New 9-aminoacridine derivatives as inhibitors of botulinum neurotoxins and P. falciparum malaria

Article abstract of DOI:10.2298/JSC130924112T

Steroidal and adamantane aminoacridine derivatives were prepared and tested as both botulinum neurotoxin (BoNT) inhibitors and antimalarials. Steroid-bound acridines provided good potency against both the BoNT/A and BoNT/B light chains (LCs). The observed

Full text of DOI:10.2298/JSC130924112T

J. Serb. Chem. Soc. 78 (12) 1847–1864 (2013)
UDC *Clostridium botulinum+547.583.5:
JSCS–4536
615.285:612.014.46+616.8+615.9+57
Original scientific paper
New 9-aminoacridine derivatives as inhibitors of botulinum

neurotoxins and P. falciparum malaria
MIKLOŠ TOT1, DEJAN M. OPSENICA2#, MILENA MITRIĆ1, JAMES C. BURNETT3,
4
5
1 #
*
LAURA GOMBA , SINA BAVARI and BOGDAN A. ŠOLAJA
1Faculty of Chemistry, University of Belgrade, Studentski trg 16, P. O. Box 51, 11158,
Belgrade, Serbia, 2Institute of Chemistry, Technology, and Metallurgy, University of
Belgrade, Njegoševa 12, 11000 Belgrade, Serbia, 3SAIC-Frederick, Inc., Frederick National
Laboratory for Cancer Research, P. O. Box B, Frederick, MD 21702, USA, 4United States
Army Medical Research Institute of Infectious Diseases, Department of Bacteriology, 1425
Porter Street, Frederick, MD 21702, USA and 5United States Army Medical Research
Institute of Infectious Diseases, Fort Detrick, 1425 Porter Street, Frederick, MD 21702, USA
(Received 24 September, revised 20 October 2013)
Abstract: Steroidal and adamantane aminoacridine derivatives were prepared
and tested as both botulinum neurotoxin (BoNT) inhibitors and antimalarials.
Steroid-bound acridines provided good potency against both the BoNT/A and
BoNT/B light chains (LCs). The observed inhibition of the BoNT/B LC by ca.
50 % is the highest attained inhibitory activity against this serotype by
acridine-based compounds to date. With respect to the antimalarial activity, the
adamantane acridines were the most potent derivatives (IC50 = 6–9 nM, SI > 326),
indicating that an adamantyl group is a better carrier than a steroidal motif for
this indication.
Keywords: antiviral, BoNT/A; BoNT/B; antimalarial; aminoacridine.
INTRODUCTION
Botulinum neurotoxins (BoNTs),** secreted by the anaerobic, spore-forming
bacterium Clostridium botulinum, are the most potent of known biological tox-
*Corresponding author. E-mail: bsolaja@chem.bg.ac.rs
# Serbian Chemical Society member.
 This manuscript is dedicated to our dear colleague and friend Prof. Branislav Nikolić on the
occasion of his 70th birthday.
doi: 10.2298/JSC130924112T
** Abbreviations: BoNT, botulinum neurotoxin; SNARE, soluble N-ethylmaleimide-sen-
sitive factor attachment protein receptor; BoNT/A, botulinum neurotoxin serotype A;
BoNT/A LC, BoNT/A light chain; SMNPIs, small molecule non-peptidic inhibitors; ACR,
9-amino-2-metoxy-7-chloroacridine; ACR2, N-(6-chloro-2-methoxyacridin-9-yl)ethane-
-1,2-diamine; ACR3, N-(6-chloro-2-methoxy-9-anthryl)propane-1,3-diamine; ACR5,
1847
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1848
TOT et al.
1,2
ins. The proteolytic activities of these enzymes are responsible for the poten-
tially fatal disease botulism, which is commonly associated with food contami-
nation, wound infection, and colonizing infection in infants. BoNTs can be easily
3
produced and disseminated, and therefore are listed as priority biological threat
4
agents. On the other hand, when dosed locally in minute concentrations, BoNTs
5
are used for the effective treatment of severe diseases.
There are seven known BoNT serotypes identified as A–G. Each cleaves a
component of the SNARE complex, which mediates the transport of acetylcho-
6,7
line into neuromuscular junctions. BoNTs are secreted as holotoxins composed
of a heavy chain (HC) and light chain (LC), which are connected by a disulfide
8,9
bridge. The HC, which transports the LC into the neuronal cytosol via an aci-
dic endosome, is comprised of two ≈50 kDa domains. The C-terminal domain
(H ), the ganglioside and protein receptor-binding domain, plays a role in bind-
C
ing to the cell membrane and internalization of a toxin into cholinergic neurons;
the N-terminal domain (H ) facilitates the release of the LC from endosomes into
N
the cytosol. The LC is a zinc-dependent metalloprotease that cleaves SNARE
proteins, thereby inhibiting the formation of the SNARE complex. BoNT sero-
10
types A and E cleave SNAP-25 (synaptosomal-associated protein (25 kDa)),
11
serotypes B, D, F and G cleave VAMP (vesicle-associated membrane pro-
tein), and serotype C cleaves both SNAP-25 and syntaxin 1. Serotype A
(BoNT/A) is the most potent of the seven serotypes. For example, it is 10 -fold
more potent than cobra toxin and 10 -fold more potent than cyanide, and its
12
13
6
11
6
–1
14
lethal dose is estimated to be between 1 and 5 ng kg for humans. Moreover,
15
the BoNT/A LC is longest acting of the LC serotypes in the neuronal cytosol.
