Synthesis and anti-HIV activity of new C2 symmetric derivatives designed as HIV-1 protease inhibitors

Article abstract of DOI:10.1016/S0014-827X(02)00016-2

The synthesis of several new anti-HIV-1 compounds is described. The new compounds contain a C₂ symmetry axis and a dihidroxyethylene moiety based on the D-tartaric acid back bone. The synthesis of these compounds was achieved in 36-69% overall

Full text of DOI:10.1016/S0014-827X(02)00016-2

Il Farmaco 58 (2003) 149ꢀ157
/
www.elsevier.com/locate/farmac
Synthesis and anti-HIV activity of new C2 symmetric derivatives
designed as HIV-1 protease inhibitors
Emerson P. Pecanha a,b, Luciana J.O. Figueiredo a,b, Rodrigo M. Brindeiro c,
¸
Amilcar Tanuri c, Alexandre R. Calazans c, O.A.C. Antunes a,b,
*
a Laboratorio 641, Ilha do Fundao, Instituto de Qu´ımica, Universidade do Brasil, Cidade Universita´ria, CT Bloco A, Rio de Janeiro, RJ 21945-970,
Brazil
b FarManguinhos/FioCruz, Rio de Janeiro, RJ 20000, Brazil
c Instituto de Biologia, Universidade do Brasil, CCS Bloco A, Rio de Janeiro, RJ 21945-970, Brazil
Received 17 March 2002; accepted 28 August 2002
Abstract
The synthesis of several new anti-HIV-1 compounds is described. The new compounds contain a C2 symmetry axis and a
dihidroxyethylene moiety based on the
yields from -tartaric acid. The protocol included: acetylation of hydroxyl groups, followed by diamide formation and deacetylation
or reduction with LiAlH4. The anti-HIV 1 activities of these substances were evaluated in PM-1 cells, using Indinavir† as standard
(IC50 2.0 and 4 mM.
0.2 mM). Two amino alcohol derivatives showed good inhibitory activity against the virus, with IC50
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
D
-tartaric acid back bone. The synthesis of these compounds was achieved in 36ꢀ69% overall
/
D
ꢁ
/
ꢁ
/
´
Keywords: HIV-1 protease; D-tartaric acid derivatives; Anti-HIV-1 activity
1. Introduction
[7]. On the other hand, the high cost of the clinically
available HIV PR inhibitors consists in an economical
drawback to the widespread implementation of these as
AIDS therapies, specially in less developed countries.
Thus, it is important to develop new HIV PR inhibitors,
effective against resistant viruses and at the same time,
available at low cost.
The acquired immunodeficiency syndrome (AIDS)
pandemic continues to be a medical, social and eco-
nomic problem of staggering dimensions. The human
immunodeficiency virus (HIV) has been identified as the
etiologic agent of this disease [1ꢀ3]. HIV protease (HIV
/
Our strategy in the development of new anti-HIV
compounds is based on the development of analogues of
known HIV PR inhibitors having C2 symmetry. This
strategy has been previously proved to be efficient for
the design of compounds with desired pharmacological
properties. Compounds which contain a C2 symmetry or
a pseudo-C2 symmetry axis would present a good
molecular complement within the enzyme and would
be less capable to be recognized by the endogenous
proteases, what should result in good bioavailability [8].
Guided by this approach we selected tartaric acid as our
starting material.
PR) processes the viral polyproteins p55gag and p160gag-
into structural proteins and enzymes, including the
pol
protease itself, playing an essential role in the assembly
and maturation of the virus [4]. Since inhibition of this
aspartic protease leads to formation of non-infectious
virions, the enzyme was identified as one of the major
targets for intervention in HIV infection [5]. In fact, the
introduction of a number of HIV PR inhibitors in
clinical practice has become the main advance in AIDS
therapy [6]. Despite the high potency and selectivity of
these drugs, selection after long-term treatment led to
the occurrence of HIV PR inhibitors resistant mutants
The use of tartaric acid backbones as the core of HIV-
1 protease inhibitors has been reported in literature
[9,10]. The compounds described, e.g. compound 1,
lacked P1 and P1? hydrophobic residues. Therefore, at
* Corresponding author.
E-mail address: octavio@iq.ufrj.br (O.A.C. Antunes).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 0 0 1 6 - 2

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¸
least, conceptually, they would not be able to interact to
S1 and S1? pockets in the catalytic site of the target
enzyme. Despite this, such compounds have high
potency in inhibiting in vitro isolated HIV-1 protease.
Unfortunately, however, these compounds [9,10] have
not proven to be effective in preventing HIV-1 infection
in living cells [10], probably due to their low ability to
penetrate the cellular membranes.
methane at room temperature. The desired diamides
(7aꢀ
78 to 95%. The selective de-esterification of the acetyl
group of derivatives (7aꢀ7c) and (8aꢀ8g), using EtOH
in the presence of catalytic H2SO4 under reflux furn-
/
7c) and (8aꢀ
/
8g) were isolated in yields ranging from
/
/
ished the inhibitors (2aꢀ
yield. Compounds (4aꢀ4c) were obtained in 55ꢀ
yield, by the reduction of derivatives (2aꢀ
/
2c) and (3aꢀ
/
3c) in 63ꢀ
/
85%
/
/
62%
/2c) using
Compounds 2aꢀ
/
2c and 4aꢀ4c, were designed as
/
LiAlH4 in THF at reflux temperature for 72 h [15]
(Fig. 1).
simple analogues of the known HIV-1 inhibitors by
molecular simplification. These compounds present low
molecular weight and thus should be more effective in
penetrating biological membranes. In addition, due to
their structures, they should have good in vivo meta-
bolic and pharmacokinetic profile.
Several procedures for the amide formation, the esters
hydrolysis (ethanolysis) and reduction were tested
before selecting the above methodologies. No epimer-
ization of any chiral center occurred using the proce-
dures described here. In particular, the ethanolysis step,
2. Results and discussion
apparently simple, was found to be crucial in maintain-
ing the chiral integrity.
