Synthesis of partially modified retro and retroinverso psi[NHCH(CF(3))]-peptides.

Article abstract of DOI:10.1021/ol005876p

[reaction: see text] Asymmetric conjugate additions of chiral alpha-amino esters to chiral 4-CF(3) Michael-acceptors were exploited to prepare a small library of enantiomerically pure partially modified retropeptides having a psi[NHCH(CF(3))] unit as a po

Full text of DOI:10.1021/ol005876p

ORGANIC
LETTERS
2000
Vol. 2, No. 13
1827-1830
Synthesis of Partially Modified Retro
and Retroinverso
ψ[NHCH(CF3)]-Peptides
,†,|
Alessandro Volonterio,*,†,‡ Pierfrancesco Bravo,§ and Matteo Zanda*
Dipartimento di Chimica del Politecnico di Milano, Via Mancinelli 7,
I-20131 Milano, Italy, and C.N.R. - Centro di Studio sulle Sostanze Organiche
Naturali, Via Mancinelli 7, I-20131 Milano, Italy
zanda@dept.chem.polimi.it
Received March 30, 2000
ABSTRACT
Asymmetric conjugate additions of chiral r-amino esters to chiral 4-CF3 Michael-acceptors were exploited to prepare a small library of
enantiomerically pure partially modified retropeptides having a ψ[NHCH(CF3)] unit as a possible mimic of the classical ψ(NHCO) retropeptide
unit. Yields were nearly quantitative, and the stereoselectivity, which is controlled mainly by the nitrogen nucleophile, was progressively
1
higher with increasing the steric bulk of the r-amino ester side-chain R .
The pharmacological properties of most peptides preclude
their use as drugs. The main problems are connected with
their poor bioavailability, bioselectivity, and biostability, and
also to the fact that very often a single conformation of a
peptide is responsible for its biological activity and hence
function. The aim of peptide modification is to discover and
produce analogues that can overcome the barriers and
drawbacks described above, while retaining selected activity,
i.e., as specific receptor antagonists or agonists. Two of the
most popular strategies to modify peptides are (a) to replace
a peptide bond with a surrogate unit X, which is usually
symbolized as ψ(X);1 (b) to reverse all or some of the peptide
bonds (NH-CO instead of CO-NH), giving rise to the so-
called retro- or partially modified retropeptides, respectively.2
When the stereochemistry of one or more amino acids of
the reversed segment is inverted, the resulting pseudopeptide
is termed as retroinverso. A malonic unit is classically
incorporated to provide partially modified retropeptides,
while the direction can be restored incorporating an additional
gem-diaminoalkyl unit.3
Our idea is to combine these strategies in a novel class of
pseudopeptides having a novel ψ[NHCH(CF3)] as a possible
mimic of the classical ψ(NHCO) unit featured by retropep-
tides (Figure 1).4 This surrogate is expected to be stable
toward proteolytic degradation, isopolar with the NH-CO
unit, and eventually the stereoelectronically demanding CF3
might introduce some conformational constraint, thus limiting
(1) (a) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bo¨s, M.; Cook, C. M.;
Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; Kahn, M.; Madison, V.
S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall, J.; Vincent, G. P.; Voss, M. E.
J. Med. Chem. 1993, 36, 3039-3049. (b) Gante, J. Angew. Chem.. Int. Ed.
Engl. 1994, 33, 1699-1720. (c) Leung, D.; Abbenante, G.; Fairlie, D. P. J.
Med. Chem. 2000, 43, 305-341.
(2) Fletcher, M. D.; Campbell, M. M. Chem. ReV. 1998, 98, 763-795.
(3) (a) Goodman, M.; Chorev, M. Acc. Chem. Res. 1979, 12, 1-7. (b)
Chorev, M.; Willson, C. G.; Goodman, M. J. Am. Chem. Soc. 1977, 99,
8075-8076.
* *To whom correspondence should be addressed.
† Dipartimento di Chimica del Politecnico di Milano.
‡ Fax: +39 (02) 2399-3080; e-mail: alessandro.volonterio@
dept.chem.polimi.it.
§ C.N.R. - Centro di Studio sulle Sostanze Organiche Naturali.
| Fax: +39 (02) 2399-3080.
