Natural product derived antiprotozoal agents: Synthesis, biological evaluation, and structure-activity relationships of novel chromene and chromane derivatives

Article abstract of DOI:10.1021/jm401007p

Various natural products with the chromane and chromene scaffold exhibit high antiprotozoal activity. The natural product encecalin (7) served as key intermediate for the synthesis of different ethers 9, amides 11, and amines 12. The chromane analogues 14 and the phenols 15 were obtained by reductive amination of ketones 13 and 6, respectively. Angelate 3, ethers 9, and amides 11 did not show considerable antiprotozoal activity. However, the chromene and chromane derived amines 12, 14, and 15 revealed promising antiprotozoal activity and represent novel lead compounds. Whereas benzylamine 12a and α-methylbenzylamine 12g were active against P. falciparum with IC ₅₀ values in the range of chloroquine, the analogous phenols 15a and 15b were unexpectedly 10- to 25-fold more potent than chloroquine with selectivity indexes of 6760 and 1818, respectively. The phenylbutylamine 14d based on the chromane scaffold has promising activity against T. brucei rhodesiense and L. donovani.

Full text of DOI:10.1021/jm401007p

Article
pubs.acs.org/jmc
Natural Product Derived Antiprotozoal Agents: Synthesis, Biological
Evaluation, and Structure−Activity Relationships of Novel Chromene
and Chromane Derivatives
Dipak Harel,† Dirk Schepmann,† Helge Prinz,† Reto Brun,‡ Thomas J. Schmidt,§ and Bernhard Wunsch*,†
̈
†Institut fur Pharmazeutische und Medizinische Chemie der Westfalischen Wilhelms-Universitat Munster, Corrensstraße 48, D-48149
̈
̈
̈
̈
Munster, Germany
̈
‡Swiss Tropical and Public Health Institute (Swiss TPH), Socinstraße 57, CH-4002 Basel, Switzerland
§Institut fur Pharmazeutische Biologie und Phytochemie der Westfalischen Wilhelms-Universitat Munster, Corrensstraße 48,
̈
̈
̈
̈
D-48149 Munster, Germany
̈
S
* Supporting Information
ABSTRACT: Various natural products with the chromane
and chromene scaffold exhibit high antiprotozoal activity. The
natural product encecalin (7) served as key intermediate for
the synthesis of different ethers 9, amides 11, and amines 12.
The chromane analogues 14 and the phenols 15 were obtained
by reductive amination of ketones 13 and 6, respectively.
Angelate 3, ethers 9, and amides 11 did not show considerable
antiprotozoal activity. However, the chromene and chromane
derived amines 12, 14, and 15 revealed promising anti-
protozoal activity and represent novel lead compounds.
Whereas benzylamine 12a and α-methylbenzylamine 12g
were active against P. falciparum with IC50 values in the range
of chloroquine, the analogous phenols 15a and 15b were unexpectedly 10- to 25-fold more potent than chloroquine with
selectivity indexes of 6760 and 1818, respectively. The phenylbutylamine 14d based on the chromane scaffold has promising
activity against T. brucei rhodesiense and L. donovani.
(Glossina spp.). It is estimated that about 20 000 people are
currently suffering from human African trypanosomiasis.6 The
absence of vaccines and the availability of only few chemo-
therapeutics, some with reduced efficacy and considerable
adverse effects, impede the efficient treatment of these diseases.
Therefore, the discovery and development of novel effective,
safe, and affordable antiprotozoal agents remain an urgent need.
Natural products can play an important role as potential lead
structures toward such novel pharmacologically active com-
pounds.7,8
Chromanes and chromenes represent a widely distributed
class of natural products. The chromenodihydrochalcone 1
isolated from Crotalaria ramosissima (a medicinal plant endemic
to India) inhibited in an in vitro assay the growth of L.
donovani. The corresponding chalcone with a double bond in
the side chain between the carbonyl moiety and the phenyl
residue was almost as active as the natural product 1.9 The
synthetic tricyclic chromene 2 with the exocyclic benzylidene
moiety also showed strong antileishmanial acitivity.10 The
medicinal plant Ageratum conyzoides L. (Asteraceae) is used in
folk medicine against various diseases including protozoan
INTRODUCTION
■
Neglected tropical diseases caused by protozoan parasites are
among the most serious public health problems in developing
countries. Some of the neglected diseases also affect the people
living in Europe and U.S. According to the World Health
Organization (WHO) World Global Burden of Disease analysis
report in 2008, around 17% of deaths worldwide are caused by
neglected tropical diseases.1 However, only very few drugs are
available for the treatment of these lethal infections.
Malaria, leishmaniasis, Chagas disease, and human African
trypanosomiasis (HAT or sleeping sickness) are among the
most serious neglected tropical diseases. Plasmodium falciparum
is one of the protozoan agents leading to malaria after a bite of
an infected female Anopheles mosquito. Malaria causes almost
800 000 deaths per year, mostly in children.2 Visceral
leishmaniasis (Kala Azar) is caused by Leishmania donovani
(India and Near East) and Leishmania infantum (around the
Mediterranean Sea), and an estimated 1.3 million new cases
and 20 000 to 30 000 deaths occur annually.3,4 There are about
10 million people worldwide suffering from Chagas disease
(American trypanosomiasis), which is caused by Trypanosoma
cruzi.5 Human African trypanosomiasis is caused by Trypano-
soma brucei rhodesiense and gambiense (East and West African
forms, respectively), which are transmitted by the tsetse fly
Received: July 8, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
infections.11 Recently, it was discovered that the dichloro-
methane extract of A. conyzoides L. displays considerable activity
against T. b. rhodesiense.12 In a search for the active constituents,
the chromene encecalol angelate (3)13 was identified in the
extract but showed only low antitrypanosomal activity when
tested after total synthesis.14 The stability of 3 was found to be
very low so that the weak bioactivity may be related to its rapid
degradation when tested as a pure compound. The instability of
3 originates from facile dissociation of the angelate anion, since
the remaining benzylic cation is stabilized by two O-
substituents. Thus, a rapid formation of the corresponding
methyl ether was observed when storing the angelate 3 in
methanolic solution14 (Figure 1).
