Design, synthesis and evaluation of amino-substituted 1H-phenalen-1-ones as anti-leishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2017.10.032

Screening of a designed collection of mono-substituted amino-1H-phenalen-1-ones against promastigote forms of L. donovani and L. amazonensis, identified seven compounds with anti-leishmanial activities comparable or better than the commonly prescribed anti-leishmanial drug, miltefosine. Structure-activity analysis revealed that appendages containing a basic tertiary nitrogen were favored, and that the position of the appendage also affected their potency. Like miltefosine, several of these active compounds significantly reduced the mitochondrial membrane potential in promastigotes. Further studies in amastigotes of L. amazonensis revealed that compounds 14, 15 and 33 were more active and more selective than miltefosine, with sub-micromolar potencies and selectivity indices >100.

Full text of DOI:10.1016/j.ejmech.2017.10.032

Accepted Manuscript
Design, synthesis and evaluation of amino-substituted 1H-phenalen-1-ones as anti-
leishmanial agents
Mónica Blanco Freijo, Atteneri López-Arencibia, José E. Piñero, Grant McNaughton-
Smith, Teresa Abad-Grillo
PII:
S0223-5234(17)30824-3
10.1016/j.ejmech.2017.10.032
EJMECH 9824
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 July 2017
Revised Date: 15 September 2017
Accepted Date: 10 October 2017
Please cite this article as: Mó.Blanco. Freijo, A. López-Arencibia, José.E. Piñero, G. McNaughton-
Smith, T. Abad-Grillo, Design, synthesis and evaluation of amino-substituted 1H-phenalen-1-
ones as anti-leishmanial agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.032.
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ACCEPTED MANUSCRIPT
Graphical Abstract
1

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of amino-substituted 1H-
phenalen-1-ones as anti-leishmanial agents.
Mónica Blanco Freijo,a Atteneri López-Arencibia,b José E. Piñero,b Grant McNaughton-Smith,c,** Teresa
Abad-Grillo,a*
aInstituto Universitario de Bio-Orgánica ‘Antonio González’, Departamento de Química Orgánica, Universidad de La Laguna, Avda.
Fco. Sánchez 2, 38206 La Laguna, Tenerife, Spain
bInstituto Universitario de Enfermedades Tropicales y Salud Pública de Las Islas Canarias, Laboratorio de Quimioterapias de
Protozoos, Universidad de La Laguna, Tenerife, Spain
cCentro Atlántico del Medicamento S.A (CEAMED S.A.), PCTT, La Laguna, Tenerife, Spain
ABSTRACT:
Screening of a designed collection of mono-substituted amino-1H-phenalen-1-ones against promastigote forms of
L. donovani and L. amazonensis, identified seven compounds with anti-leishmanial activities comparable or
better than the commonly prescribed anti-leishmanial drug, miltefosine. Structure-activity analysis revealed that
appendages containing a basic tertiary nitrogen were favored, and that the position of the appendage also affected
their potency. Like miltefosine, several of these active compounds significantly reduced the mitochondrial
membrane potential in promastigotes. Further studies in amastigotes of L. amazonensis revealed that compounds
14, 15 and 33 were more active and more selective than miltefosine, with sub-micromolar potencies and
selectivity indices >100.
Keywords: Amino-substituted 1H-phenalen-1-ones, anti-leishmanial activity, structure-activity relationship,
mitochondrial membrane potential.
* Corresponding author.
** Corresponding author.
E-mail address: tereabad@ ull.edu.es (T. Abad-Grillo).
1. INTRODUCTION
Leishmaniasis is caused by protozoan parasites of the genus Leishmania that are transmitted between mammalian
hosts by female sandfly vectors of the genera Phlebotomus and Lutzomyia during feeding [1]. Leishmanial
infections are prevalent in more than 98 countries and an estimated 1.3 million new cases occur annually [2].
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There are three main types of leishmaniasis, cutaneous, mucocutaneous and visceral. Symptoms of cutaneous
leishmaniasis include unsightly spontaneously healing ulcers on the skin, while non-healing lesions in the mucosa
are symptoms of mucocutaneous leishmaniasis. Chronic visceral leishmaniasis can lead to a debilitating infection
of the reticuloendothelial system, that if left untreated can be fatal [3]. The majority of cases of visceral
leishmaniasis are caused by L. donovani in East Africa and Asia, L. infantum in the Mediterranean region, and L.
chagasi (genetically identical to L. infantum [4]) in Latin America [5]. L. amazonensis can lead to both cutaneous
and mucosal forms of leishmaniasis.
In many geographical areas the pentavalent antimonial sodium stibogluconate (Figure 1A) is still the current
first-in-line anti-leishmanial drug (for both cutaneous and visceral forms), even though resistance to it can be
greater than 50% in some locations [6a]. A combination of daily injections for up to 28 days and side effects,
leads to low patient compliance [6b]. Because of these drawbacks, amphotericin B and miltefosine (Figure 1A)
are being prescribed more frequently. One significant advantage of miltefosine is that it is an oral drug, however
again, treatment periods are long, typically 28 days. Because of these shortcomings, new anti-leishmanial drugs
are needed. Recently there have been a number of publications that have identified new anti-leishmanial
compounds against different Leishmania strains (L. donovani, L. infantum and L. major) [7-9]. Herein we
describe the identification of potent and selective compounds against L. amazonensis.
1H-Phenalen-1-one (1) containing compounds have been isolated from plants and fungi, where they play a
crucial role in the defense of the organism. Natural and synthetic substituted 1H-phenalen-1-ones exhibit a
diverse range of biological activities, such as anti-fungal (Fusarium oxysporum [10] and Mycosphaerella fijiensis
[11]), anti-malarial [12] and cytotoxic activity against human cancer cells [13]. Previous work from our group
had demonstrated that a set of 9-phenyl-1H-phenalen-1-one based anti-fungal phytoalexins, possessed modest
leishmanicidal activity (A and B, IC50 L. donovani = 35-40 µM, Figure 1B) [14]. Preliminary studies pointed to a
disruption of the leishmanial mitochondrial respiratory chain system, as a possible mechanism of action for these
compounds [14]. Further screening of synthetic 1H-phenalen-1-ones analogues lead to the discovery of the
significantly more potent derivative C (IC50 L. donovani = 4.8 µM, Figure 1B) [15]. Using the 1H-phenalen-1-
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one as a core structure, we have investigated the anti-leishmanial activity of a small, but diverse, collection of
mono-amino substituted derivatives (Figure 1C) against cutaneous and visceral forms of leishmaniasis.
Figure 1. Anti-leishmanial compounds: A) prescribed anti-leishmanial drugs; B) previously reported 1H-phenalen-1-one derivatives
with anti-leishmanial activities; C) compounds described in this work.
2. RESULTS AND DISCUSSION
2.1. Design Strategy. Since the 1H-phenalen-1-one core (1) itself is relatively lipophilic (cLogP = 2.7), we
decided that the groups to be added should be relatively small and/or contain polar functionalities to improve
water solubility. In particular, we wished to cover an ample spectrum of chemical diversity by including
combinations of both primary and secondary amines containing different types of hydrophobic groups (alkyl or
aromatic) and/or hydrogen bond donating/accepting functionalities (ethers, alcohols, tertiary amines or
heterocyclic groups). Furthermore, in order to investigate the positional aspects we wished to incorporate each
appendage in several different positions around the core. In particular, positions 2, 3, 4 and 9 were targeted, as
many of the previously known biologically active 1H-phenalen-1-ones contain appendages in one, or more, of
these positions [10-15].
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2.2. Chemistry. The majority of the desired 2- and 3-amino-substituted compounds (Scheme 1) were prepared
following a general method previously described by Cromwell et al. [16]. The reaction between cyclic secondary
amines and 2-bromo-1H-phenalen-1-one (2) generally lead to a mixture of the C2:C3-adducts, with the C3-
adduct being favored, while non-cyclic secondary amines gave a 3:1 preference for the C2-adduct. Primary
amines under the same reaction conditions typically gave a more balanced C2:C3 ratio but with much lower
conversions. Higher yields and a much shorter reaction time could be achieved by employing microwave assisted
heating, with no significant change to the C2:C3 ratio. Interestingly the reaction between 2 and ethanolamine
generated a stable aziridine, which upon ring opening with concentrated acid gave the C2 product 18 exclusively.
Amines that were not reactive using the Cromwell conditions (such as anilines or amino-heteroaromatics) were
introduced into C2 using Büchwald-Hartwig cross-coupling methodology (Scheme 1).
Scheme 1. Synthesis of 2-amino and 3-amino-substituted 1H-phenalen-1-onesa
Br
R
O
O
R
O
a or b
+
2
3, R = N(CH3)CH2CH2OH (a, 62%)
4, R = N(CH3)CH2CH2OH (a, 17%)
c
(a, 16%)
5, R =
7, R =
9, R =
N
N
N
O
6, R =
8, R =
10, R =
N
N
N
O
(a, 68%)
R
(a, 73%)
NCH2Ph
(a, 18%)
NCH2Ph
O
N
N
N
N
N
N
( , 63%)
a
(
a, 12%)
(a, 84%)
11, R = N
19, R = NHPh (88%)
20, R = NHPh-4-CN (26%)
21, R = NH-3-pyridyl (63%)
22, R = NH-2-pyridyl (14%)
12, R = NH(CH2)2CH3 (b, 41%)
14, R = NH(CH2)2N(CH3)2 (b, 17%)
16, R = NHCH2Ph (b, 30%)
13, R = NH(CH2)2CH3 (b, 31%)
15, R = NH(CH2)2N(CH3)2 (b, 13%)
17, R = NHCH2Ph (b, 20%)
18, R = NH(CH2)2OH (b, 68%)
a Reagents and conditions: (a) secondary alkyl amines, rt, 2 days; (b) primary alkyl amines, MW, 50 oC, 10 min; (c) aryl or heteroaryl
amines, Pd(PPh3)4, NaOtBu, 80 oC, 8-24 h.
The synthesis of the 9-amino-substituted 1H-phenalen-1-one derivatives (Scheme 2) were performed by
nucleophilic substitution of either the 9-hydroxy-1H-phenalen-1-one (23) [17], prepared from 2-
methoxynaphthalene [18], or from the corresponding triflate [19], depending on the basicity of the amine (the
more basic amines generated the corresponding ammonium salts with 23, and not the desired products).
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Scheme 2. Synthesis of 9-amino-1H-phenalen-1-one derivativesa
O
O
O
24, R = NH2 (70%)
HO
R
TfO
25, R = NHCH2CH2CH3 (51%)
26, R = NHCH2CH2OH (55%)
27, R = NHCH2Ph (57%)
b
a
23
28 (86%)
c
33, R =
34, R =
(70%)
N
N
NCH2Ph
27, R = NHCH2Ph (91%)
O
29, R = NHCH2CH2N(CH3)2 (96%)
30, R = N(CH3)CH2CH2OH (98%)
N
N
N
R
(91%)
31, R =
32, R =
N
N
(96%)
(94%)
35, R = NHPh (97%)
36, R = NHPh-4-CN (91%)
37, R = NH-3-pyridyl (98%)
38, R = NH-2-pyridyl (68%)
O
a Reagents and conditions: (a) primary alkyl amines, MW, 120-180 oC, Ar; (b) Tf2O (1.3 eq), K3PO4 (30%)
water:toluene (1:1), rt, 5 h; (c) aryl or heteroaryl amines, rt-80 oC, 30 min-days.
The most potent appendages, observed from the screening of the C2, C3 and C9 substituted compounds were
also prepared in the C4 position, in a similar fashion to their C9 analogues (Scheme 3). 4-Methoxy-1H-phenalen-
1-one (42) was generated using a reaction sequence consisting of a microwave assisted Heck-Fujiwara cross-
coupling reaction, double bond reduction and a one-pot cyclization/oxidation step [20]. Heating 42 in the
presence of the selected amines gave the desired products (43-46).
Scheme 3. Synthesis of 4-amino-1H-phenalen-1-one derivativesa
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The desired 2-amino derivative 47 was not accessible via the previously described chemistry and was therefore
prepared by nitration of 1 followed by catalytic hydrogenation over palladium. Chromatographic separation gave
47, the 5-amino 48 and the 6-amino-1H-phenalen-1-one (49) in a ratio of 1:1:2.6 (Figure 2).
Figure 2. 2-, 5- and 6-amino-1H-phenalen-1-ones (47, 48 and 49)
2.3. In Vitro Anti-leishmanial Efficacy. All target compounds were initially tested against L. donovani and L.
amazonensis in their promastigote stages (Table 1) to identify compounds worthy of more advanced studies. For
a review of the different stages of Leishmania during its life cycle, see reference 21. Anti-leishmanial activity
was determined using promastigotes in their exponential growth phase and a previously described colorimetric
assay [22]. An initial test concentration of 25 µM was used as it was noted that several compounds were not
completely soluble in the assay conditions at higher concentrations (visual inspection using a microscope). The
prescribed anti-leishmanial drug miltefosine was used as the positive control.
6

