J. Med. Chem. 1994, 37, 3969-3976
`
`3969
`
`Dipeptide Phosphonates as Inhibitors of Dipeptidyl Peptidase IV
`
`Bogdan Boduszek,* Jozef Oleksyszyn,§ Chih-Min Kam,* Joe Selzler,* Robert E. Smith,t and James C. Powers*·*
`From the School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, and Enzyme
`Systems Products, 6491 Sierra Lane, Dublin, California 94568
`
`Received November 1, 1993®
`
`A series of dipeptides which contained phosphonate analogs of proline and piperidine-2-
`carboxylic acid (homoproline) have been synthesized and tested as inhibitors of DPP-IV. The
`rates of inhibition of DPP-IV by these compounds are moderate, but the inhibitors are quite
`specific. The best inhibitor in the series is Ala-PipP(OPh-4-Clh (13), which has a kinact of0.353
`s-1 and Kr of 236 ,uM. The DPP-IV inhibitors Ala-ProP(OPhh (6), Ala-ProP(OPh-4-Clh (12),
`and Ala-PipP(OPh-4-Clh (13) do not inhibit trypsin, human leukocyte elastase (HLE), porcine
`pancreatic elastase (PPE), acetylcholinesterase, papain, and cathepsin B. However, compounds
`12 and 13 inhibited chymotrypsin slowly. Most of these dipeptides containing a homoproline
`phosphonate residue (PipP) or a Pro phosphonate residue (ProP) at the P1 site are stable in a
`pH 7.8 buffer with half-lives of several hours to several days. DPP-IV inhibited by 6, 7 (Ala(cid:173)
`PipP(OPh)2), 12, or 13 is quite stable, and no enzyme activity was recovered after removal of
`excess inhibitor and incubation in buffer for 1 day. Since the phosphonate inhibitors are specific
`toward DPP-IV and the inhibited enzymes are stable, they should be useful in establishing
`the biological functions ofDPP-IV and may be useful therapeutically in the prevention of the
`rejection of transplanted tissue.
`
`Introduction
`Dipeptidyl peptidase IV (DPP-IV, 1 EC 3.4.14.5, CD26)
`is a post-proline cleaving enzyme which will remove the
`dipeptides AA-Pro (AA = amino acid residue) from the
`N-terminus of proteins or polypeptides. DPP-IV has
`been found in a variety of mammalian cells and tissues,
`including kidney, placenta, and blood plasma, and on
`the surface of certain T-lymphocyte subsets. Despite
`extensive studies, the biological role of DPP-IV in
`mammalian systems have not been established, al(cid:173)
`though a number of functions have been postulated.
`DPP-IV may participate in the metabolism and uptake
`of proline-containing peptides in the intestine and
`kidney2 and may be involved in fibronectin-mediated cell
`movement and adhesion.3 DPP-IV may also play a role
`in the metabolism or catabolism of collagen which has
`a high frequency of Gly-Pro sequences.4 DPP-IV in
`human plasma has been shown to cleave N-terminal
`Tyr-Ala from growth hormone-releasing factor and
`cause inactivation of this hormone. 5 DPP-IV is also
`involved in T-cell activation and regulation ofT-cell
`proliferation.6 Thus, inhibitors of DPP-IV may have
`therapeutic utility in the modulation of the rejection of
`transplanted tissue by the host organism.
`DPP-IV is a serine protease which has been demon(cid:173)
`strated by its complete inhibition by DFP. 7 The amino
`acid sequence of rat liver DPP-IV deduced from eDNA
`has been established, and it contains the sequence of
`Gly-Trp-Ser-Tyr-Gly corresponding to the common se(cid:173)
`quence Gly-X-Ser-X-Gly found in the active site of
`various serine proteases.8 Recent studies have shown
`that radiolabeled [3H]DFP is bound to Ser-631 ofDPP-
`
`* Corresponding author: James C. Powers, School of Chemistry and
`Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-
`0400. Tel: (404) 894-4038. Fax: (404) 894-7452.
`t Enzyme Systems Products.
`* Georgia Institute of Technology.
`! Current address: OsteoArthritis Sciences, Inc., One Kendall
`Square, Cambridge, MA 02139.
`®Abstract published in Advance ACS Abstracts, October 1, 1994.
`
`IV, and the residues Gly-629, Ser-631, and Gly-633 are
`essential for the enzyme activity ofDPP-IV.9 Interest(cid:173)
`ingly, the catalytic triad residues (Ser-624, Asp-702, and
`His-734) of mouse DPP-IV are arranged in a novel
`sequential order (e.g., Ser-Asp-His) that is different from
`that of archetypical serine proteases (His-Asp-Ser).10
`A wide variety of inhibitors of serine proteases have
`been reported; 11 however, only a few classes of com(cid:173)
`pounds are effective inhibitors ofDPP-IV. N-Peptidyl-
`0-( 4-ni trobenzoyl)hydroxylamines irreversibly inacti(cid:173)
`vate DPP-IV, but most of the inhibitor is hydrolyzed
`during the inhibition process.12 Azapeptides such as
`Ala-AzaPro-OPh form acyl enzyme derivatives with
`DPP-IV, but they are not very effective inhibitors, and
`the acyl enzyme can deacylate regenerating active
`enzyme.13 The boronic acids Ala-boroPro and Pro(cid:173)
`boroPro are specific and potent reversible inhibitors of
`DPP-IV with Kr values in the nanomolar range; how(cid:173)
`ever, both inhibitors are unstable in solution at neutral
`pH.14 Many other types of transition-state inhibitors
`would be expected to be quite unstable when incorpo(cid:173)
`rated into a dipeptide structure with a free N-terminal
`amino group. For example, dipeptide trifluoromethyl
`ketones, a-keto acids, or chloromethyl ketones would be
`expected to cyclize and be unstable in aqueous solu(cid:173)
`tion.15 Therefore, new specific and potent inhibitors of
`DPP-IV are needed.
