Total synthesis of a Pepstatin analogue incorporating two trifluoromethyl hydroxymethylene isosteres

Article abstract of DOI:10.1016/S0040-4039(00)01244-2

The total synthesis of a trifluoromethyl (Tfm) analogue of the aspartate protease inhibitor Pepstatin has been accomplished via incorporation of two α-Tfm-amino β-hydroxy peptide isosteres instead of the natural statine units. The title compound as well as several Tfm-substituted precursors did not show anti-HIV activity. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01244-2

Tetrahedron Letters 41 (2000) 7239±7243
Total synthesis of a Pepstatin analogue incorporating two
tri¯uoromethyl hydroxymethylene isosteres
Cristina Pesenti,a Alberto Arnone,b Anne Marie Aubertin,c Pierfrancesco Bravo,a,b
Massimo Frigerio,a Walter Panzeri,b Sylvie Schmidt,c Fiorenza Vianib,*
and Matteo Zandaa,*
aDipartimento di Chimica del Politecnico di Milano, via Mancinelli 7, I-20131 Milano, Italy
bCNR±Centro di Studio sulle Sostanze Organiche Naturali, via Mancinelli 7, I-20131 Milano, Italy
cINSERM U74, Institut de Virologie, 3 Rue Koeberle, 67000 Strasbourg, France
Received 31 May 2000; revised 11 July 2000; accepted 20 July 2000
Abstract
The total synthesis of a tri¯uoromethyl (Tfm) analogue of the aspartate protease inhibitor Pepstatin has
been accomplished via incorporation of two a-Tfm-amino b-hydroxy peptide isosteres instead of the
natural statine units. The title compound as well as several Tfm-substituted precursors did not show anti-HIV
activity. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: peptide mimetics; enzyme inhibitors; Pepstatin; tri¯uoromethyl group.
Pepstatin1 (Fig. 1) is a natural inhibitor of peptidic aspartate proteases, including pepsin,1,2
renin,3 HIV-1,1b,4 and HIV-2 proteases.1b,5 The central statine unit is believed to mimic the
tetrahedral intermediate of peptide hydrolysis, acting as an isostere for a restricted conformation
of a dipeptide unit,2a and its stereochemistry has a large e€ect on protease inhibition, a syn
diastereomeric relationship between the amine and hydroxyl groups being required.2a,6a However,
Pepstatin is rated only as `moderately active' by NCI in a cell-based AIDS anti-viral screen.6b
Much e€ort has been directed toward the synthesis of Pepstatin mimetics in order to discover
analogues having improved properties.1b
The list includes a few ¯uorinated derivatives containing di¯uorostatine and di¯uorostatone
units, which have been reported to be potent inhibitors of Penicillopepsin.7 Replacement of the
statine isobutyl residue in the P1 position with other groups has often led to analogues with
greatly improved features.8 However, its replacement with a ¯uoroalkyl residue has never been
reported, despite the fact that ¯uoroalkyl groups are known to deeply modify physical±chemical
properties such as lipophilicity, acidity/basicity, nucleophilicity and preferred conformation, and
* Corresponding authors. Fax: +39 02 2399-3080; e-mail: viani@dept.chem.polimi.it, zanda@dept.chem.polimi.it
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01244-2

7240
Figure 1.
that useful spectroscopic data on the binding process might be obtained by 19F NMR. This could
be due to the following reasons: (1) the requisite stereode®ned b-¯uoroalkyl b-amino alcohol
units9 have been hitherto synthetically unavailable; and (2) incorporation of a-¯uoroalkyl amino
moieties into peptidic sequences via amide bond formation is a challenging endeavour, due to the
low nucleophilicity of the NH2 function.
This is particularly true for a-tri¯uoromethyl (Tfm) amino derivatives,10a since the Tfm group
is strongly electron-withdrawing and `sterically at least as large as CH(CH3)2'.10b Recently, we
reported a stereocontrolled route to orthogonally protected syn-(3S,4R)-g-(Tfm)GABOB 2
(Scheme 1), a new hydroxymethylene (statine) dipeptide isostere.11 In this paper we describe the
total solution-phase synthesis of the Pepstatin analogue 1 (Fig. 1), having two (Tfm)GABOB
units in place of the natural syn-(3S,4S)-statines in the P1 and P30 positions, as part of a project
aiming at the investigation of the e€ect exerted by ¯uorine atoms belonging to ¯uoroalkyl groups
on the binding process to aspartyl proteases.
Scheme 1.
In our early attempts, we tried to build the peptide 1 starting from the central g-(Tfm)GABOB
(Scheme 1). Unfortunately, the dipeptide 3, prepared by standard solution methods, failed to
undergo coupling with Cbz-Val-Val-OH under a variey of conditions (for example EDCI/HOBt
or HATU/HOAt12 in DMF±TMP at 0ꢀC) and also with Val derived Leuchs anhydride,13 thus
evidencing the expected poor reactivity of the H-(Tfm)GABOB fragment. However, a partial
migration of the O-Bz protection to the amino group was occasionally observed under coupling
conditions. This suggested to us that an unprotected b-OH group might favor the coupling.14
Therefore, we decided to re-undertake the synthesis of 1 starting from O-unprotected statine
isosteres 7 and 9 (Scheme 2). The former was prepared from the ole®n 6, accessible via a C±C
bond forming reaction of the a-lithiated 3-butenyl-p-tolylsulfoxide 4 with the N-p methoxy-
phenylimine 5, followed by stereoselective SN2-type substitution of the sul®nyl with an hydroxy
group.11 Oxidative cleavage of 6 with KMnO4, which occurred with excellent chemoselectivity,
then treatment of 7 with diazomethane, followed by hydrogenolysis of the Cbz group of the
resulting ester 8, provided 9.

