Synthesis and Antiplasmodial Activity of Novel Chloroquine Analogues with Bulky Basic Side Chains

Article abstract of DOI:10.1002/cmdc.201500195

Chloroquine is commonly used in the treatment and prevention of malaria, but Plasmodium falciparum, the main species responsible for malaria-related deaths, has developed resistance against this drug. Twenty-seven novel chloroquine (CQ) analogues characterized by a side chain terminated with a bulky basic head group, i.e., octahydro-2H-quinolizine and 1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2-a][1,5]diazocin-8-one, were synthesized and tested for activity against D-10 (CQ-susceptible) and W-2 (CQ-resistant) strains of P. falciparum. Most compounds were found to be active against both strains with nanomolar or sub-micromolar IC₅₀ values. Eleven compounds were found to be 2.7- to 13.4-fold more potent than CQ against the W-2 strain; among them, four cytisine derivatives appear to be of particular interest, as they combine high potency with low cytotoxicity against two human cell lines (HMEC-1 and HepG2) along with easier synthetic accessibility. Replacement of the 4-NH group with a sulfur bridge maintained antiplasmodial activity at a lower level, but produced an improvement in the resistance factor. These compounds warrant further investigation as potential drugs for use in the fight against malaria.

Full text of DOI:10.1002/cmdc.201500195

DOI: 10.1002/cmdc.201500195
Full Papers
Synthesis and Antiplasmodial Activity of Novel
Chloroquine Analogues with Bulky Basic Side Chains
[a]
[a]
[a]
[b]
[c]
Bruno Tasso,* Federica Novelli, Michele Tonelli, Anna Barteselli, Nicoletta Basilico,
[d]
[d]
[b]
[a]
Silvia Parapini, Donatella Taramelli, Anna Sparatore, and Fabio Sparatore
Chloroquine is commonly used in the treatment and preven-
tion of malaria, but Plasmodium falciparum, the main species
responsible for malaria-related deaths, has developed resist-
ance against this drug. Twenty-seven novel chloroquine (CQ)
analogues characterized by a side chain terminated with
a bulky basic head group, i.e., octahydro-2H-quinolizine and
compounds were found to be 2.7- to 13.4-fold more potent
than CQ against the W-2 strain; among them, four cytisine de-
rivatives appear to be of particular interest, as they combine
high potency with low cytotoxicity against two human cell
lines (HMEC-1 and HepG2) along with easier synthetic accessi-
bility. Replacement of the 4-NH group with a sulfur bridge
maintained antiplasmodial activity at a lower level, but pro-
duced an improvement in the resistance factor. These com-
pounds warrant further investigation as potential drugs for use
in the fight against malaria.
1
,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2-a][1,5]diazo-
cin-8-one, were synthesized and tested for activity against D-
0 (CQ-susceptible) and W-2 (CQ-resistant) strains of P. falcipa-
1
rum. Most compounds were found to be active against both
strains with nanomolar or sub-micromolar IC50 values. Eleven
Introduction
Recent estimates suggest that more than 198 million clinical
cases of malaria occur every year worldwide, with 584000
deaths in 2013. Most of the casualties are children living in
pies) able to improve efficacy and delay the onset of resist-
ance. The different approaches are illustrated and discussed in
[
3]
some recent reviews.
[1]
sub-Saharan Africa or pregnant women. Plasmodium falcipa-
rum (Pf), which is the main species responsible for the ob-
served fatalities, has developed resistance to conventional anti-
malarials such as chloroquine (CQ) and antifolates. Resistance
even to the artemisinin derivatives has been recently reported
Particularly, the 4-aminoquinoline derivatives continue to at-
tract interest because resistance appears to be compound spe-
cific and not related to changes in the structure of the drug
[
4]
targets. Indeed, even close analogues of CQ and amodia-
quine retain potent activity against CQ-resistant (CQ-R) strains
[
2]
[5]
in southeast Asia. To overcome the drug resistance issue, ex-
tensive modification of existing antimalarial chemotypes and
the search for novel targets (and new chemotypes interacting
with them) have been undertaken and are currently being pur-
sued. Thus, many interesting compounds have been selected
for use in monotherapy or, even better, in combination chemo-
therapies (particularly artemisinin-based combination thera-
of P. falciparum, such as short side-chain compounds A–C
and metabolically stable isoquine (D), tert-butylisoquine (E),
[
6]
and 4’-fluoro-N-tert-butylamodiaquine (F) (Figure 1). Peculiar
modification of the side chain of CQ is observed in ferroquine
[
7]
(G), which incorporates a ferrocenyl moiety, and in pipera-
quine (H), an old bisquinoline analogue that has been resumed
[
8]
as an efficient partner of artemisinin derivatives.
A valuable approach to obtain novel CQ analogues is repre-
sented by the assembly of hybrid molecules containing two
pharmacophores joined through a linker that should not dis-
turb the interaction of each component with the relevant
[
a] Dr. B. Tasso, Dr. F. Novelli, Dr. M. Tonelli, Prof. F. Sparatore
Dipartimento di Farmacia, Università degli Studi di Genova
Viale Benedetto XV 3, 16131 Genova (Italy)
[
9]
E-mail: bruno.tasso@unige.it
target. The two pharmacophores may act in different ways
[
10]
[
b] Dr. A. Barteselli, Prof. A. Sparatore
on the same target, as in trioxaquine derivatives I and J,
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano
via Mangiagalli 25, 20133 Milano (Italy)
which are able to alkylate heme and to inhibit the formation
of hemozoin, or they may act on different targets, as is the
case for “reversed chloroquines” K and L; these derivatives in-
corporate different kinds of inhibitors of P. falciparum chloro-
quine resistance transporter (PfCRTs), which inhibit the forma-
tion of hemozoin and also the efflux of the drug from the di-
[
c] Dr. N. Basilico
Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche
Università degli Studi di Milano
via C. Pascal 36, 20133 Milano (Italy)
[
d] Dr. S. Parapini, Prof. D. Taramelli
Dipartimento di Scienze Farmacologiche e Biomolecolari
Università degli Studi di Milano
[11]
gestive vacuole of the parasite.
Particularly interesting hybrid antimalarial agents have been
obtained by combining the scaffold of chloroquine and prima-
via C. Pascal, 36, 20133 Milano (Italy)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500195.
[
12]
quine (PQ) through different linkers (e.g., M). Primaquine, an
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tivity against CQ-R P. falciparum by inhibiting the
[
14]
PfCRT.
The synthesized CQ–PQ hybrids display
good to excellent activity against both the liver and
blood stages of the Plasmodium infection and against
gametocytes, which thus inhibits the transmission
cycle.
Additional examples of hybrid molecules are com-
bination products of the 4-aminoquinoline scaffold
with polyarylmethyl system N; these hybrids are able
to form radical intermediates that are toxic to the
parasite, and additionally, they block the formation of
[
15]
hemozoin. Finally, CQ analogues O and P bearing
a side chain ending with bulky, nonbasic moieties are
[
16,17]
noteworthy.
Some of these compounds display
potent activity against different strains of P. falcipa-
rum, which suggests that a basic side chain is not
very essential for antiplasmodial activity, and this
opens new possibilities for the design of novel chlor-
oquine analogues. Interestingly, the former group of
[
16]
compounds inhibits (similar to CQ) the formation
[
17]
of hemozoin, whereas the latter
inhibits, albeit
weakly, falcipain 2 (a cysteine protease critical for he-
moglobin catabolism) as an alternative or additional
[
18]
target for antimalarial activity.
Over the past several years we have been engaged
in the search of novel antimicrobial agents, and re-
cently, we studied the antimalarial activity of some 4-
aminoquinoline and 9-aminoacridine derivatives (e.g.,
Q–T; Figure 2) characterized by the presence of
a bulky, strongly basic, and lipophilic bicyclic moiety,
that is, a quinolizidine (octahydro-2H-quinolizine) or
[
19–22]
pyrrolizidine (hexahydro-1H-pyrrolizine) ring.
These moieties appear as interesting structural fea-
tures that are able to overcome the resistance mech-
anism. Indeed, these novel CQ analogues exhibited
high antimalarial activity against CQ-susceptible (CQ-
S) and CQ-R strains of Pf in vitro through a CQ-like
mechanism, and some of them also showed efficacy
against P. berghei and P. yoelii if given orally or intra-
[
23,24]
peritoneally in a murine standard four-day test.
Pursuing our efforts to obtain novel antimalarial
agents containing a bulky basic head, we deemed it
interesting to investigate the effects on the activity
of the following structural changes, operated either
singularly or in combination: 1) Further elongation of
the chain between the NH group and the quinolizi-
dine nucleus (e.g., compounds 1–5; Figure 3), which
has already been shown to improve activity and/or
[
5a,19]
to alter the selectivity for CQ-R or CQ-S parasites.
) The shift in the side chain from the 1-position to
2
Figure 1. Examples of chloroquine (CQ) and amodiaquine (AQ) analogues with modified
side chains.
the 9a-position of the quinolizidine ring to avoid ste-
reochemical issues (e.g., compounds 6–8; Figure 3).
3
) The exchange of the quinolizidine nucleus with an
old 8-aminoquinoline derivative, acts against all exoerythrocyt-
ic forms of Plasmodium and displays gametocytocidal activity,
which is still the only transmission-blocking antimalarial that is
even bulkier, but less lipophilic, moiety such as that of cytisine
[(1R,5S)-(1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2-a]
[1,5]diazocin-8-one] (e.g., compounds 9–20; Figure 3). The pyri-
done ring present in the cytisine moiety might act as a Michael
[
13]
clinically available. Moreover, PQ synergizes chloroquine ac-
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the relevant compounds are
thought to be fairly resistant to
metabolic attack.
Results and Discussion
Chemistry
The preparation of the long-
chain w-(quinolizidin-1-yl)alkyla-
mines required for the synthesis
of higher homologues of com-
Figure 2. Previously studied 4-aminoquinoline and 9-aminoacridine derivatives with bulky basic head groups.
pounds of general structure Q (Figure 2) may be a difficult and
time-consuming task; thus, we resorted to the use of w-(lupi-
nylthio)alkylamines {w-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)-
methylthio]alkylamines} taking into account the well-known bi-
oisosterism of a methylene unit for a sulfur atom. This ap-
proach was already used profitably for the preparation of long-
chain N-(w-quinolizindinyl)alkanoyl- and N- or O-(w-quinolizidi-
nyl)alkyl derivatives of several aromatic and heteroaromatic
moieties (e.g., phenothiazine and other tricyclic systems, ani-
lines, 9-aminoacridines, and 6-hydroxycoumarin), to obtain
[
30,31]
[32]
muscarinic M1 and M2 ligands,
antiarrhythmics, antipri-
[
33]
on toxicity agents, and acetylcholinesterase and butyrylcholi-
[
34]
nesterase inhibitors.
[
35]
Thus, thiolupinine (28) [(1R,9aR)-(octahydro-2H-quinolizin-
-yl)methanthiol] was treated with iodoacetamide,
[
34a]
1
acryloni-
[
33]
trile, chloroacetone, and methyl vinyl ketone to obtain com-
pounds 29, 31, 33, and 36, respectively. The last two ketones
were converted into corresponding oximes 34 and 37, which
in addition to compounds 29 and 31 were reduced with LiAlH4
to required long-chain amines 30, 32, 35, and 38. These
amines were, finally, treated with 4,7-dichloroquinoline (39) or
4
-chloro-7-trifluoromethylquinoline (40) to obtain compounds
1
–5 (Scheme 1).
For the synthesis of compounds 6–8, 4,7-dichloroquinoline
was treated with w-(quinolizidin-9a-yl)alkylamines (dihydro-
chlorides) 41–43 in the presence of diisopropylethylamine
Figure 3. Structures of novel quinoline derivatives tested as antimalarial
agents (see Table 1 for structural details).
(
DIPEA).
4/6-(Octahydro-2H-quinolizin-9a-yl)butan/hexan-1-
amines (41 and 42) were recently described by Barteselli
[
36]
et al.,
and the homologue 8-(octahydro-2H-quinolizin-9a-
acceptor and, as such, could affect the thiol group of the
yl)octan-1-amine (43) was obtained in a similar way starting
[25]
[37]
active site of cysteine protease (as falcipain). Interestingly,
from quinolizidinium perchlorate through hydroxyalkylation
[
38]
some N-benzylcytisine derivatives have been recently shown
with the method of McIntosh and final amination according
[
26]
[39]
to display activity against the parasite Leishmania donovani.