`A variety of peptide phosphorus derivatives have been
`reported to be serine protease inhibitors. For example,
`DFP analogs such as peptide phosphonyl fluo.rides
`inhibit serine proteases potently, but they are extremely
`unstable in aqueous solution.16 Peptide phosphonates
`which incorporate a tetrahedr~ phosphorus moiety in
`the peptide substrate inhibit serine proteases poorlyP
`Peptidyl (o.-aminoalkyl)phosphonate diphenyl esters
`offer one potential route to effective DPP-IV inhibitors
`since phosphonate esters are relative unreactive with
`nitrogen nucleophiles (e.g., the N-terminal amino group).
`These peptide phosphonate esters are specific and
`
`0022-2623/94/183 7-3969$04.50/0
`
`© 1994 American Chemical Society
`
`AstraZeneca Exhibit 2095
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 8
`
`

`
`3970 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 23
`
`Boduszek et al.
`
`Table 1. Inhibition of Human Placenta DPP-IV by Peptidyl
`Phosphonatesa
`
`% inhibition
`[I] (mM)
`30min
`2min
`inhibitors
`33
`HCl•Ala-ProP(QPhh
`0
`0.12
`6
`AcOH•Ala-PipP(QPh)2
`100
`0
`0.12
`7
`8 AcOH·Ala-PipP(QH)(OPh)
`0
`0.12
`0
`HBrPhe-ProP(QPhh
`0
`0
`0.12
`9
`10
`0
`0
`0.12
`2HBrLys-ProP( OPhh
`88
`2HCl•Lys-PipP(QPh)2
`0.12
`35
`11
`12
`0
`100
`0.12
`HCl-Ala-ProP(QPh-4-Clh
`13
`100
`HCl•Ala-PipP(QPh-4-Cl)2
`0.12
`88
`a Percentage inhibition was measured after 2 or 30 min incuba(cid:173)
`tion in 0.05 M Tris, pH 7.8 buffer and 5% Me2SO and at 23 °C.
`TFA•Ala-Pro-AFC (0.190 mM) was used as the substrate.
`
`Table 2. Rates of Inhibition of DPP-IV by Peptide
`Phosphonates and Half-Lives for Hydrolysis of Peptide
`Phosphonatesa
`
`Figure 1. Structures of Pro- or Pip-containing dipeptide
`phosphonates, where R is the side chain of Ala, Phe, or Lys
`and Ax is phenyl, 4-chlorophenyl, or 4-fluorophenyl.
`
`Scheme 1a
`
`+HPO(OR)2
`
`a,b
`
`R' I'
`
`J.. JN~
`dore
`- - - - - - Z·NW lf '"(oR
`o 0~P,
`OR
`a Reagents: (a) heat under argon; (b) HCl gas in ether; (c)
`Z-NHCHR'COOH, DCC; (d) Pd/C, H2, W; (e) 30% HBr/AcOH.
`
`potent inhibitors of several serine proteases including
`PPE, HLE, and chymotrypsin.18 A good interaction with
`the S1 pocket19 of the enzyme is required for inactivation
`of serine proteases by these peptide phosphonates, and
`the interaction with extended substrate binding site of
`the enzyme is also essential for effective inhibition. In
`this paper we report the syntheses of several dipeptide
`phosphonates and their inhibitory activities against
`DPP-N and other proteases.
`
`Results and Discussion
`
`Chemistry. A series of dipeptides which contain
`2-pyrrolidylphosphonate (proline phosphonate, ProP) or
`2-piperidylphosphonate (homoproline phosphonate, PipP)
`(Figure 1) were synthesized using the reactions outlined
`in Scheme 1. The proline phosphonate HCl•ProP(QR)2
`was synthesized by reaction of diphenyl phosphite or
`bis(4-chlorophenyl) phosphite with 1-pyrroline trimer.20
`Subsequent coupling of HCl•ProP(OPhh (1) or HCl•
`ProP(OPh-4-Clh (3) with the N-blocked amino acid
`Z-AA-OH using the DCC method gave the dipeptide
`phosphonate Z-AA-ProP(QR)2. Deblocking of the dipep(cid:173)
`tides was accomplished by hydrogenolysis in the pres(cid:173)
`ence of acid or by the use of 30% HBr in AcOH to give
`compounds 6, 9, 10, and 12. Similarly, the homoproline
`derivative HCl·PipP(OPh)2 (2), HCl·PipP(QPh-4-Clh (4),
`or HCl·PipP(QPh-4-Fh (5) was synthesized by reaction
`of diphenyl phosphite or bis(4-halophenyl) phosphite
`with 2,3,4,5-tetrahydropyridine trimer.21 The interme(cid:173)
`diate PipP(ORh•HCl was then coupled with Z-AA-OH
`using the DCC method to give the dipeptides Z-AA-PipP(cid:173)
`(ORh. Subsequent deblocking of Z-AA-PipP(ORh with
`hydrogenolysis in the presence of acid or HBr in AcOH
`gave compounds 7, 12, 13, and 14. These dipeptide
`phosphonates were prepared as mixtures of diastereo(cid:173)
`mers. Silica gel column chromatography and prepara(cid:173)
`tive thin-layer chromatography were used to attempt
`the separation of these two isomers. In most cases, the
`column fractions contained both diastereomers with one
`isomer being the dominant species, which showed two
`
`tl/2 (h)
`23.1
`>72
`52
`>48
`5.3
`
`[I] (mM)
`0.42
`0.42
`0.42
`0.42
`0.42
`
`kob.l[l]
`(M-1 s-1)
`
`1.2
`12.6
`1.7
`4.2
`28
`
`inhibitors
`HCl•Ala-ProP( 0Phl2
`6
`AcOH•Ala-PipP(QPhh
`7
`10
`2HBrLys-ProP(QPhl2
`2HCl•Lys-PipP(QPhl2
`11
`12
`HC1-Ala-ProP(QPh-4-Cl)2
`13 AcOH•Ala-PipP(QPh-4-Clh
`156
`0.017
`(diastereomeric mixture)
`67
`(single diastereomer) >48b
`1300
`0.017
`14
`HCl-Ala-PipP(QPh-4-F)2
`12
`0.42
`>68
`a Hydrolysis and inhibition were measured in 0.05 M Tris, pH
`7.8 buffer and 8% Me2SO and at 23 oc. TFA•Ala-Pro-AFC (0.2
`mM) was used as the substrate. b 31P NMR spectra were used to
`·monitor the hydrolysis.
