Antimalarial activity of 4-amidinoquinoline and 10-amidinobenzonaphthyridine derivatives

Article abstract of DOI:10.1021/jm501809x

Chloroquine (CQ) has been used as first line malaria therapeutic drug for decades. Emergence of CQ drug-resistant Plasmodium falciparum malaria throughout endemic areas of the world has limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic drug due to its being long-acting and having a high potency against blood stage P. falciparum (Pf). However, serious CNS toxicity of MQ has compromised its clinical value as a prophylaxis drug. Therefore, new and inexpensive antimalarial drugs with no cross-resistance to CQ or CNS toxicity are urgently needed to combat this deadly human disease. In this study, a series of new 4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-AMB) derivatives were designed, prepared, and assessed to search for new therapeutic agents to replace CQ and MQ. The new derivatives displayed high activity in vitro and in vivo, with no cross-resistance to CQ, and none were toxic in mice up to 160 mpk × 3. The best compound shows IC₅₀ < 1 ng/mL against D6, W2 and C235 Pf clones, low inhibitory activity in hERG K⁺ channel blockage testing, negativity in the Ames test, and 5/5 cure @ <15 mpk × 3 in mice infected with Plasmodium berghei. In addition to these desirable pharmacological profiles, compound 13b, one of the most active compounds, is metabolically stable in both human and mouse liver microsomal preparations and has a plasma t_1/2 of 50 h in mice, which made it a good MQ replacement candidate.

Full text of DOI:10.1021/jm501809x

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Article
Antimalarial Activity of 4-Amidinoquinoline and
10-Amidinobenzonaphthyridine Derivatives
Ai J Lin, Vasiliy N Korotchenko, Ramadas Sathunuru, Lucia Gerena,
Diana Caridha, Qigui Li, Mara KreishmanDeitrick, and Philip Smith
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501809x • Publication Date (Web): 05 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Antimalarial Activity of 4-Amidinoquinoline and 10-Amidinobenzonaphthyridine
Derivatives
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Vasiliy Korotchenko+, Ramadas Sathunuru+, Lucia Gerena, Diana Caridha, Qigui Li, Mara
Kreishman-Deitrick, Philip L. Smith and Ai J. Lin*, Division of Experimental Therapeutics,
Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland
20910.
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Abstract
Chloroquine (CQ) has been used as first line malaria therapeutic drug for decades. Emergence of
CQ drug-resistant Plasmodium falciparum malaria throughout endemic areas of the world has
limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic
drug, due to its long acting and high potency against blood stage Plasmodium falciparum (Pf).
However, serious CNS toxicity of MQ has compromised its clinical value as prophylaxis drug.
Therefore, new and inexpensive antimalarial drugs with no cross-resistance to CQ or CNS
toxicity are urgently needed to combat this deadly human disease. In this study, a series of new
4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-AMB) derivatives were
designed, prepared and assessed to search for new therapeutic agents to replace CQ and MQ.
The new derivatives displayed high activity in vitro and in vivo, with no cross-resistance to CQ,
and none were toxic in mice up to 160 mpk x 3. The best compound shows IC50 < 1 ng/mL
against D6, W2 and C235 Pf clones, low inhibitory activity in hERG K+ channel blockage test,
negative in Ames test and 5/5 cure @ < 15 mpk x 3 in mice infected with P. berghei. In addition
to these desirable pharmacological profiles, compound 13b, one of the most active compounds,
is metabolically stable in both human and mouse liver microsomal preparations and has plasma
t
1/2 of 50 hours in mice which made it a good MQ replacement candidate.
*Corresponding author: Tel 301-251-0762; e-Mail: aijeng@aol.com
+Equal contributions
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INTRODUCTION
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Chloroquine (CQ), one of the 4-aminoquinoline antimalarial agents, has been used as the
first line antimalarial drug since War World II.1-2 However, the wide spread of chloroquine-
resistant Plasmodium falciparum (Pf) malaria is severely limiting its therapeutic value to combat
this deadly human disease.3-5 Mefloquine (MQ) was commonly used as a malaria prophylactic
drug,6 but drug resistance, high cost and CNS toxicity have greatly compromised its clinical
value as an antimalarial drug. Thus, there remains an urgent need for new and inexpensive
antimalarial agents.7
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Chloroquine is believed to exert its activity by inhibiting hemozoin formation in the
digestive vacuoles of the malaria parasites,8-13 although some oppose this view14 and other
possibilities have also been postulated.15-17 O'Brien and Hahnla18-20 have offered a model to
account for the antimalarial activity of chloroquine and its congeners. They suggested that:
(1) these compounds exert their antimalarial effect by intercalation with the parasite DNA,
and that the activity of a given compound depends on the stability of its complex with DNA;
(2) high activity requires an electronegative group attached to the 7-position of the quinoline
ring; and (3) the diamino side chain attached to the 4-position of quinoline ring bridges the
two DNA strands by electrostatic interactions between the two side chain nitrogens and the
DNA phosphate groups.
Additionally, the potency of chloroquine to inhibit parasite growth is greatly enhanced by
its concentration within the parasites, possibly because of the high acidity in parasitic food
vacuoles and the basicity of CQ molecule, resulting in protonation of CQ and decreasing its
efflux from parasites.21 The cause of chloroquine resistance is unknown, but it is clearly
associated with alteration in membrane-associated transport processes causing a reduction of
drug uptake into parasites and/or an increased efflux of the drug out of the bugs.22-23
Existing structure-activity relationships of 4-aminoquinoline (4-AQ) antimalarials clearly
indicate that not only both side chain nitrogens (N2 and N3), but all 3 nitrogen atoms of the CQ
molecule play an important role in CQ activity and/or toxicity. Replacement of any one of the
three CQ nitrogen atoms with a carbon results in dramatic decrease or complete abolishment of
the antimalarial activity of compounds A, B and C.24-26 (Figure 1).
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Although large numbers of 4-aminoquinoline derivatives were prepared in search for
better CQ analogs during the past decades, the majority of the medicinal chemistry efforts
focused mainly on modifications of the chain length between the two amino groups (N2 and N3)
and the size of substituents on the terminal amino group (N3).27-30 Although some attempts have
been made to change the pKa of either ring nitrogen (N1) or 4-amino (N2) of CQ, no attempt has
been made to change the basicity of both N1 and N2 nitrogens of CQ at the same time.24 In this
study, a series of new 4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-
AMB) derivatives were designed, prepared and assessed for antimalarial activities in an attempt
to explore the effect of changing the N1 and N2 basicity to the antimalarial activities (Figure 2).
The main difference between the new 4-AMQ derivatives and CQ is that the 4-amino group of
CQ is replaced by an amidine (4-NHCR=NH) functional group. Through conjugation with the
amidine group, the basicity of ring nitrogen (N1) and 4-amino group (N2) of the new molecules
will be altered significantly as shown in Figure 3. Furthermore, the new 4-AMQ analogs add an
additional amino group (N4) which could provide a potential additional binding site to the drug’s
biological receptors, resulting in significant changes in pharmacological profiles from
chloroquine and its congeners. Another advantage of the new 4-amidino analogs over the CQ is
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that amidine is a stronger base than the 4-aminoquinoline.31-32 Higher basicity of amidines (N2
and N4) may also lead to more stable DNA intercalation, and/or render the new amidines better
inhibitors of hemozoin formation, both of which are believed to be responsible for CQ
antimalarial activity.
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Extending the same approach, a series of new 10-amidinobenzonaphthyridine derivatives
(10-AMB) was also prepared and tested in this study (Figure 2). The difference between 10-
AMB and the known antimalarial azacrin is that the 10-amino group of the latter is replaced with
an amidine function. Antimalarial activities of 10-aminobenzonaphthyridine derivatives, such as
azacrin and pyronaridine, have been described previously33-37 (Figure 4). Clinical use of azacrin
was abandoned because its efficacy was not superior to mepacrine or CQ.34b Pyronaridine has
been used clinically for treatment of CQ-resistant P. falciparum infections in Africa continent.35
However, pyronaridine is a Mannich base antimalarial with a benzonaphthyridine instead of a
quinoline ring, like those found in amodiaquine, amopyroquine, tebuquine and isotebuquine.38-42
Mannich base antimalarials have attracted vast attention in malaria chemotherapy for decades,
because they are more potent than and showed no cross-resistance to CQ. Nevertheless, the
clinical use of Mannich bases has been severely restricted because of chronic hepatoxicity and
agranulocytosis side effects associated with its long term use.43-45 Recent reports indicate the
amodiaquine is metabolized by cytochrome P-450 to form a reactive quinoneimine metabolite
with subsequent conjugation of the metabolite to glutathione or cysteinyl functions of
enzymes.46-48 The report suggests that alkylation of key macromolecules, such as DNA, RNA,
glutathione and enzymes, by the reactive quinoneimine metabolite is the possible mechanism of
action and/or toxicity of Mannich base antimalarials. The new 10-AMB derivatives described in
this study, while they do contain a benzonaphthyridine ring, are not Mannich base compounds
and thus are not likely to share the same hepatotoxicity of pyronaridine.