) The replacement, in the foregoing compounds, of the NH
to Becker et al. (Scheme 2).
[
40]
4
Compound 9 was obtained by treating N-aminocytisine
group with a sulfur bridge (e.g., compounds 22–27; Figure 3),
which in addition to decreasing the basicity of the molecule
should also increase its lipophilicity. The right balance of these
physicochemical characteristics is fundamental for the accumu-
lation of the drug in the digestive vacuole and for its associa-
(48) with 4,7-dichloroquinoline, whereas compounds 10–16,
19, and 20 were obtained from the reactions of 4-[N-(w-bro-
moalkyl)amino]-7-chloroquinolines 54–58 with cytisine (59), 9-
nitrocytisine (60), and 9,11-dibromocytisine (61) eventually
in the presence of DIPEA. The reaction of 4-[N-(4-bromobutyl)a-
mino]-7-chloroquinoline with cytisine or its derivatives pro-
duced, instead of the expected compounds, 7-chloro-4-(pyrroli-
din-1-yl)quinoline (21) by intramolecular loss of hydrogen bro-
mide. The catalytic reduction of 9-nitrocytisine derivatives 15
and 16 gave corresponding amino derivatives 17 and 18
[
41]
[42]
[
4c]
tion with hematin to inhibit the formation of hemozoin. 7-
Chloro-4-(pyrrolidin-1-yl)quinoline (21), incidentally obtained
during the syntheses, was also considered. The structures of
the 27 tested compounds are collected in Figure 3. On the
[27–29]
basis of previous observations,
the bulky basic heads of
(
Scheme 3).
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Scheme 1. Reagents and conditions: a) iodoacetamide, EtOH, RT, 3 days; b) acrylonitrile, Triton B, dioxane, 608C, 1 h; c) chloroacetone, EtOH, RT, 1 h; d) methyl
vinyl ketone, dry dioxane, RT, 1 h; e) LiAlH , THF, reflux, 72 h; f) hydroxylamine hydrochloride/6n NaOH, EtOH, reflux, 2 h; g) 4-chloro-7-substituted quinoline,
4
phenol, 1808C, 4 h.
[
32,48]
[49]
nines 63 and 64
Scheme 4).
and N-(w-haloalkyl)cytisines 65 and 66
(
Antimalarial activity
Compounds 1–27 were tested in vitro against D-10 (CQ-S) and
W-2 (CQ-R) strains of P. falciparum. The antimalarial activity was
evaluated by inhibition of parasite growth, as measured by the
production of parasite lactate dehydrogenase. Table 1 shows
the IC50 values against the D-10 and W-2 strains of P. falcipa-
rum, as well as the means of the ratios between the IC50 of CQ
and that of each compound against the D-10 and W-2 strains,
calculated for each single experiment.
The ratios between the IC50 values of each compound
against the two strains are also indicated. These last values are
suggestive of the susceptibility of the drug to the resistance
mechanism (resistance factor, RF). The results listed in Table 1
indicate that many of novel 4-aminoquinoline derivatives 1–20
display high antimalarial activity with IC50 values <100 nm
against both the CQ-S and CQ-R strains, which thus resulted in
Scheme 2. Reagents and conditions: a) phenol, DIPEA, N
b) ZnBr in THF, 2-methyl-2-butene,
, dry THF, 508C!reflux, 20 h; c) 1n BH
dry THF, RT, 4.5 h; d) H O, 6n NaOH, 35% H , RT, 1 h; e) phthalimide, Ph
DEAD, dry THF, RT, 20 h; f) 6n HCl, reflux, 20 h.
2
, 1308C 5 h;
2
3
2
2
O
2
3
P,
11 compounds that were from 2.7- to 13.4-fold more active
than chloroquine against the CQ-R strain W-2. The remaining
compounds show an appreciable level of activity with sub-mi-
cromolar IC50 values with the only exception of compounds 10
and 13. On the other hand, 4-pyrrolidinylquinoline 21 and
compounds 22–27, which are six derivatives of 4-thioquinoline,
display only modest or moderate antimalarial activity with IC50
values in the low (1.4–12.5) mm range; nevertheless, these com-
pounds exhibit an interesting improvement in the resistance
factor.
Required 4-[N-(w-bromoalkyl)amino]-7-chloroquinolines 54–
8 were obtained by treating 4,7-dichloroquinoline with the
5
suitable w-hydroxyalkylamines and subsequent bromination by
means of 48% hydrobromic acid in the presence of concen-
trated sulfuric acid, as previously described for some of
[
43–46]
them.
Compounds 22 and 23 were obtained by treating thiolupi-
[
35]
nine (28) with the proper 4-chloro-7-substituted quinolines,
whereas compounds 24–27 were obtained from 7-chloro-4-thi-
With regard to the structure–activity relationships, a general
enhancement in the activity with increasing distance between
[
47]
oquinoline (62), which was treated with S-(haloalkyl)thiolupi-
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2 2 4 3
Scheme 3. Reagents and conditions: a) phenol, N , 1808C 7 h; b) hydroxyalkylamine, 1508C, 3 h; c) 48% HBr/H SO , 1608C, 3.5 h; d) CH CN, 1008C, 36 h;
e) EtOH, H , 10% Pd/C, RT.
2
9
and 25.9 nm for the CQ-S and CQ-R strains, respectively),
even if the interest in this compound is restrained by its com-
plex synthesis.
In the set of cytisinyl derivatives 9–13, elongation of the
linker produces an alternating effect on the activity, which is
initially decreasing and then steadily increasing. The minimal
activity is observed in compound 10 (linker with two methyl-
ene units), with IC50 values of 941 and 1776 nm for the CQ-S
and CQ-R strains, respectively. The maximal activity is found in
compound 12 (linker with five methylene units) with IC50
values of 32.9 and 77.1 nm for the CQ-S and CQ-R strains,
which are 28- and 23-fold enhancements in potency, respec-
tively. The introduction of substituents on the pyridone ring of
the cytisine moiety (i.e., compounds 14–20) produces a clear
increase in antimalarial activity that is cumulative with the pos-
itive effect of elongation of the linker. Thus, compounds 16,
Scheme 4. Reagents and conditions: a) Dowtherm A, 1708C, 2 h; b) KOH,
CH CN, N , RT, 24 h.
3
2
18, and 20 are the most potent among those presently report-
ed, and these derivatives are 5-, 10-, and 8-fold more potent
than chloroquine, respectively, and are from 1/3 to 2/3 as
potent as artemisinin, against the W-2 (CQ-R) strain of P. falci-
parum. Moreover, the cytisinyl derivatives are characterized by
low cytotoxicity (Table 2) versus two human cell lines (human
microvascular endothelial cells, HMEC-1, and human Caucasian
hepatocellular carcinoma, HepG2), with selectivity indices (SI)
in the ranges of 400 to 880 and of 143 to 540, respectively
(CQ, SI>120 and 62, respectively). Additionally, comparably
with CQ and artemisinin, they produced less than 10% hemol-
ysis upon incubation for 72 h at a concentration of 200 mm
with uninfected erythrocytes. Among the most potent com-
pounds, only 8 displayed some hemolytic activity with a CC50
value of (148.6Æ1.2) mm.
the quinoline ring and the basic nitrogen atom of the side
chain was observed, even if with some peculiarities in each
[
19]
subgroup of compounds. Indeed, we previously showed
that the increasing length of the methylene chain in com-
pounds of structure Q (Figure 2) influenced negatively the anti-
malarial activity, especially against the CQ-R strain (for n=0,
IC =23.3 nm; for n=3, IC =384.4 nm). Such a trend is now
50
50
inverted in compounds 1 and 2, which are characterized by
a longer linker of four and five atoms, respectively. This behav-
ior is similar to that observed in a set of diethylaminoalkyl ana-
logues of CQ that first exhibit decreasing activity in the
medium-length compounds but then recover the high activity
[5a]
if the number of methylene units is further increased.
Therefore, the cytisinyl derivatives of 4-aminoquinoline
appear worthy of further evaluation as antimalarial agents, also
in consideration of the easy availability of the starting material,
which is extracted from the seeds of the widespread Laburnum
anagyroides and of other plants (Sophora and Thermopsis spe-
cies). Particularly, the complex structure of the cytisine moiety
that characterizes the best compounds warrants thorough in-
vestigation of their mechanism of action. The high activity of
Interestingly, although stereochemical issues are overcome
by shifting the linker (between the quinoline and quinolizidine
moieties) from the 1-position to the 9a-position of the quinoli-
zidine ring, this shift produces only minor variations in the an-
timalarial potency (compare compounds 1 and 2 with 6 and
7
). However, if the linker is elongated up to eight methylene
units (as in 8), a strong increase in potency is observed (IC =
50
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Table 1. Antimalarial activity of compounds 1–27 tested in vitro against D-10 (CQ-S) and W-2 (CQ-R) P. falciparum strains.
Compd
R
X
R’
D-10 (CQ-S)
W-2 (CQ-R)
Ratio IC50
CQ-R/CQ-S
[a]
[b]
[a]
[b]
[c]
IC50 [nm]
Ratio to CQ
IC50 [nm]
Ratio to CQ
1
2
3
4
5
6
7
8
9
Cl
Cl
Cl
Cl
CF
–
–
–
–
–
–
–
–
–
–
–
–
–
NH-CH
NH-(CH
2
-CH
2
-S-CH
2
–
–
–
–
–
–
–
–
H
H
H
H
53.7Æ13.5
27.1Æ6.9
0.61
1.21
1.25
1.33
0.55
0.80
1.50
2.34
0.06
0.02
0.17
0.28
0.01
0.08
0.15
0.56
0.14
1.11
0.28
0.79
0.02
0.003
259.4Æ60.1
155.4Æ49.0
82.6Æ15.5
3.15
5.25
6.20
4.27
6.13
0.68
1.67
13.45
0.50
0.16
0.94
2.96
0.22
0.26
0.42
5.43
0.52
10.32
2.76
8.06
0.31
0.07
0.04
0.28
0.36
0.04
0.10
–
4.83
5.72
3.36
5.19
1.49
21.12
7.16
2.88
1.72
1.89
2.56
2.34
1.16
7.59
8.72
2.55
4.975
2.04
1.91
1.85
0.915
0.63
0.62
0.75
0.81
1.25
1.32
16
2
)
3
-S-CH
2
NH-CH(CH
NH-CH(CH
NH-CH(CH
NH-(CH
NH-(CH
NH-(CH
NH
NH-CH
NH-(CH
NH-(CH
3
3
3
)-CH
)-CH
)-CH
2
2
2
-S-CH
-CH
-S-CH
2
24.6Æ8.3
2
-S-CH
2
23.1Æ8.7
119.9Æ48.3
83.5Æ37.8
3
2
56.1Æ17.3
22.5Æ9.6
2
)
2
)
2
)
4
6
8
475.3Æ145.7
194.3Æ74.6
25.9Æ3.7
27.15Æ8.0
9.0Æ3.7
370.1Æ110
941.5Æ262.1
116.5Æ38.4
32.9Æ2.4
635.8Æ113
1775.9Æ304.4
298.7Æ35.7
77.1Æ22.8
1
0
2
-CH
2
1
1
2
)
)
3
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
2
5
NH-CH(CH
NH-CH -CH
3
)-CH
2
H
902.3Æ263.4
115.3Æ23.2
62.7Æ5.3
1048.6Æ360.8
874.8Æ366.2
546.6Æ174.3
42.0Æ21.1
2
2
9-NO
9-NO
9-NO
9-NH
9-NH
2
2
2
NH-(CH
NH-(CH
NH-(CH
NH-(CH
NH-(CH
NH-(CH
–
2
)
2
)
2
)
2
)
2
)
2
)
3
5
3
5
3
5
16.5Æ5.2
2
2
96.2Æ7.5
478.4Æ140.0
24.3Æ10.1
11.9Æ6.0
–
–
–
9,11-Br
9,11-Br
2
2
47.5Æ13.9
16.8Æ5.5
90.7Æ42.1
31.1Æ16.4
–
–
–
–
–
H
H
886.6Æ25.9
7190.0Æ2620
810.9Æ162.8
4530.0Æ2557
7820.0Æ2539
1818.0Æ660.1
1416.0Æ379.8
12281.0Æ400.3
5190.0Æ2028
320Æ51
Cl
CF
Cl
Cl
–
S-CH
S-CH
S-CH
2
2
2
[d]
3
>10000
NA
-CH
2
-S-CH
2
2412.0Æ192.1
1747.0Æ63.0
9841.0Æ2023
3922.0Æ162.2
20.0Æ5
0.013
0.018
0.003
0.008
–
S-(CH
S-(CH
S-(CH
2
2
2
)
)
)
3
3
4
-S-CH
2
–
chloroquine (CQ)
mefloquine
artemisinin
33.2Æ11.3
–
–
16.9Æ7.3
–
–
0.51
0.52
27.7Æ8.9
14.5Æ4.2
[
a] Results are expressed as the mean ÆSD of at least three different experiments, each performed in duplicate or triplicate. [b] Mean of ratios between
the IC50 of CQ and that of each compound against P. falciparum strains D-10 or W-2, calculated for each single experiment. [c] Resistance factor: Ratios be-
tween the IC50 values of each compound against the two strains of P. falciparum. [d] Not available.
these compounds might be related to strong association of
the molecules with hematin, similar to that observed for Solo-
pounds 1, 2, and 10 with those of corresponding methyl ho-
mologues 3, 4, and 13.
