Fluoroaromatic fragments on 1,3-disubstituted ureas enhance soluble epoxide hydrolase inhibition
A B S T R A C T
A series of soluble epoxide hydrolase (sEH) inhibitors containing 2-fluorophenyl fragment was developed. Inhibition potency of the described compounds ranges from 0.7 to 630.9 nM. 1-(Adamantan-1-ylmethyl)-3-(2- fluorophenyl) urea (3b, IC50 = 0.7 nM) and 1-(adamantan-2-yl)-3-(2-fluorophenyl) urea (3i, IC50 = 1.0 nM) were found to be the most potent sEH inhibitors within the described series. Crystal results suggest that potency is probably enhanced by extra hydrogen bond between the fluorine atom and catalytic tyrosine residues.
1.Introduction
The mammalian soluble epoxide hydrolase (sEH) is an enzyme in- volved in the metabolism of epoxides of arachidonic acid and other natural epoxy-fatty acids [1], which have numerous biological activities [2]. Through the addition of a water molecule, sEH converts epoxides into corresponding vicinal diols thus affecting inflammatory processes, pain and other disease states [2]. Thereby inhibition of sEH could be beneficial in treatment of many renal and cardiovascular diseases [3,4]. Although thousands of various sEH inhibitors were synthesized over the last few decades [5–7], they have limited bioavailability, especially toward the CNS where sEH is emerging as a potential target for neu- rological diseases [8]. Inhibitors of sEH containing fluorine atoms are among the most promising to go through the blood-brain-barrier [9]. Another structure fragment which is widely used in the design of sEH inhibitors is adamantine [10,11]. Herein, we decided to combine both adamantyl and fluoroaromatic fragments in a single molecule and test them as soluble epoxide hydrolase inhibitors, with physical properties that could allow to target sEH in the CNS.
2.Results and discussion
As a starting material we used 2-fluorophenyl isocyanate (1, Scheme 1) and various adamantyl amines 2a-j. Starting amines 1a-h have 1–3 methyl substituents in the bridgehead positions of adamantane or hydrocarbon spacers between adamantane and the amino group. In addition, the reactive amino groups of amines 2i and 2j are at the bridge position of adamantane.Structures of the obtained chemicals were assessed by NMR, while purity was assessed by GCeMS, LCeMS and elemental analysis (see Supplemental materials for details). 19F NMR spectra (δ-131.60 ± 0.03, see experimental for details) show that structural changes in the adamantyl part of urea has no detectable effect on the electron structure of the fluorine substituted aromatic ring. Physical and chemical properties of the synthesized compounds (Table 1) show that introduction of hydrocarbon spacers between the adamantane fragment and the urea group leads to a decrease in melting points. Methyl substituents in bridgehead positions of adamantane also de- crease melting points except of compound 3h with methyl in each (3, 5 and 7) vacant bridgehead positions.Calculated LogP for most of the synthesized compounds lays within the Lipinsky rule’s borders [13]. Experimental LogP values for com- pounds 3b-d, 3i and 3j are very close to the calculated and in most cases are lesser. Thus the selected method can be used to predict logP for this type of compounds. Solubilities in water (sodium phosphate buffer) of ureas 3a-j (Table 1) lie in a narrow interval of 50–90 μM and shows slight dependence on the structure of adamantane part of the molecule.
The potency of the compounds was then measured against the human sEH. Data (Table 2) confirm that introduction of a singlemethylene spacer between adamantane substituent and the urea group is one of the most activity-enhancing structural change [14]. Further enlargement of such spacer leads to dramatic drop of activity, 15-fold when 3c with ethylene spacer compared to spacerless 3a and 170-foldfor 3d with butylene spacer compared to 3a. Surprisingly compound 3e with 1,4-phenylene spacer show relatively moderate activity (94.2 nM) while previously tested diureas [15] and thioureas [16] bearing this fragment possessed low inhibitory activity of 0.7–57 μM. Moreover, the2-fluoro substituted urea 3a is 4-fold more active if compared to un- substituted 1-(adamantan-1-yl)-3-phenyl urea and 37-fold more active than 2-hydroxy substituted analog (Table 2). This is quite unexpected, and reflect the particular properties of the C–F bond, and the interac- tion of the fluorine atom with its environment. Recently, thioureas containing fluorophenyl fragment were found to inhibit the human sEH. Interestingly the ureas described herein with similar structure of the reported thioureas are more potent, confirming previous findings [16].Introduction of one methyl substituent into bridgehead position of adamantane leads to 2.5-fold increase of activity (1.5 nM for compound 3f compared to 3.7 nM of 3a, Table 2).
