Synthesis of new cyclitol compounds that influence the activity of phosphatidylinositol 4-kinase isoform, PI4K230

Article abstract of DOI:10.1021/jm001081c

The synthesis, chemical derivatization, and investigation of the inhibitory properties of novel cyclitol derivatives on the phosphatidylinositol 4-kinase enzymes PI4K55 and PI4K230 involved in the phosphatidylinositol cycle are reported. Some of the prepa

Full text of DOI:10.1021/jm001081c

J . Med. Chem. 2001, 44, 627-632
627
Syn th esis of New Cyclitol Com p ou n d s Th a t In flu en ce th e Activity of
P h osp h a tid ylin ositol 4-Kin a se Isofor m , P I4K230
Istva´n F. Pelyva´s,*,§ Zolta´n G. To´th,§ Gyo¨rgy Vereb,# Andra´s Balla,# Elza Kova´cs,§ Andra´s Gorzsa´s,§
Ferenc Sztaricskai,*,§ and Pa´l Gergely#
Research Group of Antibiotics of the Hungarian Academy of Sciences, University of Debrecen, P.O. Box 70,
Debrecen H-4010, Hungary, and Department of Medical Chemistry, Faculty of Medicine, University of Debrecen,
Bem ter 18/ B, Debrecen H-4026, Hungary
Received September 18, 2000
The synthesis, chemical derivatization, and investigation of the inhibitory properties of novel
cyclitol derivatives on the phosphatidylinositol 4-kinase enzymes PI4K55 and PI4K230 involved
in the phosphatidylinositol cycle are reported. Some of the prepared cyclitol derivatives (i.e. 9,
11, 12, and 14) proved to be very powerful and specific irreversible inhibitors of PI4K230 at or
below a concentration of 1 mM.
In tr od u ction
chain chiral cyclitols from carbohydrates and from D-
(-)-quinic acid, as well as their utilization for the
production of pseudodisaccharide-type aminocyclitol
antibiotics.15 To synthesize cyclitol analogues carrying
an exocyclic CdC bond suitable for further derivatiza-
tion, the deoxyinososes 1,16 2,17 and 314 were treated
with methoxycarbonylmethylenetriphenylphosphorane
to obtain the methoxycarbonylmethylenecyclohexanes
4-6, as single geometric isomers (TLC, NMR), in
medium to good yield (Scheme 1). The structure of
Phosphatidylinositol (PtdIns) 4-kinase (PI4K) (EC
2.7.1.67) catalyzes the formation of phosphatidylinositol
4-phosphate (PtdIns-4P), which serves as the starting
material for the biosynthesis of several regulatory
phosphoinositides, being further phosphorylated at the
D-5 and/or D-3 position of the cyclitol ring.1,2 The
diversity of physiological functions of PI4Ks located at
several subcellular compartments can involve different
phosphoinositide pools and might explain3,4 the exist-
ence of the multiple forms of PI4K. According to their
enzymological and immunological properties, domain
structures, and molecular masses, the mammalian
PI4Ks are grouped4 into forms of PI4K55, PI4K92, and
PI4K230. It is supposed that the plasma membrane-
bound PI4K55 is involved in the agonist-stimulated
activation of the phosphatidylinositol pathway, while
other forms, reported to be associated5-7 with intracel-
lular membranes, can participate in the regulation of
cytoskeletal actin-modifying proteins8 and vesicular
transport.9
PI4Ks are the targets of and possess high sensitivity
to several microbial products, such as echiguanine10 and
wortmannin, which is a fungal sterol-like toxin.5-7,11,12
Previously we have demonstrated13 the inhibitory effect
of (2S,3R,5R)-3-azido-2-benzoyloxy-5-hydroxycyclohex-
anone (1) on the enzyme D-myo-inositol-1-phosphate
phosphatase of Neurospora crassa. Supposing structural
similarities in the substrate-recognizing domain of the
inositol (Ins) and PtdIns-metabolizing enzymes, we
decided to test the effect of 1 as well as its derivatives
and structural analogues on the activity of the PI4K
enzymes (PI4K55 and PI4K230) involved in the phos-
phatidylinositol cycle.
1
compounds (E)-4-6 was deduced from the H and 13C
NMR spectra, which showed all of the signals (see
Tables 1 and 2) characteristic of the structural elements
of the exocyclic dCH-COOCH3 function. The E geom-
etry for each of the three products was concluded from
the large (∆δ ) 0.9-1.1 ppm) downfield shift of the
axially oriented H-6 proton as compared to that of the
starting ketones 1-3, indicating that the methoxycar-
bonylmethylene side chain is close to this proton.
Inosose 718 was transformed into the corresponding
branched-chain cyclitol 8 (55%) in an essentially similar
fashion. The NMR data (Tables 1 and 2) substantiated
the development of the side chain, but the doubled
signals clearly indicated the presence of two geometrical
isomers in a ca. 3:2 ratio, which could not be separated
by chromatography.
