Prolyl Oligopeptidase Inhibition by N-Acyl-pro-pyrrolidine-type Molecules†

Prolyl Oligopeptidase Inhibition by N-Acyl-pro-pyrrolidine-type Molecules†

Karoly Kanai,‡ Peter Aranyi,‡ Zsolt Bocskei,‡ Gyorgy Ferenczy,‡ Veronika Harmat,§ Kalman Simon,‡ Sandor Batori,‡

Gabor Naray-Szabo,§,¶ and Istvan Hermecz‡,*

CHINOIN, Ltd, H-1045 Budapest, To u. 1-5, Hungary, Protein Modeling Group, Hungarian Academy of Sciences, EotVos Lorand UniVersity,H-1117 Budapest, Pazmany Peter u. 1a, Hungary, and Co-operation Research Center for Molecular Design, Semmelweis UniVersity,H-1062 Budapest, Rippl-Ronai u. 37, Hungary

ReceiVed July 28, 2008

Three novel, N-acyl-pro-pyrrolidine-type, inhibitors of prolyl oligopeptidase (POP) with nanomolar activitieswere synthesized and their binding analyzed to the host enzyme in the light of X-ray diffraction and molecularmodeling studies. We were interested in the alteration in the binding affinity at the S3 site as a function ofthe properties of the N-terminal group of the inhibitors. Our studies revealed that, for inhibitors with flataromatic terminal groups, the optimal length of the linker chain is three C-C bonds, but this increases tofour C-C bonds if there is a bulky group in the terminal position. Molecular dynamics calculations indicatethat this is due to the better fit into the binding pocket. A 4-fold enhancement of the inhibitor activity uponreplacement of the 4-CH2 group of the proline ring by CF2 is a consequence of a weak hydrogen bondformed between the fluorine atom and the hydroxy group of Tyr473 of the host enzyme. There is notablygood agreement between the calculated and experimental free energies of binding; the average error in theIC50 values is around 1 order of magnitude.

Introduction

Prolyl oligopeptidase (POPa) (EC 3.4.21.26), previouslycalled prolyl endopeptidase or postproline cleaving enzyme, acytosolic serine protease, degrades a variety of proline-contain-ing peptides by cleaving the peptide bond on the carboxy sideof proline residues.1 Its active site consists of a catalyticSer554His680Asp641 triad that is common in all serine pro-teases. The enzyme is selective for oligopeptides not longer thanabout 30 amino acid residues.2 It has been identified and purifiedfrom various mammalian tissues, such as the brain, liver, muscleand kidney.3-10

POP degrades proline-containing neuropeptides involved inthe processes of memory and learning, such as vasopressin,substance P and thyrotropin-releasing hormone.11 Its activityin plasma correlates with different stages of depression,12 andit has also been suggested that the enzyme participates in thecontrol of blood pressure through the degradation of bradykinin,and angiotensin I and II.13 POP also plays a central role in moodstabilization, learning and memory modulating inositol con-centration.14,15a Inhibitors of POP are potential therapeuticagents15 in the treatment for dysfunction of the memory system,for example, Alzheimer’s disease,16 and for the improvementof general cognitive behavior in the elderly.17

The C-terminal catalytic domain of POP belongs in the R/�hydrolase fold family.18,19 Sequence homology can be detectedwith lipases,20 while it is structurally unrelated to the chymot-rypsin and subtilisin serine protease families. X-ray proteincrystallographic investigations suggested that its N-terminal

seven-bladed � propeller domain regulates access to the activesite, which is in a large cavity at the interface of the twodomains,21,22 but recent investigations revealed that an opening-closing mechanism of domains is more likely.23 The crystalstructure of the complex with a reversible transition stateinhibitor, 124 (Z-Pro-prolinal), reveals that the prolinal residueis covalently bonded to the active site serine, with the prolineand the phenyl moiety of benzyloxycarbonyl groups accom-modated in subsites S2 and S3, respectively.21 Subsite S3 ismore open than subsite S1, which allows the binding of largerresidues. The wide subsite S3 is very hydrophobic in nature.The structures of POP (both native and inactive mutant forms)complexes with substrates and substrate analogues have shedlight on various structural aspects of the catalytic mechanism,and substrate binding.25-28

Most currently available inhibitors are derivatives of thetransition-state analogue inhibitor, for example, 1 and thereversible 217 (SUAM-1221) (Figure 1). The effects of modi-fication of the five-membered rings, the shape and polarity ofthe N-terminal moiety and the linker connecting the N-terminalgroup and the proline ring on the inhibitor potency have beenextensively studied in Vitro.29

In our POP program, substitution of the 4-phenylbutanoyl of2 with hexanoyl resulted in 3a with activity similar to that of2.30-32 Substitution of the 4-CH2 group of the proline moietyof 3a with a sulfur atom resulted in a derivative 3b that was 4times more active. Introduction of carbonyl-containing N-heterocycles into the end of the alkyl chain of compounds 3led to novel types of POP inhibitors 4 with nanomolaractivities.33,34 A comparative molecular field analysis study on44 derivatives of 4 containing phthalimido (e.g., 5) or saccharinomoieties as N-terminal group suggested some steric andelectronic modifications on these N-heterocyclic end-groups35

(Figure 2).However, as the interactions at subsite S3 of POP are

nonspecific and difficult to predict, we decided to map theseinteractions in the structures of the complexes for derivatives6, 8, and 11. We determined the structures of three POP inhibitor

† Atomic coordinates and structure factors have been deposited in theProtein Data Bank with accession codes 3EQ7, +EQ8 and 3EQ9,respectively.