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CHEMISTRY
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4-Amidinoquinolines derivatives 6a-s were prepared according to the method shown in
Scheme 1. The key starting material, 4-amino-7-chloroquinoline (3), was prepared by two
approaches. The first approach involved heating 4,7-dichloroquinoline (1) with ammonia in
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0
phenol at 165 C to give compound 3 in 40% yields. The second approach used ammonium
carbonate/phenol as ammonia source to give the desired product 3 in > 90% yields. The
products prepared by both methods are identical in NMR and MS, but the second approach gave
much better yields than the first. N-(7-Chloroquinolin-4-yl)-acetamide (4) was prepared in 72%
yields by heating 4-amino-7-chloroquinoline (3) in acetic anhydride at 140 oC for 2 hrs. The key
intermediate, N-(7-chloroquinolin-4-yl)-ethanethioamide (5), was prepared by refluxing
acetamide 4 and Lawesson reagent [2,4-bis-(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane
2,4-disulfide]49 in toluene. Lawesson reagent is the most commonly used thiation reagent and is
available commercially. Initially, thiation of amide 4 failed to afford the thioacetamide 5a by
heating 4a and Lawesson’s reagent in benzene or THF. However, at higher temperature, thiation
of acetamide 4a with Lawesson reagent proceeded smoothly when the reaction was carried out in
boiling toluene to give 5a in 25 - 45% yields. Product 5a is not very stable at room temperature
and thus was used soon after it was prepared. The instability of the product 5a may account for
the low and variable yields of thiation reactions.
Two different catalysts, HgO and DCC (N, N’-dicyclohexylcarbodiimide), were used in
the preparation of the final products 6a-s (Table 1). Coupling reactions of thioacetamide 5 with
appropriate amines proceeded smoothly at room temperature under HgO catalysis to afford the
desired 4-amidinoquinoline derivatives 6 in 70 - 90 % yields. Without HgO as catalyst, the
thioacetamide failed to couple with amines even at elevated temperature. Due to high toxicity
of HgO, non-toxic DCC was employed as an alternative catalyst. The latter catalyst, however,
gave lower yields than HgO, due to tedious separation of the desired product from the thiourea
byproduct. Although HgO gave cleaner reactions and superior yields than DCC, the latter is a
preferred catalyst for the final step coupling reactions, in view of the strict FDA regulations on
residual heavy metals in foods and drugs.
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Scheme 1: Synthesis of 4ꢀAmidinoquinolines Derivatives
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Method A
R1
HN
O
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Cl
N
NH2
NH2
N
i
ii
.
iii
HCl
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Cl
Cl
N
Cl
Cl
N
1
2
3
4a R1 = ꢀCH
b R1 = -CH(3CH3)2
c R1 = -
iv
Ph
R1
HN
S
R1
NH
N
R2
v
Cl
N
Cl
N
5a R1 = ꢀCH3
6
b
c
R1 = ꢀCH(CH3)2
R1 = ꢀPh
Where R1 and R2 are defined in Table 1
Reagents and conditions:
i) Ammonium carbonate, Phenol, 110 0C, 3h; ii) 10% NaOH, CHCl3, 89.0 %; iii) Ac2O, reflux, 2h, 72.6%;
iv) Lawson's reagent, Toluene, 24h reflux, 60 %; v) Amine, HgO/DMF, Et3N, rt, 8h, 90%;
vi) Succinic acid, CH3CN/MeOH, rt, 3h, 74%.
The Method A for preparation of 5a was successfully applied to prepare (7-
chloroquinolin-4-yl)-thioisobutyramide (5b).
However, thiation of quinolin-4-yl-benzamide
(4c) using Lawesson’s reagent49 failed to give the corresponding thioamide 5c. Therefore, an
alternative Method B with shorter route and better yields was developed to prepare benzamidine
intermediates 7a-d and the final products 6 and 8 as shown in Scheme 2.
The Method B is a new and facile procedure to synthesize the target compounds with
wider choice of substituents at R1 position than Method A. In addition, Method B also avoided
the use of hazardous HgO or tedious DCC as catalyst. The approach started with N-arylation of
substituted amidines [R1-C(=NH)NH2] with 4,7-dichloroquinoline (1) to give 7a-d, followed by
N alkylation with appropriate N,N-disubstituted 3-chloropropylamines (Scheme-2, Method-B).
Copper-catalyzed arylation of amidines with aryl iodides as coupling partners was reported,
providing either arylated amidines50 or anilines51. The latter was formed as a result of hydrolysis
of amidines 6 or 7. However, with an active chloride, 4, 7-dichloroquinoline (1) does not
require copper catalyst for arylation reaction. In all coupling reactions, small amount
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of unreacted starting material 4, 7-dichloroquinine was recovered, and in some cases, formation
of 4-amino-7-chloroquinoline was observed, a result of base hydrolysis of the amidines formed.
At higher temperatures or longer reaction times, formation of multiple unknown side products
was observed. Treatment of 7a-d and N, N-disubstituted 3-chloropropylamines in anhydrous
DMSO and K2CO3 or NaH gave final products 6 and 8 in various yields, depending on amidine 7
and the ratio of the reagents used. On 1: 1 ratio of the reagents, the coupling reactions provided
predominantly product 6, whereas higher ratio of halide and amidine 7 yielded mainly the bis-
alkylation product 8. While alkylation of acetamidine 7a and isobutyrimidamide 7b preceded
smoothly using K2CO3 or NaH as base catalyst, alkylation of benzamidine (7c-d) required the
use of NaH; use of K2CO3 as catalyst provided poor yields of desired product 6. Since
compounds 6w and 6x contain an electron-withdrawing 4-chlorophenyl and pyridine ring,
respectively, they are more prone to be hydrolyzed under the strong base reaction conditions than
analogs 6t-v, resulting in lower yields of 6w-x than 6t-v.
N-alkylation of amidines 7a with N, N-diethyl-3-chloropropylamine can produce two
isomeric products with N-alkylation taking place at either terminal nitrogen (N1) to give 6b or
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Compound #
R1
R2
R3
H
H
H
H
H
Method % Yield
-CH3
-CH3
-CH3
-CH3
-CH3
-(CH2)2NEt2
-(CH2)3NEt2
-(CH2)4NEt2
-(CH2)5NEt2
81
90
86
92
72
6a
6b
6c
6d
6e
A
A and B
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A
A
A
-CH3
-CH3
-CH3
H
H
H
77
64
73
6f
6g
6h
A
A
A
-CH3
H
80
6i
A
-CH3
-CH3
H
H
H
H
H
85
74
87
87
94
71
6j
6k
6l
A
A
A
A
A
-CH3
-CH(CH3)2
-CH(CH3)2
6m
6n
-CH(CH3)2
-CH(CH3)2
-CH(CH3)2
-CH(CH3)2
-(CH2)2NEt2
-(CH2)3NEt2
-(CH2)4NEt2
H
H
H
H
6p
6q
6r
6s
A
A
A
A
98
70
-(CH2)3NMe2
-(CH2)3NEt2
H
H
H
H
H
B
B
B
B
B
50
50
49
9
6t
6u
6v
6w
6x
-(CH2)3NMe2
-(CH2)3NEt2
25
-CH3
[-(CH2)3NEt2]2
-(CH2)3NEt2
-
-(CH2)3NEt2
B
B
13
8
8a
8b
-(CH2)3NMe2
-(CH2)3NMe2
B
B
B
4
8c
8d
8e
12
+3
-(CH2)3NEt2
-(CH2)3NEt2
-(CH2)3NEt2
+14
*-(CH2)3NEt2
B
8f
+by-product of 6x synthesis; 2nd alkyl side chain on ring nitrogen.
*
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the substituted nitrogen (N2) to produce 6b’ (Scheme 3). Unambiguous identification of 6b
from 6b’ based on NMR data is difficult, due to similarity in NMR spectrum. To positively
confirm the structure of the alkylation product, both Method A and Method B were used to
prepare compound 6 as shown in Scheme 3. Method-A can provide only N1 –alkylation product
6b, but Method B produces either N1- or N2- alkylation product, 6b or 6b’. Since NMR and
LC/MS of the compounds prepared by both methods are identical, N-alkylation reaction of 7a
with N,N-diethyl-3-chloropropylamine at ratio of 1: 1 should have occurred predominantly at
terminal nitrogen N1 to yield 6b, not 6b’.
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As described above (Scheme 2), treatment of compound 7a with 3-chloro-N,N-
disubstituted- propylamine in 1: 1 ratio yielded desired product 6b in 80% yields, but a minor
bis-alkylation product 8a was also isolated in 13 % yields. Other bis-aminoalkyl analogs 8b-f
were, likewise, isolated from the synthesis of the corresponding benzamidine analogs 6t, 6u, 6v
and 6x, respectively (Table 1). When the ratio of N, N-disubstituted 3-chloropropylamines to
amidines 7 was increased to 2: 1 or higher, the N, N’-bis-alkylation products 8a-e were the major
products of the coupling reactions. Depending upon the substituent at R1 position, the follow-up
alkylation of 6 could take place at either amidine nitrogen (N1, N2) or ring nitrogen to give bis-
alkylation products 8a-f (Table 1). While compound 6b (R1 = -CH3) gave bis-alkylation product
8a with the first and the 2nd alkyl side chains attached at the same terminal nitrogen (N1) as in
8’, the second alkylation of the other 3 benzamidine analogs 6t-v (R1 = Ph) occurred at N2
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nitrogen as in 8” to give 8b-d, respectively. Compound 6x (R1 = 3-pyridyl), however, gave two
isomeric bis-alkylation products 8e and 8f with the 2nd aminoalkyl side chain located at N2 (8”)
1
and quinoline ring nitrogen (8’’’), respectively. Assignment of structures 8a-f was based on H
7
8
9
13
1
13
and C NMR spectra. There is only one set of H and C signals for both side chains of
compound 8a (8’); but both aminoalkyl chains of 8b-f display as two different sets of signals.
