Synthesis, antileishmanial activity and structure-activity relationship of 1-N-X-phenyl-3-N′-Y-phenyl-benzamidines

Article abstract of DOI:10.1016/j.ejmech.2013.06.040

Two series of N,N′-diphenyl-benzamidines were synthesized as part of a study to search potential new drugs with antileishmanial activity. These compounds were obtained by anilides in PCl₅ halogenation reaction with generation in situ of the cor

Full text of DOI:10.1016/j.ejmech.2013.06.040

Accepted Manuscript
Synthesis, Antileishmanial Activity and Structure-Activity Relationship of 1-N-X-
phenyl-3-N’-Y-phenyl-benzamidines
Clá udio Eduardo Rodrigues-Santos, Leonor L. Leon, Adailton J. Bortoluzzi, Marilene
Marcuzzo Canto-Cavalheiro, Gé rzia C. Machado, Aurea Echevarria
PII:
S0223-5234(13)00408-X
10.1016/j.ejmech.2013.06.040
EJMECH 6268
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 March 2013
Revised Date: 21 May 2013
Accepted Date: 18 June 2013
Please cite this article as: C.E. Rodrigues-Santos, L.L. Leon, A.J. Bortoluzzi, M.M. Canto-Cavalheiro,
G.C. Machado, A. Echevarria, Synthesis, Antileishmanial Activity and Structure-Activity Relationship
of 1-N-X-phenyl-3-N’-Y-phenyl-benzamidines, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.06.040.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, Antileishmanial Activity and Structure-Activity Relationship of 1-N-X-
phenyl-3-N’-Y-phenyl-benzamidines
Cláudio Eduardo Rodrigues-Santos,a Leonor L. Leon,b Adailton J. Bortoluzzi,c Marilene
Marcuzzo Canto-Cavalheiro,b Gérzia C. Machado,b and Aurea Echevarriaa,*
a Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do
Rio de Janeiro, 23890-900, Seropédica, RJ, Brazil
b
Departamento de Imunologia, Instituto Oswaldo Cruz, FIOCRUZ, 21042-900, Rio de
Janeiro, RJ, Brazil
c
Departamento de Química, Universidade Federal de Santa Catarina, 88040-900,
Florianópolis, SC, Brazil
Corresponding author: Phone: +55 21 26822807, Fax: +55 21 26822807, E-mail:
echevarr@ufrrj.br
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Abstract
Two series of N,N’-diphenyl-benzamidines were synthesized as part of a study to search
potential new drugs with antileishmanial activity. These compounds were obtained by
anilides in PCl5 halogenation reaction with generation in situ of the corresponding
benzimidoyl chlorides, and subsequently treatment with adequate anilines. The series I
showed expressive results of antileishmanial activity, highlighted the compounds 9a with
IC50 = 81.28 µM (log IC50 = 1.91 µM) against L. chagasi, 8e with IC50 = 26.30 (log IC50 =
1.52 µM) against L. braziliensis. From the results obtained from SAR study (series I), the
series II was planned from Craig 2-dimensional map, in which was possible the discovery
of the potent compounds, 9v and 9j with IC50 = 12.60 µM (log IC50 = 1.10 µM) and 13.00
µM (log IC50 = 1.11 µM), respectively, against L. amazonensis. The results obtained from
the SAR and QSAR studies indicated the best results when electron-donors groups in the
ring attached to amidinic carbon, unlike when electron-withdrawing groups at the phenyl-N
ring showing inhibitory activity increased. Furthermore, the QSAR model obtained
indicated the hydrophobicity as a fundamental property for antileishmanial activity
presented by these series.
Keywords: N,N’-diphenyl-benzamidines; Leishmania amazonenses; Leishmania chagasi;
Leishmania braziliensis; QSAR; Hansch model.
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1. Introduction
Leishmania parasites are small and intriguing organisms that cause leishmaniasis,
and the symptoms of these diseases are skin sores that erupt weeks to months after the
person affected is bitten by sandflies. Other consequences include fever, damage to the
spleen and liver [1-2]. This disease is endemic in some geographical areas of world, where
constitutes a serious public health problem [3].
Leishmania amazonensis has been isolated from patients with all the different
clinical forms of the disease. The extracellular promastigote stage of this protozoan parasite
is introduced into subcutaneous tissue in the human host during the bite of an infected
sandfly vector. It is phagocytosed by a mononuclear phagocyte, after which it converts into
the obligate intracellular amastigote form [3].
The World Health Organization considers leishmaniasis one of the most serious
diseases worldwide caused by protozoan parasites. However, the control of this disease
remains a problem; the available antileishmanial drugs still rely on the highly toxic
pentavalent antimonials (meglumine antimoniate, Glucantime and sodium stibogluconate,
Pentostam), which cause serious side effects and require long-term treatment [4].
Second-line drugs include pentamidine (1) and amphotericin B, but these drugs
have not experienced widespread use because of toxicity and cost. Recently, the oral drug
miltefosine (2), an alkylphosphocoline was approved for the treatment of human visceral
Leishmania infections, but it present high cost and a long half-life (100 - 200 h) in humans
and low therapeutic ratio, the characteristics that could encourage the development of
resistance. In addition, the miltefosine is not suitable for use during pregnancy because of
teratogenecity and also cause mild to severe gastrointestinal side effects (Figure 1) [5].
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Please, insert the Figure 1
Since the chemotherapy against leishmaniasis is still inefficient, there is an urgent
need for the development of new, efficient, and safe drugs for the treatment of this disease.
Amidines are much used in synthesis. In some cases for the preparation of acyclic
compounds, but mostly for the synthesis of heterocyclic compounds such as aziridines,
pyrroles, oxazoles, oxadiazoles, pyridines, pyrimidines, imidazoles and triazines [6]. They
combine the properties of an azomethine-like and C-N single bond having some partial
double bond character [7].
The amidines are important medical and biochemical agents. Their anti-inflammatory,
antiviral, antifungal, antibacterial, antibiotic, antihypertensive and anesthetic activities have
been reported [8]. We have previously demonstrated that N,N’-diphenyl-p-methoxy-
benzamidine (3) was effective against Leishmania amazonensis promastigotas and axenic
amastigotes and Trypanosoma evansi trypomastigotes. Besides it was the most effective
derivative in the parasite-macrophage interaction [9-11].