Table 1. Leishmanicidal activity of 1H-phenaleAn-1C-oCneE(P1)TaEndDmMonoA-sNubUstiStuCtedR1IHP-Tphenalen-1-ones at C2, C3, C4 and C9 against
promastigote forms of L. donovani and L. amazonensis.
R group
#
1
L. donov.
IC50 ± SD
(µM)
>25
L.amazo.
IC50 ± SD
(µM)
>25
#
L. donov.
IC50 ± SD
(µM)
L. amazon.
IC50 ± SD
(µM)
#
L. donov.
IC50 ± SD
(µM)
L. amazon.
IC50 ± SD
(µM)
#
L. donov.
IC50 ± SD
(µM)
L. amazon.
IC50 ± SD
(µM)
H
OH
NH2
-
-
-
-
-
-
-
-
-
-
-
-
>25
>25
>25
>25
26.2 ± 2.9
>25
23
24
25
>25
>25
47
12
>25
>25
>25
>25
13
11
-
-
>25
-
>25
-
-
-
-
-
>25
>25
>25
>25
31
35
>25
>25
19
20
>25
>25
-
-
-
-
>25
>25
36
>25
>25
>25
>25
>25
>25
8.3 ± 1.2
3.5 ± 0.7
16
17
43
27
nd
nd
-
-
-
-
>25
>25
>25
27.4 ± 2.9
>25
18
3
-
-
26
30
32
>25
>25
>25
>25
>25
>25
>25
>25
4
6
nd
nd
>25
5
45
46
26.4 ± 3.8
>25
24.0 ± 3.1
>25
24.0 ± 0.5
>25
9.7 ± 0.8
>25
26.3 ± 3.4
-
17.5 ± 4.0
-
14.0 ± 1.0
>25
11.6 ± 1.6
>25
7
9
8
33
34
10
>25
>25
-
-
-
-
>25
>25
21
37
22
14
>25
>25
-
-
-
-
38
29
>25
>25
9.6 ± 0.3
6.0 ± 1.0
4.3 ± 0.1
2.1 ± 0.1
32.7 ± 0.9
7.2 ± 0.2
2.8 ± 0.2
1.4 ± 0.1
15
44
nd = not determined; - = not synthesized; miltefosine anti-leishmanial activity L. donovani IC50 = 3.3 ± 0.3 µM, L.
amazonensis IC50 = 6.5 ± 0.2 µM.
2.4. Structure-activity relationship analysis against promastigote forms of L. amazonensis and L.
donovani. Screening at 25 µM quickly highlighted the activities of the compounds containing the N-
benzylpiperazine or the N,N-dimethylethylenediamine group. Interestingly, the N,N-dimethylethylenediamine
containing compound at C2 (14) exhibited actives in both strains of Leishmania comparable to the reference
compound, while the N-propyl (12), N-methylethanolamine (3), -NH2 (47) and 3-pyridyl (21) analogues were all
inactive at 25 µM (< 25% inhibition). This comparison highlighted the significant positive effect of the basic
tertiary amine in the N,N-dimethylethylenediamine moiety. In agreement with this observation was the activity,
although more modest, of the compound containing the N-benzylpiperazine group at C2 (7) and the lack of
7

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activity of the compounds containing a morpholino (5), benzyl (16) or a pyrimidine substituted piperazine (9). In
general, the activity of the various amines in C2, was mirrored in the other positional isomers. With respect to L.
amazonensis, the 2- and 4-positional isomers were significantly less active than the 3- and 9-positonal isomers
(Figure 3). While there was no distinct difference between the positional isomers for the N-benzylpiperazine
containing compounds against promastigotes of L. donovani, the N,N-dimethylethylenediamine containing
compounds appeared to conform to the same pattern as seen in the L. amazonensis strain. The only other amine to
exhibit significant activity was the N-benzylamine, and only in the more favored 9-position (27). Interestingly,
unlike miltefosine, the active mono-substituted phenalenones were generally more potent against L. amazonensis
than the L. donovani strain.
Figure 3. Representation of the positional effects of the appendages on anti-leishmanial activity in L. amazonensis promastigotes.
Groups in blue are favourable positions; groups in red are less favorable; groups in cyano show the difference between the N,N-
dimethylethylenediamine and N-benzylpiperazine groups.
2.5. Analysis of the effect of mono-substituted 1H-phenalen-1-ones on the mitochondrial membrane
potential (∆Ψm) of L. amazonensis promastigotes. As our previous studies with 1H-phenalen-1-ones possessing
anti-leishmanial activities suggested they modulated the mitochondrial respiratory chain (which leads to a
lowering in the mitochondrial membrane potential and induces apoptosis), we investigated whether these more
potent amino-substituted derivatives also affected this biological system. As the active compounds were generally
more potent against L. amazonensis, we focused further investigations on this strain. The ∆Ψm was measured for
each compound, at its IC90 concentration, by spectrofluorometric analysis using the mitochondrial membrane
specific cationic dye JC-1 [23]. Miltefosine was used as the positive control, as it is known to cause parasitic
death by lowering of the ∆Ψm and inducing apoptosis [9b]. While compounds 7 and 33 did not cause sizeable
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changes in the ∆Ψm, the remaining five compounds (8, 14, 15, 27 and 29) all showed statistically significant
reductions (Figure 4). In particular, the reductions caused by compounds 8, 14 and 29 were comparable to the
effect caused by miltefosine, and are indicative of a strong depolarization of the leishmanial mitochondrial
membrane.
Figure 4. Changes in the mitochondrial membrane potential (∆Ψm) (measured as the ratio of fluorescence intensity at 595/535 nm) after
24 h treatment of L. amazonensis promastigotes with IC90 concentration of 1H-phenalen-1-ones. *Indicates a P < 0.01 between vehicle
and compound measures (n = 3).
2.6. Activity and selectivity against the intracellular amastigote form of L. amazonensis. Compounds with
an IC50 value < 25 µM against L. amazonensis promastigotes, were further tested against intracellular amastigotes
of L. amazonensis in macrophages (Table 2) using the parasite-rescue and transformation assay pioneered by
Tekwani et al. [24]. To assess the selectivity of these compounds their cytotoxicity was measured in a murine
macrophage cell line, J774A.1. The selectivity index (SI) was calculated as the ratio of CC50 value for J774A.1
and the IC50 value for L. amazonensis [SI = CC50 (J774A.1)/ IC50 (L. amazonensis amastigotes)]. Again,
miltefosine was used as a comparative reference.
9

Table 2. Anti-leishmanial activity against amastiAgotCesCoEf LP. TamEaDzonMensAis,NcyUtoStoCxicRitIyPagTainst J774A.1 and the calculated SI of selected
amino-substituted 1H-phenalen-1-ones.
L. amazonensis
amastigote
J774A.1
Macrophage
Comp.
SI
CC50 ± SD (µM)
IC50 ± SD (µM)
2.4 ± 0.6
1.1 ± 0.1
0.9 ± 0.2
0.6 ± 0.1
2.3 ± 0.2
0.7 ± 0.2
0.6 ± 0.1
3.1 ± 0.3
122.1 ± 10.5
28.5 ± 0.4
122.4 ± 7.4
95.6 ± 3.4
9.4 ± 1.1
51.0
26.0
136.0
159.3
4.1
7
8
14
15
27
29
38.9 ± 0.2
91.7 ± 7.1
73.7 ± 3.1
55.6
153.0
23.8
33
Miltefosine
IC50 = Half maximal inhibitory concentration; CC50 = Half maximal cytotoxic concentration; SI =
Selectivity index representing the ratio of CC50 (macrophage)/IC50 (amastigote)
All seven compounds selected from the primary screening, exhibited higher levels of potency against the
intracellular amastigote form of L. amazonensis compared to their free-living promastigote form. This positive
shift in potency has been noted with other phenalenones [14] and other anti-leishmanial compounds, and may be
attributed to increased local concentrations within the macrophages [25]. Interestingly the N-benzylpiperazines
derivatives (7, 8 and 33) all showed larger shifts than the N,N-dimethylethylenediamine derivatives (14, 15 and
29). All compounds, except 7, possessed IC50 values better than miltefosine, with four of the compounds
possessing sub-microlar IC50 values (14, 15, 29 and 33). With respect to their activities against murine
macrophages, compounds 7, 14, 15 and 33, all possessed CC50 values higher than miltefosine. The remaining
three compounds had much lower CC50 values, especially the C9-N-benzyl compound 27. Overall compounds
14, 15 and 33 are particularly noteworthy as they possess sub-micromolar potencies against amastigotes of L.
amazonensis, SI values >100, and favorable predicted physicochemical properties (Table 3).
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Table 3. Calculated physicochemical properties for amino-substituted 1H-phenalen-1-ones (14, 15 y 33).
Oral
MW
(g/mol)
# of Lipinski
volationsa
Comp.
logPa
Bioavailability
(Veber rule)a
266.34
266.34
354.45
14
15
33
3.41
2.92
4.61
0
0
0
Good
Good
Good
a parameters predicted using FAF-Drug4 web service [26].
3. CONCLUSION
Our strategy to introduce a small but chemically diverse set of amines into various positions around a 1H-
phenalen-1-one core, has resulted in the identification of three novel compounds with interesting anti-leishmanial
profiles against L. amazonensis. These three compounds (14, 15 and 33) are 3-5 times more potent against
intracellular amastigotes (L. amazonensis) than the prescribed anti-leishmanial drug miltefosine, and are >5 times
less cyctotoxic to a murine macrophage cell line than miltefosine. These results, combined with their predicted
physiochemical properties, support their continued exploration in in vivo models of Leishmania.
4. EXPERIMENTAL SECTION
4.1. General Methods. All reagents and solvents were obtained from Aldrich Chemical Co. and used without further
purification. Reactions with sensitive reagents were performed under inert atmosphere (argon or nitrogen) and organic
solvents were dried by standard methods. Unless otherwise stated, solvents were removed under reduced pressure using a
o
rotary evaporator at 40-60 C. All reactions were monitored by analytical thin layer chromatography (TLC) on
POLIGRAM® SIL G/UV254 silica gel coated plates (0.20 mm) from MACHEREY-NAGEL. Column chromatography was
performed on silica gel 60 (0.063-0.20 mm) from MERCK. Preparative thin layer chromatography was carried out with GF
silica gel plates (1mm) with fluorescent indicator 254 nm (UNIPLATE). Compounds were visualized by ultraviolet light
(254 nm). The purity of the final compounds was determined to be
≥
95% by high pressure liquid chromatography (HPLC)
1
using a Jasco PU-2080 intelligent HPLC pump (Jasco MD 2020 Plus multiwavelength detector). H and 13C NMR spectra
were recorded at room temperature (rt) on a Bruker Avance 400 or 500 MHz NMR spectrometer in the solvent indicated.
1
Data for H NMR are reported as follows: chemical shift (
δ
ppm), integration, multiplicity and coupling constant (Hz),
11

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whereas 13C NMR analyses are reported in terms of chemical shift. IR and UV spectra were recorded on a BRUKER IFS 66
and a JASCO modelo V-560 spectrophotometer, respectively. Melting points were determined using a Stuart Scientific
SMP11 instrument. Low resolution (EIMS) and high-resolution mass spectrometry (HRMS) were performed on a
Micromass AutoSpec magnetic tri-sector (EBE geometry) mass spectromter. Microwave reactions were performed using a
Biotage® Initiator (software version 2.5). Lyophilization was performed using a CHRIST ALPHA 2-4 lyophilizer.
4.2. Synthesis
4.2.1. 2-Amino- and 3-amino-1H-phenalen-1-one derivatives. General procedure:
Method A: A solution of 2-bromo-1H-phenalen-1-one (2) in the amine was stirred at rt in the dark for 2 days. The amine
was removed under reduced pressure and EtOAc was added. The crude material was washed with H2O and dried (Na2SO4).
After filtration and evaporation of the solvent, purification using preparative thin layer chromatography or column
chromatography afforded the desired product(s).
o
Method B: A solution of 2-bromo-1H-phenalen-1-one (2) in the amine was heated at 50 C for 10 min using MW
irradiation. The amine was removed under reduced pressure and EtOAc was added. The crude material was acidified with
1N HCl (aq) to pH = 2-3 and extracted with EtOAc. The aqueous phase was then basified with 5% NaHCO3 (aq) to pH = 6-
7 and extracted with EtOAc. The combined organics were dried (Na2SO4), filtered and concentrated. Purification using
preparative thin layer chromatography or column chromatography afforded the desired product(s).
4.2.1.1. 2-[(2-Hydroxyethyl)(methyl)amino]-1H-phenalen-1-one (3) and 3-[(2-hydroxyethyl)(methyl)amino]-1H-
phenalen-1-one (4). Method A: using 2 (50.9 mg, 0.193 mmol) and 2-(methylamino)ethanol (1.0 mL). Purification using
preparative thin layer chromatography (hexane/EtOAc; 1:1), gave compound 3 (30.4 mg, 62%) as a red oil [Rf 0.30
1
(hexane/EtOAc, 1:1)] and compound 4 (8.2 mg, 17%) as an orange oil [Rf 0.22 (hexane/EtOAc, 1:1)]. Compound 3: H
NMR (400 MHz, CDCl3) δ: 8.63 (1H, dd, J = 0.7, 7.4 Hz, H-9), 8.17 (1H, d, J = 7.9 Hz, H-7), 7.83 (1H, d, J = 8.1 Hz, H-6),
7.73 (1H, t, J = 7.6 Hz, H-8), 7.60 (1H, d, J = 7.0 Hz, H-4), 7.53 (1H, dd, J = 7.4, 8.0 Hz, H-5), 6.93 (1H, s, H-3), 4.55 (s,
OH), 3.89 (2H, t, J = 5.0 Hz, H-2’), 3.36 (2H, t, J = 4.9 Hz, H-1’), 2.92 (3H, s, CH3); 13C NMR (100 MHz, CDCl3)
δ: 183.1
(s, C-1), 147.52 (s, C-2), 135.5 (d, C-7), 131.9 (s, C-6a), 131.3 (d, C-9), 130.1 (s, C-9a), 129.1 (s, C-3a), 128.9 (d, C-4),
128.7 (d, C-6), 127.2 (d, C-5), 126.9 (d, C-8), 124.7 (s, C-9b), 119.25 (d, C-3), 60.4 (t, C-2’), 56.18 (t, C-1’), 38.53 (c,
CH3); EIMS: m/z 235 [M]+ (25), 222 (100), 194 (27), 152 (18); HRMS: calcd. for C16H15NO2 253.1103, found 253.1109;
12

