Pergamon
`
`Bioorganic & Medicinal Chemistry letters, Vol. 6. No. 10, pp, H63-H66, I996
`Copyright © 1996 Elsevier Science Ltd
`Printed in Great Britain. All rights reserved
`0960-894X/96 $15.00 + 0.00
`
`PII: S0960—894X(96)00190-4
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`2-CYANOPYRROLIDIDES AS POTENT, STABLE INHIBITORS
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`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.,
`
`S016 7NP. Fax +44 (1703)766253; e-mail pdj@ferring.demon.c0.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 the N-tenninus of proteins with the sequence H-X-Pro-Y- or H-X-Ala-Y- (where X, Y: any
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`amino acid, Ya: Pro).’ DP-IV is widely distributed in mammalian tissues and is found in great abundance in the
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`kidney, liver, intestinal epithelium and placenta? In the human immune system, DP-IV is identical to the T cell
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`activation marker, CD26. Recent evidence has also shown CD26 to be an activation marker of natural killer
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`cells’ and of a main population of B cells."
`
`Our interest
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`in DP-IV was stimulated by the publication of data which showed that either simple
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`inhibitors or antibodies of the enzyme were effective as inhibitors of T cell proliferation and were thus potential
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`immunomodulators.”
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`Substrates and-inhibitors of DP-IV require a free N-terminus, which means that potential dipeptide serine
`
`protease inhibitors
`
`(e.g. C—terminal aldehydes, boronic
`
`acids,
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`ot—ketoacids,
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`trifluoromethylketones, or
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`chloromethylketones) are inherently unstable at neutral pH due to intramolecular cyclisationf‘
`
`H-Ala/Q
`
`H-Pro/“Q
`
`B(OH)2
`
`1
`
`B(OH)2
`
`2
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`The most potent DP-IV inhibitors reported to date are the boroproline analogues 1, (K,=2nM) and 2,
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`(K;=3nM). However,
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`these boronic acids are unstable at neutral pH (t.,,=30min and 90min for
`
`1 and 2
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`Sun-Amneal-IPRZO16-O1104- Ex. 1007, p. 1 of4
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`1163
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:19)(cid:26)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
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`EXHIBIT
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`Ex. 1007
`Ex. 1007
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`1164
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`D. M. AS]-{WORTH er al.
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`respectively).7 Other. more stable classes of DP-IV inhibitors have been reported. These include tripeptides;°
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`aminoacyl pyrrolidides and thiazolididesf dipeptide phosphonates;9"° azaprolinesf‘ and the irreversible
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`N-peptidyl-0—aroylhydroxylamines.‘Z Although specific for DP-IV,
`modest levels of inhibition.
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`these compounds exhibit, at best, only
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`We felt that it was necessary to develop more potent, stable inhibitors of DP-IV. These would help
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`elucidate the physiological role of the enzyme and may have therapeutic potential ir1 a number of disease states
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`such as inflammation, graft versus host disease (GVHD), cancer, or AIDS.’
`
`Table I. Inhibition of human DP—IV by aminoacyl pyrrolidides.”
`
`H-Xaa-AD
`
`
`
`coma-maN°
`Z Cyclohex 11 cine [Ch]
`j <R.s>-cyclontyllycine [C1
`
`ejZ
`
`1
`
`
`
`jij
`jj
`T1
`
`jl
`
`jj A
`
`s
`
`Our attention was drawn by a patent claiming 2-cyanopyrrolidides as inhibitors of prolyl endopeptidase‘
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`(PEP, EC 3.4.21.26), an enzyme belonging to the same subfamily of serine proteases as DP-IV. PEP differs from
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`DP-IV by being an endopeptidase but the two enzymes share the common specificity for cleaving peptides at the
`
`4
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`carboxyl side of proline peptidyl bonds.
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`We now wish to report on the synthesis and biological activity of a series of dipeptide nitriles” with
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`potencies versus human DP-IV comparable to the boroprolines 1 and 2 but with superior stability in aqueous
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`solution. One other group has recently described similar compounds as inhibitors of DP-IV” 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 0.19-1.22 p.tM).
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:19)(cid:26)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
`Sun-Amneal-|PR2016-01104- Ex. 1007, p. 2 of 4
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`

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`2—Cyanopyrrolidides
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`l 165
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`To establish an optimal N-terminal residue, we prepared a series of amino acid pyrro]idides.° These
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`compounds were prepared by reaction of the 0-succinirnide, (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
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`HC1/dioxane) afforded the inhibitor as its hydrochloride salt. As expected, from the substrate specificity of
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`DP—IV, only (S)—arnino acid derivatives showed any activity and, as can be seen in Table I, lipophilic amino
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`acids gave more potent compounds. In particular, [3-branched on-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
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`(compound 5 possessing a K value of 64 nM).
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`We then applied these findings to a series of 2—cyanopyrrolidides. The preparation of these compounds
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`required a large scale synthesis of 2—cyanopyrrolidine 4 (Scheme I). N—Boc-2—cyanopyrrolidine was readily
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`prepared from Boc-Pro-NH2 using a dehydrating mixture of phosphorous oxychloride, pyridine and irnidazole
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`but the usual acidic conditions required to remove the Boc protecting group led to decomposition of the
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`2-cyanopyrrolidide. Employment of the o-nitrophenylsulfenyl (ONPS) protecting group” however, enabled a
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`very mild deprotection to be used in the final step. Adding three equivalents of 4N HCI/dioxane to 3 in a large
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`volume of diethyl ether afforded the hydrochloride salt 4 as an off-white precipitate in excellent yield.
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`Scheme I. Preparation of dipeptide nitriles.
`
`d
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`H-Pro-OH —"'°—p ONPS-Pro-NH2 -'""-E;/T’ ONPS/A?‘
`
`94%
`
`CN
`
`g
`9°"’°
`
`I
`
`l
`H'N
`HQ] 4
`
`CN
`
`f,g
`
`l
`N
`
`-Xaa-
`
`H
`.TFA
`
`I
`
`CN
`
`Reagents: a.ONPS—Cl, 2N NaOH. b. HONSu, Water soluble carbodiimide. c conc. NH4OH, dioxane.
`
`d. imidazole (2 equiv.), POCI3 (4 equiv.), pyridine. e. 4N HCl/dioxane (3 equiv.), diethyl ether.
`f. Boc—Xaa-OH, pyBop, NEI3, CH;C1;. g. Trifluoroacetic acid.
`
`The series of dipeptide nitriles described in Table H were prepared via a pyBop” mediated coupling of 4
`
`with the required Boc protected amino acid, followed by deprotection with TFA (Scheme 1).
`
`We were gratified to find that these compounds were potent inhibitors of DP-IV. The S.A.R. for the
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`N-terminal residue developed in the pyrrolidide series correlated well for the dipeptide nitrile series and the most
`
`potent compounds 24, 25, 26 and 27 possessed activity comparable to the boroprolines, 1 and 2. Stability
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`studies” revealed excellent half-lives (ta) in aqueous solution (pH 7.4) at room temperature (Table II) with
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`several examples having tt, greater than 48h. Further work on optimisation of the pyrrolidine ring will be
`
`reported shortly.
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:19)(cid:26)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:22)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
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`1166
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`D. M. ASHWORTH et al.
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`Table H.
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`Digeggidg hjyilgs: Potency versus human DP-IV and stability in
`
`aqueous solution (pH 7.4).
`
`H-Xaa—N0‘ N
`
`-
`
`27
`
`,
`
`.
`
`L s<Z>
`
`5211.0
`
`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 10mg/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
`
`®-4QVJIJIU-)t\)\.r\.r\4sdszxaxa
`
`1) Heins, J., Weiker, P., Schonlein, C., Born, 1., Hanrodt, B., Neubert, 1(., Tsurn, D. and Earth, A., Btochim. er Biophys. Acta
`1988, 954, 161.
`Hegen, M., Niedobitek, G., Clein, C.E., Stein, H. and Fleischer. B., J. Immunol. 1990, 144, 2908.
`Buhling, F., Kunz, D., Rheinhold, D., Ulmer, A.I., Ernst, M., Flad, H.-D. and Ansorge, S., Nat. Immun. 1994, 13, 270.
`Buhling, F., Junker, D., Rheinhold, D., Neubert, K., Jager, L. and Ansorge, S., Immunology Lett. 1995, 45, 47.
`A review has recently been published, discussing the evidence that CD26 has important functions in the immune system.
`Fleischer, B., Immunology Today 1994, I5. 180.
`Schon, E., Born, 1., Demuth, H.—U., Faust, J., Neubert, K., Steinmetzer, T., Barth, A. and Ansorge, S., Biol. Chem. Hoppe-
`Seyler 1991, 372, 305.
`Flentke, G.R., Munoz, B., Huber, B.T., Plaut, A.G., Kettner, CA. and Bachovchin, W.W.. Proc. Natl. Acad. Sci. USA 1991.
`88, 1556.
`Sudmeier, J.L., Gunther, U.L., Gutheil. W.G., Coutts, S.J., Snow, R.J., Barton, RW. and Bachovchin, W.W., Biochemistry
`1994, 33. 12427.
`9) Bodusek, B., Oleksyszyn, J., Kam, C.-M., Smith, R.E. and Powers, J.C., J. Med. Chem. 1994. 37, 3969.
`10) Belyaev. A., Borloo. 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.
`ll) 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. Set‘. 1995, 2, 198.
`12) Demuth, H.-U., Fischer, G., Barth, A. and Schowen, R.L., J. Org. Chem. 1988, 54, 5880.
`13) All compounds 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 ooncentrations per inhibitor. A typical assay (total volume
`0.4 mL) comprised sodium HEPES 83.3 mM, EDTA [.67 mM, BSA 1.5 mg mL". pH 7.8, DP-IV 25 p.U 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. W0 91/18877 (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. 1.
`18) Martinez, 1., Bali, J.P., Rodriguez, M., Castro. B., Laur, J. and Lignon, M.-F., J. Med. Chem. 1988, 28, 1874.
`19) The stability of the inhibitors 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)
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:19)(cid:26)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:23)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
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