•
`
`Pergamon
`
`Bioorganic & Medicinal Chemistry Letters, Vol. 6, No. 10, pp. 1163-1166, 1996
`Copyright © 1996 Elsevier Science Ltd
`Printed in Great Britain. All rights reserved
`0960-894X/96 $15.00 + 0.00
`
`PIT: S0960-894X(96)00 190-4
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`2-CYANOPYRROLIDIDES AS POTENT, STABLE INHIBITORS
`
`OF DIPEPTIDYL PEPTIDASE IV
`
`Doreen M. Ashworth, Butrus Atrash, Graham R. Baker, Andrew J. Baxter, Paul D. Jenkins*,
`
`D. Michael Jones and Michael Szelke
`
`Ferring Research Institute, Chilworth Research Centre, Chilworth, Southampton, U.K.,
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`S016 7NP. Fax +44 ( 1703)766253; e-mail pdj@jerring.demon.co.uk
`
`Abstract: A novel series of stable, potent inhibitors of dipeptidyl peptidase IV has been developed. A
`number of dipeptide analogues, incorporating a 2-cyanopyrrolidide, were found to have Ki values of less than 5
`
`nM versus human DP-IV and half-lives of >48h in aqueous solution (pH 7.4). Copyright© 1996 Elsevier Science Ltd
`
`Dipeptidyl peptidase IV (DP-IV, EC 3.4.14.5) is a serine protease which catalyses the cleavage of
`
`dipeptides from theN-terminus of proteins with the sequence H-X-Pro-Y- or H-X-Ala-Y- (where X, Y= any
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`amino acid, Y ¢ Pro). 1 DP-IV is widely distributed in mammalian tissues and is found in great abundance in the
`kidney, liver, intestinal epithelium and placenta.2 In the human immune system, DP-IV is identical to the T cell
`
`activation marker, CD26. Recent evidence has also shown CD26 to be an activation marker of natural killer
`cells3 and of a main population of B cells.4
`
`Our interest in DP-IV was stimulated by the publication of data which showed that either simple
`
`inhibitors or antibodies of the enzyme were effective as inhibitors ofT cell proliferation and were thus potential
`immunomodulators.5
`
`7
`-
`
`Substrates and inhibitors of DP-IV require a free N-terminus, which means that potential dipeptide serine
`
`trifluoromethylketones, or
`inhibitors (e.g. C-terminal aldehydes, boronic acids, a-ketoacids,
`protease
`chloromethylketones) are inherently unstable at neutral pH due to intramolecular cyclisation.8
`
`2
`The most potent DP-IV inhibitors reported to date are the boroproline analogues 1, (1<.=2nM) and 2,
`
`1
`
`(1<.=3nM). However, these boronic acids are unstable at neutral pH (L;,=30min and 90min for 1 and 2
`
`1163
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`TEVA - EXHIBIT 1007
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`

`
`1164
`
`D. M. ASHWORTH et al.
`
`respectively).7 Other, more stable classes of DP-IV inhibitors have been reported. These include tripeptides;6
`thiazolidides;6 dipeptide phosphonates;9
`10 azaprolines; 11 and the irreversible
`aminoacyl pyrrolidides and
`'
`N-peptidyl-O-aroylhydroxylamines. 12 Although specific for DP-IV, these compounds exhibit, at best, only
`
`modest levels of inhibition.
`
`We felt that it was necessary to develop more potent, stable inhibitors of DP-IV. These would help
`
`elucidate the physiological role of the enzyme and may have therapeutic potential in a number of disease states
`such as inflammation, graft versus host disease (GVHD), cancer, or AIDS.5
`
`Table I. Inhibition of human DP-IV by aminoacyl pyrrolidides. 13
`
`H-Xaa-D
`Xaa
`Cyclohexylglycine [Chg]
`(R,S)-Cyclopentylglycine [Cpg]
`Ile
`allo-Ile
`Val
`Lys(Cbz)
`tert-Butylglycine [Tbg]
`Thr(Me)
`Om(Cbz)
`2-Aminohexanoic acid [Aha]
`Glu
`Pro
`Cyclohe~alanine [Cha]
`Glu(OBn)
`Thr
`Phenylglycine [Phg]
`Ser(Bn)
`Ala
`Asp
`
`CompoundN°
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`
`Kt ().I.M)13
`0.064 ± 0.01
`0.21 ± 0.04
`0.41 + 0.01
`0.44+0.04
`0.47 ± 0.02
`0.52 ± 0.07
`0.88 + 0.20
`0.90±0.15
`0.91 ± 0.20
`1.20 ± 0.20
`2.00 ± 0.40
`2.10 ± 0.20
`2.15 ± 0.50
`2.70 ±0.30
`4.90+ 0.90
`5.30 ± 0.10
`6.00 ± 1.50
`7.00 ± 1.00
`14.50 ± 1.90
`
`Our attention was drawn by a patent claiming 2-cyanopyrrolidides as inhibitors of prolyl endopeptidase14
`
`(PEP, EC 3.4.21.26), an enzyme belonging to the same subfamily of serine proteases as DP-IV. PEP differs from
`
`DP-IV by being an endopeptidase but the two enzymes share the common specificity for cleaving peptides at the
`
`carboxyl side of proline peptidyl bonds.
`We now wish to report on the synthesis and biological activity of a series of dipeptide nitriles15 with
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`potencies versus human DP-IV comparable to the boroprolines 1 and 2 but with superior stability in aqueous
`solution. One other group has recently described similar compounds as inhibitors of DP-IV16 but whereas they
`
`confirm that such derivatives possess good stability, their series exhibit only modest potency (four compounds
`
`with Ki values versus rat DP-IV of 0.19-1.2 J.!M).
`
`

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`2-Cyanopyrrolidides
`
`1165
`
`To establish an optimal N-terrninal residue, we prepared a series of amino acid pyrrolidides.6 These
`
`compounds were prepared by reaction of the 0-succinimide, (ONSu), ester of the required Boc protected amino
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`acid with a slight excess of pyrrolidine in dichloromethane. Subsequent acid catalysed deprotection (4N
`
`HCI/dioxane) afforded the inhibitor as its hydrochloride salt. As expected, from the substrate specificity of
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`DP-IV, only (S)-amino acid derivatives showed any activity and, as can be seen in Table I, lipophilic amino
`
`acids gave more potent compounds. In particular, [3-branched a-amino acid derivatives were the most potent
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`compounds with the non-proteinogenic amino acid, (S)-cyclohexylglycine providing the most active pyrrolidide
`
`(compound 5 possessing a Ki value of 64 nM).
`
`We then applied these fmdings to a series of 2-cyanopyrrolidides. The preparation of these compounds
`
`required a large scale synthesis of 2-cyanopyrrolidine 4 (Scheme 1). N-Boc-2-cyanopyrrolidine was readily
`
`prepared from Boc-Pro-NH2 using a dehydrating mixture of phosphorous oxychloride, pyridine and imidazole
`
`but the usual acidic conditions required to remove the Boc protecting group led to decomposition of the
`2-cyanopyrrolidide. Employment of the o-nitrophenylsulfenyl (ONPS) protecting group 17 however, enabled a
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`very mild deprotection to be used in the fmal step. Adding three equivalents of 4N HCI/dioxane to 3 in a large
`
`volume of diethyl ether afforded the hydrochloride salt 4 as an off-white precipitate in excellent yield.
`
`Scheme I. Preparation of dipeptide nitriles.
`
`H-Pro-OH
`
`a-c
`94%
`
`., ONPS-Pro-NH
`
`95% ONPS/~N
`--"t,..,_g __...., H-Xaa-~
`
`_ _.d.__+
`
`2
`
`3
`
`.TFA
`
`CN
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`Reagents: a.ONPS-Cl, 2N NaOH. b. HONSu, Water soluble carbodiimide. c cone. N~OH, dioxane.
`d. imidazole (2 equiv.), POCb (4 equiv.), pyridine. e. 4N HCI/dioxane (3 equiv.), diethyl ether.
`f. Boc-Xaa-OH, pyBop, NEt3, CH2Ch. g. Trifluoroacetic acid.
`
`The series of dipeptide nitriles described in Table ll were prepared via a pyBop 18 mediated coupling of 4
`
`with the required Boc protected amino acid, followed by deprotection with TFA (Scheme 1).
`
`We were gratified to fmd that these compounds were potent inhibitors of DP-IV. The S.A.R. for the
`
`N-terrninal residue developed in the pyrrolidide series correlated well for the dipeptide nitrile series and the most
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`potent compounds 24, 25, 26 and 27 possessed activity comparable to the boroprolines, 1 and 2. Stability
`studies19 revealed excellent half-lives (t.,) in aqueous solution (pH 7.4) at room temperature (Table II) with
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`several examples having t., greater than 48h. Further work on optimisation of the pyrrolidine ring will be
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`reported shortly.
`
`

`
`1166
`
`D. M. ASHWORTH et al.
`
`Table II.
`
`Diveptide nitrites: Potency versus human DP-/V and stability in
`
`aqueous solution (pH 7.4).
`
`CompoundN°
`24
`25
`26
`27
`28
`29
`
`Xaa
`Cpg_
`Chg
`lle
`Tbg
`Lys(Z)
`Pro
`
`K1(nM)'~
`l.l ± 0.2
`1.4 ± 0.5
`2.2 ±0.5
`3.8 ± 0.8
`5.2 ± 1.0
`22.0 ±4.0
`
`~(h)IY
`48
`>48
`48
`>48
`24
`7.5
`
`These compounds were found to be non-toxic in T cell assays up to 72h and inhibitor 26 had no acute
`
`toxicity when injected into mice (up to lOmg/Kg). We are currently exploring the effects of these compounds on
`
`lymphocytes (e.g. proliferation and cytokine release) and further details will be reported in due course.
`
`REFERENCES AND NOTES
`
`I) Heins, J., Weiker, P., Schonlein, C., Born, I., Hartrodt, B., Neubert, K., Tsuru, D. and Barth, A., Biochim. et Biophys. Acta
`1988, 954, 161.
`2) Hegen, M., Niedobitek, G., Clein, C.E., Stein, H. and Fleischer, B., J.lmmunol. 1990,144, 2908.
`3) Buhling, F., Kunz, D., Rheiuhold, D., Ulmer, A.J., Ernst, M., Flad, H.-D. and Ansorge, S., Nat. Immun. 1994, 13, 270.
`4) Buhling, F., Junker, D., Rheiuhold, D., Neubert, K., Jager, L. and Ansorge, S.,lmmunology Lett. 1995, 45, 47.
`5) A review has recently been published, discussing the evidence that CD26 has important functions in the immune system.
`Fleischer, B., Immunology Today 1994, /5, 180.
`6) Schon, E., Born, I., Demuth, H.-U., Faust, J., Neubert, K., Steinmetzer, T., Barth, A. and Ansorge, S., Bioi. Chem. Hoppe·
`Seyler 1991, 372, 305.
`7) Flentke, G.R., Munoz, E., Huber, B.T., Plaut, A.G., Kettner, C.A. and Bachovchin, W.W., Proc. Nat/. Acad. Sci. USA 1991,
`88, 1556.
`8) Sudmeier, J.L., Gunther, U.L., Gutheil, W.G., Coutts, S.J., Snow, R.J., Barton, R.W. and Bachovchin, W.W., Biochemistry
`1994, 33, 12427.
`9) Bodusek, B., Oleksyszyn, J., Karn, C.-M., Smith, R.E. and Powers, J.C., J. Med. Chem. 1994, 37, 3969.
`10) Be1yaev, A., Bor1oo, M., Augustyns, K., Lambeir, A.-M., De Meesters, 1., Scharpe, S., Blaton, N., Peeters, O.M., De Ranter, C.
`and Haemers, A., Tetrahedron Lett. 1995, 36, 3755.
`11) Borloo, M., Augustyns, K., Belyaev, A., De Meester, 1., Lambeir, A.-M., Goossens, F., Bollaet, W., Rajan, P, Scharpe, S. and
`Haemers, A., Letts. in Pep. Sci. 1995, 2, 198.
`12) Demuth, H.-U., Fischer, G., Barth, A. and Schowen, R.L., J. Org. Chem. 1988, 54, 5880.
`13) All contpounds were tested in vitro against pure human DP-IV (purchased from M&E, Copenhagen, Denmark). Inhibition was
`determined using the fluorogenic substrate, H-Ala-Pro-AFC at three concentrations per inhibitor. A typical assay (total volume
`0.4 mL) contprised sodium HEPES 83.3 mM, EDTA 1.67 mM, BSA 1.5 mg mL·', pH 7.8, DP-IV 25~tU mL·', inhibitor (in 10
`mM acetate pH 4.0). The reaction was started by the addition of substrate and readings taken every 30 sec for 7.5 min,
`excitation at 395 nm, emission 450nm. K; values were determined using Dixon plots.
`14) Patent. WO 91118877 (7 June 1990).
`15) Patent. WO 95/15309 (6 Dec. 1993).
`16) Li, J., Wilk, E. and Wilk, S., Arch. Biochem. and Biophys. 1995, 323, 148.
`17) Schroder, E. and Lubke, K., The Peptides, Academic Press, New York, 1965, Vol. I.
`18) Martinez, J., Bali, J.P., Rodriguez, M., Castro, B., Laur, J. and Lignon, M.-F., J. Med. Chem. 1988, 28, 1874.
`19) The stability of the iuhibitors in buffered, aqueous solution (100 mM Tris, pH 7.4) was monitored by reverse-phase HPLC.
`
`(Received in Belgium 21 February 1996; accepted 19 April 1996)