10.1021/ol005876p CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/27/2000

the sample reaction between L-1a and 2a. The best results
were obtained in DCM (entry 1), while a remarkable drop
of de was observed with more polar solvents such as ethanol,
acetonitrile, THF, DMF, or mixtures of them (entries 2-5).
In light of these results, DCM was used as the solvent of
choice for the preparation of pseudodipeptides 3a-j. The
facial diastereoselectivity of these reactions is mainly
controlled by R-amino esters 1. In fact, in all cases
L-configured 1 (Scheme 1 and Table 1) attacks preferentially
Scheme 1
Figure 1.
the number of stable conformational isomers, as well as
modify the binding properties acting as a hydrogen bond
acceptor or coordinative site with enzymes or receptor
subsites.5
In this communication we report the synthesis of the first,
and simplest, series of partially modified retro- and retro-
inverso ψ[NHCH(CF3)]-tripeptides, where the central unit
is a mimic of glycine (R ) H).
the Si-face of (S)-2a producing (S)-configured centers,
whereas enantiomeric D-Ala-OMe 1a (Scheme 2) attacks the
Re-face producing the other diasteromer 4, although with
lower diastereocontrol (mismatch).8 Moreover, compound 3f
was produced with good diastereocontrol when L-Val-OBn
1c was reacted with the achiral Michael-acceptor 2c (entry
10), while achiral Gly-OEt 1f added to (S)-2a (entry 14)
without stereocontrol, affording an equimolar mixture of 3j.
In this context, it is not surprising that the stereoelectronic
features of the R1 side-chain have a remarkable impact on
the degree of diastereoselectivity, which follows the trend
iso-Pr > iso-Bu > Me > Bn > H (de up to 78% in the case
of 3e, entry 9). Modest de was obtained with the cyclic
R-amino ester L-Pro-OBn 1e, but the reaction occurred also
in this case with very good yield. The R3 substituent on the
Assembling of ψ[NHCH(CF3)]-containing dipeptide units
was achieved by conjugate N-addition of a series of L-R-
amino esters 1 (Scheme 1) to the enantiomerically and
geometrically pure Michael acceptors (S)-(E)-2, prepared
according to literature methods.6 Pseudodipeptides 3 were
formed in excellent yields by mixing the hydrochlorides or
PTSA salts of 1 (2 equiv) with (S)-(E)-2 and sym-collidine
(4 equiv) in the appropriate solvent and stirring at room
temperature (rt) for 16-88 h (Table 1).7 Diastereomerically
pure 3 were smoothly isolated by flash chromatography on
silica gel. The solvent has a strong influence on the
stereoselectivity, as shown by experiments carried out on
Table 1.
entry
prod.
amino acid
oxaz
R1
Me
Me
Me
Me
Me
Me
Bn
iso-Pr
iso-Pr
iso-Pr
iso-Bu
iso-Bu
R2
R3
Bn
Bn
Bn
Bn
Bn
iso-Pr
Bn
Bn
iso-Pr
H
Bn
iso-Pr
Bn
Bn
X
solvent
DCM
T (h)
yield (%)c
de (%)d
1
2
3
4
5
6
7a
8
9
10
11a
12
13
14
3a
3a
3a
3a
3a
3b
3c
3d
3e
3f
1a
1a
1a
1a
1a
1a
1b
1c
1c
1c
1d
1d
1e
1f
2a
2a
2a
2a
2a
2b
2a
2a
2b
2c
2a
2b
2a
2a
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Et
16
16
40
40
40
64
24
64
68
68
88
68
40
40
90
80
80
ndb
>98
91
50
20
31
26
29
60
43
72
78
65
50
60
29
ca. 0
EtOH
MeCN
THF/DMF 4:1
PhH/DMF 4:1
DCM
DCM
DCM
DCM
DCM
DCM
DCM
78
92
>98
90
>98
>98
>98
>98
3g
3h
3i
-(CH2)3-
H
DCM
DCM
3j
H
a PTSA salt of the amino acid was used. b Not determined. c Overall isolated yield of both diastereomeric products. d Determined by 500 MHz 1H and 19
NMR.
