Chapter 15
Synthesis and Biological Evaluation of Inositol Derivatives as Inhibitors of Phospholipase C 1
2
Franz Kaufmann, D. James R. Massy , Wolfgang Pirson, and Pierre Wyss Pharmaceutical Research Department, F. Hoffmann—La Roche Limited, Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: May 30, 1991 | doi: 10.1021/bk-1991-0463.ch015
CH-4002 Basel, Switzerland
The synthesis and biological evaluation of analogues of phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2) is described. Inositol derivatives with and without homologation at C(1) and with and without ionic groups (phosphate or sulfate) at C(4) and C(5) were prepared. In all these compounds, palmitate ester groups were introduced in place of the diacylglyceryl group of PI or PIP2. All compounds with ionic groups (sulfates or phosphates) were found to inhibit phospholipase C (PLC) in vitro significantly. In contrast, compounds without ionic groups showed only a slight degree of inhibition. Some of the compounds with ionic groups have also been tested for PLC inhibition in intact cells and were found to be inactive in this assay. Introduction.- A number of studies have documented the pivotal role of myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) in the intracellular signalling pathway [1-4]. Thus, it has been established that extracellular signals can trigger the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to IP3 and DG, a reaction which is catalysed by phospholipase C (PLC) in the cell membrane. It seems likely that this initial event is associated with both inflammatory responses via the arachidonic-acid cascade [5] and the regulation of cell proliferation [6]. As part of a programme aimed at obtaining substances controlling the functions of the second messengers mentioned above, we have synthesised a number of compounds, analogues of both phosphatidylinositol (PI) and PIP2, as potential inhibitors of PLC. All compounds described are either meso or racemic mixtures. In the latter case the structures portrayed here correspond, for convenience, to one enantiomer, but the DL-form is implied. Numbering and nomenclature are in accordance with IUPAC conventions [7], except for the abbreviations PI, PIP2, IP3, and DG which are based on the 1984 Chilton Conference system [8].
1
Current address: School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, England Corresponding author
2
0097-6156/91/0463-0202S06.00/0 ©1991 American Chemical Society In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
15.
inositol Derivatives as Inhibitors of Phospholipase C 203
K A U F M A N N ET A L
PLC
° Y ^ ( C H (HO) (0)PŒ
)
1
5CH3
"OH
2
OP(0)(OH)
2
PIP Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: May 30, 1991 | doi: 10.1021/bk-1991-0463.ch015
2
2
OP(0)(OH).
k
(ΗΟ) (0)Ρσ** ^ 2
'"OH OP(0)(OH)
IP3
°Y^(CH ) 2
2
1 5
CH
3
Ο CXB
We report here the synthesis and biological evaluation of PI and PIP2 analogues in which a CH2 group isosterically replaces the oxygen attached to C-l of the inositol ring, and the lipid moiety is replaced by a palmitoyloxy group attached to this CH2 group. The function of PLC being to cleave a lipid/phosphate bond, we anticipated that such homologated inositol carboxylates would be effective inhibitors. To ascertain the importance of the PIP2 phosphate groups at C(4) and C(5), we prepared and evaluated C(l) homologated myo-inositol palmitates both with and without ionic groups (phosphates or sulfates) at C(4) and C(5), (4, 5 and 6). The corresponding chiroinositol compounds were also synthesised and evaluated (1,2 and 3) as well as two nonionic deoxy /wyo-inositol compounds (7 and 8). Finally, wyo-inositol palmitates with ionic groups at C(4) and C(5) but without homologation at C(l), were prepared for comparison (9 and 10). Biological evaluation.- 7. Inhibition of phospholipase C (PLC) in vitro. Phospholipase C was prepared from human platelets as crude cytosolic enzyme obtained after sonication and centrifugation (100000 χ g supernatant) of platelets isolated fromfreshhuman blood. This enzyme preparation was stored in aliquots at -75° C. Phospholipase C activity was determined by measuring the hydrolysis of phos-
In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
204
INOSITOL PHOSPHATES
HO
HO
OH
AND DERIVATIVES
OH [CH OCO(CH ) CH< 2
RO
J CH OCO(CH ) CH OH 2
2
1 4
OH
R=Η
4
R=H
2
R= SQjNa
5
FUSOaNa
3
R = Ρ(0)(ΟΗ) · 1 . 6 Μ θ Ν
6
Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: May 30, 1991 | doi: 10.1021/bk-1991-0463.ch015
HO
2
HO
l_JÇH OCO(CH )
R
R - Ρ(0)(ΟΗ) · 1.6Μθ Ν
2
1 4
CH
R = H,
8
R = OH,
OH
LJ0C0(CH ) CH 2
1 4
3
3
OH
1
7
3
3
R 2
14
3
1
2
2
R =OH 1
R =H
9 10
R = S03Na Ρ = Ρ(0)(ΟΗ) · 2 . 5 Μ θ Ν 2
3
phatidyl-[^H]inositol (PI) as a substrate at pH 5.5 without any detergent. Watersoluble ^H-inositolphosphate was determined as product in the water phase after extraction of ^H-PI with chloroform. The assay was carried out in 0.1 ml for 10 min at 37°C in the presence of 100 mM Tris-acetate, pH 5.5, 0.5 mM CaCl2 and 250 μΜ sonicated 3 -PI. The substrate suspension was prepared by mixing ^H-phosphatidylinositol (^H-PI from Amersham) and unlabelled PI (Sigma), removing the solvent chloroform under N2, and sonicating the residue in 200 mM Tris-acetate buffer, pH 5.5, containing 1 mM CaCl2 for 20 min. For the assay, 10 ml DMSO (with or without test compound) was mixed with 40 uL of enzyme solution (containing about 10 μg protein). After preincubation for 15 min the reaction was started with 50 μΐ. substrate suspension. The assay was run for 10 min. Thereafter the reaction was stopped by addition of 1.5 mL of an ice-cold mixture of chloroform/butanol/conc. H Q (10:10:0.6) and 0.45 mL of 1 N HC1. After vigorous mixing the phases were separated by centrifugation for 10 min (1000 rpm). An aliquot of 0.2 mL from the upper water phase was counted for radioactivity. Under the assay conditions 10-20% of the PI was hydrolyzed. In this range the assay was linear with time and enzyme. For each concentration of the test compound the percent inhibition was calculated relative to controls with vehicle only. The concentration for 50% inhibition (IC50) was determined graphically. The synthetic compounds described above were evaluated according to the procedure just described. The results are summarized in the following Table. The compounds without ionic groups showed only a slight degree of inhibition. In contrast, all compounds with ionic groups (sulfates or phosphates) were found to H
In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
15.