[
16]
mon’s compounds (e.g., O, Figure 1) bearing a side chain
with a bulky, highly lipophilic head. However, owing to the Mi-
chael acceptor characteristics of the pyridone ring embodied
in the cytisine moiety, interaction with the thiol group of the
active site of falcipain cannot be excluded. Actually, even com-
pound P (Figure 2) inhibited, albeit weakly, falcipain 2, which
Replacement of the chlorine atom in the quinoline ring with
a trifluoromethyl group decreases the activity on the CQ-S
strain, but it does not affect the activity on the CQ-R strain
(compare compounds 3 and 5). Such a decrease in activity was
[
19]
already observed by us
in compounds of structure Q
(Figure 2) and by several authors in other 4-aminoquinoline de-
[17]
[4c]
should attack one of its carboamide groups.
Notably, pyridone-derived falcipain inhibitors have been de-
rivatives. Kaschula et al.
correlated the decreased activity
with the stronger electron-withdrawing character of the tri-
fluoromethyl group, which decreases the basicity of both nitro-
gen atoms of the side chain. However, in the case of the CQ-R
strain the enhanced lipophilicity, and subsequent cell penetra-
tion, of the fluorinated compounds might compensate the
[
50]
scribed, for which, however, the pyridone ring served mainly
as a rigid peptidomimetic backbone, and additional more
potent electrophilic groups were included in the structures. In
both sets of quinolizidinylalkyl and cytisinylalkyl derivatives,
branching of the linker produces a slight increase in activity, as
shown by comparison of the activities of unbranched com-
[
51]
negative effect of the decreased basicity on the activity.
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Conclusions
Table 2. Cytotoxicity against human microvascular endothelial cells (HMEC-1)
and human Caucasian hepatocyte carcinoma (HepG2), and selectivity index of
the most potent and other representative compounds.
Twenty-seven novel chloroquine analogues characterized
by a side chain bearing a bulky basic head (i.e., octahy-
dro-2H-quinolizine and 1,2,3,4,5,6-hexahydro-1,5-meth-
ano-8H-pyrido[1,2-a][1,5]diazocin-8-one) were synthe-
sized and tested for antimalarial activity against the D-10
[
a]
[b]
Compd
IC50 [nm]
SI
HMEC-1
HepG2
W-2 (CQ-R) HMEC- HepG2
1
3
6
7
8
18673Æ5915
9773Æ1912
NT
82.6Æ15.5
226
118.3
–
–
(
CQ-susceptible) and W-2 (CQ-resistant) strains of P. falci-
>26000
475.3Æ145.7 >54.7
9211Æ938
5495Æ697
NT
194.3Æ74.6
25.9Æ3.7
47.4
212.2
710.8
547.8
880.5
399.1
41.4
parum. Most compounds were active against both strains
with nanomolar or sub-micromolar IC50 values; in particu-
lar, 11 compounds were 2.7- to 13.4-fold more active
than chloroquine against the chloroquine-resistant strain.
The influence on activity of some structural features
was analyzed, and in each subset of compounds, the
positive effect of increasing the length of the spacer be-
tween the quinoline and the bulky basic head was con-
firmed. Moreover, exchange of the 4-NH group with
a sulfur bridge maintained the antimalarial activity but at
a lower level of potency; however, this structural modifi-
1869Æ628
11165Æ2263
10113 Æ217
13142Æ1402
4454Æ1211
NT
72.2
1
1
1
2
2
2
6
8
0
4
54800Æ14547
23010Æ6797
21388Æ5245
12413Æ2066
75282Æ11 316
77.1Æ22.8
42.0Æ21.1
24.3Æ10.1
31.1Æ16.4
1818.0Æ660.1
144.8
240.8
540.8
143.8
–
CQ
Arte
>38000
NT
19942Æ1643 320Æ51
>106260
>118.8
–
62.3
14.5Æ4.2
>7328.3
[
a] Results are the mean ÆSD of three experiments each performed in dupli-
cate; NT: not tested. [b] Selectivity index: [IC50 (HMEC-1 or HepG2)]/[IC50 (W-
2
(CQ-R)].
Finally, in compounds 22–27 the replacement of the 4-NH
group by a sulfur bridge should increase the lipophilicity of
the molecule but still allow resonance of the positive charge
from the protonated quinoline nitrogen atom to the sulfur
bridge, as suggested by the UV spectra of other 7-chloro-4-thi-
Table 3. Calculated logD and pK
a
values for representative compounds
(quinolizidine derivatives: Q-8 and 22–25; cytisine derivatives: 11–20 and
26, 27).
[b]
[c]
[d]
[e]
[f]
Compd
logD7.4
logD5.2
pKa1
pKa2
pKa3
[
52]
oquinoline derivatives described by Clinton and Suter. The
presence of this resonance should favor interaction of the mol-
NH bridged
Q (n=1)
1
2
3
5
8
11
1.98
2.67
3.01
3.02
3.70
4.13
2.40
2.88
2.82
2.32
4.47
0.73
1.41
1.73
1.79
2.84
10.18
10.15
10.16
10.15
6.35
6.30
6.43
6.23
À4.48
À4.69
À4.13
À3.99
III
[4a]
ecules with ferriprotopophirin IX (Fe PPIX) and should there-
fore increase antimalarial activity, even if no activity was ob-
served upon screening the above 4-S analogues of CQ in vivo
[h]
[h]
[h]
NC
NC
NC
[52]
against avian malaria.
Actually, the lack of in vivo activity
2.92
10.59
8.80
8.92
8.88
8.78
8.08
6.51
6.32
6.48
6.48
6.48
6.48
À3.66
À4.94
À3.86
À3.86
À3.84
À3.83
may be related to inadequate dosage or unfavorable pharma-
cokinetic characteristics of the particular compounds.
À0.35
1
1
1
2
6
8
0.23
À0.12
À0.43
1.44
Indeed, S-bridged compounds 22–27 display, in vitro, mod-
erate antimalarial activity against both of the CQ-S and CQ-R
strains of P. falciparum, with most IC50 values in the low-micro-
molar range. This activity is in line with that exhibited by a set
of 7-chloro-4-(S-diethylaminoalkyl)thioquinolines (described by
[g]
20
S bridged
2
2
24
25
2
2
2
3
2.81
2.99
3.31
3.65
3.21
3.33
2.13
2.32
2.79
3.15
1.29
1.51
9.91
9.91
10.13
10.15
8.07
3.30
2.49
3.25
3.37
3.28
3.37
–
–
–
–
–
–
[
53]
Natarajan et al. ), despite the largely different bulkiness of the
terminal heads of the respective side chains.
6
7
In both series of compounds, the activity improves some-
what with elongation of the linker, in conformity with the
trend observed for the respective NH analogues. However, dif-
ferently from the latter, most of these sulfur-containing com-
8.38
[
a] Calculated with ACD/Labs ver. 7.00. [b] LogD at plasma pH. [c] LogD
at P. falciparum digestive vacuole pH. [d] pK of side chain N atom. [e] pK
of 4-NH group. [g] pK of 9-NH group: 3.61.
a
a
of quinoline N atom. [f] pK
a
a
2
pounds exhibit a resistance factor (RF=IC CQ-R/IC CQ-S) sig-
5
0
50
[h] Not calculated.
nificantly below 1 (RF=0.62–0.81 for compounds 22–25),
which suggests low susceptibility of the S-bridged compounds
to resistance mechanisms. Thus, once more, within a given
type of compounds the enhanced lipophilicity seems to bal-
ance the negative effect of the reduced basicity on the activity
against the CQ-R strain. However, comparing the S-bridged
compounds with the 4-aminoquinoline derivatives, the re-
duced activities of the former appear to be mainly due to the
strongly decreased basicity of the quinoline nitrogen atom
cation produced an interesting improvement in the resistance
factor (RF=0.6–0.8) that appears worthy of further investiga-
tion. The introduction of a cytisinyl head and a spacer contain-
ing five methylene units produced the most interesting com-
pounds endowed with high activity, low cytotoxicity on two
human cell lines (HMEC-1 and HepG2), and nonhemolytic at
concentrations up to 200 mm. Therefore, these compounds de-
serve further investigation to define their ADME profiles and
their in vivo antimalarial activities in a murine standard four-
day test.
(
mean pK =6.40!pK =3.18), not balanced by the increased
a2
a2
lipophilicity (Table 3, values calculated with ACD/Labs 7.00).
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N of Q+2H, SCH +2H, CH S), 3.72–4.08 (m, 1H, CH-CH ), 5.33 (s,
Experimental Section
2
2
3
1
H, NH, collapsed with D O), 6.45 (d, J=5.4 Hz, 1H, arom), 7.39
2
Chemistry
(dd, J=8.8, 2.0 Hz, 1H, arom), 7.72 (d, J=8.8 Hz, 1H, arom), 7.97
d, J=2.0 Hz, 1H, arom), 8.55 ppm (d, J=5.4 Hz, 1H, arom); ele-
mental analysis calcd (%) for C H ClN S (404.01): C 65.40, H 7.48,
(
General: All commercially available solvents and reagents were
used without further purification unless otherwise stated. Melting
22
30
3
N 10.40, S 7.94, found: C 65.26, H 7.54, N 10.37, S 8.06.
1
points were determined with a Büchi apparatus. H NMR and
13
C NMR spectra were recorded with a Varian Gemini 200 or Varian
Mercury 300VX spectrometer in CDCl or [D ]DMSO with Me Si as
7-Chloro-4-(N-{4-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)me-
3
6
4
thylthio]but-2-yl}amino)quinoline (4): Yield: 9%; mp: 77–828C;
an internal standard; in the assignments, Q denotes the quinolizi-
dine ring and bisp denotes the bispidine moiety. HRMS was per-
formed with a FT-Orbitrap mass spectrometer in positive electro-
spray ionization (ESI). Elemental analyses were performed with
a Carlo Erba EA-1110 CHNS-O instrument in the Microanalysis Labo-
ratory of the Department of Pharmacy of Genoa University. All final
compounds and some intermediates were characterized by
1
H NMR (200 MHz, CDCl ): d=0.95–2.14 (14H, Q+2H, CHCH CH +
3
2
2
d superimposed at 1.36, J=6.4 Hz, 3H, CH-CH ), 2.42–2.96 (m, 2Ha
3
near N of Q+2H, SCH +2H, CH S), 3.74–4.08 (m, 1H, CH-CH ), 5.20
2
2
3
(
(
(
s, 1H, NH, collapsed with D O), 6.50 (d, J=5.6 Hz, 1H, arom), 7.37
dd, J=9.0, 1.6 Hz, 1H, arom), 7.72 (d, J=9.0 Hz, 1H, arom), 7.97
d, J=2.0 Hz, 1H, arom), 8.53 ppm (d, J=5.6 Hz, 1H, arom); ele-
2
mental analysis calcd (%) for C H ClN S (418.04): C 66.08, H 7.72,
23
32
3
1
H NMR spectroscopy and elemental analyses or HRMS, which indi-
N 10.05, S 7.67, found: C 66.15, H 7.89, N 10.08, S 7.67.
cated that their purity was always ꢀ95%. The remaining inter-
1
mediates were characterized by either H NMR spectra or elemen-
4-(N-{3-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methylthio]prop-
tal analyses. For the most interesting compounds (i.e., 12, 16, 18,
and 20) the C NMR spectroscopy data are also included.