Currently, there are no therapies available for the treatment of BoNT LC-
mediated paralysis post-neuronal intoxication, although several different appro-
aches are currently under development. Two of these approaches include anti-
16
body-based therapeutics and small molecule-based therapeutics. However, anti-
bodies cannot reverse the proteolytic effects of the toxins after the LC component
2
has entered the neuronal cytosol. Hence, the development of small molecules
that will effectively inhibit proteolytic activity of BoNT LC after post-neuronal
intoxication is of special interest, with the BoNT/A LC being the main enzyme of
study. At this time, one of the best known inhibitors of the BoNT/A LC is mpp-
RATKML, which consists of the oligopeptide RATKML covalently bonded to
17
2-mercapto-3-phenylpropionyl (mpp) group (K = 330 nM). However, peptides
i
are readily hydrolyzed by proteases, thus limiting their application in the therapy.
N-(6-chloro-2-methoxyacridin-9-yl)pentane-1,5-diamine; ACR6, N-(6-chloro-2-methoxy-
acridin-9-yl)hexane-1,6-diamine; CQR, chloroquine resistant strain; CQS, chloroquine
susceptible strain; MDR, multi-drug resistant strain; BS, binding site; QA, quinacrine;
CQ, chloroquine; MFQ, mefloquine; ART, artemisinin; EA, ethyl acetate; DCM, dichloro-
methane; PCC, pyridinium chlorochromate; TFA, trifluoroacetic acid.
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
1849
For this reason, the development of small molecule inhibitors is crucial. To this
18
end, many inhibitors of the BoNT/A LC have been reported, and are composed
19
of compounds that competitively coordinate the catalytic zinc ion, as well as
20
allosteric inhibitors. The most potent possess K values ranging from 0.1–10
i
2+
21
μM and include zinc (Zn ) bidentate coordinating hydroxamic acid 1, 2,5-
diphenyl-thiophene derivative 2, and diazachrysene 3 (Chart 1).
22
23
In contrast to BoNT/A LC inhibitors, a paucity of small molecules that inhi-
24
bit the BoNT/B LC has been reported. These include isocoumarine 4, aryl-
25
26
-phosphoramidic acid ICD 2821 , bis-benzimidazole-5-carboximidamide 5
(which bidentately coordinates the catalytic Zn ion of enzymes), and L-chicoric
27
acid 6 (which is reported to bond to an exosite of the enzyme). In addition, it
has been reported that the reducing agent TCEP (tris(2-carboxyethyl)phosphine
hydrochloride) reduces the disulfide bond connecting the HC–LC bond of the
enzyme both in vitro and in neuronal cells, and that it works prophylactically
28
with a maximum activity at 1 mM. In this way, the LC cannot undergo endo-
cytosis, and therefore cannot cleave VAMP.
It is well known that many 4-aminoquinoline antimalarials act as inhibitors
29,30
of the BoNT/A LC.
In contrast, little has been reported about the efficacy of
acridine-based (ACR) compounds as BoNT inhibitors. However, it was found
that quinacrine (QA, Chart 2), effectively prolonged the time of BoNT/A block-
31
ing of nerve-elicited muscle twitch in isolated mouse diaphragms. Furthermore,
it was found that QA was four times more efficacious than CQ, with a muscle
twitch protective index of 2.85 at 10 μM.
Malaria* is a devastating disease and a global health problem. It is estimated
32
to cause 300–500 million clinical cases and ca. one million deaths per annum.
The most successive drugs appeared to kill the parasite either by producing toxic
33
free radicals or by blocking the polymerization of heme, as in the case of the
34
4-amino-7-chloroquinolines (ACQs). The development of widespread drug-
resistance to CQ has resulted in an urgent need for new drug modalities, for
35
example, synthetic peroxide antimalarials, as well as for the development of
36
novel molecules that prevent heme polymerization.
Quinacrine was the first synthetic antimalarial used clinically that acts as a
37
blood schizonticide, but later it was replaced with the more efficient CQ. How-
ever, due to the widespread development of CQ resistant (CQR) strains, there is
renewed interest for evaluating acridine-based compounds as antimalarials. For
example, N-sulfonamide derivatives, obtained from various simple 9-aminoacri-
38
dines, show high antimalarial activity against both CQS and CQR strains. They
do not lose activity even when the amino group is stripped of its basic character,
39
which is in contrast to most CQs or other quinoline or acridine derivatives. Qui-
*Malaria is caused by five Plasmodium species, P. falciparum, P. ovale, P. vivax, P. malariae
and P. knowlesi, of which P. falciparum causing cerebral malaria is the major death threat.