K A U F M A N N E T AL.
Inositol Derivatives as Inhibitors of Phospholipase C 205
Table. Evaluation of Inositol Derivatives ï>LCInhî binon
Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: May 30, 1991 | doi: 10.1021/bk-1991-0463.ch015
Compound
%, 500 μΜ*
1 (nonionic) 39 4 27 7 26 8 17 2 (ionic) 3 5 6 9 10 Inhibition too feeble for determination of IC50.
IC50 (μΜ)
30 14.5 22 14 18 230
inhibit the enzyme significantly. The results, however, do not seem to show any other significant correlation between structure and activity. 2. Formation of inositol phosphates in GH3 cells. Some of the compounds have been tested for PLC inhibition in intact cells by measuring their effect on the formation of inositol phosphates in GH3 cells on TRH (Thyrotropin releasing hormone) induction. 3H -Inositol labelled cells were incubated for 30 min with a solution of the test compound in dimethylsulfoxide (DMSO), followed by stimulation with TRH (25 nM) for 30 min at 37 C. The maximal DMSO concentration in the incubation medium was 0.1%. The ^H-inositol phosphates liberated in the cell were extracted and determined by anion exchange chromatography as described elsewhere [9]. The compounds tested in this assay (3, 5, 6 and 10) did not inhibit the formation of inositol phosphates up to a concentration of 10 mM. We suspect that the lack of activity in this system might be due to a poor penetration of the charged compounds into the cells. In order to overcome this potential problem, we are now concentrating our efforts in the synthesis of prodrugs. Chemistry. Full details on the synthetic work discussed here have recently been published [10]. 1. Homologation atC(l) without Ionic Groups in the Molecule. The initial target of this work was the homologation of inositol at C(l) without the introduction of ionic groups, i.e. compounds 1 and 4 . These compounds which are analogues of PI were prepared by the route shown in Scheme I. Thus, wyo-inositol was reacted with excess cyclohexanone in DMF/toluene, catalysed by TsOH, to give the monocyclohexylidene compound 11. Benzylation of 11 yielded the tetrabenzyl compound 12, which was hydrolysed to give c/s-diol 13. We adopted the following route to convert this to the desired 1-alcohol 16: stannylidene-activated allylation [11] (13—>14), benzylation (14—>15), and deallylation. Oxidation of 16 to ketone 17 proceeded satisfactorily by the use of pyridinium chlorochromate. Homologation of 17 by means of a Wittig reaction, followed by hydroboration of the methylidene compound 18 afforded the epimeric alcohol mixture 19. This mixture was readily esterified with palmitoyl chloride, and the resulting esters 20 and 21 could be separated cleanly by MPLC. The ratio of c/i/roester 20 to myo-ester 21 was ca. 2.5:1. The corresponding deprotected compounds 1 and 4 were obtained as pure, crystalline compounds. Ketone 17 was reacted with (methoxymethyl)triphenylphosphonium chloride to yield enol ethers 22a and 22b (Scheme 2). Conversion of these enol ethers to the
In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
OBn
71%
RO
^
OR
2
2
14
CH OCO(CH ) CH
OR
20 (56%)R = Bn
RO
InO*""f
OBn
R = Bn
12
BnO
R = H
11
OR
3
OH
OBn
2
18
OBn
JnO^~[
BnO
13
OBn
5nO^—Γ
BnO
k)
82% ^
2
( 4
2
14
CH 2
OR
R= H
2
(çH OCO(CH ) CH
OR
Q
21 (21%)R = Bn
/OR
RO
JnO^I
/OBn \
OBn
R = Bn
15
BnO
R= H
OBn
14