* To whom correspondence should be addressed. Tel: +36-136-90-151.Fax: +36-137-05-597. E-mail: [email protected].

‡ CHINOIN Ltd.§ Protein Modeling Group, Eotvos Lorand University.¶ Co-operation Research Center for Molecular Design, Semmelweis

University.a Abbreviations: POP prolyl oligopeptidase; MD Molecular Dynamics;

DAST (diethylamino)sulfur trifluoride.

J. Med. Chem. 2008, 51, 7514–75227514

10.1021/jm800944x CCC: $40.75 2008 American Chemical SocietyPublished on Web 11/12/2008

complexes (POP-6, POP-8 and POP-11) and carried out amolecular dynamics-based study on the binding of theseinhibitors and their homologues 5, 7, 9, and 10. Below, we reporton our results and attempt to find a relationship between inhibitorstructures and activities.

Chemistry

All compounds investigated in this paper were synthesizedby acylation of the known prolylpyrrolidinine 16a36 or itsdifluoro derivative 16b with acids 12,37 14 and 15,38 using thestandard mixed anhydride N-acylation method (Scheme 1).

Compounds 14 were prepared by alkylation of quinoxalin-2-one 1339 with ethyl ω-bromoalkanoates in DMF in the presenceof NaH, followed by acidic hydrolysis.

Replacement of methylene by a CF2 moiety may enhancethe biological activity,40 and we therefore decided to synthesizeand investigate 6. The starting difluoro derivative 16b wasprepared from (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyr-rolidine-2-carboxylic acid 1741 as outlined in Scheme 2.Compound 17 was coupled with pyrrolidine by the classicalmixed anhydride method to give 18. Subsequent Swern oxida-tion of the secondary hydroxy group of 17 provided thecorresponding oxo derivative 18, which could be transformedto the difluoro derivative via a known protocol with DAST.42

A deprotective step with HCl/EtOAc gave access to 16b.Compounds 5-11 exhibit cis-trans isomerism about the

prolyl amide bond in solution.43

Results and Discussion

From the aspect of inhibitor design, it is important to knowthe main driving forces that influence the position of theN-terminal group at subsite S3 of the enzyme. A further questionis why the optimal linker chain length is three C-C bonds forsome N-terminal groups and four C-C bonds for others. The4-fold enhancement of the inhibitor activity upon substitutionof the 4-CH2 of the P2 proline ring by CF2, a group of similarsize, was a further interesting result. It was decided to attemptto clarify the structural basis of this finding. We carried outX-ray crystallographic studies of enzyme-inhibitor complexes,as representatives of the inhibitor families (6, 8 and 11), in orderto try to answer these questions.

The Binding Pattern at Subsites S1 and S2 is Well-Defined. The accommodation of molecules 6, 8 and 11 in theircomplex structures with POP is very similar to that of 1. Thereare only minor differences in the interacting side chainconformations of POP, in spite of the shape diversity of theN-terminal residue and the P2 proline ring of these inhibitors.Figure 3 depicts the superposition of inhibitors at the activesite, while Table 1 lists contacting residues. The complexstructures preserve the hydrogen bonds tying the carbonyloxygen atoms of the linker between P3 and P2 groups, and theproline moiety (P2) to the Trp595 and Arg643 side chains,respectively.

In the substrate specificity pocket (S1), the C-terminalpyrrolidine moieties of the studied inhibitors form severalcontacts with various side chains and also with the active siteserine, Ser554 (Table 1, Figure 3). However, as they are unableto bind covalently to the active site serine, the five-memberedring of the C-terminal pyrrolidine can be slightly rotated aboutthe N-C bond, while it retains its stacking interaction with theindole ring of Trp595. Subsite S2, lined by Cys255, Tyr473,Phe476, Trp595 and Arg643, has a slightly polar character andis partially open toward the cavity of the enzyme. Thehydrophobic side chain of Pro forms relatively few contactswith it.

We found that replacement of the 4-CH2 moiety by 4-CF2

results in a 4 times lower IC50 value (Table 2, 5 vs 6).Comparison of the crystal structures of the complex formedbetween POP and molecule 6 and the other complexes revealsa weak hydrogen bond between the fluorine atom (cis to theproline carboxyl) of 6 and the hydroxy group of Tyr473 (3.5Å, Figure 3). We suggest that this extra hydrogen bond isresponsible for the stronger inhibitory effect of 6. The otherfluorine atom is solvent-exposed.

Figure 1

Figure 2

Prolyl Oligopeptidase Inhibition Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7515

There Are Three Types of Inhibitor Binding at SubsiteS3. At the border of the two domains of POP, the substratebinding groove broadens, generating the flat and open subsiteS3, lined by residues Phe173, Met235, Gly236, Ala594 andbackbone atoms of Ser174 below and by Gly254, Cys255 andside chain carbon atoms of Arg252 on one side. At the borderof subsites S2 and S3, besides the hydrogen bond with Trp595,plane-to-plane contacts with the guanidino group of Arg643stabilize the P2-P3 amide bond for all inhibitors examined.