Then, considering the previous results of compound 3 against trypanosomatid parasites
and the importance of the discovery of new compounds for leishmaniasis treatment, our
research group describes the synthesis and the L. amazonensis inhibitory activity of two
new series of amidine derivatives. The series I was based in the methoxy moiety
importance considering the compound 3, the most active in the N,N’-diphenyl-p-X-
benzamidine series [9-11] led us to prepare 8 derivatives with mono, di and tri-methoxy
substituents on para and meta position in the N-phenyl groups. The results of
antileishmania assays supplied a Structure-Activity Relationship (SAR) study and the Craig
Graphic [12] allowed the synthesis of series II with 14 derivatives (Figure 2).
Please, insert the Figure 2
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2. Chemistry
The route adapted [8] for the synthesis of the two series is outlined in Scheme 1.
Initially the benzanilides (6) were prepared from of the corresponding aniline (4) and the
appropriately substituted acid chlorides (5) supplying excellent yields (85-90%). The
benzanilides (6) were purified by ethanol recrystallization.
After, the benzanilides were converted to benzimidoyl chlorides, in situ, by
treatment with halogenating reagent (PCl5) under reflux in dry toluene for 8h, that
subsequently reacts with aniline dissolved in dry toluene to furnish the compounds 8a-v in
good yields (60-85%). The reactions were monitored by thin layer chromatography (TLC)
and the benzamidinic chlorides (8a-v) were purified from acetone. Following, 8a-v were
treated with aqueous solution of NaHCO3 (5%, m/v) submitted to CHCl3 extraction
affording the target compounds 9a-h (series I) and 9i-v (series II).
The formation of the benzimidoyl chloride in situ using dry toluene [13-14]
demonstrated to be a more effective way than solvent-free [8, 15] to obtain N,N’-diphenyl-
benzamidine derivatives, because it generated in high purity (it was verified by TLC). This
small change was significant to affording the target compounds with high degree of purity
(≥ 95%, CG-MS). The compounds that were obtained with less than 95% of purity were
recrystallized from methanol afforded needle crystals.
Please, insert the Scheme
The infrared spectra show the disappearance of the benzanilide (6) ν (C=O) band at
1646-1655 cm-1 and the new ν (C=N) band at 1587-1632 cm-1 resulted from benzamidine
evidence in agreement with the literature [15].
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1
The values of H and 13C NMR spectra (supplementary data) permitted the full
characterization of all compounds. The 13C NMR chemical shifts indicated peak at 154-158
ppm corresponding to C=N. As the expected the central carbon atom in benzamidines
resonates at higher field than in the corresponding benzanilides (164-165 ppm) and
benzamidinium cations (161-163 ppm).
The structures of compounds 9b and 9o were determined by single crystal X-ray
diffraction. Asymmetric unit of 9o contains two independent molecules, which are different
rotamers (A and B) with different methoxy group position (Figure 3).
The solid-state structure reveals a noncentrosymmetric molecule, with an E
configuration around the C=N double bond. The crystal structures 9b, 9o-rotamer A and 9o
-rotamer B, respectively, shows lengthened C=N (1.282, 1.283 and 1.281) and shortened C-
N (1.371, 1.364 and 1.372) bonds, a feature of n-π conjugation [15], an important
characteristic of this moiety. In the crystal structures, molecules are linked via N-H….O
interactions. It is already known that the difference between the C-N and C=N distances is
related to the degree of delocalization in the N-C=N skeleton. An interesting fact that can
be observed in these results was the electronic delocalization also depends of conformation,
the compound 9o-rotamer A, 0.081Å and 9o-rotamer B, 0.091 Å.
Please, insert the Figure 3.
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3. Results and Discussion
3.1. Antileishmanial Assays
The 50 % growth inhibitory activity value, IC50, of each compound (9a-v) was
determined using L. amazonensis in the evolutive form of promastigotes. For compounds
9a, 9e and 9f, and its salt forms (8a, 8e and 8f), the L. braziliensis and L. chagasi inhibitory
activities were evaluated. The remaining parasites were counted in a Naubauer chamber,
and the IC50 /24h values were determined by linear regression, relating percentage and log
of drug concentration in µg/mL and µM, as shown in Tables 1, 2 and 3.
The compound with one methoxy moiety (9a) and two methoxy moieties (9e) in phenyl
group attached at amidinic carbon, and with two methoxy moieties (9f) in phenyl groups
linked to N and N´ atoms were evaluated against promastigotas of L. amazonenesis, L.
braziliensis and L. chagasi, and also against macrophages in order to evaluate the toxicity
(Table 1). The results for 9a, in neutral form, IC50 = 1.91 µM presented the best
antileishmanial activity against L. chagasi, in similar potency of the pentamidine (IC50
=
1.76 µM), reference drug, already assayed against L. braziliensis the compound 9e, both
neutral (IC50 = 1.52 µM) and salt form 8e (IC50 =1.42 µM) presented comparable values
with the pentamidine (IC50 = 1.36 µM). However, the results against L. amazonensis to 8f
(IC50 = 1.62 µM) presented the best antileishmanial activity (pentamidine, IC50 = 1.05 µM).
Interesting, the macrophage toxicity presented of 9a and 9f was the 15% and 0%,
respectively, while the pentamidine presented 100%, at 320 µg/mL of concentration. This
SAR study made possible to verify that the substitutions on two N-phenyl rings do not
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increase the antileishmanial activity against L. amazonensis and furthermore cause toxicity
increase (Table 1). Then faced this data, this series I (9a-h) was proposed and evaluated
against L. amazonensis. The results obtained demonstrated that the 9a and 9h highlighted
among the compounds substituted with methoxy groups presenting values of IC50 = 1.85
µM and IC50 = 1.60 µM, respectively (Table 2).
Please insert the Tables 1 and 2
From the results obtained of SAR study, the new series II was planned, with
methoxy group linked in the para position of the phenyl ring attached to amidinic carbon,
because as it can be seen, the compound 9a presented low toxicity, as well as the compound
9f. Thus, it was selected the appropriate substituent groups with a wide range of both
lipophilicity (π) and Hammett electronic (σ) parameters from 2-dimensional map proposed
by Craig [12].