The preliminary anti-HIV profile of the derivatives
The initial step in the synthesis of inhibitors 2aꢀ
and 3aꢀ3g from -tartaric acid was the protection of
secondary hydroxyl groups, employing acetyl chloride
under reflux for 48 h [11ꢀ13]. This furnish the key
/
2c
/
D
(2aꢀ
/
2c), (3aꢀ
/
3g), (4aꢀ4c), (7a) and (8e) was performed
/
in a PM-1 cell culture plate assay [16]. We used standard
HIV-1 isolate Z2Z6 (subtype D, highly infectious in
/
PM-1 culture) with 3.96ꢂ
/
103 TCID50 mlꢃ1 in a 96
2ꢂ
10ꢃ3
intermediate (5) in 85% yield. The coupling step of
derivative (5) with benzylamine, (S)-a-methyl-benzyla-
mine, (R)-a-methyl-benzylamine and the ethyl esters of
alanine, valine, leucine, isoleucine, phenylalanine, tyr-
osine and tryptophan were performed through the
formation of diacyl chloride intermediate (6), generated
in situ using oxalyl chloride [14], and its addition over
the amines, in presence of triethylamine in dichloro-
holes plate, 104 cell per hole, and MOIÂ
/
/
.
Indinavir (Crixivan†) was employed as standard; one
plate line was maintained as infection control. The
infection was maintained under 5% CO2 at 37 8C into
a 5% CO2 incubator, and daily verified by optical
microscopy for 6 days. After this period the plates
were revealed with 3-(4,5-dimethylthiazol-2-yl)-2,5-di-

E.P. Pecanha et al. / Il Farmaco 58 (2003) 149ꢀ
/157
151
¸
Fig. 1. (a) AcCl, reflux, 48 h, 85%; (b) oxalyl chloride, dichloromethane, DMF, r.t. 2 h; (c) primary amines, dichloromethane, triethylamine, r.t., 30
min; (d) EtOH, H2SO4, reflux, 4 h; (e) LiAlH4, THF, reflux, 72 h.
cytotoxic effects and are able to prevent cell infections
by HIV-1. In particular, compounds (4b) and (4c)
Table 1
Anti-HIV activity of derivatives (2aꢀ
(8e) in PM-1 cells
/
2c), (3aꢀ
/
3g), (4aꢀ4c), (7a) and
/
(IC50ꢁ
/
2.0 and 4 mM) have shown activities in the log
range of Indinavir (Crixivan†), IC50ꢁ
/
0.2 mM. The
Comp.
IC50(mM)
anti-HIV activities of these compounds can be attrib-
uted to a series of factors, including their simple design
and their low molecular weight. The concept of low
molecular weight compounds is well exemplified in
literature and here again is successful.
Indinavir
0.2
10
50
2
3c
3f
4b
4c
8e
4
50
2a, 2b, 2c, 3a, 3b, 3d, 3e, 3g, 4a and 7a
ꢀ100 mM
/
3. Conclusions
phenyl-tetrazolium bromide (MTT) and read on ELISA
reader with 490l filter for the measurement of cellular
viability [17]. Each plate was made in triplicate for
statistical treatment.
The pharmacological data obtained from PM1 cells
have demonstrated the relevant anti-HIV-1 activity for
this new class of compounds. Derivatives (4b) and (4c)
have shown activity in the log range of Indinavir
(Crixivan†) [15]. These results confirm the utility of
tartaric acid as an abundant chiral building block in the
synthesis of anti-HIV compounds. In view of the fact
that these compounds were synthesized as analogous of
HIV PR inhibitors, and regarding their structural
similarity, kinetic studies using HIV PR are underway
in order to determine the Ki of these compounds against
HIV-1 PR, and thereby confirm the probable mechan-
ism of action of these new compounds. The present and
other similar synthesized compounds will be tested
against mutant HIV-1 strains.
Initial screening was performed at 100 mM and
compounds (3c), (3f), (4b), (4c) and (8e) which demon-
strated no inhibitory effect on cell viability at this
concentration were submitted to the same assay in two
different experiments using decreasing drug concentra-
tions, starting with 100ꢀ
/
6.25 mM (by 2ꢂ
/
decreasing
decreasing steps)
steps) and with 10ꢀ0.039 mM (also 2ꢂ
/
/
in order to determine the IC50, as described elsewhere
[1,16]. The results of these experiments are shown in
Table 1.
As shown in Table 1, the new compounds are able to
penetrate cellular membranes, do not exhibit any drastic

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/
¸
4. Experimental
may be obtained by recrystallization in AcOEtꢀ
m.p. 184ꢀ185 8C; [a]Dꢁ 4.8 (cꢁ1.12, CH3OH); H
NMR (CDCl3, 200 MHz) d 7.26 (m, 5H), 6.43 (m, 1H),
5.69 (s, 1H), 4.52 (dd, Jꢁ6.5, 14.8 Hz, 1H), 4.29 (dd,
Jꢁ
5.1, 14.8 Hz, 1H), 2.05 (s, 3H); 13C NMR (CDCl3,
50 MHz) d 169.1, 166.1, 137.6, 128.8, 127.7, 72.5, 43.5,
20.4; IR (cmꢃ1) 3321, 3088, 3033, 2942, 1474, 1683,
1660, 1533, 1239, 1089, 749, 702; HR MS m/z Calc. for
C22H24N2O6: 412.44. Found: 412.39%.
/
hexane:
1
/
/
ꢃ
/
/
Thin layer chromatography was performed using
aluminum sheets precoated with silica gel 60 (HF-254,
Merck) to a thickness of 0.25 mm and detected by
ultraviolet light or iodine. Column chromatography was
/
/
performed on silica gel 60, 230ꢀ400 mesh (Merck).
/
NMR (1H, 13C) spectra were recorded in a Bruker
AMX-200 or WX-200 Fourier transform spectrometer.
Coupling constants (J) are reported in hertz (Hz), and
chemical shifts are reported in parts per million (d)
relative to tetramethylsilane (TMS, 0.00 ppm). Splitting
patterns are as follows: br, broad; s, singlet; d, doublet;
dd, double doublet; t, triplet; q, quartet; m, multiplet.