To obtain substituted and modified derivatives of the
inososes 1-3 for structure-activity relationship (SAR)
studies, the corresponding 5-O-acetyl derivatives 9-11
and the unsaturated ketones 12, 13,14 14, and 1519 were
prepared. To avoid â-elimination of the C-5 hydroxyl
group of the deoxyinososes 1-3, the acetylations were
carried out with the acetic-trifluoroacetic mixed anhy-
dride in dichloromethane. For the preparation of the R,â-
unsaturated ketones 12-15, the desired â-elimination
reaction was facilitated by means of the introduction
and elimination of an 5-O-methanesulfonyl leaving
group. Incorporation of the double bond into the mol-
ecules of the resulting R,â-unsaturated ketones was
shown by the appearance of carbon signals at δ ) 148-
195 ppm and of proton signals in the region δ ) 6.10-
7.20 ppm characteristic of related structures.14
Resu lts a n d Discu ssion
Ch em istr y. In recent papers we have described the
preparation14 of novel azido, anhydro, and branched-
* Corresponding authors. Tel: 36-52-512-900. Fax: 36-52-512-914.
E-mail: antibiotics@tigris.klte.hu.
§ Research Group of Antibiotics.
#
Department of Medical Chemistry.
10.1021/jm001081c CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/17/2001

628 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pelyva´s et al.
Sch em e 1a
a
(a) Ph3PdCHCOOCH3/toluene; (b) (CF3CO)2O/CH3COOH; (c) CH3SO2Cl/pyr.
Sch em e 2
To extend the scale of the samples for the SAR
investigations, the aminodeoxyinosose 16, derived20
from D-glucosamine, the unsaturated ketone 17,17 and
the 3-azidodeoxyinoseses 1817 and 19,15 carrying the
carbonyl group in protected form and prepared earlier
in our laboratory, were employed. The 5-O-acetylated
derivative (20) of the dithioketal 19 was obtained by
means of conventional acetylation of 19 with acetic
anhydride in pyridine.
at δ ) 8.46 ppm, and the carbons of the CCl3 and the
CdNH blocks of the ester moiety were assigned at δ )
137.71 and 161.89 ppm, respectively. Thermal Overman
rearrangement22 of 22 in boiling xylene, or that per-
formed at room temperature in dichloromethane under
mercury(II) trifluoroacetate catalysis, did not give the
desired unsaturated aminocyclitol 23. Instead, a very
small portion of the starting ester 22 was recovered, and
extensive ester hydrolysis of the labile 22 to lead to 21
proceeded under the employed experimental conditions.
The failure of the desired rearrangement may be at-
tributed to the lability of the imidate ester 22 and the
With the goal of synthesizing diazidocyclitol and
diaminocyclitol derivatives, the latter being structurally
related to the neuraminidase enzyme inhibitory amino-
cyclitols GS 4071 and GS 4104,21 the well-known Over-
man rearrangement22 of an appropriate trichloroace-
timidate ester, such as the (1S,5R,6S)-allyl alcohol 21,14
was attempted (Scheme 2). Treatment of 21 with
trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) in dichloromethane gave 97% of the target
trichloroacetimidate ester 22. The incorporation of the
fact that it does not adopt a T6 twist-boat conformation
5
required14 for an easy steric approach of the ester side
chain to the â-position. In contrast, the thermal Claisen
rearrangement of the allyl alcohol 21 (possessing an
undistorted 5T6 twist-boat conformation) with N,N-
dimethylacetamide dimethyl acetal readily provided14
the corresponding branched-chain cyclitol derivative,
carrying a dimethylacetamide side chain, with a com-
1
ester moiety was clearly proved by the H and 13C NMR
spectral data: the proton of the imine function appeared

Synthesis of New Cyclitol Compounds
Ta ble 1. 200-MHz 1H NMR Data for Compounds 4-6, 8-14, 20, 22, 25, and 26
chemical shifts (δ, ppm)a
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 629
compd
solvent
CDCl3
H-1
H-2
H-3
H-4
H-5 H-6
dCH-
OH
2.73
others
-OCH3 3.69
4
5.49
(9.5)
4.07
4.09
1.78(a)
4.35
2.33(e)
5.99
(11.33, 4.75) 2.33(e)
(6.58, 3.29)
4.10
3.97(a)
2.62(e)
3.52(a)
2.64(e)
3.88(a)
5
6
8
acetone-d6
CDCl3
3.90
1.77(a)
2.13(e)
2.04(a)
2.51(e)
4.16
6.15
6.10
2.82
2.57
3.02
3.02
-OCH2Ph 4.67
-OCH3 3.67
-OCH3 3.69
5.81
(8.77)
2.22-3.15 4.47
5.67
4.41
(4.4)
4.04
(9.14, 4.4)
CDCl3
2.22-3.15 5.78
-C(CH3)2 1.33, 1.45
-OCH3 3.58
CDCl3
2.72-3.15 4.49
4.10
3.94
4.70
4.38
4.58
2.22-3.15 5.76
-C(CH3)2 1.33, 1.47
-OCH3 3.70
9
10
11
12
13
14
20
22
25
26
acetone-d6
acetone-d6
CDCl3
5.56
4.18
5.78
4.40
4.02
5.94
2.18(a)
2.42(e)
1.94(a)
2.12(e)
2.20(a)
2.61(e)
2.78(a)
3.05(e)
2.44(a)
2.88(e)
3.01(a)
2.56
2.95
2.46(a)
2.78(e)
2.85
-COCH3 2.10
-COCH3 2.05
-COCH3 2.06
acetone-d6
acetone-d6
acetone-d6
acetone-d6
CDCl3
6.14
(10.0)
6.02
7.15
4.52
5.68
(12.0)
4.49
(6.0, 2.5)
7.00
(12.0, 5.5)
4.50
-OCH2Ph 4.72, 5.08
6.21
(9.87)
5.39
(9.13)
5.76
7.20
5.81
6.01
(2.56, 5.85) 3.24(e)
4.16 2.22
(10.92, 4.34) 1.98
(9.50, 5.85, 11.70) (11.70)
5.12
2.63
-S(CH2)2S- 3.18, 3.34
-COCH3 2.08
-OCH2Ph 4.87
-CdNH 8.46
5.68
(7.0)
5.76
2.14(a)
2.54(e)
3.61
3.80
3.69
(10.5)
3.64
(10.5, 6.0)
1.90-2.07
(3.5)
acetone-d6 3.92
(3.5)
acetone-d6 3.60
4.02
(8.0)
4.20
3.80
(9.0)
3.78
4.28, 4.51 -OCH2Ph 4.86
(2.5)
3.54-3.62 1.92 1.98
3.54-3.62
4.41, 4.48 -OCH2Ph 4.79
(9.5, 9.5)
a
Coupling constants (in parentheses) are given in Hz.