The difference in location of the 2nd aminoalkyl side chain of 8e and 8f was reflected in changes
in chemical shifts of 2-H and 3-H of quinoline ring. The chemical shift of 2-H was up field
shifted from 8.35 ppm for 8e to 7.01 ppm for 8f. Similarly, the proton at 3 position of 8e was up
field shifted from 6.12 ppm for 8e to 5.50 ppm for compound 8f.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4: Synthesis of Benzonaphthyridine Derivatives 12 and 13
R R
R1
N
R1
R1
2
2
Cl
R2
HN
N
N
N
N
N
H2N
N
N
OMe
OMe
N
OMe
N
OMe
i or ii
iii
+
Cl
N
Cl
Cl
Cl
N
11a-e
12a-f
12h-m
and
10
12g
R1
+
R3
R2
N
N
N
OMe
Cl
N
R R R
and 3 are defined in Table 2
2
Where
,
1
13a-b
Reagents and conditions:
(ii) R1C(=NH)NH2 × HCl, NaH, DMSO, rt, 68 h; (iii) RN(CH2)nCl, NaH, DMSO, 100 °C, 2ꢀ6 h.
(i) R1C(=NH)NH2 × HCl, K2CO3, DMSO, 120 °C, 4ꢀ24 h;
Method B used for the synthesis of 4-amidinoquinoline derivatives was adapted to
prepare 10-amidinobenzonaphthyridine analogs 12 and 13 as shown in Scheme 4. Like the
synthesis of 4-amidinoquinolines, treatment of 7,10-dichlorobenzonaphthyridine (10) with
amidines in dried DMSO using K2CO3 or NaH as catalyst gave good yields of amidines 11a-e,
which on treatment with N,N-disubstituted 3-chloropropylamine provided final products 12 and
13 in various yields and products distribution, depending upon the conditions and ratio of
starting materials and reagents used. Alkylation of amidine 11a-e gave predominantly mono-
alkylation products 12a-m with alkylation took place at the terminal nitrogen N1 of amidine plus
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Table 2: List of 10-Amidinobenzonaphthyridine Derivatives
Method
Compound #
12a
R1
-CH3
R2
-(CH2)3NMe2
R3
-H
% Yield
43
B
7
-CH3
-CH3
-(CH2)3NEt2
-H
-H
B
B
47
44
12b
12c
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-CH(CH3)2
-CH(CH3)2
-CH(CH3)2
-(CH2)3NEt2
-(CH2)3NMe2
-H
-H
-H
B
B
B
70
61
61
12d
12e
12f
-CH(CH3)2
-[(CH2)3NEt2]2
-(CH2)3NMe2
-
-H
B
B
8
51
12g
12h
-(CH2)3NEt2
-H
-H
-H
B
B
B
49
23
25
12i
12j
12k
-(CH2)3NEt2
-(CH2)3NMe2
-H
-H
B
B
28
63
12l
12m
-CH3
-CH3
-(CH2)3NMe2
-(CH2)3NEt2
-(CH2)3NMe2
-(CH2)3NEt2
B
B
*20
**11
13a
13b
* by-product of 12a synthesis, ** by-product of 12b synthesis
minor bis-alkylation products with which the 2nd alkylation occurred at either N1 or N2 nitrogen
of amidine to give 12g or 13, respectively. Like the quinoline analogs 8a-f, assignments of the
1
13
structures 12g and 13a-b were based on H and C NMR spectra. Compound 12g possesses
only one set of signals for both side chain protons, indicating both aminoalkyl side chains are
located on N1 amidine nitrogen. The protons and carbons of the two aliphatic chains of analogs
13a-b appear as two different sets of signals, indicative of different alkylation sites for both
alkylamino side chains as shown in Scheme 4. The new 10–amidinobenzonaphthyridine (10-
AMB) compounds are listed in Table 2.
RESULTS AND DISCUSSION:
Each of the two new classes of 4-amidinoquinoline (4-AMQ) and 10-amidinobenzo-
naphthyridine (10-AMB) antimalarial contains acetimidamide, isobutyrimidamide and
benzimidamide analogs (Figure 2, R1 = -CH3, -CH(CH3)2, -Ph, 4-Cl-Ph and 3-pyridyl). The
majorities of the 4-acetimidamidoquinoline derivatives (6b-l) are metabolically stable in both
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7
8
9
human and mouse microsomal preparations with t1/2 > 60 min, except 6a, 6e, 6g, and 6k.
However, among the 6 isobutyrimidamide analogs 6m-s, only compound 6q is metabolically
stable and all 5 benzimidamide analogs (6t-x) are metabolically unstable. Piperidine,
morpholine, and pyridine rings which are vulnerable to microsomal oxidation are partially
responsible for the metabolic instability of the new compounds 6e, 6g, and 6k. It is interesting
to note that compounds 8a-f with bis-alkyl side chains are metabolically stable. Steric hindrance
may play a role in reducing the susceptibility of 8a-f to microsomal hydroxylation and
subsequent degradation of the hydroxylated metabolites (Table 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Most of the new compounds exhibited moderate to potent cell growth inhibition in both
CQ sensitive (D6) and CQ resistant P. falciparum clones (W-2 and C-235) with IC50 <100
ng/mL, except 6g, 6k, 8e and 8f which contain morpholine or pyridine rings. Benzimidamide
derivatives (6t-w) are more active than the isobutyrimidamide and acetimidamide analogs. The
IC50s of the most active compounds, 6t and 6u, are less than 1 ng/mL against the CQ sensitive
clone D6, with a W2/D6 ratio of 4. This is much improved compared to that of CQ, which has a
W2/D6 ratio of 35, suggesting that compounds of this series have little or no cross resistance to
chloroquine. Against the CQ resistant clone W-2, compounds 6t and 6u are 50-60 folds more
active than CQ with IC50s of 3.4 and 2.8 ng/mL, respectively. Furthermore, the 4-chlorophenyl
analog 6w is equally potent against both CQ sensitive and CQ resistant cell lines, D6, W2 and
C235, with IC50s of 1.1, 1.5 and 0.59 ng/mL, respectively. Although metabolically stable and
highly active against the CQ sensitive cell line D6, compounds 8a-d with bis-aminoalkyl side
chains showed significant CQ cross resistance with W2/D6 ratio of 8 – 17.
Table 3: In vitro Biological Activities of 4-Amidinoquinoline Derivatives
P. falciparum IC : ng/mL(nM)
HepG2
IC
Met. Stab.
(t , min)
Clog-P *hERG
%@
50
#
1/2
50
10 ꢀM
(ng/mL)
12020
1322
D6
W2
C235
W2/D6
35
*HLM *MLM
4.8 (15)
60 (189)
46 (138)
8.6 (24.9)
45 (125)
8.6 (25)
22.3 (62)
168 (525)
94 (296)
75 (226)
34 (98.3)
164 (455)
12.5 (36)
45 (126)
66 (206)
58 (182)
44 (133)
9.0 (26)
63 (175)
4.9 (14)
23 (64)
>60
26
>60
18
3.73
2.94
3.12
3.53
3.96
3.14
3.66
71
#NA
NA
CQ
6a
6b
6c
6d
6e
6f
1.6
1.63
3.95
3.64
1.45
2.02
>10000 >60
>60
>60
>60
25
2683
12469
23273
9814
>60
56
NA
NA
>60
>60
27.67
NA
>60
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7
8
9
113 (327)
24 (76)
102 (295)
87 (275)
57 (166)
78 (199)
200 (617)
200 (578)
28 (89)
0.9
3.6
>30300
25
22
2.07
2.24
NA
NA
6g
6h
>30300 >60
>60
37 (108)
22 (56)
46 (134)
34 (87)
1.54
3.54
0.67
5.3
5710
8072
>60
>60
47
>60
>60
13
3.01
3.96
3.02
3.74
4.38
4.90
4.18
4.24
4.76
5.24
4.06
4.77
4.91
4.66
3.56
3.97
5.82
4.39
6.09
4.60
5.09
NA
NA
NA
NA
74.5
33
6i
6j
298 (920)
233 (719)
25.9 (64)
21 (56)
11270
6726
6k
6l
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14.8 (36.4) 78.6 (193)
>60
29
>60
43
12.7 (34)
2.9 (7.5)
18.6 (54)
7.9 (22)
34 (91)
7.2 (18.6)
33.7 (97)
15.3 (42)
95.2 (254)
24.7 (58.7)
3.4 (9.2)
2.7
>24242
5715
6m
6n
6p
6q
6r
6s
4.7 (12)
2.5
29
31
18 (52)
1.8
12699
>60
12
NA
NA
54.6
51.8
90.6
100
93
9.3 (25.8)
47 (125)
1.93
4.14
3.92
4.3
>24242 >60
>60
42
23 (61)
5008
5154
5130
5085
5123
1926
7867
3002
4092
5773
2449
2944
14106
55
25
6.3 (15)
13 (30.9)
1.25 (3.4)
1.3 (3.3)
30
0.79 (2.1)
0.71 (1.8)
2.02 (5.2)
1.07 (2.0)
>60
21
52
6t
2.84 (7.2)
6.4 (15.7)
1.5 (2.9)
4
16
6u
6v
6w
6x
8a
8b
8c
8d
8e
8f
2.3 (5.7)
3.2
12
18
0.59 (1.1)
31.5 (79.5)
15.1 (33.9)
17.1 (33.7)
20.5 (45.4)
3.7 (6.95)
147.5 (290)
1.4
48
35
115
101
16.2
21.9
16.4
21.7
NA
NA
15.4 (38.9) 80.4 (203)
5.22
7.94
12.5
17
36
>60
>60
>60
>60
>60
>60
6.8 (15)
3.1 (6.1)
3.2 (7.1)
1.0 (1.9)
54 (121)
39 (76.8)
54.7 (121)
8.5 (16)
>60
>60
>60
31
8.5
13.7 (34.8) 195 (383)
14
>60
>60
984 (2497) 1994 (3917) 1356 (2664)
2.0
>60
#
*HLM = human liver microsomes, MLM = mouse liver microsomes; NA = not tested
Among the 4-amidinoquinolines, the in vitro activity and Clog-P of the new compounds
appear to correlate with the side chain length of the molecules. As the side chain length of the 4-
AMQ increases from compounds 6a (Scheme 1, n = 2), to 6b (n = 3) and 6c (n = 4), the
lipophilicity increases and so does their cell growth inhibitory activity (Table 3). However, as
the chain length of 6c (n = 4) increases further to 6d (n = 5), the Clog-P increases from 3.58 to
3.99, and its activity decreases more than 5 fold. Thus, it appears that the optimal side chain
length for activity of this 4-AMQ is n = 3 – 4.