F
1828
Org. Lett., Vol. 2, No. 13, 2000

The results above can be rationalized if one considers that
in the absence of chelating agents N-(E)-enoyl-oxazolidin-
2-ones 2 are known to exist in s-trans conformation,9 with
the R3 substituent being pointed away from the CdC bond,
thus exerting little control of the facial selectivity.10 In
contrast, the R1 side-chain of R-amino esters 1 should be
spatially close to the forming stereogenic center in the
transition state; therefore, its influence is much more
important. Reaction time has no effect on the diastereose-
lectivity; in fact 3g was formed with the identical 50% de
after 16 h (ca. 50% conversion) or after 88 h as well (entry
11), as shown by 500 MHz NMR of the crude reaction
mixture. This strongly supports the conclusion that adducts
3 are formed irreversibly, and the stereochemical outcome
is under kinetic control.
Scheme 2
oxazolidinone residue has lower effect on the stereoselec-
tivity. In fact, (S)-configured products 3e (R3 ) iso-Pr, entry
9), 3d (R3 ) Bn, entry 8), and 3f (R3 ) H, entry 10) were
obtained from L-Val-OBn 1c in 78%, 72%, and 65% de
respectively, with little variation and the same sense of facial
selectivity.
It is worth noting that stereocontrol is totally absent in
the 1,4-addition of L-Ala-OMe 1a to methyl 4,4,4-trifluo-
rocrotonate 5, which was chosen initially as the Michael
acceptor (Scheme 3), and the reaction is much slower (1
(4) A conceptually related peptide bond surrogate ψ[CH(CN)NH] has
been reported: (a) Herrero, S.; Sua´rez-Gea, M. L.; Gonza´lez-Mun˜iz, R.;
Garc´ıa-Lo´pez, M. T.; Herranz, R.; Ballaz, S.; Barber, A.; Fortun˜o, A.; Del
R´ıo, J. Bioorg. Med. Chem. Lett. 1997, 7, 855-860. For some recent
examples of fluorine-containing peptidomimetics: (b) Garrett, G. S.;
McPhail, S. J.; Tornheim, K.; Correa, P. E.; McIver, J. M. Bioorg. Med.
Chem. Lett. 1999, 9, 301-306. (c) Poupart, M.-A.; Fazal, G.; Goulet, S.;
Mar, L.-T. J. Org. Chem. 1999, 64, 1356-1361. (d) Eda, M.; Ashimori,
A.; Akahoshi, F.; Yoshimura, T.; Inoue, Y.; Fukaya, C.; Nakajima, M.;
Fukuyama, H.; Imada, T.; Nakamura, N. Bioorg. Med. Chem. Lett. 1998,
8, 919-924 and references therein. (e) Hoffman, R. V.; Tao, J. Tetrahedron
Lett. 1998, 39, 4195-4198. (f) Bartlett, P. A.; Otake, A. J. Org. Chem.
1995, 60, 3107-3111. (g) Boros, L. G.; Decorte, B.; Gimi, R. H.; Welch,
J. T.; Wu, Y.; Handschumacher, R. E. Tetrahedron Lett. 1994, 35, 6033-
6036. (h) Revesz, L.; Briswalter, C.; Heng, R.; Leutwiler, A.; Mueller, R.;
Wuethrich, H.-J. Tetrahedron Lett. 1994, 35, 9693-9696. (i) Robinson, R.
P.; Donahue, K. M. J. Org. Chem. 1992, 57, 7309-7314.
Scheme 3
(5) See for example: Scolnick, L. R.; Clements, A. M.; Liao, J.;
Crenshaw, L.; Hellberg, M.; May, J.; Dean, T. R.; Christianson, D. W. J.
Am. Chem. Soc. 1997, 119, 850-851.
month, rt). This is likely to be a consequence of the lower
conformational rigidity and electrophilicity of 5 with respect
to the oxazolidinone acceptors 2.
(6) (a) Shibuya, A.; Kurishita, M.; Ago, C.; Taguchi, T. Tetrahedron
1996, 52, 271-278. See also: (b) Yamazaki, T.; Shinohara, N.; Kitazume,
T.; Sato, S. J. Fluorine Chem. 1999, 97, 91-96.