Ta ble 2. 200-MHz 13C NMR Data for Compounds 4-6, 8, 14, 22, 25, and 26
chemical shifts (δ, ppm)
compd
solvent
CDCl3
C-1
C-2
C-3
C-4
C-5
C-6
dCH-
others
4
151.42
77.14
61.62
35.20
66.13
36.75
115.86
-OCH3 51.50
-CdO 165.08, 167.04
-OCH3 51.25
5
acetone-d6
155.31
84.10
63.40
35.54
66.57
37.39
116.98
-OCH2Ph 73.01
-CdO 167.15
6
8
CDCl3
CDCl3
CDCl3
151.21
155.27
155.08
75.64
31.90
29.47
72.43
72.20
71.91
35.38
76.51
76.65
66.63
67.65
68.35
36.71
35.35
36.13
117.28
116.62
117.10
-OCH3 51.67
-CdO 165.50, 165.62, 167.07
-OCH3 50.79
-C(CH3)2 24.22, 26.62
-OCH3 50.80
-C(CH3)2 24.38, 26.89
12
14
20
acetone-d6
acetone-d6
acetone-d6
170.24
192.14
67.05
148.87
148.08
80.02
134.57
134.26
60.58
32.50
32.11
34.38
61.05
71.17
68.83
79.26
77.59
44.14
-COCH3 21.34
-COCH3 165.58
-S(CH2)2S- 39.34, 41.77
-OCH2Ph 74.79;
-CdNH 161.89
-CCl3 137.71
-OCH2Ph 75.11
-OCH2Ph 73.37
22
CDCl3
80.93
128.36
124.18
31.11
60.26
79.90
25
26
acetone-d6
acetone-d6
82.25
67.94
72.39
78.49
71.69
70.36
60.54
57.99
30.38
30.21
60.55
67.93
plete chirality transfer from carbon C-1 of the enol to
the â-position.
isolated by means of column chromatography. The large
values of the J 2,3 and J 3,4 coupling constants (8 and 9
3
Hz, respectively) indicated the C6 chair conformation
As an alternative route for synthesizing diazido (and
thus diamino) cyclitols, (1R,2S,3R,5S,6R)-3-azido-2-
benzyloxy-5,6-anhydrocyclohexan-1-ol (24) prepared14
earlier in our laboratory was applied. We have re-
ported14 that treatment of 24 with allyl alcohol in the
presence of boron trifluoride etherate resulted in a
diaxial opening of the oxirane ring with excellent
regiospecificity and yield.
of the major product 25, which carries the newly
introduced 1-OH and 6-N3 groups in a trans-diaxial
relationship (J 1,2 ) 3.5 Hz, J 1,6 ) 2.5 Hz, and J 5,6 ) 3.5
3
Hz). The minor product 26 also exists in the C6 chair
conformation, and this compound is a result of the trans-
diequatorial ring opening (J 1,2 ) J 1,6 ) 9.5 Hz) of the
oxirane function of 24 with sodium azide.
When the anhydro ring of 24 was opened with sodium
azide in hot N,N-dimethylformamide, a ca. 13:1 mixture
of the 4,6-diazido- (25) and 1,4-diazidocyclitol (26) with
1S,2S,3S,4R,6R and 1R,2S,3S,4R,6S configuration, re-
spectively, was obtained and the pure isomers could be
Biology. The cyclitol derivatives influenced the activ-
ity of the enzyme PI4K230 very differently. Some
compounds, such as 9 and 11, proved to be powerful
inhibitors at a concentration of 1 mM exerting a clear-
cut time-dependent inhibition (Figure 1). Other sub-

630 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pelyva´s et al.
appear to be critical in the inhibition of the enzyme
PI4K230 (compare e.g. the data in entries 5, 7; 11, 12;
16, 17; and 18, 19). (d) Acetylation of the 5-OH hydroxyl
group (see compounds 9 and 11 in entries 16 and 17) or
elimination of the C-5 substituent, resulting in the
cyclitols 14 and 12 with a conjugated unsaturated
system (entries 18 and 19), furnished the inosose
derivatives possessing the highest inhibitory activity.