The linker between the P2 amide nitrogen and the ring systemof the N-terminal group is three C-C bonds long for inhibitors6 and 8, similarly as for 1. These molecules with three linkerC-C bonds stack on the flat bottom of subsite S3, forming

several contacts with Phe173 (see Figure 4A and Table 1). Theplanes of their rings are in very similar orientations: the planeangle with the P3 group’s plane of 6 is -4.9° for 8, and -2.2°for 1. However, the positions of these ring systems in thecomplexes differ significantly from that of the benzyl group in1 (Figure 4A). While the phenyl group in 1 is close to the wallof subsite S3, it is mainly stabilized by Ala594 and Cys255besides Met235 and Ile591, forming few contacts with Phe173.The ring systems of the imido and amido group-containinginhibitors 6 and 8, respectively, with more polar characters, areshifted closer to Phe173. The vicinity of the NCO group toArg643 and the similarity of the conformations of the linkerchains of 6 and 8, and of the position of the ring N atoms,suggest that the N-terminal groups of these molecules participatein a similar hydrogen-bonding network, though waters mediatingthese hydrogen bonds could not be localized in electron densitymaps. Possibly for better binding of the phenyl group in 8, theimido group in that inhibitor is shifted as compared with thosein 6, but the guanidino group in Arg643 is also shifted. (Thedistances of the guanidine NH1 and NH2 atoms from the imido

Scheme 1a

a Reagents and conditions: a, Br(CH2)nCO2Et/NaH/DMF; b, HCl/∆; c, Piv-Cl, Et3N, 16a or 16b.

Scheme 2a

a Reagents and conditions: a, Piv-Cl, Et3N, pyrrolidine; b, (COCl)2/DMSO; c, DAST/CH2Cl2; d, HCi(g)/EtOAc.

Figure 3. Binding of the P1-P2 moiety of the inhibitors in the crystalstructures. The ribbon model of the protein is colored gray, while theinhibitor molecules and POP binding sites of the P1-P2 moieties aremagenta, red and green for the POP-6, POP-8 and POP-11 complexes,respectively. Hydrogen bonds are shown as shaded lines. The Figurewas made with the program MOLSCRIPT.67

Table 1. Number of Contacts (d < 4.0 Å) Formed by POP andInhibitors 6, 8 and 11 in the Crystal Structures

inhibitorresidue POP residue 6 8 11

P1 Phe476 3 5 6P1 Ser554 2 4 4P1 Asn555 1 3 1P1 Val580 1 1P1 Val588 1P1 Trp595 11 9 14P1 Tyr599 1 1 1P1 Val644 1 2 2P2 Cys255 1 1P2 Tyr473 1 1P2 Phe476 4 2 1P2 Ile478 1P2 Trp595 7 2 2P2 Arg643 8 4 5P2 His680 1P3 Phe173 13 22 6P3 Ser174 2P3 Met235 4 6P3 Arg252 9P3 Gly254 3 4P3 Cys255 3 3 7P3 Ile591 1 1P3 Ala594 1 1P3 Trp595 4 2 4P3 Arg643 3 3P3 H-bonds to water 1 1

7516 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 Kanai et al.

oxygen of 6 are 4.4 and 3.7 Å, while the amido one of 8 are4.3 and 4.8 Å, respectively. The plane angles of guanidine andoxo groups are 70.7° and 49.0° for 6 and 8, respectively, withoxy group-guanidine plane distances of 3.5 Å in both com-plexes.) This relates to the hydrogen-bonded connection withArg643. Although the ring systems in 6 and 8 have verydifferent sizes and shapes, their imido moieties are still bondedin a similar way. This suggests that the building-up of thehydrogen-bonding network, including the imido oxygen in theinhibitor and the guanidino group in Arg643, is a very importantfactor for stabilization of the complex.

The N-heterocyclic (aromatic) moieties in 6 and 8 coverdifferent regions of subsite S3. While that in 6 binds betweenthe side chains of Met235 and Phe173, those in 8 areaccommodated mainly by the side chain of Met235 and are closeto the edge of the “wall” at the side of subsite S3 and contactCys255. The phenyl group in 8 undergoes a stacking interactionwith the amide plane of the Phe173-Ser174 peptide bond. Theother side of the phenyl group is solvent-exposed due to thebroadening of subsite S3. As the nearby groups of the protein

are polar, replacement of the phenyl group by a polar aromaticgroup would facilitate further favorable interactions with thebinding site.

