trans-Dihydroxypiperidines: Synthesis, Stereochemistry, and Anti-HIV Activity

Full text of DOI:10.1023/A:1025307617288

Doklady Chemistry, Vol. 391, Nos. 4–6, 2003, pp. 195–199. Translated from Doklady Akademii Nauk, Vol. 391, No. 4, 2003, pp. 487–491.
Original Russian Text Copyright © 2003 by Grishina, Borisenko, Nosan’, Veselov, Ashkinadze, Karamov, Kornilaeva, Zefirov.
CHEMISTRY
trans-Dihydroxypiperidines: Synthesis, Stereochemistry,
and Anti-HIV Activity
G. V. Grishina*, A. A. Borisenko*, Z. G. Nosan’*, I. S. Veselov*, L. D. Ashkinadze*,
E. V. Karamov**, G. V. Kornilaeva, and Academician N. S. Zefirov*
Received October 2, 2002
The search for medicines for HIV and AIDS treat-
ment is among the most acute problems of modern sci-
ence all over the world. However, the experience of
using the vast majority of anti-HIV preparations shows
that this virus soon develops resistance to the drugs
OH
OH
OH
HO
OH
OH
HO
OH
H
HO
OH
OH
OH
N
R
N
N
H
(
actually, within several months after the beginning of
1
2
3: R = Me,
the therapy). Therefore, a continuous search for new
classes of organic compounds exhibiting anti-HIV
activities is required [1, 2].
4
5
: R = Et,
: R = Bu.
The biological activities of castanospermin 1, one of
the 32 possible optically active stereoisomers, are quite
diverse, but inhibition of HIV replication and replica-
tion of other viruses is most significant [2]. The HIV
inhibition is thought to take place upon glycosylation of
the viral shell protein in which the cell glucosidase uses
Data on the structure of the main HIV-1 enzymes,
namely, reverse transcriptase and protease, are widely
used in the search for appropriate inhibitors. Several
nucleoside (azidothymidine, zalcitabine, stavudine)
and nonnucleoside (nevirapine, delavirdine, efavirenz) an iminosugar molecule instead of D-glucose. This
substitution results in incompleteness of the HIV pro-
tein shell and in the inhibition of replication of the
immunodeficiency virus.
In this study, while working on the quest for and
preparation of new anti-HIV compounds using a biomi-
preparations are now widely used for inhibiting HIV
replication at the reverse transcriptase step. Virus pro-
tease is inhibited by sakvinavir, ritanovir, indinavir, and
other drugs [3].
In the late 1990s, data concerning the discovery of metic approach, we isolated a common structural frag-
ment, trans-3,4-dihydroxypiperidine, from the family
of polyhydroxylated alkaloids 1–5 containing several
chiral centers and suggested that this fragment may
prove to be an anti-HIV pharmacophore. To verify this
assumption, we chose target compounds, namely, racemic
an absolutely new class of HIV replication inhibitors,
polyhydroxylated alkaloids isolated from the fruits of
the Australian chestnut Castanospermus australe, were
published. These include castanospermin 1, 2-deoxy-
nojuerymycin 2 and its N-alkylated analogues 3–5, and chiral C- and N-substituted trans-3,4-dihydroxy-
swansonine, and other compounds [2–4], which are piperidines 1–12, and carried out a stereoselective syn-
thesis and conformational study of these compounds.
The compounds were tested in vitro for anti-HIV activ-
ity and toxicity in cell lines of human origin infected
with different strains of HIV-1. The results were highly
successful and promising. The simplest azasugars,
imino- or azasugars in which the oxygen atom of the
pyranose ring is replaced by nitrogen. The report on this
alkaloid family suppressing the development of HIV
infection triggered a stream of studies dealing with the
synthesis of their homologues and analogues, most trans-dihydroxypiperidines 1–12, containing only two
often, on the basis of natural sugars (see for example, hydroxy groups, were found to be relatively nontoxic
and to suppress the development of HIV infection to
different extents. Within this group, we chose the most
promising leading compounds that can be recom-
mended for in-depth study [6]. The new group of anti-
HIV substances we found is especially promising for
further studies because the anti-HIV activity of enanti-
omers of the leading compounds can be increased
manyfold and because their synthesis is rather simple
and economical.
[
5]), and evaluation of their ability to inhibit enzyme
systems.