7241
Scheme 2.
Assembling of the tripeptide fragment H-(Tfm)GABOB-Ala-(Tfm)GABOB with Iva-Val-Val-
OH was envisaged as a viable strategy to accomplish the synthesis of 1, therefore the synthesis of
the ¯uorinated tripeptide 13 was undertaken ®rst (Scheme 3). Coupling of 7 with H-Ala-OMe
(HATU/HOAt, DMF±TMP) a€orded 10, which was hydrolyzed with LiOH to the corresponding
acid 11. Satisfactorily, the key assembling of 11 with 9 a€orded good yields of 12,15 which was
hydrogenolyzed to the H2N-tripeptide 13.
Scheme 3.
The ®nal assembling of 13 with Iva-Val-Val-OH (14), prepared by standard solution-phase
technique, proved to be quite challenging (Scheme 3). In fact, under a variety of conditions (for
example HATU/HOAt both in DMF and AcOEt, or iso-BuCO2Cl/NMM in DMF) epimerization
of the second Val unit took place, a€ording the target 15 as a mixture of epimers. This trouble
was solved by using the exact conditions reported by Bartlett for the synthesis of a phosphorus-
containing analogue of Pepstatin (iso-BuCO2Cl/NMM in AcOEt)16 which provided the stereo-
chemically pure pentapeptide 15, which was hydrolyzed with LiOH in excellent yield to the ®nal
target Pepstatin analogue 1.17
The target 1 as well as precursors 7±13 were evaluated for their capacity to inhibit HIV-1
multiplication. The virus production was measured, in the presence or absence of test
compounds, by quanti®cation of the reverse transcriptase activity associated with virus particles