Con clu sion
The prepared cyclitol compounds influenced the activ-
ity of the phosphatidylinositol 4-kinase enzyme PI4K230
very differently. The observed time-dependent inhibition
suggests an irreversible chemical modification of the
enzyme decreasing its catalytic activity. The most
powerful inhibitors contain a C-1 oxo, a C-2 benzoyloxy,
and a C-5 acetoxy group or an unsaturated (cyclohexene)
ring, suggesting the role of the multiple conjugated
unsaturated system providing a close to planar struc-
ture for the inhibitory compounds. Quite interestingly,
another type of the PI4K enzymes (such as PI4K55) do
not show sensitivity to the cyclitol compounds which
inactivated PI4K230. According to this finding, the
inhibitory effect of the reported new cyclitol derivatives
seems to be rather specific for PI4K230.
F igu r e 1. Time dependency of the inhibiton of PI4K230 with
the cyclitol derivatives. The PI4K230 preparation was incu-
bated with 1 mM cyclitol derivatives at 25 °C. At the indicated
time points the samples were supplemented with the sub-
strate, and the remaining activities were determined as
described in the Experimental Section. The activities are
expressed as a percent (%) of the appropriate controls run
without the cyclitol derivatives. The compounds are numbered
as shown in the schemes.
Ta ble 3. Inactivation of the Enzyme PI4K230 by 19 Selected
Cyclitol Derivativesa
The structural basis of this difference is not clear
since the amino acid sequence of PI4K55 is not yet
known. However, several lipid kinases show structural
similarity to PI4K230, such as PI4K92, type II â-phos-
phatidylinositol phosphate kinase, and phosphoinositide
3-kinases.23,24 Among them PI4K92, PI4K230, and
especially phosphoinositide 3-kinases proved to be
sensitive to inhibition by wortmannin, while PI4K55 is
not sensitive to this microbial toxin.3,25
Supposing similarities in the accessibility to wort-
mannin and cyclitol derivatives, our compounds might
efficiently influence hormonal and mitogenic signaling
mediated by phosphoinositide 3-kinases25 or the func-
tions of PI4K92 exerted in Golgi complex,26 as well. It
is noteworthy that PI4K230 seems to be involved
predominantly in the vesicular traffic of neuronal
cells;27,28 however, its exact role has not yet been
clarified.
remaining
activity (%)
remaining
activity (%)
entry
compd
entry
compd
1
2
3
4
5
6
7
8
9
19
20
2
13
3
10
1
7
96
96
85
85
84
82
80
80
70
66
11
12
13
14
15
16
17
18
19
6
4
64
63
57
56
51
20
10
8
15
16
17
11
9
14
12
5
18
5
10
a
The PI4K230 preparation was incubated with 1 mM of the
compounds for 10 min at 25 °C, and the remaining activity was
determined immediately as described in the Experimental Section.
stances (e.g. 1 and 3) caused only about 20% inhibiton
without any consistent time dependency. A third group
possessed about 40-50% largely time-dependent inhibi-
tion such as 4 and 17. Finally, some compounds were
ineffecive within a 5% limit. The inhibition exerted by
the compounds showed a concentration dependence, as
well (not documented). To compare the efficiency of
various derivatives the inactivation was performed
regularly for 10 min.
By comparing the structure and the inhibitory effect
of 19 selected cyclitols (Table 3) the following conclu-
sions can be drawn. (a) The presence of a keto functional
group, or an other group attached with a double bond
to the cyclohexane ring, is a prerequirement for inhibi-
tion (see entries 3-18). Thus, the inososes 19 and 20
carrying the C-1 keto functions in a protected, dithioket-
al form are completely ineffective (entries 1 and 2). (b)
An ester group (benzoyl or acetyl) attached to the 2-OH
function can greatly enhance the inhibitory power, while
the inosose derivatives carrying an ether substituent
(e.g. a benzyl group) at the same position are not so
effective. This is most significantly demonstrated by the
huge differences between the inhibitory potencies of 12
and 13 (entries 19 and 4) or 9 and 10 (entries 17 and
6). (c) The substituents at carbons C-3 and C-4 do not
Exp er im en ta l Section
Ch em istr y. Gen er a l. Melting points were determined on
a Kofler hot-stage apparatus and are uncorrected. Optical
rotations were measured with a Perkin-Elmer 283 B instru-
ment. 1H (200 MHz) and 13C NMR spectra (50.3 MHz) were
recorded with a Bruker 200 SY spectrometer (internal TMS).
Mass spectra were recorded with AEI-MS 902 and VG-7035
instruments. TLC and column chromatography were per-
formed on Kieselgel 60 F254 (Merck) and silica gel 60 (Merck),
using (A) 1:1 hexanes-ethyl acetate, (B) 6:4 hexanes-ethyl
acetate, (C) 7:3 hexanes-ethyl acetate, (D) 9:1 dichlo-
romethane-methanol, (E) 9:1 toluene-methanol, (F) 10:0.1
dichloromethane-methanol, (G) 8:2 hexanes-ethyl acetate,
(H) 3:7 hexanes-ethyl acetate as the developing system/eluent.