The compounds of series II were assayed against L. amazonensis, the results are
showed in the Table 3. The most active compounds were the 9j (R3 = H; R5= Cl; IC50 =
1.11 µM); 9m (R3 = Br; R5 = H; IC50 = 1.34 µM); 9o (R3 = H; R5 = NO2; IC50 = 1.22 µM)
and highlighted the compound 9v (R3 = Cl; R5 = NO2; IC50 = 1.10 µM) that presented
inhibitory activity close to reference drug, pentamidine (IC50 = 1.05 µM). The results
obtained from series II indicated that unlike the series I, when the best results were
observed to electron donors moieties attached to benzamidine ring (methoxy group), the
inhibitory activity increased when electron-withdrawing groups at the phenyl-N ring.
Please, insert the Table 3.
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3.2. Hansch Model
To try to understand the intrinsic multivariate nature of the results of the
antileishmanial activity presented by compounds, it decided to calculate the electronic,
steric and lipophilicity properties represented for polarizability (POLZ), superficial tension
(ST), volume molar (VM), molar refractivity (MR) and log P descriptors (Table 4). All
these parameters were calculated from the ACDLabs software package (version 12.0),
because Spessard [17] demonstrated that this is a good software to simulate these
parameters. From the results was established the Hansh model [18-19].
Please, insert the Table 4.
The models of 2D-QSAR were obtained from of multiple linear regression (MLR)
utilized BuildQSAR software [20].
After to analyze several models, it was possible to indicate a model with statistics
parameters expressive, so much in the quality of the adjustment of the data in the model (r2
= 0.85; F = 24.90) as in the predictability of this (q2 = 0.74), according to the works of
Erikssons et al [21] (Equation 1).
Equation 01.
log 1/IC50 = 1.04 (± 0.29)log P2 – 10.80 (± 3.23)log P- 0.04 (± 0.02)MR + 29.04 (± 9.98)
n = 18; r2 = 0.85; s = 0.26; F = 24.90; q2 = 0.74
The 2D-QSAR model (Equation 01) demonstrates a parabolic relationship with log
P, and the linear dependence of MR. The lipophilicity presents larger importance that the
refractivity molar due to your largest coefficient. The model indicates that the N-phenyl-
N’-phenyl-benzamidines reaches good results of antileishmanial activity with molecules
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with small volume and it lowers polarity, since the refractivity molar is an ambiguous
descriptor, that combines as much volume effect as of polarizability, according to Verma &
Hansch [22]. The proposed model presented 4 outliers, the compounds 9a, 9k, 9n and 9o.
4. Conclusions
The new series of N,N’-diphenyl-4-methoxybenzamidine derivatives showed
important antileishmanial activity, especially when compared with the licensed drug,
pentamidine, highlighted the compounds 9a with IC50 = 81.28 µM against L. chagasi, 8e
with IC50 = 26.30 µM against L. braziliensis, and 9v and 9j with IC50 = 12.60 and 13.00
µM respectively, against L. amazonensis. Also, it was observed that the inhibitory activity
increase when electron-withdrawing groups are attached on the N-phenyl ring, furthermore,
the QSAR model obtained were satisfactory showing that the hydrophobicity is the
fundamental property for antileishmanial activity. The results of the antileishmanial activity
presented by N,N’-diphenyl-benzamidines indicated a promising new class of
leishmanicidal drugs.
5. Experimental Section
5.1. Chemistry
5.1.1. Reagents and Instruments
Melting points were determined on a Kofler hot plate apparatus and are uncorrected.
Infrared spectra were recorded on a Perkin-Elmer 1600 FTIR spectrophotometer using
potassium bromide tablets. 1H and 13C NMR spectra were obtained in a Brucker Avance-II
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400 or Avance-III 500 spectrometers, with tetramethysilane as the internal reference, in
DMSO-d6 as solvent; the chemical shifts were reported in ppm. Mass spectrums were
recorded using Saturn GC-MS - CP-SIL8CB (30m x 25mm x 25mm). Reactions were
monitored on Merck silica gel 60 F254 aluminum sheets. TLC spots were visualized by
inspection of plates under UV light (254 and 365 mm). All commercial reagents were
obtained from Aldrich or Across Co. and used without any further purification, only aniline
was distillated.
5.1.2. N-R2-phenyl-R,R1-benzamides (6a-d)
These compounds were prepared and characterized according to the literature [23-
27].
5.1.3. General Procedure for the Preparation of the N-R2-phenyl-N’-R3,R4-phenyl-R,R1-
benzamidine (9a-v).
A mixture of N-R2-phenyl-R,R1-benzamide (6a-d) (2.25 mmol), and phosphorous
pentachloride (PCl5) (2.25 mmol) was refluxed in dry toluene for 8 h under N2 atmosphere
furnished, in situ, the benzimidoyl chlorides. Subsequently, was added drop wise to mixture
the corresponding aniline (4) (2.25 mmol) in dry toluene reflux. After, the mixture was
stirred for 3 h; was cooled and the precipitate was filtered afforded the corresponding
benzamidinic chlorides (8a-v). The 8a-v were sequentially washed with acetone (5 x 5
mL), and neutralized with NaHCO3 5% (m/v) under stirred at room temperature for 4 h.
Finally, the mixture was extracted with CHCl3, dried and concentrated to afford the target
compounds (9a-v) purified by recrystallization from ethanol.
5.1.3.1. N,N’-Diphenyl-4-methoxybenzamidine (9a). Yield 87%; mp 122-124 °C (118-120
1
°C [8]); IR (KBr): ν 3312, 2927, 2835, 1627, 1591, 1533, 1252, 1030, 789 cm-1; H NMR
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(DMSO-d6): δ 9.10 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 1H), 7.21 (d, J =
8.0 Hz, 2H), 7.04 (t, J = 8.0 and 4.0 Hz, 2H), 6.95 (t, J = 8.0 and 4.0 Hz, 1H), 6.84 (t, J =
13
8.0 Hz, 2H), 6.75 (t, J = 8.0 and 4.0 Hz, 1H), 6.57 (t, J = 4.0 Hz, 2H), 3.71 (s, 3H); C
NMR (DMSO-d6): δ 159.5, 154.4, 150.8, 141.4, 130.5, 128.3, 126.9, 122.1, 121.7, 119.5,
113.5, 55.1. MS, m/z (%): 302 (M+., 13), 286 (1), 210 (100), 77 (15), 51 (9).