*
Moscow State University, Vorob’evy gory, Moscow,
19992 Russia
1
*
* Ivanovsky Research Institute of Virology, Russian
Academy of Medical Sciences, ul. Gamalei 16, Moscow,
1
23098 Russia
0
012-5008/03/0008-0195$25.00 © 2003 åÄIä “Nauka /Interperiodica”

1
96
GRISHINA et al.
Synthesis of target compounds 1–12. All C- and (S)-phenylethylamine with subsequent reduction of the
N-substituted trans-3,4-dihydroxypiperidines 1–12 resulting new pyridinium salts (THPs 23, 24) with
were synthesized by trans-hydroxylation of two series NaBH in ethanol at 0°ë. THP 13, unsubstituted at
4
of tetrafluoroborates or trifluoroacetates of C- and N- nitrogen, was prepared by removing the N-benzyl
substituted 1,2,5,6-tetrahydropyridines (THPs) 13–24 group from THP 18 by hydrogenolysis over palladium
by treatment with trifluoroperacetic acid or free THP
bases by treatment with performic acid. Under these
conditions, the nitrogen atom is blocked, which pre-
vents the formation of N oxides deactivating the double
bond.
black. THP 22 was produced upon dehydration of
-benzyl-4-hydroxy-4-phenylpiperidine, which was, in
turn, synthesized by the addition of phenyllithium to
-benzylpiperidin-4-one. The individual trans isomers
1
1
of 3,4-dihydroxy derivatives 1–12 were isolated as free
bases by column chromatography on silica gel or by
salt crystallization. The chemical purity of the target
compounds obtained in each step was checked by
GC/MS and elemental analyses; the spatial structure
and the diastereomeric composition were determined
R1
R1
OH
HO
[
RCOOOH]
N
N
R
1
13
using high-resolution ç and C NMR spectra, and the
presence of an intramolecular hydrogen bond was
established from the data of IR spectroscopy [6].
R
1
3–24
1–12
1
1
7, 19. R = Bn, R = 6-Me;
8, 20. R = Bn, R = 3-Me;
9, 21. R = Bn, R = 4-Me;
10, 22. R = Bn, R = 4-Ph;
11, 23. R = (CH ) OH, R = H;
12, 24. R = MeCHPh, R = H.
1
2
3
4
5
6
, 13. R = R = H;
, 14. R = Me, R = H;
, 15. R = Et, R = H;
, 16. R = n-Pr, R = ç;
, 17. R = n-Bu, R = H;
, 18. R = Bn, R = H;
1
1
Conformational analysis. The trans geometry of
3,4-dihydroxypiperidines 1–6 and 11, without substitu-
1
1
1
1
1
13
ents in the piperidine ring, was confirmed by ç and C
1
1
1
2 2
NMR data. The ç NMR spectral patterns and the mag-
1
1
nitudes of vicinal spin–spin coupling constants are sim-
ilar for different diols (Table 1). A typical example is
the spectrum of 1-benzyl-3,4-dihydroxypiperidine 6.
The great magnitudes of vicinal spin–spin coupling
The first series of THPs 14–21 was prepared by
reduction of 1-benzylpyridinium or 1-benzyl-2-, -3-,
and -4-picolinium chlorides with NaBH in ethanol at
4
3
3
3
constants ( J
, J
, and J
) are indica-
3H , 4H
2
H , 3H
5H , 4H
0
°ë. The second series of THPs 25 and 26 was synthe-
a
a
a
sized by transamination of 1-(2,4-dinitrophenyl)pyri- tive of the diaxial orientation of the protons at C-3 and
dinium chloride (Zincke salt) by ethanolamine and C-4, which implies the diequatorial orientation of the
Table 1. Vicinal spin–spin coupling constants of protons at C-2, C-3, C-4, and C-5 in diols
3J, Hz
2
3
4
5
6
7
10
11
3
3
3
3
3
3
3
3
3
J2
11.64
4.27
11.44
4.35
10.88
4.19
11.23
4.22
9.60
4.40
7.86
4.17
1.94
9.29
4.64
H , 3H
a
a
a
e
a
a
e
e
e
J2
J3
J3
J4
J4
J5
J5
J6
H , 3H
e
4.8
H , 4H
e
9.03
12.02
4.84
8.93
11.81
4.89
9.14
11.70
4.98
8.87
11.87
–
8.40
8.7
–
–
–
9.29
H , 4H
a
10.6
8.97
10.7
H , 5H
a
4.80
–
H , 5H
a
5.26
3.91
6.60
H , 6H
e
4.2
H , 6H
a
H , 6-CH
e
3
DOKLADY CHEMISTRY Vol. 391 Nos. 4–6 2003

trans-DIHYDROXYPIPERIDINES
197
3
,4-hydroxy groups. Thus, it follows that the conforma- Table 2. Stretching vibrations of the hydroxy group in 3,4-di-
–
1
hydroxypiperidines 2–6 and 8–10, ν , cm
tion equilibrium Ä ⇔ Ç for diols 1–6 and 11 is shifted
almost completely toward the diequatorial conformer Ä.