Herein the synthesis, antiprotozoal activity, and relationships
between the structure and the antiprotozoal activity of
chromenes and chromanes related to the lead compounds 1−
3 are reported.
RESULTS AND DISCUSSION
■
Chemistry. The natural product encecalin (7)15 was the
central intermediate for the synthesis of various ethers 9,
amides 11, and amines 12 (Scheme 1). In contrast to the first
reported synthesis of 715 we followed our previously reported
more efficient synthetic strategy,14 which started with resorcinol
(4). Formic acid catalyzed annulation of 4 with 2-methylbut-3-
en-2-ol provided a chromanol derivative, which was acetylated
to give acetylchromanol 5. Dehydrogenation of 5 afforded
chromenol 6, which upon methylation yielded the methyl ether
encecalin (7).14
For the synthesis of ethers 9, encecalin (7) was reduced with
NaBH4 to afford encecalol (8). In order to obtain compounds
structurally related to the lead compounds 1−3, the alcohol 8
was deprotonated with NaH and subsequently treated with
different arylalkyl halides to form the ethers 9a−c in good
yields. The arylalkyl substructure of the ethers 9 should imitate
the arylalkyl residues of the lead compounds 1 and 2 as well as
the alkenoyl substructure of angelate 3. In contrast to 3 the
ethers 9 are very stable against hydrolytic degradation.
Reaction of the ketone 7 with ammonium acetate and
NaBH3CN16 led to the primary amine 10 in 65% yield. The
primary amine 10 was converted into amides 11 by acylation
with various acids in the presence of carbonyl diimidazole
(CDI). The acyl residues were selected because of their
similarity to the lead compounds 1−3.
Figure 1. Chromenes with antiprotozoal activity.
In order to study the antiprotozoal properties of chromene
derivatives related to 1−3, we planned to synthesize more
stable analogues of the angelate 3. In particular the hydrolyti-
cally very labile ester group of 3 should be replaced by ether
(9), amide (11), and amine (12, 14, 15) moieties, taking the
arylalkyl side chains of the chromenes 1 and 2 into account.
Reductive amination of ketone 7 with benzylamine in the
presence of NaBH(OAc)3 failed to give the secondary amine
12a. However, microwave assisted condensation of ketone 7
a
Scheme 1
a
Reagents and conditions: (a) HCO2H, 100 °C; (b) CH3COCl, AlCl3, CH2Cl2, 0 °C; (c) DDQ, toluene, 110 °C; (d) CH3I, K2CO3, DMF, rt; (e)
NaBH4, THF, rt; (f) R-Br, NaH, DMF, rt, residues R are defined in Table 2; (g) NH4OAc, NaBH3CN, MeOH, rt; (h) RCO2H, CDI, THF, rt,
residues R are defined in Table 2; (i) (1) R2NH, MeOH, microwave irradiation, (2) NaBH4, rt, the substituents of the amino moiety NR2 are defined
in Table 3. (j) oOnly for synthesis of 12o: (1) PhCH2CH2CH2NH2, MeOH, microwave irradiation, NaBH4, rt; (2) HCHO, NaBH(OAc)3, THF, rt.
B
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
with various primary amines led to formation of imines, which
were reduced subsequently with NaBH4 to give the secondary
amines 12a−l in 52−75% yields. In order to synthesize the
cyclic amines 12m and 12n an intermediate iminium ion was
formed upon microwave irradiation, which was reduced with
NaBH4. Methylation of the secondary amine 12c with
formaldehyde and NaBH(OAc)3 resulted in the tertiary
amine 12o.
against nonpathogenic lizard-infecting L. tarentolae (strain P10,
Jena Biosciences) promastigotes, which can be cultured cost-
efficiently with hemin-supplemented BHI medium and which
rapidly grow in vitro. This relatively simple L. tarentolae system
has recently been propagated as a model for the in vitro
screening of compounds for antileishmanial activity.18−20 In
Table 1 the activity of reference compounds (positive controls)
In order to broaden the structure−activity relationships in
this compound class, chromanamines 14 without a double
bond in the heterocycle were taken into consideration. After
methylation of the phenol 5 without the endocyclic double
bond, the resulting ketone 13 was reacted with primary amines
assisted by microwave irradiation. Subsequent reduction with
NaBH4 led to the secondary amines 14a−e in 56−68% yields
(Scheme 2). The selection of the amines for the synthesis of
Table 1. In Vitro Antiprotozoal Activity of Reference
Compounds (Positive Controls) and Cytotoxicity of
Podophyllotoxin (IC50 [μM])
parasite
P. falciparum
reference drug
IC50 [μM]
chloroquine
melarsoprol
benznidazole
miltefosine
0.263
0.005
1.573
0.361
0.030
0.010
T. brucei rhodesiense
T. cruzi
L. donovani
a
L. tarentolae
pentamidine
podophyllotoxin
Scheme 2
L6 cells (cytotoxicity)
toward the different protozoan parasites is summarized.
Moreover, the cytotoxic effect of podophyllotoxin (used as a
positive control) toward L6 rat skeletal myoblasts is included.
The ester 3, ethers 9a−c, and amides 11a−c generally show
only low activity against the protozoan parasites. Only the
benzyl ether 9a reveals an IC50 value against Pfc below 10 μM.
The compounds also display low selectivity indices (SI < 5)
indicating a small gap between the desired antiprotozoal activity
(anti-Pfc, anti-Tbr) and the undesired cytotoxicity.