`peaks with different ratios in the 31 P NMR spectra of
`the phosphonates. Only one compound (13, CH3-
`COOH·Ala-PipP(OPh-4-Clh) showed one peak in the 31P
`NMR spectrum after chromatography. It is likely that
`this isomer is L,L (see Kinetics section), although we
`cannot exclude the possibility that both diastereomers
`are present.
`Inhibition Kinetics. The results of initial inhibition
`studies ofDPP-N by a series of dipeptide phosphonates
`are shown in Table 1. At inhibitor concentrations of
`0.12 mM and with a 2 min incubation time, only Lys(cid:173)
`PipP(OPh)2 (11) and Ala-PipP(OPh-4-Clh (13) effectively
`inhibited DPP-N. With a 30 min incubation time, five
`dipeptide phosphonates (6, 7, 11, 12, 13) showed some
`inhibitory potency. The monoester Ala-PipP(QH)(OPh)
`(8), compound 9 with Phe at the P2 site, and 10 did not
`show any inhibition of DPP-N under these conditions.
`The second-order inhibition rate constants kobJ[I] for
`the better inhibitors are shown in Table 2. All the
`phosphonates in Table 2 are mixtures of both diaster(cid:173)
`eomers. Compound 13 was also initially obtained as a
`mixture of diastereomers. Upon further chromatogra(cid:173)
`phy, one diastereomer (probably L,L) was obtained
`which showed one peak in the 31P NMR spectrum.
`The best inhibitor in the series is Ala-PipP(OPh-4-Clh
`(13) which has a kobs value of 1300 M-1 s-1. The k;nact
`(0.353 s-1) andKr (236 ,uM) values of this compound are
`obtained from Kitz and Wilson plot [kobs = k;nact[l]I(Kr
`+ [1])].22 The inhibitory potency of the single diastere(cid:173)
`omer of 13 was 8-fold higher than the diastereomer
`mixtures. Substitution of phenoxy by a 4-chlorophenoxy
`group improves the inhibition rate by 12-23~fold (12 >
`6; 13 > 7). However, the inhibition rate of DPP-N by
`compound 14 containing a 4-fluorophenyl group is
`similar to the inhibition rate for the unsubstituted
`
`Page 2 of 8
`
`

`
`Dipeptide Phosphonates at Inhibitors of DPP-IV
`
`Journal of Medicinal Chemistry, 1994, Vol. 37, No. 23 3971
`
`Table 3. Inhibition of Proteases and Esterases by Dipeptide Phosphonatesa
`
`Ala-PipP(QPh-4-Clh
`Ala-ProP(QPhh
`Ala-ProP(QPh-4-Clh
`(13)
`(12)
`(6)
`enzymes
`18
`Nib
`chymotrypsin
`26
`NI
`NI
`NI
`trypsin
`NI
`NI
`NI
`HLE
`NI
`PPE
`NI
`NI
`acetylcholinesterase
`NI
`NI
`NI
`NI
`papain
`NI
`NI
`NI
`cathepsin B
`NI
`NI
`a Inhibition was measured in 0.1 M Hepes, 0.5 M NaCl, pH 7.5 buffer (chymotrypsin, PPE, HLE), 0.1 M Hepes, 0.01 M CaCh, pH 7.5
`(trypsin), 0.1 M phosphate, pH 7.5 (acetylcholinesterase), 0.05 M Tris, 2 mM EDTA, 5 mM cysteine, pH 7.5 (papain), or 0.1 M phosphate,
`1.33 mM EDTA, 2.7 mM cysteine, pH 6.0 (cathepsin B), 8-9% Me2SO and at 23 •c. Substrates were Suc-Phe-Thr-Phe-pNA (0.48 mM)
`for chymotrypsin, Z-Phe-Gly-Arg-pNA (0.09 mM) for trypsin, MeO-Suc-Ala-Ala-Pro-Val-pNA (0.24 mM) for HLE, Suc-Ala-Ala-Ala-pNA
`(0.44 mM) for PPE. Inhibitor concentrations were 0.42 mM. b NI, no inhibition after 30 min of incubation of enzyme with inhibitor.