To further explore the relationship of
lipophilicity and antimalarial activity, a series of new analogs with side chain length fixed at n =
3 and the methyl group of 6a-l (R1 = -CH3 in Scheme 1 and 2) being replaced with isopropyl
(6m-s), phenyl (6t-w and 8c-d) or pyridyl (6x and 8e-f) groups were prepared and evaluated.
The results confirm the previous observation that lipophilicity plays an important role in
antimalarial activity (Table 3). Notably, replacement of the piperidine group of 6e with a
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morpholine ring resulted in analog 6g which has a lower Clog-P as well as diminished
antimalarial activity. Compounds 6g-i, and 6k with low Clog-P (< 3), showed weak in vitro
antimalarial activity (IC50 > 100 ng/mL). When the side chain length of the isopropyl and
phenyl analogs (6m-6x) are fixed at n = 3, the relationships of Clog-P and activity of methyl
(6b), isopropyl (6q), and phenyl (6u) analogs are rather clear and straightforward. As the methyl
group of 6b (R1 = -Me) was replaced with a larger isopropyl [6q, R1 = -CH(CH3)2] or phenyl
(6u, R1 = Ph) group, Clog-P increased substantially from 3.12 to 4.24 and 4.77 for 6b, 6q and 6u,
respectively. Parallel to Clog-P, antimalarial activity of the three analogs increased 6 fold from
methyl 6b to isopropyl 6q analog and 11 fold from isopropyl 6q to phenyl 6u analogs.
Introduction of 4-Cl to the phenyl ring resulted in 4-chlorophenyl analog 6w which is equally
active to phenyl analog 6u against CQ sensitive D6, but is almost twice as active as 6u against
CQ resistant W-2 and C235 clones. However, when phenyl group of 6u was replaced with a 3-
pyridyl ring, the Clog-P and activity of the resulting pyridine analog 6x decrease simultaneously.
Despite the fact that compounds with bis-aminoalkyl side chains 8a-c show good activity against
D-6 with IC50 < 10 ng/ml, they are much less active against W-2 clone with IC50 > 40 ng/ml,
indicating cross-resistance to CQ. The pyridyl analog 8f, in which the 2nd aminoalkyl side chain
is attached to the ring nitrogen, loses nearly all of its antimalarial activity. The result is of no
surprise from the SAR point of view, since the aminoalkyl side chain of CQ is attached to the 4-
amino group, not on ring nitrogen. Nevertheless, it is a surprise to note that compound 6k, an
analog of 6a with the terminal amino group being replaced with a pyridine ring nitrogen, instead
of disubstituted amino group, shows moderate growth inhibitory activity against both CQ-
sensitive and CQ-resistant Pf clones with IC50 in the range of 200 - 300 ng/ml, making it a
potential new lead warranting further exploration.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4: In vitro Biological Activities of Benzonaphthyridine Derivatives
HepG2
IC50
(ng/ml)
P. falciparum
IC50: ng/mL (nM)
Metab. Stab.
(t1/2, min)
W2/D6 *HLM *MLM
Clog-P hERG
%@
#
D6
CQ 4.8 (15)
W-2
C235
10 ꢀM
71
168 (525)
66 (206)
21 (51.3)
11 (26.6)
7.9 (13.1)
1.67 (3.8)
1.78 (4.3)
35
8.5
2.3
3.1
4.3
4.4
>60
>60
33
>60
42
29
15
22
46
3.73
3.91
3.93
4.07
5.14
4.43
10.7 (26) 41 (100)
7.3 (17.6) 17 (41)
18052
2057
2809
2063
3562
91.45
100
12a
12b
12c
12d
12e
4.8 (8.0)
0.9 (2.0)
0.9 (2.2)
15 (24.9)
3.9 (8.8)
4 (9.7)
14
94.55
86.53
72.12
20
33
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8
9
0.5 (1.1) 1.8 (3.96) 0.88 (1.94)
0.62 (1.1) 3.4 (6.1) 1.8 (3.2)
0.8 (1.8) 0.9 (1.98) 0.78 (1.7)
3.6
5.7
9
>60
18
16
9
8
>60
44
26
12
29
2007
668
5.28
6.2
69.61
32.46
48.89
NA
12f
12g
12h
12i
1.1
3.4
5778
3699
2564
8603
5.04
5.75
5.89
4.54
0.8 (1.7)
2.6 (5.1)
3.5 (7.2)
2.7 (5.6)
1.6 (3.3)
10 (20.6)
1.34 (2.8)
2.2 (4.3)
4.00 (8.2)
0.62
2.9
80
63
12j
12k
32
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.3 (2.8)
3.2 (6.6)
2.1 (4.3)
15 (29.7)
24 (51)
2.4
2.7
6.9
7.3
32
>60
>60
>60
>60
2510
>24242
1696
5.65
6.73
3.52
4.94
77
10
12l
12m
13a
13b
11 (21.8) 30.8 (61)
5.5 (11.7) 38 (80.8)
>60
>60
>60
18.26
13.78
0.7 (1.3)
5.1 (9.5)
2.7 (5.1)
3045
*HLM = human liver microsomes, MLM = mouse liver microsomes.
A relationship between lipophilicity and antimalarial activity exists not only in the 4-AMQ
class, but also among 10-AMB derivatives. In general, 10-AMB derivatives are more lipophilic
and more active than 4-AMQ derivatives in tests against Pf clones. In comparison to 4-A
MQ derivative 6b, the corresponding 10-AMB analog 12b is 6.3 times more active and the Clog-
P is also more than one log higher than 6b. The same relationship also exists between 6q and
12d, with the latter about 8 fold more active than the former and more than a log higher in Clog-
P. The relationship between Clog-P and activity appears to diminish when the R1 is a phenyl
group, as in the case of 6u and 12i. Even though Clog-P of 12i is still a log higher than 6u, the in
vitro activities of both compounds are about equal with IC50 ~ 0.8 ng/mL. Figure 5 shows the
correlation between Clog-P of 10-AMB derivatives and their in vitro antimalarial activity (IC50)
against Pf clones, W2, D6 and C235. As the Clog-P of the 10-AMB analogs increases, so does
their antimalarial activity (smaller IC50) until an optimal Clog-P value is reached around 5 – 5.5,
at which point the compounds show maximal antimalarial activity and minimum CQ cross-
resistance.
Five of the methyl analogs (R1 = -Me) of 4-AMQ (6c, 6e, 6f, 6i, and 6j) with good
metabolic stability (t1/2 > 60 min) and potent in vitro activity were selected for assessment in
mice infected with CQ resistant P. berghei-infected erythrocytes (ANKA strain). Chloroquine
was used as a positive control which gave 5/5 cures at 160 MPK (milligram per kilogram). The
results are shown in Table 5. All 5 test compounds are active at 160 mg/kg x 3, po. The treated
mice were parasite free 2-4 days after treatment, except 6e which did not completely clear
parasites during the treatment, but showed 95.4% parasitemia reduction 2 days after the
treatment. None of the five methyl analogs cured mice at 160 mpk, but the treated mice stayed
parasite free for 5 to 7 days before recrudescence took place. Among the 5 compounds tested,
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compounds 6c, 6f, 6i and 6j are metabolically stable with t1/2 > 60 minutes and showed
comparable in vivo and in vitro antimalarial activity. The 5th compound 6e is the most active
(IC50, 8.6 ng/mL) among the five compounds tested, but is the least active in the Thompson test
and the least stable metabolically. The poor metabolic stability of 6e in mice may have led to
shorter plasma half-life and consequently weaker in vivo activity than analogs 6c, 6f, 6i and 6j.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5: Correlations of Clog-P and In vitro Antimalarial Activities of 10-AMB
50.00
D6
W2
C235
W2
40.00
Poly. (D6)
Poly. (W2)
Poly. (C235)
30.00
D6
D6:
y = 6.3232x2 - 66.54x + 176.58
R² = 0.9214
C235
W2
W2:
y = 23.486x2 - 247.25x + 651.47
R² = 0.9642
W2
20.00
D6
C235:
y = 12.746x2 - 133.36x + 349.56
R² = 0.9675
C235
10.00
W2
W2
C235
D6
D6
W2
W2
D6
C235
W2
C235
C235
D6
C235
W2
C235
W2
D6
D6
C235
D6
C235
D6
D6
0.00
3
3.5
4
4.5
5
5.5
6
6.5
CLogP
Five isopropyl analogs 6m, 6n, 6p, 6q, and 6r [R1 = -CH(CH3)2] were also selected for
assessment in mice. All showed better efficacy than methyl analogs 6c, 6e, 6f, 6i and 6j with
cures at 160 mpk x 3, po, although the in vitro activity of these isopropyl analogs are at best
comparable to that of the above mentioned 5 methyl analogs. Since Clog-P values (> 4.2) of
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isopropyl analogs are more than one log higher than the average methyl analogs tested (Table 3),
positive correlations between Clog-P and in vivo activity in mice are apparent. However, no
correlation between the metabolic stability and in vivo antimalarial activity was observed.