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
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1852
TOT et al.
nolizidinyl and quinolizidinyl-alkyl derivatives of 9-amino-6-chloro-2-methoxy-
acridine were almost 10 times more active than CQ against CQR (W2) strain, and
40
were as active as CQ against the CQS (D10) strain; a series of 9-amino-
41
acridines showed high inhibitory potency. Most active derivatives have diami-
nopropylidene side chains and exert low nM activity (EC in range 1–4.1 nM).
50
Similar to AQ antimalarials, 9-aminoacridines inhibit polymerization of
hemozoin. In a β-hematin inhibitory assay (BHIA), it was found that ACR deri-
42
vatives formed complexes with hematin in a 3:2 molar ratio (hematin:drug). It
was shown that the presence of 2-methoxy, 6-chloro substituents and an ionizable
terminal amino group are essential for good antimalarial activity. It was also
39,43
discovered that ACRs inhibit DNA topoisomerase II,
parasite folate meta-
44
45
46
bolism and plasmepsin II. However, chimeric derivatives with ART or
47
39,48
AQ, as well as bis-ACR derivatives,
showed poor activities.
In addition to their significant antimalarial activity, ACR-based compounds
exhibit other biological activities, including nuclear localization signal (NLS)-
49
50
-labeling agents for plasmid DNA, photocleavage reagents for DNA, anti-
51
52
leishmania agents and anti-prion compounds.
Previously, we reported on the inhibitory activities of steroidal 4-amino-
quinolines 7–9 and adamantyl-aminoquinolines (for example, 10) against the
30
53
BoNT/A LC and as antimalarial agents were reported (Chart 2). Specifically,
these derivatives provided a high degree of inhibition of BoNT/A metallopro-
tease, as well as significant inhibition of CQS and more important, CQR strains
of P. falciparum. Herein, the synthesis and biological evaluation of new ACR
derivatives possessing steroid and adamantane carrier component are presented.
RESULTS AND DISCUSSION
Synthesis
The syntheses of the ACR derivatives examined in this study are provided in
Scheme 1, and the corresponding structures are given in Chart 3. First, aminoalk-
CARRIER
( )nNH
2
Cl
HN
( )Nn H
HN
OMe
ii
OMe
i
OMe
Cl
N
Cl
N
Cl
N
11
12: TMACR2: n = 1
13: TMACR3: n = 2
14: TMACR5: n = 4
15: TMACR6: n = 5
16 - 24
i) diamine, ;
ii) ketone, NaBH3CN/ MeOH/CH3CN, or NaBH4 / MeOH or NaBH4 / Ti(OPri)4 / EtOH, or NaB(OAc)3H / DCM
Scheme 1. Synthesis of acridine derivatives 12–24.
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
1853
AcO
OCH3
H
AcO
O
H
N( )n
N
N
OMe
OAc
( )Nn H
H
H
N
N
AcO
Cl
OAc
H
OMe
20 TM27: n = 1
21 TM28: n = 2
Cl
16 MM18c: n = 1, -isomer
17 MM18d: n = 1, -isomer
18 MM17a: n = 2, mixture of isomers with excess of -isomer
19 MM17b: n = 2, mixture of isomers with  / -ratio = 1:1.5
( )NnH
HN
OMe
NH
HN
Cl
N
OMe
22 TM36-2: n = 1
23 TM37-1: n = 2
Cl
N
24 TM48/1
Chart 3. Structures of examined derivatives 16–24.
ylamino acridines 12–15 were obtained by the heating of 6,9-dichloro-2-meth-
50
oxyacridine 11 with neat diamines. The spectra of the isolated products were in
agreement with literature data, and the compounds were used in the next reaction
step without additional purification. Derivatives 16–24 were synthesized using
30,53
procedures described earlier.
The 3-keto steroidal derivative (7α,12α-diace-
54
toxy-3-oxo-5β-cholan-24-oate) was coupled with aminoalkylamino acridines
12 and 13 to afford the corresponding 3α-and 3β-diastereomers (16–19). The
crude reaction product obtained from 12 afforded diastereomers 16 and 17 via
chromatographic separation. However, derivatives obtained from 13 were only
partially separated to afford 18 containing ca. 20 % of 19, and the second fraction
consisting of both isomers in a 1:1.5 ratio. The configuration at C(3) of the
1
derivatives 16–19 was deduced from the respective H-NMR spectra. The cor-
responding H–C(3) atom of derivative 16 had a broad multiplet at 2.50–2.34
ppm, while that of derivative 17 had a narrow signal at 2.93 ppm. Therefore, 16
has an axial H–C(3) (in the β-orientation) and acridine substituent in an α-orien-
tation (α-isomer). Consequently, 17 is the β-isomer since it has H –C(3) and the
α
acridine substituent in the β-orientation. Based on the above correlations, it was
possible to establish the approximate composition of mixtures obtained from par-
tially separated isomers 18 and 19. Steroidal alcohol 3α,7α,12α-triacetoxy-5β-
30
-cholan-24-ol was oxidized to the corresponding aldehyde, which was further
coupled to 12 and 13 to give the corresponding steroidal acridine derivatives 20
53
and 21. Following the general approach, 2-(adamantyl)ethanal gave the ada-
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1854
TOT et al.
mantyl–acridine derivatives 22 and 23, and the benzaldehyde afforded N-benzyl
acridine 24. The structures of all synthesized compounds were confirmed by
spectral and analytical techniques, with all details given in the Supplementary
material to this paper.