2-yl}amino)-7-trifluoromethylquinoline (5): Yield: 59%; mp: 91–
1
3
1
938C; H NMR (200 MHz, CDCl ): d=0.92–2.15 (14H, Q+d superim-
3
posed at 1.46, J=6.6 Hz, 3H, CH-CH ), 2.47–2.96 (m, 2H near N of
3
a
General method for the preparation of 4-(N-{[(1R,9aR)-(octahy-
dro-2H-quinolizin-1-yl)methylthio]alkyl}amino)-7-substituted
quinolines 1–5: Amino compound 30, 32, 35, or 38 (6.5 mmol)
was mixed with equimolar amounts of 4,7-dichloroquinoline
Q+2H, SCH +2H, CH S), 3.78–4.12 (m, 1H, CH-CH ), 5.41 (s, 1H,
2
2
3
NH, collapsed with D O), 6.55 (d, J=5.4 Hz, 1H, arom), 7.62 (dd, J=
2
8
.8, 1.6 Hz, 1H, arom), 7.91 (d, J=8.8 Hz, 1H, arom), 8.29 (s, 1H,
arom), 8.65 ppm (d, J=5.4 Hz, 1H, arom); elemental analysis calcd
%) for C H F N S (425.55): C 63.13, H 6.91, N 9.60, S 7.33, found:
(
(
1.28 g) or 4-chloro-7-trifluoromethylquinoline (1.50 g) and phenol
4 g). The mixture was heated at 1808C for 4 h. After cooling, the
(
23
30
3
3
C 63.04, H 7.03, N 9.70, S 7.15.
mixture was treated with 2n NaOH solution until strongly alkaline
and was extracted with Et O (3”40 mL). The Et O solution was
2
2
General method for the preparation of 7-chloro-4-{[w-octahy-
washed with 2n NaOH solution, then with H O, and finally with
2
dro-2H-quinolizin-9a-yl)alkyl]amino}quinolines 6–8: A mixture of
5
% acetic acid solution (2”20 mL). The acid solution was alkalized
[36]
amine 41, 42,
or 43 as the dihydrochloride or free base
with 2n NH solution (to pH 8) and was extracted with CH Cl (3”
3
2
2
(0.88 mmol), 4,7-dichloroquinoline (192 mg, 0.97 mmol), phenol
415 mg, 4.41 mmol), and DIPEA (0.25 mL, 1.76 mmol) was heated
5
0 mL) to yield a light-brown solid that was washed with boiling
(
dry Et O (5 mL), which left the title compound as white crystals. By
2
under a N atmosphere for 5 h at 1308C. After cooling, the mixture
2
concentration of the Et O solution, some more compound was re-
2
was diluted with CH Cl , and the resulting solution washed with
2
2
covered. In the cases of compounds 4 and 5, the extracted raw
compounds were chromatographed on neutral alumina (1:20) elut-
ing with CH Cl .
2
n NaOH solution (3”5 mL) and then with brine (10 mL). After
drying (Na SO ), the solvent was evaporated, and the crude solid
was purified by column chromatography (silica gel, different mix-
tures CH Cl /MeOH as indicated for each compound). For the prep-
aration of compound 8, it was more convenient to use the free
base (obtained preliminary from the dihydrochloride), which avoid-
ed the use of DIPEA and heating the mixture for only 3 h.
2
4
2
2
2
2
7
-Chloro-4-(N-{2-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)me-
thylthio]ethyl}amino)quinoline (1): Yield: 37%; mp: 160–1658C;
1
H NMR (200 MHz, CDCl ): d=1.03–2.14 (m, 14H, Q), 2.64–2.91 (m,
3
2
3
6
7
8
H, 2Ha near N of Q+2H, SCH ), 2.93 (t, J=6.2 Hz, 2H, CH S),
2 2
.42–3.61 (m, 2H, Ar-NH-CH ), 5.66 (s, 1H, NH, collapsed with D O),
2
2
7
-Chloro-4-{[4-(octahydro-2H-quinolizin-9a-yl)butyl]amino}quino-
.44 (d, J=5.4 Hz, 1H, arom), 7.41 (dd, J=9.0, 2.0 Hz, 1H, arom),
.50 (d, J=9.0 Hz, 1H, arom), 7.98 (d, J=2.0 Hz, 1H, arom),
.56 ppm (d, J=5.4 Hz, 1H, arom); elemental analysis calcd (%) for
line (6): Column chromatography (CH Cl /MeOH 94:6); solid
washed with PE. Yield: 64%; mp: 157.8–159.38C; H NMR
2
2
1
(
300 MHz, CDCl ): d=1.19–1.84 (m, 14H, Q+4H, CH CH ), 2.42–
3
2
2
C H ClN S (389.99): C 64.68, H 7.24, N 10.77, S 8.22, found: C
21
28
3
2
.64 (m, 2H, 2H near N of Q+2H, CH C), 3.31–3.37 (m, 2H, Ar-NH-
a 2
6
4.76, H 7.36, N 10.81, S 8.27.
CH ), 4.96 (s, 1H, NH), 6.43 (d, J=5.5 Hz, 1H, arom), 7.36 (dd, J=
2
2
2
.2, 8.8 Hz, 1H, arom), 7.65 (d, J=8.8 Hz, 1H, arom), 7.95 (d, J=
.2 Hz, 1H, arom), 8.54 ppm (d, J=5.5 Hz, 1H, arom); elemental
7
-Chloro-4-(N-{3-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)me-
thylthio]prop-1-yl}amino)quinoline (2): Yield: 38%; mp: 95–
1
analysis calcd (%) for C H ClN (371.95): C 71.04, H 8.13, N 11.30,
found: C 70.89, H 8.28, N 11.45.
1
008C; H NMR (200 MHz, CDCl ): d=1.04–2.14 (m, 14H, Q+2H,
22 30
3
3
CH CH CH ), 2.69 (t, J=6.4 Hz, 2H, CH S), 2.76–2.93 (m, 2H, 2H
a
2
2
2
2
near N of Q+2H, SCH ), 3.49–3.58 (m, 2H, Ar-NH-CH ), 5.60 (s, 1H,
2
2
7
-Chloro-4-{[6-(octahydro-2H-quinolizin-9a-yl)hexyl]amino}qui-
NH, collapsed with D O), 6.44 (d, J=5.4 Hz, 1H, arom), 7.36 (dd, J=
2
noline (7): Column chromatography (CH Cl /MeOH 92:8); amor-
phous solid crystallized and washed with Et O. Yield: 46%; mp:
2
2
9
2
.0, 2.0 Hz, 1H, arom), 7.61 (d, J=9.0 Hz, 1H, arom), 7.96 (d, J=
.0 Hz, 1H, arom), 8.53 ppm (d, J=5.4 Hz, 1H, arom); elemental
2
1
1
44.6–146.08C; H NMR (300 MHz, CDCl ): d=1.12–1.82 (m, 14H,
3
analysis calcd (%) for C H ClN S (404.01): C 65.40, H 7.48, N 10.40,
2
2
30
3
Q+8H, CH CH CH CH ), 2.41–2.65 (m, 2H, 2H near N of Q+2H,
CH C), 3.29–3.35 (m, 2H, Ar-NH-CH ), 4.96 (s, 1H, NH), 6.42 (d, J=
2
2
2
2
a
S 7.49, found: C 65.22, H 7.75, N 10.34, S 7.89%.
2
2
7
-Chloro-4-(N-{3-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)me-
5.5 Hz, 1H, arom), 7.36 (dd, J=2.2, 8.8 Hz, 1H, arom), 7.65 (d, J=
8.8 Hz, 1H, arom), 7.96 (d, J=2.2 Hz, 1H, arom), 8.53 ppm (d, J=
5.2 Hz, 1H, arom); elemental analysis calcd (%) for C H ClN
3
thylthio]prop-2-yl}amino)quinoline (3): Yield: 50%; mp: 110–
1
112 8C; H NMR (200 MHz, CDCl ): d=0.88–2.12 (m, 14H, Q+d su-
3
24 34
perimposed at 1.44, J=6.2 Hz, 3H, CH-CH ), 2.48–2.96 (m, 2H near
(400.00): C 72.06, H 8.57, N 10.51, found: C 72.23, H 8.81, N 10.19.
3
a
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Full Papers
7
-Chloro-4-{[8-(octahydro-2H-quinolizin-9a-yl)octyl]amino}quino-
6.4 Hz, 1H, bisp), 4.13–4,18 (d, J=15.6 Hz, 1H, bisp), 4.97 (s, 1H,
line (8): Column chromatography (CH Cl /MeOH 94:6); amorphous
NH, collapses with D O), 6.01 (d, J=6.9 Hz, 1H, a-pyr), 6.20 (d, J=
2
2
2
solid crystallized and washed with Et O. Yield: 55%; mp: 116.8–
5.5 Hz, 1H, arom), 6.46 (d, J=9.0 Hz, 1H, a-pyr), 7.27–7.42 (m, 1H,
a-pyr+1H, arom), 7.56 (dd, J=8.8, 1.9 Hz, 1H, arom), 7.91 (d, J=
1.9 Hz, 1H, arom), 8.45 ppm (d, J=5.5 Hz, 1H, arom); elemental
analysis calcd (%) for C H ClN O (408.92): C 67.55, H 6.16, N 13.70,
2
1
1
18.38C; H NMR (300 MHz, CDCl ): d=1.12–1.77 (m, 14H, Q+12H,
3
CH CH CH CH CH CH ), 2.47–2.63 (m, 2H, 2H near N of Q+2H,
2
2
2
2
2
2
a
CH C), 3.28–3.32 (m, 2H, Ar-NH-CH ), 4.95 (s, 1H, NH), 6.42 (d, J=
2
2
23 25
4
5
8
5
.5 Hz, 1H, arom), 7.36 (dd, J=2.2, 8.8 Hz, 1H, arom), 7.65 (d, J=
.8 Hz, 1H, arom), 7.96 (d, J=2.2 Hz, 1H, arom), 8.54 ppm (d, J=
found: C 67.65, H 6.45, N 13.38.
7
-Chloro-4-{N-[5-(citisin-3-yl)pentyl]amino}quinoline (12): Yield:
.2 Hz, 1H, arom); elemental analysis calcd (%) for C H ClN
26
38
3
1
8
0%; mp: 61–648C; H NMR (200 MHz, CDCl ): d=0.86–1.58 (m,
3
(
428.05): C 72.95, H 8.95, N 9.82, found: C 73.14, H 9.08, N 9.56. The
2
H, bisp+6H, CH CH CH ), 1.62–2.06 (m, 2H, bisp), 2.12–2.53 (m,
2
2
2
compound was converted into the corresponding dihydrochloride;
mp: 250–2528C (dec.); H NMR (300 MHz, [D ]DMSO): d=1.05–1.91
1
2H, NH-CH +3H, bisp), 2.71–3.32 (m, 2H, Ar-NH-CH +1H, bisp),
2 2
6
3
5
6
7
.84 (dd, J=15.2, 6.4 Hz, 1H, bisp), 4.19 (d, J=15.2 Hz, 1H, bisp),
.85 (s, 1H, NH, collapsed with D O), 6.04 (d, J=6.8 Hz, 1H, a-pyr),
.34 (d, J=5.14 Hz, 1H, arom), 6.45 (dd, J=9.2, 1.4 Hz, 1H, a-pyr),
.21–7.37 (m, 1H, a-pyr+1H, arom), 7.39 (dd, J=8.8, 1.8 Hz, 1H,
(
m, 26H), 2.86–3.09 (m, 2H), 3.35–3.40 (m, 2H), 3.49–3.52 (m, 2H),
2
6
1
.85 (d, J=7.2 Hz, 1H, arom), 7.76 (d, J=8.8 Hz, 1H, arom), 8.06 (s,
H, arom), 8.52 (d, J=7.2 Hz, 1H, arom), 8.70 (d, J=8.8 Hz, 1H,
arom), 9.62 (brs, 1H, collapsed with D O), 10.20 ppm (brs, 2H, col-
lapsed with D O); elemental analysis calcd (%) for C H ClN ·2HCl
2
arom), 7.96 (d, J=1.8 Hz, 1H, arom), 8.12 (d, J=8.6 Hz, 1H, arom),
2
26 38
3
13
8
1
1
4
.49 ppm (d, J=5.4 Hz, 1H, arom); C NMR (50 MHz, CDCl ): d=
3
(
500.98): C 62.33, H 8.05, N 8.39, found: C 62.01, H 8.32, N 8.15.