Metabolically more stable compounds are expected to maintain longer plasma half-life and thus
should be more active than those with shorter plasma half-life. However, the data in Tables 5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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32
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38
39
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50
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60
Table 5: Antimalarial Activity of New Compounds in Mice Infected with P. berghei
Log-P Met. Stab (min)
(mpk x 3)
Compd. Oral Dose
#
Results
# Mice (Day Died)
Remark
*HLM
>60
*MLM
>60
5/5 (D14)
160
3.53
3.14
active
6c
6e
2/5 (D10), 2/5 (D12),
1/5 (D17)
2/5 (D14), 2/5 (D17), 1/5 (D21)
160
>60
25
suppressive
160
160
160
160
120
3.66
3.01
3.96
4.38
4.90
>60
>60
>60
29
>60
>60
>60
43
active
active
active
cure
6f
6i
17 (3/5), 19 (1/5), 21 (1/5)
4/5 (D17), 1/5 (D19)
3/5C, 1/5 (D19), 1/5 (D21)
1/5C, 4/5 (D18)
6j
6m
6n
29
31
cure
2/5C, 3/5 (D7)
4/5C, 1/5 (D24)
160
160
160
120
4.18
4.24
4.76
>60
>60
55
12
>60
42
cure/toxic
cure
cure
6p
6q
5/5C
6r
1/5 (D5), 1/5 (D19), 1/5 (20),
1/5 (D24), 1/5 (D26)
12a
3.91
>60
42
active
1/5 (D4), 1/5 (D6), 1/5 (D10),
1/5 (D14), 1/5 (D21)
120
120
12b
3.93
4.07
33
14
29
15
active
cure
2/5C, 1/5 (D19),
2/5 (D21)
12c
12h
4/5C, 1/5 (D5)
5/5C
120
60
5.04
18
18
9
44
44
12
cure
cure
cure
5/5C
120
5.85
5.65
12j
12l
5/5C
5/5C
5/5C
60
15
60
15
32
32
>60
>60
>60
>60
cure
cure
cure
cure
6.73
6.73
>60
>60
12m
13b
1/5C, 1/5 (D19), 1/5 (D24), 1/5
(D25), 1/5 (D26)
5/5C
120
60
4.94
3.73
>60
>60
>60
>60
cure
cure
4/5C, 1/5 (D24)
5/5C
160
0
>60
>60
valid
valid
CQ
2/5 (D6), 2/5 (D7), 1/5 (D11)
Vehicle
*HLM = human liver microsomes, MLM = mouse liver microsomes.
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showed that metabolically stable methyl analogs 6c, 6e, 6f, 6i and 6j displayed much weaker in
vivo activity than the metabolically less stable isopropyl analogs 6m, 6n, 6p, 6q, and 6r,
implying that metabolites may also contribute to the observed antimalarial activity. 10-
Amidinobenzonaphthyridines (10-AMB) are higher in Clog-P and more active in vitro and in
vivo than the corresponding 4-amidinoquinoline (4-AMQ) derivatives. However, the former are
generally less stable metabolically than the latter, except compounds with branched aminoalkyl
(12m) and bis-aminoalkyl side chains (12g, 13a and 13b). This observation indicates that steric
hindrance may play a role in reducing the susceptibility of 12m, 12g, 13a and 13b to microsomal
hydroxylation and subsequent degradation of the hydroxylated metabolites.
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A clear correlation between Clog-P and in vivo antimalarial activities exists not only
among 4-AMQ derivatives, but also among the 10-AMB analogs. As shown in Table 5, none of
the compounds with Clog-P < 4 showed cure in the Thompson test and all compounds with
Clog-P > 4 showed cures at 15, 60, 120 or 160 mpk x 3 by oral dosing, and this is the case for
both 4-AMQ or 10-AMB derivatives. Like 4-AMQ, the metabolic stability of the 10-AMB
derivatives does not correlate with their antimalarial activity, suggesting again that metabolites
may be, at least partially, responsible for the observed antimalarial activity.
One of the major goals of this study is to search for a potent, safe and long acting
antimalarial compound to replace mefloquine as malaria prophylactic drug. Among the 10-AMB
analogs, compound 13b showed potent in vitro (IC50 < 1 ng/mL) and in vivo activities (TT: 4/5c
at 60 mpk), good metabolic stability (t1/2 >60 min.) and a very long plasma half-life (t1/2 ~ 50 hr)
in mouse PK studies (Table 6). Furthermore, compound 13b also showed weak inhibitory
activity in hERG (the human Ether-à-go-go-Related Gene) K+ channel blockage test and was
negative in an Ames mutagenicity study (data not shown, assays performed by WuXi App Tech,
Inc., St. Paul, MN; see also further discussion below on hERG inhibition testing). The results
indicate that 13b is a promising potential candidate to replace mefloquine.
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Table 6: Compounds 6q and 13b PK parameters after single PO at 80 mg/kg in mice
plasma
6
Compound Regimen Half-life Tmax Cmax
AUClast AUCINF_obs Vz/F_obs Cl/F_obs MRTlast
7
8
9
#
(hr)
6.74
6.58
6.66
0.118
1.8
(hr) (ng/ml) (hr*ng/ml) (hr*ng/ml) (ml/kg) (ml/hr/kg) (hr)
oral-1
oral-2
Mean
SD
0.5 695.7 3805.73
0.5 696.8 3222.6
0.5 696.3 3514.2
3916.64
3315.58
198725 20425.7
228918 24128.5
7.73
7.55
6q
10
11
12
13
14
15
16
17
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22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
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60
3616.107 213822 22277.1 7.641
425.014 21349.7 2618.31 0.133
0
0
0.778 412.332
0.1 11.7
CV%
11.8
10
11.8
1.7
oral-1
oral-2
Mean
SD
40.6
54.43
47.515
9.785
20.6
2
2
2
0
0
279.3 5815.35
163.8 4043.45
221.6 4929.4
8374.75
9517.05
559467 9552.53 23.65
660128 8405.97 20.61
13b
8945.898 609797 8979.25 22.127
81.67 1252.923 807.727 71177.5 810.738 2.149
36.9 25.4 11.7 9.7
CV%
9
9
The hERG potassium channels are essential for normal electrical activity in the heart.
Inherited mutations in the hERG gene cause long QT syndrome, a disorder that predisposes
individuals to life-threatening arrhythmias. Arrhythmia can also be induced by blockage of
hERG channels by a surprisingly diverse group of drugs 52-53, including antimalarial agents, such
54-56
57
and Halofantrine . This side effect is a common reason for drug failure in
as quinidine
58
preclinical safety trials. Chloroquine and mefloquine
59-60
also exhibited the same cardiac
toxicity as quinidine, but to a lesser extent. Selected 4-AMQ and 10-AMB analogs were
submitted for hERG channel inhibition test and the results are shown in Tables 3 and 4. Among
the fourteen 4-AMQ derivatives tested, the mono-alkylated analogs [R1 = -Me or –CH(CH3)2, 6e,
6m, 6n, 6q, and 6r] showed comparable to or weaker inhibition than CQ at 10 ꢀM
concentration. While all 5 aromatic analogs (R1 = Ph or pyridyl, 6t, 6u, 6v, 6w, and 6x), showed
higher hERG inhibitory activity than CQ, another four analogs with bis-alkyl side chains, 8a-d,
displayed much weaker hERG inhibitory activity (6 – 20 %) than chloroquine (71 [Tables 3 and
4]. At the same concentration, mefloquine showed 95%, whereas Halofantrine and quinidine
showed 100% inhibition. Likewise, among the 10-AMB derivatives tested, mono-alkylated
analogs (12a-c and 12h) have stronger proarrhythmic potential than CQ, while bis-alkylated
compounds (12g, 12k, 13a and 13b) displayed much weaker potential than CQ. Bis-alkylated
analogs of 4-AMQ and 10-AMB are not only much less toxic in the hERG test, but are also more
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active than mono-alkylated analogs. Furthermore, the new compounds showed no in vitro
toxicity in HepG2 (human hepatocellular carcinoma) with IC50 > 2000 ng/mL and no host
toxicity in mice up to 160 mpk/day x 3 days. More encouraging discovery on toxicity of the
amidine analogs is that all 4 selected compounds (6t, 6w, 12l and 13b) are negative in Ames
mutagenicity test performed by WuXi App Tech, Inc., St. Paul, MN.