Evaporations were carried out under diminished pressure at
35-40 °C (bath temperature).
(2S,3R,5R)-3-Azid o-2-b en zoyloxy-5-h yd r oxy-1-(m et h -
oxyca r bon ylm eth ylen e)cycloh exa n e (4). A mixture of the
azidodeoxyinosose 116 (102.5 mg, 0.37 mmol) and methoxycar-
bonylmethylenetriphenylphosphorane (150 mg, 0.44 mmol) in
dry toluene (3.0 mL) was stirred at 70 °C for 23 h, when TLC
(A) showed that all of the starting 1 had disappeared. The
reaction mixture was evaporated to dryness and the residue

Synthesis of New Cyclitol Compounds
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 631
purified by flash-column chromatography (B) to obtain pure
crystalline 4 (99.7 mg, 81.3%): mp 91-93 °C; [R]2D0 ) -127 (c
) 0.77 CHCl3); Rf 0.36 (A). Anal. (C16H17N3O5) C, H, N.
(2S,3R,5R)-3-Azid o-2-ben zyloxy-5-h yd r oxy-1-(m eth oxy-
ca r bon ylm eth ylen e)cycloh exa n e (5). To a stirred solution
of 215 (96.1 mg, 0.37 mmol) in dry toluene (3.5 mL) methoxy-
carbonylmethylenetriphenylphosphorane (156.1 mg, 0.47 mmol)
was added, the mixture was refluxed for 2 h, and then stirred
at 70 °C for 22 h. Following evaporation, the residue was
purified by column chromatography (AfC) to obtain 53.0 mg
(45.3%) of pure crystalline 5: mp 68-69 °C; [R]2D0 ) -154 (c )
1.0 CHCl3); Rf 0.72 (A), 0.24 (C). Anal. (C16H19N3O4) C, H, N.
(2S,3R,5R)-2,3-Dib en zoyloxy-5-h yd r oxy-1-(m et h oxy-
ca r bon ylm eth ylen e)cycloh exa n e (6). A mixture of 317 (100
mg, 0.28 mmol) and methoxycarbonylmethylenetriphenyl-
phosphorane (114 mg, 0.34 mmol) in dry toluene (2.0 mL) was
stirred at 50 °C for 16 h, when TLC (A) showed that all of the
starting material had disappeared. The reaction mixture was
concentrated and the residue was submitted to flash-column
chromatography to furnish pure crystalline 6 (100 mg,
87.2%): mp 127-129 °C; [R]2D0 ) -91 (c ) 0.74 CHCl3); Rf
0.44 (A). Anal. (C23H22O7) C, H, N.
(3R,4S,5R)-3,4-Isopr opyliden edioxy-5-h ydr oxy-1-(m eth -
oxyca r bon ylm eth ylen e)cycloh exa n e (8). A mixture of 718
(434 mg, 2.33 mmol) and methoxycarbonylmethylenetri-
phenylphosphorane (935 mg, 4.79 mmol) in dry toluene (10.0
mL) was stirred at 100 °C until all of the starting material
had disappeared [19 h, TLC (D)]. The reaction mixture was
then concentrated, and the residue was submitted to flash-
column chromatography (D) to obtain pale yellow syrupy 8
(310 mg, 55%), as a 3:2 mixture of two isomers: Rf 0.62 (D).
Anal. (C12H18O5) C, H, N.
chloride (0.1 mL) and then the reaction mixture was allowed
to warm to room temperature. After 4-5 h, it was diluted with
dichloromethane (30 mL) and washed with saturated aqueous
NaHCO3 solution (2 × 15 mL) and water (2 × 15 mL), dried
(MgSO4) and concentrated. Coevaporation with dry toluene (2
× 15 mL), followed by column chromatography (dichlo-
romethane) resulted in pure crystalline 14 (279 mg, 98%): mp
106-108 °C; [R]2D0 ) -146.5 (c ) 1.08 CHCl3); Rf 0.76 (F).
Anal. (C20H16O5) C, H.
(2S,3R,5R)-3-Azid o-5-a cet oxy-2-b en zoyloxycycloh ex-
a n on e-eth ylen ed ith ioa ceta l (20). To a cold solution of 1915
(697 mg, 0.2 mmol) in dry pyridine (5.0 mL) acetic anhydride
(21 mL, 0.22 mmol) was added, and the solution was stirred
at room temperature for 24 h. It was then diluted with
dichloromethane (15 mL), washed with saturated aqueous
NaHCO3 solution (2 × 10 mL) and water (2 × 5 mL), dried
(MgSO4) and concentrated to afford pure syrupy 20 (52.3 mg,
68%): mp 120-121 °C. Anal. (C17H19N3O4S2) C, H, N, S.
(1S,5R,6S)-5-Azid o-6-b en zyloxy-1-(t r ich lor oa cet im i-
d oyloxy)cycloh ex-2-en e (22). To a stirred solution of 2114
(590 mg, 2.40 mmol) in dry dichloromethane (25 mL) trichlo-
roacetonitrile (2.41 mL, 24.04 mmol) and DBU (179 mL, 1.202
mmol) were added. The colorless mixture first became yellow
and then brown, and no starting material was detected after
3 h by TLC (E). It was concentrated and the residue chro-
matographed (E containing 1% of triethylamine) to give 913.4
mg (97%) of pure syrupy 22: Rf 0.80 (E); [R]20 +94.5 (c ) 0.93
CHCl3). Anal. (C15H15N4O2Cl3) C, H, N, Cl. D
P r ep a r a tion of (1S,2S,3S,4R,6R)-3-Ben zyloxy-4,6-d ia zi-
d o-1,2-d ih yd r oxycycloh exa n e (25) a n d (1R,2S,3S,4R,6S)-
3-Ben zyloxy-1,4-d ia zid o-2,6-d ih yd r oxycycloh exa n e (26).