`
`s,
`I ("',I Ser195
`l.. .. J.. d
`N
`P-QPh
`Enz-CO£----•H3N ~O 8
`R
`Figure 2. Inhibition mechanism of DPP-IV by the dipeptide phosphonate, Ala-PipP(QPh)2. The mechanism involves the
`nucleophilic substitution at the phosphorus atom by the active site Ser-195 through a pentavalent intermediate to form a
`phosphonylated enzyme.
`
`H-HISs7
`
`phenoxy derivative 7. Replacing the Pro phosphonate
`by a homoproline phosphonate (PipP) also enhanced the
`inhibition by 2-10-fold (7 > 6; 11 > 10; 13 > 12).
`Previous studies with synthetic substrates demon(cid:173)
`strated that DPP-IV hydrolyzed the dipeptide p-nitroa(cid:173)
`nilides AA-Pro-pNA faster when the P2 site contained
`a Pro, Abu, Leu, Val, or Ala rather than Phe or Lys.23
`In the Pro or homoproline-containing phosphonate
`inhibitors, Ala is preferred at the P2 site rather than
`Lys or Phe. For example, Ala-ProP(QPhh (6) but not
`Phe-ProP(QPh)2 (9) or Lys-ProP(QPhh (10) inhibited
`DPP-IV at 0.12 mM and 30 min incubation (Table 1).
`Similarly, Ala-PipP(QPhh (11) inhibited DPP-IV more
`potently than Lys-PipP(OPhh (7). Interestingly, both
`Pro and homoproline-containing dipeptide phosphonates
`inhibited DPP-IV and the substitution of a Pro phos(cid:173)
`phonate by a homoproline phosphonate enhanced the
`inhibition rates. This indicates that the S1 pocket of
`DPP-IV is bigger than a proline ring and can accom(cid:173)
`modate the larger homoproline structure.
`The specificity of these dipeptide phosphonates for
`DPP-IV was examined by measuring inhibition rates
`with other proteases and esterases. The results were
`shown in Table 3. Three inhibitors, Ala-ProP(QPhh (6),
`Ala-ProP(OPh-4-Clh (12), and Ala-PipP(OPh-4-Clh (13),
`inhibited DPP-IV but not six other proteases and
`esterases. Two chlorophenoxy phosphonates 12 and 13
`inhibited chymotrypsin very slowly, which is surprising
`since chymotrypsin does not hydrolyze peptide sub(cid:173)
`strates with Pro at the P1 site. We postulate that one
`of the two 4-chlorophenoxy groups in inhibitors 12 and
`13 is fitting into the large hydrophobic sl site of
`chymotrypsin, and this result shows in the inhibition
`rates. With this exception, the inhibitors are highly
`specific for DPP-IV.
`Spontaneous Hydrolysis of Dipeptide Phospho·
`nates. Peptide phosphonates are known to be stable
`in buffer and plasma.18 Half-lives for hydrolysis of
`seven phosphonates are shown in Table 2. These
`
`inhibitors are quite stable with half-lives of several
`hours to several days. Phosphonates with homoproline
`at the P1 site are more stable than those with Pro Ctv2:
`7 > 6; 13 > 12). The 31P NMR spectra were used to
`monitor the hydrolysis of compound 13 in 50 mM Tris,
`pH 7.8 buffer containing 10% DMSO. The spectra show
`only one peak at 19.8210 ppm initially, and no extra
`peaks appear during a period of 48 h. This result
`indicates that the inhibitor is stable in the pH 7.8 buffer
`and does not react with Tris as in the earlier studies
`with different phosphonates under different condi(cid:173)
`tions.24 The inhibition of DPP-IV by CH3COOH·Ala(cid:173)
`PipP(OPh-4-Clh went to completion in a few min giving
`a stable phosphonylated enzyme Ct112 > 24 h). Thus, no
`reaction of the phosphonylated enzyme with Tris oc(cid:173)
`curred in our case. The 31P NMR also indicates the
`cyclization of the free amino group onto the phosphorus
`does not occur at this pH. Phosphonate are relatively
`unreactive to nitrogen nucleophiles and the amine group
`is protonated at this pH.
`Inhibition Mechanism. The proposed inhibition
`mechanism of DPP-IV by the dipeptide phosphonate
`Ala-PipP(QPhh (7) is similar to that previously described
`for other serine proteases (Figure 2).18 It involves the
`nucleophilic substition at the phosphorus atom by the
`active site Ser-195 through a pentavalent intermediate
`to form a phophonylated enzyme. The leaving group
`in these dipeptide phosphonates is an electronegative
`phenoxy or 4-halophenoxy group. The Pro or Pip
`residue fits into the S1 pocket. The DPP-IV which was
`inhibited by compounds 6, 7, 12, or 13 was stable and
`did not regain enzyme activity after 24 h. Excess
`inhibitors in the inhibited enzyme solution were re(cid:173)
`moved by centrifugation of the diluted enzyme solution
`twice using Amicon microconcentrators. These results
`are consistent with the formation of a stable phospho(cid:173)
`nylated enzyme derivative. A similar mechanism has
`also been proposed for the inhibition of class C P-lacta(cid:173)
`mase by m-carboxyphenyl [(phenylacetamido)methyl]-
`
`Page 3 of 8
`
`

`
`3972 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 23
`
`Boduszek et al.
