H2O (15 mL) was added, and the aqueous
–d
,
–j
phase was extracted several times with EtOAc. The combined organic layers were washed with 1 M
NaHCO3, dried over Na2SO4, and concentrated in vacuo to yield the crude products, which were used
without further purification.

Molecules 2018, 23, 2902
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PEG 400-assisted condensation. A mixture of tyramine
anhydride (1.9 mmol) in PEG 400 (0.3 mL) was heated under stirring at 140 C for 4 h. After cooling to
ambient temperature, a large quantity of ice-water (~30 mL) was added. The resultant precipitate was
8
(274 mg, 2.0 mmol) and the corresponding
◦
filtered, washed several times with cold water, and dried under reduced pressure (7a
, 7e). For 7b–d
and 7f , the aqueous phase was extracted several times with EtOAc, the combined organic layers
–j
were dried over Na2SO4, and evaporated to dryness. The crude products were purified by flash
chromatography (7a–c and 7e–j) or recrystallization from EtOAc/acetone/EtOH (30:5:5) (7d).
4-(Thexyldimethylsilyloxy)phenethyl bromide (
anhydrous CH2Cl2 (10 mL), DDQ (1.1 g, 5.0 mmol) was added slowly at room temperature. Then,
(n-butyl)4NBr (1.6 g, 5.0 mmol) and 1.2 g (4.2 mmol) of alcohol (dissolved in an additional 5 mL of
anhydrous CH2Cl2) were added in 10 min intervals to the thick, beige-coloured mixture. After the
addition of , the colour of the reaction mixture immediately changed to deep red. The reaction
9). To a stirred solution of PPh3 (1.3 g, 5.0 mmol) in
5
5
was stirred for 50 min at an ambient temperature until TLC showed complete consumption of the
starting material. The solvent was evaporated to dryness, and the crude product was purified by flash
chromatography using CH/EtOAc (1:1) to obtain 1.4 g (81%) of
9 as yellow oil. Rf = 0.70 (CH:EtOAC
= 1:1); 1H-NMR (400 MHz, CDCl3)
δ
7.05 (d, J = 8.4 Hz, 2H, H-2/6), 6.77 (d, J = 8.5 Hz, 2H, H-3/5),
3.52 (t, J = 7.8 Hz, 2H, H-8), 3.08 (t, J = 7.8 Hz, 2H, H-7), 1.72 (hept, J = 6.9 Hz, 1H, CH-(CH3)2),
0.94 (d, J = 6.9 Hz, 6H, (CH3)2-CH), 0.94 (s, 6H, (CH3)2-C), 0.21 (s, 6H, (CH3)2-Si) ppm; 13C-NMR
(100 MHz, CDCl3)
δ 154.4 (C-4), 131.5 (C-1), 129.6 (C-2/6), 120.2 (C-3/5), 38.8 (C-7), 34.1 (CH-(CH3)2),
33.3 (C-8), 25.0 (C-(CH3)2), 20.1 ((CH3)2-C), 18.6 ((CH3)2-CH),
for C16H27SiOBr [M]+ = 342.1014; Found: 342.1017.
−
2.5 ((CH3)2-Si) ppm; HRMS (EI) Calcd.
3.2.2. General Procedure for the Synthesis of the Compounds, 11a–11d
A. Nitrogen alkylation
Conventional method. 6 mmol of the respective secondary amine (11a: isoindoline, 11b: pyrrolidine,
11c: morpholine, 11d: octahydroisoindole) were dissolved in anhydrous EtOH (3 mL). Then, 206 mg
(0.6 mmol) of 9 (dissolved in 1 mL anhydrous EtOH) were added and the mixture was refluxed for 72 h.
The solvent was evaporated to dryness to give a residue (10a–10d), which was used in the following
deprotection step without further purification.
Proton-sponge® method. 275 mg (0.8 mmol) of
9 (dissolved in 4 mL anhydrous CH3CN) were mixed with
a stirred solution of 120 mg (0.8 mmol) of NaI and 171 mg (0.8 mmol) of proton-sponge® in anhydrous
CH3CN (3 mL). Then, 1.6 mmol of the respective secondary amine were added and the mixture was
refluxed for 20 h. The solvent was evaporated to dryness to give a residue (10a–10d), which was used
in the following deprotection step without further purification.
B. Removal of the TDS-protecting group
LiOH-hydrate (126 mg, 3.0 mmol) was added to a solution of the TDS ether (10a–d, 1 mmol) in
anhydrous DMF (2 mL) and the mixture was stirred at ambient temperature until TLC showed
complete consumption of the starting material (3–17 h). The reaction mixture was then diluted with
H2O (15 mL), neutralised with phosphate buffer (pH = 6.4), and extracted several times with EtOAc.