A stirred mixture of the anhydrocyclitol 2414 (190.5 mg, 0.73
mmol) and sodium azide (142.2. mg, 2.18 mmol) in dry N,N-
dimethylformamide (7 mL) was boiled under reflux. After
completion of the reaction (3 h) the solvent was distilled off
by means of coevaporation with toluene. The residue was
dissolved in dichloromethane (15 mL) and the solution washed
with water (2 × 5 mL). The organic layer was dried (MgSO4),
concentrated, and the residue was submitted to column
chromatography (GfB) to give, as the major product, crystal-
line 25 (145.2 mg, 65.4%) and syrupy 26 (10.6 mg, 4.8%) as
(2S,3R,5R)-3-Azid o-2-b en zoyloxy-5-a cet oxycycloh ex-
a n e (9). To a cold (0 °C) solution of trifluoroacetic anhydride
(3.2 mL, 23.0 mmol) and glacial acetic acid (0.6 mL, 10.48
mmol) the deoxyinosose 116 (400 mg, 1.45 mmol) was added
and the reaction mixture was stirred for 16 h at room
temperature. It was then poured into dilute aqueous NaHCO3
solution and extracted with chloroform (2 × 15 mL). The
organic layer was washed with water (2 × 10 mL), dried
(Na2SO4) and concentrated. The residual syrupy product was
purified by means of flash-column chromatography (E) to
the minor component. Compound 25: mp 82-83 °C; [R]D20
)
afford pure
(C15H15N3O5) C, H, N.
9
(300 mg 65%): mp 115-117 °C. Anal.
+24.2 (c ) 1.02 CHCl3); Rf 0.6 (H). Anal. (C13H16N6O3) C, H,
N. Compound 26: [R]D20 ) +10.2 (c ) 0.8 CHCl3); Rf 0.8 (H).
Anal. (C13H16N6O3) C, H, N.
(2S,3R,5R)-3-Azid o-2-b e n zyloxy-5-a ce t oxycycloh e x-
a n e (10). Acetylation of 215 (366 mg, 1.20 mmol) with the
mixed anhydride generated from trifluoroacetic anhydride (3.2
mL, 23.0 mmol) and glacial acetic acid (0.6 mL, 10.08 mmol)
was carried out as described above for 9 to obtain, after column
chromatography, amorphous 10. Anal. (C15H17N3O4) H, N; C:
calcd, 59.35; found, 58.33.
(2S,3R,5R)-2,3-Diben zoyloxy-5-acetoxycycloh exan e (11).
Acetylation of 317 (105 mg, 0.29 mmol) with the mixed
anhydride generated from trifluoroacetic anhydride (0.6 mL,
4.31 mmol) and glacial acetic acid (0.12 mL, 2.09 mmol) was
carried out as described above for 9 to give, after column
chromatography, crystalline 11: mp 128-130 °C. Anal.
(C22H20O7) H; C: calcd, 66.60; found, 65.33.
En zym e In h ibitor y Assa y. P r ep a r a tion of P I4K55 a n d
P I4K230. The enzymes PI4K55 and PI4K230 were isolated
and purified from bovine brain membranes as described by
Gehrmann et al.29 The specific activity of the PI4K230
preparation was 3000 U‚mg-1 and SDS-PAGE showed a
major protein band of 200 kDa. The PI4K55 preparation
showed a specific activity of about 40 U‚mg-1 and was free
from other isoforms as revealed by using immunoblots.27
Assa y of P I4K Activity. PI4K activity was routinely tested
at 25 °C in a 38-µL volume containing 0.83 mg/mL PtdIns, 5
mM [γ32-P]ATP (400-800 Bq/nmol), 27 mM MgCl2, 116 mM
KCl, 116 mM HEPES/KOH, 1 mM EDTA, 1 mM EGTA, 1 mM
DTE and 0.4% Triton X-100, pH 7.5, as described by Varsa´nyi
et al.30 One unit of enzyme activity was defined as the amount
of the enzyme which catalyzes the formation of 1 nmol PtdIns-
4P/min at 25 °C.
In a ctiva tion of P I4Ks w ith Cyclitol Der iva tives. The
cyclitol derivatives were dissolved in acetonitrile or dimethyl
sulfoxide at a concentration of 100 mM. This resulting solution
was further diluted with ethylene glycol to 26 mM. Routinely,
1 µL of the cyclitol derivative was added to 25 µL of the PI4K
preparation (10-30 U/mL in 10 mM K-phosphate/150 mM
NaCl/0.5 mM dithioerythritol/0.05% Triton X-100, pH 7.0) and
incubated for 10 min at 25 °C. Then the reaction mixture was
supplemented with the substrate solution and incubated for
a further 5-10 min at 25 °C to determine the residual activity.