Inhibition of BoNT/A LC
The inhibitory efficacies of synthesized derivatives 16–21 and 24 at 20 μM
concentration were obtained using a well-established HPLC-based assay for
55
BoNT/A LC inhibition. These data are presented in Table I. The activities of
30
corresponding aminoquinoline derivatives 7–9 were established previously,
and are provided for comparison. In this study, the inhibitory effects of CQ were
also examined. It exhibited only marginal inhibition and thus clearly emphasized
the necessity of the cholic acid component for superior inhibitory activity.
TABLE I. In vitro inhibitory activities (%, at 20 μM concentration) of the tested compounds
against BoNT/A LC and BoNT/B LC; N.T. – not tested
Compound
NSC 240898
BoNT/A-LC
81.43
58.34
58.25
77.04
59.38
15.07
61.25
5.82
BoNT/B-LC
77.38
57.03
52.04
47.40
48.10
31.94
33.63
N.T.
16
17
18
19
20
21
24
12
13
14
15
7a
42
20
8
10
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
74
8a
47.22
56.26
6.51
9a
CQb
MQc
28.06
N.T.
a
31 b
c
Results published earlier;
tested as diphosphate salt; tested as HCl salt
The results given in Table I indicate that 9-aminoacridine derivatives
coupled to cholic acid derivative as the carrier exhibited moderate to good acti-
vities against the BoNT/A LC, with potencies ranging from 58 to 77 % inhi-
bition. The exception is derivative 20, which provided only 15 % inhibition. The
most active derivative was compound 18, which contains the acridine moiety in
the β-orientation. However, the results obtained for derivatives 18 and 19 are not
so easily interpretable, since both samples were mixtures of diastereomers: 18
contained ≈20 % of the α-isomer, and 19 was a mixture of both isomers in 1:1.5
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
1855
α:β ratio. Nevertheless, from the obtained results, it is plausible to assume that
the pure β-isomer would have higher activity versus the pure α-isomer (since the
former sample showed 20 % higher potency – 18 77.04 % inhibition vs. 19 59.38 %
inhibition).
The compounds incorporating acridine 12, i.e., 16 and 17, were equally
potent, suggesting that the configuration at position C(3), in this instance, is not
crucial for BoNT/A LC inhibition. Comparison of the inhibitory activities for
steroidal acridines with the corresponding data for steroidal aminoquinolines 7–9
indicates that these compounds expressed approximately the same level of inhi-
bitory capacity. This suggests that both acridine and quinoline moieties within
the small molecule inhibitors engage in similar types of interactions with the
active site of the BoNT LC metalloprotease. However, more reliable correlations
will be made on pure isomers after measuring the corresponding K values.
i
The simple 9-aminoacridines 12–15 exhibited poor inhibitory activity (8–20 %
inhibition) with 12 being an exception (42 % inhibition). In addition, the results
showed that elongation of the aminoalkyl chain leads to decreased activity for
this class of inhibitor. The low activities of 9-aminoacridines 12–15 clearly indi-
cate that for good inhibitory activity, a carrier is necessary in order to enable
additional interactions within the active site of a metalloprotease. The poor acti-
vity of N-benzyl derivative 24 further supports this observation.
Derivatives 16–21 were also tested for inhibition of BoNT/B LC, giving rise
to some of the most potent inhibitors of this serotype reported to date (Table I).
The results indicate that steroidal C(3) derivatives are slightly more active in
comparison to C(24) derivatives. Moreover, the results showed no differences
between the stereoisomers, i.e., the H –C(3) isomers exhibited the same level of
β
activity as the H –C(3) isomers. The most active derivative was 16, which pro-
α
vides 57 % inhibition at 20 μM concentration.
As steroidal 9-aminoacridines inhibited Zn proteases BoNT/A and B LCs,
the next step was to examine the activity of synthesized derivatives against a
sampling of human metalloprotease – to evaluate selectivity. The four derivatives
16–19 were tested at 20 μM concentration against human metalloprotease ther-
molysin, serine protease trypsin, and cysteine protease papain. The results are
given in Table II. The data indicate that the compounds are poor inhibitors of
generic human proteases. The exception was derivative 18, which meaningfully
inhibited papain. Nevertheless, in general, it could be concluded that these deri-
vatives selectivity inhibit BoNT LC metalloproteases (as opposed to human pro-
teases). In addition, the low activities of the compounds against the Zn-dependent
protease thermolysin indicates the inhibitory potencies against BoNT LCs were
not the result of general preferences for interaction with the Zn ion, but that addi-
tional specific interactions with the enzymes were involved.
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1856
TOT et al.