62.80, 150.94, 150.26, 149.46, 147.58, 137.95, 133.89, 126.81,
24.12, 121.37, 116.30, 115.20, 103.92, 97.56, 59.71, 58.98, 55.70,
9.22, 42.55, 34.62, 27.06, 26.94, 24.93, 24.61, 23.92 ppm; elemental
7
-Chloro-4-(cytisin-3-yl)aminoquinoline (9): A mixture of N-ami-
[40]
nocytisine
(332 mg, 1.62 mmol), 4,7-dichloroquinoline (333 mg,
analysis calcd (%) for C H ClN O (436.98): C 68.71, H 6.69, N 12.82,
1
.62 mmol), and phenol (1.03 g, 4.41 mmol) was heated under a N2
25 29
4
found: C 68.89, H 6.90, N 12.81.
atmosphere for 7 h at 1808C. After cooling, the mixture was taken
up with 2n NaOH to strongly alkaline pH and then with Et O (3”
2
7
-Chloro-4-{N-[2-(citisin-3-yl)-1-methylethyl]amino}quinoline
2
0 mL). The insoluble compound was filtered and washed with 2n
1
(13): Yield: 53%; mp: 221–2238C; H NMR (200 MHz, CDCl ): d=
3
NaOH (3 mL), H O (2”3 mL), and thoroughly with Et O (3”5 mL)
2
2
1
.13 (d, J=6.4 Hz, 3H, CHCH ), 1.63–2.02 (m, 3H, bisp), 2.33–2.66
3
and finally crystallized from EtOH. Yield: 94%; mp: 296–2988C;
(
m, 2H, NH-CH +2H, bisp), 2.77–3.12 (m, 3H, bisp), 3.55–3.78 (m,
2
1
H NMR (300 MHz, [D ]DMSO) d=1.70–1.98 (m, 2H, bisp), 2.54 (s,
6
1
H, CHCH ), 3.82–4.04 (m, 2H, bisp), 5.27 (s, 1H, NH, collapsed with
3
1
1
1
H, bisp), 2.92–3.05 (m, 2H, bisp), 3.08–3.25 (m, 2H, bisp), 3.30 (s,
H, bisp), 3.75 (dd, J=5.5, 14.6 Hz, 1H, bisp) 4.04 (d, J=14.6 Hz,
H, bisp), 6.02 (d, J=4.4 Hz, 1H, a-pyr), 6.13 (d, J=6.3 Hz, 1H, a-
D O), 6.06 (d, J=6.8 Hz, 1H, a-pyr), 6.34 (d, J=5.6 Hz, 1H, arom),
2
6
.53 (dd, J=9.2, 1.4 Hz, 1H, a-pyr), 7.18–7.44 (m, 2H, arom,+1H,
a-pyr), 7.94 (d, J=0.8 Hz, 1H, arom), 8.49 ppm (d, J=5.4 Hz, 1H,
arom); elemental analysis calcd (%) for C H ClN O (408.92): C
pyr), 6.33 (d, J=8.8 Hz, 1H, arom), 7.30–7.53 (m, 1H, a-pyr+1H,
arom), 7.74 (s, 1H, arom), 8.09 (d, J=8.8 Hz, 1H, arom), 8.14 (d, J=
23
25
4
6
7.55, H 6.16, N 13.70, found: C 67.72, H 6.56, N 13.42.
5
Hz, 1H, arom), 8.47 ppm (s, 1H, NH collapsed with D O); HRMS
2
+
(
ESI): m/z: calcd for C H ClN O: 367.13256 [M+H] , found:
General method for the preparation of 7-chloro-4-{[w-(9-nitrocy-
tisin-3-yl)alkyl]amino}quinolines 14–16 and 7-chloro-4-{[w-(9,11-
2
0
20
4
3
67.13245.
dibromocytisin-3-yl)alkyl}quinolines 19 and 20: In an Aldrich
General method for the preparation of 7-chloro-4-{N-[w-(citisin-
-yl)alkyl]amino}quinolines 10–13: In an Aldrich pressure tube,
[41,42]
pressure tube, the substituted cytisine
(0.61 mmol) was dis-
3
solved in CH CN (4 mL). Subsequently, equimolar amounts of
3
cytisine (0.19 g, 1 mmol) was dissolved in CH CN (3 mL) and 4-[(w-
3
DIPEA and 4-[(w-bromoalkyl)amino]-7-chloroquinoline 54–58 were
bromoalkyl)amino]-7-chloroquinoline 54–58 (0.5 mmol) was
[43–46]
sequentially added;
the closed tube was heated at 1008C for
[43–46]
added;
the closed tube was heated at 1008C for 36 h. The
2
4 h. After cooling, the solvent was evaporated, and the residue
cooled mixture was filtered from cytisine hydrobromide, and the
organic phase was evaporated to dryness. The residue was taken
was taken up with H O (10 mL), alkalized, (2n NaOH until strongly
2
alkaline), and extracted with CH Cl (3”40 mL). The organic phase
2
2
up in H O (10 mL), alkalized (2n NaOH until strongly alkaline), and
2
was dried (Na SO ) and evaporated to dryness; the residue was fi-
2
4
extracted with CH Cl (3”40 mL). After drying (Na SO ), the solvent
2
2
2
4
nally chromatographed on alumina (1:30) eluting with CH Cl . The
2
2
was removed, and the oily residue was subjected to chromatogra-
phy on alumina (1:25) eluting with CH Cl .
obtained compound was, eventually, further purified by trituration
2
2
with dry Et O.
2
7
-Chloro-4-{N-[2-(citisin-3-yl)ethyl]amino}quinoline (10): Yield:
7
-Chloro-4-{[2-(9-nitrocytisin-3-yl)ethyl]amino}quinoline
(14):
1
1
7
3
8%; mp: 79–818C; H NMR (200 MHz, CDCl ): d=1.77–2.10 (m,
3
Yield: 66%; mp: 182–1848C; H NMR (200 MHz, CDCl ): d=1.86–
2.12 (m, 2H, bisp), 2.43–2.90 (m, 2H, NH-CH +2H, bisp), 2.98–3.37
3
H, bisp), 2.34–2.92 (m, 2H, NH-CH +3H, bisp), 2.94–3.33 (m, 2H,
2
2
Ar-NH-CH +2H, bisp), 4.01 (dd, J=15.6, 6.4 Hz, 1H, bisp), 4.23 (d,
2
(m, 2H, Ar-NH-CH +3H, bisp), 4.07 (dd, J=16.2, 6.6 Hz, 1H, bisp),
2
J=15.6 Hz, 1H, bisp), 5.36 (s, 1H, NH, collapses with D O), 5.95 (d,
2
4.32 (d, J=16.2 Hz, 1H, bisp), 5.16 (s, 1H, NH, collapsed with D O),
2
J=6.8 Hz, 1H, a-pyr), 6.28 (d, J=5.4 Hz, 1H, arom), 6.50 (d, J=
6
.04 (d, J=8.2 Hz, 1H, a-pyr), 6.28 (d, J=5.4 Hz, 1H, arom), 7.07 (d,
9
.2 Hz, 1H, a-pyr), 7.04 (d, J=9.2 Hz, 1H, a-pyr), 7.12–7.23 (m, 1H,
J=9.0 Hz, 1H, arom), 7.38 (dd, J=8.0, 2.0 Hz, 1H, arom), 7.92 (d,
J=2.0 Hz, 1H, arom), 8.17 (d, J=8.0 Hz, 1H, a-pyr), 8.49 ppm (d,
J=5.4 Hz, 1H arom); elemental analysis calcd (%) for C H ClN O
3
arom), 7.46 (dd, J=8.8, 1.6 Hz, 1H, arom), 7.91 (d, J=1.6 Hz, 1H,
arom), 8.49 ppm (d, J=5.4 Hz, 1H, arom); elemental analysis calcd
22
22
5
(
%) for C H ClN O·0.75H O (408.41): C 64.69, H 6.05, N 13.79,
22 23 4 2
(439.90): C 60.07, H 5.04, N 15.92, found: C 60.17, H 5.33, N 15.96.
found: C 64.85, H 6.25, N 13.56.
7
-Chloro-4-{[3-(9-nitrocytisin-3-yl)propyl]amino}quinoline (15):
1
7
-Chloro-4-{N-[3-(citisin-3-yl)propyl]amino}quinoline (11): Yield:
Yield: 77%; mp: 103–1058C; H NMR (200 MHz, CDCl ): d=1.65–
2.08 (m, 2H, bisp+2H, CH CH CH ), 2.32–2.62 (m, 2H, NH-CH +
2H, bisp), 2.91–3.22 (m, 2H, Ar-NH-CH +3H, bisp), 4.01 (dd, J=
16.0, 6.4 Hz, 1H, bisp), 4.24 (d, J=16.0 Hz, 1H, bisp), 4.98 (s, 1H,
3
1
7
4
2
4%; mp: 143–1458C; H NMR (300 MHz, CDCl ): d=1.65–2.10 (m,
3
2
2
2
2
H, bisp+2H CH CH CH ), 2.24–2.61 (m, 2H, NH-CH +2H, bisp),
2
2
2
2
2
.86–3.23 (m, 2H, Ar-NH-CH +2H, bisp), 3.86–3.93 (dd, J=15.6,
2
ChemMedChem 2015, 10, 1570 – 1583
www.chemmedchem.org
1578
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
NH, collapsed with D O), 6.13 (d, J=8.2 Hz, 1H, a-pyr), 6.24 (d, J=
4-{N-[3-(9-Aminocytisin-3-yl)propyl]amino}-7-chloroquinoline
2
1
5
9
1
.4 Hz, 1H, arom), 7.36 (dd, J=9.0, 2.2 Hz, 1H, a-pyr), 7.64 (d, J=
.0 Hz, 1H, arom), 7.96 (d, J=2.2 Hz, 1H, arom), 8.33 (d, J=8.2 Hz,
H, a-pyr), 8.51 ppm (d, J=5.4 Hz, 1H, arom); elemental analysis
(17): Yield: 86%; mp: 173–1758C; H NMR (300 MHz, [D ]DMSO):
6
d=0.78–1.98 (m, 4H, bisp+2H, CH CH CH ), 2.04–2.45 (m, 2H, NH-
2
2
2
CH +1H, bisp), 2.69–3.58 (m, 2H, Ar-NH-CH +3H, bisp), 3.74 (dd,
2
2
calcd (%) for C H ClN O ·0.5H O (462.93): C 59.67, H 5.44, N 15.13,
J=16.0, 6.2 Hz, 1H, bisp), 3.95 (d, J=16.0 Hz, 1H, bisp), 4.67 (s, 1H,
2
3
24
5
3
2
found: C 59.94, H 5.75, N 14.75.