In contrast to the human pathogens, the nonpathogenic L.
tarentolae promastigotes were much more sensitive to these
compounds, with IC50 values generally lower than 10 μM. The
cinnamyl ether 9c (IC50 = 0.86 μM) and the phenylethyl ether
9b (IC50 = 1.17 μM) represent the most potent anti-Ltr
compounds of this series, although their activity is still about
30-fold lower than the activity of the reference compound
pentamidine (IC50 = 0.030 μM; see Table 1).
a
Reagents and conditions: (a) CH3I, K2CO3, DMF, rt; (b) (1) RNH2,
MeOH, microwave irradiation, NaBH4, rt.
chromenamines 12 and chromanamines 14 was stimulated by
the corresponding substituents in the lead compounds 1−3
and, moreover, by the biological activity obtained during the
synthetic work.
Finally the chromene derived phenols 15a and 15b were
synthesized (Scheme 3). For this purpose a two-step reductive
a
Scheme 3
In contrast to the ester, ether, and amide derivatives 3, 9, and
11, the chromen- and chromanamines 12, 14, and 15 show
much better antiprotozoal activity. Some of the amines also
reveal activity against Tbr. As summarized in Table 3, the
antiprotozoal activity in the chromenamine series 12 decreases
in the order anti-Pfc > anti-Tbr > anti-Tcr > anti-Ldo. In
contrast to the chromenamines 12, the chromanamines 14
display promising anti-Ldo activity.
The phenylalkylamines 12a, 12c, 12e, 12f, and 12g display
IC50 values against Pfc below 1 μM and can hence be classified
as potent antiplasmodial agents with activity in the same range
as the positive control chloroquine (Table 3). Phenylalkyl-
amines are generally more active against Pfc than aliphatic
amines (e.g., 12i, 12j). With the exception of 12c, the
phenylalkylamines 12a, 12e, 12f, and 12g also show relatively
low cytotoxicity indicating a big difference between the
antiplasmodial effect and potential side effects so that these
compounds deserve further attention as potent and safe
antimalarials.
a
Reagents and conditions: (a) RNH2, Ti(OEt)4,THF, 65 °C, then
NaBH4, rt.
amination of ketone 6 (see Scheme 1) with benzylamine and 3-
phenylpropan-1-amine was performed. At first the ketone 6 was
reacted with the primary amines in the presence of Ti(OEt)4 to
form imine intermediates, which were subsequently reduced
17
with NaBH4 to afford the secondary amines 15a and 15b.
Antiprotozoal Activity. In a search of compounds with
improved stability and antiprotozoal activity in comparison with
the natural product 3, derivatives with ether (9), amide (11),
and amino (12, 14, 15) groups instead of the labile ester moiety
of 3 were synthesized.
The activities of the compounds 3, 9, 11, 12, 14, and 15 were
evaluated in vitro against four pathogenic protozoan parasites:
P. falciparum (Pfc, erythrocytic stage, K1 strain, which is
chloroquine and pyrimethamine resistant), T. b. rhodesiense
(Tbr, trypomastigote stage, STIB 900 strain), T. cruzi (Tcr,
intracellular amastigote stage, Tulahuen C4 strain), and L.
donovani (Ldo, axenic amastigote stage, MHOM-ET-67/L82)
(Table 2). A preliminary antileishmanial assay was performed
Replacement of the methoxy group of 12 with a phenolic
hydroxy moiety led to 15 with surprisingly high activity against
P. falciparum. The benzylamine 15a and the phenylpropylamine
15b show IC50 values of 0.02 and 0.01 μM, respectively. This
activity is about 10- to 25-fold higher than the activity of the
standard compound chloroquine (IC50 = 0.263 μM). Moreover,
the cytotoxicity of both phenols 15a and 15b is very low
C
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Activity of Ester 3, Ethers 9a−c, and Amides 11a−c against the Protozoan Parasites P. falciparum (Pfc), T. brucei
rhodesiense (Tbr), T. cruzi (Tcr), L. donovani (Ldo), and L. tarentolae (Ltr), Cytotoxicity against L6 Rat Skeletal Myoblasts, and
a
Selectivity Indices (SI) for Pfc and Tbr
antiprotozoal activity IC50 [μM]
compd
R
Pfc
Tbr
Tcr
Ldo
Ltr
cytotoxicity IC50 [μM], L6 cells
SI (Pfc)
SI (Tbr)
SI (LDo)
312
18.9
9.48
59.9
nd
157
76.1
180
94.7
23.4
103
35.9
61.4
45.0
56.7
73.0
36.8
44.7
23.1
46.2
21.2
63.5
23.0
26.3
53.8
26.3
21.3
1.53
1.17
0.86
8.61
7.69
1.78
139
43.9
123
64.4
34.8
86.5
107
7.35
4.64
2.08
nd
0.89
0.58
0.69
0.68
1.45
0.83
2.99
3.0
2.1
1.9
2.8
1.3
1.6
4.1
9a
PhCH2
9b
PhCH2CH2
PhCHCHCH2
Ph
9c
11a
11b
11c
11.5
31.0
nd
3.01
2.79
nd
PhCH2
PhCHCH
a
nd: not determined.