TABLE II. Inhibitory activities (%,at 20 μM concentration) of ACR SMNPIs against human
proteases; A: trypsin; B: thermolysin; C: papain
O
H2N
N
H
NH
NSC 240898
NH2
HN
Compound
A
B
C
NSC 240898
52.07
12.36
17.24
22.94
7.48
8.53
–11.78
5.19
20.76
78.54
–7.85
16
17
18
19
13.98
15.42
22.37
12.68
Antimalarial activity
It was previously shown that aminoquinoline SMNPIs exerted both BoNT
30,53
inhibiting effects and were simultaneously excellent antimalarials.
Based on
this observation, acridine derivatives were elected for screening against three P.
falciparum strains: D6 (a CQ and MFQ susceptible strain), W2 (a CQ-resistant,
susceptible to MFQ strain), and TM91C235 (Thailand), a multi-drug-resistant
55
strain, using a well-established protocol. The gathered in vitro antimalarial acti-
vities (Table III) show that all tested derivatives had significantly better activities
than CQ against the CQR strain W2 (16–53 times) and the multi-drug resistant
TM91C235 strain (2–24 times). Against the CQS strain D6, the steroidal deriva-
tives were less active than CQ; however, the other derivatives were as active as
CQ. The steroidal acridines 20 and 21 were less active in comparison to 7 against
all the tested strains. However, at the same time, the adamantyl derivatives 22
and 23 were more active when compared to 10 against the CQR W2 and the
multi-drug-resistant TM91C235 strains. Importantly, all the new derivatives were
38
39
significantly more active than N-sulfonamides, 9-amino-polyamide acridines,
42
aminoalkylamino acridines, and had activities to those of quinolizidinyl and
quinolizidinylalkyl derivatives, which are the second best known acridine anti-
40
malarials. The most active of the derivatives was 22, with IC (W2) = 8.6 nM,
50
IC (D6) = 9.3 nM and IC (TM91C235) = 5.8 nM. Its in vitro activity is com-
50
50
parable to that of artemisinin (Table III). In addition, compound 22 had similar
activity to those of the most potent diaminopropylidene-aminoacridine deriva-
41
tives described in the literature.
It should be emphasized that steroidal/adamantane carriers of the 9-amino-
alkylamino acridine moiety had significantly improved antimalarial activities.
Comparatively, simple acridines such as 12 and 15 showed much lower anti-
malarial activity against the CQR W2 strain versus new derivatives (12 (IC
=
50
42
= 250 nM) and 15 (IC = 180 nM)). Thus, the attachment of these simple
50
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
1857
ACRs to either cholic acid or the adamantyl moiety significantly increased
antimalarial activity against CQR strains. The same trend was observed against
the CQS strain. The results reported herein indicate that steroidal and adamantyl
acridines are highly effective antimalarials.
TABLE III. In vitro IC50 antimalarial activities (nM) and cytotoxicity of the compounds tested
against P. falciparum strains; N.D. – not determined
Compd.
W2a
D6b TM91C235c
RId
HEPG2
SIe
20
21
22
23
23.8
23.3
8.6
41.5
39.0
9.3
51.8
53.0
5.8
12.2
15.6
1.25 / 0.57
1.36 / 0.60
0.62 / 0.92
0.78 / 0.71
0.56 / 1.56
1.64 / 0.67
4281
1030
>3030
3261
1183
N.D.
180 / 103 / 83
44 / 26 / 19
>352 / >326 / >522
291 / 208 / 267
42 / 66 / 76
–
11.2
28.0
11.4
15.66
250h
180h
456.20
4.93
6.70
15.7
17.9
16.9
8.59
N.D.
N.D.
12.27
15.70
9.00
24
7f
27.7
10g
12
17.76
N.D.
N.D.
138.82
36.50
13.40
–
–
N.D.
N.D.
N.D.
N.D.
–
–
–
–
–
15
CQ
MFQ
23.17 / 43.32
3.43 / 0.39
1.36 / 0.90
ARTi
N.D.
c
a
b
P. falciparum Indochina W2 clone; P. falciparum African D6 clone; P. falciparum multi-drug resistant
d
TM91C235 strain (Thailand); resistance index, defined as the ratio of the IC for the resistant versus sensitive
strain, TM91C235/D6 and W2/D6, respectively; selectivity index, defined as the ratio of the IC for
HepG2/W2, HepG2/D6 and HepG2/ TM91C235, respectively; results published earlier;
earlier;
50
e
50
results published
f
30 g
53 h 42 i
results taken from literature; average of more than eight replicates
The toxicities of the new derivatives were estimated using the human liver
carcinoma cell line HEPG2, and the results are given in Table III. The HEPG2
cell line is frequently used for cytotoxicity examination of xenobiotics, since this
cell line has low levels of CYP450 enzymatic activities and possible toxic effects
56
are the result of particular molecules and not metabolites. In addition, since all
xenobiotics pass through the liver, it is essential to know if they provide heapto-
toxic side effects. In this study, it was found that steroidal derivative 20, con-
taining aminoacridine part 12, was the least cytotoxic of the series, possessing an
IC = 4281 nM. However, the adamantyl derivatives had a much better selec-
50
tivity index (SI) than the other antimalarials. Thus, 22 has SI values 352/326/522
vs. 180/103/83 for 20, and thus have a higher therapeutic potential. It was also
observed that elongation of aminoalkylamino chains by only one methylene
group in 21 significantly increased cytotoxicity when compared to 20.