The complexes with inhibitors 6 and 8 reveal the reason forthe different optimal linker chain length in molecules 10 and11. These also possess the imido oxygen at the junction of theirN-terminal moiety and the linker chain, but they cannot adoptthe same position as can 6 and 8 in their complexes, becausethis requires a flat N-terminal group stacked tightly to the bottomof subsite S3. The N-terminal group in 10 and 11 is stericallydemanding. Its large substituents (i.e., phenyl groups) lie outof the plane of the central imido ring of the N-terminal group.Indeed, the crystal structure of the POP-11 complex shows theN-terminal group in a different position, with contacts differentfrom those of the other complex structures (cf. Table 1). Itsconnecting chain with the extra CH2 group bends toward the“wall” of subsite S3 (cf. Figure 4B and Table 1). The hydrogenbond of the imido oxygen is lost and the hydantoin moietycontacts the side chains of Met235 from one side and Cys255from the other. Its plane is rotated by 41.9°, allowing theformation of a hydrogen bond with the carbonyl oxygen of

Table 2. Experimental and Calculated Binding Free Energies and their Components (kcal/mol)

code X n ∆Eele a ∆Evdw b ∆Econf IC50c ∆Gexp ∆Gcalc

d ∆Gexp - ∆Gcalc

5 CH2 1 19.4 -21.0 0.3 3.0 -10.6 -10.9 +0.36e CF2 1 -- -- -- 0.81 -11.8 -- --7 CH2 2 18.0 -21.4 0.8 40 -9.1 -11.6 +2.58e CH2 1 13.6 -26.5 6.1 0.88 -11.3 -13.6 +2.39 CH2 2 23.0 -22.2 1.7 59 -8.9 -9.0 +0.110 CH2 1 36.1 -26.3 6.2 - -4.0f -2.1 -1.911e CH2 2 20.8 -25.3 7.2 41 -9.1 -7.7 -1.4

a ∆Eele ) Eele(L - E) - Eele(L - W). b ∆Evdw ) Evdw(L - E) - Evdw(L - W). c IC50 values are in nM. d ∆Gcalc ) ∆G4 in eq 1. e X-ray structureavailable. f No inhibition at 0.1 µM. Estimated ∆G corresponds to an IC50 of 25 µM.

Figure 4. Binding at subsite S3 in the crystal structures. Inhibitors with three C-C bond linkers in their P3 moiety (A) bind in significantlydifferent orientations from those with four C-C linkers (B). The ribbon model of the protein is colored gray, while 1 covalently bound to Ser554of Protein Data Bank entry 1QFS is shown in dark-gray as reference. The inhibitor molecules and POP binding sites of the S3 moieties of thePOP-6, POP-8 and POP-11 complexes are colored magenta, red and green, respectively. Hydrogen bonds are shown as shaded lines. The Figurewas made with the program MOLSCRIPT.67


Gly254. A dipole-dipole interaction is also established betweenthe carbonyl groups of the N-terminal group and Gly254 inreverse orientation. One of the phenyl rings lies in the extensionof subsite S3, packed between the side chain of Met235 andside chain carbon atoms of Arg252, while the other is accom-modated above subsite S3 by Arg252 side chain carbon atomsand Cys255.

Molecular Dynamics (MD). Calculations were performedto study the structural and energetic features of some representa-tive complexes. The calculated free energies of binding, togetherwith the experimental values, are presented in Table 2 andFigure 5. The mean unsigned error is 1.37 kcal/mol, whichcorresponds to an error of approximately 1 order of magnitudein IC50. This agreement between the calculated and experimentalvalues is remarkably good with regard to the simple modelapplied (see Computations within the Experimental Section).The pyrrolidine head of the ligands resides at the hydrophobicS1 binding site, while other regions are partially exposed towater in the central cavity. In the course of the MD simulationsof the enzyme-ligand complexes, the pro-pyrrolidine moietyexhibited little flexibility and adopted similar conformations inall complexes. Moreover, the bound conformations of thesefragments are very close to that obtained in water.

For all the studied molecules, a favorable van der Waals andan unfavorable electrostatic energy component (cf. Table 2) werefound to accompany the change of the environment from waterto enzyme. The conformational strain proved to be significantin the binding of molecules 8, 10 and 11. These are the structureswith the largest N-terminal groups. In spite of the open natureof the S3 binding site, the bulky substituents are unable to adopta low-energy conformation. The excess conformational energyis close to the higher limit of the conformational strain thattypically occurs in ligand binding.44

It is interesting that in the higher homologue of 8, that is, 9,the conformational strain almost disappears (cf. Table 2). Thiscan occur because the increased length of the alkyl chain givesmore flexibility to the molecule and allows the P3 group topenetrate further into the large water-filled pocket of the�-propeller region. In spite of the decreased conformationalstrain, 9 binds to POP with lower affinity than does 8, sincethe latter interacts more favorably with the enzyme.

A comparison of molecules 10 and 11 reveals better bindingof the latter, which has a longer alkyl chain (n ) 2). This is in

contrast with molecules with other P3 groups, where those withthe shorter chain (n ) 1) exhibit higher binding affinities.Structures 10 and 11 have the largest P3 group, and thus, theyare unable to simultaneously adopt a low energy conformationand undergo favorable interactions with the enzyme when thealkyl chain is short (n ) 1, 10). The insertion of an extramethylene group (n ) 2, 11) allows the P3 group to fit betterinto the binding pocket. Although the conformational strain isstill significant, the lack of the highly unfavorable electrostaticinteractions demonstrated by the MD simulation in 10 (cf. Table2) means that 11 binds to the enzyme with increased affinity ascompared with 10.