(7) This reaction is extraordinarily simple and efficient, if one considers
that examples of 1,4-additions by chiral R-amino esters to 4-substituted
Michael-acceptors are very scarce in the literature: (a) Urbach, H.; Henning,
R. Tetrahedron Lett. 1984, 25, 1143-1146. (b) Eckert, H. G.; Badian, M.
J.; Gantz, D.; Kellner, H.-M-; Volz, M. Arzneim. Forsch. 1984, 34, 1435-
1447. For Michael-type reactions of amines with 4-substituted acceptors:
(c) Burke, A. J.; Davies, S. G.; Hedgecock, C. J. R. Synlett 1996, 621-
622. (d) d′Angelo, J.; Maddaluno, J. J. Am. Chem. Soc. 1986, 108, 8112-
8114. (e) Cardillo, G.; Di Martino, E.; Gentilucci, L.; Tomasini, C.;
Tomasoni, L. Tetrahedron: Asymmetry 1995, 6, 1957-1963. (f) Amoroso,
R.; Cardillo, G.; Sabatino, P.; Tomasini, C.; Trere`, A. J. Org. Chem. 1993,
58, 5615-5619. (g) Baldwin, S. W.; Aube´, J. Tetrahedron Lett. 1987, 28,
179-182. (h) Hirama, M.; Shigemoto, T.; Yamazaki, Y.; Ito, S. J. Am.
Chem. Soc. 1985, 107, 1797-1798. (i) Bunnage, M. E.; Davies, S. G.;
Goodwin, C. J.; Walters, I. A. S. Tetrahedron: Asymmetry 1994, 5, 35-
36. (j) Davies, S. G.; Walters, I. A. S. J. Chem. Soc., Perkin Trans. 1 1994,
1129-1139. (k) Davies, S. G.; Ichihara, O.; Walters, I. A. S. J. Chem.
Soc., Perkin Trans. 1 1994, 1141-1147. (l) Hawkins, J. M.; Lewis, T. A.
J. Org. Chem. 1994, 59, 649-652. (m) Asao, N.; Shimada, T.; Sudo, T.;
Tsukada, N.; Yazawa, K.; Gyoung, Y. S.; Uyehara, T.; Yamamoto, Y. J.
Org. Chem. 1997, 62, 6274-6282. (n) Rudolf, K.; Hawkins, J. M.;
Loncharich, R. J.; Houk, K. N. J. Org. Chem. 1988, 53, 3879-3882. (o)
Hawkins, J. M.; Fu, G. C. J. Org. Chem. 1986, 51, 2820-2822. (p)
Liebeskind, L. S.; Welker, M. E. Tetrahedron Lett. 1985, 26, 3079-3082.
(q) De, A.; Basak, P.; Iqbal, J. Tetrahedron Lett. 1997, 38, 8383-8386. (r)
Yamamoto, Y.; Asao, N.; Uyehara, T. J. Am. Chem. Soc. 1992, 114, 5427-
5429. (s) Dumas, F.; Mezrhab, B.; d’Angelo, J.; Riche, C.; Chiaroni, A. J.
Org. Chem. 1996, 61, 2293-2304. (t) d’Angelo, J.; Maddaluno, J. J. Am.
Chem. Soc. 1986, 108, 8112-8114. (u) Mezrhab, B.; Dumas, F.; d’Angelo,
J.; Riche, C. J. Org. Chem. 1994, 59, 500-503. (v) Cardillo, G.; Casolari,
S.; Gentilucci, L.; Tomasini, C. Angew. Chem., Int. Ed. Engl. 1996, 35,
1848-1849.
With a number of pseudo-dipeptides 3 in hand we
addressed the next issue, namely the chemoselective cleavage
of the oxazolidinone auxiliary. This result was achieved in
55-82% yields upon treatment of 3 with LiOH/H2O2 (30
min, 0 °C) (Scheme 4).11 The resulting pseudodipeptides
having a terminal CO2H group were purified by FC and then
coupled with another R-amino ester (HATU/HOAt, sym-
(8) The stereochemistry of 3a was assigned by X-ray diffraction (X-ray
data will be published in a full-paper), while the stereochemistry of the
other major diastereomers 3b-j was assigned on the basis of their spectral
and chemical-physical similarities with 3a. The stereochemistry of 6,
derived from D-Ala-OMe 1a was determined by chemical correlation with
3a, after cleavage of the oxazolidinone auxiliary.