However, the addition of a second methyl group sets the activity back to the value of unsubstituted analog. Activity of compound 3h with three methyl substituents in each available bridgehead position of adamantane is 25-fold less than ac- tivity of 3a. Such decrease of activity could be explained by the lack of space for the bulky 3,5,7-trimethyladamantane in the most appropriate conformation available for these compounds.An interesting result was obtained when adamantyl fragment was linked to the urea group by its bridge position. The activity of com- pound 3i is 3.7-fold better than activity of 3a and the only difference between them is the type of carbon atom in adamantane linked with urea nitrogen. To understand the origins of this activity difference be- tween 1- and 2-adamantyl containing ureas the X-ray analysis of a single crystal was made (Fig. 1). The asymmetric unit of the 3i contains three molecules. The molecules are connected via six strong classical intermolecular hydrogen bonds, N2A–H2A···O1C, N3A–H3A···O1C, N2B–H2B···O1A, N3B–H3B···O1A, N2C–H2C···O1B, N3C–H3C···O1B. TheH···A distances are 2.09, 2.20, 2.12, 2.10, 2.42 and 2.06 Å respectivelyand the angles are 157.8, 152.5, 150.3, 154.9, 132.9 and 148.0° re- spectively. There are also three unusual intramolecular hydrogen bonds, C36A–H36 A···O1A, C36B–H36B···O1B, C22C–H22C···O1C with H···A distances 2.27, 2.37 and 2.53 Å respectively and the angles of 120.2, 113.5 and 117.9° respectively. Symmetry codes: (i) –x, 1/2 + y, 1/2 – z. The crystallographic data for the investigated compound have been deposited in the Cambridge Crystallographic Data Centre as sup- plementary publication number CCDC 1543475.The 2-fluorophenyl ring is twisted in position in which fluorine atom positioned closely to oxygen atom of the urea group. Suchorientation of fluorine allows it to form hydrogen bonds with Tyr383 or Tyr466 at the sEH active site and does not interfere the formation of hydrogen bonds between the NH’s and Asp335. Such unpredicted or- ientation of fluorine atom gives very good explanation for the high activity of compound 3i.
3.Conclusions
A series of soluble epoxide hydrolase (sEH) inhibitors containing a 2-fluorophenyl fragment was developed. Inhibition potency of the de- scribed compounds ranges from 0.7 to 630.9 nM. 1-(Adamantan-1-yl- methyl)-3-(2-fluorophenyl) urea (3b, IC50 = 0.7 nM) and 1-(ada- mantan-2-yl)-3-(2-fluorophenyl) urea (3i, IC50 = 1.0 nM) were found to be the most potent sEH inhibitors within the described series.
4.Experimental
The mass spectra were obtained on a Thermo Scientific Incos 50 mass spectrometer and on an Agilent 7820/5975 GC/MS system (HP- 5MS quartz capillary column, 30 m; carrier gas helium; oven tempera- ture programming from 80 to 280 °C; injector temperature 250 °C). The 1H NMR spectra were recorded on a Bruker DRX-500 spectrometer at500.13 MHz using DMSO-d6 as solvent and tetramethylsilane as re- ference. The elemental compositions were determined on a Perkin Elmer 2400 Series II analyzer. 2-Fluorophenyl isocyanate was com- mercial product (Sigma Aldrich). Initial adamantyl amines were syn- thesized according to the known procedures [8]. The solvents were dried according to standard procedures. To 1 equiv. of corresponding amine 2a-j in 40 equiv. of DMF compound 3i was added 1 equiv. of 2-fluorophenyl isocyanate and 1 equiv. of Et3N (2 equiv. when amine used in form of hydrochloride) at 0 °C. The reaction mixture was stirred at room temperature overnight. After adding 1 N HCl and water, the resulting white precipitates were collected by suc- tion filtration.