EXPERIMENTAL
Melting points were determined on a Boetius PMHK or a Mel-Temp apparatus and were
not corrected. IR spectra were recorded on a Perkin-Elmer spectrophotometer FT-IR 1725X.
NMR spectra were recorded on Varian Gemini-200, Varian XL-300, and Bruker AM-250
spectrometers in the indicated solvent using TMS as an internal standard. Chemical shifts are
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recorded in parts per million (ppm), and coupling constants J are expressed in Hz. ESI-MS
spectra were recorded on an Agilent Technologies 6210 Time-Of-Flight LC-MS instrument in
positive ion mode with CH3CN/H2O–0.2 % HCOOH as eluent. The samples were dissolved in
CH3CN or MeOH (HPLC grade purity). The elemental analysis was performed on the Vario
EL III – C, H, N, S/O elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Ger-
many). Thin-layer chromatography (TLC) was performed on pre-coated Merck silica gel 60
F254 and RP-18 F254 plates. For column chromatography, Lobar LichroPrep Si 60 (40–63 µm),
RP-18 (40–63 µm) columns coupled to a Waters RI 401 detector were used, Biotage SP1
system with UV detector.
Methyl 7α,12α-diacetoxy-3α-{N-{2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl}amino}-
-5β--cholan-24-oate (16) and methyl 7α,12α-diacetoxy-3β-(N-{2-[(6-chloro-2-methoxy-
acridin-9--yl)amino] ethyl}amino)-5β-cholan-24-oate (17)
To a solution of methyl 7α,12α-diacetoxy-3-oxo-5β-cholan-24-oate (300 mg, 0.59 mmol)
in CH3CN (6.0 mL), 12 (357.9 mg, 1.19 mmol) in MeOH (3 mL) was added, and after stirring
for 30 min at r.t., NaBH3CN (48.39 mg, 0.77 mmol) was added in one portion. The resulting
mixture was further stirred for 2 h at r.t., followed by the addition of glac. AcOH (4 drops)
and stirring was continued until TLC indicated the consumption of all starting ketone (ca. 1
h). The reaction was quenched with Et3N, the solvents were evaporated to dryness, and after
chromatographic purification (dry-flash, SiO2, gradient EA  EA/MeOH/NH3; Lobar RP-18,
eluent MeOH/H2O = 9:1; Biotage flash SP, gradient EA/Hex = 6:4  EA/Hex = 9:1) 16 (45.0
mg) and 17 (40 mg) were isolated (9.6 and 8.5 % yield, respectively).
Methyl 7α,12α-diacetoxy-3β-(N-{3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl}amino)-
-5β-cholan-24-oate (18) and methyl 7α,12α-diacetoxy-3α-(N-{3-[(6-chloro-2-methoxyacridin-
-9-yl)amino]propyl}amino)-5β-cholan-24-oate (19)
To a solution of methyl 7α,12α-diacetoxy-3-oxo-5β-cholan-24-oate (500 mg, 0.99 mmol)
in CH3CN (9.5 mL), 13 (124.3 mg, 0.39 mmol) in MeOH (5 mL) was added, and after stirring
for 30 min at r.t., NaBH3CN (82.8 mg, 1.32 mmol) was added in one portion. The resulting
mixture was stirred for 2 h at r.t. followed by the addition of glac. AcOH (4 drops) and stirring
was continued until TLC indicated the consumption of all starting ketone. The reaction was
quenched with Et3N, the solvents were evaporated to dryness and after chromatographic
purification (dry-flash, SiO2, gradient EA  EA/MeOH/NH3; Lobar RP-18, eluent MeOH/
/H2O = 9:1; Biotage flash SP, gradient EA/Hex = 6:4  EA/Hex = 9:1) 18 and 19 were
isolated (120.0 mg, 15 % and 90 mg, 11 %, respectively). 18 contained ca. 20 % of 3α-isomer
19 and 19 was a mixture of a ca. 1:1.5 ratio of the β- and α-isomers.
3α,7α,12α-Triacetoxy-24-{N-{2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl}amino}-5β-
-cholan (20)
The alcohol 3α,7α,12α-triacetoxy-5β-cholan-24-ol (540 mg, 1.04 mmol) was dissolved
in DCM (50 mL), PCC (185 mg, 1.56 mmol) was added and the mixture was stirred at r.t. for
3.5 h. The reaction mixture was filtered through a short SiO2 column (eluent CH2Cl2/EA =
= 95/5). Obtained crude aldehyde (500 mg) was dissolved in MeOH (10.0 mL), followed by
12 (290 mg, 0.96 mmol) and mixture was stirred at r.t. After 12 h, NaBH4 (73 mg, 1.92 mmol)
was added in one portion and stirring was continued at r.t. for 2 h. The solvent was evaporated
to dryness and after chromatographic purification (dry-flash, SiO2, eluent EA/MeOH = 8/2),
the desired product was isolated as an orange solid. Yield: 325 mg (42 %).