OH
Vibrations of the hydroxyl
involved in involved in
an intermo- an intramo-
lecular bond lecular bond
H
OH
Compound
N
free
R
H
OH
OH
R
H
N
R1
R1
H
OH
1-Me-3-hydroxy- 3050–3580
piperidine
3540
–
3625
3618
A
B
1
-Me-4-hydroxy- 3020–3580
piperidine
The predominant 3e,4e,6a-conformation for 6-
methyl-3,4-dihydroxypiperidine 7 was established in
line with the diequatorial orientation of the hydroxy
2
3
3010–3570 3510 (vw) 3600; 3627
3025–3570 3515 (vw) 3598; 3628
3020–3580 3520 (vw) 3600; 3628
3030–3570 3515 (vw) 3600; 3627
3040–3560 3525 (vw) 3600; 3625
3
3
groups at ë-3 and ë-4 (large J
, J
, and
4H , 5H
3
H , 4H
a
a
4
3
J4H, 5H spin–spin coupling constants, equal to 8.7,
5
3
3
8
.07, and 8.15 Hz, and small J3
and J
con-
6H , 5H
e
6
H , 4H
e
stants, equal to 4.81 and 4.05 Hz, respectively) and
preferentially axial orientation of the 6-methyl group
8
3110–3570
3110–3470
3130–3580
3510
3520
3510
3600, 3633
3615
9
3
3
(the small J
, J
constants correspond to the
5H , 6H
5
H , 6H
10
3605
a
e
equatorial orientation of the proton at ë-6) (Table 1).
The introduction of substituents into the 3(Me) and
The low intensity of this band may be indicative of
essential predominance of conformer A in the Ä ⇔
4
(Me, Ph) positions of the piperidine ring in diols 8–10
Ç
shifts the conformational equilibrium Ä ⇔ Ç toward
the diaxial conformer B. For instance, the diaxial orien-
tation of the 3,4-dihydroxy groups in diol 9 was estab-
lished in double resonance experiments on the basis of
1
equilibrium. This is in line with H NMR data for diols
–6. The situation changes sharply for compounds 9
2
and 10: in particular, in dilute solutions, a clearly
–
1
defined band appears at 3510–3520 cm , which is due
3
3
the J
(3.7 Hz) and J
(2.1 Hz) values, to stretching vibrations of the hydroxy group involved
2
H , 3H
2H , 3H
a
e
in an intramolecular bond. Simultaneously, the long-
wavelength absorption band for the free hydroxyl either
disappears (in the spectra of 9 and 10) or becomes less
intense (in the spectrum of 8). According to the above
which correspond to the equatorial orientation of the
proton at C-3 and, hence, the axial orientation of the
3
-hydroxy group. The orientation of the 4-hydroxy
group should also be axial, as stipulated by the trans-
hydroxylation conditions. The trans-diaxial orienta-
tions of the 3,4-hydroxy groups in diols 8 and 10, con-
taining quaternary centers at C-3 and C-4, were estab-
3
1
H NMR data, the hydroxy groups in diols 8–10 are
diaxial. In this case, the orientation of the axial 3-OH
group is convenient for the formation of a nonstrained six-
membered ring due to an intramolecular hydrogen bond.
lished in a similar way. For diol 8, the J
(4.17 Hz)
H , 3H
a
2
Anti-HIV activity of the hydroxylated pipe-
ridines. The antiviral and cytotoxic properties of trans-
orientation of the proton at C-3 and, hence, the axial hydroxylated piperidines 1–12 were studied. The cyto-
orientation of the hydroxy group at C-3. A similar situ- toxic properties were determined with respect to the
3
and J
(1.94 Hz) values point to the equatorial
2
H , 3H
e
3
transplantable T-lymphoblastoid CEM SS cell line. The
CEM SS cell line is a biologically cloned variant capa-
ble of virus-induced syncytium formation. It is widely
used to study HIV and its inhibitors. The antiviral prop-
erties were studied with respect to the acute infection
model CEM SS/HIV-1-BRU. The reference strain HIV-
1BRU is actively replicated in T-lymphoblastoid cell
lines. To assay the antiviral activity of the piperidine
ation is observed for diol 10 ( J2
, 4.10 Hz;
H , 3H
a
3
J2
, 1.9 Hz). In view of the trans-hydroxylation
H , 3H
e
conditions, the second hydroxy group at C-4 should
also be axial in both diol 8 and diol 10. This pattern of
conformations is supplemented by the data of IR spec-
tra (Table 2).
