Ureidopyridazine Derivatives as Acyl-CoA : cholesterol acyltransferase Inhibitors

A series of N-(2,4-difluorophenyl)-N’-heptyl-N’4-[(substituted)-pyridazin-3yl)thio]pentyl urea derivatives having a phenyl ring at positions 5 and/or at position 6 of the heterocycle, as well as the corresponding sulfones, were synthesized. Their inhibitory activity against acyl-CoA:cholesterol acyltransferase (ACAT) was tested on the enzyme prepared from rat liver microsomes. Theoretical studies were performed to correlate their activity to their structural features.


Introduction
Hypercholesterolemiah as been identified as one of the major risk factors for coronary heart diseases.Many efforts have been directed towards the discoveryo f newa nd effectiveh ypocholesterolemic drugs [ 1,2].A cyl-CoA: cholesterol acyltransferase (ACAT)i samicrosomial enzyme that catalyzes the formation of long chain fatty acidc holesterole sters [ 3,4,5].It represents an attractive targett o design novel hypolipidemic and anti-atherosclerotic drugs.Its inhibition produces a reduction in intestinal absorption of cholesterol, liver secretion of veryl ow-density lipoprotein (VLDL) particles, and reduced accumulation of cholesteryl estersi nt he arterial wall cells, the latter beingakey step in the atherosclerotic process [6].
We have now undertaken aS AR studyo nt he linker, in ordert og et information on thee ssential requirements of this moietyf or activity.As af irst approach, branching of the linker was considered.Thisp aper reports on the synthesis and ACAT inhibition activity of compounds 1a-c and 2a-c.I na ddition,a ttempts to correlate their activity to their structural features through theoretical calculations are presented.

Resultsand Discussion
All compoundsw ere tested for theiri nhibitory properties towardsA CATe xtracted from rat liver microsomes.Their activity, expressed as inhibitionp ercentage at 50 g/ml, is shown in Table 1.Amongst the thioderivatives, the 5-phenyl derivative ( 1b)s howed the best activity with an inhibition percentage of 78.Both the 5,6diphenyl ( 1a)a nd the6 -phenyl derivative ( 1c)w ere weaker inhibitors (67% and 62%, respectively).Oxidation of 1b to its corresponding sulfone ( 2b)l ed to a significant loss of potency.In thec ase of 1c,t he same transformation did not bring abouta ny significantc hange( 2c inhibition value was 64%).B yc ontrast, oxidation of 1a to thec orresponding sulfone 2a,l ed to ac ompound provided with much higherinhibitoryactivity (78%).Scheme 1 .Synthetic pathway to compounds 1a-c,2a-c.Amodeling study of compounds 1a-c and 2a-c wasperformed in ordertoattempt a rationalization of thei nhibitory activityo ft he two series of compounds on geometrical grounds.As the substituted ureido group was invarianti na ll the cases, this groupw as omittedi nt he theoretical calculations and the simplified structures 8a-c and 9a-c werem odeled.The energy profiles for rotation around the C3-X (X=S, SO 2 )b ond were initially determined on models unsubstituted at the 5a nd 6 positions of the heterocyclic ring.Then, after addition of the 5-and/or the 6-phenyl groups to the minima located in the profiles, the structures were optimized allowing to determine the minimum energyc onformations reported in  In the hypothesisthat the ureido function of all the compounds interactsinthe same way at theb inding site inducing as imilar orientation of the alkyl chain, we performed ac omparison of theg eometryo ft he conformation 8aA of 8a with the two conformations 9aA and 9aC of 9a by superimposing the alkyl chain and evaluating the differences in the orientation of the diphenylheterocyclic moiety (Figure1 ).The distances between the centroids of the phenyl groups of the molecules in comparison were measured and are reportedi nF igure 1.The comparison of 8aA with 9aA (Figure 1A) as well as with 9aC (Figure 1B) shows a correspondence of the6-phenyl group of the twomolecules.On thecontrary, the5phenyl group is oriented in different ways.

Enzyme assays
In vitro assayagainst ratACAT.Microsomesp repared from rat liverwere used as as ource of thee nzyme.The activity of the ACAT inhibitors against rat ACAT was measured accordingt oap reviously described method [15].G ERI-BP001M wasusedasreference compound.

Computational methods
All calculations were carried out using the Gaussian 03 [16] program package.
The conformational space of compounds 8a-c , 9a-c was explored through optimizationsa tt he B3LYPl evel with the 6-31G* basis set.The energy profiles for rotation around the C3-X bond were initially determined on simplified molecules unsubstituteda tt he 5a nd 6p ositions of theh eterocyclicr ing.Then,a fter addition of the 5-and/or 6-phenyl groups to the minima located in the profiles, the structures were fully optimizeda llowing to determine the minimum energy conformations of compounds 8-9.

a)
In vitro %A CAT inhibition determined in ratl iverm icrosomes.All values are means from three experiments, which differ by less than 10%.b) From 7a-c .c) From 1a-c .

Table 2 .
The data showt hat the presence of thep henyl groups at C5 andC 6d oes not influence the as of 9 .F or the sulfides 8 the differences in energy between the twoo rientations (see,f or example, 8aA vs. 8aC)i sl arge enough (about 3.8k cal/mol) to ensure that onlyo ne orientation gives conformers significantly populated, those characterized by 1 3°; in the case of the sulfones 9 ,i nstead, the second orientation (see, for example, 9aC ), characterized by 1 -50°gives an ot negligible contribution to the overallp opulation being onlya bout 1.2k cal/mol lesss tablet hant he preferred one characterized by 1 45°(see, for example, 9aA).Tab. 2. Relative energy ands electedg eometrical data of the preferred conformationsofcompounds 8a-c and 9a-c.