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
1859
3α-7α,12α-Triacetoxy-24-{N-{3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl}amino}-5β-
-cholan (21)
The alcohol 3α,7α,12α-triacetoxy-5β-cholan-24-ol (540 mg, 1.04 mmol) was dissolved
in DCM (50 mL), PCC (185 mg, 1.56 mmol) was added and the mixture was stirred at r.t. for
3.5 h. The reaction mixture was filtered through a short SiO2 column (eluent CH2Cl2/EA =
= 95/5). The obtained crude aldehyde (500 mg) was dissolved in MeOH (10.0 mL) followed
by 13 (303 mg, 0.96 mmol), and the mixture was stirred at r.t. After 12 h, NaBH4 (73 mg, 1.92
mmol) was added in one portion and stirring was continued at r.t. for 2 h. The solvent was
evaporated to dryness and after chromatographic purification (dry-flash, SiO2, eluent EA/
/MeOH = 8/2), product was isolated as an orange solid. Yield 230 mg (29 %).
N-[2-(1-Adamantyl)ethyl]-N'-(6-chloro-2-methoxyacridin-9-yl)ethane-1,2-diamine (22)
Into stirred mixture of 2-(1-adamantyl)acetaldehyde (140 mg, 0.86 mmol) and 12 (260
mg, 0.86 mmol) in abs. EtOH (10 mL), Ti(OPri)4 (381 µL, 1.29 mmol) was added dropwise.
After stirring for 1 h at r.t., NaBH4 (65 mg, 1.72 mmol) was added in one portion and when all
the aldehyde had been consumed, the reaction was quenched with 0.1 M NaOH (10mL). The
reaction mixture was extracted with CH2Cl2 (3×30 mL), the combined organic layers were
washed with brine and dried over anh. Na2SO4. The solvent was removed under reduced pres-
sure and the crude product was purified by dry-flash chromatography (SiO2, eluent hexane/EA =
= 1/1). Yield 40 mg (10 %).
N-[2-(1-Adamantyl)ethyl]-N'-(6-chloro-2-methoxyacridin-9-yl)propane-1,3-diamine (23)
According to the procedure described for 22, 2-(1-adamantyl)acetaldehyde (140 mg, 0.86
mmol) was transformed into 23 using 13 (271 mg, 0.86 mmol). The product was isolated after
dry-flash chromatography (SiO2, eluent EA/MeOH = 8/2). Brown–orange amorphous solid.
Yield: 190 mg (46 %).
N-Benzyl-N'-(6-chloro-2-methoxyacridin-9-yl)ethane-1,2-diamine (24)
To a stirred mixture of benzaldehyde (100 µL, 0.98 mmol) and 12 (312.0 mg, 1.03
mmol) in CH2Cl2 (15 mL), NaB(OAc)3H (313.0 mg, 1.47 mmol) was added. After stirring for
20 h at r.t., the reaction was quenched with 1 M NaOH (10 mL). The mixture was extracted
with CH2Cl2 (3×30 mL), the combined organic layers were washed with brine and dried over
anh. Na2SO4. The solvent was removed under reduced pressure and the crude product was
purified by dry-flash chromatography (SiO2, eluent EA/MeOH = 95/5). Yield 230.0 mg (60 %).
N-(6-Chloro-2-methoxyacridin-9-yl)ethane-1,2-diamine (12)57
A solution of 6,9-dichloro-2-methoxyacridine 11 (600 mg, 2.15 mmol) in 1,2-diamino-
ethane (10 mL, 140 mmol) was stirred at 70 °C under an Ar atmosphere. After 1 h, the mix-
ture was poured into ice cold water, the powder was filtered and dried under reduce pressure.
The product was obtained as a yellow powder and used in the next reaction step without
further purification. Yield 580 mg (89 %).
N-(6-Chloro-2-methoxy-acridin-9-yl)propane-1,3-diamine (13)57
According to procedure described for 12, 6,9-dichloro-2-methoxyacridine (1 g, 3.59
mmol) was transformed into 13 using 1,3-diaminopropane (20 mL, 232 mmol). The product
was obtained as a yellow powder and used in the next reaction step without further puri-
fication. Yield 1.08 mg (95 %).
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1860
TOT et al.
N-(6-Chloro-2-methoxyacridin-9-yl)pentane-1,5-diamine (14)49
According to procedure described for 12 6,9-dichloro-2-methoxyacridine (500 mg, 1.79
mmol) was transformed into 14 using 1,5-diaminopentane (13 mL, 11.3 g) at 80 °C. The
product was obtained as yellow powder and used in next reaction step without further puri-
fication.