Table 3. Antiprotozoal Activity of Amino Substituted Chromenes 12a−o, Chromanes 14a−e, and Phenols 15a,b: Activity
against the Parasites P. falciparum, T. brucei rhodesiense, T. cruzi, L. donovani, and L. tarentolae, Cytotoxicity against L6 Rat
a
Skeletal Myoblasts, and Selectivity Indices (SI) for Pfc and Tbr
antiprotozoal activity IC50 [μM]
compd
R2N
Pfc
Tbr
Tcr
Ldo
Ltr
cytotoxicity IC50 [μM], L6 cells SI (Pfc) SI (Tbr) SI (Ldo)
12a
12b
12c
12d
12e
12f
PhCH2NH
0.64
2.42
0.85
1.41
0.93
0.72
0.37
4.41
8.12
4.18
2.39
2.46
9.92
16.4
1.24
1.51
2.49
2.13
1.34
3.22
0.02
0.01
2.38
2.22
1.03
3.15
2.42
2.02
5.10
5.84
22.8
8.19
1.86
2.16
96.2
41.1
3.17
4.98
4.15
2.27
1.84
7.82
6.18
5.31
33.9
9.10
13.6
4.19
33.1
nd
159
127
101
71.7
225
nd
2.40
1.04
0.22
0.77
9.80
8.61
9.01
3.26
8.76
6.25
3.24
0.65
12.4
18.6
9.01
270
21.0
15.4
1.08
4.73
14.9
15.9
16.5
14.6
176
32.8
6.34
1.27
3.35
16.0
22.1
44.4
3.32
22.0
12.6
17.5
5.88
15.9
10.8
13.5
10.1
5.80
1.23
3.94
18.3
6760
1818
8.83
6.93
1.05
1.50
6.13
7.85
3.23
2.50
7.71
6.41
22.5
6.69
1.63
4.31
5.12
3.08
3.48
1.15
2.87
7.54
17.7
5.08
0.13
0.12
0.11
0.07
0.07
PhCH2CH2NH
PhCH2CH2CH2NH
PhCH2CH2CH2CH2NH
p-MeOPhCH2NH
(R)-PhCH(CH3)NH
(S)-PhCH(CH3)NH
PhCHCHCH2NH
CH3CH2NH
12g
12h
12i
nd
nd
8.13
91.5
25.2
35.5
12.9
110
68.2
9.17
13.3
12.1
5.35
5.03
38.4
nd
62.4
365
nd
0.23
0.48
12j
CH3CH2CH2CH2NH
Et2N(CH2)3CH(CH3)NH
C6H11CH2NH
52.5
41.9
14.5
157
12k
12l
nd
139
148
270
84.3
3.70
1.73
nd
0.10
1.06
0.66
0.19
4.1
12m
12n
12o
14a
14b
14c
14d
14e
15a
15b
O(CH2CH2)2N
(CH2CH2)2N
177
PhCH2CH2CH2NCH3
PhCH2NH
16.3
15.3
14.5
2.63
5.28
59.0
109
PhCH2CH2NH
126
8.4
PhCH2CH2CH2NH
PhCH2CH2CH2CH2NH
CH3CH2CH2CH2NH
PhCH2NH
129
0.57
10.8
nd
90.6
nd
9.3
5.5
12.4
6.20
PhCH2CH2CH2NH
nd
nd
27.0
a
nd: not determined.
leading to selectivity indices of 6760 and 1818, respectively.
This extraordinarily high anti-Pfc activity was completely
unexpected, since the starting point of this project was the
optimization of the antitrypanosomal activity, which is rather
low for both phenols 15.
With respect to antitrypanosomal potency, the homologous
phenylalkylamines 12a−d show increasing activity with
increasing number of methylene moieties in the side chain
from the benzylamine 12a to the phenylpropylamine 12c,
which represents the most active anti-Tbr compound with an
IC50 value of 1.03 μM. The phenylbutylamine 12d is 3-fold less
D
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
active than 12c against Tbr but about 3 times more active
against Tcr, indicating different optimal chain lengths for the
two related Trypanosoma species. Quite interestingly, the
activity against Pfc shows an opposite behavior with respect
to the chain length in the phenylalkyl series than observed for
the antitrypanosomal potency. Here, activity decreases with
increasing number of carbon atoms in the side chain (exception
12b). Thus, benzylamine 12a has the highest antimalarial
activity with an IC50 value of 0.64 μM, which is only 2-fold
higher than the IC50 value of the reference compound
chloroquine (IC50 = 0.26 μM). The Pfc selectivity index of
12a with a value of 32.8 also appears quite promising and
indicates a specific activity.
The p-methoxybenzylamine 12e (IC50 = 0.93 μM) shows a
slightly reduced antimalarial activity compared to the
unsubstituted benzylamine 12a. Introduction of a methyl
group in α-position of the benzyl group led to the α-
methylbenzylamines 12f and 12g which display comparable or
slightly increased antiplasmodial activity compared with
benzylamine 12a. The (S)-configured α-methylbenzylamine
12g (IC50 = 0.37 μM) represents the most potent and safe (SI
= 44) compound of the series of methyl ethers. It is noteworthy
that the (R)-enantiomer 12f is about 2 times less active than
the (S)-configured enantiomer 12g. The influence of the
stereochemistry on the antiplasmodial activity of these
compounds indicates the interaction with a particular but yet
unknown target in the parasite Pfc.
Within the series of alkylamines, 12k with a chloroquine-like
diamine side chain displays higher anti-Pfc and anti-Tbr activity
(IC50 = 2.4 and 1.9 μM, respectively) compared to other
alkylamines like ethylamine 12i and butylamine 12j.
In order to compare the antiparasitic effect of secondary and
tertiary amines, some tertiary amines 12m−o were prepared.
The morpholine 12m and the pyrrolidine derivative 12n
display very low antiparasitic activity but also very low
cytotoxicity. N-Methylation of the secondary amine 12c
resulted in 12o with slightly decreased antiprotozoal activity
against the parasites (exception anti-Tcr activity). It can be
concluded that secondary amines are generally more potent
antiprotozoal agents than tertiary amines.
With exception of the anti-Ldo activity, the antiprotozoal
activity of the chromane derivatives 14a−e is in most cases
lower than the activity of the corresponding chromene
derivatives 12a−d and 12j. Obviously, the Δ3,4-double bond
of the chromene ring increases the antiprotozoal activity. As
discussed for the chromene derivatives 12a−d, the anti-Tbr
activity of the phenylalkylamines 14a−d correlates nicely with
the number of methylene moieties in the side chain. The
phenylbutylamine 14d displays the highest anti-Pfc and anti-Tbr
activity but also reveals higher cytotoxicity than the benzyl-
amine 14a and phenylethylamine 14b. The benzylamine 14a
has the highest selectivity index of 10.1 for Pfc in this series of
compounds. The aliphatic butylamine 14e possesses an even
higher SI of 18.3, but its antiplasmodial activity is rather low.