10
11
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Rationale for the preparation of amidine analogs in this study, as stated in the
introductory section of this report, is that replacement of the 4-amino group of CQ with an
amidine function will alter or enhance the basicity of both ring nitrogen (N1) and the 4-amino
group (N2) of CQ, resulting in changes in pharmacological profiles and antimalarial activities of
the new amidine analogs (Figure 3). As was postulated, the pKa of the ring nitrogen (N1) and the
4-amino (N2) or 10-amino (N2) groups of the amidine analogs are much higher than that of CQ
(Table 7). However, no or little changes in pKa (9.27 – 10.32) of the disubstituted aminoalkyl
side chain (N3) of CQ and the amidine analogs were observed and the side chain amino groups
(N3) remain the most basic nitrogen in CQ and all amidine analogs; thus, the results ruled out
the possibility that pKa change of amidine analogs is the primary cause of their better
antimalarial activities than CQ against the CQ-resistant P. falciparum clone (W2). On the other
hand, the pKa of the amino group at the 4-position of 4-AMQ and 10-position of 10-AMB are
substantially higher than that of the 4-amino group of CQ, from -5.56 for CQ to average of 8 and
6 for 4-AMQ and 10-AMB, respectively. The results indicate a clear correlation between pKa of
the 4- or 10-amino group of the amidine analogs and their antimalarial activity against CQ
resistant cell line. While there is little change in pKa of the 10-AMB and CQ ring nitrogen,
substantial differences in pKa of the ring nitrogen between 4-AMQ and CQ were observed. The
relationship between the observed differences in pKa of the ring nitrogen between 4-AMQ and
10-AMB and their antimalarial activity is not clear.
The mechanism of action of CQ has been the subject of extensive studies in many
laboratories around the world. The literature reports on this subject are complex and far from
clear,8-26 including DNA intercalation, drug accumulation in the food vacuole of the parasites,
inhibition of hemozoin formation or PfCRT (P. falciparum Chloroquine-Resistance Transporter).
Since all 3 amino groups of CQ are essential for its antimalarial activity, alteration of pKa of N1
and N2 of amidine analogs will result in alteration of binding affinity of the new analogs to the
listed targets and change the antimalarial activity. The antimalarial activity results confirm our
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postulation. However, it is not the intention of this paper to determine the mechanism of action
of this new class of antimalarial which is in progress and the results will be reported elsewhere.
Table 7: *pKa, Clog-P and in Vitro Activity of Selected Compounds
7
8
9
Compd
Structure and pKa of Amino groups
Clog-P
IC50 (ng/ml)
W2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
D6
4.8
RI=
W2/D6
35
CQ
3.73
168
75
2.43
N
10.12
NEt2
7.88
N
9.53
NEt2
9.69
NEt2
3.00
46
1.6
N
HN
8.67
N
N
H
6b
Cl
N
H
4.07
Cl
N
5.35
Cl
N
1.77
4.24
4.77
7.9
0.7
15.3
2.8
1.9
4
6q
6u
4.22
5.14
7.3
0.9
17.0
3.9
2.3
4.3
12b
12d
5.75
4.94
0.8
0.7
2.7
5.1
3.4
7.3
12i
13b
*pKa and Clog-P values were calculated using physico-chemical property predictors
from ChemAxon’s Calculators and Calculator Plugins, ChemAxon, Cambridge, MA.
http://www.chemaxon.com/products/calculator-plugins/property-predictors/
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CONCLUSION
5
6
7
8
9
4-AMQ and 10-AMB derivatives prepared in this study represent two new classes of
antimalarial agents with potent in vitro growth inhibitory activity against CQ resistant Pf clones
W-2 and C235 and demonstrated cures in mice infected with P. berghei. The high activity of the
new amidine analogs against CQ resistant parasites correlates well with the increase in pKa of
the nitrogen at 4- or 10-position of the 4-AMQ and 10-AMB. Further, the results also indicate a
positive correlation between the Clog-p and the antimalarial activity of the new compounds
10
11
12
13
14
15
16
17
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22
23
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32
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59
60
prepared in this study.
No overt toxicity was observed in HepG2 culture or in mice.
Preliminary data from hERG channel blockage testing also showed much weaker hERG
inhibitory activity than CQ and are negative in Ames test, indicating analogs of this new class
have good potential to be developed as CQ replacement drugs to combat malaria infection.
Furthermore, compound 13b, a 10-AMB analog with bis-aminoalkyl side chains, showed not
only potent activity in mice, low in vitro and in vivo toxicity, negative in Ames test and also
possessed a long plasma half-life of about 50 hours in mouse PK studies, suggesting it could be a
good potential candidate to replace mefloquine as a weekly or biweekly malaria prophylactic
drug.
EXPERIMENTAL SECTION
Melting points were determined in open capillary tubes on an OptiMelt melting point
1
apparatus (Standard Research Systems, USA) and are uncorrected. H NMR and 13C NMR
spectra were recorded using Bruker Avance-600 and Bruker Avance 300 spectrometers (Bruker
Instrument, Inc., Wilmington, DE). Chemical shifts are given in ppm (δ) relative to
tetramethylsilane (TMS) as internal standard. Analytical thin layer chromatography (TLC) was
performed using HPLC-HLF normal phase 150 ꢀm silica gel plates (Analtech, Newark, DE).
Visualization of the developed chromatogram was performed with UV254 lamp or iodine
chamber. Flash chromatography was conducted with silica gel 60, 0.060-0.2mm (70-230 mesh)
from Sigma-Aldrich Co. Solvents and reagents obtained from commercial sources were used
without purification unless noted. Reactions were carried out under an inert atmosphere of
nitrogen. Elemental analysis was performed by Atlantic Microlab, Inc. (Norcross, GA). Where
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analyses are indicated by symbols of the elements, the analytical results obtained were within
0.4% of the theoretical values. An LC/UV – Vis/Trap MS was employed for purity analysis and
chromophore properties. The system consisted of an Agilent 1100 series LC-UV/VIS system
online with a ThermoFinnigan (now Thermo-Fisher Scientific, Waltham, MA) LCQ MS
equipped with electro spray ionization (ESI) source. Samples were analyzed using shallow
10
11
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13
14
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CH3CN:1% HCOOH/H2O gradients (pH 2.5) at low rate. The purity of the final products is
95%. Log- P values are obtained from ChemDraw Ultra software version 12.0.
A. Chemistry
Synthesis of 4-Amino-7-Chloroquinoline (3)61: 4,7-Dichloroquinoline (10.0g, 50 mmol)
was dissolved in phenol (47.51g, 0.5 mol.) with stirring and heating at 110.0C. Ammonium
carbonate (24.2g, 0.25 mol.) was added in portions as rapidly as the frothing would permit.
After a further 2hr at 165 0C, the mixture was cooled and macerated with diethyl ether (500mL).
The crystalline white HCl salt compound 2 was collected, washed with ether, and dried. The
free amine was obtained by shaking a suspension of 2 (HCl salt) for an hr in an excess of 10%
NaOH solution. The free amine 3 was collected and recrystallized from EtOH to give 8.0 g
(89.0%) of the desired product as bright white needles, mp 148 0C (lit 62, mp 148.5-149.5 0C). 1H
NMR (300 MHz, CD3OD): δ 6.63 (d, J = 5.4 Hz, 1 H), 7.41 (dd, J = 9.0, and 2.1 Hz, 1 H), 7.79
(d, J = 2.1, 1 H), 8.09 (d, J = 9.0, 1 H), 8.29 (d, J = 5.4 Hz, 1 H). 13C NMR (75 MHz, CD3OD):
δ 102.51, 116.92, 123.51, 124.36, 126.02, 135.07, 148.61, 150.54, 152.90. MS (ESI): m/z 179.09
[M + 1] +.
Synthesis of N-(7-chloroquinolin-4-yl)-acetamide (4a): A solution of 4-amin-7-
chloroquinoline (3) (5.0 g, 28 mmol) in acetic anhydride (20 mL) was refluxed for 2h. After
cooling to rt, the reaction mixture was treated with brine to precipitate the product, which was
filtered off and re-dissolved in water. The aqueous solution was basified with 10% NaOH and
extracted with EtOAc (50 mL x 3). The extracts were combined, dried over anhydrous Na2SO4
0
and evaporated to afford 3.58g (72.6 %) of the desired product as white crystals, mp, 195.6 C
(lit 63, mp 195 0C). 1H NMR (600 MHz, CD3OD): δ 2.34 (s, 3 H), 7.62 (dd, J = 9.0 and 2.4 Hz, 1
H), 8.01 (d, J = 2.4 Hz, 1 H), 8.18 (d, J = 4.8 Hz, 1 H), 8.27 (d, J = 9.0 Hz, 1 H), 8.77 (d, J =
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13
4.8 Hz, 1 H); C NMR (150 MHz, CD3OD): δ 24.32, 113.86, 121.08, 124.90, 128.36, 128.56,
137.03, 144.11, 150.23, 153.08, 172.72. MS (ESI): m/z 221.10 [M + 1] +.
7
8
9
Synthesis of N-(7-chloroquinolin-4yl)-isobutyramide (4b): A solution of 4-amino-7-
chloroquinoline (3), (1.0g, 5.6 mmol) in butyric anhydride (4.64 mL, 28 mmol) was refluxed for
4h. After cooling to rt the reaction mixture was poured into ice cold water and extracted with
ethyl acetate (20 x 3). The combined extracts were dried over anhydrous Na2SO4 and evaporated
to afford the product as a light yellow solid which was recrystallized from EtOH to give pure
10
11
12
13
14
15
16
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1
product as white solid (1.0g, 71.83%), mp 173.9 0C. H NMR (300 MHz, CD3OD): δ 1.31 (d, J
= 6.60 Hz, 6H), 2.95 (m, 1H), 7.65 (dd, J = 9.00 and 2.10 Hz, 1H) 8.03 (d, J = 2.10 Hz, 1H),
8.16 (d, J = 5.40 Hz, 1H), 8.26 (d, J =9.00Hz, 1H), 8.80 (d, J = 5.40 Hz, 1H). 13C NMR (75
MHz, CD3OD): δ 20.04, 37.12, 114.46, 121.51, 125.05, 128.40, 128.58, 137.07, 144.28, 150.30,
153.06, 179.60. MS (ESI): m/z 249.08 [M + 1] +.