The composition of the inactivation reaction was routinely 1
mM cyclitol derivative, 1% acetonitrile or DMSO, and 3%
ethylene glycol. By varying the concentrations of the inhibitory
(5R,6S)-5-Azid o-6-ben zoyloxy-2-cycloh exen -1-on e (12).
To a stirred, cold (0 °C) solution of 116 (0.25 g, 0.908 mmol) in
dry pyridine (10 mL) was added dropwise methanesulfonyl
chloride (0.10 mL, 1.20 mmol) and then the reaction mixture
was allowed to warm to room temperature. After 2 h, it was
diluted with dichloromethane (10 mL) and washed with
saturated aqueous NaHCO3 solution (2 × 10 mL) and water
(2 × 10 mL), dried (Na2SO4) and concentrated. Coevaporation
with dry toluene (2 × 15 mL) resulted in crude syrupy 12,
which was submitted to column chromatography (C) to furnish
pure syrupy 12 (0.185 g, 79%): [R]2D0 ) -124.4 (c ) 3.3
CHCl3); Rf 0.6 (C); IR λmax (KBr) 2106 (N3), 1694 (CdO) cm-1
Anal. (C13H11N3O3) C, H, N.
.
(5R,6S)-5,6-Diben zoyloxy-2-cycloh exen -1-on e (14). To
a stirred, cold (0 °C) solution of 317 (300 mg, 0.85 mmol) in
dry pyridine (15 mL) was added dropwise methanesulfonyl

632 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pelyva´s et al.
(15) Pelyva´s, I. F.; Puska´s, M. M.; To´th, Z. G.; Varga, Z.; Hornya´k,
M.; Batta, G.; Sztaricskai, F. Synthesis of new pseudodisaccha-
ride aminoglycoside antibiotics from carbohydrates. J . Antibiot.
1995, 48, 683-695.
(16) Pelyva´s, I. F.; Sztaricskai, F.; Bogna´r, R. Novel approach to
aminocyclitol analogues from azidotrideoxyhex-5-enopyrano-
sides. J . Chem. Soc., Chem. Commun. 1984, 104-105.
(17) La´szlo´, P.; Pelyva´s, I. F.; Sztaricskai, F.; Szila´gyi, L.; Somogyi,
AÄ . Novel aspects of the Ferrier carbocyclic ring-transformation
reaction. Carbohydr. Res. 1988, 175, 227-239.
compounds the composition of the solvent mixture was kept
constant. The control experiments carried out without the
cyclitol compounds resulted in 10-15% loss of the enzyme
activity during the experimental period. The enzyme activities
were expressed as a percent (%) of the appropriate controls.
The reactions were run in duplicate or triplicate, and the
average of two or three independent experiments was calcu-
lated. SEM was less than 5% of the means.
(18) Trost, B. M.; Romero, A. G. Synthesis of optically active
isoquinuclidines utilizing a diastereoselectivity control element.
J . Org. Chem. 1986, 51, 2332-2342.
(19) Audia, J . E.; Boisvert, L.; Patten, A. D.; Villalobos, A.; Danishef-
sky, S. J . Synthesis of two useful, enantiomerically pure deriva-
tives of (S)-4-hydroxy-2-cyclohexenone. J . Org. Chem. 1989, 54,
3738-3740.
(20) Ma´di-Puska´s, M.; Pelyva´s, I. F.; Bogna´r, R. Carbocyclic ring
closure of an aminodeoxy hex-5-enopyranoside. J . Carbohydr.
Chem. 1985, 4, 323-331.
Ack n ow led gm en t. This work was financially sup-
ported by the National Research Foundation (Budapest,
Hungary) with Grants OTKA T029027, T029075,
T017695, and T026541 and also by the Hungarian
Ministry of Education (MKM FKP 0784/1977). We
thank Mrs. J . Hunyadi for her excellent technical
assistance.
(21) Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.;
Swaminathan, S.; Bischofberger, N.; Chen, M. S.; Mendel, D.
B.; Tai, C. Y.; Laver, W. G.; Stevens, R. C. Influenza Neuramini-
dase inhibitors possessing a novel hydrophobic interaction in the
enzyme active site: Design, synthesis, and structural analysis
of carbocyclic sialic acid analogues with potent anti-influenza
activity. J . Am. Chem. Soc. 1997, 119, 681-690.
(22) Overman, L. E. A general method for the synthesis of amines
by the rearrangement of allylic trichloroacetimidates. 1,3-
Transposition of alcohol and amine functions. J . Am. Chem. Soc.
1976, 98, 2901-2910.
(23) Rao, V. D.; Misra, S.; Boronenkov, I. V.; Anderson, R. A.; Hurley,
J . H. Structure of type II â-phosphatidylinositol phosphate
kinase: a protein kinase fold flattened for interfacial phospho-
rylation. Cell 1998, 94, 829-839.
(24) Walker, D. H.; Perisic, O.; Rled, C.; Stephens, L.; Williams, R.
L. Structural insights into phosphoinositide 3-kinase catalysis
and signaling. Nature 1999, 402, 313-318.
(25) Wymann, M. P.; Pirola, L. Structure and function of phospho-
inositide 3-kinases. Biochim. Biophys. Acta 1998, 1436, 127-
150.