The IR spectra of diols 2–6 exhibit intense bands at derivatives, the CEM SS cells were incubated at 37°ë
–
1
about 3600 cm (stretching vibrations of the free 4-OH with different concentrations of the preparations (100,
–
1
group) and 3625–3630 cm (vibrations of the free 10, 1, 0.1, and 0.01 µg/ml) and infected with a virus
3
3
3
-OH group) and a low-intensity band at 3510– with a multiplicity of 10 TCID (Tissue Culture Infec-
50
–
1
525 cm . In the spectra of dilute solutions, we tious Dose-50). After 24-hour incubation, the unbound
assigned this band to vibrations of the hydroxy group virus was removed by low-velocity centrifugation
involved in an intramolecular bond with nitrogen [7]. (1000 rpm, 7 min), and the cell precipitate was re-sus-
DOKLADY CHEMISTRY Vol. 391 Nos. 4–6 2003

1
98
GRISHINA et al.
Table 3. Anti-HIV activity, toxicity, and chemotherapeutical index for some piperidine derivatives
%
of infection for the HIV-1BRU cell line
at the concentration (µg/ml)
ED50,
µg/ml
CD50,a Therapeuti-
Compound
µg/ml
cal index
1
00
10
1
0.1
0.01
1
2
3
4
5
6
17.0
93.9
87.7
89.9
87.6
97.7
15.0
93.2
14.0
–
18.0
94.4
80.9
89.7
84.7
98.1
16.0
97.1
48.0
45.0
95.7
100.3
95.2
52.0
95.1
73.0
95.8
87.5
94.4
80.0
73.2
76.0
77.3
37.0
108.7
72.8
89.8
94.9
100.0
84.3
115.1
101.2
91.3
–
–
2.7
>100
>100
>100
>100
>100
2.8
310
195
115
<2
–
190
<2
98.3
–
210
<2
110.6
71.0
–
750
<7.5
<16
460
<10
1080
85
–
1600
1300
1030
650
6
6
7
7
8
9
0
1
2
/1b
/2c
93.0
–
98.0
–
>100
0.6
102.0
109.0
92.6
–
/1b
100
–
8.6
725
93.8
95.7
57.7
21.5
>100
>100
160
410
<4
99.1
–
740
<7
1
1
1
95.8
–
450
3
102.0
101.8
–
12
1100
1670
90
94.8
d
–
>100
<16
400
1000
Stavudine
d
Azidothymidine (AZT)
a
For Sup T1 cells; b as the hydrochloride; c as the 3,4-diacetate; d published data.
pended in a fresh growth medium with the correspond- with diaxial orientation of both hydroxy groups should
ing concentrations of the compounds under study. The also be noted. These data should be taken into account
infection development was monitored for 5 days with in considering the conformation–activity relationships.
estimation of the cytopathogenic effect (cytolysis, syn-
cytium formation), and the concentration of the p24
EXPERIMENTAL
viral antigen in the culture broth was determined using
1
13
enzyme immunoassay (EIA) (Innogenetics, Belgium).
The ç and C spectra were recorded on a Varian
The results of testing of the anti-HIV activity and tox- VXR-400 spectrometer operating at 400 MHz using
icity and the chemotherapeutical index (selectivity CDCl and as the solvent and TMS as the internal stan-
3
index) for some of the compounds are listed in Table 3. dard. The chemical shifts are given in the δ scale. The
The greatest chemotherapeutical indices were noted for GC/MS spectra were measured on an HP-5990 instru-
the leading compounds 6/1 and 7. Judging by published ment with an HP-5972 mass selective detector (a 30 m ×
data, these values are quite comparable with the values 0.2 mm quartz capillary column with the HP-5MS sta-
for anti-HIV preparations that are currently in use.