Infrared (IR) spectra were recorded with a Nicolet 505
Magna spectrophotometer by using sodium chloride cell
or potassium bromide plates. High Resolution mass
spectra were obtained in a Bruker Reflex III MALDI-
ToF instrument. Optical rotations were recorded on a
4.3. 1N,4N-Di[1-phenyl-(1S)-ethyl]-2,3-diacetoxy-
(2S,3S)-butanediamide (7b)
Compound 7b (1.69 g, 3.84 mmols) was obtained
from 2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g,
4.27 mmols) and (S)-a-methylbenzylamine (1.25 g, 10.3
mmols), by the same method described for obtaining 7a,
with 90% yield, as a white solid. Analytically pure
samples may be obtained by recrystallization in acet-
Perkinꢀ
/
Elmer 241 polarimeter. Solvents used in the
oneꢀ
/
water: m.p. 226ꢀ
/
227 8C; [a]Dꢁ
/
ꢃ
/
53.7 (cꢁ1.08,
/
1
CH2Cl2). H NMR (CDCl3) 200 MHz) d 7.25 (m, 5H),
reactions were redistilled prior to use and stored over 3ꢀ
˚
4 A molecular sieves.
/
7.08 (br s, 1H), 5.64 (s, 1H), 4.99 (m, 1H), 1.94 (s, 3H),
1.42 (d, Jꢁ
6.7 Hz, 3H); 13C NMR (CDCl3, 50 MHz) d
/
4.1. 2,3-Diacetoxy-(2S,3S)-butanedioic (5) [13]
168.2, 164.1, 141.4, 127.2, 126.0, 124.8, 71.3, 47.8, 20.0,
19.0; IR (cmꢃ1) 3255, 3063, 3029, 2978, 1755, 1649,
1544, 1208, 1055, 756, 698; HR MS m/z Calc. for
C24H28N2O6: 440.49. Found: 440.71%.
A refluxing solution of D-tartaric acid (20.00 g, 133
mmol) in acetyl chloride (200 ml) was stirred for 48 h.
After this period, the reaction mixture was evaporated.
Recrystallization of the residue AcOEtꢀ
/
hexane gave
compound (5) (26.52 g, 113 mmols) at 85% yield as a
4.4. 1N,4N-Di[1-phenyl-(1R)-ethyl]-2,3-diacetoxy-
(2S,3S)-butanediamide (7c)
white hygroscopic crystalline solid: melting point (m.p.)
109ꢀ
/
110 8C; [a]Dꢁ
/
ꢃ
/
95.0 (cꢁ
/
1.00, H2O); 1H NMR
Compound 7c (1.69 g, 3.84 mmols) was obtained from
2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g, 4.27
mmols) and (R)-a-methylbenzylamine (1.25 g, 10.3
mmols), by the same method described for obtaining
(7a), with 90% yield, as a white solid. Analytically pure
(CDCl3) 200 MHz) d 5.72 (s, 1H), 2.21 (s, 3H); 13C
NMR (CDCl3, 50 MHz) d 169.8, 163.4, 72.2, 20.2; IR
(cmꢃ1) 3300, 2942, 1743, 1693, 1239, 1089.
4.2. 1N,4N-Dibenzyl-2,3-diacetoxy-(2S,3S)-
butanediamide (7a) [14]
samples may be obtained by recrystallization in AcOEtꢀ
hexane: m.p. 212ꢀ213 8C; [a]Dꢁ 41.8 (cꢁ0.98,
CH2Cl2); 1H NMR (CDCl3, 200 MHz) d 7.27 (m,
5H), 6.77 (d, Jꢁ8.0 Hz, 1H), 5.70 (s, 1H), 5.05 (m, 1H),
1.94 (s, 3H), 1.44 (d, Jꢁ
7.0 Hz, 3H); 13C NMR (CDCl3,
50 MHz) d 169.4, 165.5, 142.5, 128.9, 127.7, 126.4, 72.7,
48.9, 21.6, 20.6; IR (cmꢃ1) 3359, 3087, 3031, 2986, 2942,
1756, 1660, 1529, 1208, 1057, 765, 701; HR MS m/z
Calc. for C24H28N2O6: 440.49. Found: 440.71%.
/
/
/
ꢄ
/
/
Oxalyl chloride (1.62 g, 12.8 mmols) was added for a
period of 10 min to a solution of compound (5) (1.00 g,
4.27 mmols), and DMF (0.2 ml) of anhydrous dichlor-
omethane at 0 8C under magnetic stirring in an argon
atmosphere. After a period of 2 h, at room temperature
(r.t.), the solution was evaporated under vacuum, and
the yellowish solid residue was extracted with dichlor-
omethane (20 ml), and added over a period of 20 min to
a mixture of benzylamine (1.10 g, 10.3 mmols) and
triethylamine (1.29 g, 12.8 mmols), at r.t. After 30 min
of magnetic stirring, the mixture was concentrated under
vacuum, extracted with AcOEt (100 ml) and treated
/
/
4.5. 1N,4N-Di[1-carboethoxy-3-methyl-(1S)-butyl]-
2,3-diacetoxy-(2S,3S)-butanediamide (8c)
Compound 8c (2.05 g, 3.97 mmols) was obtained from
2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g, 4.27
mmols) and the ethyl ester of leucine (1.64 g, 10.3
mmols), by the same method described for obtaining
(7a), with 93% yield, as a white solid. Analytically pure
with aqueous HCl (2ꢂ70 ml). The organic phase was
/
washed with a saturated solution of sodium chloride (50
ml), dried with sodium sulfate, and evaporated under
vacuum, providing the diamide (7a) (1.69 g, 4.10 mmols)
at 96% yield, as a white solid. Analytically pure samples
samples may be obtained by recrystallization in AcOEtꢀ
/
hexane: m.p. 156ꢀ157 8C; [a]Dꢁ 14.0 (cꢁ1.00,
/
/
ꢃ
/
/

E.P. Pecanha et al. / Il Farmaco 58 (2003) 149ꢀ
/157
153
¸
CH2Cl2); 1H NMR (CDCl3, 200 MHz) d 6.64 (d, Jꢁ
Hz, 1H), 5.65 (s, 1H), 4.59 (m, 1H), 4.19 (q, Jꢁ7.1 Hz,
2H), 2.19 (s, 3H), 1.62 (m, 3H), 1.28 (t, Jꢁ7.1 Hz, 3H),
0.95 (d, Jꢁ
5.4 Hz, 6H); 13C NMR (CDCl3, 50 MHz) d
172.7, 169.1, 165.9, 72.9, 61.8, 51.1, 41.9, 24.9, 23.0,
22.1, 20.7, 14.3; IR (cmꢃ1) 3308, 2961, 2873, 1758, 1656,
1541, 1203, 1058; HR MS m/z Calc. for C24H40N2O10:
516.58. Found: 516.32%.
/8.4
pure samples may be obtained by recrystallization in
AcOEtꢀhexane: m.p. 159ꢀ160 8C; [a]Dꢁ 55.2 (cꢁ
1.16, CH2Cl2). 1H NMR (CDCl3, 200 MHz) d 7.45
(m, 5H), 6.98 (d, Jꢁ7.5 Hz, 1H), 5.86 (s, 1H), 5.02 (m,
1H), 4.32 (q, Jꢁ7.2 Hz, 2H), 3.35 (m, 2H), 2.33 (s, 3H),
1.43 (t, Jꢁ
7.2 Hz, 3H); 13C NMR (CDCl3, 50 MHz) d
170.9, 169.1, 165.8, 136.8, 129.5, 128.7, 127.3, 72.2, 61.8,
53.5, 38.7, 20.6, 14.2; IR (cmꢃ1) 3353, 3333, 3086, 3033,
2983, 1755, 1663, 1536, 1211, 1066, 748, 701; HR MS m/
z Calc. for C30H36N2O10: 584.62. Found: 584.13%.
/
/
/
/
ꢄ
/
/
/
/
/
/
/
4.6. 1N,4N-Di[1-carboethoxy-3-methyl-(1S)-ethyl]-
2,3-diacetoxy-(2S,3S)-butanediamide (8a)
4.9. 1N,4N-di[1-carboethoxy-2-methyl-(1S)-propyl]-
2,3-diacetoxy-(2S,3S)-butanediamide (8b)
Compound 8a (1.57 g, 3.63 mmols) was obtained
from 2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g,
4.27 mmols) and the ethyl ester of
mmols), by the same method described for obtaining 7a,
with 85% yield, as a white solid. Analytically pure
L
-alanine (1.21 g, 10.3
Compound 8b (1.94 g, 3.97 mmols) was obtained
from 2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g,
4.27 mmols) and the ethyl ester of L-valine (1.49 g, 10.3
samples may be obtained by recrystallization in AcOEtꢀ
hexane: m.p. 170ꢀ171 8C; [a]Dꢁ 2.5 (cꢁ0.99,
CH2Cl2); H NMR (CDCl3, 200 MHz) d 6.97 (d, Jꢁ
6.8 Hz, 1H), 5.63 (s, 1H), 4.48 (m, 1H), 4.17 (q, Jꢁ7.1
Hz, 2H), 2.14 (s, 3H), 1.37 (d, Jꢁ7.1 Hz, 3H), 1.24 (t,
Jꢁ
7.1 Hz, 3H); 13C NMR (CDCl3, 50 MHz) d 172.6,
169.0, 165.6, 72.3, 61.9, 48.4, 20.6, 18.4, 14.2; IR (cmꢃ1
3372, 3318, 2968, 1748, 17 390, 1663, 1537, 1261, 1202,
1032; HR MS m/z Calc. for C18H28N2O10:432.42.
Found: 432.17%.
/
mmols), by the same method described for obtaining 7a,
with 93% yield, as a white solid. Analytically pure
/
/
ꢃ
/
/
1
/
samples may be obtained by recrystallization in AcOEtꢀ
hexane: m.p. 147ꢀ148 8C; [a]Dꢁ 5.5 (cꢁ0.99,
CH2Cl2). H NMR (CDCl3) 200 MHz) d 6.85 (d, Jꢁ
7.5 Hz, 1H), 5.56 (s, 1H), 4.44 (m, 1H) 4.14 (q, Jꢁ7.1
7.1 Hz,
/
/
/
/
ꢄ
/
/
1
/
/
/
/
)
Hz, 2H), 2.15 (s, 3H), 2.13 (m, 1H), 1.24 (t, Jꢁ
/
3H), 0.89 (m, 6H); 13C NMR (CDCl3, 50 MHz) d 171.4,
169.1, 166.1, 72.5, 71.0, 61.6, 57.4, 31.6, 20.7, 19.0, 17.9
14.3; IR (cmꢃ1) 3373, 3318, 2966, 1747, 1740, 1666,
1537, 1261, 1202, 1032; HR MS m/z Calc. for
C22H36N2O10: 488.53. Found: 488.62%.
4.7. 1N,4N-di[1-carboethoxy-2-(1H-3-indoil)-(1S)-
ethyl]-2,3-diacetoxy-(2S,3S)-butanediamide (8g)
4.10. 1N,4N-di[1-carboethoxy-2-methyl-(1S, 2S)-
butyl]-2,3-diacetoxy-(2S,3S)-butanediamide (8d)
Compound 8g (2.20 g, 3.33 mmols) was obtained
from 2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g,
4.27 mmols) and the ethyl ester of
10.3 mmols), by the same method described for obtain-
ing 7a, with 78% yield, after flash chromatography with
L
-tryptophane (2.39 g,
Compound 8d (2.07 g, 4.01 mmols) was obtained
from 2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g,
4.27 mmols) and the ethyl ester of L-isoleucine (1.64 g,
silica gel (AcOEtꢀ
103ꢀ105 8C; [a]Dꢁ
(CDCl3, 200 MHz) d 8.58 (s, 1H), 7.37 (d, Jꢁ
1H), 7.08 (d, Jꢁ7.2 Hz, 1H), 6.99 (m, 4H), 5.53 (s, 1H),
4.67 (m, 1H), 3.79 (q, Jꢁ7.0 Hz, 2H), 3.11 (m, 2H), 1.62
(s, 3H), 0.91 (t, Jꢁ
7.0 Hz, 3H); 13C NMR (CDCl3, 50
MHz) d 171.5, 169.7, 166.1, 136.2, 127.5, 123.6, 122.0,
119.4, 118.4, 111.5, 109.2, 72.2, 61.8, 53.0, 27.7, 20.2,
13.9; IR (cmꢃ1) 3406, 3058, 2981, 1740, 1673, 1525,
1205, 744; HR MS m/z Calc. for C34H38N4O10: 662.69.