5.3.1.2. N,N’-Diphenyl-3-methoxybenzamidine (9b). Yield 65%; mp 210-213 °C; IR (KBr):
1
ν 3349, 2839, 1625, 1590, 1528, 1325, 1269, 1022 cm-1; H NMR (DMSO-d6): δ 9.21 (s,
1H), 7.88 (sl, 2H), 7.21 (dd, J = 6.0 Hz, 3H), 7.06 (d, J = 8.0 Hz, 3H), 6.88 (t, J = 8.0 Hz,
13
4H), 6.61 (d, J = 8.0 Hz, 2H), 3.65 (s, 3H); C NMR (DMSO-d6): δ 158.7, 154.3, 136.0,
129.1, 128.3, 121.8, 121.1, 119.6, 114.7, 114.5, 55.0. MS, m/z (%): 302 (M+., 100), 287
(1), 210 (55), 75 (5), 51 (3). Anal. Calcd. for C20H18N2O: C, 79.44, H, 6.00, N, 9.26.
Found: C, 80.02, H, 5.90, N, 9.30.
5.3.1.3. N-(4-methoxyphenyl)-N’-phenyl-benzamidine (9c). Yield 80%; mp 110-111 °C
1
(114 °C [27]); IR (KBr): ν 2935, 2839, 1632, 1513, 1242, 1026, 730, 639 cm-1; H NMR
(DMSO-d-6): δ 9.09 (s, 0.5H), 9.03 (s, 0.5 H), 7.76 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz,
3H), 7.01 (t, J = 8.0 and 4.0 Hz, 1H), 6.85 (d, J = 4.0 Hz, 1H), 7.2 (d, J = 4.0 Hz, 1H), 6.60
13
(s, 1H), 6.60 (s, 1H), 6.55 (s, 1H), 6.49 (s, 1H), 3.71 (s, 1.7H), 3.60 (s, 1.3H); C NMR
(DMSO-d6): δ 161.9, 158.1, 136.2, 133.0, 132.3, 130.5, 128.6, 126.8, 125.0, 122.2, 114.2,
55.4. MS, m/z (%): 302 (M+., 50), 286 (1), 180 (75), 77 (32), 51 (16).
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5.3.1.4. N-(4-methoxyphenyl)-N’-phenyl-3-methoxybenzamidine (9d). Yield 70%; mp 127-
128 °C; IR (KBr): ν 3349, 2948, 2834, 1634, 1593, 1541, 1335, 1240, 1034 cm-1; 1H NMR
(DMSO-d6): δ 9.05 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 6.0 Hz, 1H), 7.20 (m, J
=8.0 Hz, 2H), 7.03 (m, J = 8.0 Hz, 2H), 6.84 (m, J = 8.0 Hz, 5H), 6.58 (m, J = 4.0 Hz, 2H),
3.71 (s, 3H), 3.64 (sl, 3H); 13C NMR (DMSO-d6): δ 158.7, 154.4, 136.2, 129.2, 128.3,
122.9, 122.2 121.2, 119.2, 114.6, 55.0. MS, m/z (%): 332 (M+., 63), 240 (100), 91 (1), 77
(28), 51 (15). Anal. Calcd. for C21H20N2O2: C, 75.88, H, 6.06, N, 8.43. Found: C, 76.05, H,
5.91, N, 8.60.
5.3.1.5. N-(4-methoxyphenyl)-N’-phenyl-4-methoxybenzamidine (9e). Yield 65%; mp 121-
122 °C (124°C [28]) ; IR (KBr): ν 3334, 3037, 2950, 2833, 1626, 1592, 1533, 1505, 1244,
1
1030, 832 cm-1, H NMR (DMSO-d6): δ 8.98 (s, 0.56H), 8.93 (s, 0.44H), 7.84 (d, J = 10.0
Hz, 1H), 7.75 (d, J = 5.0 Hz, 1H), 7.24 (d, J = 5.0 Hz, 1H), 7.19 (d, J = 10.0 Hz, 2H), 7.02
(t, J = 10.0 and 5.0 Hz, 1H), 6.93 (t, J = 10.0 and 5.0 Hz, 0.5H), 6.84 (sl, 3H), 6.73 (sl,
0.5H), 6.61 (d, J = 10.0 Hz, 1H), 6.55 (d, J = 10.0 Hz, 1H), 6.49 (d, J = 10.0 Hz, 1H), 3.72
(s, 5H), 3.62 (s, 1H); 13C NMR (DMSO-d6): δ 159.4, 154.2, 134.0, 130.7 128.2, 127.1,
122.8, 122.2, 121.0, 120.5, 119.3, 113.6 113.4, 113.3, 55.12. MS, m/z (%): 332 (M+., 47),
240 (100), 211 (90), 77 (21), 51 (11).
5.3.1.6. N-(4-methoxyphenyl)-N’-(4-methoxyphenyl)-benzamidine (9f). Yield 60%; mp 119-
120 °C (125 °C [28]); IR (KBr): ν 3352, 3055, 2933, 2834, 1625, 1505, 1241, 1032, 831
1
cm-1; H NMR (DMSO-d6): δ 8.92 (s, 1H), 7.78 (d, J = 10.0 Hz, 2H), 7.30 (t, J = 5.0 Hz,
3H), 7.25 (dd, J = 5.0 Hz, 2H), 6.84 (d, J = 5.0 Hz, 2H), 6.59 (d, J = 10.0 Hz, 2H), 6.45 (d,
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J = 5.0 Hz, 2H), 3.71 (s, 3H), 3.59 (s, 3H); 13C NMR (DMSO-d6): 162.4, 157.8, 136.0,
132.3, 130.3, 128.6, 126.8, 121.1, 113.9, 55.4. MS, m/z (%): 332 (M+., 33), 210 (100), 77
(10), 51 (5).
5.3.1.7. N-(4-methoxypheny)-N’-(4-methoxyphenyl)-4-methoxybenzamidine (9g). Yield
70%; mp 110-113 °C (105 °C [28]); IR (KBr): ν 3414, 3065, 2837, 1606, 1551, 1512,
1458, 1253, 1027 cm-1; 1H NMR (DMSO-d6): 7.62 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.8 Hz,
4H), 6.82 (d, J = 8.6 Hz, 4H), 6.70 (sl, 2H); 3.69 (sl, 6H), 3.63 (sl, 3H); 13C NMR (DMSO-
d6): δ 161.9, 157.7, 132.7, 128.3, 126.6, 119.8, 114.1, 55.7, 55.4. MS, m/z (%): 362 (M+.,
100), 241 (100), 226 (30), 197 (10), 123 (15), 77 (27).