Conclusions

Our X-ray diffraction study has shed light on various aspectsof the binding of N-acyl-pro-pyrrolidine-type inhibitors of POP.Analysis of the structures of the complexes provides an efficienttool in drug design. The enhanced inhibitory activity of theγ-CF2-substituted proline-containing derivatives proved to bedue to the formation of a new hydrogen bond and a subsequentslight shift of the N-acyl-pro-pyrrolidine moiety. The crystalstructures available allowed us to study the change in bindingpower at the S3 site, depending on the size, shape and polarityof the N-terminal group of the inhibitors. Inhibitors with flatring systems are stacked on the bottom of the S3 site. For theseinhibitors, the optimal linker chain length is three C-C bonds.Insertion of an imido moiety into the junction of the ring systemallows hydrogen bond formation with water molecules of thefirst solvent shell of the enzyme, indicated by a shift of thering position in the complex as compared with the hydrophobicbenzyloxycarbonyl group. We have provided an explanation forthe change in optimal length of the linker chain for a derivativewith a bulky N-terminal group. The binding site of this groupoverlaps only partially with those of the flat ones.

Experimental Section

Chemistry. Compounds 12,37 1538 and 16a36 were preparedaccording to literature methods. All other reagents and solventswere purchased from Aldrich or Fluka and were used as received.Solvents were evaporated under reduced pressure on a Buchi RE111rotavapor. Flash chromatography was performed on Merck Kie-selgel gel 60 (230-400 mesh) silica, with CH2Cl2/MeOH as mobilephase. Melting points were determined on a Buchi-530 melting pointapparatus and were uncorrected. Proton nuclear magnetic resonance(1H NMR) spectra were recorded on a Bruker Avanence DRX-200 instrument [200 MHz, δ ) 0 (TMS), in DMSO-d6 or CDCl3

as solvent]. Chemical shifts are expressed in ppm, and signalmultiplicities are described as s (singlet), d (doublet), t (triplet), q(quartet) or br (broad). Combustion analysis was performed on aCarlo Erba Mod 1110 for C, H and N, while F was determined bytitration after Schoniger oxidation. Combustion analyses for C, H,N and F gave results within 0.4% of theory.

tert-Butyl (2S,4R)-4-Hydroxy-2-(pyrrolidinocarbonyl)pyrro-lidine-1-carboxylate. Compound 1741 (6.92 g, 30.0 mmol) wasreacted with pyrrolidine (2.13 g, 30.0 mmol) according to generalprocedure (see below) (18 h), and the reaction residue was purifiedby flash chromatography to yield 6.48 g (76%) of tert-butyl (2S,4R)-4-hydroxy-2-(pyrrolidinocarbonyl)pyrrolidine-1-carboxylate as awhite powder; mp 106-107 °C. 1H NMR (DMSO-d6): δ 1.30 +1.37 (s + s, 9H), 1.62-1.97 (m, 5H), 2.06 (m, 1H), 3.19-3.56(m, 6H), 4.26 (br s, 1H), 4.42 (m, 1H), 5.00 (d, 1H, exchangeablewith D2O). Anal. (C14H24N2O4) C, H, N.

tert-Butyl (2S)-4-Oxo-2-(pyrrolidinocarbonyl)pyrrolidine-1-carboxylate (18). To a solution of oxalyl chloride (3.81 g, 30 mmol)in CH2Cl2 (120 mL) was gradually added DMSO (4.26 mL, 60mmol), followed by a solution of tert-butyl (2S,4R)-4-hydroxy-2-(pyrrolidinocarbonyl)pyrrolidine-1-carboxylate (5.68 g, 20.0 mmol)

Figure 5. Calculated versus experimental binding free energies. Theindicated line corresponds to perfect matching of the calculated andexperimental values.


in CH2Cl2 (30 mL) at -78 °C under an argon atmosphere. Afterstirring for 90 min at this temperature, Et3N was added and thereaction mixture was left to warm up to room temperature. Themixture was then diluted with CH2Cl2 (50 mL), washed successivelywith water and brine, dried over MgSO4, filtered and evaporatedin Vacuo. The residue was purified by flash chromatography to give4.5 g (79.7%) of 18 as a white solid; mp 148-150 °C. 1H NMR(DMSO-d6): δ 1.36 + 1.40 (s + s, 9H) 1.67-1.96 (m, 4H), 2.36(d, 1H), 3.01 (m, 1H), 3.14-3.68 (m, 4H), 3.74 (m, 2H), 4.86 (t,1H). Anal. (C14H22N2O4) C, H, N.

(2S)-4,4-Difluoro-2-(pyrrolidinocarbonyl)pyrrolidine (16b).To a solution of 18 (2.26 g, 8 mmol) in dry benzene (20 mL),DAST (2.44 g, 15.1 mmol) was carefully added at -5 °C under anargon atmosphere. After 30 min, the reaction mixture was warmedup to room temperature and was stirred overnight. The mixturewas then poured onto ice-water and the separated aqueous layerwas extracted 3 times with EtOAc. The combined organic phaseswere washed successively with water and brine, dried over MgSO4,filtered and evaporated under reduced pressure. The residue waspurified by column chromatography (silica gel, 30% n-hexane inacetone) to give 1.24 g (52%) of tert-butyl (2S)-4,4-difluoro-2-(pyrrolidinocarbonyl)pyrrolidine-1-carboxylate as an off-white solid;mp 106-107 °C. 1H NMR (DMSO-d6): δ 1.36 + 1.39 (s + s,9H), 1.71-1.96 (m, 4H), 2.30 (m, 1H), 2.87 (m, 1H), 3.21-3.62(m, 4H), 3.75 (m, 2H), 4.61 (m, 1H). Anal. (C14H22F2N2O3) C, H,N, F.