ACCEPTED MANUSCRIPT: 3394, 1615, 1564, 1328, 1052, 843 cm-1.
UV-Vis (EtOH) λmax: 475, 448, 365, 329, 269, 237, 213, 202 nm; IR (CHCl3) νmax
Compound 4: 1H NMR (400 MHz, CDCl3)
δ: 8.49 (1H, d, J = 7.2 Hz, H-9), 8.24 (1H, d, J = 7.6 Hz, H-7), 8.06 (1H, d, J =
8 Hz, H-6), 7.96 (1H, d, J = 8 Hz, H-4), 7.65 (1H, dd, J = 7.6, 7.8 Hz, H-8), 7.56 (1H, dd, J = 7.7, 8.0 Hz, H-5), 6.25 (1H, s,
13
H-2), 5.83 (s, OH), 3.97 (2H, t, J = 5.6 Hz, H-2’), 3.52 (2H, t, J = 5.6 Hz, H-1’), 3.03 (3H, s, CH3); C NMR (100 MHz,
CDCl3) δ: 184.7 (s, C-1), 162.7 (s, C-3), 134.2 (d), 132.6 (s), 132.1 (d), 129.2 (d), 129.1 (s), 128.9 (d), 128.7 (s), 126.7 (d),
125.8 (d), 125.8 (s), 113.4 (d), 59.6 (t), 58.0 (t), 41.2 (c, CH3); EIMS: m/z 253 [M]+ (18), 222 (100), 209 (33), 151 (27);
HRMS: calcd. for C16H15NO2 253.1103, found 253.1109; UV-Vis (EtOH) λmax: 372, 345, 237 nm; IR (CHCl3) νmax: 3349,
1629, 1555, 1353, 1023, 830 cm-1
4.2.1.2. 2-(Morpholin-4-yl)-1H-phenalen-1-one (5) and 3-(morpholin-4-yl)-1H-phenalen-1-one (6). Method A: using 2
(50.0 mg, 0.196 mmol) and morpholine (0.5 mL). Purification using preparative thin layer chromatography (CH2Cl2/EtOAc,
4:1), gave product 5 (8.0 mg, 16%) as a red solid [Rf 0.46 (CH2Cl2/EtOAc, 4:1)] and product 6 (35.0 mg, 68%) as an orange
solid [Rf 0.38 (CH2Cl2/EtOAc, 4:1)]. Compound 5: 1H NMR (500 MHz, CDCl3)
δ: 8.63 (1H, dd, J = 1.0, 7.4 Hz, H-9), 8.17
(1H, d, J = 8 Hz, H-7), 7.86 (1H, d, J = 8.1 Hz, H-6), 7.75 (1H, dd, J = 7.5, 8.0 Hz, H-8), 7.63 (1H, d, J = 7.0 Hz, H-4), 7.54
(1H, dd, J = 7.5, 8.0 Hz, H-5), 6.94 (1H, s, H-3), 3.95 (4H, t, J = 3.6, 7.6 Hz, H-2’, H-6’), 3.26 (4H, t, J = 4.0, 7.6 Hz, H-3’,
H-5’); 13C NMR (125 MHz, CDCl3)
δ: 182.0 (s, C-1), 147.5 (s, C-2), 135.0 (d, C-7), 131.9 (s, C-6a), 131.0 (d, C-9), 130.3
(s, C-3a), 129.2 (d, C-4), 129.1 (d, C-6), 128.9 (s, C-9a), 127.1 (d, C-5), 127.1 (d, C-8), 125.1 (s, C-9b), 120.8 (d, C-3), 67.0
(t, 2 x C, C-2’, C-6’), 50.2 (t, 2 x C, C-3’, C-5’); EIMS: m/z 265 [M]+ (72), 208 (100), 180 (36), 152 (34); HRMS: calcd. for
1
C17H15NO2 265.1103, found 265.1094. Compound 6: H NMR (400 MHz, CDCl3)
δ: 8.53 (1H, dd, J = 1.2, 7.2 Hz, H-9),
8.10 (1H, dd, J = 0.8, 8.0 Hz, H-7), 8.02 (1H, d, J = 7.6 Hz, H-4), 7.99 (1H, d, J = 8 Hz, H-6), 7.68 (1H, t, J = 7.6 Hz, H-8),
7.58 (1H, t, J = 7.6 Hz, H-5), 6.22 (1H, s, H-2), 3.96 (4H, t, J = 4.5 Hz, H-2’, H-6’), 3.23 (4H, t, J = 4.5 Hz, H-3’, H-5’); 13C
NMR (100 MHz, CDCl3) δ: 185.1 (s, C-1), 161.8 (s, C-3), 134.3 (d, C-7), 132.6 (s, C-6a), 132.1 (d, C-6), 129.4 (d, C-9),
129.0 (s, C-9a), 128.4 (s, C-9b), 128.0 (d, C-4), 126.8 (d, C-8), 125.9 (d, C-5), 125.3 (s, C-3a), 113.9 (d, C-2), 66.8 (t, 2 x C,
C-2’, C-6’), 53.1 (t, 2 x C, C-3’, C-5’); EIMS: m/z 265 [M]+ (76), 208 (100), 180 (36), 152 (33), 151 (19); HRMS: calcd.
for C17H15NO2 265.1103, found 265.1110.
4.2.1.3. 2-(4-Benzyl-piperazin-1-yl)-1H-phenalen-1-one (7) and 3-(4- benzylpiperazin-1-yl)-1H-phenalen-1-one (8).
Method A: using 2 (50.6 mg, 0.193 mmol) and 1-benzylpiperazine (1.0 mL). Purification using column chromatography
(hexane/EtOAc; 2:3), gave compound 7 (12.4 mg, 18%) as an orange oil [Rf 0.53 (hexane/EtOAc, 2:3)] and compound 8
13

ACCEPTED MANUSCRIPT
(50.0 mg, 73%) as a yellow oil [Rf 0.36 (hexane/EtOAc, 2:3)]. Compound 7: 1H NMR (400 MHz, CDCl3)
δ: 8.63 (1H, d, J
= 6.4 Hz, H-9), 8.17 (1H, d, J = 8 Hz, H-7), 7.85 (1H, d, J = 8.2 Hz, H-6), 7.75 (1H, t, J = 7.6 Hz, H-8), 7.62 (1H, d, J = 7.2
Hz, H-4), 7.54 (1H, t, J = 7.6 Hz, H-5), 7.40-7.29 (5H, m, Ar), 6.94 (1H, s, H-3), 3.67 (2H, s, H-1’’), 3.32 (4H, s, H-2’, H-
13
6’), 2.77 (4H, s, H-5’, H-3’); C NMR (100 MHz, CDCl3)
δ: 182.1 (s, C-1), 147.8 (s, C-2), 138.0 (s), 134.9 (d), 131.9 (s),
130.9 (d), 130.4 (s), 129.5 (d, 2 x C), 129.1 (s), 128.9 (d), 128.8 (d), 128.4 (d, 2xC), 127.3 (d), 127.1 (d), 127.0 (d), 125.1
(s), 120.8 (d), 63.2 (t, C-1’’), 53.1 (t, 2 x C, C-2’, C-6’), 49.7 (t, 2 x C, C-3’, C-5’); EIMS: m/z 355 (24), 354 [M]+ (96), 235
(22), 221 (27), 208 (71), 146 (59), 91 (100); HRMS: calcd. for C24H22N2O 354.1732, found 354.1725); UV-Vis (EtOH)
1
λmax: 364, 349, 263, 241, 210, 206, 203 nm; IR (CHCl3) νmax: 3190, 1618, 1566, 1450, 1007, 847 cm-1. Compound 8: H
NMR (400 MHz, CDCl3) δ: 8.47 (1H, dd, J = 0.8, 7.2 Hz, H-9), 8.02 (1H, d, J = 8.4 Hz, H-7), 7.95 (1H, d, J = 7.2 Hz, H-4),
7.91 (1H, d, J = 8 Hz, H-6), 7.61 (1H, dd, J = 7.6, 8.0 Hz, H-8), 7.49 (1H, dd, J = 7.6, 8.0 Hz, H-5), 7.29-7.22 (5H, m, Ar),
6.15 (1H, s, H-2), 3.57 (2H, s, H-1’’), 3.22 (4H, s, H-2’, H-6’), 2.66 (4H, s, H-5’, H-3’); 13C NMR (100 MHz, CDCl3)
δ:
184.9 (s, C-1), 162.1 (s, C-3), 137.7 (s), 134.1 (d, C-7), 132.5 (s, C-6a), 131.9 (d, C-6), 129.6 (d, 2 x C), 129.3 (d, C-9),
129.2 (s, C-9a), 129.0 (s, C-9b), 128.4 (d, 2 x C), 128.1 (d, C-4), 127.4 (d), 126.6 (d, C-8), 125.8 (d, C-5), 125.50 (s, C-3a),
113.6 (d, C-2), 63.09 (t, C-1’’), 52.96 (t, 2 x C, C-2’, C-6’), 52.65 (t, 2 x C, C-3’, C-5’). EIMS: m/z 354 [M]+ (61), 146 (85),
91 (100); HRMS: calcd. for C24H22N2O 354.1732, found 354.1722); UV-Vis (EtOH) λmax: 372, 346, 242, 205, 202 nm; IR
(CHCl3) νmax: 3422, 1629, 1570, 1353, 1209, 834 cm-1.
4.2.1.4. 2-(4-Pyrimidin-2-yl-piperazin-1-yl)-1H-phenalen-1-one (9) and 3-(4-pyrimidin-2-yl-piperazin-1-yl)-1H-
phenalen-1-one (10). Method A: using 2 (50.0 mg, 0.193 mmol) and 2-(piperazin-1-yl)pyrimidine (0.82 mL, 5.79 mmol).
Purification using preparative thin layer chromatography (toluene/EtOAc; 4:1), gave compound 9 (8.1 mg, 12%) as an
orange oil [Rf 0.40 (toluene/EtOAc, 4:1)] and compound 10 (31.7, 63%) as a yellow solid [Rf 0.15 (toluene/EtOAc; 4:1)].
1
Compound 9: H NMR (400 MHz, CDCl3)
δ: 8.67 (1H, d, J = 7.2 Hz, H-9), 8.35 (2H, d, J = 4.7 Hz), 8.18 (1H, d, J = 8.0
Hz), 7.87 (1H, d, J = 8.2 Hz), 7.77 (1H, t, J = 7.6 Hz), 7.64 (1H, d, J = 7.0 Hz), 7.55 (1H, dd, J = 7.2, 8.0 Hz), 6.98 (1H, s,
H-3), 6.51 (1H, t, J = 4.7 Hz), 4.08 (4H, t, J = 5.2 Hz, H-3’, H-5’), 3.32 (4H, t, J = 5.2 Hz, H-2’, H-6’); 13C NMR (100
MHz, CDCl3) δ: 182.1 (s, C-1), 161.9 (s, C-2), 157.9 (d, 2 x C), 147.7 (s), 135.0 (d), 131.9 (s), 131.0 (d), 130.3 (s), 129.2
(d), 129.1 (d), 128.9 (s), 127.1 (d, 2 x C), 125.2 (s), 121.9 (d), 110.2 (d), 49.8 (t, 2 x C, C-3’, C-5’), 43.8 (t, 2 x C, C-2’, C-
6’); EIMS: m/z 366 (26), 365 [M + Na]+ (100), 355 (9); HRMS: calcd. for C21H18N4ONa 365.1378, found 365.1382; UV-
Vis (EtOH) λmax: 450, 363, 271, 243, 209, 206, 203 nm; IR (CHCl3) νmax: 3377, 1583, 1505, 1359, 1252 cm-1. Compound
1
10: H NMR (400 MHz, CDCl3)
δ: 8.56 (1H, d, J = 7.1 Hz, H-9), 8.36 (2H, d, J = 4.7 Hz), 8.14 (1H, d, J = 6.8 Hz), 8.13
14