NH, collapsed with D O), 5.89 (d, J=7.4 Hz, 1H, a-pyr), 6.12 (d, J=
2
5
.5 Hz, 1H, arom), 6.43 (d, J=7.4 Hz, 1H, a-pyr), 7.15 (s, 2H, NH2,
7
-Chloro-4-{[5-(9-nitrocytisin-3-yl)pentyl]amino}quinoline
(16):
collapsed with D O) 7.40 (dd, J=9.0, 1.8 Hz, 1H, arom), 7.72 (d, J=
1
2
Yield: 74%; mp: 84–868C; H NMR (200 MHz, CDCl ): d=0.82–2.04
3
1
5
.8 Hz, 1H, arom), 8.21 (d, J=9.0 Hz, 1H, arom), 8.34 ppm (d, J=
.5 Hz, 1H, arom); elemental analysis calcd (%) for C H ClN O
(
m, 2H, bisp+6H, CH CH CH ), 2.08–2.62 (m, 2H, NH-CH +2H,
2 2 2 2
23
26
5
bisp), 2.82–3.35 (m, 2H, Ar-NH-CH +3H, bisp), 3.94 (dd, J=16.0,
2
(
423.94): C 65.16, H 6.18, N 16.52, found: C 64.72, H 6.38, N 16.29.
6
.4 Hz, 1H, bisp), 4.22 (d, J=16.0 Hz, 1H, bisp), 5.44 (s, 1H, NH, col-
lapsed with D O), 6.11 (d, J=8.2 Hz, 1H, a-pyr), 6.34 (d, J=5.4 Hz,
4-{N-[5-(9-Aminocytisin-3-yl)pentyl]amino}-7-chloroquinoline
2
1
1
H, arom), 7.36 (dd, J=9.0, 2.2 Hz, 1H, a-pyr), 7.82–7.99 (m, 2H,
(18): Yield: 76%; mp: 137–1398C; H NMR (300 MHz, [D ]DMSO):
6
arom), 8.32 (d, J=8.2 Hz, 1H, a-pyr), 8.51 ppm (d, J=5.4 Hz, 1H,
d=0.78–2.01 (m, 4H, bisp+6H, CH CH CH ), 2.11–2.42 (m, 2H, NH-
CH +1H, bisp), 2.64–3.49 (m, 2H, Ar-NH-CH +3H, bisp), 3.73 (dd,
2 2
2
2
2
1
3
arom); C NMR (50 MHz, CDCl ): d=159.87, 154.30, 149.90, 149.41,
3
1
1
2
47.07, 136.73, 134.12, 133.54, 126.54, 124.29, 121.02, 116.08,
01.76, 97.66, 58.77, 58.54, 55.66, 50.39, 42.24, 35.52, 28.63, 27.22,
6.57, 24.89, 24.22, 23.62 ppm; elemental analysis calcd (%) for
J=16.0, 6.2 Hz, 1H, bisp), 3.90 (d, J=16.0 Hz, 1H, bisp), 4.76 (s, 1H,
NH, collapsed with D O), 5.84 (d, J=7.4 Hz, 1H, a-pyr), 6.32–6.47
2
(m, 1H, arom+1H, a-pyr), 7.19 (s, 2H, NH , collapsed with D O)
2
2
C H ClN O (481.98): C 62.30, H 5.86, N 14.53, found: C 62.33, H
7.40 (dd, J=9.0, 2.2 Hz, 1H, arom), 7.74 (d, J=2.0 Hz, 1H, arom),
8.24 (d, J=9.0 Hz, 1H, arom), 8.36 ppm (d, J=5.5 Hz, 1H, arom);
25
28
5
3
5
.87, N 14.25.
13
C NMR (50 MHz, CDCl ): d=157.23, 150.27, 149.43, 147.58, 138.19,
3
7
-Chloro-4-{[3-(9,11-dibromocytisin-3-yl)propyl]amino}quinoline
1
6
2
33.89, 133.20, 126.86, 124.10, 121.17, 116.31, 112.67, 103.59, 97.59,
0.13, 59.03, 55.31, 49.52, 42.44, 33.99, 28.63, 27.16, 26.87, 25.55,
4.50, 23.61 ppm; elemental analysis calcd (%) for C H ClN O
1
(
19): Yield: 68%; mp: 103–1058C; H NMR (300 MHz, CDCl ): d=
3
1
.47–1.98 (m, 4H, bisp+2H, CH CH CH ), 2.24–2.53 (m, 2H, NH-
2 2 2
2
5
30
5
CH +2H, bisp), 2.94–3.13 (m, 2H, Ar-NH-CH +1H, bisp), 3.96 (dd,
2
2
(
451.99): C 66.43, H 6.69, N 15.49, found: C 66.72, H 6.62, N 15.12.
J=16.0, 6.2 Hz, 1H, bisp), 4.17 (d, J=16.0 Hz, 1H, bisp), 4.86 (s, 1H,
NH, collapsed with D O), 6.21 (d, J=5.5 Hz, 1H, arom), 7.37 (dd, J=
General method for the preparation of 4-[(1R,9aR)-(octahydro-
2H-quinolizin-1-yl)methyl)thio]-7-substituted quinolines 22 and
23: A suspension of 4,7-dichloroquinoline (0.51 g, 2.6 mmol) or 4-
chloro-7-trifluoromethylquinoline (0.6 g) in Dowtherm A (4 mL) was
heated at 1008C until a clear solution was obtained, and then a so-
lution of thiolupinine (0.48 g, 2.6 mmol) in Dowtherm A (1 mL)
was added. The tube was closed and the temperature was raised
and maintained at 1708C for 2 h. After cooling, the pasty material
2
8
.8, 2.0 Hz, 1H, arom), 7.58 (d, J=8.8 Hz, 1H, arom), 7.82 (s, 1H, a-
pyr), 7.95 (d, J=2.0 Hz, 1H, arom), 8.54 ppm (d, J=5.5 Hz, 1H,
arom); elemental analysis calcd (%) for C H Br ClN O (566.72): C
2
3
23
2
4
4
8.75, H 4.09, N 9.89, found: C 48.67, H 4.19, N 9.63.
[35]
7
-Chloro-4-{[5-(9,11-dibromocytisin-3-yl)pentyl]amino}quinoline
1
(
20): Yield: 69%; mp: 104–1068C; H NMR (300 MHz, CDCl ): d=
3
0
.92–2.01 (m, 4H, bisp+6H, CH CH CH ), 2.08–2.52 (m, 2H, NH-
2 2 2
was partitioned between Et O and 0.5n HCl (30 mL each). The
2
CH +1H, bisp), 2.74–3.52 (m, 2H, Ar-NH-CH +3H, bisp), 3.92 (dd,
2
2
aqueous phase was extracted with Et O (2”10 mL), alkalized, (2n
2
J=16.0, 6.2 Hz, 1H, bisp), 4.20 (d, J=16.0 Hz, 1H, bisp), 5.41 (s, 1H,
NaOH (20 mL) and extracted again with Et O (3”40 mL). The dried
2
NH, collapsed with D O), 7.21 (d, J=5.5 Hz, 1H, arom), 7.39 (dd, J=
2
Et O solution was evaporated to leave an oil that slowly crystal-
2
9
.0, 2.2 Hz, 1H, arom), 7.86 (s, 1H, a-pyr), 7.95 (d, J=2.0 Hz, 1H,
lized.
1
3
arom), 8.50 ppm (d, J=5.5 Hz, 1H, arom); C NMR (50 MHz, CDCl3):
d=157.92, 149.68, 147.66, 146.92, 142.69, 134.27, 126.49, 124.39,
7-Chloro-4-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl)thio]-
1
1
4
21.07, 116.09, 111.29, 97.70, 96.03, 58.49, 55.91, 55.64, 51.72,
2.47, 33.89, 27.25, 26.82, 24.86, 23.79 ppm; elemental analysis
quinoline (22): Yield: 61%; mp: 114–1158C; H NMR (200 MHz,
CDCl ): d=1.16–2.28 (m, 14H, Q), 2.75–3.02 (m, 2H, CH S), 3.18–
3
2
calcd (%) for C H Br ClN O·0.5H O (603.78): C 49.72, H 4.67, N
3.54 (m, 2H near N of Q), 7.21 (d, J=5.0 Hz, 1H, arom), 7.47 (dd,
2
5
27
2
4
2
a
9
.18, found: C 49.60, H 5.04, N 8.92.
J=9.0, 2.2 Hz, 1H, arom), 8.03–8.08 (m, 2H, arom), 8.67 ppm (d, J=
5
.0 Hz, 1H, arom); elemental analysis calcd (%) for C H ClN S
19 23 2
7
-Chloro-4-(pyrrolidin-1-yl)quinoline (21): Reaction of 4-[(4-bro-
(
346.92): C 65.78, H 6.68, N 8.07, found: C 66.02, H 6.78, N 8.11. Ele-
mobutyl)amino]-7-chloroquinoline with cytisine or 9-nitrocytisine,
either in the presence or absence of DIPEA, only the intramolecular
loss of hydrogen bromide was obtained to give. Yield: 88%; mp:
mental analysis calcd (%) for C H ClN S·HCl·0.5H O (392.39): C
5
19
23
2
2
8.16, H 6.42, N 7.14, found: C 58.45, H 6.36, N 7.13.
1
9
3
0–918C; H NMR (200 MHz, CDCl ): d=1.82–2.21 (m, 4H, CH CH ),
.42–3.56 (m, 4H, N-CH ), 6.44 (d, J=5.4 Hz, 1H, arom), 7.28 (dd,
4-{[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]thio}-7-trifluor-
3
2
2
omethylquinoline (23): Yield: 69%; mp: 78–798C (pentane);
2
1
J=9.2, 2.2 Hz, 1H, arom), 7.95 (d, J=2.2 Hz, 1H, arom), 8.16 (d, J=
.2 Hz, 1H, arom), 8.47 ppm (d, J=5.4 Hz, 1H, arom); elemental
analysis calcd (%) for C H ClN (232.71): C 67.10, H 5.63, N
H NMR (200 MHz, CDCl ) d=1.06–2.24 (m, 14H, Q), 2.70–2.99 (m,
3
9
2H, CH S), 3.14–3.50 (m, 2H near N of Q), 7.32 (d, J=5.5 Hz, 1H,
2
a
arom), 7.69 (dd, J=8.8, 2.0 Hz, 1H, arom), 8.25 (d, J=8.8 Hz, 1H,
arom), 8.35 (s, 1H, arom), 8.77 ppm (d, J=5.5 Hz, 1H, arom); ele-
mental analysis calcd (%) for C H F N S (380.47): C 63.14, H 6.09,
1
3
13
2
1
2.04%, found: C 67.31, H 5.88, N 12.10%.
20
23
3
2
General method for the preparation of 4-{N-[w-(9-aminocytisin-
3
1
N 7.36, found: C 63.18, H 6.10, N 7.35.
-yl)alkyl]amino}-7-chloroquinolines 17 and 18: Nitro compound
5 or 16 (0.25–0.44 mmol) was dissolved in EtOH (40 mL), and
General method for the preparation of S-(7-Chloro-4-quinolinyl)-
w-{[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]thio}alkane-
thiols 24 and 25: A mixture of 7-chloro-4-thioquinoline (0.21 g,
1
0% palladium on charcoal (20 mg) was added. The mixture was
[47]
hydrogenated at atmospheric pressure. The catalyst was filtered,
and the solvent was removed under vacuum. The residue was
1.09 mmol) and KOH (ground pellets, 61 mg, 1.09 mmol) in CH CN
3
rinsed with dry Et O (3 mL) to give the title compound as a white
powder that rapidly became pale yellow.
(5 mL) was stirred for 30 min under an atmosphere of nitrogen. A
solution of an equimolar amount of S-(2-chloroethyl)thiolupinine
2
[
32]
ChemMedChem 2015, 10, 1570 – 1583
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[48]
(
63) or S-(3-bromopropyl)thiolupinine (64) in CH CN (3 mL) was
sis calcd (%) for C H ClN OS (440.00): C 65.51, H 5.96, N 9.55, S
3
24 26
3
dropwise added slowly, and the solution was stirred at room tem-
perature for 24 h. The solvent was removed, and the residue was
taken up in 1n HCl (10 mL). The acid solution was extracted with
7.29, found: C 65.42, H 5.86, N 9.54, S 7.43.
w-(Octahydro-quinolizin-1/9a-yl)alkylamines, intermediates for
compounds 1–8: Amines 29, 32, 41, and 42 were prepared as pre-
Et O (2”10 mL), alkalized (2n NaOH, 15 mL), and extracted with
2
[
32]
[33]
viously described by Tasso et al.,
Villa et al.,
and Barteselli
CH Cl (3”10 mL). The dried solution was evaporated, and the oily
2
2
[36]
et al., whereas amine 35, 38, and 43 were prepared as follows.
residue was subjected to chromatography on alumina (1:30) elut-
ing with CH Cl .