To a solution of tert-butyl (2S)-4,4-difluoro-2-(pyrrolidinocar-bonyl)pyrrolidine-1-carboxylate (1.0 g, 0.33 mmol) in EtOAc (3mL), 3 M HCl in EtOAc (1.0 mL) was added dropwise at roomtemperature. After stirring for 20 h, the precipitated crystals werefiltered off, washed with EtOAc, dried and used in the followingstep without further purification.

3-(2-Oxo-3-phenylquinoxalin-1(2H)-yl)propanoic acid (14a).To a suspension of NaH (220 mg, 5.4 mmol) in dry MeCN (20mL) under argon, 1339 (999 mg, 4.5 mmol) was carefully added,followed by the dropwise addition of a MeCN solution of ethyl2-bromopropionate (825 mg, 4.5 mmol). The reaction mixture washeated at reflux for 12 h. After cooling to room temperature, thesolvent was removed under reduced pressure, and the residue wasextracted with EtOAc. The combined organic solvent was washedsuccessively with water and brine, and dried over anhydrousMgSO4. Removal of the solvent, followed by flash chromatographicpurification gave 1.05 g (72%) of the ethyl ester of 14a. The crudeethyl ester of 14a (0.72 g, 2 mmol) in 6 N HCl (25 mL) was stirredat reflux for 10 h, and the reaction mixture was then concentratedunder reduced pressure to give 14a (370 mg, 28%) as a white solid;mp 174-177 °C. 1H NMR (DMSO-d6 + CDCl3): δ 2.77 (t, 2H),4.60 (t, 2H), 7.38 (t, 1H), 7.52 (m, 3H), 7.60 (t, 1H), 7.62 (d, 1H),7.91 (d, 1H), 8.30 (m, 2H), 12.35 (br s, 1H). Anal. (C17H14N2O3)C, H, N.

4-(2-Oxo-3-phenylquinoxalin-1(2H)-yl)butanoic acid (14b).Compound 14b (350 mg, 57%) was prepared from 1339 (999 mg,4.5 mmol) and ethyl 3-bromobutyrate (1053 mg, 5.4 mmol)similarly as for 14a (18 h), as a white solid; mp 172-176 °C. 1HNMR (DMSO-d6): δ 1.94 (m, 2H), 2.42 (t, 2H), 4.33 (t, 2H), 7.38(t, 1H), 7.50 (m, 3H), 7.68 (m, 2H), 7.86 (d, 1H), 8.26 (m, 2H),12.17 (br s, 1H). Anal. (C18H16N2O3) C, H, N.

General Procedure for Preparation of Compounds 5-11. Thesynthesis of 5 is described to illustrate the general method. Asolution of 12a37 (876 mg, 4 mmol) in CH2Cl2 (10 mL) was cooledto -15 °C under argon and treated with NEt3 (445 mg, 4.4 mmol),followed by pivaloyl chloride (431 mg, 4.4 mmol), and the mixturewas stirred for 30 min. A solution of 16a (818 mg, 4 mmol) inCH2Cl2 (5 mL) was added and the reaction mixture was allowedto warm up to room temperature. After stirring for 5 h, the mixturewas washed successively with water and brine, and dried overanhydrous MgSO4. Removal of the solvent, followed by flashchromatographic purification, gave 5 (1.16 g, 83%) as a white solid;mp 121-122 °C. 1H NMR (DMSO-d6): δ 1.72-2.14 (m, 8H), 2.63(m, 2H), 3.18-3.59 (m, 6H), 3.75 (m, 2H), 4.49 + 4.63 (two dd,1H), 7.86 (m, 4H). Anal. (C20H23N3O4) C, H, N.

2-{3-[(2S)-4,4-Difluoro-2-(pyrrolidinocarbonyl)pyrrolidin-1-yl]-3-oxopropyl}isoindole-1,3(2H)-dione (6). Compound 6 (970mg) was prepared in 73% yield from 12a37 (723 mg, 3.3 mmol)and 16b (794 mg, 3.3 mmol) according to general method, mp 129°C. 1H NMR (DMSO-d6): δ 1.72-1.91 (m, 4H), 2.30-3.05 (m,4H), 3.19-3.42 (m, 4H), 3.57 (m, 1H), 3.74 (m, 1H), 3.93 (m,1H), 4.08 (m, 1H), 4.74 + 4.99 (two dd, 1H), 7.86 (m, 4H). Anal.(C20H21F2N3O4) C, H, N, F.