`
`phosphonate where m-hydroxybenzoate was released
`stoichiometrically. 25 The phosphonylated enzymes have
`also been observed in the crystal structures of a.-lytic
`protease with two stereoisomers of Boc-Ala-Ala-Pro(cid:173)
`ValP(OPh)-Lac-Ala-OMe.24·26 In the complex of one
`isomer, the phenyl ester is displaced by the active site
`serine to form a tetrahedral adduct. In the complex of
`the other isomer, the same tetrahedral adduct and an
`adduct with both ester groups hydrolyzed were ob(cid:173)
`served.
`
`Conclusion
`A series of dipeptide phosphonates which contained
`a Pro or a homoproline analog (PipP) at the P 1 site are
`specific irreversible inhibitors ofDPP-IV. The dipeptide
`phosphonates are moderate inhibitors of DPP-IV with
`kobsi1I] values of 1-1300 M- 1 s- 1 and the best inhibitor
`is Ala-PipP(OPh-4-Clh (13). Since the 4-chlorophenoxy
`group is a better leaving group than the phenoxy
`substituent, it is expected that the dipeptide phospho(cid:173)
`nates with 4-chlorophenoxy groups would hydrolyze
`faster in buffer and inhibit DPP-IV more potently than
`those with phenoxy groups. The phosphonates are quite
`specific, and Ala-ProP(OPhh (6), Ala-ProP(OPh-4-Clh
`(12), and Ala-PipP(OPh-4-Cl)2 (13) did not inhibit pro(cid:173)
`teases and esterases such as trypsin, HLE, PPE, ace(cid:173)
`tylcholinesterase, papain, and cathepsin B, although 12
`and 13 inhibited chymotrypsin fair slowly. Most of
`these dipeptide phosphonates are stable in pH 7.8 buffer
`with half-lives of several hours to several days. DPP(cid:173)
`IV inhibited by 6, 7, 12, or 13 is quite stable, and no
`enzyme activity was regained after removal of excess
`inhibitors and incubation in the buffer for 1 day. Due
`to their high specificity and stability, these dipeptide
`phosphonates should be useful in establishing the
`biological roles of DPP-IV and may have therapeutic
`utility in preventing organ transplant rejection.
`
`Experimental Section
`Synthesis. Benzyl carbamate, diphenyl phosphite, pyrro(cid:173)
`lidine, piperidine, DCC, and all common chemicals were
`obtained from Aldrich Co., Milwaukee, WI. (Benzyloxycarbo(cid:173)
`nyl)proline (Z-Pro) and Na ,N•-bis(benzyloxycarbonyl)lysine
`were obtained from Bachem Fine Chemicals, CA. The purity
`of each new synthesized compound was checked by TLC, 1H
`NMR, mass spectroscopy (FAB), and elemental analysis. In
`the case of multistep synthesis, the first and final products
`were checked by 1H NMR, FAB spectra, and elemental
`analysis. The solvent system used for TLC was chloroform(cid:173)
`acetone (9:1). Preparative thin-layer chromatography was
`performed with plates precoated with silica gel (Merck). The
`NMR spectra were recorded on a Varian Gemini 300 MHz
`instrument in CDC13, DMSO-da, or D20 solutions. Mass
`spectra (FAB) were recorded on a VG 70-SE mass spectrom(cid:173)
`eter. Elemental analyses were performed by Atlantic Micro(cid:173)
`Lab Inc., Norcross, GA.
`Bis(4-chlorophenyl) phosphite and bis(4-fluorophenyl) phos(cid:173)
`phite were prepared from tris(4-chlorophenyl) phosphite and
`tris(4-fluorophenyl) phosphite, respectively, using a previously
`described procedure. 27 Tris( 4-chlorophenyl) phosphite was
`prepared from 4-chlorophenol and phosphorus trichloride with
`3 equiv of triethylamine as a base using a modification of a
`previous procedure.28 Similarly, tris(4-fluorophenyl) phosphite
`was prepared from 4-fluorophenol and phosphorus trichloride
`using 1 equiv of triethylamine as a base.
`Diphenyl Pyrrolidine-2-phosphonate Hydrochloride
`(HCl·ProP(QPhh, 1). This compound was synthesized from
`1-pyrroline trimer29 and diphenyl phosphite using the proce(cid:173)
`dure previously described for the synthesis of the diethyl
`
`ester.20 A mixture of 1-pyrroline trimer (17 mmol, 3.5 g) and
`diphenyl phosphite (50 mmol, 11.7 g) was heated at 85 oc for
`1.5 h under argon to give crude diphenyl pyrrolidine-2-
`phosphonate which was dissolved in 100 mL of dry diethyl
`ether, filtered, and saturated with dry gaseous HCI. The
`precipitated hydrochloride 1 was collected by filtration, washed
`with ether, and recrystallized from acetone to give the pure
`product as a white solid in 49% yield: mp 146-148 oc; 1H(cid:173)
`NMR (D20) o (ppm) 2.0-2.5 (m, 4H), 3.40 (m, 2H), 4.25 (m,
`1H), 6.90-7.30 (m, 10H); MS (FAB) m!e (rei intensity) 304 (M
`- Cl)+ (100). Anal. CC1aH19NOaPCl): C, H, N, Cl.