Found: 662.44%.
/
hexane 4:6), as a violet solid, m.p.
41.7 (cꢁ
1.07, CH2Cl2); 1H NMR
7.3 Hz,
10.3 mmols), by the same method described for obtain-
ing 7a, with 94% yield, as a white solid. Analytically
pure samples may be obtained by recrystallization in
/
/
ꢃ
/
/
/
/
AcOEtꢀ
0.98, CH2Cl2); H NMR (CDCl3, 200 MHz) d 7.50 (d,
Jꢁ8.0 Hz, 1H), 4.80 (d Jꢁ7.9, 1H), 4.48 (m, 1H), 4.35
(d, Jꢁ7.9, 1H), 4.19 (q, Jꢁ7.1 Hz, 2H), 2.0 (s, 3H),
/
hexane: m.p. 137ꢀ
/
138 8C; [a]Dꢁ
/
ꢃ
/
8.53 (cꢁ
/
1
/
/
/
/
/
/
1.88 (m, 1 H), 1.30 (m, 5H), 0.89 (m, 6H); 13C NMR
(CDCl3, 50 MHz) d 173.7, 171.0, 169.8, 70.9, 61.5, 56.4,
37.9, 25.1, 15.5, 14.3, 11.7; IR (cmꢃ1) 3331, 2970, 2937,
1759, 1747, 1665, 1536, 1260, 1202, 1056; HR MS m/z
Calc. for C24H40N2O10: 516.58. Found: 516.62%.
4.8. 1N,4N-di[1-carboethoxy-2-phenyl-(1S)-ethyl]-2,3-
diacetoxy-(2S,3S)-butanediamide (8e)
4.11. 1N,4N-di[2-(hydroxyphenyl)-1-carboethoxy-
(1S)-ethyl]-2,3-diacetoxy-(2S, 3S)-butanediamide (8f)
Compound 8e (2.37 g, 3.06 mmols) was obtained from
2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g, 4.27
mmols) and the ethyl ester of L-phenylalanine (1.99 g,
10.3 mmols), by the same method described for obtain-
ing 7a, with 95% yield, as a white solid. Analytically
Compound 8f (2.34 g, 3.80 mmols) was obtained from
2,3-diacetoxy-(2S,3S)-butanedioic acid (5) (1.00 g, 4.27
mmols) and the ethyl ester of L-tyrosine (2.15 g, 10.3
mmols), by the same method described for obtaining 7a,

154
E.P. Pecanha et al. / Il Farmaco 58 (2003) 149ꢀ157
/
¸
with 94% yield, after flash chromatography with silica
gel (CH2Cl2:CH3OH 96:4), as a crystalline white solid,
crystalline solid, m.p. 144ꢀ
/
146 8C; [a]Dꢁ
1.12, CH2Cl2), H NMR (CDCl3, 200 MHz) d 7.34 (d,
Jꢁ7.8 Hz, 1H), 7.21 (m, 5H), 5.32 (br s, 1H), 5.04 (m,
1H), 4.31 (s, 1H), 1.50 (d, Jꢁ
7.0 Hz, 3H); 13C NMR
/
ꢃ
/
46.5 (cꢁ
/
1
1
1.02, CH3OH); H
m.p. 210ꢀ
/
211 8C; [a]Dꢁ
/
ꢃ
/
35.8 (cꢁ
/
/
NMR(DMSO-d6, 200 MHz) d 9.24 (s, 1H), 8.35 (d, Jꢁ
7.7, 1H), 6.97 (d, Jꢁ8.2 Hz, 2H), 6.64 (d, Jꢁ8.2, 2H),
5.50 (s, 1H), 4.36 (m, 1H), 4.00 (q, Jꢁ7.0 Hz, 2H), 2.85
(m, 2H), 1.94 (s, 3H), 1.09 (t, Jꢁ
7.0 Hz); 13C NMR
(DMSO-d6, 50 MHz) d 171.4, 169.7, 166.2, 156.5, 130.4,
127.4, 115.5, 72.1, 61.2, 54.3, 36.9, 21.0, 14.5; IR (cmꢃ1
/
/
/
/
(CDCl3, 50 MHz) d 173.1, 142.3, 128.9, 127.6, 126.0,
70.5, 48.8, 22.0; IR (cmꢃ1) 3386, 3342, 3194, 2985, 2964,
1661, 1642, 1532, 1443, 1139, 1077, 763, 700; HR MS m/
z Calc. for C20H24N2O4: 356.42. Found: 356.81%.
/
/
)
3353, 3333, 3301, 3086, 3033, 2983, 1755, 1663, 1536,
1211, 1066, 748, 701; HR MS m/z Calc. for
C30H36N2O12: 616.62. Found: 616.68%.