The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give a residue,
which was purified by flash chromatography.
N-(4-Hydroxyphenethyl)isoindoline (11a). Compound 11a was prepared from
9 via 10a as a white
amorphous solid and purified by flash chromatography using CH/EtOAc (1:3). Yield: 60%
(proton-sponge® method), 0% (conventional method); Rf = 0.30 (CH:EtOAC = 1:3); 1H-NMR (400 MHz,
DMSO-d6)
2H, H-3/5), 3.87 (s, 4H, CH2-N), 2.86–2.81 (m, 2H, H-8), 2.69 (t, J = 7.7 Hz, 2H, H-7) ppm; 13C-NMR
(100 MHz, DMSO-d6) 155.9 (C-4), 140.5 (ArC), 130.7 (C-1), 129.9 (C-2/6), 127.0 (ArC), 122.6 (ArC),
δ 9.15 (s, 1H, 4-OH), 7.24–7.16 (m, 4H, ArH), 7.05 (d, J = 8.4 Hz, 2H, H-2/6), 6.67 (d, J = 8.3 Hz,
δ

Molecules 2018, 23, 2902
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115.4 (C-3/5), 58.9 (CH2-N), 57.9 (C-8), 34.3 (C-7) ppm; HRMS (EI) calcd. for C16H17NO [M]+ = 239.1310;
Found: 239.1303.
N-(4-Hydroxyphenethyl)pyrrolidine (11b). Compound 11b was prepared from
9 via 10b as a white
amorphous solid and purified by flash chromatography using CHCl3/MeOH (1:1). Yield: 81%
1
(proton-sponge® method), 86% (conventional method); Rf = 0.22 (CHCl3:MeOH = 1:1); H-NMR
(400 MHz, CDCl3)
δ
6.99 (d, J = 8.3 Hz, 2H, H-2/6), 6.63 (d, J = 8.3 Hz, 2H, H-3/5), 2.75 (s, 4H, H-7/8),
155.2
2.69–2.62 (m, 4H, CH2-N), 1.88–1.79 (m, 4H, CH2-CH2-N) ppm; 13C-NMR (100 MHz, CDCl3)
δ
(C-4), 130.6 (C-1), 129.5 (C-2/6), 115.7 (C-3/5), 58.6 (C-8), 54.0 (CH2-N), 34.2 (C-7), 23.3 (CH2-CH2-N)
ppm; HRMS (EI) calcd. for C12H17NO [M]+ = 191.1310; Found: 191.1304.
N-(4-Hydroxyphenethyl)morpholine (11c). Compound 11c was prepared from
9 via 10c as a white
amorphous solid and purified by flash chromatography using CHCl3/MeOH (15:1). Yield: 87%
1
(proton-sponge® method), 87% (conventional method); Rf = 0.27 CHCl3:MeOH (15:1); H-NMR
(400 MHz, DMSO-d6)
δ 9.14 (s, 1H, 4-OH), 6.99 (d, J = 8.5 Hz, 2H, H-2/6), 6.65 (d, J = 8.5 Hz, 2H,
H-3/5), 3.56 (t, J = 4.6 Hz, 4H, CH2-O), 2.62–2.57 (m, 2H, H-7), 2.44–2.39 (m, 2H, H-8), 2.41–2.35 (m, 4H,
CH2-N) ppm; 13C-NMR (100 MHz, DMSO-d6)
δ 155.9 (C-4), 130.7 (C-1), 129.9 (C-2/6), 115.5 (C-3/5),
66.6 (CH2-O), 61.1 (C-8), 53.8 (CH2-N), 32.1 (C-7) ppm; HRMS (EI) calcd. for C12H17NO2 [M]+ =
207.1259; Found: 207.1255.
N-(4-Hydroxyphenethyl)octahydroisoindole (11d). Compound 11d was prepared from
9 via 10d as a
white amorphous solid and purified by flash chromatography using CHCl3/EtOH (5:2). Yield: 77%
1
(proton-sponge® method), 79% (conventional method); Rf = 0.19 (CHCl3:EtOH = 5:2); H-NMR
(400 MHz, CDCl3) δ 6.93 (d, J = 8.3 Hz, 2H, H-2/6), 6.77 (d, J = 8.3 Hz, 2H, C-3/5), 3.18 (dd, J = 10.7,
6.3 Hz, 2H, CH2(a)-N), 3.06–3.00 (m, 2H, H-8), 2.93–2.87 (m, 2H, CH2(b)-N), 2.86–2.80 (m, 2H, H-7),
2.34–2.24 (m, 2H, CH-CH2), 1.67–1.58 (m, 2H, CH2(a)-CH), 1.54–1.45 (m, 2H, CH2(a)-CH2-CH), 1.52–1.43
(m, 2H, CH2(b)-CH), 1.39–1.31 (m, 2H, CH2(b)-CH2-CH) ppm; 13C-NMR (100 MHz, CDCl3)
δ 156.4
(C-4), 129.5 (C-2/6), 128.1 (C-1), 115.9 (C-3/5), 59.0 (C-8), 57.1 (CH2-N), 36.7 (CH-CH2), 32.3 (C-7), 25.9
(CH2-CH), 22.5 (CH2-CH2-CH) ppm; HRMS (EI) calcd. for C16H23NO [M]+ = 245.1780; Found: 245.1772.