(26) Godi, A.; Pertile, P.; Meyers, R.; Marra, P.; Di Tullio, G.; Iurisci,
C.; Luini, A.; Corda, D.; De Matteis, M. A. ARF mediates
recruitment of PtdIns-4-OH kinase-â and stimulates synthesis
of PtdIns(4,5)P2 on the Golgi complex. Nat. Cell Biol. 1999, 1,
280-287.
(27) Balla, A.; Vereb, G.; Gu¨lkan, H.; Gehrmann, T.; Gergely, P.;
Heilmeyer, L. M. G., J r.; Antal, M. Immunohistochemical
localisation of two phosphatidylinositol 4-kinase isoforms, PI4K230
and PI4K92, in the central nervous system of rats. Exp. Brain.
Res. 2000, 134, 279-288
(28) Zo´lyomi, A.; Zhao, X.; Downing, G. J .; Balla, T. Localization of
two type III phosphatidylinositol 4-kinase enzyme mRNAs in
the rat. Am. J . Physiol. Cell. Physiol. 2000, 278, C914-C920.
(29) Gehrmann, T.; Vereb, G.; Schmidt, M.; Klix, D.; Meyer, H. E.;
Varsa´nyi, M.; Heilmeyer, L. M. G., J r. Identification of a 200
kDa polypeptide as type 3 phosphatidylinositol 4-kinase from
bovine brain by partial protein and cDNA sequencing. Biochim.
Biophys. Acta 1996, 1311, 53-63.
Refer en ces
(1) Fruman, D. A.; Meyers, R. E.; Cantley, L. C. Phosphoinositide
kinases. Annu. Rev. Biochem. 1998, 67, 481-507.
(2) Martin, T. F. Phosphoinositide lipids as signaling molecules:
common themes for signal transduction, cytoskeletal regulation,
and membrane trafficking. Annu. Rev. Cell. Dev. Biol. 1998, 14,
231-264
(3) Balla, T. Phosphatidylinositol 4-kinases. Biochim. Biophys. Acta
1998, 1436, 69-85.
(4) Gehrmann, T.; Heilmeyer, L. M., J r. Phosphatidylinositol 4-ki-
nases. Eur. J . Biochem. 1998, 253, 357-370.
(5) Nakagawa, T.; Goto, K.; Kondo, H. Cloning and characterization
of a 92 kDa soluble phosphatidylinositol 4-kinase. Biochem. J .
1996, 320, 643-649.
(6) Nakagawa, T.; Goto, K.; Kondo, H. Cloning, expression, and
localization of 230-kDa phosphatidylinositol 4-kinase. J . Biol.
Chem. 1996, 271, 12088-12094.
(7) Meyers, R.; Cantley, L. C. Cloning and characterization of a
wortmannin-sensitive human phosphatidylinositol 4-kinase. J .
Biol. Chem. 1997, 272, 4384-4390.
(8) Hinchliffe, K.; Irvine, R. Inositol lipid pathways turn turtle.
Nature 1997, 390, 123-124.
(9) De Camilli, P.; Emr, S. D.; McPherson, P. S.; Novick, P.
Phosphoinositides as regulators in membrane traffic. Science
1996, 271, 1533-1539.
(10) Nishioka, H.; Sawa, T.; Nakamura, H.; Iinuma, H.; Ikeda, D.;
Sawa, R.; Naganawa, H.; Hayashi, C.; Hamada, M.; Takeuchi,
T. Isolation and structure determination of novel phosphatidyli-
nositol kinase inhibitors, echiguanines A and B, from Strepto-
myces sp. J . Nat. Prod. 1991, 54, 1321-1325.
(11) Downing, G. J .; Kim, S.; Nakanishi, S.; Catt, K. J .; Balla, T.
Characterization of a soluble adrenal phosphatidylinositol 4-ki-
nase reveals wortmannin sensitivity of type III phosphatidyli-
nositol kinases. Biochemistry 1996, 35, 3587-3594.
(12) Balla, T.; Downing, G. J .; J affe, H.; Kim, S.; Zolyomi, A.; Catt,
K. J . Isolation and molecular cloning of wortmannin-sensitive
bovine type III phosphatidylinositol 4-kinases. J . Biol. Chem.
1997, 272, 18358-18366.
(13) Szabo´, G.; Schablik, M.; Kiss, AÄ .; Koncz, C.; Bogna´r, R.; Pelyva´s,
I. F.; Sztaricskai, F. A new cyclitol derivative influences inositol
metabolism in Neurospora crassa. FEMS Microbiol. Lett. 1991,
82, 15-18.
(30) Varsa´nyi, M.; Messer, M.; Brandt, N. R. Intracellular localization
of inositol-phospholipid-metabolizing enzymes in rabbit fast-
twitch skeletal muscle. Can D-myo-inositol 1,4,5-trisphosphate
play a role in excitation-contraction coupling? Eur. J . Biochem.
1989, 179, 473-479.
(14) To´th, Z. G.; Pelyva´s, I. F.; Szegedi, C.; Benke, P.; Magyar, E.;
Miklovicz, T.; Batta, G.; Sztaricskai, F. Synthesis of new anhydro
and branched-chain cyclitols. Carbohydr. Res. 1997, 300, 183-
189.
J M001081C