(9) (a) Evans, D. A.; Chapman, K. T.; Bisaha, J. J. Am. Chem. Soc. 1988,
110, 1238-1256. (b) Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow,
R. L. J. Am. Chem. Soc. 1990, 112, 4011-4030. See also: (c) Soloshonok,
V. A.; Cai, C.; Hruby, V. J. Org. Lett. 2000, 2, 747-750.
(10) The use of Lewis acids for achieving higher stereocontrol via
prechelation of oxazolidin-2-ones 2 was tried with little success. For
example, treatment of 2a with Sc(OTf)3 in CH2Cl2 at rt for 30 min., followed
by addition of L-1c and sym-collidine, produced 3d in 58% de and ca. 85%
yield (determined by 19F NMR of the crude reaction mixture) after 30 h at
rt. For a very recent successful example of use of Sc(OTf)3 in related
reactions: Mero, C. L.; Porter, N. A. J. Org. Chem. 2000, 65, 775-781.
(11) Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. J. Am.
Chem. Soc. 1990, 112, 4011-4030. Oxazolidin-2-one auxiliaries were
usually recovered in nearly quantitative yields after cleavage.
Org. Lett., Vol. 2, No. 13, 2000
1829

synthesis and biological activity evaluation of the new retro
ψ[NHCH(CF3)]-peptides are currently in progress.
Scheme 4
Acknowledgment. We thank M.U.R.S.T. COFIN 1998
(Nuove metodologie e strategie di sintesi di composti di
interesse biologico) and C.N.R. - C.S.S.O.N. for financial
support.
OL005876P
(13) Experimental Section. Michael Addition. Typical procedure. To
a stirred solution of 2a (80 mg, 0.27 mmol) and 1c (131 mg, 0.54 mmol)
in dry CH2Cl2 (3.4 mL) was added neat sym-collidine (0.14 mL, 1.08 mmol).
After 64 h at rt the solvent was removed in vacuo, the crude dissolved in
AcOEt and washed once with 1 N HCl. The organic layer was dried over
anhydrous Na2SO4 and filtered, the solvent removed in vacuo, and the crude
purified by FC (hexane/diethyl ether 65:35) affording 136 mg (92%) of
diastereomerically pure 3d and its minor diastereomer in 6:1 ratio. 3d (major
diastereoisomer): [R]23D ) + 28.3° (c ) 1.0, CHCl3); FT IR (film): νmax
) 3449, 1794, 1700, 1385 cm-1; 1H NMR (250 MHz, CDCl3): δ ) 7.38-
7.20 (m, 10H), 5.13 (d, J ) 12.1 Hz, 1H), 5.06 (d, J ) 12.1 Hz, 1H), 4.70
(m, 1H), 4.24 (m, 1H), 4.16 (dd, J ) 8.8, 2.8 Hz, 1H), 3.62 (m, 1H), 3.42
(dd, J ) 15.3, 3.6 Hz, 1H), 3.36 (m, 2H), 3.15 (dd, J ) 15.3, 9.6 Hz, 1H),
2.77 (dd, J ) 13.7, 10.1 Hz, 1H), 2.01 (m, 1H), 1.88 (br s, 1H), 0.96 (d, J
) 6, 7 Hz, 3H), 0.85 (d, J ) 6, 7 Hz, 3H); 19F NMR (250 MHz, CDCl3):
δ ) -77.5 (d, J ) 6.7 Hz, 3F); 13C (250 MHz, CDCl3): δ ) 174.8, 169.3,
153.6, 135.6, 135.4, 129.4, 129.0, 128.6, 128.4, 128.2, 127.4, 126.0 (q, J
) 282.3 Hz), 66.8, 66.6, 66.5, 55.7 (q, J ) 29.3 Hz), 55.5, 37.9, 36.3,
31.8, 19.3, 17.5; MS (70 eV): e/z (%): 507 (8) [M+ + 1], 371 (90), 91
(100). Cleavage of the Oxazolidinone from 3. Typical procedure. To a
cooled solution of 3d (97 mg, 0.21 mmol) in THF/H2O 4: 1 (1.5 mL) at 0
°C under nitrogen atmosphere was added a 30% aqueous H2O2 solution
(0.86 mL, 0.85 mmol), followed by solid LiOH (5 mg, 0.21 mmol). After