7242
released from HIV-1 Lai-infected CEM-SS in culture medium or the cytotoxicity induced by
HIV-1 IIIB replication in MT-4 cells. The cytotoxicity of the compounds was evaluated in
parallel on uninfected cells.18 At the highest concentration tested, 10 mg/ml in the case of 1, there
was no measurable antiviral or cytotoxic activity. The precursors 7±13 were used at 100 mg/ml or
less but no activity was detected.
In summary, the challenging incorporation of an a-Tfm-amino b-hydroxy peptide isostere into
a complex peptidic sequence has been successfully accomplished, producing the Tfm-Pepstatin
analogue 1 on a hundredth of a milligram scale and very good overall yield. The solution and
solid-phase synthesis of further ¯uorinated analogues of Pepstatin, as well as the elucidation of
the e€ect of ¯uoroalkyl groups on aspartyl proteases inhibition are currently under investigation.19
Acknowledgements
MURST COFIN 1998 (Nuove metodologie e strategie di sintesi di composti di interesse
biologica) and CNR±CSSON are gratefully acknowledged for ®nancial support. C.P. thanks
Politecnico di Milano for a biennial scholarship.
References
1. (a) Umezawa, H.; Aoyagi, T.; Morishima, H.; Matsuzaki, M.; Hamada, M.; Takeuchi, T. J. Antibiot. 1970, 23,
259±262. (b) Babine, R. E.; Bender, L. E. Chem. Rev. 1997, 97, 1359±1472.
2. (a) Rich, D. H. J. Med. Chem. 1985, 28, 263±273. (b) Kratzel, M.; Schlichtner, B.; Kirchmayer, R.; Bernkop-
Schnurk, A. J. Med. Chem. 1999, 42, 2041±2045.
3. (a) Boger, J.; Lohr, N. S.; Ulm, E. H.; Poe, M.; Blaine, E. H.; Fanelli, G. M.; Lin, T.-H.; Payne, L. S.; Schorn,
T. W.; LaMont, B. I.; Vassil, T. C.; Stabilito, I. I.; Veber, D.; Rich, D. H.; Bopari, A. S. Nature (London) 1983,
303, 81±84. (b) Guegan, R.; Diaz, J.; Cazaubon, C.; Beaumont, M.; Carlet, C.; Clement, J.; Demarne, H.; Mellet,
M.; Richaud, J.-P.; Segondy, D.; Vedel, M.; Gagnol, J.-P.; Roncucci, R.; Castro, B.; Corvol, P.; Evin, G.; Roques,
B. P. J. Med. Chem. 1986, 29, 1152±1159.
4. Richards, A. D.; Roberts, R.; Dunn, B. M.; Graves, M. C.; Kay, J. FEBS Lett. 1989, 247, 113±117, and references
cited therein.
5. Richards, A. D.; Broadhurst, A. V.; Ritchie, A. J.; Dunn, B. M.; Kay, J. FEBS Lett. 1989, 253, 214±216, and
references cited therein.
6. (a) X-Ray di€raction analysis of the Rhizopus chinensis carboxyl proteinase complexed with Pepstatin has been
achieved: Bott, R.; Subramanian, E.; Davies, D. R. Biochemistry 1982, 21, 6956±6962. (b) Information about the
anti-HIV screen on Pepstatin (NSC number 272671) can be obtained at the Internet page: http://dtp.nci.nih.gov/.
7. James, M. N. G.; Sielecki, A. R.; Hayakawa, K.; Gelb, M. H. Biochemistry 1992, 31, 3872±3886.
8. Rich, D. H.; Bernatowicz, M. S.; Agarwal, N. S.; Kawai, M.; Salituro, F. G.; Schmidt, P. G. Biochemistry 1985,
24, 3165±3173.
9. For a review on synthesis and biological activity of b-¯uoroalkyl b-amino alcohols: Bravo, P.; Crucianelli, M.;
Ono, T.; Zanda, M. J. Fluorine Chem. 1999, 97, 27±49.
10. (a) Fluorine-Containing Amino Acids: Synthesis and Properties; Kukhar, V. P.; Soloshonok, V. A., Eds.; Wiley:
Chichester, 1994. (b) Banks, R. E.; Tatlow, J. C.; Smart, B. E. Organo¯uorine Chemistry: Principles and
Commercial Applications; Plenum Press: New York, 1994, p. 81.
11. Bravo, P.; Corradi, E.; Pesenti, C.; Vergani, B.; Viani, F.; Volonterio, A.; Zanda, M. Tetrahedron: Asymmetry
1998, 9, 3731±3735.
12. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem. Soc., Chem. Commun. 1994, 201±203.
13. (a) Leuchs, H. Ber. Dtsch. Chem. Ges. 1906, 39, 857±861. (b) Wilder, R.; Mobashery, S. J. Org. Chem. 1992, 57,
2755±2756.
14. To this end, O-debenzoylation of the dipeptide 5 with MeOH, K2CO3 was attempted, but the desired H-
(Tfm)(OH)GABOB-Ala-OPh was not achieved.