N-(4-Hydroxyphenethyl)-4,5,6,7-tetrahydroisoindole (11e). A mixture of 11a (239 mg, 1 mmol), ammonium
formate (631 mg, 10 mmol), and palladium hydroxide on carbon, 20 wt. % loading (64 mg) in anhydrous
MeOH (8 mL) was refluxed for 16 hrs. The mixture was filtered through celite®, diluted with H2O,
and extracted three times with EtOAc. The combined organic layers were dried over Na2SO4 and
concentrated in vacuo to give 241 mg of crude 11e as a slightly yellow oil in quantitative yields, which
can be used without further purification. Rf = 0.69 (EtOAc); 1H-NMR (400 MHz, CDCl3)
δ 7.00 (d, J =
8.2 Hz, 2H, H-2/6), 6.75 (d, J = 8.2 Hz, 2H, H-3/5), 6.31 (s, 2H, CH-N), 3.97–3.91 (m, 2H, H-8), 3.01–2.90
(m, 2H, H-7), 2.59–2.52 (m, 4H, CH2-C=), 1.76–1.68 (m, 4H, CH2-CH2-C=) ppm; 13C-NMR (100 MHz,
CDCl3) δ 154.3 (C-4), 130.7 (C-1), 129.8 (C-2/6), 119.4 (C=C(H)-N), 115.9 (CH-N), 115.4 (C-3/5), 51.3
(C-8), 37.6 (C-7), 24.2 (CH2-CH2-C=), 22.0 (CH2-C=) ppm; HRMS (EI) calcd. for C16H19NO [M]+ =
241.1467; Found: 241.1465.
3.2.3. General Procedure for the Reduction of Heterocyclic N-Imides
The corresponding imide, 7a/7h, (2 mmol) was dissolved in 8 mL of anhydrous THF and then
treated dropwise with 4 mL LiAlH4 (1 M solution in THF) at 0 ◦C. After the addition of LiAlH4 was
complete, the reaction was allowed to reach an◦ambient temperature for 2 h. Then, a 2 M aqueous
solution of Na/K tartrate (50 mL) was added at 0 C to quench the reaction and the mixture was stirred
for 1 h. The solution was extracted four times with MTBE, the combined organic layers were dried over
Na2SO4, and concentrated in vacuo to give a residue, which was purified by flash chromatography.

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N-(4-Hydroxyphenethyl)isoindoline (11a). Compound 11a was prepared from 7a as a white amorphous
solid and purified by flash chromatography using CH/EtOAc (1:3). Yield: 72%; Rf = 0.30 (CH:EtOAC
= 1:3).
3-Hydroxy-N-(4-hydroxyphenethyl)octahydroisoindole-1-one (12). Compound 12 was prepared from 7h as
a white amorphous solid and purified by flash chromatography using CH/EtOAc (1:3). Yield: 88%;
Rf = 0.28 (CH:EtOAc = 1:3); HRMS (EI) calcd. for C16H21NO3 [M]+ = 275.1521; Found: 275.1527.
3.2.4. Procedures for the Synthesis of Dienones, 13a–j, 14a, 14c, 15
Conventional method. A solution of the cyclic imide, 7a
–
j, or amide, 12, (1.5 mmol) in CH3CN (20 mL)
◦
and H2O (8 mL) at 0 C was treated with PIDA (644 mg, 2.0 mmol), and stirred for 7 min at this
temperature. The reaction mixture was diluted with EtOAc and washed with a 1 M aqueous solution
of NaHCO3. The aqueous phase was re-extracted three times with EtOAc. The combined organic
layers were dried over Na2SO4 and concentrated in vacuo to give a residue, which was purified by
flash chromatography.