60 min the reaction was quenched with saturated aqueous Na2SO3, warmed
to room temperature, and extracted three times with AcOEt, and the
combined organic layers were dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. Purification by FC (1:1 hexane/AcOEt until complete
recovery of the oxazolidin-2-one and then MeOH) afforded 50 mg of the
acid (68%). Synthesis of Retropeptides 7. Typical procedure. To a stirred
solution of the acid above (30 mg, 0.08 mmol) and 1a (12 mg, 0.08 mmol)
in dry DMF (1 mL) was added, at 0 °C under nitrogen atmosphere, neat
sym-collidine (0.032 mL, 0.24 mmol), followed by solid HOAt (11 mg,
0.08 mmol) and solid HATU (31 mg, 0.08 mmol). After 40 min the solution
was quenched with 1 N HCl, warmed to room temperature, and extracted
with AcOEt, and the combined organic layers dried over anhydrous Na2-
SO4, filtered, and concentrated in vacuo. Purification by FC (75: 25 hexane/
AcOEt) afforded 35 mg of 7b (quantit). 7b: Rf ) 0.35 (hexane/AcOEt 70:
30); [R]23D ) - 15.1° (c ) 0.7, CHCl3); FTIR (film): νmax ) 3335, 1742,
1657, 1536, 1384 cm-1; 1H NMR (250 MHz, CDCl3): δ ) 7.35 (m, 5H),
7.23 (br d, J ) 6.4 Hz, 1H), 5.15 (s, 2H), 4.60 (q, J ) 7.2 Hz, 1H), 3.74
(s, 3H), 3.67 (q, J ) 7.2 Hz, 1H), 3.54 (m, 1H), 2.64 (dd, J ) 15.4, 3.4 Hz,
1H), 2.40 (dd, J ) 15.4, 9.4 Hz, 1H), 2.02 (br s, 1H), 1.78 (m, 1H), 1.5 (m,
2H), 1.44 (d, J ) 7.2 Hz, 3H), 0.91 (d, J ) 4.1 Hz, 3H), 0.88 (d, J ) 4.1
Hz, 3H); 19F NMR (250 MHz, CDCl3): δ ) -76.4 (d, J ) 7.6 Hz, 3F);
13C (250 MHz, CDCl3): δ ) 175.4, 173.5, 168.2, 135.5, 128.6, 128.4, 128.2,
126.0 (q, J ) 282.2 Hz), 66.8, 58.4, 55.3 (q, J ) 27.7 Hz), 52.4, 48.2,
42.9, 35.9, 24.6, 22.9, 21.6, 17.9; MS (70 eV): e/z (%): 447 (7) [M+ + 1],
311 (93), 208 (30), 166 (30), 91 (100).
collidine, DMF). The final retro- and retroinverso tripeptides
7a-d, orthogonally protected at the carboxy end-groups and
therefore suitable for further selective elongation, were
obtained in quantitative yields as solid materials.12
In conclusion, we have described the synthesis of a novel
class of retropeptides incorporating a ψ[NHCH(CF3)] unit
as a surrogate of the ψ[NH-CO] retropeptide bond. This
result has been achieved by exploiting uncommon conjugate
additions of chiral R-amino esters to 4-substituted Michael-
acceptors, which take place in excellent yields and moderate
to good stereocontrol. Both this novel class of retropeptides
and the synthetic approach appear to be particularly suitable
for solid-phase/combinatorial chemistry. Indeed, the central
R group (in this paper R ) H), as well as the R1, R2, ...., Rn
amino acidic side-chains, and even the fluorinated RF residue
can be modified in order to prepare a wide library of
structures for biological screening. Solid-phase/combinatorial
(12) Only proline derived pseudo-tripeptide 7d, obtained as a ca. 3:1
mixture of isomers at the peptide bond, was isolated as a foam.
1830
Org. Lett., Vol. 2, No. 13, 2000