`Diphenyl Piperidine-2-phosphonate Hydrochloride
`(HCl•PipP(OPhh, 2). This compound was prepared from the
`trimer of 2,3,4,5-tetrahydropyridine30 and diphenyl phosphite
`using the procedure previously described for the synthesis of
`the diethyl ester.21 A mixture of the trimer (10 mmol, 2.5 g)
`and diphenyl phosphite (30 mmol, 7.0 g) was heated for 1.5 h
`at 100 oc under argon. The resulted crude diphenyl piperi(cid:173)
`dine-2-phosphonate was dissolved in 100 mL of dry ether,
`undisolved material was removed by filtration, and the solu(cid:173)
`tion was saturated with gaseous HCI. The precipitated
`hydrochloride 2 was collected by filtration, washed with ether,
`dried, and recrystallized from acetone to give a white solid in
`41% yield: mp 172-174 oc; 1H-NMR (D20), o (ppm) 1.5-2.4
`(m, 6H), 3.05 (m, 1H), 3.45 (m, 1H), 4.10 (m, 1H), 6.9-7.4 (m,
`10H); MS (FAB) mle (rei intensity) 318 (M- Cl)+ (100). Anal.
`(C17H21NOaPCl): C, H, N, Cl.
`Bis(4-chlorophenyl) Pyrrolidine-2-phosphonate Hy(cid:173)
`drochloride (HCl·ProP(QPh-4-Cl)2, 3). A mixture of 1-pyr(cid:173)
`roline trimer and bis(4-chlorophenyl) phosphite was reacted
`using the procedure described for compound 1. Hydrochloride
`3 was obtained by dissolving the crude phosphonate in ether
`saturated with gaseous HCl (white solid in 30% yield): mp
`160-165 oc dec; 1H-NMR (D20) o (ppm) 1.8-2.5 (m, 4H), 3.4
`(m, 2H), 4.30 (m, 1H), 7.0 (m, 4H), 7.2 (m, 4H); MS (FAB) m/e
`(rei intensity) 372 (M - Cl)+ (100). Anal. (C1aH17NOaPCla):
`C,H,N.
`Bis(4-chlorophenyl) Piperidine-2-phosphonate Hy(cid:173)
`drochloride (HCl·PipP(OPh-4-Cl)2, 4). The trimer of 2,3,4,5-
`tetrahydropyridine and bis(4-chlorophenyl) phosphite were
`reacted using the procedure described for compound 2. The
`phosphonate hydrochloride 4 was obtained by reaction with
`HCI as described above in 55% yield: mp >140 °C dec; 1H(cid:173)
`NMR CD20) o (ppm) 1.3-2.1 (m, 6H), 3.2-3.5 (m, 2H), 4.0 (m,
`1H), 6.86 (m, 4H), 7.1 (m, 4H); MS (FAB) mle (rei intensity)
`386 (M - Cl)+ (70); HRMS calcd for C17H19N10aP1Cb mle
`386.0479, found 386.0468.
`Bis(4-fluorophenyl) Piperidine-2-phosphonate Hydro(cid:173)
`chloride (HCl·PipP(QPh-4-F)2, 5). A mixture of piperidine
`trimer ( 4.5 g, 54 mmol) and his( 4-fluorophenyl) phosphite (14.8
`g, 55 mmol) was heated at 90-100 oc for 3 h under nitrogen.
`The resulted oil was cooled and dissolved in a mixture of 50
`mL ofCH2Cb and 50 mL of ether. The solution was saturated
`with dry HCI, and the oil was separated and solidified after
`several hours. The solid was filtered, washed with ether, and
`dried. The hygroscopic material was stirred in 200 mL of dry
`ether for several hours, and the yellowish solid was filtered
`and dried. The product was obtained in 54% yield (11.6 g)
`and used for subsequent reaction: mp 155-165 oc dec; 1H
`NMR (D20) o 7.2-6.8 (m, 8H), 3.6-2.9 (m, 3H), 2.2-1.4 (m,
`6H); MS (FAB) m/e (rel intensity) 354 (M - Cl)+ (100); HRMS
`calcd for C17H19N10aP1F2 mle 354.1070, found 354.1098.
`Dipeptide Synthesis: General Procedure. The hydro(cid:173)
`chloride of the phosphonates (1, 2, 3, 4 or 5) (5 mmol) and
`triethylamine (5 mmol) were dissolved in 25 mL ofCH2Cl2 and
`cooled to -10 oc. A Z-blocked amino acid (5 mmol) was added,
`and the mixture was stirred at -10 oc for 15 min. The
`coupling reagent DCC (6 mmol) in 25 mL ofCH2Cb was added,
`and the mixture was stirred at -10 oc for 2 h and 20 h at
`room temperature. The DCU precipitate was removed by
`filtration, and the filtrate was evaporated. The residue was
`dissolved in 100 mL of ethyl acetate and filtered. The organic
`layer was washed subsequently with 50 mL of 1M HCl, water,
`6% NaHC03, and water and dried over MgS04. The filtrate
`was evaporated to give the crude dipeptide. Traces of DCU
`was removed by filtration of the crude dipeptide dissolved in
`
`Page 4 of 8
`
`

`
`Dipeptide Phosphonates at Inhibitors of DPP-N
`
`Journal of Medicinal Chemistry, 1994, Vol. 37, No. 23 3973
`
`25 mL of ether. The dipeptide was dried in vacuo and
`recrystallized from hexane-ether.