ACCEPTED MANUSCRIPT
(1H, d, J = 6.8 Hz), 8.03 (1H, d, J = 8.1 Hz), 7.72 (1H, dd, J = 7.6, 8.0 Hz), 7.64 (1H, dd, J = 7.6, 8.0 Hz), 6.56 (1H, t, J =
4.7 Hz), 6.27 (1H, s, H-2), 4.10 (4H, t, J = 4.8 Hz, H-3’, H-5’), 3.32 (4H, t, J = 4.8 Hz, H-2’, H-6’); 13C NMR (100 MHz,
CDCl3) δ: 185.1 (s, C-1), 162.0 (s), 161.8 (s), 157.9 (d, 2 x C), 134.4 (d), 132.7 (s), 132.1 (d), 129.5 (d), 129.1 (s), 128.5 (s),
128.1 (d), 126.8 (d), 126.0 (d), 125.6 (s), 114.3 (d), 110.6 (d), 52.7 (t, 2 x C, C-3’, C-5’), 43.8 (t, 2 x C, C-2’, C-6’); EIMS:
m/z 365 (59), 343 [M]+ (100); HRMS: calcd. for C21H18N4O 343.1559, found 343.1569; UV-Vis (EtOH) λmax: 375, 347,
246, 201 nm; IR (CHCl3) νmax: 1572, 1494, 1351, 1195, 956, 833 cm-1; mp: 202-204 ºC.
4.2.1.5. 3-(Piperidin-1-yl)-1H-phenalen-1-one (11). Method A: using 2 (102.4 mg, 0.395 mmol) and piperidine (1
mL); the crude material was acidifed with 1N HCl (aq) (8 mL) to pH = 2-3 and then extracted with EtOAc (2 x 8 mL). The
aqueous phase was then basified with a solution of 5% NaHCO3 to pH = 6-7 and extracted with EtOAc (2 x 8 mL). The
combined organics were dried, filtered and concentrated. Purification using preparative thin layer chromatography (CH2Cl2)
1
gave compound 11 (83.2 mg, 80%) as an orange solid. H NMR (400 MHz, CDCl3)
δ: 8.51 (1H, dd, J = 1.2, 6 Hz, H-9),
8.06 (1H, dd, J = 0.8, 6.4 Hz, H-7), 7.98 (1H, d, J = 6 Hz, H-4), 7.95 (1H, d, J = 6.5 Hz, H-6), 7.65 (1H, t, J = 6.0 Hz, H-8),
7.55 (1H, t, J = 6.0 Hz, H-5), 6.19 (1H, s, H-2), 3.19 (4H, t, J = 4 Hz, H-2’, H-6’), 1.81 (4H, m, H-3’, H-5’), 1.69 (2H, m,
H-4’); 13C NMR (100 MHz, CDCl3)
δ: 185.0 (s, C-1), 163.2 (s, C-3), 134.0 (d), 132.5 (s), 131.7 (d), 129.2 (s), 129.1 (d),
128.5 (s), 128.2 (d), 126.6 (d), 125.9 (s), 125.8 (d), 113.3 (d, C-2), 54.0 (t, 2 x C, C-2’, C-6’), 26.1 (t, 2 x C, C-3’, C-5’),
24.5 (t, C-4’); EIMS: m/z 263 [M]+ (84), 262 (100), 180 (59), 152 (20), 151 (21); HRMS: calcd for C18H17NO 263.1310,
found 263.1315; UV-Vis (EtOH) λmax: 375, 347, 243, 223, 203 nm; IR (CHCl3) νmax: 3396, 1634, 1576, 1360, 1223, 826 cm-
1; mp: 108-110 ºC.
4.2.1.6. 2-(Propylamino)-1H-phenalen-1-one (12) and 3-(propylamino)-1H-phenalen-1-one (13). Method B: using 2
(46.5 mg, 0.179 mmol) and 1-propanamine (0.7 mL). Purification using column chromatography using (hexane/EtOAc; 1:1)
gave compound 12 (17.5 mg, 41%) as a red solid [Rf 0.90 (hexane/EtOAc, 1:1)] and compound 13 (13.0 mg, 31%) as a
yellow solid [Rf 0.25 (hexane/EtOAc, 1:1)]. Compound 12: 1H NMR (400 MHz, CDCl3)
δ: 8.68 (1H, dd, J = 1.0, 7.4 Hz, H-
9), 8.18 (1H, d, J = 8.0 Hz, H-7), 7.75-7.71 (2H, m), 7.54-7.47 (2H, m), 6.43 (1H, H-3), 5.19 (1H, br s, NH), 3.19 (2H, c, J
= 6.8, H-1’), 1.76 (2H, sext, J = 7.2 Hz, H-2’), 1.06 (3H, t, J = 7.4 Hz, H-3’); 13C NMR (100 MHz, CDCl3)
δ: 180.8 (s, C-
1), 142.0 (s, C-2), 135.7 (d), 132.1 (s), 130.5 (d), 130.4 (s), 128.2 (s), 127.3 (d), 127.2 (d), 126.8 (d), 126.5 (d), 123.6 (s),
105.3 (d, C-3), 44.91 (t), 105.3 (d), 44.9 (t, C-1’), 22.3 (t, C-2’), 11.9 (c, C-3’); EIMS: m/z 237 [M]+ (54), 209 (15), 208
(100), 167 (16); HRMS: calcd. for C16H15NO 237.1154, found 237.1160; UV-Vis (EtOH) λmax: 373, 341, 269, 240, 211, 202
15

ACCEPTED MANUSCRIPT
nm; IR (CHCl3) νmax: 3307, 1613, 1508, 839, 821 cm-1; mp: 62-64 °C. Compound 13: 1H NMR (400 MHz, CDCl3)
δ: 8.54
(1H, d, J = 7.2 Hz, H-9), 8.03-7.99 (2H, m, H-7, H-6), 7.84 (1H, d, J = 7.4 Hz, H-4), 7.67 (1H, dd, J = 7.6, 8.0 Hz, H-8),
7.55 (1H, dd, J = 7.6, 8.0 Hz, H-5), 5.86 (1H, s, H-2), 5.49 (1H, br s, NH), 3.29 (2H, c, H-1’), 1.80 (2H, sext, H-2’), 1.05
(3H, t, J = 7.4 Hz, H-3’); 13C NMR (100 MHz, CDCl3)
δ: 183.2 (s, C-1), 154.1 (s, C-3), 133.0 (d, C-7), 132.3 (s, C-6a),
132.2 (d, C-6), 129.5 (s, C-9a), 128.6 (d, C-9), 127.9 (s, C-9b), 127.5 (d, C-8), 126.1 (d, C-5), 124.5 (s, C-3a), 122.9 (d, C-
4), 99.5 (d, C-2), 45.3 (t, C-1’), 21.9 (t, C-2’), 11.7 (c, C-3’); EIMS: m/z 237.11 [M]+ (95), 208 (77), 195 (100), 151 (35);
HRMS: calcd. for C16H15NO 237.1154, found 237.1156; UV-Vis (EtOH) λmax: 373, 355, 340, 236, 219, 202 nm; IR (CHCl3)
νmax: 3271, 2920, 1546, 1356, 1270 cm-1; mp: 165-167 °C.
4.2.1.7. 2-[(2-Dimethylamino)ethyl]amino-1H-phenalen-1-one (14) and 3-[(2-dimethylamino)ethyl]amino-1H-
phenalen-1-one (15). Method B: using 2 (51.0 mg, 0.197 mmol) and N,N-dimethylethane-1,2-diamine (0.64 mL).
Purification using column chromatography (CH2Cl2/MeOH; 9:1 followed by CH2Cl2/MeOH/NH3, 90:9:1) gave compound
14 (8.7 mg, 17%) as a red oil [Rf 0.30 (CH2Cl2/MeOH, 9:1)] and compound 15 (6.7 mg, 13%) as a yellow oil [Rf 0.33
1
(CH2Cl2/MeOH/NH3, 90:9:1)]. Compound 14: H NMR (500 MHz, CDCl3)
δ: 8.68 (1H, dd, J = 0.8, 7.3 Hz, H-9), 8.17
(1H, d, J = 7.8 Hz, H-7), 7.72-7.71 (2H, m, H-6, H-8), 7.54-7.47 (2H, m, H-4, H-5), 6.45 (1H, s, H-3), 5.54 (1H, br s, NH),
3.32-3.29 (2H, m, H-1’), 2.66 (2H, t, J = 6.3 Hz, H-2’), 2.31 (6H, s, 2 x CH3); 13C NMR (125 MHz, CDCl3)
δ: 179.7 (s, C-
1), 141.0 (s, C-2), 134.7 (d, C-7), 131.1 (s, C-6a), 129.5 (d, C-9), 129.3 (s, C-9a), 127.2 (s, C-3a), 126.3 (d), 126.2 (d), 125.8
(d), 125.5 (d), 122.7 (s, C-9b), 104.6 (d, C-3), 56.7 (t, C-1’), 44.5 (c, 2 x C, CH3), 39.7 (t, C-2’); EIMS: m/z 266 [M]+ (7),
208 (6), 58 (100); HRMS: calcd. for C17H18N2O 266.1419, found 266.1405; UV-Vis (EtOH) λmax: 339, 268, 237, 211, 202
1
nm; IR (CHCl3) νmax: 3384, 3274, 1614, 1509, 1462, 1357, 843 cm-1. Compound 15: H NMR (400 MHz, CDCl3)
δ: 8.56
(1H, dd, J = 7.2 Hz, H-9), 8.02 (2H, dd, J = 1.2, 8.2 Hz), 8.01 (1H, dd, J = 0.5, 8.1 Hz), 7.89 (1H, dd, J = 0.5, 7.1 Hz), 7.66
(1H, dd, J = 7.2, 8.0 Hz), 7.56 (1H, dd, J = 7.5, 8.0 Hz), 6.38 (1H, br s, NH), 5.82 (1H, s, H-2), 3.33 (2H, m, H-1’), 2.69
(2H, t, J = 5.8 Hz, H-2’), 2.32 (6H, s, 2 x CH3); 13C NMR (100 MHz, CDCl3)
132.3 (s), 132.2 (d), 129.6 (s), 128.7 (d), 128.0 (s), 126.9 (d), 125.5 (d), 124.5 (s), 123.1 (d), 99.9 (d, C-2), 56.74 (t, C-1’),
δ: 183.3 (s, C-1), 153.9 (s, C-3), 133.1 (d),
45.05 (c, 2 x C, CH3), 39.92 (t, C-2’); EIMS: m/z 266 [M]+ (1), 262 (5), 220 (19), 219 (15), 58 (100); UV-Vis (EtOH) λmax
371, 339, 236 nm; IR (CHCl3) νmax: 3271, 1550, 1365, 1272, 819 cm-1.
:
4.2.1.8. 2-(Benzylamino)-1H-phenalen-1-one (16) and 3-(benzylamino)-1H-phenalen-1-one (17). Method B: Using 2
(93.0 mg, 0.36 mmol,) and benzylamine (2.0 mL). Purification using column chromatography (hexane/EtOAc; 1:2) gave
16

ACCEPTED MANUSCRIPT
compound 16 (30.0 mg, 30%) as a red solid [Rf 0.95 (hexane/EtOAc, 1:2)] and compound 17 (20.0 mg, 20%) as an orange
1
solid [Rf 0.40 (hexane/EtOAc, 1:2)]. Compound 16: H NMR (400 MHz, CDCl3)
δ: 8.70 (1H, dd, J = 0.8, 7.2 Hz, H-9),
8.18 (1H, d, J = 8 Hz, H-7), 7.75-7.71 (2H, m), 7.48-7.45 (2H, m), 7.39-7.26 (5H, m, Ar), 6.44 (1H, s, H-3), 5.64 (1H, br s,
NH), 4.47 (2H, d, J = 5.6 Hz, H-1’); 13C NMR (100 MHz, CDCl3)
δ: 180.7(s, C-1), 141.7 (s, C-2), 138.5 (s), 135.8 (d),
132.1 (s), 130.6 (d), 130.1 (s), 128.8 (d, 2 x C), 128.1 (s), 127.6 (d), 127.5 (d, 3 x C), 127.3 (d), 127.2 (d), 126.6 (d), 123.8
(s), 106.5 (d, C-3), 47.3 (t, C-1’); EIMS: m/z 286 (21), 285 [M]+ (100), 284 (24), 91 (28); HRMS: calcd. for C20H15NO
285.1154, found 285.1159; UV-Vis (EtOH) λmax: 350, 270, 239, 211, 205, 201 nm; IR (CHCl3) νmax: 3370, 1609, 1490, 831
cm-1; mp: 124-126 ºC. Compound 17: 1H NMR (400 MHz, CDCl3)
δ: 8.56 (1H, dd, J = 1.0, 7.3 Hz, H-9), 8.05 (2H, m, H-7,
H-6), 7.81 (1H, d, J = 7.2 Hz, H-4), 7.69 (1H, dd, J = 7.6, 7.6 Hz, H-8), 7.56 (1H, dd, J = 7.6, 8.4 Hz, H-5), 7.35-7.28 (5H,
m, Ar), 5.94 (1H, s, H-2), 5.51 (1H, br s, NH), 4.51 (2H, d, J = 5.0 Hz, H-1’); 13C NMR (100 MHz, CDCl3)
δ: 182.6 (s, C-
1), 152.3 (s, C-3), 136.0 (s), 132.3 (d), 131.5 (s), 131.4 (d), 128.5 (s), 128.2 (d, 2 x C), 128.0 (d), 127.3 (d), 127.2 (d, 2 x C),
127.1 (s), 126.1 (d), 124.5 (d), 123.3 (s), 121.6 (d), 99.9 (d, C-2), 47.0 (t, C-1’); EIMS: m/z 285 [M]+ (100), 284 (39), 91
(63); HRMS: calcd. for C20H15NO 285.1154, found 285.1151; UV-Vis (EtOH) λmax: 340, 237, 236 nm; IR (CHCl3) νmax
:
3267, 1550, 1362, 1266, 841 cm-1; mp: 218-220 ºC.
4.2.1.9. 2-[(2-Hydroxyethyl)amino]-1H-phenalen-1-one (18). Method B: Using 2 (51 mg, 0.197 mmol) in 2-
aminoethanol (0.7 mL). Purification using preparative thin layer chromatography (CH2Cl2/MeOH; 9:1) gave the
intermediate 8-(2-Hydroxyethyl)-8,8a-dihydrophenalen[1,2-b]aziren-7(7aH)-one (14 mg, 33%) as a pink oil: 1H NMR (400
MHz, CDCl3) δ: 8.32 (1H, dd, J = 0.9, 7.2 Hz, H-9), 8.07 (1H, d, J = 8.1 Hz, H-7), 7.84 (1H, d, J = 8.3 Hz, H-6), 7.72 (1H,
d, J = 6.9 Hz, H-4), 7.59 (1H, dd, J = 7.9, 7.6 Hz, H-8), 7.50 (1H, dd, J = 7.2, 7.1 Hz, H-5), 3.79 (2H, s, H-2’), 3.38 (1H, d,
J = 5.8 Hz, H-8a), 2.98 (1H, d, J = 5.8 Hz, H-7a), 2.86 (1H, m, H-1’), 2.73 (1H, m, H-1’); 13C RMN (100 MHz, CDCl3 )
δ:
193.5 (s, C-1), 134.7 (d), 133.5 (s), 130.5 (s), 129.6 (s), 128.3 (d), 127.7 (d), 127.5 (d), 126.3 (d), 126.0 (d), 62.3 (d, C-2),
62.0 (d, C-3), 46.8 (t, C-2’), 46.3 (t, C-1’); EIMS: m/z 239 [M]+ (100), 208 (30), 195 (26), 168 (69), 139 (34); HRMS:
calcd. for C15H13NO2 239.0946, found 239.0954; UV-Vis (EtOH) λmax: 334, 321, 250, 213 nm; IR (CHCl3) νmax: 3410, 1672,
1346, 1113 cm-1. To a solution of 8-(2-hydroxyethyl)-8,8a-dihydrophenalen[1,2-b] aziren-7(7aH)-one (13.5 mg, 0.056
mmol) in CH2Cl2 (1.0 mL) was added conc. HCl (0.5 mL). The reaction mixture was stirred for 30 min at room temperature
after which a 10% NaOH solution (aq) (0.5 mL) was added and the mixture was extracted with EtOAc (3 x 5 mL), dried
(MgSO4), filtered and concentrated. Purification using preparative thin layer chromatography (CH2Cl2/MeOH; 9:1) gave 18
(9 mg, 68%) as a red oil with Rf 0.5 (CH2Cl2/MeOH, 9:1). 1H NMR (400 MHz, CDCl3)
δ: 8.66 (1H, dd, J = 0.96, 7.4 Hz, H-
17