4.15. 1N,4N-di[1-carboethoxy-3-methyl-(1S)-butyl]-
2,3-dihydroxy-(2S,3S)-butanediamide (3c)
Compound 3c (0.64 g, 1.01 mmol) was obtained from
the derivative (8c) (0.55 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 81% yield as a
4.12. 1N,4N-dibenzyl-2,3-dihydroxy-(2S,3S)-
butanediamide (2a)
colorless oil. [a]Dꢁ
(CDCl3, 200 MHz) d 7.35 (d, Jꢁ
1H), 4.51 (m, 1H), 4.33 (s, 1H), 4.17 (q, Jꢁ
1.62 (m, 3H), 1.25 (t, Jꢁ
7.1 Hz, 3H), 0.92 (m, 6H); 13
/
ꢃ
/
21.6 (cꢁ
/
1.76, CH2Cl2); 1H NMR
8.0 Hz, 1H), 4.80 (br s,
7.1 Hz, 2H),
Sulfuricacid (0.5 ml) was added to a diamide solution
(7a) (0.52 g, 1.25 mmol) in absolute etnanol (50 ml) at
r.t. and the mixture was kept under magnetic stirring at
reflux temperature for 4 h. After cooling, the solvent
was concentrated under vacuum to half the original
volume, and had AcOEt (70 ml) added. The mixture was
extracted with aqueous NaOH at 5% (20 ml) and
aqueous 1 N HCl (20 ml). The organic phase was
washed with NaCl, dried with anhydrous sodium sulfate
and evaporated under vacuum, providing the diol (2a)
/
/
/
C
NMR (CDCl3, 50 MHz) d 173.4, 172.0, 70.7, 61.5, 50.6,
41.3, 24.8, 22.7, 21.9, 14.1; IR (cmꢃ1) 3388, 2960, 2872,
1724, 1651, 1533, 1357, 1202, 1154; HR MS m/z Calc.
for C20H36N2O8: 432.51. Found: 432.85%.
4.16. 1N,4N-di[1-carboethoxy-(1S)-ethyl]-2,3-
dihydroxy-(2S,3S)-butanediamide (3a)
(0.34 g, 10.0 mmol) at 83% yield, as a crystalline solid,
1
1.20, CH3OH); H
m.p. 198ꢀ
/
200 8C; [a]Dꢁ
/
ꢄ
/
15.2 (cꢁ
/
Compound 3a (0.26 g, 0.75 mmol) was obtained from
the derivative (8a) (0.54 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 60% yield as a
NMR (DMSO-d6, 200 MHz) d 8.04 (m, 1H), 7.08 (m,
7.0 Hz), 4.16 (m, 2H), 3.13 (s, 1H); 13
5H), 5.52 (d, Jꢁ
/
C
NMR (DMSO-d6) 50 MHz) d 172.6, 139.9, 128.6, 127.6,
127.1, 73.2, 42.4; IR (cmꢃ1) 3362, 3316, 3086, 3034,
2927, 1628, 1546, 1095, 742, 697; HR MS m/z Calc. for
C18H20N2O4: 328.36. Found: 328.10%.
crystalline solid, m.p. 102ꢀ
/
103 8C; [a]Dꢁ
0.98, CH2Cl2); H NMR (CDCl3, 200 MHz) d 7.58 (d,
Jꢁ7.4 Hz, 1H), 4.48 (m, 1H), 4.39 (m, 1H), 4.17 (q, Jꢁ
7.1 Hz, 2H), 3.92 (br s, 1H), 1.38 (d, Jꢁ7.1 Hz, 3H),
7.1 Hz, 3H); 13C NMR (CDCl3, 50 MHz) d
/
ꢃ
/
2.53 (cꢁ
/
1
/
/
/
1.24 (t, Jꢁ
/
4.13. 1N,4N-di[1-phenyl-(1S)-ethyl]-2,3-dihydroxy-
(2S,3S)-butanediamide (2b)
173.0, 172.3, 71.1, 61.8, 48.2, 18.2, 14.2; IR (cmꢃ1) 3388,
2960, 2872, 1724, 1651, 1533, 1357, 1202, 1154; HR MS
m/z Calc. for C14H24N2O8: 348.35. Found: 340.00%.
Compound 2b (0.36 g, 1.03 mmol) was obtained from
the derivative (7b) (0.55 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 82% yield as a
4.17. 1N,4N-di[1-carboethoxy-2-phenyl-(1S)-ethyl]-
2,3-dihydroxy-(2S,3S)-butanediamide (3e)
crystalline solid, m.p. 130ꢀ
1.03, CH2Cl2); 1H NMR (CDCl3, 200 MHz) d 7.29
(m, 6H), 5.20 (br s, 1H), 5.06 (dq, Jꢁ7.4, 7.0 Hz, 1H),
4.23 (s, 1H), 1.51 (d, Jꢁ
7.0 Hz, 3H); 13C NMR (CDCl3,
/
131 8C; [a]Dꢁ
/
ꢃ
/
91.3 (cꢁ
/
/
Compound 3e (0.51 g, 1.02 mmol) was obtained from
the derivative (8e) (0.75 g, 1.28 mmol), by the same
procedure described for obtaining 2a with 80% yield as a
/
50 MHz) d 173.0, 142.3, 128.8, 127.7, 126.1, 70.3, 49.0,
21.9; IR (cmꢃ1) 3413, 3332, 3087, 3032, 2973, 1658,
1526, 1140, 1063, 763, 698; HR MS m/z Calc. for
C20H24N2O4: 356.42. Found: 356.18%.
crystalline solid, m.p. 138ꢀ
/
140 8C; [a]Dꢁ
1.08, CH2Cl2); H NMR (CDCl3, 200 MHz) d 7.41 (d,
Jꢁ7.5 Hz, 1H), 7.20 (m, 5H), 4.75 (m, 1H), 4.57 (br s,
1H), 4.26 (br s, 1H) 4.07 (q, Jꢁ7.2 Hz, 2H), 3.00 (m,
2H), 1.14 (t, Jꢁ
7.2 Hz, 3H); 13C NMR (CDCl3, 50
/
ꢄ
/
78.5 (cꢁ
/
1
/
/
4.14. 1N,4N-Di[1-phenyl-(1R)-ethyl]-2,3-dihydroxy-
(2S,3S)-butanediamide (2c)
/
MHz) d 173.2, 170.8, 135.7, 129.6, 128.8, 127.4, 71.0,
61.9, 53.2, 38.3, 14.3; IR (cmꢃ1) 3448, 3384, 3347, 3062,
3030, 2979, 1729, 1658, 1625, 1531, 1209, 1138, 700, 609;
HR MS m/z Calc. for C26H32N2O8: 500.54. Found:
500.72%.