`The Z group of the dipeptides were removed by hydro(cid:173)
`genolysis or treatment with 30% HBr in acetic acid. The
`Z-blocked dipeptide (1-2 mmol) was dissolved in 100 mL of
`methanol, 1 equiv of concentrated hydrochloric acid (1-2
`mmol) and 5% Pd on carbon (0.5-1.0 g) were added, and the
`mixture was hydrogenated at room temperature for 2-3 h.
`After hydrogenation, the catalyst was removed and the filtrate
`was evaporated to give the deblocked dipeptide hydrochloride,
`which was recrystallized from methanol-ether or ether. The
`Z-blocked dipeptide (1 mmol) can also be treated with 1 mL of
`30% HBr/AcOH and stirred at room temperature for 1 h. The
`mixture was protected against moisture during stirring. The
`solution was diluted with 50 mL of dry ether and kept at 0 °C
`for 1-2 h. The hydrobromide of the dipeptide precipitated and
`was filtered, washed with dry ether, and dried as a yellow(cid:173)
`brown solid.
`Diphenyl Alanylpyrrolidine-2-phosphonate Hydro(cid:173)
`chloride (HCl·Ala-ProP(OPh)2, 6). Z-Ala-ProP(OPhh was
`obtained as a thick oil in 90% yield: 1H-NMR (CDC13) o (ppm)
`1.3 (dd, 3H), 1.5-2.5 (m, 5H), 3.3-3.8 (m, 2H), 4.1 (m, 1H),
`4.5 (m, 1H), 5.10 (m, 2H), 5.7 (dd, 1H), 7.0-7.4 (m, 15H).
`Hydrogenolysis of Z-Ala-ProP(OPh)2 gave the product 6 as a
`hygroscopic solid in 65% yield: mp 80-85 oc; MS (FAB) m/e
`(rei intensity) 375 (M - Cl)+ (100). The product was further
`purified by silica gel column chromatography eluted with
`CHCia:MeOH:CHaCOOH, 8:2:0.1: 1H-NMR (DMSO) o (ppm)
`1.20-1.35 (d, 3H, J = 6.8 Hz), 1.9-2.4 (m, 4H), 3.6-3.75 (m,
`2H), 3.9-4.05 (m, 1H), 4.8-4.9 (m, lH), 7.05-7.5 (m, 10H);
`31P-NMR (DMSO, ppm), 19.2433, 19.1841 (1:0.5); MS (FAB)
`mle (rel intensity) 375 (M- CHaCOO)+ (100); HRMS calcd for
`C19H2~204P1 mle 375.1474, found 375.1473.
`Diphenyl Alanylpiperidine-2-phosphonate Acetate
`(CH3COOH·Ala-PipP(OPh)2, 7). Z-Ala-PipP(OPh)2 was ob(cid:173)
`tained as a thick oil in 76% yield: 1H-NMR (CDCI3) o (ppm)
`1.2-1.3 (dd, 3H), 1.5-2.4 (m, 6H), 3.6-3.8 (m, 2H), 4.5 (m, 1H),
`5.10 (s, 2H), 5.6 (m, 1H), 5.8 (dd, 1H), 7.0-7.4 (m, 15H).
`Hydrogenolysis ofZ-Ala-PipP(OPh)2 using 1 equiv of acetic acid
`gave the product 7 as a glass-like solid in 82% yield: mp 60-
`70 oc; MS (FAB) mle (rei intensity) 389 (M- CH3COO)+ (100).
`The product was further purified by silica gel column chro(cid:173)
`matography eluted with CHCia:MeOH:CH3COOH, 8:2:0.1: 1H(cid:173)
`NMR (DMSO) o (ppm) 1.0 (d, 3H, J = 6.5 Hz), 1.4-2.3 (m,
`6H), 1.9 (s, 3H), 3.4-3.5 (t, 2H, J = 13 Hz), 3.8-4.0 (m, 2H),
`5.45-5.50 (m, 1H), 7.0-7.4 (m, 10H); 31P-NMR (DMSO, ppm)
`18.9337, 18.7177 (1:0.1); HRMS calcd for C2oH2sN204P1 mle
`389.1630, found 389.1639.
`Monophenyl Alanylpiperidine-2-phosphonate Acetate
`(CHaCOOH·Ala-PipP(OH)(OPh), 8). A small amount of the
`monophenyl ester 8 was isolated in 10% yield during the
`workup of product 7 as a white solid: mp 175-180 oc dec;
`MS (FAB) mle (rel intensity) 313 (M - CH3COO)+ (100). The
`product was further purified by silica gel column chromatog(cid:173)
`raphy eluted with CHCI3:MeOH:CH3COOH, 8:2:0.1: 1H-NMR
`(DMSO) o (ppm) 1.1-1.3 (d, 3H, J = 6.5 Hz), 1.4-2.2 (m, 6H),
`1.9 (s, 3H), 3.1-3.2 (t, 1H, J = 13Hz), 4.1-4.2 (m, 1H), 4.25-
`4.42 (m, 2H), 6.9-7.3 (m, 5H); 31P-NMR (DMSO, ppm) 13.2251,
`13.1494 (1:0.5); HRMS calcd for C1,H22N20~1 m/e 313.1317,
`found 313.1314.