ACCEPTED MANUSCRIPT
9), 8.18 (1H, d, J = 7.9 Hz, (1H, t, J = 7.7 Hz, H-8), 7.55 (1H, d, J = 6.6 Hz, H-4), 7.50 (1H, t, J = 7.2 Hz, H-5), 6.53 (1H, s,
H-3), 3.89 (2H, t, J = 5.4 Hz, H-2'), 3.39 (2H, t, J = 5.1 Hz, H -1'); 13C NMR (100 MHz, CDCl3)
δ: 180.9 (s, C-1), 141.8 (s,
C-2), 135.9 (d, C-7), 131.9 (s, C-9), 129.8 (s, C-9a), 127.9 (s, C-9c), 127.7 (d, C-8), 127.3 (d, 2 x C, C-5, C-4), 126.5 (d, C-
6), 123.5 (s, C-9b), 106.6 (d, C-3), 60.5 (t, C-1'), 45.1 (t, C-2'); EIMS: m/z 239 [M]+ (40), 208 (100), 167 (28), 152 (21);
HRMS: calcd. for C15H13NO2 239.0946, found 239.0939; UV-Vis (EtOH) λmax: 496, 371, 338, 295, 267, 236, 209, 204 nm;
IR (CHCl3) νmax: 3415, 3367, 1597, 1513, 1488, 1053, 840 cm-1.
4.2.2. 2-Arylamino-1H-phenalen-1-one derivatives. General procedure. To a solution of 2-bromo-1H-phenalen-1-one
(2), NaOtBu and Pd(PPh3)4 in anhydrous toluene was added the arylamine. The reaction mixture was heated at 80 °C for 2-
96 h in the dark, under an atmosphere of argon. The reaction was cooled to rt, CH2Cl2 (10 mL) was added and the mixture
was filtered through Celite®. The solvent was removed and the product was purified by column chromatography or
preparative thin layer chromatography.
4.2.2.1. 2-Anilino-1H-phenalen-1-one (19). Using 2 (50 mg, 0.193 mmol), NaOtBu (26 mg, 0.270 mmol), Pd(PPh3)4
(22.3 mg, 0.02 mmol), anhydrous toluene (0,75 mL) and aniline (40
µ
L, 0.232 mmol); 80 °C for 2 h. Purification using
1
column chromatography (hexane/EtOAc; 4:1) gave compound 19 (45.9 mg, 88%) as a red solid. H NMR (400 MHz,
CDCl3) δ: 8.67 (1H, dd, J = 1.2, 7.6 Hz, H-9), 8.15 (1H, d, J = 8 Hz, H-7), 7.74 (1H, d, J = 8.0 Hz, H-6), 7.70 (1H, dd, J
=7.6, 8.0 Hz, H-8), 7.50-7.43 (2H, m, H-5, H-4), 7.34-7.30 (2H, m, H-3’, H-5’), 7.26-7.22 (2H, m, H-2’, H-6’), 7.19 (1H, s,
H-3), 6.99 (1H, t, J = 7.2 Hz, H-4’); 13C NMR (100 MHz, CDCl3)
δ: 180.6 (s, C-1), 140.9 (s, C-1’), 137.8 (s, C-2), 136.1
(d), 132.0 (s), 130.9 (d), 129.62 (d, 2 x C, C-3’, C-5’), 129.4 (s), 128.6 (d), 128.1 (d), 128.0 (s), 127.3 (d), 126.8 (d), 124.1
(s), 122.7 (d, C-4’), 120.5 (d, 2 x C, C-2’, C-6’), 109.3 (d, C-3); EIMS: m/z 272 (19), 271 [M]+ (100), 270 (34); HRMS:
calcd. for C19H13NO 271.0997, found 271.1008.
4.2.2.2. 4-[(1-Oxo-1H-phenalen-2-yl)amino]benzonitrile (20). Using 2 (102.0 mg, 0.393 mmol), NaOtBu (52.4 mg,
0.545 mmol), Pd(PPh3)4 (45.5 mg, 0.039 mmol), anhydrous toluene (1.5 mL) y 4-aminobenzonitrile (58.1 mg); 80 ºC for 17
h. Purification using column chromatography (toluene/EtOAc; 95:5) gave compound 20 (30.0 mg, 26%) as a dark red solid.
1H NMR (400 MHz, CDCl3)
δ: 8.76 (1H, dd, J = 1.2, 7.6 Hz, H-9), 8.28 (1H, dd, J = 0.8, 8 Hz, H-7), 7.92 (1H, d, J = 8 Hz),
7.82 (1H, t, J = 7.6 Hz), 7.69 (1H, d, J = 7.0 Hz), 7.70-7.58 (4H, m), 7.52 (1H, s, H-3), 7.32 (2H, d, J = 8.8 Hz, H-2’, H-6’);
13C NMR (100 MHz, CDCl3)
δ: 180.3 (s, C-1), 145.3 (s, C-4’), 136.5 (d), 135.7 (s, C-2), 133.9 (d, 2 x C, C-2’, C-6’), 132.1
(s), 131.4 (d), 129.9 (d), 129.5 (d), 128.3 (s), 127.7 (s), 127.4 (d), 127.1 (d), 124.5 (s), 119.5 (s, CN), 118.3 (d, 2 x C, C-3’,
18

ACCEPTED MANUSCRIPT
C-5’), 113.5 (d, C-3), 103.9 (s, C-1’); EIMS: m/z 297 (22), 296 [M]+ (100), 295 (35), 279 (20); HRMS: calcd. for
C20H12N2O 296.0950, found 296.0954; UV-Vis (EtOH) λmax: 483, 271, 319, 242, 213, 208, 203 nm; IR (CHCl3) νmax: 3306,
2209, 1587, 1509, 1168, 820 cm-1; mp: 217-219 ºC.
4.2.2.3. 2-(Pyridin-3-ylamino)-1H-phenalen-1-one (21). Using 2 (51.4 mg, 0.198 mmol), NaOtBu (26.3 mg, 0.274
mmol), Pd(PPh3)4 (24.8 mg, 0.021 mmol), anhydrous toluene (0.75 mL) and pyridin-3-amine (27.6 mg, 0.293 mmol); 80 °C
for 3 h. Purification using column chromatography (hexane/EtOAc; 3:2) gave compound 21 (30.9 mg, 63%) as a red solid.
1H NMR (400 MHz, CDCl3)
δ: 8.68 (1H, d, J = 7.6 Hz, H-9), 8.62 (1H, d, J = 2.4 Hz, H-2’), 8.28 (1H, dd, J = 1.0, 4.1 Hz),
8.18 (1H, d, J = 8 Hz, H-7), 7.81 (1H, d, J = 8 Hz, H-6), 7.73 (1H, t, J = 7.6 Hz, H-5), 7.59-7.48 (3H, m), 7.29-7.24 (3H,
m); 13C NMR (100 MHz, CDCl3)
δ: 180.2 (s, C-1), 143.5 (d), 142.4 (d), 137.6 (s), 137.0 (s), 136.2 (d), 131.9 (s), 131.1 (d),
129.1 (d), 128.7 (d), 128.7 (s), 127.7 (s), 127.3 (d), 126.8 (d), 126.6 (d), 124.2 (s), 123.9 (d), 110.3 (d, C-3); EIMS: m/z 273
(18), 272 [M]+ (100), 271 (61), 255 (40); HRMS: calcd. for C18H12N2O 272.0950, found 272.0962; UV-Vis (EtOH) λmax
486, 369, 351, 289, 245, 236, 210, 202 nm; IR (CHCl3) νmax: 3305, 1617, 1517, 839 cm-1; mp: 170-172 ºC.
:
4.2.2.4. 2-(Pyridin-2-ylamino)-1H-phenalen-1-one (22). Using 2 (100 mg, 0.386 mmol), NaOtBu (52.1 mg, 0.542
mmol), Pd(PPh3)4 (52.1 mg, 0.041mmol), anhydrous toluene (1.5 mL) and pyridin-2-amine (260.1 mg, 0.638 mmol); 80 °C
for 2 days and 17 h. Purification using column chromatography (toluene/EtOAc; 95:5) gave compound 22 (15.1 mg, 14%)
as a red solid. 1H NMR (400 MHz, CDCl3)
δ: 8.96 (1H, s, H-3), 8.76 (1H, d, J = 7.2 Hz, H-9), 8.39 (1H, d, J = 4.1 Hz),
8.25 (1H, d, J = 8.0 Hz), 8.02 (1H, br s, NH), 7.88 (1H, d, J = 8.2 Hz), 7.82-7.77 (2H, m), 7.61-7.56 (2H, m), 6.87 (1H, d, J
= 8.3 Hz), 6.85-6.81 (1H, m); 13C NMR (100 MHz, CDCl3)
δ: 180.4 (s, C-1), 155.1 (s, C-2’), 147.8 (d, C-6’), 137.4 (d),
136.1 (d), 135.3 (s), 132.0 (s), 131.1 (d), 130.4 (d), 129.4 (s), 128.9 (d), 127.8 (s), 127.4 (d), 126.7 (d), 124.5 (s), 117.0 (d),
115.7 (d), 112.4 (d, C-3); EIMS: m/z 296 (20), 295 [M + Na]+ (100), 273 (31); HRMS: calcd. for C21H17NO 273.1028,
found 273.1033; UV-Vis (EtOH) λmax: 480, 367, 352, 284, 237, 210, 203 nm. IR (CHCl3) νmax: 3272, 1590, 1476, 1334, 839
cm-1; mp: 235-237 ºC.
4.2.3. 9-amino-1H-phenalen-1-one derivatives.
4.2.3.1. 9-Amino-1H-phenalen-1-one (24). A solution of 23 (30 mg, 0.153 mmol) in 25% aqueous ammonia (0.61 mL)
was heated at 125 °C for 1 h using MW irradiation. Cooled to rt and H2O was added (10 mL). The resulting mixture was
extracted with CH2Cl2 (3x10 mL), dried (Na2SO4), filtered and concentrated. Purification using column chromatography
19

ACCEPTED MANUSCRIPT
1
(alumina) (toluene/CH2Cl2; 9:1 to 7:3) gave compound 24 (21 mg, 70%) as a yellow solid. H NMR (400 MHz, CDCl3) δ:
10.95 (1H, br s, NH), 7.93 (1H, d, J = 9.0 Hz), 7.89 (1H, d, J = 7.8 Hz), 7.85 (2H, d, J = 9.0 Hz), 7.45 (1H, dd, J = 7.6, 7.6
Hz, H-5), 7.01 (1H, d, J = 9.0 Hz), 6.95 (1H, d, J = 9.5 Hz), 5.66 (1H, br s, NH); 13C NMR (100 MHz, CDCl3)
δ: 185.6 (s,
C-1), 155.8 (s, C-9), 139.2 (d), 137.7 (d), 131.7 (d), 131.5 (d), 129.2 (d), 128.6 (s), 125.6 (s), 125.2 (s), 122.3 (d), 119.7 (d),
108.6 (s); EIMS: m/z 197 (19), 203 (31), 196 [M + 1]+ (100); HRMS: calcd. for C13H9NO 196.0762, found 196.0759.
4.2.3.2. 9-(Propylamino)-1H-phenalen-1-one (25). A solution of 23 (31.7 mg, 0.161 mmol) in 1-propanamine (1 mL)
was heated at 125 °C for 1 h using MW irradiation. Cooled to rt and H2O was added (10 mL). The resulting mixture was
extracted with CH2Cl2 (3x10 mL), dried (Na2SO4), filtered and concentrated. Purification using column chromatography
(alumina) (toluene/CH2Cl2; 9:1) gave compound 25 (19.6 mg, 51%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
δ: 12.3
(1H, br s, NH), 7.99 (1H, d, J = 9.4 Hz), 7.89-7.83 (3H, m), 7.43 (1H, dd, J = 8.8, 6.8 Hz), 7.22 (1H, d, J = 9.3 Hz), 7.00
(1H, d, J = 9.4 Hz), 3.53 (2H, q, J = 6.7 Hz, H-1'), 1.84 (2H, sext, J = 7.2 Hz, H-2'), 1.14 (3H, t, J = 7.3 Hz, H-3'); EIMS:
m/z 239 (21), 238 [M + 1]+ (100), 227 (20); HRMS: calcd. for C16H16NO 238.1232, found 238.1229.
4.2.3.3. 9-[(2-Hydroxyethyl)amino]-1H-phenalen-1-one (26). A solution of 23 (32 mg, 0.163 mmol) in 2-aminoethanol
(16.6 mmol, 1 mL) was heated at 150 °C for 20 min using MW irradiation. Cooled to rt and H2O was added (10 mL). The
resulting mixture was extracted with CH2Cl2 (3x10 mL), dried (Na2SO4), filtered and concentrated. Purification using
preparative thin layer chromatography (CH2Cl2/MeOH; 9.8:0.2) gave 26 (21.3 mg, 55%) as an orange solid. 1H NMR (400
MHz, (CD3)2CO) δ: 12.38 (1H, br s, NH), 8.14 (1H, d, J = 9.6 Hz, H-3), 8.00 (1H, d, J = 8 Hz), 7.96-7.92 (2H, m), 7.48
(1H, d, J = 9.6 Hz, H-2), 7.46 (1H, t, J = 8 Hz, H-5), 6.85 (1H, d, J = 9.2 Hz), 4.22 (1H, t, J = 5.6 Hz, OH), 3.92 (2H, c, J =
5.6 Hz, H-2’), 3.77 (2H, c, J = 5.6 Hz, H-1’); 13C NMR (100 MHz, (CD3)2CO)
δ: 184.7 (s, C-1), 157.2 (s, C-9), 139.0 (d),
138.8 (d), 132.4 (d), 132.2 (d), 129.7 (d), 129.3 (s), 125.9 (s), 125.3 (s), 122.5 (d), 115.9 (d), 108.7 (s), 61.6 (t, CH2), 46.0 (t,
CH2); EIMS: m/z 303 (31), 262 [M + Na]+ (100); HRMS: calcd. for C15H13NO2Na 262.0844, found 262.0843; UV-Vis
(EtOH) λmax: 473, 445, 420, 355, 340, 269, 252, 203 nm; IR (CHCl3) νmax: 3261, 1635, 1524, 1234, 1145, 839 cm-1; mp:
153-155 °C.
4.2.3.4. 1-Oxo-1H-phenalen-9-trifluromethylsulfonate (28). To a solution of 23 (400 mg, 2.04 mmol) in toluene (20 mL)
was added a solution of K3PO4 30% (20 mL, 8.6 g, 40.8 mmol). The mixture was cooled to -10 °C and then triflic anhydride
(0.42 mL, 2.448 mmol) was added slowly and stirred at room temperature for 5 h. The aqueous phase was extracted with
toluene (3 x 15 mL) and the combined organics were dried (MgSO4), filtered and concentrated. Purification using column
20

ACCEPTED MANUSCRIPT
chromatography (hexane/EtOAc; 9:1 to 7:3) gave 28 (575 mg, 86%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
δ: 8.29
(1H, d, J = 8.8 Hz), 8.07 (1H, d, J = 8.2 Hz), 7.87 (1H, d, J = 7 Hz), 7.76 (1H, d, J = 9.8 Hz), 7.70 (1H, dd, J = 7.8, 7.5 Hz),
7.57 (1H, d, J = 8.8, Hz), 6.74 (1H, d, J = 9.8 Hz); EIMS: m/z 139 (100), 166 (31), 195 (35), 196 (16). 236 (32) 327 (73),
328 [M]+ (14); HRMS: calcd. for C14H7F3O4S 328.0017, found 327.0019; UV-Vis (EtOH) λmax: 203, 239, 250, 256, 263,
307, 340, 355, 382 nm.
4.2.3.5. 9-(Benzylamino)-1H-phenalen-1-one (27). Using 23: A solution of 23 (20.0 mg, 0.10 mmol) in benzylamine
(0.6 mL) was heated at 120 °C for 6 min using MW irradiation. Cooled to rt and 10% HCl (aq) was added until pH = 7 was
attained. The mixture was extracted with EtOAc (2 x 3 mL), washed with brine (2 x 3 mL), dried (Na2SO4), filtered and the
concentrated. Purification using column chromatography (hexane/EtOAc; 4:1) gave 27 (16.7 mg, 57%) as a yellow solid.
From 28: A solution of 28 (62 mg, 0.188 mmol,) and benzylamine (1 mL) was stirred at room temperature for 2 h. Cooled
to rt and the amine was removed under reduced pressure. Purification as above, gave 27 (49.3 mg, 91%) as a yellow solid.
1H NMR (400 MHz, CDCl3)
(1H, d, J = 10.0 Hz), 7.10 (1H, d, J = 9.6 Hz), 4.48 (2H, d, J = 5.6 Hz, H-1’); 13C NMR (100 MHz, CDCl3)
δ: 12.3 (1H, br s, NH), 7.99 (1H, d, J = 10.0 Hz), 7.86-7.25 (3H, m), 7.50-7.25 (6H, m), 7.18
δ: 182.6 (s, C-
1), 152.3 (s, C-9), 136.0 (s), 132.3 (d), 131.4 (s), 131.3 (d), 128.5 (s), 128.2 (s, 2 x C), 128.0 (d), 127.3 (d), 127.2 (d, 2 x C),
127.0 (s), 126.1 (d), 124.5 (d), 123.3 (s), 121.6 (d), 99.8 (d), 47.0 (t, C-1’); EIMS: m/z 286 (22), 285 [M]+ (100), 268 (25);
HRMS: calcd. for C20H15NO 285.1154, found 285.1157.
4.2.3.6. 9-{[2-(Dimethylamino)ethyl]amino}-1H-phenalen-1-one (29). A solution of 28 (62 mg, 0.188 mmol) and N,N-
dimethylethane-1,2-diamine (0.5 mL) was stirred at room temperature for 1 h, after which H2O (5 mL) was added. The
mixture was lyophilized. Purification using column chromatography (CH2Cl2/MeOH /NH4OH; 90:9:1) gave compound 29
1
(48.5 mg, 96%) as an orange oil. H NMR (500 MHz, CDCl3)
δ: 12.24 (1H, br s, NH), 7.98 (1H, d, J = 9.3 Hz), 7.88-7.84
(2H, m), 7.82 (1H, d, J = 9.4 Hz, H-3), 7.41 (1H, t, J = 7.5 Hz, H-5), 7.19 (1H, d, J = 9.3 Hz), 6.99 (1H, d, J = 9.4 Hz, H-2),
3.64 (2H, td, J = 6.7, 5.5 Hz, H-1’), 2.73 (2H, t, J = 6.7 Hz, H-2’), 2.37 (6H, s, H-1’’); 13C NMR (125 MHz, CDCl3)
δ:
184.4 (s, C-1), 156.0 (s, C-9), 138.6 (d), 138.2 (d), 131.8 (d), 131.4 (d), 129.1 (d), 128.5 (s), 125.3 (s), 124.4 (s), 121.8 (s),
114.5 (d), 108.3 (s), 58.3 (t, C-1’), 45.7 (c, 2 x C, CH3), 41.1 (t, C-2’); EIMS: m/z 266 [M]+ (2), 208 (6), 58 (100); HRMS:
calcd. for C17H18N2O 266.1419, found 266.1416; UV-Vis (EtOH) λmax: 473, 445, 420, 355, 341, 251, 203 nm; IR (CHCl3)
νmax: 3478, 1636, 1593, 1528, 848 cm-1.
21

4.2.3.7. 9-[(2-Hydroxyethyl)(methyl)amiAnoC]-1CHE-pPhTenEalDen-M1-AonNe U(30S)C. ARIsPoTlution of 28 (55 mg, 0.168 mmol) and 2-
(methylamino)ethanol (0.6 mL) was stirred at room temperature for 30 min. Cooled to rt, and H2O (5 mL) was added.
Lyophilization was used to remove the solvents. Purification using column chromatography (EtOAc/MeOH; 10:1) gave 30
(42 mg, 98%) as an orange oil. 1H NMR (400 MHz, CDCl3)
δ: 7.94 (1H, d, J = 9.3 Hz), 7.87 (1H, d, J = 7.8 Hz, H-6), 7.80
(2H, m), 7.56 (1H, d, J = 9.3 Hz), 7.46 (1H, dd, J = 7.4, 7.6 Hz, H-5), 6.86 (1H, d, J = 9.4 Hz, H-2), 3.85 (2H, m, H-2’),
3.79 (2H, m, H-1’), 3.02 (3H, s, H-1’’); 13C NMR (100 MHz, CDCl3)
δ: 182.9 (s, C-1), 157.2 (s, C-9), 138.7 (d), 135.3 (d),
131.0 (d), 130.7 (d), 129.8 (d), 129.7 (s), 126.3 (s), 126.3 (s), 123.1 (d), 122.2 (d), 114.2 (s), 58.2 (t, C-2’), 56.6 (t, C-1’),
41.4 (C, C-1’’); EIMS: m/z 253 [M]+ (37), 222 (100), 206 (62), 196 (66); HRMS: calcd. for C16H15NO2 253.1103, found
253.1100; UV-Vis (EtOH) λmax: 472, 447, 362, 348, 284, 255 nm; IR (CHCl3) νmax: 3345, 1628, 1533, 1216, 841cm-1
4.2.3.8. 9-(Piperidin-1-yl)-1H-fenalen-1-one (31). A solution of 28 (60 mg, 0.183 mmol) and piperidine (0.6 mL) was
stirred at room temperature for 30 min. Cooled to rt, and H2O (5 mL) was added. Lyophilization was used to remove the
solvents. Purification using column chromatography (hexane/EtOAc; 1:4) gave 31 (46 mg, 96%) as a red oil. 1H NMR (400
MHz, CDCl3) δ: 7.90 (1H, d, J = 9.3 Hz, H-7), 7.82 (1H, d, J = 7.3 Hz, H-6), 7.73 (1H, d, J = 7.1 Hz, H-4), 7.68 (1H, d, J =
9.4 Hz, H-3), 7.43 (1H, d, J = 9.3 Hz, H-8), 7.38 (1H, t, J = 7.6 Hz, H-5), 6.81 (1H, d, J = 9.4 Hz, H-2), 3.46 (4H, m, H-2’,
H-6’), 1.80 (4H, m, H-3’, H-5’), 1.75 (2H, m, H-4’); 13C NMR (100 MHz, CDCl3 )
δ: 182.1 (s, C-1), 155.8 (s, C-9), 137.8
(d), 135.6 (d), 130.9 (d), 130.6 (d), 130.6(d), 130.3 (s), 126.5 (s), 126.0 (s), 122.5 (d), 120.4 (d), 113.3 (s), 53.2 (t, 2 x C, C-
2’, C-6’), 26.5 (t, 2 x C, C-3’, C-5’), 24.3 (t, C-4’); EIMS: m/z 263 [M]+ (100), 246 (46), 234 (25), 206 (23), 180 (17), 152
(37); HRMS: calcd. for C18H17NO 263.1310, found 263.1299; UV-Vis (EtOH)
λ max: 494, 475, 366, 351, 282, 258, 230,
203, 201 nm; IR (CHCl3) ν max: 3523, 1629, 1594, 1236, 1031, 841 cm-1
4.2.3.9. 9-(Morpholin-4-yl)-1H-phenalen-1-one (32). A solution of 28 (61 mg, 0.186 mmol) and morpholine (0.5 mL)
was stirred at room temperature for 1 h, after which the solvent was removed. Purification using column chromatography
(hexane/EtOAc; 1:1) gave 32 (46.4 mg, 94%) as an orange solid. 1H NMR (500 MHz, CDCl3)
δ: 8.04 (1H, d, J = 9.2, H-7),
7.90 (1H, dd, J = 7.9, 1.0 Hz ), 7.79 (1H, dd, J = 7.3, 1.0 Hz), 7.72 (1H, d, J = 9.5 Hz, H-3), 7.46 (1H, dd, J = 7.6, 7.5 Hz,
H-5), 7.43 (1H, d, J = 9.2 Hz), 6.80 (1H, d, J = 9.5 Hz, H-2), 4.04 (4H, t, J = 4.6 Hz, H-3’, H-5’), 3.45 (4H, t, J = 4.6 Hz, H-
2’, H-6’); 13C NMR (125 MHz, CDCl3)
δ: 182.9 (s, C-1), 155.6 (s, C-9), 138.4 (d), 136.5 (d), 131.5 (d), 131.2 (d), 130.7
(d), 130.1 (s), 127.0 (s), 126.8 (s), 123.4 (d), 119.7 (d), 114.8 (s), 67.2 (t, 2 x C, C-3’, C-5’), 52.4 (t, 2 x C, C-2’, C-6’);
EIMS: m/z 265 [M]+ (91), 208 (100), 206 (37), 152 (56); HRMS: calcd. for C17H15NO2 265.1103, found 265.1098; UV-Vis
22