2-{4-Oxo-4-[(2S)-2-(pyrrolidinocarbonyl)pyrrolidin-1-yl]bu-tyl}-1H-isoindole-1,3(2H)-dione (7). Compound 7 (850 mg) wasprepared in 74% yield from 12b37 (699 mg, 3.0 mmol) and 16a(614 mg, 3.0 mmol) according to general method, mp 115-116°C. 1H NMR (DMSO-d6): δ 1.62-2.18 (m, 10H), 2.26 (m, 2H),3.18-3.54 (m, 6H), 3.50 (m, 2H), 4.30 + 4.55 (two dd, 1H), 7.85(m, 4H). Anal. (C21H25N3O4) C, H, N.

1-{3-Oxo-3-[(2S)-2-(pyrrolidinocarbonyl)pyrrolidin-1-yl]pro-pyl}-3-phenylquinoxalin-2(1H)-one (8). Compound 8 (153 mg)was prepared in 43% yield from 14a (247 mg, 0.8 mmol) and 16a(146 mg, 0.8 mmol) according to general method, mp 223-226°C. 1H NMR (CDCl3): δ 1.87-2.30 (m, 8H), 2.83 (m, 2H),3.42-3.85 (m, 6H), 4.57 (m, 1H), 4.70 (m, 1H), 4.75 (m, 1H),7.34 (t, 1H), 7.48-7.64 (m, 5H), 7.95 (d, 1H), 8.31 (m, 2H). Anal.(C26H28N4O3) C, H, N.

1-{4-Oxo-4-[(2S)-2-(pyrrolidinocarbonyl)pyrrolidin-1-yl]bu-tyl}-3-phenylquinoxalin-2(1H)-one (9). Compound 9 (140 mg) wasprepared in 38% yield from 14b (230 mg, 0.78 mmol) and 16a(160 mg, 0.88 mmol) according to general method, mp 159-160°C. 1H NMR (CDCl3): δ 1.83-2.38 (m, 10H), 2.53 (m, 2H),3.35-3.90 (m, 6H), 4.42 (m, 2H), 4.68 (m, 1H), 7.36 (t, 1H), 7.48(m, 3H), 7.59 (t, 1H), 7.67 (d, 1H), 7.94 (d, 1H), 8.30 (m, 2H).Anal. (C27H30N4O3) C, H, N.

3-{3-Oxo-3-[(2S)-2-(pyrrolidinocarbonyl)pyrrolidin-1-yl]pro-pyl}-5,5-diphenylimidazolidine-2,4-dione (10). Compound 10 (273mg) was prepared in 36% yield from 15a38 (518 mg, 1.6 mmol)and 16a (337 mg, 1.6 mmol) according to the general method, mp129 °C (recrystallized from aqueous MeOH). 1H NMR (DMSO-d6): δ 1.70-2.19 (m, 8H), 2.41-2.71 (m, 2H), 3.19-3.69 (m, 8H),4.41 + 4.43 (two dd, 1H), 7.36 (m, 10H), 9.64 (s, 1H). Anal.(C27H30N4O4) C, H, N.

3-{4-Oxo-4-[(2S)-2-(pyrrolidinocarbonyl)pyrrolidin-1-yl]bu-tyl}-5,5-diphenylimidazolidine-2,4-dione (11). Compound (646mg) 11 was prepared from 15b38 (700 mg, 2.07 mmol) and 16a(430 mg, 2.1 mmol) according to general method, mp 230-232°C. 1H NMR (DMSO-d6): δ 1.70-1.95 (m, 10H), 2.04 (m, 1H),2.17 (t, 1H), 3.15-3.58 (m, 8H), 4.41-4.44 (m, 1H), 7.37 (m,10H), 9.62 + 9.65 (s + s, 1H). Anal. (C28H32N4O4) C, H, N.

Biological Assays. After removal of the cerebellum, the wholebrains of male rats (Sprague-Dawley, 180-220 g) were homog-enized in 0.1 M Tris-HCl, 1 mM EDTA buffer, pH ) 7.5. Thehomogenate was centrifuged and the 40 000g supernatant was usedat 300 times final dilution in the reaction mixture. Enzyme reactionswere performed at room temperature for 15 min in the presence of62.5 nM Z-glycyl-prolyl-7-amino-4-methylcoumarin as a highlyspecific synthetic substrate of POP. The inhibitory effects of thecompounds were tested at inhibitor concentrations of 100-0.001nM. The formation of 7-amino-4-methylcoumarin was detected byspectrofluorometry at an excitation wavelength of 370 nm and anemission wavelength of 440 nm. The concentrations of thecompounds producing 50% inhibition (IC50) were calculated bycurve fitting of the % inhibition versus inhibitor concentration (M)plot, using the Hill equation.

X-ray Crystallography. POP purified from porcine muscle waspurchased from Laszlo Polgar in the Institute of Enzymology,Hungarian Academy of Sciences.45,46 Crystals were grown bycocrystallization at 4 °C, using the hanging drop vapor diffusionmethod. The reservoir composition was as described by Fulop etal.21 Drops were prepared by mixing equal amounts of proteinsolution (10 mg/mL) and the reservoir solution containing theinhibitor (1 mM). The largest crystals were obtained by micro-seeding. Data were collected at room temperature, using a RigakuR-AXIS IIC image plate detector attached to a Rigaku RU-H2R