5.3.1.8. N-(4-methoxyphenyl)-N’-phenyl-3,4-methylenedioxybenzamidine (9h). Yield 80%;
mp 146-148 °C; IR (KBr): ν 3349, 2964, 2840, 1633, 1592, 1533, 1348, 1245, 1037 cm-1;
1H NMR (DMSO-d6): δ 8.95 (sl, 1H), 7.78 (sl, 1H), 7.15 (sl, 3H), 6.82 (t, J = 8.0 Hz, 6H),
6.57 (sl, 2H), 5.99 (s, 2H), 3.67 (sl, 3H); 13C NMR (DMSO-d6): δ 155.1, 147.5, 146.7,
128.7, 128,3, 123,2, 107.9, 113,5, 101.5, 55.0. MS, m/z (%): 346 (M+., 55), 254 (93), 224
(100), 91 (1), 77 (22), 51 (13). Anal. Calcd. for C21H18N2O3: C, 72.82, H, 5.24, N, 8.09.
Found: C, 72.30, H, 5.54, N, 8.30.
5.3.1.9. N-(4-chorophenyl)-N’-phenyl-4-methoxybenzamidine (9i). Yield 55%; mp 168-170
1
°C; IR (KBr): ν 3124, 2963, 2839, 1620, 1591, 1534, 1340, 1252, 1030 cm-1; H NMR
(DMSO-d6): δ 9.24 (d, 1H), 7.86 (dd, J = 6.0 Hz, 2H), 7.22 (m, J = 8.0 Hz, 3H), 7.07 (d, J
13
= 10.0 Hz, 2H), 6.89 (d, J = 6.0 Hz, 4H), 6.57 (d, J = 8.0 Hz, 2H), 3.72 (s, 3H); C NMR
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(DMSO-d6): δ 159.7, 152.11, 149.9, 136.9, 130.5, 128.3, 128.2, 1256.6, 123.8, 122.0,
119.6, 120.9, 113.4, 55.1. MS, m/z (%): 337 (M+., 100), 301 (2), 210 (92), 92 (3), 77 (6),
51 (5). Anal. Calcd. for C20H17ClN2O: C, 71.32, H, 5.09, N, 8.32. Found: C, 70.80, H, 5.25,
N, 8.48.
5.3.1.10. N-(3-chorophenyl)-N’-phenyl-4-methoxybenzamidine (9j). Yield 67%; mp 215-
218 °C; IR (KBr): ν 3422, 2841, 1586, 1512, 1327, 1257, 1025 cm-1; 1H NMR (DMSO-d6):
δ 9.29 (d, 1H), 7.66 (m, J = 6.0 Hz, 2H), 7.24 (m, J = 6.0 Hz, 4H), 7.03 (m, J = 6.0 Hz,
3H), 6.87 (m, J = 10.0 Hz, 3H), 6.82 (m, J = 4.0 Hz, 1H), 3.72 (s, 3H); 13C NMR (DMSO-
d6): δ 162.1, 158.1, 152.1, 149.3, 135.0, 131.9, 130.7, 129.6, 128,0, 123.8, 122.9, 121.3,
114.5, 55.7. MS, m/z (%): 337 (M+., 100), 301 (2), 210 (85), 92 (3), 77 (6), 51(5). Anal.
Calcd. for C20H17ClN2O: C, 71.32, H, 5.09, N, 8.32. Found: C, 71.40, H, 5.05, N, 8.55.
5.3.1.11. N-(4-fluorophenyl)-N’-phenyl-4-methoxybenzamidine (9k). Yield 89%; mp 149-
150 °C; IR (KBr): ν 3300, 2932, 2843, 1627, 1596, 1535, 1335, 1254, 1031 cm-1; 1H NMR
(DMSO-d6): δ 9.15 (s, 1H), 7.80 (m, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 4H), 7.07 (d, J =
13
10.0 Hz, 2H), 6.88 (m, J = 8.0 Hz, 3H), 6.57 (d, J = 8.0 Hz, 2H), 3.72 (s, 3H); C NMR
(DMSO-d6): δ 159.6, 154.8, 150.8, 147.4, 141.4, 130.5, 128.3, 123.4, 122.2, 121.9, 120.9,
115.0, 114.6, 113.4, 55.1. MS, m/z (%): 320 (M+., 20), 305 (5), 210 (85), 91 (1), 77 (20),
51 (9). Anal. Calcd. for C20H17FN2O: C, 74.98, H, 5.35, N, 8.74. Found: C, 74.71, H, 5.23,
N, 8.33.
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5.3.1.12. N-(3-fluorophenyl)-N’-phenyl-4-methoxybenzamidine (9l). Yield 85%; mp 95-97
1
°C; IR (KBr): ν 3329, 2960, 2839, 1628, 1597, 1531, 1336, 1251, 1031 cm-1; H NMR
(DMSO-d6): δ 9.27 (d, 1H), 7.87 (m, J = 6.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.25 (dd, J
= 6.0 Hz, 3H), 7.06 (m, J = 6.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 3H), 6.59 (d, J = 8.0 Hz, 1H),
6.39 (d, J = 8.0 Hz, 1H), 3.72 (s, 3H); 13C NMR (DMSO-d6): δ 164.6, 159.8, 154.6, 153.4,
150.5, 141.2, 130.9, 129.8, 128.5, 126.7, 122.9, 119.6, 115.4, 112.5, 107.8, 55.3. MS, m/z
(%): 320 (M+., 20), 304 (1), 210 (68), 91 (2), 77 (20), 51 (8). Anal. Calcd. for C20H17FN2O:
C, 74.98, H, 5.35, N, 8.74. Found: C, 75.80, H, 5.44, N, 8.28.