tionary phase and temperature programming from 70 to
50°ë with a heating rate of 30 K/min). Thin-layer
2
The most important outcome of this study is identi-
fication of a new, promising and relatively nontoxic
group of anti-HIV agents, namely, (±)-trans-3,4-dihy-
droxypiperidines. In the series of these compounds, a
chromatography was carried out on DC-Alufolien-Alu-
mina oxide plates (Merck, Germany). Column chroma-
tography was carried out on columns packed with silica
gel 60 (Merck, Germany). IR spectra were recorded for
certain relationship was found between the structure, saturated solutions in CCl . Then, the solutions were
4
the preferred conformation, and the anti-HIV activity diluted, usually tenfold, until the spectra recorded on a
(
toxicity). Indeed, the variation of the anti-HIV activity UR-20 spectrometer stopped changing. Due to the very
in the “free base–hydrochloride” pairs for diols 6 and 7 low solubility of hydroxylated pyridines in ëCl , the
4
was found to be substantially dissimilar (Table 3); the concentration of the solute was not determined. To
hydrochloride of diol 6 was nearly 30 times more active study the cytotoxic properties, CEM SS cells were cul-
than the free base, whereas the activity of the free base tivated in the presence of different doses of the prepara-
of 7 was 13 times as high as the activity of its hydro- tion for 5–7 days. After completion of the specified
chloride. A sharp increase in the toxicity in the case of period, the cell viability was determined by the MTT
compounds that occur predominantly in a conformation method. This method is based on measuring the ability
DOKLADY CHEMISTRY Vol. 391 Nos. 4–6 2003

trans-DIHYDROXYPIPERIDINES
199
of the cells under study to transform the readily soluble
yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium (MTT) bromide into insoluble intracellular
crystals of MTT formazan. The efficiency of this trans-
formation reflects the general level of dehydrogenase
activity of the cells under study and, within certain lim-
its, is directly proportional to the living cell concentra-
tion. The amount of formazan that can be optically
determined (540 nm) is directly proportional to the
amount of living cells. The cell control containing no
preparation was taken as 100%.
2. Fleet, G.W.J., Karpas, A., Dwek, R.A., et al., FEBS Lett.,
988, vol. 237, pp. 128–132.
1
3. Rajni Garg, Satya P. Gupta, Hua Gao, et al., Chem. Rev.,
1999, vol. 99, pp. 3525–3601.
4. Polt, R., Sames, D., and Chruma, J., J. Org. Chem., 1999,
vol. 64, pp. 6147–6158.
5. Pistia-Brueggeman, G. and Hollingsworth, R.I., Tetrahe-
dron, 2001, vol. 57, pp. 8773–8778.
6. Grishina, G.V., Karamov, E.V., Kornilaeva, G.V., et al.,
RF Patent 2 198 658, Byull. Izobret., 2003, no. 5.
7. Maksimova, T.N., Mochalin, V.B., and Unkovskii, B.V.,
Khim. Geterotsikl. Soedin., 1980, no. 6, pp. 783–786.
1
,2,5,6-TRPs 14–18 and 20–22 were synthesized by
the reduction of the corresponding pyridinium salts
8. Lukes, R., Collect. Czechosl. Chem. Commun., 1947,
vol. 12, pp. 71–74.
(
25 mmol) with sodium tetrahydroborate (60 mmol) in
ethanol (60 mmol) at 0°ë. THPs 23 and 24 were pre-
9. Leonard, N.E. and Gash, V.W., J. Am. Chem. Soc., 1954,
vol. 76, pp. 2781–2784.
pared according to published procedures [14, 15]. The
structures of the resulting compounds 14–18 and 20–22 10. Judin, L.G., Zh. Obshch. Khim., 1957, vol. 27, pp. 3021–
3
024.
data [8–14]. Diols 1–5 and 10 were obtained by oxida- 11. Kost, A.N., Vest. Mosk. Univ., 1956, vol. 11, no. 1,
pp. 209–211.
ide solution in 98% formic acid for seven days at 30°C. 12. Oediger, H. and Joop, N., Justus Liebigs Ann. Chem.,
Diols 6, 7, and 12 were prepared by the oxidation of the
1972, vol. 764, pp. 21–27.
were confirmed by spectroscopy resorting to published
tion of THPs 13–17 and 22 by a 25% hydrogen perox-
corresponding THPs 18, 19, and 24 with trifluoroacetic 13. Schmidle, C.J. and Mansfield, R.C., J. Am. Chem. Soc.,
acid in dichloromethane as described previously [15].
1956, vol. 78, pp. 425–428.
1
4. Chabrier, P., Najer, H., Giudicelli, M., and Joannic, M.,
Bull. Soc. Chim. France, 1957, vols. 11–12,
pp. 1365−1369.
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DOKLADY CHEMISTRY Vol. 391 Nos. 4–6 2003