N-(6-Chloro-2-methoxyacridin-9-yl)hexane-1,6-diamine (15)39
According to procedure described for 12 6,9-dichloro-2-methoxyacridine (1g, 3.59
mmol) was transformed in 15 using 1,6-diaminohexane (23 g, 197.9 mmol) at 90 °C. Product
was obtained as yellow powder and used in next reaction step without further purification.
In vitro BoNT LC metalloprotease activity
Determination of BoNT/A LC percent inhibition by SMNPIs was performed as
previously described.55
For BoNT/B LC percent inhibition, the HTS-assay utilizes a commercially available
fluorogenic substrate from List Biological Laboratories (Campbell, CA). Briefly, a multi-
channel pipette was used to add fluorescent substrate (final concentration 20 µM) and small
molecules (final concentration 20 µM) to a 96 well microplate. The reactions were initiated by
adding recombinant BoNT/B LC (final concentration 40 nM) to each well. All reactions were
conducted in a buffered solution consisting of 40 mM Hepes pH 7.2, 1 mM DTT and 100 µM
ZnCl2. The change in fluorescence intensity over time was continuously monitored in each
well using a 96 well plate fluorimeter (Saphire 2, Tecan, Männedorf, Switzerland). The assays
were run at 37 °C, quenched by the addition of TFA, and analyzed using reverse-phase HPLC.
Potential SMNPIs of the BoNT B LC were identified by comparing the reaction velocities
(change in fluorescence intensity over time) of samples assayed in the presence of compounds
versus control samples.
In vitro antimalarial activity and toxicity
The in vitro antimalarial drug susceptibility and toxicity screening were realized at the
Walter Reed Army Institute of Research.23
CONCLUSIONS
Aminoacridine derivatives possessing adamantane and steroid carriers were
prepared and tested for BoNT/A LC, BoNT/B LC, and antimalarial activity.
Acridines with the cholic acid-derived carrier showed good potency against both
the BoNT/A LC and BoNT/B LC. The inhibition of the BoNT/B LC by ca. 50 %
is the highest inhibitory activity of any acridine reported to date. The antimalarial
potency of adamantane acridines (IC in range 6–9 nM), combined with their
50
low potencies (IC ca. 3000 nM) is a good starting point for further research in
50
this area.
SUPPLEMENTARY MATERIAL
Spectral and analytical data of all the synthesized compounds are available electronically
at http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
Acknowledgements. This research was supported by National Institute of Allergy and
Infectious Diseases (USA) grant 5-U01AI082051-02, by the Ministry of Education, Science
and Technological Development of the Republic of Serbia (Grant No. 172008) and the
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AMINOACRIDINE DERIVATIVES AS INHIBITORS OF BOTULINUM NEUROTOXINS
1861
Serbian Academy of Sciences and Arts. Furthermore, for J.C.B., in compliance with SAIC-
-Frederick, Inc. contractual requirements: this project was funded in whole or in part with
federal funds from the National Cancer Institute, National Institutes of Health (USA), under
Contract No. HHSN261200800001E. The content of this publication does not necessarily
reflect the views or policies of the Department of Health and Human Services (USA), or the
US Army, nor does the mention of trade names, commercial products, or organizations imply
endorsement by the US Government, or the US Army. B.Š. is grateful to WRAIR for pro-
viding the in vitro antimalarial results.
И З В О Д
НОВИ ДЕРИВАТИ 9-АМИНОАКРИДИНА КАО ИНХИБИТОРИ БОТУЛИНУМ
НЕУРОТОКСИНА И P. falciparum ПАРАЗИТА МАЛАРИЈЕ
МИКЛОШ ТОТ1, ДЕЈАН М. ОПСЕНИЦА2, МИЛЕНА МИТРИЋ1, JAMES C. BURNETT3, LAURA GOMBA4,
SINA BAVARI5 и БОГДАН А. ШОЛАЈА1
1Хемијски факултет, Универзитет у Београду, Студентски трг 16, п. пр. 51, 11158 Београд,
2Институт за хемију, технологију и металургију, Његошева 12, 11000 Београд, 3SAIC-Frederick, Inc.,
Frederick National Laboratory for Cancer Research, P. O. Box B, Frederick, MD 21702, USA, 4United States
Army Medical Research Institute of Infectious Diseases, Department of Bacteriology, 1425 Porter Street,
Frederick, MD 21702, USA и 5United States Army Medical Research Institute of Infectious Diseases, Fort
Detrick, 1425 Porter Street, Frederick, MD 21702, USA
Синтетисани су деривати стероидних и адамантил-акридина и испитана је њихова
инхибиторна активност према ботулинум неуротоксинима (BoNT) и паразиту маларије.
Стероидни акридини показују добру инхибицију према кратком низу (LCs) BoNT/A и
BoNT/B. Остварена инхибиција BoNT/B LC од око 50 % је највша постигнута вредност
акридинских деривата према овом серотипу. Адамантил–акридински деривати су пока-
зали највећу антималаријску активност (IC50 у опсегу 6–9 nM, SI > 326), показујући да је
адамантил-група бољи носач фармакофоре у поређењу са стероидним, према овој инди-
кацији.
(Примљено 24. септембра, ревидирано 20. октобра 2013)
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