5.3.1.13. N-(4-bromophenyl)-N’-phenyl-4-methoxybenzamidine (9m). Yield 65%; mp 175-
178 °C; IR (KBr): ν 3120, 2963, 2838, 1618, 1591, 1534, 1341, 1251, 1030 cm-1; 1H NMR
(DMSO-d6): δ 9.23 (d, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.22 (m, J =
8.0 Hz, 4H), 7.05 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 6.54 (t, J = 8.0 Hz, 2H),
3.73 (s, 3H); 13C NMR (DMSO-d6): δ 159.7, 154.9, 150.3, 141.2, 140.8, 131.1, 130.5,
128.3, 126.6, 124.4, 122.6, 122.0, 121.3, 119.8, 113.3, 55.2. MS, m/z (%): 381 (M+., 50),
350 (1), 210 (100), 91 (5), 77 (15), 51 (5). Anal. Calcd. for C20H17BrN2O: C, 63.00, H,
4.49, N, 7.35. Found: C, 62.85, H, 4.55, N, 7.77.
5.3.1.14. N-(3-methoxyphenyl)-N’-phenyl-4-methoxybenzamidine (9n). Yield 65%; oil; IR
-1
1
(KBr): ν 3384, 2932, 2842, 1603, 1266, 1023 cm ; H NMR (DMSO-d6): δ 7.75 (d, J =
8.0 Hz, 2H), 7.28 (m, J = 10.0 Hz, 8H), 6.77 (d, J = 8.0 Hz, 3H), 3.88 (s, 3H), 3.78 (s, 3H);
13C NMR (DMSO-d6): δ 161.7, 138.1, 135.9, 133.1, 131, 130.1, 126.8, 125.2, 121.9, 115.3,
114.1, 111.0, 55.9, 55.24. MS, m/z (%): 332 (M+., 20), 317 (1), 210 (80), 91 (3), 77 (27),
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51 (10). Anal. Calcd. for C21H20N2O2: C, 75.88, H, 6.06, N, 8.43. Found: C, 74.11, H, 5.94,
N, 8.50.
5.3.1.15. N-(4-nitrophenyl)-N’-phenyl-4-methoxybenzamidine (9o). Yield 60%; mp 150-
153 °C; IR (KBr): ν 3375, 2930, 2838, 1625, 1582, 1527, 1322, 1251, 1028 cm-1; 1H NMR
(DMSO-d6): δ 9.72 (d, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.95 (d, J =
8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.28 (m, J = 4.0 Hz, 4H), 6.90 (d, J = 8.0 Hz, 2H),
7.05 (m, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 3.73 (s, 3H);
13C NMR (DMSO-d6): δ 159.9, 158.0, 149.9, 140.6, 130.8, 128.5, 124.6, 122.8, 122.6,
122.0, 120.2, 113.7, 113.7, 55.27. MS, m/z (%): 347 (M+., 10), 300 (1), 210 (30), 91 (1), 77
(6), 51 (6). Anal. Calcd. for C20H17N3O3: C, 69.15, H, 4.93, N, 12.10. Found: C, 68.77, H,
4.85, N, 11.86.
5.3.1.16. N-(3-nitrophenyl)-N’-phenyl-4-methoxybenzamidine (9p). Yield 76%; oil; IR
1
(KBr): ν 3410, 2923, 2850, 1602, 1527, 1349, 1260, 1031 cm-1; H NMR (DMSO-d6): δ
9.52 (d, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.27 (m, J = 8.0 Hz, 4H),
7.03 (m, J = 8.0 Hz, 3H), 6.90 (m, J = 8.0 Hz, 3H), 6.63 (d, J = 6.0 Hz, 1H), 3.72 (s, 3H);
13C NMR (DMSO-d6): δ 160.0, 156.0, 152.4, 140.8, 147.9, 150.1, 130.8, 129.5, 128.4,
122.5, 122.2, 120.0, 115.5, 113.6, 55.2. MS, m/z (%): 303 (30), 210 (100), 91 (1), 77 (20),
,51 (10). Anal. Calcd. for C20H17N3O3: C, 69.15, H, 4.93, N, 12.10. Found: C, 68.85, H,
4.87, N, 11.82.
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5.3.1.17. N-(4-hydroxyphenyl)-N’-phenyl-4-methoxybenzamidine (9q). Yield 80%; mp 160-
162 °C; IR (KBr): ν 3202, 2923, 2852, 1626, 1605, 1536, 1361, 1236, 1028 cm-1; 1H NMR
(DMSO-d6): δ 7.66 (d, J = 2.0 Hz, 2H), 7.10 (m, J = 6.0 Hz, 10H), 6.80 (d, J = 8.0 Hz, 2H),
3.83 (s, 3H); 13C NMR (DMSO-d6): δ 154.6, 152.3, 130.7, 128.4, 127.1, 122.2, 121.2,
120.9, 115.0, 55.1. MS, m/z (%): 318 (M+., 30), 226 (100), 210 (100), 91 (2), 77 (26), 51
(20). Anal. Calcd. for C20H18N2O2: C, 75.45, H, 5.70, N, 8.80. Found: C, 75.05, H, 5.58, N,
8.76.
5.3.1.18. N-(3-hydroxyphenyl)-N’-phenyl-4-methoxybenzamidine (9r). Yield 75%; mp 190-
192 °C; IR (KBr): ν 3118, 2918, 1604, 1536, 1347, 1266, 1025 cm-1; 1H NMR (DMSO-d6):
δ 7.34 (dd, J = 8.0 Hz, 2H), 7.17 (t, J = 8.0 Hz, 3H), 7.01 (d, J = 8.0 Hz, 2H), 6.88 (m, J =
8.0 Hz, 5H), 6.35 (dd, J = 8.0 Hz, 1H), 3.77 (s, 3H); 13C NMR (DMSO-d6): δ 157.6, 154.7,
154.6, 151.6, 131.8, 130.1, 129.5, 122.8, 115.1, 114.3, 113.1, 111.1, 55.7. MS, m/z (%):
318 (M+., 20), 226 (100), 210 (90), 91 (3), 77 (18), 51 (10). Anal. Calcd. for C20H18N2O2:
C, 75.45, H, 5.70, N, 8.80. Found: C, 74.80, H, 5.62, N, 8.23.