`Diphenyl Phenylalanylpyrrolidine-2-phosphonate Hy(cid:173)
`drobromide (HBrPhe-ProP(OPh)2, 9). Z-Phe-ProP(OPhh
`was obtained as a thick oil which partially solidified in 61%
`yield: 1H-NMR (CDCla) o (ppm) 1.0-2.0 (m, 4H), 3.25 (d, 2H),
`3.0-3.5 (m, 3H), 4.7 (m, lH), 5.05 (m, 1H), 5.15 (s, 2H), 5.35
`(m, 1H), 7.0-7.4 (m, 20H). Deblocking of Z-Phe-ProP(OPh)2
`with 30% HBr/AcOH gave the product 9 as a yellow-brown
`hygroscopic solid in 33% yield: mp >140 oc dec; 1H-NMR
`CD20) o 1.0-2.0 (m, 4H), 3.0-3.4 (m, 3H), 3.3 (d, 2H), 4.3 (m,
`1H), 4.7 (m, 1H), 7.0-7.4 (m, 15H); MS (FAB) mle 451 (M(cid:173)
`Br)+ (100); HRMS calcd for C2sH2sN204P1 mle 451.1786, found
`451.1769.
`Diphenyl Lysylpyrrolidine-2-phosphonate Dihydro(cid:173)
`bromide (2HBrLys-ProP(OPh)2, 10). The lysine derivative
`Z-Lys(Z)-OH was reacted with phosphonate 1 to give Z-Lys(cid:173)
`(Z)-ProP(OPh)2 as a thick oil in 86% yield: 1H-NMR (CDCla) o
`
`(ppm) 1.0-2.5 (m, 8H), 3.0-4.0 (m, 4H), 4.5 (m, 1H), 4.9 (m,
`1H), 5.1 (2s, 4H), 5.5-5.8 (dd, 2H), 7.0-7.4 (m, 20H). Treat(cid:173)
`ment of Z-Lys(Z)-ProP(OPhh with 30% HBr/AcOH gave the
`product 10 as a yellow brown hygroscopic solid in 64% yield:
`mp >85 oc dec; 1H-NMR (D20) o (ppm) 1.2-2.5 (m, 8H), 2.8-
`3.0 (m, 4H), 3.55 (t, 1H), 4.1-4.5 (m, 2H), 7.0-7.5 (m, 10H);
`MS (FAB) m/e (rei intensity) 432 (M- H- 2Br)+ (100); HRMS
`calcd for C22Ha1Na04P1 m/e 432.2052, found 432.2059.
`Diphenyl Lysylpiperidine-2-phosphonate Dihydro(cid:173)
`chloride (2HCl•Lys-PipP(OPh)2, 11). The lysine derivative
`Z-Lys(Z)-OH was reacted with phosphonate 2 to give diphenyl
`Z-Lys(Z)-PipP(OPh)2 as a thick oil in 65% yield: 1H-NMR
`(CDCI3) o (ppm) 1.0-2.0 (m, 12H), 3.0-3.2 (m, 2H), 3.4-3.8
`(m, 2H), 4.4-4.8 (m, lH), 5.1 (2s, 4H), 5.5-5.9 (m, 2H), 7.0-
`7.5 (m, 20H). Hydrogenolysis ofZ-Lys(Z)-PipP(OPhh gave the
`product 11 as a white solid in 54% yield: mp 110-115 oc dec;
`1H-NMR (D20) o (ppm) 1.3-2.1 (m, 12H), 2.8-3.0 (m, 2H),
`3.3-3.7 (m, 2H), 4.3-4.5 (m, 1H), 7.0-7.4 (m, 10H); MS (FAB)
`m/e (rei intensity) 446 (M - H - 2Cl)+ (100); HRMS calcd for
`C2aHaaNa04P1 m/e 446.2208, found 446.2213.
`Bis(4-chlorophenyl) Alanylpyrrolidine-2-phosphonate
`Hydrochloride (HCl·Ala-ProP(OPh-4-Cl)2, 12). (Benzy(cid:173)
`loxycarbonyl)alanine (Z-Ala) was reacted with phosphonate 3
`to give Z-Ala-ProP(OPh-4-Cl)2 (12a) as a semisolid in 41%
`yield: 1H-NMR (CDCla) o (ppm) 1.25-1.35 (dd, 3H), 1.8-2.5
`(m, 4H), 3.3-3.9 (m, 2H), 4.55 (m, lH), 5.0 (m, 1H), 5.1 (s,
`2H), 5.5-5.7 (dd, lH), 7.0-7.4 (m, 13H); MS (FAB) m/e (rei
`intensity) 577 (M + 1)+ (100). Hydrogenolysis of Z-Ala-ProP(cid:173)
`(0Ph-4-Cl)2 gave the product 12 as a hygroscopic white solid
`in 53% yield: mp 88-91 °C; MS (FAB) mle (rei intensity) 443
`(M - Cl)+ (100). The product was further purified by silica
`gel column chromatography eluted with CHCla:MeOH:CHa(cid:173)
`COOH, 8:2:0.1: 1H-NMR (DMSO) o (ppm) 1.25-1.35 (d, 3H,
`J = 7.1 Hz), 2.0-2.4 (m, 4H), 3.6-3.8 (m, 2H), 4.1-4.3 (m,
`1H), 4.8-4.9 (m, 1H), 7.2-7.5 (m, 8H); 31P-NMR (DMSO, ppm),
`19.8748, 19.6918 (1:0.33); MS (FAB) mle 443 (M- CHaCOO)+;
`HRMS calcd for C1gH~20,CbP1 m/e 443.0694, found 443.0693.
`Bis(4-chlorophenyl) Alanylpiperidine-2-phosphonate
`Hydrochloride (HCl·Ala-PipP(OPh-4-Cl)2, 13). (Benzyloxy(cid:173)
`carbonyl)alanine (Z-