Phosphate buffer method. 1 mmol of the respective tertiary amine, 11a/11c, was dissolved in 1.5 mL HCl
(1 M in dioxane) and concentrated in vacuo to yield the corresponding hydrochloride. A solution◦of
the hydrochloride in CH3CN (12 mL), H2O (3 mL) and phosphate buffer (1 M, pH = 6.4, 2 mL) at 0 C
was then treated with PIDA (644 mg, 2.0 mmol) and stirred for 7 min at this temperature. The solvent
was evaporated to dryness, and the crude product was purified by flash chromatography.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]phthalimide (13a). Compound 13a was prepared from
7a as white crystals and purified by flash chromatography using CH/EtOAc (1:3). Yield: 67%; Rf = 0.42
(CH:EtOAc = 1:3); m.p.: 161–162 ◦C; 1H-NMR (300 MHz, DMSO-d6)
δ
7.88–7.80 (m, 4H, ArH), 6.97 (d,
J = 10.2 Hz, 2H, H-2/6), 6.10 (d, J = 10.2 Hz, 2H, H-3/5), 5.88 (s, 1H, 1-OH), 3.68–3.50 (m, 2H, H-8),
2.08–1.88 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
185.0 (C-4), 167.7 ((CO)N), 152.2 (C-2/6),
δ
134.4 (ArC), 131.7 (ArC), 127.1 (C-3/5), 123.0 (ArC), 67.5 (C-1), 37.8 (C-7), 33.1 (C-8) ppm; HRMS (EI)
calcd. for C16H13NO4 [M]+ = 283.0845; Found: 283.0845.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]maleimide (13b). Compound 13b was prepared from
7b as yellow crystals and purified by flash chromatography using CH/EtOAc (1:5). Yield: 17%; Rf =
0.40 (CH:EtOAc = 1:5); m.p.: 151–152 ◦C; 1H-NMR (300 MHz, DMSO-d6)
δ
6.99 (s, 2H, CH-(CO)N),
6.91 (d, J = 10.1 Hz, 2H, H-2/6), 6.08 (d, J = 10.1 Hz, 2H, H-3/5), 5.85 (s, 1H, 1-OH), 3.45–3.38 (m, 2H,
H-8), 1.93–1.85 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
185.0 (C-4), 170.8 ((CO)N), 152.1
δ
(C-2/6), 134.6 (CH-(CO)N), 127.1 (C-3/5), 67.4 (C-1), 37.9 (C-7), 32.8 (C-8) ppm; HRMS (EI) calcd. for
C12H11NO4 [M]+ = 233.0688; Found: 233.0686.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]succinimide (13c). Compound 13c was prepared from
7c as a white solid and purified by flash chromatography using CHCl3/CH3CN (1:3). Yield: 55%; Rf =
◦
0.51 (CHCl3:CH3CN = 1:3); m.p.: 128–129 C; 1H-NMR (400 MHz, DMSO-d6)
δ
6.93 (d, J = 10.2 Hz, 2H,
H-2/6), 6.10 (d, J = 10.2 Hz, 2H, H-3/5), 5.86 (s, 1H, 1-OH), 2.57 (s br, 4H, CH2-(CO)N), 3.38–3.31 (m,
2H, H-8), 1.89–1.77 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
185.5 (C-4), 178.0 ((CO)N),
δ
152.6 (C-2/6), 127.5 (C-3/5), 67.9 (C-1), 37.5 (C-7), 33.9 (C-8), 28.4 (CH2-(CO)N) ppm; HRMS (EI) calcd.
for C12H13NO4 [M]+ = 235.0845; Found: 235.0826.
4,5-Dichloro-N-[2-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]phthalimide (13d). Compound 13d was
prepared from 7d as white crystals and purified by◦flash chromatography using CH/EtOAc (1:1).
Yield: 40%; Rf = 0.22 (CH:EtOAc = 1:1); m.p.: 213–214 C; 1H-NMR (400 MHz, DMSO-d6)
δ 8.17 (s, 2H,
ArH), 6.96 (d, J = 10.1 Hz, 2H, H-2/6), 6.10 (d, J = 10.1 Hz, 2H, H-3/5), 5.89 (s, 1H, 1-OH), 3.62–3.56
(m, 2H, H-8), 2.02–1.95 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
δ
185.5 (C-4), 166.4 ((CO)N),
152.6 (C-2/6), 137.7 (C(Cl)=), 132.1 (C=C(CO)N), 127.6 (C-3/5), 125.6 (ArC), 67.9 (C-1), 38.0 (C-7), 34.0
(C-8) ppm; HRMS (EI) calcd. for C16H11Cl2NO4 [M]+ = 351.0065; Found: 351.0090.

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3,4-Dichloro-N-[2-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]maleimide (13e). Compound 13e was
prepared from 7e as yellowish crystals and purified by fla◦sh chromatography using CH/EtOAc (1:1).