5.3.1.19. N-(4-methylphenyl)-N’-phenyl-4-methoxybenzamidine (9s). Yield 60%; mp 140-
142 °C; IR (KBr): ν 3290, 2926, 2857, 1624, 1591, 1535, 1336, 1250, 1031 cm-1; 1H NMR
(DMSO-d6): δ 9.09 (sl, 1H), 7.77 (dd, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.05 (d, J =
10.0 Hz, 2H), 6.85 (d, J = 12.0 Hz, 5H), 6.65 (d, J = 6.0 Hz, 2H), 371 (s, 3H), 2.18 (d, 3H);
13C NMR (DMSO-d6): δ 159.5, 154.3, 151.0, 141.5, 139.0, 130.5, 128.7, 128.3, 127.0,
122.1, 119.6, 113.3, 55.1. MS, m/z (%): 316 (M+., 20), 224, (100), 210 (85), 91 (5), 77
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(20), 51 (6). Anal. Calcd. for C21H20N2O: C, 79.72, H, 6.37, N, 8.85. Found: C, 78.67, H,
6.33, N, 8.54.
5.3.1.20. N-(3-methylphenyl)-N’-phenyl-4-methoxybenzamidine (9t). Yield 86%; mp 234-
236 °C; IR (KBr): ν 3391, 2851, 1603, 1547, 1334, 1263, 1024 cm-1; 1H NMR (DMSO-d6):
δ 9.02 (sl, 1H), 7.83 (d, J = 12.0 Hz, 1H), 7.63 (d, J = 10.0 Hz, 1H), 7.22 (m, J = 8.0 Hz,
3H), 7.10 (m, J = 8.0 Hz, 1H), 6.85 (m, J = 6.0 Hz, 4H), 6.56 (d, J =6.0 Hz, 2H), 6.32 (m, J
= 8.0 Hz, 1H), 3.71 (s, 3H), 2.18 (sl, 3H); 13C NMR (DMSO-d6): 159.6, 154.2, 150.7,
141.4, 137.3, 128.3, 128.1, 122.9, 122.2, 121.6, 122.5, 119.1, 116.8, 113.32, 55.15, 30.7.
MS, m/z (%): 316 (M+., 25), 224, (100), 210 (80), 91 (12), 77 (15), 51 (8). Anal. Calcd. for
C21H20N2O: C, 79.72, H, 6.37, N, 8.85. Found: C, 78.87, H, 6.24, N, 8.77.
5.3.1.21. N-(4-sulfonylaminophenyl)-N’-phenyl-4-methoxybenzamidine (9u). Yield 60%;
1
mp 159-161 °C; IR (KBr): ν 3199, 2913, 1602, 1552, 1337, 1262, 1014 cm-1; H NMR
(DMSO-d6): δ 9.67 (s, 0.2H), 9.43 (s, 0.6H), 8.73 (s, 0.3H), 7.34 (d, J = 8.0 Hz, 1H), 7.25
(d, J = 8.0 Hz, 2H), 7.21 (d, J = 4.0 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz,
1H). 6.90 (d, J = 10.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 1H), 6.78 (dd, J = 8.0 and 4.0 Hz, 1H),
6.58 (d, J = 8.0 Hz, 2H), 371 (s, 2.2H), 3.68 (0.7H); 13C NMR (DMSO-d6): δ 162.24,
151.2, 143.5, 129.5, 132, 127.6, 123.9, 122.6, 114.6, 55.2. MS, m/z (%): 301, (13), 210
(100), 91 (1), 77 (18), 51 (10). Anal. Calcd. for C20H19N3O3S: C, 62.98, H, 5.02, N, 11.02.
Found: C, 61.76, H, 4.89, N, 11.20.
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5.3.1.22. N-(4-choro-3-nitrophenyl)-N’-phenyl-4-methoxybenzamidine (9v). Yield 50%; mp
1
128-130 °C; IR (KBr): ν 3418, 2920, 2849, 1603, 1537, 1336, 1265, 1026 cm-1; H NMR
(DMSO-d6): δ 9.45 (s, 1H), 8.00 (d, J = 6.0 Hz, 2H), 7.39 (m, 7H), 6.86 (d, J = 6.0 Hz, 2H),
6.65 (s, 1H), 3.75 (s, 3H); 13C NMR (DMSO-d6): δ 160.1, 154.4, 151.3, 147.1, 140.7,
131.4, 130.8, 128.4, 127.6, 125.8, 122.6, 120.1, 115.7, 113.7, 55.2. MS, m/z (%): 381 (M+.,
15), 289, (100), 210 (37), 91 (1), 77 (13), 51 (6). Anal. Calcd. for C21H16ClN3O3: C, 62.91,
H, 4.22, N, 11.01. Found: C, 61.89, H, 4.15, N, 10.92.
5.2. Biology.
5.2.1. Parasite Culture. L. amazonensis promastigotes MHOM/BR/77/LTB0016 strain
were grown at 25 °C in LIT medium supplemented with 10% (v/v) heat-inactivated fetal
calf serum (FCS). Cells were harvested in the late log phase, resuspended in fresh medium,
counted in Neubauer’s chamber, and adjusted to a final concentration of 4 x 106/mL. This
strain has been characterized by molecular and immunological techniques [9].
5.2.2. Antileishmanial Assays. The assays were carried out in 96-well plates in volume of
180 µL/well. The drugs were added to a parasite culture in a concentration range from 160
to 5 µg/mL, solubilized in DMSO (the highest percentage used was 1.6%, v/v, which was
not hazardous to the parasites). After 24 h incubation, the remaining parasites were counted
and the percentage of inhibition was calculated, comparing to the controls (DMSO without
the drugs and with the parasites alone). The IC50 values were determined by linear
regression from these percentages of inhibition using statistical error limits up to 10%. All
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tests were done in triplicate and pentamidine isethionate (May & Baker Lab., England) was
used as reference drug.
5.3. Molecular Descriptors. The molecular structures were drawn by ACD/ChemSketch
software (ACDLabs software package, version 12.0) and log P, polarizability, superficial
tension, volume molar and molar refractivity and log P descriptors were calculated for each
compound.
5.4. QSAR Analysis. QSAR models were derived by multiple regression analyses that
were performed using the BuildQSAR program to determine the coefficients of the
correlation equations. In all equations in this paper, the numbers in parentheses represent
the 95% confidence intervals of the coefficients, n is number of data points, r is the
correlation coefficient, s is the standard deviation, q2 is the cross-validated and F is Fisher
value; measures for the statistical significance.