Compound 2c (0.36 g, 1.03 mmol) was obtained from
the derivative (7c) (0.55 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 82% yield as a

E.P. Pecanha et al. / Il Farmaco 58 (2003) 149ꢀ
/157
155
¸
4.18. 1N,4N-Di[1-carboethoxy-2-(1H-3-indoil)-(1S)-
ethyl]-2,3-dihydroxy-(2S,3S)-butanediamide (3g)
9.27 (s, 1H), 7.70 (d, Jꢁ
2H), 6.67 (d, Jꢁ8.3, 2H), 5.87 (d, Jꢁ
(m, 1H),4.27 (d, Jꢁ7.0, 1H), 4.04 (q, Jꢁ
2.92 (m, 2H), 1.12(t, Jꢁ
7.0 Hz); 13C NMR (DMSO-d6,
50 MHz) d 172.2, 1716, 156.7, 130.8, 126.9, 115.7, 73.0,
61.2, 53.8, 36.9, 14.5; IR (cmꢃ1) 3353, 3333, 3302, 3086,
3033, 2983, 1664, 1532, 1211, 1066, 748, 701; HR MS m/
z Calc. for C26H32N2O10: 532.54. Found: 532.72%.
/
7.7, 1H), 6.99 (d, Jꢁ
/
8.3 Hz,
/
/
7.0 Hz, 1H), 4.51
/
/7.0 Hz, 2H),
Compound 3g (0.42 g, 0.73 mmol) was obtained from
the derivative (8g) (0.83 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 55% yield as a
/
crystalline solid, m.p. 114ꢀ
/
116 8C; [a]Dꢁ
0.89, CH3OH); H NMR (CDCl3, 200 MHz) d 10.92
(s, 1H), 7.78 (d, Jꢁ7.7 Hz, 1H), 7.50 (d, Jꢁ7.2Hz, 1H),
6.99 (m, 4H), 5.99 (d, Jꢁ7.1 Hz, 1H), 4.63 (m, 1H), 4.33
(d, Jꢁ7.1 Hz, 1 H), 3.97 (q, Jꢁ7.0 Hz, 2H), 3.21 (m,
2H), 1.07 (t, Jꢁ
7.0 Hz, 3H); 13C NMR (CDCl3, 50
/
ꢄ
/
17.9 (cꢁ
/
1
/
/
/
/
/
4.22. 1,4-di(benzylamine)-(2R,3R)-butane-2,3-diol
/
(4a) [15]
MHz) d 172.2, 171.8, 136.5, 127.7, 124.5, 121.5, 118.9,
118.6, 111.9, 109.0, 73.0, 61.2, 52.9, 27.8, 14.3; IR
(cmꢃ1) 3389, 3316, 3060, 2977, 2928, 1727, 1650, 1538,
1220, 1106, 736; HR MS m/z Calc. for C30H34N4O8:
578.62. Found: 578.60%.
A solution of compound 7a (1.20 g, 2.91 mmols) in
anhydrous THF (20 ml), was added to a suspension of
LiAlH4 (1.30 g, 34.0 mmols) in anhydrous THF (20 ml),
under argon at r.t. The reaction mixture was kept under
magnetic stirring at reflux temperature for 48 h. After
cooling at 0 8C, water (2 ml) and aqueous NaOH at 10%
(3 ml) were carefully added, and the mixture was
maintained under stirring at r.t. for 1 h, when it was
then evaporated under vacuum. The solid residue
obtained was dissolved in HCl 1 N (100 ml), extracted
4.19. 1N,4N-di[1-carboethoxy-2-methyl-(1S)-propyl]-
2,3-dihydroxy-(2S,3S)-butanediamide (2b)
Compound 3b (0.41 g, 1.03 mmol) was obtained from
the derivative (8b) (0.61 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 82% yield as a
with AcOEt (2ꢂ
added to the aqueous phase, until pH 10, and was
extracted with AcOEt (5ꢂ100 ml). This organic phase
/
20 ml). Aqueous NaOH at 50% was
1
colorless oil. [a]Dꢁ
(CDCl3, 200 MHz) d 7.51 (d, Jꢁ
1H), 4.41 (m, 2H), 4.07 (q, Jꢁ
1H), 1.24 (t, Jꢁ
/
ꢃ
/
2.3 (cꢁ
/
1.29, CH2Cl2); H NMR
/
7.5 Hz, 1H), 4.96 (br s,
/
/
7.1 Hz, 2H), 2.13 (m,
was washed with saturated NaCl (50 ml), dried with
anhydrous sodium sulfate and evaporated under va-
cuum. Flash column chromatography with silica gel
(NH4OH conc.:CH3OH:CH2Cl2 0.25:4.75:95) of the
residue obtained the amino alcohol (4a) (0.48 g, 1.60
/
7.1 Hz, 3H), 0.87 (m, 6H); 13C NMR
(CDCl3, 50 MHz) d 173.4, 171.3, 71.5, 61.5, 57.1, 31.2,
19.0, 17.8, 14.2; IR (cmꢃ1) 3404, 2968, 2938, 1737, 1662,
1530, 1206, 1150, 1025; HR MS m/z Calc. for
C18H32N2O8: 404.46. Found: 405.10%.
mmol) at 55% yield, as a crystalline solid, m.p. 77ꢀ
79 8C; [a]Dꢁ 42.3 (cꢁ
1.08, CH2Cl2); 1H NMR
(CDCl3, 200 MHz) d 7.27 (m, 5H), 3.82 (d, Jꢁ2.3
Hz, 1H), 3.76 (d, Jꢁ5.9 Hz, 1H), 3.08 (dd, Jꢁ3.4, 11.2
Hz, 1H), 3.05 (br s, 1H), 2.71 (d, Jꢁ
11.2 Hz, 1H); 13C
/
/
ꢃ
/
/
4.20. 1N,4N-di[l-carboethoxy-2-methyl-(1S, 2S)-
/
butyl]-2,3-dihydroxy-(2S,3S)-butanediamide (2d)
/
/
/
Compound 3d (0.44 g, 1.01 mmol) was obtained from
the derivative (8d) (0.64 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 81% yield as a
NMR (CDCl3, 50 MHz) d 139.5, 128.7, 128.4, 127.4,
73.1, 54.1, 53.3; IR (cmꢃ1) 3334, 3289, 3086, 3028, 2928,
2873, 1644, 1454, 1254, 1055, 740, 701 HR MS m/z
Calc. for C18H24N2O2: 300.40. Found: 300.54%.