2
2
1-{[(1R,9aR)-(Octahydro-2H-quinolizin-1-yl)methyl]thio}-2-oxo-
S-(7-Chloroquinolin-4-yl)-2-{[(1R,9aR)-(octahydro-2H-quinolizin-1-
yl)methyl]thio}ethanethiol (24): Yield: 43%. Yellow oil; H NMR
propane (33): Chloroacetone (0.75 mL, 6.5 mmol) was added to
a solution of thiolupinine (1 g, 5.4 mmol) in EtOH (5 mL) under
1
[35]
(
300 MHz, CDCl ): d=1.04–2.12 (m, 14H, Q), 2.66–2.98 (m, 2H,
protection from air, and the mixture was stirred at room tempera-
ture for 1 h. The solvent was removed under reduced pressure,
3
CH S+2H, ArSCH S+2H, SCH ), 3.14–3.50 (m, 2H, 2H near N of
2
2
2
a
Q), 7.19 (dd, J=4.7, 1.6 Hz, 1H, arom), 7.49 (dd, J=9.0, 2.0 Hz, 1H,
arom), 7.96–8.15 (m, 2H, arom), 8.72 ppm (dd, J=4.7, 1.6 Hz, 1H,
arom); elemental analysis calcd (%) for C H ClN S (407.04): C
and the residue was partitioned between Et
(20 mL). The acid solution was alkalized with 2n NaOH (15 mL) and
extracted with Et O (4”10 mL). The Et O solution was dried
(Na SO ) and concentrated to leave a pale-yellow oil (1.23 g, 95%);
elemental analysis calcd (%) for C13 23NOS (241.39): C 64.68, H 9.60,
2
O (10 mL) and 1n HCl
2
1
27
2 2
2
2
6
1
1.97, H 6.69, N 6.88, S 15.76, found: C 61.80, H 6.75, N 6.83, S
5.28.
2
4
H
N, 5.80, found: C 64.81, H 9.61, N 5.65.
S-(7-Chloroquinolin-4-yl)-3-{[(1R,9aR)-(octahydro-2H-quinolizin-1-
yl)methyl]thio}propanethiol (25): Yield: 40%; mp: 81–838C (dry
2-Hydroxyimino-1-{[(1R,9aR)-(Octahydro-2H-quinolizin-1-yl)me-
thyl]thio}propane (34): 6n NaOH (1.1 mL) was added to a solution
of ketone 33 (1.23 g, 5.1 mmol) and hydroxylamine hydrochloride
(0.43 g, 6.12 mmol) in EtOH (4 mL), and the solution was heated at
reflux for 2 h and then evaporated under reduced pressure. The
1
Et O); H NMR (300 MHz, CDCl ): d=0.95–2.21 (m, 14H, Q+2H,
2
3
CH CH CH ), 2.56–2.95 (m, 2H, CH S+2H, ArSCH S+2H, SCH ),
2
2
2
2
2
2
3
7
8
.11–3.35 (m, 2H, 2H near N of Q), 7.21 (d, J=5.0 Hz, 1H, arom),
.49 (dd, J=8.8, 2.0 Hz, 1H, arom), 7.97–8.12 (m, 2H, arom),
.70 ppm (d, J=5.0 Hz, 1H, arom); elemental analysis calcd (%) for
a
residue was taken up in H O (10 mL) and extracted with CH Cl (3”
2 2 2
C H ClN S (421.06): C 62.75, H 6.94, N 6.65, S 15.23, found: C
10 mL). After removing the solvent, a pale-yellow oil (1.28 g, 97%)
22
29
2 2
6
2.51, H 7.19, N 6.68, S 15.38. Elemental analysis calcd (%) for
that crystallized on standing was recovered; mp: 79–808C (pen-
C H ClN S ·HCl·1.25H O (405.90): C 55.07, H 6.77, N 5.84, S 13.36,
tane); elemental analysis calcd (%) for C13
H N OS (256.41): C 60.89,
24 2
22
29
2
2
2
found: C 55.21, H 6.93, N 5.56, S 11.98.
H 9.43, N 10.93, found: C 61.15, H 9.44, N 10.86.
General method for the preparation of 7-chloro-4-{S-[w-(citisin-
2-Amino-1-{[(1R,9aR)-(Octahydro-2H-quinolizin-1-yl)methyl]thio}-
propane (35): A solution of oxime 34 (0.90 g, 3.5 mmol) in dry
3
-yl)alkyl]thio}quinolines 26 and 27: A mixture of 7-chloro-4-thio-
[47]
quinoline (0.28 g, 1.43 mmol) and KOH (ground pellets, 80 mg,
.43 mmol) in CH CN (5 mL) was stirred for 30 min under an atmos-
Et
pension of lithium aluminum hydride (0.65 g, 17 mmol) in dry Et
(20 mL), and then the suspension was heated at reflux for 20 h.
After cooling, H O (1 mL), 15% NaOH solution (1 mL), and H
(1.5 mL) were sequentially added. The precipitate was filtered and
washed thoroughly with Et O (3”10 mL), and the organic solution
O (20 mL) was added dropwise to a stirred and ice-cooled sus-
2
1
O
2
3
phere of nitrogen. A solution of an equimolar amount of N-(2-
[49]
[49]
chloroethyl)cytisine
(65) or N-(4-chloropropyl)cytisine
(66) in
2
O
2
CH CN (5 mL) was dropwise slowly added, and the solution was
3
stirred at room temperature for 72 h. The solvent was removed,
and the residue was taken up in 1n HCl (5 mL). The acid solution
was filtered, alkalized (2n NaOH until strongly alkaline) and ex-
tracted with CH Cl (3”20 mL). The dried solution was concentrat-
2
was dried with KOH pellets. After elimination of the solvent, the
oily residue was distilled in vacuo (0.07 hPa) collecting a colorless
oil (0.73 g, 86%) at 1408C (air bath temperature); elemental analy-
2
2
ed, and the oily residue was subjected to chromatography on alu-
mina (1:30) eluting with CH Cl2 and CH Cl /MeOH (98:2). Finally,
sis calcd (%) for C13
found: C 64.57, H 10.97, N 11.52.
H N S (242.42): C 64.40, H 10.81, N 11.56,
26 2
2
2
2
the solid was crystallized with dry Et O.
2
1-{[(1R,9aR)-(Octahydro-2H-quinolizin-1-yl)methyl]thio}-3-oxobu-
7
-Chloro-4-{S-[3-(citisin-3-yl)propyl]thio}quinoline (26): Yield:
tane (36): A solution of methyl vinyl ketone (1.4 mL, 17.5 mmol) in
1
5
3
3
4%; mp: 140–1418C; H NMR (200 MHz, CDCl ): d=1.78–2.16 (m,
dry dioxane (1.5 mL) was added dropwise to a solution of thiolupi-
3
[35]
H, bisp+2H, CH CH CH ), 2.41–2.72 (2H, S-CH +3H, bisp), 2.84–
nine (3.04 g, 16.3 mmol) in dry dioxane (2.5 mL). The solution
was stirred at room temperature for 1 h and then maintained at
708C for 30 min, always under an atmosphere of nitrogen. After
2
2
2
2
.21 (m, 2H, NH-CH +2H, bisp), 4.04 (dd, J=15.6, 6.4 Hz, 1H,
2
bisp), 4.29 (d, J=15.6 Hz, 1H, bisp), 6.12 (d, J=6.8 Hz, 1H, a-pyr),
6
1
.51 (d, J=5.4 Hz, 1H, arom), 7.11 (d, J=5.4 Hz, 1H, arom), 7.32 (m,
H, arom), 7.64 (dd, J=8.8, 1.6 Hz, 1H, arom), 8.07–8.28 (m, 2H,
cooling, the solution was diluted with Et
with 0.5n HCl (3”15 mL). The acid solution was strongly alkalized
(2n NaOH (30 mL)) and extracted with Et O (3”20 mL). After re-
2
O (20 mL) and extracted
arom), 8.84 ppm (d, J=5.0 Hz, 1H, arom); elemental analysis calcd
%) for C H ClN OS (425.97): C 64.85, H 5.68, N 9.86, S 7.53,
2
(
moving the solvent, the oily residue was distilled at 0.07 hPa col-
lecting a pale-yellow oil (3.92 g, 93%) at 1408C (air bath tempera-
ture); elemental analysis calcd (%) for C H NOS (255.42): C 65.86,
2
3
24
3
found: C 64.66, H 5.66, N 10.05, S 7.53.
14
25
7
-Chloro-4-{S-[4-(citisin-3-yl)butyl]thio}quinoline (27): Yield: 43%;
H 9.87, N 5.48, found: C 65.76, H 9.99, N 5.63.
1
mp: 100–1018C; H NMR (200 MHz, CDCl ): d=1.56–2.17 (m, 2H,
3
bisp+4H, CH CH CH CH ), 2.33–2.66 (2H, S-CH +3H, bisp), 2.88–
3-Hydroxyimino-1-{[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)me-
thyl]thio}butane (37): Ketone 35 (3.48 g, 13.6 mmol) was convert-
ed into the corresponding oxime by treatment with hydroxylamine
hydrochloride (1.14 g, 16.3 mmol) and 6n NaOH solution (2.7 mL)
under the conditions used for oxime 34. Thus, a pale-yellow oil
(3.55 g, 96%) was obtained. Elemental analysis calcd (%) for
2
2
2
2
2
3
.19 (m, 2H, NH-CH +2H, bisp), 4.02 (dd, J=15.6, 6.4 Hz, 1H,
2
bisp), 4.22 (d, J=15.6 Hz, 1H, bisp), 6.11 (d, J=6.8 Hz, 1H, a-pyr),
6
7
.56 (d, J=9.0 Hz, 1H, arom), 7.22 (d, J=5.0 Hz, 1H, arom), 7.26–
.42 (m, 1H, arom), 7.65 (dd, J=9.0, 2.2 Hz, 1H, arom), 8.15–8.31
(
m, 2H, arom), 8.84 ppm (d, J=5.0 Hz, 1H, arom); elemental analy-
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C H N OS (270.43): C 62.17, H 9.69, N 10.36, found: C 62.22, H
washed with ice-cold H O (2”5 mL). The aqueous solution was
14
26
2
2
9
.78, N 10.36.
concentrated under vacuum to dryness to afford a brownish, very
1
hygroscopic powder (398 mg, 84%); H NMR (300 MHz, [D ]DMSO):
6
3
-Amino-1-{[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]thio}-
d=1.01–1.93 (m, 24H), 2.72–3.45 (m, 8H), 7.99 (brs, 2H, collapsed
butane (38): Following the procedure described for the prepara-
tion of amino compound 35, oxime 37 (2.25 g, 8.3 mmol) was re-
duced with lithium aluminum hydride (1.57 g, 41 mmol). Thus,
after distillation at 0.07 hPa (1408C, air bath temperature) amine
with D O), 10.27 ppm (brs, 2H, collapsed with D O). Free base:
2
2
1
pale yellow oil; H NMR (300 MHz, [D ]DMSO): d=0.94–1.03 (m,
6
4
H), 1.25–1.51 (m, 20H), 2.24–2.52 (m, 8H), 3.33 ppm (brs, 2H, col-
lapsed with D O).
2
3
8 (1.92 g, 90%) was obtained. Elemental analysis calcd (%) for
C H N S (256.45): C 65.57, H 11.00, N 10.93, found: C 65.33, H
General method for the preparation of 7-chloro-4-[(w-hydroxyal-
kyl)amino]quinolines 49–53: 4,7-Dicholoroquinoline (0.6 g,
14
28
2
11.34, N 10.74.
3
mmol) was mixed with the w-hydroxyalkylamine (9 mmol) and
9
a-(Oct-7-enyl)-octahydro-2H-quinolizine (45): A solution of 8-
heated in an Aldrich pressure tube at 1508C for 3 h. After cooling,
bromo-1-octene (3 g, 15.7 mmol) in anhydrous THF (4.5 mL) was
added dropwise to a mixture of granular magnesium (15.7 mmol)
and a catalytic amount (30 mg) of zinc bromide in dry THF (9 mL).