1
Yield: 64%; Rf = 0.30 (CH:EtOAc = 1:1); m.p.: 168–169 C; H-NMR (400 MHz, DMSO-d6)
δ 6.95
(d, J = 10.1 Hz, 2H, H-2/6), 6.11 (d, J = 10.1 Hz, 2H, H-3/5), 5.91 (s br, 1H, 1-OH), 3.53–3.46 (m, 2H,
H-8), 1.95–1.90 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
δ
185.4 (C-4), 163.3 ((CO)N), 152.5
(C-2/6), 132.9 (C(Cl)=), 127.6 (C-3/5), 67.8 (C-1), 37.9 (C-7), 34.8 (C-8) ppm; HRMS (EI) calcd. for
C12H9Cl2NO4 [M]+ = 300.9909; Found: 300.9914.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]pyridine-2,3-dicarboximide (13f). Compound 13f was
prepared from 7f as white crystals and purified by flash chromatography using CHCl3/CH3CN (1:1).
Yield: 19%; Rf = 0.37 (CHCl3:CH3CN = 1:1); m.p.: 167–168 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ 8.95
(dd, J = 5.0, 1.5 Hz, 1H, ArH), 8.27 (dd, J = 7.7, 1.5 Hz, 1H, ArH), 7.77 (dd, J = 7.7, 5.0 Hz, 1H, ArH),
6.98 (d, J = 10.1 Hz, 2H, C-2/6), 6.11 (d, J = 10.1, 2H, H-3/5), 5.88 (s, 1H, 1-OH), 3.69–3.57 (m, 2H, C-8),
2.05–1.94 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
δ 185.5 (C-4), 166.6 ((CO)N), 155.2 (ArC),
152.6 (C-2/6), 152.0 (ArC), 131.6 (ArC), 128.3 (ArC), 127.7 (ArC), 127.6 (C-3/5), 68.0 (C-1), 38.1 (C-7),
33.7 (C-8) ppm; HRMS (EI) calcd. for C15H12N2O4 [M]+ = 284.0797; Found: 284.0792.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]morpholine-3,5-dione (13g). Compound 13g was
prepared from 7g as a yellowish solid and purified by flash chromatography using CH/EtOAc
(1:5). Yield: 18%; Rf = 0.33 (CHCl3:EtOAc = 1:5); m.p.: 142–143 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
6.94 (d, J = 10.1 Hz, 2H, H-2/6), 6.12 (d, J = 10.0 Hz, 2H, H-3/5), 4.36 (s, 4H, CH2-(CO)N), 3.70–3.56 (m,
2H, H-8), 1.89–1.78 (m, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
δ
185.5 (C-4), 170.1 ((CO)N),
152.7 (C-2/6), 127.5 (C-3/5), 68.0 (C-1), 67.4 (CH2-(CO)N), 37.8 (C-7), 33.8 (C-8) ppm; HRMS (EI) calcd.
for C12H13NO5 [M]+ = 251.0794; Found: 251.0794.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]hexahydrophthalimide (13h). Compound 13h was
prepared from 7h as yellow crystals and purified by flas◦h chromatography using CH/EtOAc (1:3).
1
Yield: 79%; Rf = 0.29 (CH:EtOAc = 1:3); m.p.: 135–136 C; H-NMR (400 MHz, DMSO-d6)
δ 6.94
(d, J = 10.1 Hz, 2H, H-2/6), 6.10 (d, J = 10.1 Hz, 2H, H-3/5), 5.84 (s, 1H, 1-OH), 3.40–3.35 (m, 2H,
H-8), 2.93–2.82 (m, 2H, CH-(CO)N), 1.85–1.80 (m, 2H, H-7), 1.71 (s, 2H, CH2(a)-CH), 1.60–1.51 (m, 2H,
CH2(b)-CH), 1.42–1.32 (m, 2H, CH2(a)-CH2-CH), 1.31–1.21 (m, 2H, CH2(b)-CH2-CH) ppm; 13C-NMR (100
MHz, DMSO-d6)
δ 185.4 (C-4), 179.7 ((CO)N), 152.7 (C-2/6), 127.5 (C-3/5), 67.9 (C-1), 39.3 (CH-(CO)N),
37.6 (C-7), 33.8 (C-8), 23.5 (CH2-CH), 21.6 (CH2-CH2-CH) ppm; HRMS (EI) calcd. for C16H19NO4
[M]+ = 289.1314; Found: 289.1310.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]-3,4,5,6-tetrahydrophthalimide (13i). Compound 13i
was prepared from 7i as an orange solid and purified by flash chromatography using CH/EtOAc
(1:1). Yield: 88%; Rf = 0.18 (CH:EtOAc = 1:1); m.p.: 93–94 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ 6.92 (d,
J = 10.1 Hz, 2H, H-2/6), 6.08 (d, J = 10.1 Hz, 2H, H-3/5), 5.85 (s, 1H, 1-OH), 3.42–3.36 (m, 2H, H-8),
2.24–2.17 (m, 4H, CH2-C=), 1.86 (dd, J = 8.5, 6.8 Hz, 2H, H-7), 1.69–1.62 (m, 4H, CH2-CH2-C=) ppm;
13C-NMR (100 MHz, DMSO-d6)
δ 185.5 (C-4), 170.9 ((CO)N), 152.6 (C-2/6), 141.5 (C=C(CO)), 127.5
(C-3/5), 67.9 (C-1), 38.6 (C-7), 33.0 (C-8), 21.3 (CH2-CH2-C=), 19.9 (CH2-C=) ppm; HRMS (EI) calcd. for
C16H17NO4 [M]+ = 287.1158; Found: 287.1160.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]-1,2,3,6-tetrahydrophthalimide (13j). Compound 13j
was prepared from 7j as white crystals and purified b◦y flash chromatography using CH/EtOAc (1:1).