1
colorless oil. [a]Dꢁ
(CDCl3, 200 MHz) d 7.50 (d, Jꢁ
Jꢁ7.9, 1H), 4.48 (m, 1H), 4.35 (d, Jꢁ
Jꢁ7.1 Hz, 2H), 1.88 (m, 1H), 1.30 (m, 5H), 0.89 (m,
/
67.3 (cꢁ/0.98, CH2Cl2); H NMR
/
8.0 Hz, 1 H), 4.80 (d,
7.9, 1H), 4.19 (q,
/
/
/
4.23. 1,4-di[1-phenyl-(1S)-ethylamine]-(2R,3R)-
butane-2,3-diol (4b)
6H); 13C NMR (CDCl3, 50 MHz) d 173.7, 171.0, 70.9,
61.5, 56.4, 37.9, 25.1, 15.5, 14.3, 11.7; IR (cmꢃ1) 3404,
2968, 1736, 1660, 1530, 1203, 1148, 1024; HR MS m/z
Calc. for C20H36N2O8: 432.51. Found: 432.72%.
Compound 4b (0.55 g, 1.69 mmol) was obtained from
the derivative (7b) (1.20 g, 2.73 mmol), by the same
procedure described for obtaining 4a with 62% yield as a
4.21. 1N,4N-di[2-(4-hydroxyphenyl)-1-carboethoxy-1-
(1S)-ethyl]-2,3-dihydroxy-(2S,3S)-butanediamide (2f)
colorless oil. [a]Dꢁ
(CDCl3, 200 MHz) d 7.09 (m, 5H), 4.03 (br s, 1H), 3.74
(s, 1H), 4.50 (q, Jꢁ6.6 Hz, 1H), 3.08 (dd, Jꢁ3.4, 12.0
Hz, 1H), 2.71 (d, Jꢁ12.0 Hz, 1H), 1.17 (d, Jꢁ6.6 Hz,
/
ꢄ
/
81.3 (cꢁ
/
1.13, CH2Cl2); 1H NMR
/
/
Compound 3f (0.51 g, 0.96 mmol) was obtained from
the derivative (8f) (0.77 g, 1.25 mmol), by the same
procedure described for obtaining 2a with 77% yield as a
/
/
3H); 13C NMR (CDCl3, 50 MHz) d 144.6, 128.7, 127.3,
126.5, 73.1, 58.3, 51.7, 23.6; IR (cmꢃ1) 3302, 3084, 3027,
2967, 2858, 1493, 1452, 1117, 1078, 763, 701; HR MS m/
z Calc. for C20H28N2O2: 328.45. Found: 328.30%.
crystalline solid, m.p. 104ꢀ
/
105 8C; [a]Dꢁ
/
ꢃ
/
13.7 (cꢁ
/
0.95, CH3OH); 1H NMR (DMSO-d6, 200 MHz) d

156
E.P. Pecanha et al. / Il Farmaco 58 (2003) 149ꢀ
/157
¸
4.24. 1,4-di[1-phenyl-(1R)-ethylamine]-(2R,3R)-
butane-2,3-diol (4c)
counter, with a 490l, absorption filter. The results were
analyzed using a Microsoft EXCEL matrix, with correc-
tion of the blanks, and plotting of the emission
frequency graph of the assay (in percentage, using as
the 100% standard the emission from the viable cells of
the wells without infection) as measurement of cellular
viability. The value of 50% of emission of the standard
was considered as cut-off point for the IC50 calculation.
This value was attained, after plotting on the graph of
the logarithmic regression curve equation, the points
obtained from the IC curve prior to the formation of the
plateau of the curve.
Compound 4c (0.55 g, 1.69 mmol) was obtained from
the derivative (7c) (1.20 g, 2.73 mmol), by the same
procedure described for obtaining 4a with 62% yield as a
colorless oil. [a]Dꢁ
(CDCl3, 200 MHz) d 7.19 (m, 5H), 4.06 (br s, 1H), 3.60
(m, 2H), 2.88 (dd, Jꢁ3.0, 12.0 Hz, 1H), 2.49 (d, Jꢁ
12.0 Hz, 1H), 1.31 (d, Jꢁ
6.5 Hz, 3H); 13C NMR
/
ꢃ
/
98.0 (cꢁ
/
0.95, CH2Cl2); 1H NMR
/
/
/
(CDCl3, 50 MHz) d 144.5, 128.8, 127.4, 126.9, 73.1,
58.9, 51.9, 24.8; IR (cmꢃ1) 3310, 3084, 3027, 2966, 2854,
1493, 1452, 1118, 1079, 763, 701; HR MS m/z Calc. for
C20H28N2O2: 328.45. Found: 328.70%.
Acknowledgements
5. Pharmacological evaluation [16,17]
Financial support from Far-Manguinhos/FioCruz,
FAPERJ, FUJB-UFRJ, CNPq, CAPES, FINEP and
PRONEX is acknowledged.
The pharmacological evaluation of the derivatives
obtained was undertaken using test plates with a PM1
strain cell culture, lymphocytic strain established in
culture, expressing the receptors CD4ꢄ and co-recep-
/
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/
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mM, with the subsequent dilutions being as follows:
10, 5, 1.25, 0.625, 0.312, 0.156, 0.078 and 0.039 mM.
The last and tenth well was kept as a control of the
infection, without the presence of the drug. Each line of
ten wells was produced in triplicate, for posterior
statistical treatment. INDINAVIR was used as control,
in the same dilutions as the compounds being tested, as
described, for cytotoxic analysis. The infection was kept
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