Synthesis of N-(7-chloroquinolin-4-yl)-ethanethioamide (5a): A mixture consisting
Lawesson reagent49 (0.91 g, 2.3 mmol), N-(7-chloroquinolin-4-yl)-acetamide (4a, 0.5g, 2.3
mmol) and toluene (15 mL) was refluxed with stirring for 24 hr. The solvent was removed under
reduced pressure. The residue was purified with a flash silica gel column using CCl3: MeOH (20
1
: 1 v/v) as eluent to give 0.32g (60%) of thioamide 5a as a yellow gum. H NMR (300 MHz,
CD3OD): δ 2.78 (s, 3 H), 7.61 (dd, J = 9.0 and 2.4 Hz, 1 H) 7.88 (d, J = 2.4 Hz, 1 H), 8.01 (d, J
= 4.8 Hz, 1 H), 8.05 (d, J =9.0 Hz, 1H), 8.89 (d, J = 4.8 Hz, 1 H). 13C NMR (75 MHz, CD3OD):
δ 34.8, 120.1, 124, 126, 128.7, 129.1, 137.4, 146.3, 150.7, 153, 205.9. MS (ESI): m/z 237.0 [M
+ 1] +.
Synthesis of N-(7-chloro-quinolin-4yl)-2-methylpropanethioamide (5b): To a stirred
solution of N-(7-chloro-quinolin-4yl)-isobutyramide (4b), (0.5 g, 2 mmol) in 1,4-dioxane
(10mL) was added Lawson’s reagent49 (2.43g, 6.0308 mmol). The reaction mixture was
refluxed with stirring for 24h, concentrated under reduced pressure and purified by flash
chromatography (CHCl3: MeOH 20: 1 v/v) to provide thioamide as yellow gum (0.35g, 66%
yields). 1H NMR (300 MHz, CD3OD): δ 1.42 (d, J = 6.60 Hz, 6H), 3.28 (m, 1H), 7.65 (dd, J =
9.00 and 2.10 Hz, 1H) 7.82 (d, J = 2.10 Hz, 1H), 7.99 (d, J = 5.40 Hz, 1H), 8.10 (d, J =9.00 Hz,
13
1H), 8.93 (d, J = 5.40 Hz, 1H). C NMR (75 MHz, CD3OD): δ 23.62, 44.92, 120.70, 124.43,
126.11, 128.74, 129.03, 137.34, 146.51, 150.68, 153.05, 216.96. MS (ESI): m/z 264.98 [M + 1]+
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Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[2-(diethylamino)-ethyl]-acetimidamide
(6a): Triethylamine (0.6 mL, 5.8 mmol) was added drop wise to a stirring suspension consisting
N, N-diethylethylenediamine (0.1 g, 0.93 mmol), thioamide 5a (0.2 g, 0.85 mmol), HgO (0.2 g,
0.92 mmol) and anhydrous DMF (10 mL). The stirring was continued at rt. for 10 hr after the
triethylamine addition was completed. The mixture was filtered through a pad of Celite and the
filtrate was concentrated under reduced pressure. Water (15 mL) was added to the residue and
the mixture was extracted with CHCl3 (10 mL) 3 times. The organic extracts were combined,
washed with brine, dried over anhydrous Na2SO4 and evaporated to dryness under reduced
pressure. The crude product was purified with a silica gel flash column and eluted with 10%
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MeOH in CHCl3 to give a colorless gum (0.22 g, 81.4% yields). H NMR (600 MHz, CD3OD):
δ 1.08 (t, J = 7.2 Hz, 6 H), 1.79 (s, 3 H), 2.64 (m, 4 H), 2.73 (t, J = 7.2 Hz , 2 H), 3.52 (t, J = 7.2
Hz , 2 H), 6.75 (d, J = 4.8 Hz, 1 H) 7.43 (dd, J = 9.0 and 2.1 Hz, 1 H), 7.87 (d, J = 2.1 Hz, 1 H),
8.0 (d, J = 9.0 Hz, 1 H), 8.55 (d, J = 4.8 Hz, 1 H). 13C NMR (150 MHz, CD3OD): δ 10.19,
16.10, 38.13, 46.73, 50.75, 112.25, 123.69, 125.78, 125.93, 126.14, 135.24, 148.71, 151.22,
157.02, 157.81. MS (ESI): m/z 319.3 [M + 1] +. Anal (C17H23ClN4): C, H, Cl, N.
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The same procedure was used to prepare new compounds 6b-s. The yield, HNMR,
13CNMR and MS data of the new analogs were described as follows.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[3-(diethylamino)-propyl]-acetimidamide
(6b): The title compound was prepared by the same procedure for 6a from 5a and 3-
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diethylamino-propylamine to give 6b as a colorless gum (0.27 g, 90.0% yields). H NMR (600
MHz, CD3OD): δ 1.11 (t, J = 7.2 Hz, 6 H), 1.83 (s, 3 H), 1.9 (m, 2 H), 2.67 (m, 6 H), 3.45 (t, J
= 6.6 Hz, 2 H), 6.81 (d, J = 4.8 Hz, 1 H) 7.48 (dd, J = 9.0 and 2.1 Hz, 1 H), 7.92 (d, J = 2.1 Hz,
1 H), 8.04 (d, J = 9.0 Hz, 1 H), 8.6 (d, J = 4.80 Hz, 1 H). 13C NMR (150 MHz, CD3OD): δ 9.91,
16.10, 25.23, 39.40, 46.32, 50.16, 112.54, 123.74, 125.84, 125.92, 126.10, 135.29, 148.69,
151.24, 157.92. MS (ESI): m/z 333.22 [M + 1] +. Anal (C18H25ClN4): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[3-(diethylamino)-propyl]acetimidamide
succinate salt. The gum 6b (0.2g, 0.6 mmol) was dissolved in CH3CN (5mL) and treated with a
solution of succinic acid (71 mg, 0.6 mmol) in MeOH (0.5 mL) and CH3CN (2 mL) mixed
0
solvent to give white succinate salt of 6b (0.2g, 74.0% yields), mp 116.5 C. H NMR (600
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MHz, CD3OD): δ 1.30 (t, J = 7.20 Hz, 6 H), 1.89 (s, 3 H), 2.18 (m, 2 H), 2.55 (s, 12 H, CH2 of 3
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succinic acid), 3.24 (m, 6 H), 3.56 (t, J = 6.6 Hz, 2 H), 6.85 (d, J = 4.8 Hz, 1 H), 7.51 (dd, J =
9.0 and 2.1 Hz, 1 H), 7.95 (d, J = 2.1 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1 H), 8.62 (d, J = 4.8 Hz, 1
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H). C NMR (150 MHz, CD3OD): δ 7.59, 16.21, 23.42, 29.63, 38.13, 46.58, 49.45, 112.17,
123.44, 125.07, 126.02, 126.33, 136.08, 147.28, 150.00, 158.32, 158.55, 175.97. MS (ESI): m/z
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333.22 [M + 1] +. Anal (C30H43ClN4O12).0.1 H2O: C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[4-(diethylamino)-butyl]-acetimidamide
(6c): Compound 6c was prepared by the same procedure from 5a and 4-(diethylamino)-
butylamine to give a colorless gum (0.25 g, 86.2% yields). 1H NMR (600 MHz, CD3OD): δ 1.04
(t, J = 7.2 Hz, 6 H), 1.62 (m, 4 H), 1.82 (s, 3 H), 2.53 (t, J = 8.40 Hz , 2 H), 2.58 (m, 4 H), 3.41
(t, J = 6.6 Hz, 2 H), 6.76 (d, J = 4.8 Hz, 1 H) 7.44 (dd, J = 9.0 and 2.1 Hz, 1 H), 7.87 (d, J = 2.1
Hz, 1 H), 7.98 (d, J = 9.0 Hz, 1 H), 8.55 (d, J = 4.8 Hz, 1 H). 13C NMR (150 MHz, CD3OD): δ
11.31, 17.63, 24.89, 28.27, 42.28, 47.76, 53.59, 114.07, 125.26, 127.32, 127.40, 127.62, 136.74,
150.19, 152.72, 158.75, 159.44. MS (ESI): m/z 347.31 [M + 1] +. Anal (C19H27ClN4).0.7 H2O:
C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[5-(diethylamino)pentyl]-acetimidamide
(6d): Compound 6d was prepared by the same procedure from compound 5a and 5-
diethylaminopentylamine to give 0.7 g (92.1%) of the desired product as colorless gum. 1H NMR
(300 MHz, CD3OD): δ 1.08 (t, J = 7.20 Hz, 3H), 1.49 (m, 2H), 1.59 (m, 2H), 1.75 (m , 2H),
1.84 (s, 3H), 2.61 (m, 6H), 3.45 (t, J = 6.90 Hz, 2H), 6.82 (d, J = 4.8 Hz, 1H), 7.49 (dd, J = 9.0
and 2.1 Hz, 1H), 7.93 (d, J = 2.1 Hz, 1 H), 8.04 (d, J = 9.0 Hz, 1H), 8.61 (d, J = 4.8 Hz, 1H).