The anti-Ldo activity of the chromanamines 14a−d with a
phenylalkyl residue at the N-atom is remarkable. An increased
number of methylene moieties between the N-atom and the
phenyl ring leads to increased anti-Ldo activity. The phenyl-
butylamine 14d represents the most potent anti-Ldo compound
of the whole series of test compounds. The IC50 value of 0.57
μM is in the same range as the IC50 value of the reference
compound miltefosine (IC50 = 0.36 μM; see Table 1) and more
than 100-fold lower than the IC50 value of the corresponding
chromene derivative 12d. The SI for Ldo of 14d is about 9.3,
which makes this phenylbutylamine an interesting starting
compound for the development of novel anti-Ldo compounds.
The phenylpropylamine 12c, the phenylbutylamine 12d, and
the cyclohexylmethylamine 12l show submicromolar activity
against the nonpathogenic parasite L. tarentolae. However, the
activities of these compounds are still 10- to 30-fold lower than
the activity of the reference compound pentamidine (IC50
=
0.03 μM). The high anti-Ldo activity of the chrmomane
derivatives 14a−d was not observed for L. tarentolae, indicating
a different sensitivity of the different Leishmania species.
The data in Table 3 do not reveal an obvious correlation
between the activity against the Ldo axenic amastigotes and Ltr
promastigotes. Different activities of structurally related
compounds against different L. ssp. are well-known and may
be in general due to species differences.21 Furthermore, data
from pro- and amastigote stages of Leishmania cannot easily be
compared, not even among the same L. ssp. L. donovani axenic
amastigotes express significant levels of amastigote-specific
proteins and enzymes,22 whereas the expression of several
promastigote form enzymes is diminished.23,24 The L. donovani
axenic amastigote model is widely used as an initial test for new
agents against visceral leishmaniasis, whereas L. tarentolae
promastigotes neither represent a relevant parasite stage nor a
human pathogen. The obvious lack of correlation between the
activity data obtained with the two models indicates that the
results obtained with the L. tarentolae promastigote model have
to be treated with caution, when new agents for the treatment
of the human disease are to be developed.
CONCLUSION
■
This study unraveled a new class of potent antiprotozoal
compounds, starting from the unstable natural product 3 found
in an extract of Ageratum conyzoides L. The high activity of the
crude extract from this plant against T. brucei rhodesiense
prompted us to search primarily for stable analogues of 3 with
increased antritrypanosomal activity. This goal was achieved
with some of the phenylalkylamine substituted chromene and
chromane derivatives; i.e., the phenylpropylamine 12c and the
phenylbutylamine 14d show the highest anti-Tbr activity of this
series of compounds with IC50 values of 1.03 and 1.84 μM,
respectively.
In addition to antitryoanosomal activity, the chromanamines
14 exhibit high anti-Ldo activity. The phenylbutylamine 14d
represents the most potent compound of this class with an IC50
value of 0.57 μM, which is close to the IC50 value of the
reference compound miltefosine.
The systematic variation of the natural products 3 and 7 led
to unexpected high in vitro antiplasmodial activity and
selectivity indices against P. falciparum. Whereas the benzyl-
amine 12a with the methoxy moiety possesses an anti-Pfc
activity (IC50 = 0.64 μM) in the range of chloroquine, the
corresponding phenol 15a (IC50 = 0.02 μM) is >10-fold more
potent than chloroquine. The selectivity index of 6760 indicates
a high safety of 15a. Altogether, the amino substituted ethers
12 and phenols 15 represent the first members of a new
promising class of potent and safe natural product derived
antimalarials.
EXPERIMENTAL PART
General, Chemistry. For flash chromatography, silica gel 60, 40−
64 μm (Merck) was used. Results in parentheses include diameter of
the column, length of column, fraction size, eluent, and Rf value.
■
E
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Melting point apparatus SMP 3 (Stuart Scientific) was used, and
N-[1-(7-Methoxy-2,2-dimethylchroman-6-yl)ethyl]-4-phenyl-
butan-1-amine (14d). According to general procedure C, ketone 13
(80 mg, 0.34 mmol) was reacted with phenylbutylamine (63 mg, 0.51
mmol) and the product was purified by flash chromatography
(dichloromethane/MeOH = 95/5, ⌀ = 2.0 cm, h = 15 cm, Rf =
0.26). Colorless oil, yield 59 mg (51%). 1H NMR (CDCl3): δ (ppm) =
1.21 (d, J = 6.7 Hz, 3H, CH3), 1.26 (s, 6H, CH3), 1.39−1.60 (m, 5H,
PhCH2CH2CH2CH2NH), 1.70 (t, J = 6.7 Hz, 2H, 3-CH2), 2.34−2.49
(m, 2H, PhCH2CH2CH2CH2NH), 2.51 (t, J = 6.7 Hz, 2H,
PhCH2CH2CH2CH2NH), 2.61 (t, J = 6.7 Hz, 2H, 4-CH2), 3.66 (s,
3H, OCH3), 3.89 (q, J = 6.7 Hz, 1H, CH3CHNH), 6.24 (s, 1H, 8-
CH), 6.85 (s, 1H, 5-CH), 7.05−7.20 (m, 5H, Ph).