Yield: 49%; Rf = 0.28 (CH:EtOAc = 1:1); m.p.: 145–146 C; 1H-NMR (400 MHz, DMSO-d6)
δ 6.90 (d, J =
10.1 Hz, 2H, H-2/6), 6.09 (d, J = 10.1 Hz, 2H, H-3/5), 5.87 (s, 1H, 1-OH), 5.85–5.82 (m, 2H, CH=CH),
3.31–3.36 (m, 2H, H-8), 3.11–3.06 (m, 2H, CH-(CO)N), 2.39–2.32 (m, 2H, CH2(a)-CH), 2.21–2.13 (m, 2H,
CH2(b)-CH), 1.79 (td, J = 7.5, 1.5 Hz, 2H, H-7) ppm; 13C-NMR (100 MHz, DMSO-d6)
δ 185.5 (C-4), 180.3
((CO)N), 152.5 (C-2/6), 128.1 (CH=CH), 127.6 (C-3/5), 67.8 (C-1), 38.9 (CH-(CO)N), 37.8 (C-7), 34.1
(C-8), 23.5 (CH2-CH) ppm; HRMS (EI) calcd. for C16H17NO4 [M]+ = 287.1158; Found: 287.1154.

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N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]isoindoline (14a). Compound 14a was prepared from
11a applying the phosphate buffer method as a brownish solid and purified by flash chromatography
◦
1
using CHCl3/EtOH (1:5). Yield: 16%; Rf = 0.50 (CHCl3:EtOH = 1:5); m.p.: 103–104 C; H-NMR
(400 MHz, DMSO-d6) 7.24–7.12 (m, 4H, ArH), 6.99 (d, J = 10.1 Hz, 2H, H-2/6), 6.06 (d, J = 10.1 Hz,
2H, H-3/5), 3.81–3.77 (m, 4H, CH2-N), 2.70–2.62 (m, 2H, H-8), 1.93–1.85 (m, 2H, H-7) ppm; 13C-NMR
δ
(100 MHz, DMSO-d6) δ 185.3 (C-4), 153.2 (C-2/6), 139.8 (ArC), 126.5 (ArC), 126.3 (C-3/5) 122.0 (ArC),
68.0 (C-1), 58.3 (CH2-N), 50.0 (C-8), 38.6 (C-7) ppm; HRMS (EI) calcd. for C16H17NO2 [M]+ = 255.1259;
Found: 255.1251.
N-[2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]morpholine (14c). Compound 14c was prepared from
11c applying the phosphate buffer method as a brownish solid and purified by flash chromatography
◦
using EtOAc/EtOH (1:1). Yield: 28%; Rf = 0.37 (EtOAc:EtOH = 1:1); m.p.: 98–99 C; 1H-NMR (400 MHz,
DMSO-d6)
δ 6.95 (d, J = 10.0 Hz, 2H, H-2/6), 6.04 (d, J = 10.0 Hz, 2H, H-3/5), 3.56–3.46 (m, 4H, CH2-O),
2.32–2.26 (m, 4H, CH2-N), 2.25–2.20 (m, 2H, H-8), 1.79 (t, J = 7.6 Hz, 2H, H-7) ppm; 13C-NMR (100 MHz,
DMSO-d6)
δ 185.8 (C-4), 153.8 (C-2/6), 126.8 (C-3/5), 68.5 (C-1), 66.6 (CH2-O), 53.7 (CH2-N), 53.4 (C-8),
37.0 (C-7) ppm; HRMS (EI) calcd. for C12H17NO3 [M]+ = 223.1208; Found: 223.1201.