rotating anode generator from one crystal for each complex. Theresolutions of data sets were 3.0, 2.8 and 2.5 Å for the complexeswith 6, 8 and 11, respectively. Data processing was carried outwith the programs MOSFLM47 (complexes with inhibitors 8 and11), BioteX48 (POP-6 complex) and SCALA.49 The scaled dif-fraction intensities were converted to structure factor amplitudesby using the program Truncate.50 The structures were isostructuralwith those of POP complexes and mutants to be found in the ProteinData Bank. Rigid body fitting was carried out with the programAMoRe51 of the Collaborative Computing Project 4 suite,52 withthe protein part of Protein Data Bank53 entry 1QFS. The modelbuilding steps were carried out with program O54 and the resultingmodel was refined with X-PLOR, version 3.851.55 During therefinement, we used torsion angle dynamics (slow cooling from4000 K), resolution-dependent weighting, overall anisotropic B-factor refinement, bulk solvent correction and grouped B-factorrefinement. Atomic coordinates have been deposited in the ProteinData Bank with accession codes 3EQ7, 3EQ8 and 3EQ9 for 6, 8and 11, respectively. Table 3 shows data collection and refinementstatistics.

Computations. Molecular dynamics simulations were performedto study the binding of the ligands in Table 2 and to make estimatesof the free energies of binding. A modified version of the LinearInteraction Energy (LIE) method of Åqvist et al.56 was applied.The free energy change accompanying ligand binding was calcu-lated as

∆G) 1⁄2{Eele(L-E)-Eele(L-W)}+ {Evdw(L-E)-

Evdw(L-W)}+ {Esolute(LE)-Esolute(LW)} (1)

where Eele(L - E) is the electrostatic interaction energy betweenthe ligand and the enzyme, and Eele(L - W) is that between theligand and water. The van der Waals components are designatedanalogously. Esolute(LE) is the internal energy of the ligand boundto the enzyme, and Esolute(LW) is the internal energy of the freeligand in water. This equation differs from that of Åqvist et al. bythe presence of the last term, which can be identified as the ligandstrain accompanying complex formation. In the equation, the factor1/2 for the electrostatic term corresponds to the ‘ideal’ value ofthe LIE method, and it was found that the experimental values couldbe reasonably reproduced without multiplying the van der Waalsterm by a factor different from 1.

The setup of the MD simulations was as follows. The ligandand the enzyme/ligand systems were immersed in a previouslyequilibrated TIP3P57 water droplet with 20 Å radius. Watermolecules within 2.5 Å heavy atom separations from any ligand

or enzyme atom were deleted. Evaporation of water was preventedby half-harmonic constraints. Residues within 18 Å from the centerof the sphere were allowed to move, while residues in an outer 2Å shell were fixed. The rest of the system was not included in thesimulations. The AMBER force field of Cornell et al.58 was applied.Charges were calculated by the multiple fitting procedure59,60 froman ab initio 6-31G* wave function obtained with the Gaussian 98program.61 A nonbonded cutoff of 15 Å was used and the SHAKEalgorithm62,63 was applied for bonds involving hydrogen atoms.NVT simulations with coupling to an external heat bath64 wereperformed.

The ligand-enzyme systems were first gradually heated from10 to 298 K. We applied positional constraints on the heavy atomsat low temperature and the constraints were gradually released inthe course of heating. Simulations were then performed for 100 pswith a time step of 1 fs at a temperature of 298 K, using the SYBYLsuite of programs.65 Data collection was performed in the final 70ps.

The experimental ∆G values were obtained as ∆G ) RT ln Ki,where Ki, the dissociation constant of a complex, was calculated66

as Ki ) IC50/(1 + S/Km), where S is the substrate concentrationand Km is the Michaelis constant of the substrate.

Acknowledgment. The authors are grateful to Drs AgnesPapp-Behr, Edit Susan and David Durham for valuable contri-butions to the synthetic work, the biological evaluations ofcompounds, and linguistic improvement of the manuscript,respectively. This work was supported in part by a grant fromthe National Scientific Research Foundation (OTKA) NK67800,F67937 and NI68466 (V.H., G.N.-S.).

References

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Table 3. X-ray Diffraction Data Collection and Refinement Statistics for POP Complexes with Inhibitors 6, 8 and 11, Respectively

6 8 11

Data Collection

Space group P212121 P212121 P212121

Cell dimensions (Å) a ) 72.44, b ) 101.44, c ) 112.25 a ) 72.44, b ) 101.57, c ) 112.17 a ) 72.55, b ) 101.46, c ) 112.33Resolution range (Å) 2.89-74.53 2.73-34.38 2.47-34.50Completeness (%) 96.5 93.5 94.1No. of reflections, redundancy 17989 2.7 21070 4.2 28405 3.4

Refinement

R factor 0.1779 0.1768 0.1744Rfree (5% of data) 0.2570 0.2533 0.2438No. of atoms (protein/inhibitor/water) 5574/29/52 5656/33/99 5675/36/132Average B factors (Å2)POP main chain 39.44 35.04 24.58POP side chain 41.31 38.06 28.10Inhibitor molecule 46.35 58.43 41.12Water molecules 41.91 40.24 30.51

rms Deviation from Ideal Geometry

Bond lengths (Å) 0.003 0.002 0.006Bond angles (deg) 0.809 0.718 1.182Dihedral angles (deg) 28.14 27.99 27.95


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