6-[1-(Benzylamino)ethyl]-2,2-dimethyl-2H-chromen-7-ol
(15a). Under N2, phenol 6 (100 mg, 0.46 mmol) was dissolved in dry
THF (10 mL). Benzylamine (59 mg, 0.55 mmol) and Ti(OEt)4 (126
mg, 0.55 mmol) were added, and the reaction mixture was heated to
reflux for 12 h. The reaction mixture was cooled to 0 °C. NaBH4 (26
mg, 0.69 mmol) was added, and the mixture was stirred for 3 h at
room temperature. Water was added. The reaction mixture was
extracted with EtOAc (3 × 100 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuum, and the residue was purified
by flash chromatography (petroleum ether/EtOAc = 70/30, ⌀ = 2.0
cm, h = 12 cm, Rf = 0.27). Colorless oil, yield 85 mg (60%). 1H NMR
(CDCl3): δ (ppm) = 1.34 (s, 6H, CH3), 1.36 (d, J = 6.7 Hz, 3H,
HNCHCH3), 3.58 (d, J = 13.1 Hz, 1H, HNCH2Ph), 3.77 (d, J = 13.1
Hz, 1H, HNCH2Ph), 3.82 (q, J = 6.7 Hz, HNCHCH3), 5.36 (d, J = 9.8
Hz, 1H, 3-CH), 6.16 (d, J = 9.8 Hz, 1H, 4-CH), 6.24 (s, 1H, 8-CH),
6.49 (s, 1H, 5-CH), 7.14−7.34 (m, 5H, Ph).
2,2-Dimethyl-6-{1-[(3-phenylpropyl)amino]ethyl}-2H-chro-
men-7-ol (15b). Under N2, phenol 6 (100 mg, 0.46 mmol) was
dissolved in dry THF (10 mL). 3-Phenylpropylamine (74 mg, 0.55
mmol) and Ti(OEt)4 (126 mg, 0.55 mmol) were added, and the
reaction mixture was heated to reflux for 12 h. The reaction mixture
was cooled to 0 °C. NaBH4 (26 mg, 0.69 mmol) was added, and the
mixture was stirred for 3 h at room temperature. Water was added.
The reaction mixture was extracted with EtOAc (3 × 100 mL). The
organic layer was dried (Na2SO4) and concentrated in vacuum, and
the residue was purified by flash chromatography (petroleum ether/
EtOAc = 70/30, ⌀ = 2.0 cm, h = 12 cm, Rf = 0.29). Colorless oil, yield
75 mg (48%). 1H NMR (CDCl3): δ (ppm) = 1.32 (s, 6H, CH3), 1.34
1
melting points were uncorrected. For H NMR (400 MHz) and 13C
NMR (100 MHz), a Mercury Plus 400 spectrometer (Varian) was
used. δ in ppm is referenced to tetramethylsilane. Coupling constants
are given with 0.5 Hz resolution. Where necessary, the assignment of
the signals in the 1H NMR and 13C NMR spectra was performed using
1H−1H and 1H−13C COSY NMR spectra. The purity of all test
compounds was determined by HPLC analysis (purity of >95%).
General Procedure A for the Synthesis of Ethers 9. Under N2
alcohol 8 (80 mg, 0.34 mmol) was dissolved in dry DMF (2.5 mL). At
0 °C NaH (19 mg, 0.40 mmol) and then the respective halogen alkane
(0.38 mmol) were added dropwise, and the reaction mixture was
stirred at room temperature for 4 h. Then water was added and the
solution was extracted with ethyl acetate (3 × 10 mL). The organic
layer was dried (Na2SO4) and concentrated in vacuum, and the residue
was purified by fc.
General Procedure B for the Synthesis of Amides 11. Under
N2 commercially available carboxylic acid (1 equiv.) was dissolved in
dry THF (5 mL). CDI (67 mg, 0.41 mmol) was added slowly and the
reaction mixture was stirred for 10 min at rt. A solution of primary
amine 10 (80 mg, 0.33 mmol) in dry THF was added and the reaction
mixture was stirred at rt for 4 h. Then water was added and the
solution was extracted with ethyl acetate (3 x10 mL). The organic
layer was dried (Na2SO4), concentrated in vacuum and the residue was
purified by flash chromatography.
General Procedure C for the Synthesis of Amines 12 and 14.
Under N2, ketone 7 (100 mg, 0.43 mmol) or ketone 13 (80 mg, 0.34
mmol) was dissolved in dry MeOH (2 mL) in a microwave vial. Amine
(0.65 mmol) was added, and the reaction mixture was treated under
microwave irradiation at 80 °C for 10 min. The reaction mixture was
cooled to 0 °C. NaBH4 (30 mg, 0.78 mmol) was added slowly, and the
mixture was stirred at room temperature for 2 h. Then it was
concentrated in vacuum. The residue was dissolved in water, and the
solution was extracted with ethyl acetate (3 × 10 mL). The organic
layer was dried (Na2SO4) and concentrated in vacuum, and the residue
was purified by flash chromatography.
Synthetic Procedures. 6-[1-(Benzyloxy)ethyl]-7-methoxy-
2,2-dimethyl-2H-chromene (9a). According to general procedure
A, alcohol 8 (80 mg, 0.34 mmol) was reacted with benzyl bromide (65
mg, 0.38 mmol) and the product was purified by flash chromatography
(petroleum ether/EtOAc = 80/20, ⌀ = 1.5 cm, h = 15 cm, Rf = 0.29).
Colorless oil, yield 62 mg, (56%). 1H NMR (CDCl3): δ (ppm) = 1.40
(d, J = 6.4 Hz, 3H, CHCH3), 1.44 (s, 6H, CH3), 3.77 (s, 3H, OCH3),
4.32 (d, J = 12.0 Hz, 1H, OCH2Ph), 4.47 (d, J = 11.9 Hz, 1H,
OCH2Ph), 4.86 (q, J = 6.4 Hz, 1H, OCHCH3), 5.47 (d, J = 9.8 Hz,
1H, 3-CH), 6.30 (d, J = 9.8 Hz, 1H, 4-CH), 6.36 (s, 1H, 8-CH), 7.08
(s, 1H, 5-CH), 7.29−7.23 (m, 2H, Ph), 7.30−7.37 (m, 3H, Ph).