3-Hydroxy-N-[2-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethyl]octahydroisoindole-1-one (15). Compound 15
was prepared from 12 as a beige solid and purified by flash chromatography using EtOAc. Yield: 65%;
◦
Rf = 0.14 (EtOAc); m.p.: 127–128 C; 1H-NMR (400 MHz, DMSO-d6)
δ
6.97–6.91 (m, 2H, H-2/6), 6.08 (d,
0
0
J = 11.0 Hz, 2H, H-3/5), 5.90 (d, J = 6.6 Hz, 1H, 9 -OH), 5.82 (s, 1H, 1-OH), 4.55 (d, J = 6.6 Hz, 1H, H-9 ),
3.39–3.31 (m, 1H, H-8(a)), 3.05–2.96 (m, 1H, H-8(b)), 2.63–2.56 (m, 1H, H-30), 2.06–1.98 (m, 1H, H0-80),
0
1.89–1.82 (m, 1H, H-7(a)), 1.83–1.77 (m, 1H, H-4 (a)), 1.80–1.72 (m, 1H, H-7(b)), 1.72–1.66 (m, 1H, H-7 (a)),
1.49–1.35 (m, 3H, H-40(b)/50(a)/60(a)), 1.19–1.06 (m, 1H, H-60(b)), 0.95–0.89 (m, 1H, H-50(b)), 0.91–0.83
(m, 1H, H-70(b)) ppm; 13C-NMR (100 MHz, DMSO-d6) 185.6 (C-4), 175.2 (C-20), 153.2 (C-2/6), 127.3
δ
(C-3/5), 85.6 (C-90), 68.1 (C-1), 40.8 (C-80), 38.5 (C-30), 38.1 (C-7), 35.2 (C-8), 26.3 (C-70), 23.3 (C-60), 23.2
(C-40), 23.1 (C-50) ppm; HRMS (EI) calcd. for C16H21NO4 [M]+ = 291.1471; Found: 291.1469.
3.3. Cytotoxicity against Human (Cancer) Cells
3.3.1. Cell Culture
Human CCRF-CEM leukemia and MDA-MB-231 breast cancer cells lines were kept in RPMI1640
medium (Gibco®, ThermoFisher Scientific Inc., New York, NY, USA), supplemented with 2 mM
L-glutamine (Gibco®), 10% heat-inactivated foetal bovine serum (FBS, Gibco®), 100 units/mL Penicillin
(PAA), and 100 µ
g/mL Streptomycin (Gibco®) (1% Pen/Strep). HCT-116 and U251 cells were cultured
in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco®) containing 2 mM L-glutamine,
10% FBS, and 1% Pen/Strep. MRC-5 cells were grown in Minimum Essential Medium (MEM, Gibco®)
supplemented with 2 mM L-glutamine, 10% FBS, and 1% Pen/Strep. All cells were kept in a humidified
5% CO2 atmosphere at 37 ◦C and passaged at 90% confluence.
3.3.2. XTT Viability Assay
A Cell Proliferation Kit II (XTT) was purchased from Sigma-Aldrich and performed as described
previously [65] and in accordance with the manufacturer’s protocol. In brief, adherent cell lines were
seeded at a density of 50,000 cells/mL or 100,000 cells/mL (MRC-5) in 96 well plates (100 µL, flat
bottom) and grown for 24 h before test compounds were added. Suspension cells (CCRF-CEM) were
seeded at 100,000 cells/mL and test compounds were added immediately. After 72 h, XTT solution
was added for another 90 min or 4 h (CCRF-CEM cells) and absorbance was measured at 490 nm with
a reference wave length of 650 nm (Hidex Sense Microplate Reader 425-301, Hidex, Turku, Finland).
Results are expressed as a percentage of the vehicle-treated (0.5% DMSO) control cells. Vinblastine
served as the positive control (0.01 µg/mL).

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3.4. In Vitro Growth Inhibition Assay of Plasmodium Falciparum NF54
In vitro activity against erythrocytic stages of P. falciparum was determined by a modified
[3H]-hypoxanthine incorporation assay [66] using the drug-sensitive NF54 strain and the standard
drug, chloroquine (Sigma C6628). Briefly, parasite cultures incubated in RPMI 1640 medium with
5% AlbuMAXTM (without hypoxanthine) were exposed to serial drug dilutions in microtiter plates.
After 48 h of incubation at 37 ◦C in a reduced oxygen atmosphere, 0.5 Ci [3H]-hypoxanthine was
µ
added to each well of the plate. Cultures were incubated for a further 24 h before they were harvested
onto glass-fiber filters and washed with distilled water. The radioactivity was counted using a
BetaplateTM liquid scintillation counter (Wallac, Zurich). The results were recorded as counts per
minute (CPM) per well at each drug concentration and expressed as a percentage of the untreated
controls. IC50 values were calculated from the sigmoidal inhibition curves using Microsoft Excel.