ACCEPTED MANUSCRIPT
(EtOH) λmax: 490, 467, 366, 351, 282, 256, 230, 201 nm; IR (CHCl3) νmax: 3542, 3044, 1359, 1066, 977, 834 cm-1; mp: 140-
142 °C.
4.2.3.10. 9-(4-Benzyl-piperazin-1-yl)-1H-phenalen-1-one (33). A solution of 28 (60 mg, 0.183 mmol) and 1-
benzylpiperazine (1 mL) was stirred at room temperature for 30 min. Cooled to rt, and H2O (5 mL) was added.
Lyophilization was used to remove the solvents. Purification using column chromatography (EtOAc/MeOH; 10:1) gave 33
(45 mg, 70%) as an orange oil. 1H NMR (400 MHz, CDCl3)
δ: 7.94 (1H, d, J = 9.2 Hz), 7.83 (1H, d, J = 7.8 Hz), 7.73 (1H,
d, J = 6.8 Hz), 7.67 (1H, d, J = 9.5 Hz, H-3), 7.42-7.24 (6H, m), 6.79 (1H, d, J = 9.5 Hz, H-2), 3.60 (2H, s, H-1’’), 3.48
(4H, t, J = 4.6 Hz, H-2’, H-6’), 2.74 (4H, t, J = 4.8 Hz, H-3’, H-5’); 13C NMR (100 MHz, CDCl3)
δ: 182.5 (s, C-1), 155.5
(s, C-9), 138.1 (d), 137.9 (s), 136.0 (d), 131.2 (d), 130.8 (d), 130.6 (d), 130.1 (s), 129.4 (d, 2 x C), 128.4 (d, 2 x C), 127.3
(d), 126.7 (s), 126.4 (s), 122.9 (d), 120.0 (d), 114.2 (s), 63.1 (t), 53.3 (t, 2 x C, C-2’, C-6’), 51.9 (t, 2 x C, C3’, C5’); EIMS:
m/z 354 [M]+ (21), 221 (19), 146 (100), 91 (55); HRMS: calcd. for C24H22N2O 354.1732, found 354.1730; UV-Vis (EtOH)
λmax: 491, 471, 366, 352, 279, 256, 203 nm; IR (CHCl3) νmax: 3476, 1630, 1539, 1295, 843 cm-1.
4.2.3.11. 9-[(4-Pyrimidin-2-yl)piperazin-1-yl]-1H-phenalen-1-one (34). A solution of 28 (60 mg, 0.183 mmol) and 2-
piperazin-1-yl-pyrimidine (1 mL) was stirred at room temperature for 30 min. Cooled to rt, and H2O (5 mL) was added.
Lyophilization was used to remove the solvents. Purification using column chromatography (hexane/EtOAc; 2:3 to EtOAc)
gave compound 34 (57 mg, 91%) as an orange solid. 1H RMN (400 MHz, CDCl3)
δ: 8.35 (2H, d, J = 4.7 Hz, H-4’’, H-6’’),
8.04 (1H, d, J = 9.2 Hz), 7.90 (1H, d, J = 7.9 Hz, H-4), 7.80 (1H, d, J = 7.1 Hz, H-3), 7.75 (1H, d, J = 9.5 Hz, H-3), 7.49
(1H, d, J = 9.2 Hz), 7.47 (1H, t, J = 7.5 Hz, H-5), 6.84 (1H, d, J = 9.5 Hz, H-2), 6.53 (1H, t, J = 4.7 Hz, H-2’, H-5’’), 4.17
(4H, t, J = 4.9 Hz), 3.58 (4H, t, J = 4.7 Hz); 13C RMN (100 MHz, CDCl3)
δ: 182.8 (s, C-1), 161.3 (s, C-9), 157.8 (d, 3 x C),
155.8 (s), 138.7 (d), 136.6 (d), 131.7 (d), 131.2 (d), 129.8 (s), 129.7 (s), 126.4 (s), 126.3 (s), 123.1 (d), 119.4 (d), 113.6 (s),
110.2 (d), 51.2 (t, 2 x C), 43.5 (t, 2 x C); EIMS: m/z 342 [M]+ (57), 234 (40), 221 (63), 208 (98), 206 (33), 152 (35), 134
(100); HRMS: calcd. for C21H18N4O 342.1481, found. 342.1476; UV-Vis (EtOH) λmax: 479, 366, 351, 280, 249, 202 nm; IR
(CHCl3) νmax: 1585, 1496, 1234, 964, 843 cm-1; mp: 223-225 °C.
4.2.3.12. 9-(Phenylamino)-1H-phenalen-1-one (35). A solution of 28 (48.5 mg, 0.148 mmol) and aniline (0.6 mL) was
stirred at room temperature for 4 h. Purification using column chromatography (hexane/CH2Cl2; 1:1 to 1:9) gave 35 (39 mg,
97%) as an orange solid. 1H NMR (500 MHz, CDCl3)
δ: 13.78 (1H, br s, NH), 7.92 (1H, d, J = 9.3 Hz), 7.91-7.88 (3H, m),
7.52-7.45 (4H, m), 7.41-7.39 (2H, m), 7.29 (1H, t, J = 7.3 Hz, H-5), 7.03 (1H, d, J = 9.4 Hz, H-2).
23

ACCEPTED MANUSCRIPT
4.2.3.13. 4-[(1-Oxo-1H-Phenalen-9-yl)amino]benzonitrile (36). To a solution of 28 (50 mg, 0.152 mmol) in anhydrous
THF (1 mL) was added 4-aminobenzonitrile (75 mg, 0.635 mmol) and the reaction was heated at 50 ºC for 24 h. Solvent
removal followed by purification using column cromatography (hexane/EtOAc; 9:1 to 4:1) gave 36 (41.0 mg, 91%) as an
orange solid. 1H NMR (500 MHz, CDCl3)
δ: 13.93 (1H, br s, NH), 8.07 (1H, d, J = 9.2 Hz), 7.98 (1H, d, J = 7.6 Hz), 7.94
(1H, d, J = 7.1 Hz), 7.93 (1H, d, J = 9.4 Hz, H-3), 7.73 (2H, d, J = 8.5, H-3’, H-5’), 7.67 (1H, d, J = 9.2 Hz), 7.54 (1H, t, J =
7.5 Hz, H-5), 7.52 (2H, d, J = 8.5 Hz, H-2’, H-6’), 7.00 (1H,d, J = 9.5 Hz, H-2); 13C NMR (125 MHz, CDCl3)
δ: 185.9 (s,
C-1), 151.8 (s, C-9), 143.5 (s), 140.0 (d), 138.4 (d), 133.9 (d, 2 x C, C-3’, C-5’), 132.7 (d), 132.0 (d), 128.9 (d), 128.3 (s),
126.1 (s), 126.0 (s), 123.7 (d, 2 x C, C-2’, C-6’), 123.5 (d), 118.9 (s, CN), 115.6 (d), 110.2 (s), 108.3 (s).
4.2.3.14. 9-(Pyridin-3-ylamino)-1H-phenalen-1-one (37). To a solution of 28 (50 mg, 0.152 mmol) in anhydrous THF
(1.25 mL) was added pyridin-3-amine (42 mg, 0.446 mmol) and the mixture was heated at 50 ºC for 9 h followed by solvent
removal. Addition of EtOAc (2 mL) gave a precipitate that was collected by filtration. Recrystallization from EtOAc gave
1
37 (40.6 mg, 98%) as a yellow solid. H NMR (500 MHz, CDCl3)
δ: 14.60 (1H, br s, NH), 8.46 (1H, d, J = 8.4 Hz), 8.14
(1H, d, J = 8.3 Hz), 8.02 (1H d, J = 1.4 Hz), 7.92 (1H, d, J = 7.0 Hz), 7.77 (2H, d, J = 9.8 Hz, H-3), 7.76-7.70 (3H, m), 7.63
(1H, dd, J = 5.6, 8.8 Hz),), 6.49 (1H, d, J = 9.8 Hz, H-2); 13C NMR (125 MHz, CDCl3)
δ: 183.6 (s, C-1), 149.2 (s, C-9),
142.4 (d), 142.1 (s), 137.3 (s), 134.4 (d), 133.8 (d), 132.8 (d), 130.8 (d), 129.2 (d), 129.1 (d), 129.0 (d), 128.4 (s), 127.7 (d),
127.6 (s), 125.3 (d), 121.5 (s); EIMS: m/z 272 [M]+ (21), 195 (100), 168 (43), 94 (80); HRMS: calcd. for C18H12N2O
272.0950, found 272.0951; UV-Vis (EtOH) λmax: 371, 357, 301, 258, 249, 220, 203 nm; IR (CHCl3) νmax: 3430, 1642, 1260,
1144, 1026, 838 cm-1; mp: 185-187 °C.
4.2.3.15. 9-(Pyiridin-2-ylamino)-1H-phenalen-1-one (38). To a solution of 28 (50 mg, 0.152 mmol) in anhydrous THF (1
mL) was added pyridin-2-amine (94.4 mg, 1 mmol) and the mixture was heated at 80 ºC for 7 days. Solvent removal
followed by purification using column chromatography (hexane/EtOAc; 9:1 to 4:1) gave 38 (28 mg, 68%) as an orange
solid. 1H NMR (500 MHz, CDCl3)
δ: 14.60 (1H, br s, NH), 9.31 (1H, d, J = 9.3 Hz, H-7), 8.44 (1H, dd, J = 4.8, 1.3 Hz, H-
6’), 8.11 (1H, d, J = 9.4 Hz, H-8), 7.97 (1H, d, J = 7.8 Hz), 7.88-7.86 (2H, m), 7.68 (1H, td, J = 8.1, 1.9 Hz, H-4’) , 7.50
(1H, t, J = 7.5 Hz, H-5), 7.13 (1H, d, J = 8.1 Hz, H-8), 7.00 (1H, td, J = 5.2, 2.1 Hz, H-1’), 6.98 (1H, d, J = 9.4 Hz, H-2);
13C NMR (125 MHz, CDCl3)
δ: 185.9 (s, C-1), 154.0 (s, C-9), 151.3 (s), 147.9 (d), 139.6 (d), 138.1 (d), 137.9 (d), 132.1 (d),
131.8 (d), 128.8 (d), 127.7 (s), 126.0 (s), 125.8 (s), 123.0 (d), 119.0 (d), 118.4 (d), 115.8 (d), 110.0 (s); EIMS: m/z 272 [M]+
24

ACCEPTED MANUSCRIPT
(82), 271 (89), 255 (100), 121 (22). HRMS: calcd. for C18H12N2O 272.0950, found 272.0943; UV-Vis (EtOH) λmax: 486,
459, 373, 365, 313, 287, 277, 253, 205, 203 nm; IR (CHCl3) νmax: 3136, 1581, 1505, 1292, 1133, 840 cm-1; mp: 152-154 °C.
4.2.4. 4-amino-1H-phenalen-1-one derivatives.
4.2.4.1. 3-(2-Methoxynaphthalen-1-yl)propanoic acid (41). To a solution of palladium acetate (II) (8 mg, 0.035 mmol),
tri(o-tolyl)phosphine (46 mg, 0.151 mmol) in DMF (1 mL, 12.9 mmol) was added Et3N (0.611 mL, 4.38 mmol), 1-bromo-2-
methoxynaphthalene (39) (342 mg, 1.44 mmol) and prop-2-enoic acid (0.41 mL, 5.98 mmol). The mixture was heated at
o
160 C for 20 min under argon using MW irradiation. Water (5 mL) was added and the solvents were removed by
lyophilization. EtOAc (10 mL) was added and the mixture was filtered through Celite®. The filtrate was washed with 1N
NaOH (aq) (5 x 3 mL) and the aqueous phases were combined, acidified with 1N HCl (aq) to pH = 2, and extracted with
EtOAc (5 x 4 mL). The combined organics were dried (MgSO4) and the solvent was removed under reduced pressure.
Addition of 1,4-dioxane (5 mL) followed by lyophilization gave intermediate (40), as a mixture of E/Z isomers, (250 mg,
1
71%) as a beige solid. H NMR (400 MHz, CDCl3)
δ: 8.51 (1H, d, J = 16 Hz, H-3), 8.23 (1H, d, J = 8.8 Hz, H-8’), 7.89
(1H, d, J = 9.2 Hz, H-4’), 7.82 (1H, d, J = 8 Hz, H-5’), 7.56 (1H, t, J = 7.4, 8.0 Hz, H-7’), 7.41 (1H, t, J = 7.2, 7.8 Hz, H-
6’), 7.32 (1H, d, J = 9.2 Hz, H-3’), 6.87 (1H, d, J = 16 Hz, H-2), 4.04 (3H, s, OCH3). To a solution of (40) (250.0 mg, 1.096
mmol) in MeOH (10 mL) was added Pd/C (10%, 31.2 mg). The reaction mixture was stirred for 24 h under an atmosphere
of hydrogen. The material was filtered through Celite® and the filtrate was concentrated. Crystallization (hexane/CH2Cl2),
gave 41 (200 mg, 80%) as colourless crystals.
4.2.4.2. 4-Methoxy-1H-phenalen-1-one (42). The 3-(2-methoxynaphthalen-1-yl)propanoic acid (41) (103.2 mg, 0.448
mmol) was dissolved in thionyl dichloride (1 mL, 13,785 mmol) and the mixture was heated at 30 °C for 1 hour under
argon. Then the reaction mixture was concentrated under reduced pressure. This process was repeated again. The resulting
crude was dissolved in anhydrous CH2Cl2 (5 mL) and AlCl3 was added (181.5 mg, 1.34 mmol). After 2 h water (10 mL)
was added to the reaction and the mixture was extracted with CH2Cl2 (3 x 15 mL). The combined organics were washed
with brine (10 mL), dried (MgSO4), filtered and concentrated. DDQ (134.4 mg, 0.582 mmol) was added to a solution of the
crude mixture in anhydrous CH2Cl2 (5 mL) and the reaction mixture was heated at 30 °C for 15 min. 1N HCl (aq) (10 mL)
was added and the mixture was extracted with CH2Cl2 (3 x 15 mL). The combined organics were washed with brine (10
mL), dried (MgSO4), filtered and concentrated. Purification using column chromatography (hexane/EtOAc; 3:1 to 1:1) gave
42 (30.9 mg, 33%) as an orange solid. 1H NMR (400 MHz, CDCl3)
δ: 8.66 (1H, d, J = 7.6 Hz, H-9), 8.23 (1H, d, J = 10 Hz,
25

ACCEPTED MANUSCRIPT
H-3), 8.09 (1H, d, J = 8 Hz, H-7), 8.01 (1H, d, J = 9.2 Hz, H-6), 7.63 (1H, t, J = 7.6 Hz, H-8), 7.28 (1H, d, J = 9.2 Hz, H-5),
6.69 (1H, d, J = 10 Hz, H-2), 4.10 (3H, s, OCH3).
4.2.4.3. 4-(Benzylamino)-1H-phenalen-1-one (43). A solution of 42 (11 mg, 0.05 mmol) in benzylamine (0.2 mL) was
heated at 110 °C for 3 h and then cooled to rt. Purification using column chromatography (hexane/EtOAc; 1:1) gave 43 (7.5
1
mg, 50%) as an orange oil. H NMR (500 MHz, CDCl3)
δ: 8.68 (1H, dd, J1 = 1.3 Hz, J2 = 7.6 Hz, H-9), 7.99 (1H, dd, J =
1.1, 7.6 Hz, H-7), 7.90 (1H, d, J = 10.0 Hz, H-3), 7.86 (1H, d, J = 9.1 Hz, H-5 or H-6), 7.54 (1H, m, H-8), 7.55-7.36 (4H,
m), 7.35-7.26 (1H, m), 7.05 (1H, d, J = 9.1 Hz, H-5 or H-6), 6.71 (1H, dd, J = 10.0 Hz, H-2), 5.62 (1H, br s, NH), 4.66 (2H,
d, J = 5.4); 13C NMR (125 MHz, CDCl3)
δ: 184.5 (s, C-1), 148.9 (s, C-4), 137.4 (s), 135.7 (d), 134.8 (d), 132.8 (d), 131.5
(d), 129.1 (d, 2 x C), 129.0 (s), 128.9 (s), 128.1 (d), 127.4 (d, 2 x C), 126.7 (s), 125.5 (d), 123.6 (d), 114. 5 (d), 107.2 (s),
47.8 (t, C-1’); EIMS: m/z 286 (42), 285 [M]+ (88), 139 (25), 91 (100); HRMS: calcd. for C20H15NO 285.1154, found
285.1141.
4.2.4.4. 4-{[2-(Dimethylamino)ethyl]amino}-1H-phenalen-1-one (44). A solution of 42 (9 mg, 0.04 mmol) in N,N-
dimethylethane-1,2-diamine (0.2 mL) was heated at reflux for 4 h, and then cooled to rt. Purification using column
chromatography (CH2Cl2/MeOH; 20:1) gave 44 (10 mg, 86%) as a red oil. 1H NMR (500 MHz, CDCl3)
δ: 8.67 (1H, d, J =
7.5 Hz, H-9), 7.98-7.95 (2H, m, H-7, H-3), 7.87 (1H, d, J = 9.2 Hz, H-5 or H-6) 7.51 (1H, m, H-8), 7.00 (1H, d, J = 9.2 Hz,
H-6 or H-5), 6.72 (1H, d, J = 9.8 Hz, H-2), 6.38 (1H, br s, NH), 3.45 (2H, m, H-1’), 2.74 ( 2H, t, J = 5.8 Hz, H-2’), 2.37
(6H, s, CH3); 13C NMR (125 MHz, CDCl3)
δ: 184.5 (s, C-1), 149.3 (s, C-4), 135.8 (d), 134.6 (d), 133.4 (d), 131.5 (d), 129.1
(s), 128.8 (s), 126.5 (s), 125.0 (d), 123.3 (d), 114.5 (d), 107.0 (s), 57.1 (d, C-1’), 44.9 (t, 2 x CH3), 40.0 (d, C-2’); EIMS: m/z
151 (6), 152 (5), 180 (4), 266 [M]+ (11); HRMS: calcd. for C17 H18 N2O 266.1419, found 266.1412.
4.2.4.5. 4-(Morpholin-4-yl)-1H-phenalen-1-one (45). A solution of 42 (17 mg, 0.08 mmol) in morpholine (0.25 mL) was
heated at reflux for 12 h, and then cooled to rt. Purification using column chromatography (hexane/EtOAc; 3:2) gave 45
(10 mg, 47%) as an orange oil. 1H NMR (500 MHz, CDCl3)
δ: 8.65 (1H, dd, J = 1.2, 7.4 Hz, H-9), 8.13 (1H, d, J = 10.0 Hz,
H-3), 8.11 (1H, dd, J = 1.2, 9.5 Hz, H-7) 7.99 (1H, d, J = 8.9 Hz, H-5 or H6), 7.67 (1H, m, H-8), 7.38 (1H, d, J = 8.9 Hz, H-
5 or H-6) 6.72 (1H, d, J = 10.0 Hz, H-2), 3,98 (4H, m, H-3’, H-5’), 3.29 (4H, m, H-2’, H-6’); 13C NMR (125 MHz, CDCl3)
δ: 185.4 (s, C-1), 154.1 (s, C-4), 137.1 (d), 134.7 (d), 133.7 (d), 131.1 (d), 129.4 (s), 129.1 (s), 129.0 (s), 127.3 (d), 125.5
(d), 119.8 (d), 118.1 (s), 67.1 (t, C x 2, C-3’, C-5’), 54.1 (t, C x 2, C-2’, C-6’); EIMS: m/z 266 (30), 265 [M]+ (100), 207
(66), 152 (32); HRMS: calcd. for C17 H15 NO2 265.1103, found 265.1114.
26

ACCEPTED MANUSCRIPT
4.2.4.6. 4-(4-Benzyl-piperazin-1-yl)-1H-phenalen-1-one (46). A solution of 42 (30 mg, 0.143 mmol) and 1-
benzylpiperazine (0.4 mL) was heated at 100 oC for 12 h, and then cooled to rt. Purification using column chromatography
(hexane/EtOAc; 1:1) gave 46 (22 mg, 43%) as an orange oil. 1H NMR (500 MHz, CDCl3)
δ: 8.63 (1H, dd, J = 1.2, 7.4 Hz,
H-9), 8.11 (1H, d, J = 10.0 Hz, H-3), 8.08 (1H, dd, J = 1.0, 7.9 Hz, H-7), 7.95 (1H, d, J = 8.9 Hz, H-5 or H-6), 7.63 (1H, m,
H-8), 7.43-7.28 (6H, m), 6.7 (1H, d, J = 10.0 Hz, H-2), 3.65 (2H, s, H-1’’), 3.34 (4H, t, J = 4.8 Hz, H-2’, H-6’), 2.74 (4H,
m); 13C NMR (125 MHz, CDCl3)
δ: 185.4 (s, C-1), 154.7 (s, C-4), 137.7 (s), 137.4 (d), 134.6 (d), 133.6 (d), 131.0 (d), 129.4
(s), 129.3 (d, 2 x C), 129.2 (s), 128.8 (s), 128.4 (d, 2 x C), 127.3 (d), 126.8 (d), 125.2 (d), 120.0 (d), 117.5 (s), 63.1 (t, C-
1’’), 53.8 (d, C x 2, C-2’, C-6’), 53.2 (d, C x 2, C-3’, C-5’); EIMS: m/z 355 (49), 354 [M]+ (100), 353 (28), 263 (24), 208
(58); HRMS: calcd. for C24 H22 N2O 354.1732, found 354.1750.
4.2.5. 2-Amino-1H-phenalen-1-ona (47), 5-amino-1H-phenalen-1-ona (48) y 6-amino-1H-phenalen-1-ona (49). To a
solution of 1H-phenalen-1-one (1) (1g, 5.5 mmol) in H2SO4 (98%, 20 mL) at 0 °C was added dropwise a mixture of HNO3
(65%, 0.42 mL) and H2SO4 (98%, 1.2 mL). The reaction mixture was stirred for 3 h at room temperature. H2O (100 mL)
was added and the mixture was extracted with CH2Cl2 (3 x 80 mL). The combined organics were dried (Na2SO4), filtered
and concentrated. MeOH (90 mL) and 10% Pd/C (232 mg) were added to the crude mixture. The mixture was stirred for 1
hour at rt under H2. The solvent was removed and replaced with CH2Cl2 (20 mL). Filtration through over Celite® followed
by purification using column chromatography (hexane/EtOAc; 1:1) gave three compounds: 47 (218.4 mg, 21%), 48 (219.0
1
mg, 21%) and 49 (574.0 mg, 55%) as red solids. Compound 47: H NMR (400 MHz, CDCl3)
δ: 8.69 (1H, dd, J = 1.0, 7.3
Hz, H-9), 8.19 (1H, dd, J = 0.8, 6.4 Hz), 7.80 (1H, dd, J = 2.0, 7.2 Hz), 7.75 (1H, t, J = 7.6 Hz), 7.56-7.40 (2H, m), 6.84
(1H, s, H-3). Compound 48: 1H NMR (400 MHz, CDCl3)
δ: 8.37 (1H, dd, J = 7.3, 0.7 Hz, H-9), 7.95 (1H, d, J = 8.1 Hz, H-
7), 7.67 (1H, dd, J = 7.7, 7.6 Hz, H-8), 7.65 (1H, d, J = 9.8 Hz, H-2), 7.22 (1H, d, J = 2.0 Hz, H-6), 7.16 (1H, d, J = 2.0 Hz,
H-4), 6.70 (1H, d, J = 9.7 Hz, H-3), 4.00 (2H, br s, NH2). Compound 49: 1H NMR (500 MHz, (CD3)2CO)
δ: 8.60 (1H, d, J =
8.2 Hz, H-7), 8.54 (1H, d, J = 7.3 Hz, H-9), 7.70 (1H, dd, J = 7.9, 7.7 Hz, H-8), 7.67 (1H, d, J = 9.5 Hz, H-3), 7.62 (1H, d, J
= 8.0 Hz, H-4), 6.80 (1H, d, J = 8.0 Hz, H-5), 6.34 (1H, d, J = 9.5 Hz, H-2). 1H NMR (400 MHz, CDCl3)
δ: 8.72 (1H, d, J
= 7.3 Hz, H-9), 8.20 (1H, d, J = 8.1 Hz), 7.75 (1H, dd, J = 7.9, 7.7 Hz, H-8), 7.64 (1H, d, J = 9.6 Hz, H-3), 7.56 (1H, d, J =
7.8 Hz), 6.74 (1H, d, J = 7.7 Hz), 6.57 (1H, d, J = 9.6, H-2), 4.84 (2H, br s, NH2); 13C NMR (125 MHz, (CD3)2CO)
δ: 184.8
(s, C-1), 151.7 (s, C-6), 142.9 (d, C-3), 136.1 (d, C-4), 131.4 (s, C-9a), 130.9 (d, C-9), 129.6 (s, C-9b), 129.2 (d, C-7), 125.7
(d, C-8), 123.7 (d, C-2), 123.2 (s, C-6a), 117.4 (s, C-3a), 108.8 (d, C-5); EIMS: m/z 195 [M]+ (100), 167 (42), 139 (29), 83
(13); HRMS: calcd. for C13H9NO 195.0684, found 195.0689.
27

ACCEPTED MANUSCRIPT
4.3. Biology
4.3.1. Parasite cultures. Experiments were carried out with L. amazonensis (MHOM/BR/77/LTB0016) and L. donovani
(MHOM/IN/90/GE1F8R) strains. Promastigotes of both strains were cultured in Schneider’s medium (Sigma-Aldrich,
Madrid, Spain) supplemented with 10% foetal bovine serum at 26 °C and were grown to the log phase previous use in
further experiments. For some of the assays, the parasites were also cultured in RPMI 1640 medium (Gibco®), with or
without phenol red.
The murine macrophages J774A.1 (ATCC# TIB-67) cell line was cultured in RPMI 1640 medium supplemented with
10% foetal bovine serum at 37 ºC and 5% CO2 atmosphere.
4.3.2. Activity assays against the promastigote stage of Leishmania spp. The activity of the amino-phenalenones was
tested in vitro against the promastigote stage of L. amazonensis and L. donovani using a colorimetric assay based on
alamarBlue® reagent (Invitrogen, Life Technologies, Madrid, Spain) as previously described [22]. Briefly, amino-
phenalenones were serially diluted in 100
µl RPMI 1640 medium (Gibco, Life Technologies, Madrid, Spain) without
phenol red and supplemented with 10% heat-inactivated foetal bovine serum in 96-well plates. After that, parasites in log
phase growth were counted, diluted (105/well) and added to these wells. Finally, 10% of alamarBlue® was added to the
plates, and these were incubated at 27 °C for 72 h in the dark. The plates were then analysed using an EnSpire® Multimode
Plate Reader (Perkin Elmer, Madrid, Spain) using a test wavelength of 560 nm and a reference wavelength of 590 nm.
Percentage of inhibition and 50% inhibitory concentrations (IC50) were calculated by non-linear regression analysis with
95% confidence limits using Sigma Plot 12.0 statistical analysis software (Systat Software). All experiments were
performed three times each in duplicate, and the mean values were also calculated.
4.3.3. Activity assays against intracellular amastigotes. Macrophages were plated in 96-well plates (2.5 x 105
cells/ml), infected with metacyclic stage promastigotes (~5-6 days old culture) of L. amazonensis in a ratio 1:20 and
incubated overnight for internalization. After removal of non-internalized promastigotes by extensive washing with RPMI
1640 medium, parasitized cells were incubated with medium (untreated control), or a range of amino-phenalenones
concentrations as described above for the promastigote stage assays, and incubated for 24 h at 37 ºC and 5% CO2 in the
dark. For the parasite-rescue, a controlled lysis of L. amazonensis amastigotes-infected macrophages was performed as
previously described with minor modifications [24]. Briefly, the 96-well plate was washed with serum-free RPMI-1640
28