N-[1-(7-Methoxy-2,2-dimethyl-2H-chromen-6-yl)ethyl]-
benzamide (11a). According to general procedure B, primary amine
10 (80 mg, 0.33 mmol) was reacted with benzoic acid (40 mg, 0.33
mmol) and the product was purified by flash chromatography
(petroleum ether/EtOAc = 60/40, ⌀ = 2 cm, h = 12 cm, Rf =
0.26). Colorless solid, mp 91 °C, yield 105 mg (94%). 1H NMR
(CDCl3): δ (ppm) = 1.41 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.53 (d, J
= 6.9 Hz, 3H, CHCH3), 3.87 (s, 3H, OCH3), 5.34 (dq, J = 8.7/6.9 Hz,
1H, CH3CHNH-CO), 5.47 (d, J = 9.8 Hz, 1H, 3-CH), 6.25 (d, J = 9.8
Hz, 1H, 4-CH), 6.40 (s, 1H, 8-CH), 6.88 (s, 1H, 5-CH), 7.07 (d, J =
8.7 Hz, 1H, NHCO), 7.50−7.38 (m, 3H, Ph), 7.77−7.72 (m, 2H, Ph).
N-Benzyl-1-(7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-
ethanamine (12a). According to general procedure C, ketone 7 (100
mg, 0.43 mmol) was reacted with benzylamine (69 mg, 0.65 mmol)
and the product was purified by flash chromatography (dichloro-
methane/MeOH = 95/5, ⌀ = 1.5 cm, h = 15 cm, Rf = 0.26). Colorless
oil, yield 85 mg (61%). 1H NMR (CDCl3): δ (ppm) = 1.34 (d, J = 6.7
Hz, 3H, CHCH3), 1.43 (s, 6H, CH3), 1.73 (bs, 1H, NH), 3.60 (d, J =
12.9 Hz, 1H, PhCH2NH), 3.66 (d, J = 12.9 Hz, 1H, PhCH2NH), 3.77
(s, 3H, OCH3), 4.05 (q, J = 6.7 Hz, 1H, CH3CH-NH), 5.46 (d, J = 9.8
Hz, 1H, 3-CH), 6.29 (d, J = 9.8 Hz, 1H, 4-CH), 6.37 (s, 1H, 8-CH),
6.97 (s, 1H, 5-CH), 7.36−7.19 (m, 5H, Ph).
(d,
J = 6.7 Hz, 3H, HNCHCH3), 1.65−1.88 (m, 2H,
HNCH2CH2CH2Ph), 2.42−2.69 (m, 4H, HNCH2CH2CH2Ph), 3.73
(q, J = 6.7 Hz, HNCHCH3), 5.35 (d, J = 9.8 Hz, 1H, 3-CH), 6.13 (d, J
= 9.8 Hz, 1H, 4-CH), 6.20 (s, 1H, 8-CH), 6.46 (s, 1H, 5-CH), 6.90−
7.16 (m, 3H, Ph), 7.18−7.26 (m, 2H, Ph).
Investigation of the Antiprotozoal Activity. In vitro assays for
activity against Trypanosoma brucei rhodesiense (bloodstream trypo-
mastigote stage, STIB 900 strain), T. cruzi (intracellular amastigote
stage, Tulahuen C4 strain), Leishmania donovani (axenic amastigote
stage, MHOM-ET-67/L82), and Plasmodium falciparum (erythrocytic
stage, K1 strain) as well as cytotoxicity determinations against L6 rat
skeletal myoblasts were carried out at the Swiss Tropical and Public
Health Insitute according to established standard protocols described
earlier.24 Compounds used as positive controls were of commercial
origin, with the exception of melarsoprol, which was a gift from WHO.
Their purity (generally >95%) was specified by the manufacturers.
Investigation of Anti Leishmania tarentolae Activity. The
assay is described in detail in the Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
Physical and spectroscopic data of all new compounds, purity
data, general chemistry methods, and description of the anti L.
tarentolae assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
F
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(11) Kamboj, A.; Saluja, A. K. Ageratum conyzoides L.: a review on its
phytochemical and pharmacological profile. Int. J. Green Pharm. 2008,
2, 59−68.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49-251-8333311. Fax: +49-251-8332144. E-mail:
wuensch@uni-muenster.de.
Notes
■
(12) Nour, A. M. M.; Khalid, S. A.; Kaiser, M.; Brun, R.; Abdalla, W.
E.; Schmidt, T. J. The antiprotozoal activity of methylated flavonoids
from Ageratum conyzoides L. J. Ethnopharmacol. 2010, 129, 127−130.
(13) Bohlmann, F.; Tsankova, E.; Jakupovic, J.; King, R. M.;
Robinson, H. Dimeric chromenes and mixed dimers of a chromene
from Encelia canescens. Phytochemistry 1983, 22, 557−560.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was done within the framework of the NRW
International Graduate School of Chemistry, Munster,
Germany, which is funded by the Government of the State
Nordrhein-Westfalen and Westfalische Wilhelms-Universitat
Munster. The financial support is deeply acknowledged. The
biological part of this study is an activity of the Research
Network Natural Products against Neglected Diseases (ResNet
NPND, http://www.uni-muenster.de/ResNetNPND).
■
(14) Harel, D.; Khalid, S. A.; Kaiser, M.; Brun, R.; Wunsch, B.;
̈
Schmidt, T. J. Encecalol angelate, an unstable chromene from
Ageratum conyzoides L.: total synthesis and investigation of its
antiprotozoal activity. J. Ethnopharmacol. 2011, 137, 620−625.
(15) Steelink, C.; Marshall, G. P. Structures, syntheses, and
chemotaxonomic significance of some new acetophenone derivatives
from Encelia farinosa Gray. J. Org. Chem. 1979, 44, 1429−1433.
(16) Borch, R. F.; Hassid, A. I. New method for the methylation of
amines. J. Org. Chem. 1972, 37, 1673−1674.