13C NMR (75 MHz, CD3OD): δ 11.32, 17.66, 26.39, 26.96, 30.06, 42.37, 47.82, 53.80, 114.06,
125.31, 127.34, 127.43, 127.69, 136.77, 150.27, 152.75, 158.75, 159.50. MS (ESI): m/z 361.46
[M + 1] +. Anal (C20H29ClN4): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[3-(piperidin-1-yl)-propyl]-acetimid-
amide (6e): The title compound was prepared by the same procedure from 5a and N-(3-
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aminopropyl)-piperidine to give 6e in 72.1% yields as colorless gum. H NMR (600 MHz,
CD3OD): δ 1.08 (m, 2 H), 1.61 (m, 4 H), 1.79 (s, 3 H), 1.88 (m, 2 H), 2.47 (m, 6 H), 3.28 (t, J =
1.8 Hz, 2 H), 6.77 (d, J = 4.8 Hz, 1 H), 7.44 (dd, J =9.0 and 2.1 Hz, 1 H), 7.88 (d, J = 2.1 Hz, 1
H), 8.0 (d, J = 9.0 Hz, 1 H), 8.56 (d, J = 4.8 Hz, 1 H). 13C NMR (150 MHz, CD3OD): δ 16.12,
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23.82, 25.18, 25.49, 39.45, 54.15, 56.84, 112.50, 123.74, 125.84, 125.95, 126.13, 135.28, 148.71,
151.23, 157.19, 157.92. MS (ESI): m/z 345.1 [M + 1] +. Anal (C19H25ClN4): C, H, Cl, N.
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Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[4-(piperidin-1-yl)-butyl]-acetimidamide
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(6f): The title compound was prepared from 5b and 4-piperidin-1-yl-butylamine in 77.0% yields
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as colorless gum. H NMR (300 MHz, CD3OD): δ 1.51 (m, 2 H), 1.61 (m, 8 H), 1.84 (s, 3 H),
2.45 ( m, 6 H), 3.46 (t, J = 1.8 Hz, 2 H), 6.81 (d, J = 4.8 Hz, 1 H), 7.49 (dd, J = 9.0 and 2.1 Hz,
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1 H), 7.93 (d, J = 2.1 Hz, 1 H), 8.03 (d, J = 9.0 Hz, 1 H), 8.61 (d, J = 4.8 Hz, 1 H). C NMR
(75 MHz, CD3OD): δ 16.16, 23.84, 25.11, 26.85, 40.78, 54.09, 58.81, 112.56, 123.77, 125.84,
125.91, 126.17, 135.26, 148.75, 151.24, 157.25, 157.96. MS (ESI): m/z 359.24 [M + 1] +. Anal
(C20H27 ClN4): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[3-(morpholino)-propyl]-acetimidamide
(6g): Compound 6g was prepared by the same procedure from 5a and 3-morpholinopropylamine
in 64.3% yields as colorless gum. 1H NMR (300 MHz, CD3OD): δ 1.80 (s, 3 H), 1.86 (m, 2 H),
2.50 (m, 6 H), 3.45 (t, J = 6.90 Hz, 2 H), 3.70 (m, 4 H), 6.77 (d, J = 4.8 Hz, 1 H), 7.45 (dd , J =
9.0 and 2.1 Hz, 1 H), 7.89 (d, J = 2.1 Hz, 1 H), 8.01 (d, J = 9.0 Hz, 1 H), 8.57 ( d, J = 4.8 Hz, 1
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H). C NMR (75 MHz, CD3OD): δ 16.20, 25.25, 39.31, 53.42, 56.50, 66.29, 78.15, 112.47,
123.76, 125.85, 125.97, 126.17, 135.27, 148.71, 151.24, 157.15, 157.88. MS (ESI): m/z 347.16
[M + 1] +. Anal (C18H23ClN4O): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-(1-methylpiperidin-4-yl)-acetimidamide
(6h): Compound 6h was prepared from 5a and 4-amino-1-methylpiperidine in 73.0% yields as
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light brown solid, mp 168.7 C. H NMR (600 MHz, CD3OD): δ 1.62 (m, 2 H), 1.78 (s, 3 H),
2.16 (m, 4 H), 2.28 (s, 3 H), 2.89 (m, 2 H), 3.92 (m, 1 H), 6.77 (d, J = 4.8 Hz,1 H), 7.46 (dd, J =
9.0 and 2.1 Hz, 1 H), 7.89 (d, J = 2.1 Hz, 1 H), 7.96 (d, J = 9.0 Hz, 1 H), 8.57 (d, J = 4.8 Hz, 1
H). 13C NMR (150 MHz, CD3OD): δ 16.24, 30.92, 50.50, 52.06, 62.68, 112.42, 123.70, 125.86,
125.89, 126.21, 127.05, 127.92, 129.37, 135.27, 137.08, 148.75, 151.22, 156.27, 157.88. MS
(ESI): m/z 317.25 [M + 1] +. Anal (C17H21ClN4): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-(1-isopropylpiperidin-4-yl)-acetimid-
amide (6i): The title compound was prepared from 5a and 4-amino-1-isopropylpiperidine in 80%
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yields as light white solid. mp 177 C. H NMR (300 MHz, CD3OD): δ 1.14 (d, J = 6.6 Hz, 6
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H), 1.66 (m, 2 H), 1.81 (s, 3 H), 2.20 (m, 2 H), 2.48 (m, 2 H), 2.87 (m, 1 H), 3.03 (m, 2 H), 3.96
(m, 1 H), 6.79 (d, J = 4.8 Hz,1 H), 7.48 (dd, J = 9.0 and 2.1 Hz, 1 H), 7.91 (d, J = 2.1 Hz, 1 H),
7.98 (d, J = 9.0 Hz, 1 H), 8.59 (d, J = 4.8 Hz, 1 H). 13C NMR (75 MHz, CD3OD): δ 16.15,
16.85, 30.65, 38.76, 46.07, 55.07, 112.50, 123.67, 125.86, 125.93, 128.46, 135.31, 137.08,
148.69, 151.25, 156.41, 157.85. MS (ESI): m/z 345.95 [M + 1] +. Anal (C19H25ClN4): C, H, Cl,
N.
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Synthesis of (Z)-N-(1-benzylpiperidin-4-yl)-N’-(7-chloroquinolin-4-yl))-acetimidamide
(6j): Compound 6j was prepared from 5a and 4-amino-1-benzylpiperidine in 84.8% yields as
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light brown gum. H NMR (600 MHz, CD3OD): δ 1.71 (m, 2 H), 1.87 (s, 3 H), 1.79 (s, 3 H),
2.19 (m, 2 H), 2.28 (t, J = 10.8 Hz, 2 H), 3.01 (m, 2 H), 3.63 (s, 2 H), 4.01 (m, 1 H), 6.85 (d, J =
4.8 Hz,1 H), 7.34 (dd, J = 9.0 and 2.1 Hz, 1 H), 7.41 (m, 4 H), 7.53 ( dd, J = 9.0 and 2.1 Hz, 1
H), 7.98 (d, J = 2.1 Hz, 1 H), 8.03 (d, J = 9.0 Hz, 1 H), 8.65 (d, J = 4.8 Hz, 1 H). 13C NMR
(150 MHz, CD3OD): δ 16.24, 30.92, 50.50, 52.06, 62.68, 112.42, 123.70, 125.86, 125.89,
126.21, 127.05, 127.92, 129.37, 135.27, 137.08, 148.75, 151.22, 156.27, 157.88. MS (ESI): m/z
393.29 [M + 1] +. Anal (C23H25ClN4): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-[2-(pyridin-4-yl)-ethyl]-acetimidamide
(6k).
Compound 6k was prepared by the same procedure from 5a and 2-(pyridin-4-yl)-
ethylamine to give the product as brown solid which was recrystallized from EtOH as colorless
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needles (0.2 g, 74.0% yields), mp 165.0 0C. H NMR (300 MHz, CD3OD): δ 1.80 (s, 3H), 3.00 (
t, J = 6.90 Hz, 2H), 3.77 ( t, J = 6.90 Hz, 2H), 6.75 (d, J = 4.8 Hz, 1H), 7.44 (d, J = 6.00 Hz,
2H), 7.48 (dd, J = 9.0 and 2.1 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 2.1 Hz, 1H), 8.49
(d, J = 6.0 Hz, 2H), 8.60 (d, J = 4.8 Hz, 1H). 13C NMR (75 MHz, CD3OD): δ 16.02, 34.33,
40.98, 112.35, 123.80, 124.80, 125.80, 125.87, 126.11, 135.28, 148.52, 150.51, 151.20, 157.00,
157.72. MS (ESI): m/z 325.25 [M + 1] +. Anal (C18H17Cl N4): C, H, Cl, N.
Synthesis of (Z)-N-(7-chloroquinolin-4-yl)-N’-2-[1-(pyridin-4-yl)-piperidin-4-yl]ethyl-
acetimidamide (6l): Compound 6l was prepared from 5a and 2-[1-(pyridin-4-yl)-piperidin-4-yl]-
ethylamine as brown solid which was recrystallized from EtOH to give in 87.2 % yields as
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colorless needles, mp 196.5 C. 1H NMR (300 MHz, CD3OD): δ 1.33 (m, 3H), 1.69 (m, 2H),
1.85 (s, 3H), 1.95 (m, 2H), 2.96 (m, 2H), 3.54 (m, 2H), 4.05 (m, 2H), 6.82 (d, J = 4.8 Hz, 1H),
6.85 (d, J = 6.9 Hz, 2H), 7.49 (dd, J = 9.0 and 2.1 Hz, 1H), 7.94 (d, J = 2.1 Hz, 1H), 8.04 (d, J =
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