After the addition of a crystal of iodine to trigger the reaction, the
mixture was stirred for 4 h at 508C under an atmosphere of nitro-
gen. Once the Grignard compound was completely formed, dry
the mixture was taken up with H O (10 mL), and the insoluble title
2
compound was filtered and washed with H O (2”5 mL) and finally
2
crystallized from MeOH. Compounds 49–51 and 53 are already
[43–46]
known.
7-Chloro-4-[(2-hydroxyethyl)amino]quinoline (49): Yield: 86%;
[37]
[43]
THF (30 mL) and octahydroquinolizidinium perchlorate (1.89 g,
mp: 214–2158C (lit. 2148C).
7
2
.85 mmol) were added, and the mixture was heated at reflux for
0 h. After cooling, the mixture was diluted with saturated aque-
7
-Chloro-4-[(3-hydroxypropyl)amino]quinoline (50): Yield: 73%;
[44]
mp: 149–1508C (lit. 148–148.58C).
ous solution of NH Cl (9 mL) and acidified with 6n HCl (to pH 2).
4
The aqueous layer was extracted with Et O (3”10 mL), then alkal-
7-Chloro-4-[(4-hydroxybutyl)amino]quinoline (51): Yield: 68%;
mp: 176–1788C (lit. 171–1748C).
2
[45]
ized and saturated with K CO3 and extracted several times with
2
Et O (4”20 mL). The collected organic layer was dried (Na SO )
2
2
4
7
-Chloro-4-[(5-hydroxypentyl)amino]quinoline (52): Yield: 82%;
and concentrated to dryness to give a yellow oil (790 mg, 54%);
1
1
mp: 148–1498C; H NMR (200 MHz, CDCl
CH CH CH ,+1H, OH) 3.36 (m, 2H, NHCH ), 3.75 (t, J=6.0 Hz, 2H,
CH O), 5.13 (s, 1H, NH, collapsed with D O), 6.42 (d, J=5.4 Hz, 1H,
): d=1.42–1.98 (m, 6H,
3
H NMR (300 MHz, CDCl ): d=1.20–2.26 (m, 22H), 2.39–2.92 (m,
3
2
2
2
2
6
H), 4.91–5.03 (m, 2H), 5.77–5.82 ppm (m, 1H).
2
2
8
-(Octahydro-2H-quinolizin-9a-yl)octan-1-ol (46): A solution of
arom), 7.37 (dd, J=9.0, 2.2 Hz, 1H, arom), 7.69 (d, J=9.0 Hz, 1H,
arom), 7.98 (d, J=2.0 Hz, 1H, arom), 8.54 ppm (d, J=5.4 Hz, 1H,
compound 45 (750 mg) in anhydrous THF (13.5 mL) was added
dropwise over 30 min to an ice-cooled mixture of 1m borane–THF
complex solution (12.6 mL, 12.6 mmol) and 2-methyl-2-butene
arom); elemental analysis calcd (%) for C14H17ClN O (264.75): C
2
63.51, H 6.47, N 10.58, found: C 63.39, H 6.70, N 10.43.
(
1.76 g, 70.1 mmol) diluted with dry THF (2.4 mL). The mixture was
7
-Chloro-4-[(2-hydroxy-1-methylethy)amino]quinoline (53): Yield:
stirred at room temperature for 4.5 h and then diluted with H O
(
[46]
2
8
2%; mp: 207–2098C (lit. 208–2108C).
3.7 mL), 6n NaOH (12.5 mL), and H O (35% solution, 3.1 mL). The
2 2
mixture was stirred at room temperature for 1 h and then saturat-
ed with K CO and extracted with Et O (4”20 mL). The collected
organic layer was concentrated to dryness, and the residue was di-
General method for the preparation of 4-[(w-bromoalkyl)amino]-
7-chloroquinolines 53–58: An Aldrich pressure tube was sequen-
tially charged with 7-chloro-4-(hydroxyalkylamino)quinoline
(2 mmol), 48% hydrobromic acid (1.5 mL), and concentrated sulfu-
ric acid (0.5 mL). The closed tube was heated at 1608C for 3.5 h.
2
3
2
luted with Et O (50 mL) and extracted with 4n HCl (3”30 mL). The
2
aqueous layer was alkalized, saturated with K CO , and extracted
2
3
with Et O (4”20 mL). The organic phase was dried (Na SO ) and
After cooling, the mixture was poured on H O and ice and then
2
2
4
2
concentrated to dryness to give an oil that was purified by column
chromatography (neutral Al O , grade IV; PE/Et O in gradient). Oil
was alkalized with 2n NaOH solution (20 mL). The mixture was ex-
2
3
2
tracted with CH Cl2 (3”30 mL); the organic phase was dried
2
1
(
380 mg, 48%); H NMR (300 MHz, CDCl ): d=1.08–1.69 (m, 26H),
(Na SO ) and concentrated. The residue was finally crystallized
3
2
4
2
.38–2.42 (m, 2H), 2.55–2.64 (m, 2H), 3.62–3.66 ppm (m, 2H). One
from toluene. Compounds 54, 55, and 58 are already
known.
[43,44,46]
H is not visible, probably because of its interaction with the sol-
vent.
4
-[(2-Bromoethyl)amino]-7-chloroquinoline (54): Yield: 74%; mp:
[43]
2
-[8-(Octahydro-2H-quinolizin-9a-yl)octyl]isoindoline-1,3-dione
139–1408C (lit. 139–1408C).
(
47): Diethylazodicarboxylate (DEAD; 0.42 mL diluted with 1.1 mL
4
-[(3-Bromopropyl)amino]-7-chloroquinoline (55): Yield: 71%;
of anhydrous THF) was added dropwise to an ice-cooled mixture
of the above alcohol (340 mg, 1.27 mmol), phthalimide (374 mg,
[44]
mp: 158–1608C (lit. 159–1608C).
2
(
2
.54 mmol), triphenylphosphine (700 mg, 2.67 mmol), and dry THF
3.9 mL). The resulting mixture was stirred at room temperature for
0 h. The solvent was evaporated, and the crude oil was purified
by column chromatography (silica gel; CH Cl /MeOH/conc. NH
4-[(4-Bromobutyl)amino]-7-chloroquinoline (56): Yield: 66%; mp:
220–2228C; H NMR (200 MHz, CDCl3): d=1.42–1.98 (m, 4H,
1
CH CH ), 3.30–3.56 (m, 2H, NHCH +2H, CH Br), 5.09 (s, 1H, NH, col-
2
2
2
2
2
2
1
3
lapsed with D O), 6.44 (d, J=5.4 Hz, 1H, arom), 7.38 (dd, J=9.0,
2
9
6:3.6:0.4). Pale-yellow oil (490 mg, 96%); H NMR (300 MHz,
2.0 Hz, 1H, arom), 7.71 (d, J=9.0 Hz, 1H, arom), 8.01 (d, J=2.0 Hz,
1H, arom), 8.59 ppm (d, J=5.2 Hz, 1H, arom); elemental analysis
calcd (%) for C H BrClN (313.62): C 49.79, H 4.50, N 8.93, found:
CDCl ): d=1.09–1.67 (m, 24H), 2.52–2.66 (m, 4H), 3.48–3.70 (m,
4
3
H), 7.69–7.73 (m, 2H arom), 7.81–7.85 ppm (m, 2H, arom).
13
14
2
C 46.69, H 4.33, N 9.18.
8
-(Octahydro-2H-quinolizin-9a-yl)octane-1-amine dihydrochlor-
ide (43): The above phthalimide derivative 47 (478 mg, 1.20 mmol)
was dissolved in 6n HCl (4.7 mL) and heated at reflux for 20 h.
After ice cooling, the precipitated phthalic acid was filtered and
4-[(5-Bromopentyl)amino]-7-chloroquinoline (57): Yield: 63%;
1
mp: 112–1138C; H NMR (200 MHz, CDCl ): d=1.42–1.98 (m, 6H,
3
CH CH CH ), 3.29–3.58 (m, 2H, NHCH +2H, CH Br), 5.02 (s, 1H, NH,
2
2
2
2
2
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1581
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
collapsed with D O), 6.45 (d, J=5.4 Hz, 1H, arom), 7.40 (dd, J=9.2,
2
Keywords: antiprotozoal agents
derivatives · medicinal chemistry · quinolizidine derivatives
· chloroquine · cytisine
1
1
.8 Hz, 1H, arom), 7.69 (d, J=9.2 Hz, 1H, arom), 8.00 (d, J=1.8 Hz,
H, arom), 8.57 ppm (d, J=5.4 Hz, 1H, arom); elemental analysis
calcd (%) for C H BrClN (327.65): C 51.32, H 4.92, N 8.55, found:
1
4
16
2
C 51.30, H 5.28, N 8.20.
[1] WHO, World Malaria Report 2014, World Health Organization, Geneva,
4
-[(2-Bromo-1-methylethyl)amino]-7-chloroquinoline (58): Yield:
Switzerland, 2014.
[46]
[2] a) E. A. Ashley, M. Dhorda, R. M. Fairhurst, C. Amaratunga, P. Lim, S.
5
3%; mp: 128–1308C (lit. 132–1338C).
Suon, S. Sreng, J. M. Anderson, S. Mao, B. Sam, et al., N. Engl. J. Med.
2014, 371, 411 –423; b) K. M. Tun, M. Imwong, K. M. Lwin, A. A. Win,
T. M. Hlaing, T. Hlaing, K. Lin, M. P. Kyaw, K. Plewes, M. A. Faiz, et al.,
Lancet Infect. Dis. 2015, 15, 415–421.
[
3] a) R. A. Jones, S. S. Panda, C. D. Hall, Eur. J. Med. Chem. 2015, 97, 335–
Biological methods
355; b) F. Calderón, D. M. Wilson, F.-J. Gamo, Progr. Med. Chem. 2013,
5
2, 97–151; c) M. A. Biamonte, J. Wanner, K. G. Le Roch, Bioorg. Med.
Parasite cultures and drug susceptibility assays: P. falciparum cultures
[54]
Chem. Lett. 2013, 23, 2829–2843; d) P. M. O’Neill, V. E. Barton, S. A.
Ward, J. Chadwick in Treatment and Prevention of Malaria (Eds.: H. M.
Staines, S. Krishna), Springer, Basel, 2012, pp. 19–44; e) S. Bawa, S.
Kumar, S. Drabu, R. Kumar, J. Pharm. BioAllied Sci. 2010, 2, 64–71;
f) T. N. C. Wells, P. L. Alonso, W. E. Gutteridge, Nat. Rev. Drug Discovery
2009, 8, 879–891; g) M. Schlitzer, ChemMedChem 2007, 2, 944–986.
were performed according to Trager and Jensen
with slight
modifications. The CQ-susceptible strain D10 and the CQ-resistant
strain W2 were maintained at 5% hematocrit (human type A-posi-
tive red blood cells) in RPMI 1640 (EuroClone, Celbio) medium with
the addition of 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypo-
xanthine, 20 mm HEPES, and 2 mm glutamine. All the cultures were
maintained at 378C in a standard gas mixture consisting of 1% O2,
[4] a) T. J. Egan, R. Hunter, C. H. Kaschula, H. M. Marques, A. Misplon, J.
Walden, J. Med. Chem. 2000, 43, 283–291; b) T. J. Egan, Mini-Rev. Med.
Chem. 2001, 1, 113–123; c) C. H. Kaschula, T. J. Egan, R. Hunter, N. Basili-
co, S. Parapini, D. Taramelli, E. Pasini, D. Monti, J. Med. Chem. 2002, 45,
5
% CO , and 94% N . Compounds were dissolved in either water
2
2
or DMSO and then diluted with medium to achieve the required
concentrations (final DMSO concentration <1%, which is nontoxic
to the parasite). Drugs were placed in 96-well flat-bottomed micro-
plates (COSTAR) and serial dilutions made. Asynchronous cultures
with parasitemia of 1–1.5% and 1% final hematocrit were aliquot-
ted into the plates and incubated for 72 h at 378C. Parasite growth
was determined spectrophotometrically (OD650) by measuring the
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