̈
̈
̈
̈
(17) Bhattacharyya, S. A high throughput synthesis of N,N-dimethyl
tertiary amines. Synth. Commun. 2000, 30, 2001−2008.
ABBREVIATIONS USED
■
(18) Morgenthaler, J. B.; Peters, S. J.; Cedeno, D. L.; Constantino, M.
̃
WHO, World Health Organization; HAT, human African
trypanosomiasis; Pfc, Plasmodium falciparum; Tbr, Trypanoso-
ma brucei rhodesiense; Tcr, Trypanosoma cruzi; Ldo, Leishmania
donovani; Ltr, Leishmania tarentolae; SI, selectivity index
H.; Edwards, K. A.; Kamowski, E. M.; Passini, J. C.; Butkus, B. E.;
Young, A. M.; Lash, T. D.; Jones, M. A. Carbaporphyrin ketals as
potential agents for a new photodynamic therapy treatment of
leishmaniasis. Bioorg. Med. Chem. 2008, 16, 7033−7038.
́
(19) Taylor, V. M.; Munoz, D. L.; Cedeno, D. L.; Velez, I. D.; Jones,
̃
̃
M. A.; Robledo, S. M. Leishmania tarentolae: utility as an in vitro model
for screening of antileishmanial agents. Exp. Parasitol. 2010, 126, 471−
475.
REFERENCES
■
(1) Global Health Risks: Mortality and Burden of Disease
Attributable to Selected Major Risks. http://www.who.int/
healthinfo/global_burden_disease/GlobalHealthRisks_report_full.
pdf. The Global Burden of Disease: 2004 Update. http://www.who.
int/healthinfo/global_burden_disease/GBD_report_2004update_full.
pdf
(2) World Malaria Report 2011; World Health Organization: Geneva,
2011; http://www.who.int/malaria/world_malaria_report_2011/en/.
(3) Stuart, K.; Brun, R.; Croft, S.; Fairlamb, A.; Gurtler, R. E.;
McKerrow, J.; Reed, S.; Tarleton, R. Kinetoplastids: related protozoan
pathogens, different diseases. J. Clin. Invest. 2008, 118, 1301−1310.
(4) Leishmaniasis; Fact Sheet No. 375; World Health Organization:
Geneva, February 2013; http://www.who.int/mediacentre/factsheets/
fs375/en/.
(20) St. George, S.; Bishop, J. V.; Titus, R. G.; Selitrennikoff, C. P.
Novel compounds active against Leishmania major. Antimicrob. Agents
Chemother. 2006, 50, 474−479.
(21) Debrabant, A.; Joshi, M. B.; Pimenta, P. F. P.; Dwyer, D. M.
Generation of Leishmania donovani axenic amastigotes: their growth
and biological characteristics. Int. J. Parasitol. 2004, 34, 205−217.
(22) Zilberstein, D.; Blumenfeld, N.; Liveanu, V.; Gepstein, A.; Jaffe,
C. L. Growth at acidic pH induces an amastigote stage-specific protein
in Leishmania promastigotes. Mol. Biochem. Parasitol. 1991, 45, 175−
178.
(23) Chavali, A. K.; Whittemore, J. D.; Eddy, J. A.; Williams, K. T.;
Papin, J. A. Systems analysis of metabolism in the pathogenic
trypanosomatid Leishmania major. Mol. Syst. Biol. 2008, 4, 177.
(24) Nour, A. M. M.; Khalid, S. A.; Brun, R.; Kaiser, M.; Schmidt, T.
J. The antiprotozoal activity of sixteen Asteraceae species native to
Sudan and bioactivity-guided isolation of xanthanolides from Xanthium
brasilicum. Planta Med. 2009, 75, 1363−1368.
̈
(5) Rassi, A., Jr.; Rassi, A.; Marin-Neto, A. J. Chagas disease. Lancet
2010, 375, 1388−1402.
(6) Trypanosomiasis, Human African (Sleeping Sickness); Fact Sheet
No. 259; World Health Organization: Geneva, October 2012; http://
www.who.int/mediacentre/factsheets/fs259/en/.
(7) Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M. A.;
Biavatti, M. W.; Brun, R.; Da Costa, F. B.; de Castro, S. L.; Ferreira, V.
F.; de Lacerda, M. V. G.; Lago, J. H. G.; Leon, L. L.; Lopes, N. P.; das
Neves Amorim, R. C.; Niehues, M.; Ogungbe, I. V.; Pohlit, A. M.;
Scotti, M. T.; Setzer, W. N.; de N. C. Soeiro, M.; Steindel, M.;
Tempone, A. G. The potential of secondary metabolites from plants as
drugs or leads against protozoan neglected diseasespart I. Curr. Med.
Chem. 2012, 19, 2128−2175.
(8) Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M. A.;
Biavatti, M. W.; Brun, R.; Da Costa, F. B.; de Castro, S. L.; Ferreira, V.
F.; de Lacerda, M. V. G.; Lago, J. H. G.; Leon, L. L.; Lopes, N. P.; das
Neves Amorim, R. C.; Niehues, M.; Ogungbe, I. V.; Pohlit, A. M.;
Scotti, M. T.; Setzer, W. N.; de N. C. Soeiro, M.; Steindel, M.;
Tempone, A. G. The potential of secondary metabolites from plants as
drugs or leads against protozoan neglected diseasespart II. Curr.
Med. Chem. 2012, 19, 2176−2228.
(9) Narender, T.; Shweta; Gupta, S. A convenient and biogenetic
type synthesis of few naturally occurring chromeno dihydrochalcones
and their in vitro antileishmanial activity. Bioorg. Med. Chem. Lett.
2004, 14, 3913−3916.
(10) Alizadeh, B. H.; Foroumadi, A.; Ardestani, S. K.; Poorrajab, F.;
Shafiee, A. Leishmanicidal evaluation of novel synthetic chromenes.
Arch. Pharm. 2008, 341, 787−793.
G
dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX