Synthetic study of VLA-4/VCAM-1 inhibitors: Synthesis and structure-activity relationship of piperazinylphenylalanine derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.12.014

To improve the poor pharmacokinetic characteristics of VLA-4 inhibitors, novel piperazinylphenylalanine derivatives were designed. This structure is expected to improve physicochemical properties by increasing overall basicity. By changing components at t

Full text of DOI:10.1016/j.bmcl.2007.12.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1053–1057
Synthetic study of VLA-4/VCAM-1 inhibitors: Synthesis
and structure–activity relationship of
piperazinylphenylalanine derivatives
Osamu Saku,* Kiminori Ohta, Eri Arai, Yuji Nomoto, Hiroko Miura,
Hiroaki Nakamura, Eiichi Fuse and Yoshisuke Nakasato*
Pharmaceutical Research Center, Kyowa Hakko Kogyo Co., Ltd, 1188 Shimotogari,
Nagaizumi-cho, Suntou-gun, Shizuoka 411-8731, Japan
Received 6 August 2007; revised 27 November 2007; accepted 7 December 2007
Available online 14 December 2007
Abstract—To improve the poor pharmacokinetic characteristics of VLA-4 inhibitors, novel piperazinylphenylalanine derivatives
were designed. This structure is expected to improve physicochemical properties by increasing overall basicity. By changing com-
ponents at the 4-position of piperazine and the terminal group of the amido bond, 12t was found to be the most potent of this series
of compounds. In addition, dichlorobenzoyl derivative 12aa exhibited better oral availability and showed efficacy in an in vivo mod-
el after oral administration.
Ó 2007 Elsevier Ltd. All rights reserved.
Adhesion with cells or extra-cellular matrices plays an
important role in chemotaxis into inflammation sites,
adhesion of leukocytes, and movement of stem cells at
the time of ontogeny. In such processes, involvement
of various adhesion molecules is evident, and integrin,
an adhesion molecule that interacts with ligands ex-
pressed on cells, plays an important role in regulation
of adhesion with cells or extra-cellular matrix.1
to be useful in the treatment of inflammatory disease.
Anti-VLA-4 antibody has been demonstrated to be
effective in various inflammation models,2 with the
humanized monoclonal antibody, Tysabri (natal-
izumab),3 recently relaunched for the treatment of
relapsing forms of multiple sclerosis. Therefore, non-
peptide small molecule VLA-4 inhibitors are also an
attractive target for the treatment of chronic inflamma-
tory disease, with several reports on orally active inhib-
itors published in recent years.4 Preclinical tests of
peptide mimetic BIO-1272 (Biogen) derived from con-
necting segment 1 (CS-1) of fibronectins were performed
for the first time as a small molecule inhibitor, showing a
marked effect in inhibiting antigen induced respiratory
hypersensitivity in sheep models of asthma.5
VLA-4 is a member of the integrin superfamily, com-
posed of a4- and b1-heterodimers. VLA-4 is mainly ex-
pressed on eosinophils and lymphocytes. VCAM-1,
expressed in vascular endothelial cells and fibronectins,
ingredients of extra-cellular matrix, is the ligand for
VLA-4. Binding of VLA-4 and VCAM-1 is one of the
most important mechanisms for tight adhesion of leuko-
cytes with vascular endothelial cells, and is strongly re-
lated to infiltration of inflammatory cells, such as
lymphocytes and eosinophils, into inflammation sites.
As VLA-4 also functions as a sub-signal in the activa-
tion and multiplication of T-cells, agents that inhibit
adhesion of VLA-4 and VCAM-1 interaction are likely
Although several phenylalanine derivatives with differ-
ent chemotypes have been reported to exhibit oral bio-
availability, there is still room for improvement in
discovery and development of antagonists with im-
proved pharmacokinetic properties. Therefore, improve-
ment of the pharmacokinetic properties of these
derivatives was attempted to identify novel inhibitors.
Previous studies of the SAR of phenylalanine derivatives
suggest that the para-position of the phenyl group on
phenylalanine is important for activity.6 Several
Keywords: VLA-4 inhibitors; Piperazinylphenylalanine derivatives.
*
Corresponding authors. Tel.: +81 55 989 2014; fax: +81 55 986
7430; e-mail: osamu.saku@kyowa.co.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.014

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O. Saku et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1053–1057
examples in published studies have directed their focus
to phenylalanine derivatives in which the benzene ring
is linked by amide or ether at the para-position
(Fig. 1).7,8 As part of this group’s effort to develop novel
VLA-4 inhibitors, replacement of the linkers with a
piperazine ring was attempted, with the aim of produc-
ing a novel advantageous surrogate for these linkers.
Introduction of two nitrogens reduces lipophilicity and
is generally expected to contribute to improving the
PK profile.
A, after deprotection of commercially available N-Boc
tyrosine derivatives, amidation was performed with car-
boxylic acids to produce 3. Subsequently 3 was converted
to a triflated tyrosine 4. A subsequent Pd-coupling reac-
tion with piperazine 6 produced 9, followed by hydrolysis
to the acid 12.9 In Method B, triflate 5 was converted to
piperazinylphenylalanine 7, followed by deprotection
and amidation to 9. Carboxylic acid 12 was obtained
by hydrolysis of 9. Introduction of functional groups at
the 4-position of piperazine was performed using Method
C, which utilized 11 prepared by the deprotection of ben-
zylpiperazine 7n obtained in Method B. Although a race-
mic mixture was produced in the Pd-coupling reaction,
optimized conditions using 1310 as the ligand in THF
could be applied to avoid this problem.
Piperazinylphenylalanine derivatives were prepared
according to the methods shown in Scheme 1. In Method
O
Compounds were assayed for VLA-4 antagonist activity
using ELISA, in which VLA-4 derived from Jurkat cells
was allowed to compete for bound human VCAM-1 in
the presence of test compounds.
Cl
N
H
O
H
N
H
N
H
N
HO
O
O
O
O
O
H
N
O
Cl O
N
N
H
N
H
O
O
O
COOH
BIO-1272 (Biogen)
O
R-411 (Roche)
O
Compound 12a substituted by tosylproline, demon-
strated to improve potency in several derivatives,
showed modest potency as shown in Table 1. Introduc-
tion of piperazine as a linking group was demonstrated
to be tolerated. Subsequently, substitution at the 4-posi-
tion of the piperazine ring was briefly explored.
O
O
Cl
O
F
O
N
H
N
H
CO2H
CO2H
Cl
Cl
TR-14035 (Tanabe)
SB-683699 (GSK)
Comparison of all three positional isomers (12b–d) indi-
cated that the position of substitute groups had little ef-
Figure 1. Structure of VLA-4 antagonists.
OH
OH
(b) or (c)
O
Method A
(a)
OR3
OR3
R2
N
H
TFA•H2N
O
O
(d)
2
3
OH
OTf
O
Boc
OR3
(d)
OR3
R2
N
H
N
H
O
O
1
4
(e)
R1
6
R1
R1
N
N
N
N
N
OTf
N
Method B
(e)
O
(a)
(b) or (c)
OR3
OR3
R2
N
H
Boc
OR3
Boc
N
OR3
TFA•H2N
N
O
R1
N
NH
O
H
H
O
O
9
8
5
6
7
Bn N
6n
NH
(e)
(g) or (h)
(i) or (j)
Bn
Method C
N
NH
R1
N
O
Cl
N
N
Cl
O
N
O
Boc
N
OR3
10
OR3
O
R2
N
H
O
PtBu2
(f)
H
O
OH
R2
N
H
13
O
7n
11
12
Scheme 1. Reagents and conditions: (a) TFA, CH2Cl2; (b) R2COOH, WSCÆHCl, HOBtÆH2O, Et3N, DMF; (c) R2COCl, iPr2NEt, CH2Cl2; (d) Tf2O,
pyridine, CH2Cl2; (e) 6, 13, Pd(OAc)2, Cs2CO3, THF; (f) 1—10, CH2Cl2; 2—MeOH, reflux; (g) R4COCl, Et3N, CHCl3; (h) ArX, iPr2NEt, DMF; (i)
LiOHÆH2O, THF, H2O; (j) TFA, CH2Cl2.

O. Saku et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1053–1057
1055
Table 1. Inhibitory effect of N-tosylproline derivatives
Table 2. Inhibitory effect of N-phenylsulfonylproline derivatives
R1
R1
Me
N
N
N
N
O
O
S
O
O
O
OH
N
S
N
H
O
OH
N
O
N
H
O
Compound
R1
Jurkat/VCAM-1 IC50 (nmol/L)
Compound
R1
Jurkat/VCAM-1 IC50 (nmol/L)
76 18
12a
123
5
12p
12b
12c
12d
12e
12f
12g
12h
12i
135
4
O2N
NC
MeO
12q
12r
12s
12t
69 12
67 29
OMe
170 35
152 30
353 78
327 59
OMe
101
19
9
2
CN
Me
Me
NC
NC
Me
O
450
6
12u
12v
12w
12x
67 10
S
Me
143 18
NO2
N
124 17
N
99
8
NC
N
O
O
80
6
12j
239 85
75 10
N
N
Et
200 12
12k
12l
N
N
O
H
IC50 values are means SEM of at least two separate assays with three
replicates at each concentration.
N
179 40
12m
12n
–Me
235 20
222 19
The phenylsulfonyl group appeared to be a suitable
replacement for the tosyl group (12p vs 12a). Thus, the
effect of replacing this group is shown in Table 2. Elec-
tron-withdrawing groups on the phenyl group at the 4-
position of piperazine, such as NO2 (12q) in the ortho-
position and CN (12r,s) in the ortho- or para-position,
were better tolerated. Introduction of two cyano groups
in the ortho-position (12t) produced a 4-fold improve-
ment in potency over compound 12p, and heterocyclic
rings such as 2-thienyl (12u) and pyrazinyl (12v) were
well tolerated. N-acyl piperazine (12w) was also toler-
ated, but a reduction in potency was observed for the
urea analog (12x).
12o
130 42
OEt
IC50 values are means SEM of at least two separate assays with three
replicates at each concentration.
fect on potency. However, a 3-fold decrease in potency
was observed when a methyl or phenyl group was intro-
duced (12e–g). Para-nitro (12h) and ortho-cyano (12i)
analogs were equipotent to 12a. Although the pyrimi-
dine (12j) and pyridine (12l) analogs were slightly less
potent than 12a, 1,4-pyrazine analog 12k exhibited
greater activity (IC50 = 75 nmol/L). This finding indi-
cates that the position of basic nitrogen in heterocyclic
groups on piperazine is important for potency. An alkyl
group on piperazine increases the basicity of nitrogen,
and marked changes in properties were expected. How-
ever, N-methyl piperazine 12m and N-benzyl piperazine
12n were only 2-fold less potent than 12a. The most po-
tent analog in the N-alkyl piperazine derivatives was es-
ter 12o. From these results, it is concluded that this
region has good tolerability.
The left side of the molecule was examined next.
Prompted by the finding that replacement of the tosyl
group with a phenylsulfonyl group produced a 2-fold
improvement in potency, this position was further char-
acterized (Table 3). 3,5-Dichlorophenyl-substituted
compound 12z exhibited the most potent activity with
an IC50 of 28 nmol/L, while introduction of the thiazol-
idine ring as a linker was detrimental (12y). Although
benzoyl analogs showed a significantly reduced activity
(12aa–ac), the dichloropyridine analog 12ad displayed

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O. Saku et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1053–1057
Table 3. Inhibitory effect of prolinyl amide replacements
structural modifications of proline derivatives did not
improve oral bioavailability.
N
N
In contrast, the PK profiles of benzoyl derivatives in rats
were excellent (Fig. 2). Oral bioavailability of com-
pounds 12aa and 12ad was 86% and 32%, respectively
(Table 4). Incorporation of the piperazine linker was
demonstrated to be a useful approach. Furthermore,
compound 12aa reduced permeation of eosinophils to
40% in a pleurisy model induced by compound 48/80,
a Ca2+-dependent histamine releaser, in rats (100 mg/
kg, po, t.i.d.). This inhibitory effect was nearly identical
to that of prednisolone (Fig. 3).
O
OH
R2
N
H
O
Compound R2
Jurkat/VCAM-1 IC50 (nmol/L)
123
O
S N
O
Me
12a
5
O
S N
O
12p
76 18
O
S
Me
Cl
S N
O
12y
196 30
O
12z
S N
28
3
O
Cl
Cl
12aa
12ab
12ac
12ad
12ae
1122 250
6807 1455
4318 1976
Cl
F
F
CF3
Cl
F
510
6
N
Cl
Figure 2. In vivo pharmacokinetic profile of compounds 12aa and
12ad. Compounds were orally administered to rats (10 mg/kg), then
plasma samples were withdrawn at the time points indicated.
Cl
42 10
HOOC
Cl
IC50 values are means SEM of at least two separate assays with three
replicates at each concentration.
approximately 2-fold higher potency compared to 12aa.
TR-14035 analogs6 demonstrated that the carboxy ana-
log 12ae, with H-bond donors at the 4-position, exhib-
ited much higher potency. In particular, potency of
12ae was comparable to those of proline derivatives
(IC50 = 42 nmol/L).
Initial pharmacokinetic screening studies were con-
ducted to investigate oral bioavailability. PK profiles
of proline derivatives in mice were characterized at a
low plasma concentration. Several examinations indi-
cated that the reason for low oral bioavailability of pro-
line derivatives was a high biliary excretion rate. This
efflux is likely due to a transporter-mediated process. Al-
most all of the compounds showed a high rate of excre-
tion that exceeded 50% within 30 min. Consequently,
Figure 3. Effect of 12aa on eosinophil infiltration into BAL fluid, 24 h
after compound 48/80 challenge in a pleurisy model in rats.
Table 4. Pharmacokinetic profile for compounds 12aa and 12ad
Compound
AUC0–1 (ng h/mL)a (iv)
Clp (L/h/kg)a
Vdss (L/kg)a
t1/2b (h)a
AUC0–1 (ng h/mL)b (po)
12aa
12ad
1290
721
0.775
1.39
0.754
6.23
1.44
7.91
11,100
2290
a Study in fed male Sprague–Dawley rats (1 mg/kg in DMSO).
b Study in fed male Sprague–Dawley rats (10 mg/kg) as a suspension in 0.5% methylcellulose.

O. Saku et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1053–1057
1057
8. Archibald, S. C.; Head, J. C.; Gozzard, N.; Howat, D. W.;
Parton, T. A. H.; Porter, J. R.; Robinson, M. K.; Shock,
A.; Warrellow, G. J.; Abraham, W. M. Bioorg. Med.
Chem. Lett. 2000, 10, 997.
In summary, a series of potent piperazinylphenylalanine
VLA-4 antagonists was prepared. Optimization at the 4-
position of piperazine produced highly potent com-
pound 12t (IC50 = 19 nmol/L), while oral bioavailability
was improved by alteration to the amide. Introduction
of piperazine as a linking group resulted in a significant
improvement in the PK profile. In addition, 2,6-dic-
hlorobenzoyl derivative 12aa demonstrated efficacy in
an in vivo pleurisy model after oral administration.
9. Preparation of 12i: To a solution of N-(toluene-4-sulfo-
nyl)-L-prolyl-L-tyrosine tert-butyl ester 3a11 (32.9 g,
67.3 mmol) in dichloromethane (200 mL), pyridine
(16.0 mL, 198 mmol) and trifluoromethanesulfonic anhy-
dride (14.6 mL, 86.8 mmol) were added. The reaction
mixture was then stirred at room temperature for 4 h,
acidified with 1 mol/L HCl, and then extracted with
dichloromethane. The organic extract was washed succes-
sively with saturated NaHCO3 solution and saturated
NaCl solution, then dried over MgSO4, filtered, and
concentrated in vacuo to produce the crude product.
Purification by silica gel chromatography (gradient: 25%
EtOAc in hexane) produced N-(toluene-4-sulfonyl)-L-
prolyl-O-(trifluoromethanesulfonyl)-L-tyrosine tert-butyl
ester 4a (36.5 g, 87%) as a white solid. 1H NMR
(300 MHz, CDCl3): 1.40–1.56 (m, 3 H), 1.45 (s, 9H),
2.00–2.05 (m, 1H), 2.21 (s, 3H), 3.03–3.38 (m, 4H), 4.03–
4.08 (m, 1H), 4.70–4.78 (m, 1H), 7.17 (d, J = 8.4 Hz, 2H),
7.28–7.36 (m, 5H), 7.84 (d, J = 8.3 Hz, 2H). 4a (623 mg,
1.00 mmol) was dissolved in THF (2 mL) under Ar and 1-
(2-cyanophenyl)piperazine (229 mg, 1.22 mmol), 2-(di-
tert-butylphosphino)biphenyl (23.0 mg, 0.0771 mmol),
palladium(II) acetate (11.0 mg, 0.0490 mmol), and cesium
carbonate (462 mg, 1.42 mmol) were added, then the
mixture was stirred at 60 °C for 4 h. Saturated NaCl
solution was added and extracted with EtOAc. The
organic extract was washed with saturated NaCl solution,
then dried over MgSO4, filtered, and concentrated in
vacuo to produce the crude product. Purification by silica
gel chromatography (gradient: 50% EtOAc in hexane)
produced N-(toluene-4-sulfonyl)-L-prolyl-4-(4-(2-cyano-
phenyl)piperazine-1-yl)-L-phenylalanine tert-butyl ester 9i
(230 mg, 35%) as a white solid. 1H NMR (300 MHz,
CDCl3): 1.42–1.47 (m, 4H), 1.48 (s, 9H), 2.43 (s, 3H), 2.98
(dd, J = 7.0, 14.0 Hz, 1H), 3.07–3.13 (m, 1H), 3.16 (dd,
J = 5.7, 13.8 Hz, 1H), 3.35–3.36 (m, 9H), 4.07–4.09 (m,
1H), 4.65–4.72 (m, 1H), 6.89 (d, J = 8.4 Hz, 2H), 7.01–7.07
(m, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.30–7.31 (m, 1H), 7.32
(d, J = 8.4 Hz, 2H), 7.51 (t, J = 7.6 Hz, 1H), 7.58 (dd,
J = 0.8, 8.1 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H). Trifluoro-
acetic acid (1.00 mL, 13.0 mmol) was added to a solution
of 9i (230 mg, 0.350 mol) in dichloromethane (5.0 mL) at
room temperature under Ar. After 12 h, the mixture was
concentrated, then dissolved in 1 mol/L NaOH solution.
The mixture was acidified with acetic acid, then the
precipitate was collected by filtration to produce N-
(toluene-4-sulfonyl)-L-prolyl-4-(4-(2-cyanophenyl)pipera-
zine-1-yl)-L-phenylalanine 12i (149 mg, 71%) as a white
solid. 1H NMR (300 MHz, CDCl3): 1.14–1.17 (m, 1H),
1.33–1.37 (m, 2H), 1.75–1.78 (m, 1H), 2.33 (s, 3H), 2.77–
2.98 (m, 2H),3.25–3.28 (m, 10H), 4.05–4.10 (m, 1H), 4.64–
4.74 (m, 1H), 6.81 (d, J = 7.6 Hz, 2H), 6.98–7.08 (m, 4H),
7.19 (d, J = 6.5 Hz, 2H), 7.49 (t, J = 8.1 Hz, 1H), 7.55 (d,
J = 7.6 Hz, 2H), 7.69 (d, J = 7.3 Hz, 2H).
References and notes
1. Elices, M. J. In Cell Adhesion Molecules and Matrix
Proteins: Role in Health and Diseases; Mousa, S. A., Ed.;
Springer-Verlag: Berlin, 1998; p 133.
2. Lobb, R. R.; Helmer, M. E. J. Clin. Invest. 1994, 94,
1722.
3. (a) Steinman, L. Nat. Rev. Drug Discov. 2005, 4, 510;
(b) Rice, G. P. A.; Hartung, H.-P.; Calabresi, P. A.
Neurology 2005, 64, 1336; (c) Miller, D. H.; Khan, O.
A.; Sheremata, W. A.; Blumhardt, L. D.; Rice, G. P.