7243
15. To a solution of 11 (600 mg, 1.53 mMol) and 9 (308 mg, 1.53 mMol) in DMF (13 mL, stored overnight over 3 A
molecular sieves), TMP (371 mg, 0.406 mL, 3.06 mMol) was added and the resulting mixture was cooled with an
ice-water bath. HATU (582 mg, 1.53 mMol) and HOAt (208 mg, 1.53 mMol) were added and the mixture was
stirred for 25 min. A 1 M solution of HCl was added until pH 1±2 was reached, and the reaction mixture was
extracted with ethyl acetate (3Â10 mL). The collected organic layers were washed with a 5% aqueous NaHCO3
solution, and then with brine. After drying over anhydrous Na2SO4, and ®ltration, the solvent was removed in
vacuo, and the residue was puri®ed by ¯ash chromatography (n-hexane:AcOEt, 1:1) to give 12 in 74% yield after
20
D
crystallization from n-hexane:AcOEt, 1:1: m.p. 205±206ꢀC; ‰ꢀŠ ^52.04 (c 1.0, MeOH); H NMR (acetone-d6) ꢁ
1
7.65 (d, J=6.6 Hz, 1H), 7.56 (d, J=9.9 Hz, 1H), 7.50±7.25 (m, 5H), 6.72 (d, J=9.9 Hz, 1H), 5.16 (br d, J=12.5
Hz, 1H), 5.13 (br d, J=12.5 Hz, 1H), 4.86 (d, J=4.9 Hz, 1H), 4.76 (d, J=5.8 Hz, 1H), 4.70 (ddq, J=9.9, 1.6 and
8.2 Hz, 1H), 4.57 (m, 1H), 4.50 (dq, J=6.6 and 7.2 Hz, 1H), 4.48 (m, 1H), 4.45 (ddq, J=9.9, 1.6 and 8.2 Hz, 1H),
3.62 (s, 3H), 2.51 (d, J=6.7 Hz, 2H), 2.49 (d, J=6.6 Hz, 2H), 1.33 (d, J=7.2 Hz, 3H); 19F NMR (acetone-d6) ꢁ
^71.72 (br d, J=8.2 Hz), ^71.41 (br d, J=8.2 Hz); 13C NMR (acetone-d6) ꢁ 173.9, 171.8, 171.7, 157.5, 137.7, 129.3,
128.8, 128.6, 126.1 (q, J=282.3 Hz), 126.0 (q, J=282.6 Hz), 67.6, 66.0, 65.3, 56.6 (q, J=28.7 Hz), 53.8 (q, J=28.7
Hz), 51.8, 50.3, 40.1, 39.1, 18.1. MS (DIS EI, 70 eV) m/z (%): 576 [(M+H)+, 7], 575 [M+Á, 9], 557 [(M^H2O)+Á, 11].
16. Bartlett, P. A.; Hanson, J. E.; Giannousis, P. P. J. Org. Chem. 1990, 55, 6268±6274.
17. (a) Coupling between 13 and 14. A suspension of 14 (76 mg) in 3.6 ml of dry AcOEt was treated with 1.2 equiv. of
NMM, cooled to ^10ꢀC, then 1.2 equiv. of iso-BuO2CCl were added under stirring. After 5 min a suspension of 13
(1 equiv.) in 2.5 ml of AcOEt was added. The mixture was stirred 4 days at rt, then centrifuged and the solid was
washed several times with AcOEt, MeOH and ®nally n-hexane to provide 100 mg of 15 (65%) as an amorphous
20
solid. (b) Selected data for 1: m.p. 265±270ꢀC (dec.); ‰ꢀŠ ^32.16 (c 0.36, DMSO); 1H NMR (DMSO-d6) ꢁ 8.08 (d,
D
J=7.0 Hz, 1H), 8.06 (d, J=9.5 Hz, 1H), 8.00 (d, J=9.5 Hz, 1H), 7.90 (d, J=9.0 Hz, 1H), 7.79 (d, J=9.0 Hz, 1H),
5.35 (br signal, 2H), 4.65±4.50 (m, 2H), 4.44 (dq, J=7.0 and 7.1 Hz, 1H), 4.31 (dd, J=9.0 and 7.2 Hz, 1H), 4.35±
4.25 (m, 2H), 4.20 (dd, J=9.0 and 7.1 Hz, 1H), 2.31 and 2.28 (m, 2H), 2.25 (dd, J=14.9 and 8.5 Hz, 1H), 2.19 (dd,
J=14.9 and 4.4 Hz, 1H), 2.10±1.85 (m, 5H), 1.24 (d, J=7.1 Hz, 3H), 0.90±0.80 (m, 18H); 19F NMR (DMSO-d6) ꢁ
^71.20 (br d, J=8.3 Hz), ^71.08 (br d, J=8.2 Hz); 13C NMR (DMSO-d6) ꢁ 173.3, 171.8, 171.62, 171.59, 171.3,
169.5, 124.9 (q, J=284.3 Hz, 2C), 64.2, 63.8, 57.8, 57.7, 52.9 (q, J=26.9 Hz), 52.4 (q, J=27.7 Hz), 48.2, 44.4, 39.5,
38.5, 30.1, 29.9, 25.6, 22.2 (2C), 19.19, 19.17, 18.23, 18.18, 17.7. MS (DIS EI, 70 eV) m/z (%): 710 [(M+H)+, 4], 692
1
[(M+H^H2O)+, 6]. (c) Epimerization of the second Val under di€erent coupling conditions was assessed by H
and 19F NMR, as witnessed by the fact that splitting of most of the signals of 15 was detected. For example,
the undesired epimer showed the following 19F NMR (DMSO-d6) ꢁ ^71.15 (br d, J=8.3 Hz) and ^70.95 (br d,
J=8.3 Hz).
18. (a) Moog, C.; Wick, A.; Le Ber, P.; Kirn, A.; Aubertin, A. M. Antiviral Res. 1994, 24, 275±288. (b) Compound 1
was tested as a mixture of epimers at the second Val center.
19. Abbreviations: GABOB, g-amino-b-hydroxybutyric acid; EDCI, N-Ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride; HOBt, 1-Hydroxybenzotriazole; HATU, N,N,N0,N0-tetramethyl-O-(7-azabenzotriazol-1-yl)-uronium
hexa¯uorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; TMP, 2,4,6-trimethylpyridine (sym-collidine); NMM,
N-methylmorpholine.