5.5. X-ray diffraction Analyses. A crystal of each sample was selected from representative
crystalline mass. The intensity data for compounds 9b and 9o were collected with an Enraf-
Nonius CAD4 diffractometer, at room temperature, with graphite-monochromated Mo Kα
radiation. The unit cell parameters were determined on the setting angles of 25 centered
reflections. All data were corrected for Lorentz and polarization effects. The structure was
solved by direct methods and refined by full-matrix least-squares methods using SIR97
[29] and SHELXL97 [30] programs, respectively. All non-hydrogen atoms were refined
anisotropically. H atoms attached to C atoms were placed at their idealized positions, with
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C-H distances and Ueq values taken from the default settings of the refinement program. H
atoms of the secondary amine groups were located from Fourier difference maps and
treated as free atoms. ORTEP plots and cif validate procedure were performed by using
PLATON software [31]. Further data of crystallographic analysis for compounds 9b and 9o
are summarized in Table S1 (supplementary data). Crystallographic data (without structure
factors) have been deposited at the Cambridge Crystallographic Data Centre (CCDC) as
supplementary publication no. CCDC-837978 and CCDC-837979. Copies of the data may
be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK;
Tel.: +44-1223-336408; Fax: +44-1223-336003; e-mail: deposit@ccdc.cam.ac.uk;
http://www.ccdc.cam.ac.uk).
Acknowledgment. The authors thank CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior) for financial support and fellowships received. The authors also thank Prof.
Dr. Anderson Coser Gaudio for the freeware use of the BuildQSAR program.
Appendix. Supplementary data
Supplementary data related to this article can be found at http…
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25

ACCEPTED MANUSCRIPT
Table 1. IC50 values for compounds 9a, 9e and 9f neutral and salts forms (8a, 8e and 8f) assayed against L. amazonesis, L. brazilensis and L. chagasi
R4
R3
Promastigotes and macrophage toxicity.
R3
R2
NH
NH2Cl
N
N
8a, 8e and 8f
9a, 9e and 9f
R1
R
Compound
Substituents
R2
L. amazonensis
L. braziliensis
L. chagasi
Toxicity (%)
R
R3
H
IC50 (µg/mL) IC50 (
µM)
IC50 (µg/mL) IC50 (
µM)
IC50 (µg/mL)
IC50 (µM)
OCH3
H
No toxic
9a
9e
1.34 ± 0.18 1.85 ± 0.25 1.48 ± 0.04 1.90 ± 0.14 1.41 ± 0.05
1.91 ± 0.66
OCH3 OCH3
H
15
100
-
2.33 ± 0.15 2.80 ± 0.19 1.05 ± 0.21 1.52 ± 0.31
1.48 ± 0.09
1.95 ± 0.12
2.25 ± 0.25
2.76 0.21
H
OCH3 OCH3
9f
1.77 ± 0.11 2.24 ± 0.14 1.77 ± 0.66 2.25 ± 0.14 1.75 ±0.067
1.43 ± 0.07 1.90 ± 0.10 1.52 ± 0.11 2.05 ± 0.06 2.25 ± 0.17
8aa
8ea
OCH3
H
H
-
OCH3 OCH3
-
1.39 ± 0.27 1.82 ± 0.35 0.99 ± 0.36 1.42 ± 0.52
1.61± 0.12
2.04 ± 0.16
8fa
1.19 ± 0.31 1.62 ± 0.42 1.70 ± 0.16 2.13 ± 0.20
0.82 ± 0.19 1.05 ± 0.25 1.12 ± 0.11 1.36 ± 0.14
H
-
OCH3 OCH3
> 2.5
>3.0
-
Pentamidineb
-
-
100
1.53 ± 0.11
1.76 ± 0.12
a benzamidine hydrochorides ; b Reference drug
26

ACCEPTED MANUSCRIPT
Table 2. IC50 values for compounds 9a-h (series I) assayed against L. amazonensis
promastigotes.
R4
OCH3
R3
NH
NH
N
N
9a-g
9h
O
R1
O
R2
Comp
Substituent
L. amazonensis
R1
R2
H
R3
H
R4
H
IC50 (µg/L)
1.34 ± 0.18
IC50 (µM)
OCH3
9a
1.85 ± 0.25
H
H
OCH3
H
H
H
2.0 ± 0.11
2.28 ± 0.21
2.10 ± 0.01
2.33 ± 0.16
1.77 ± 0.11
> 2.50
2.56 ± 0.14
2.80 ± 0.26
2.57 ± 0.020
2.80 ± 0.19
2.24 ± 0.14
> 3.0
9b
9c
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
-
H
OCH3
H
9d
OCH3
H
H
H
9e
H
OCH3
OCH3
H
9f
OCH3
OCH2O
-
H
OCH2O
-
9g
1.13 ± 0.08
-
1.60 ± 0.11
1.05 ± 0.25
9h
Pent.a
-
aPentamidine: reference drug
27

ACCEPTED MANUSCRIPT
Table 3. IC50 values for compounds 9i-v (series II) assayed against L. amazonensis
promastigotes.
R3
NH
N
R5
9i-v
CH3O
Compound
Substituents
L. amazonensis
R3
R5
IC50 (µg/ml)
IC50 (µM)
Cl
H
H
Cl
9i
9j
1.43 ± 0.43
0.64 ± 0.0
1.90 ± 0.57
1.11 ± 0.0
1.43 ± 0.0
1.62 ± 0.0
1.34 ± 0.0
1.68 ± 0.17
1.22 ± 0.18
2.59 ± 0.2
2.51 ± 0.34
2.65 ± 0.09
2.42 ± 0.27
2.42 ± 0.41
1.79 ± 0.14
F
H
9k
9l
0.94 ± 0,0
H
F
1.13 ± 0,0
Br
H
9m
9n
9o
9p
9q
9r
9s
0.92 ± 0.0
H
OCH3
H
1.20 ± 0.12
0.76 ± 0.11
2.13 ± 0.17
2.01 ± 0.27
2.15 ± 0.076
1.92 ± 0.21
1.92 ± 0.32
1.37 ± 0.11
NO2
H
NO2
H
OH
H
OH
H
CH3
H
CH3
H
9t
SO2NH2
9u
Cl
-
NO2
-
9v
0.69 ± 0.0
1.10 ± 0,0
Pentamidinea
0.82 ± 0.19
1.05 ± 0.25
aPentamidine: reference drug.
28