A.; Libonati, M. A.; Willmer-Hulme, A. J.; Dalton, C.
M.; Miszkiel, K. A.; O’Connor, P. W. N. Engl. J.
Med 2003, 348, 15.
4. (a) Yang, G. X.; Hagmann, W. K. Med. Res. Rev 2003, 23,
369; (b) Hagmann, W. K. Curr. Top. Med. Chem. 2004,
1461; (c) Sing, J.; Adams, S.; Carter, M. B.; Cuervo, H.;
Lee, W.-C.; Lobb, R. R.; Pepeinsky, B.; Petter, R.; Scott,
D. Curr. Top. Med. Chem. 2004, 1497; (d) Holland, G. W.;
Biediger, R. J.; Vanderslice, P.. In Annual Reports in
Medicinal Chemistry; Doherty, A. M., Ed.; Academic
Press: New York, 2002; Vol. 37, pp 65–74; (e) Li, B.; de
Laszlo, S. E.; Kamenecka, T. M.; Kopka, I. E.; Durette, P.
L., ; Lanza, T., Jr.; MacCoss, M.; Tong, S.; Mumford, R.
A.; McCauley, E. D.; van Riper, G.; Schmidt, J. A.;
Hagmann, W. K. Bioorg. Med. Chem. Lett. 2002, 12, 2141;
(f) Yang, G. X.; Chang, L. L.; Truong, Q.; Doherty, G.
A.; Magriotis, P. A.; deLaszlo, S. E.; Li, B.; MacCoss, M.;
Kidambi, U.; Egger, L. A. Bioorg. Med. Chem. Lett. 2002,
12, 1497; (g) Sidduri, A.; Tilley, J. W.; Lou, J. P.; Chen, L.;
Kaplan, G.; Mennona, F.; Campbell, R.; Guthrie, R.;
Huang, T. N.; Rowan, K.; Schwinge, V.; Renzetti, L. M.
Bioorg. Med. Chem. Lett. 2002, 12, 2479; (h) Withering-
ton, J.; Bordas, V.; Gaiba, A.; Green, P. M.; Naylor, A.;
Parr, N.; Smith, D. G.; Takle, A. K.; Ward, R. W. Bioorg.
Med. Chem. Lett. 2006, 16, 2256; (i) Singh, J.; Vlihmen, H.
V.; Liao, Y.; Lee, W.-C.; Corbenise, M.; Harris, M.; Shu,
I.-H.; Gill, A.; Cuervo, J. H.; Abraham, W. M.; Adams, S.
P. J. Med. Chem. 2002, 45, 2988.
5. Abraham, W. M.; Gill, A.; Ahmed, A.; Sielczak, M. W.;
Lauredo, I. T.; Botinnikova, Y.; Lin, K.-C.; Pepinsky, B.;
Leone, D. R.; Lobb, R. R.; Adams, S. P. Am. J. Respir.
Crit. Care Med. 2000, 162, 603.
6. Sircar, I.; Gudmundsson, K. S.; Martin, R.; Liang, J.;
Nomura, S.; Jayakumar, H.; Teegarden, B. R.; Now-
lin, D. M.; Cardarelli, P. M.; Mah, J. R.; Connell, S.;
Griffith, R. C.; Lazarides, E. Bioorg. Med. Chem.
2002, 10, 2051.
10. Wolfe, J. P.; Singer, R. A.; Yang, B. H.; Buchwald, S. L.
J. Am. Chem. Soc. 1999, 121, 9550.
7. Chen, L.; Tilley, J. W.; Guthrie, R. W.; Mennona, F.;
Huang, T.-N.; Kaplan, G.; Trilles, R.; Miklowski, D.;
Huby, N.; Schwinge, V.; Wolitzky, B.; Rowan, K. Bioorg.
Med. Chem. Lett. 2000, 10, 729.
11. Lombardo, L. J.; Semko, C. M.; Thorsett, E. D.; Ashwell,
S.; Kreft, A.; Baudy, A. R. B.; Dressen, D. B.; Grant, F.
S.; Konradi, A. W.; Pleiss M. A.; Sarantakis D. PCT Int.
Appl. WO9906390, 1999.