Chloroquine was used as the control.
3.5. In vitro Growth Inhibition Assay of Trypanosoma Brucei Rhodesiense
Trypanosoma brucei rhodesiense, STIB 900 strain, and the standard drug, melarsoprol, were used for
the assay. Minimum Essential Medium (50
glucose, 1% MEM non-essential amino acids (100
and 15% heat-inactivated horse serum was added to each well of a 96-well microtiter plate [67].
Serial drug dilutions of 11 three-fold dilution steps covering a range from 100 to 0.002 g/mL were
prepared. Then, 4 L were added to each
103 bloodstream forms of T. b. rhodesiense (STIB 900) in 50
well and the plate was incubated at 37 ◦C under a 5% CO2 atmosphere for 72 h. 10
L Alamar Blue
µL) supplemented with 25 mM HEPES, 1g/L additional
×
), 0.2 mM 2-mercaptoethanol, 1 mM Na-pyruvate,
µ
×
µ
µ
(resazurin, 12.5 mg in 100 mL double-distilled water) was then added to each well and incubation
continued for a further 2–4 h [68]. Then, the plates were read with a Spectramax Gemini XS microplate
fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of
536 nm and an emission wavelength of 588 nm. The IC50 values were calculated from the sigmoidal
inhibition curves using the microplate reader software, Softmax Pro (Molecular Devices Cooperation,
Sunnyvale, CA, USA). Melarsoprol was used as the control.
3.6. Cytotoxicity against L6 Cells
Assays were performed in 96-well microtiter plates, each well containing 100 µL of RPMI 1640
medium supplemented with 1% L-glutamine (200 mM) and 10% foetal bovine serum, and 4000 L6
cells (a primary cell line derived from rat skeletal myoblasts). Serial drug dilutions of 11 threefold
dilution steps covering a range from 100 to 0.002
the plates were inspected under an inverted microscope to assure growth of the controls and sterile
conditions. 10 L of Alamar Blue solution was then added to each well and the plates incubated
µg/mL were prepared. After 72 h of incubation,
µ
for another 2 h. Then, the plates were read with a Spectramax Gemini XS microplate fluorometer
(Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm
and an emission wavelength of 588 nm. The IC50 values were calculated by linear regression from
the sigmoidal dose inhibition curves using the microplate reader software, Softmax Pro (Molecular
Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxin (Sigma P4405) was used as the control.
4. Conclusions
In conclusion, we have synthesized a series of jacaranone imides and amines with promising
antiproliferative activities from commercially available methyl hydroxyphenyl acetate (
). Although the substances showed beneficial physicochemical properties, antiprotozoal effects
remained comparatively weak. Imide 13i showed the highest activity against P. falciparum NF54 with
an IC50 of 1.28 M, while the lowest IC50 against T. b. rhodesiense was displayed by the morpholine
derivate 14c with 0.27 M. The conjugated imides, 13b 13e, and 13i, exhibited the overall highest
cytotoxicity against all tested cancer cell lines.
4) and tyramine
(8
µ
µ
,

Molecules 2018, 23, 2902
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We are aware that some of the performed chemical modifications did not result in druggable
chemical entities. However, we consider that the exploration of a multifaceted chemical lead as
present in the jacaranone scaffold through systematic derivatizations may open doors to unexpected
biological properties.
Supplementary Materials: Supplementary material associated with this article are available online. Contents:
Compared overall yields of the key products. Calculated physicochemical parameters and models. Full results of
the XTT viability assay. Data and NMR spectra of the prepared compounds.
Author Contributions: Conceptualization, A.P.; Data curation, A.P., N.K., W.S., R.S., M.K. and M.-M.K.; Formal
analysis, A.P. and R.S.; Investigation, G.L., N.K., W.S. and M.-M.K.; Methodology, A.P., G.L., N.K., W.S., R.S., M.K.
and M.-M.K.; Project administration, A.P.; Resources, A.P.; Supervision, A.P.; Validation, A.P., N.K., W.S., R.S. and
M.K.; Writing—original draft, A.P. and M.-M.K.; Writing—review & editing, N.K., W.S., R.S. and M.K.
Funding: This research received no external funding.
Acknowledgments: The authors are grateful to Rudolf Bauer for supporting the XTT experiments and Sara
Crockett for help in English editing. The authors acknowledge the financial support by the University of Graz.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 13a–j, 14a, 14c and 15 are available from the authors.
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