β-aryl-succinic acid hydroxamates as dual inhibitors of matrix metalloproteinases and tumor necrosis factor alpha converting enzyme

Article abstract of DOI:10.1021/jm0110993

Novel hydroxamate inhibitors of tumor necrosis factor converting enzyme (TACE) and matrix metalloproteases (MMPs) have been synthesized via the Claisen-Ireland rearrangement. Aryl residues have been introduced to fill the enzyme's P1′ specificity pocket. The best compound inhibits MMPs and TACE with nanomolar potency and inhibits the release of TNFα from cells with an IC₅₀ of 48 nM. Oral administration to rats inhibits the LPS-induced plasma TNFα levels with an ED₅₀ of 1 mg/kg.

Full text of DOI:10.1021/jm0110993

J . Med. Chem. 2002, 45, 2289-2293
2289
â-Ar yl-Su ccin ic Acid Hyd r oxa m a tes a s Du a l In h ibitor s of Ma tr ix
Meta llop r otein a ses a n d Tu m or Necr osis F a ctor Alp h a Con ver tin g En zym e
Georg Kottirsch, Guido Koch, Roland Feifel, and Ulf Neumann*
Preclinical Research Novartis Pharma AG, CH-4002 Basel, Switzerland
Received November 9, 2001
Novel hydroxamate inhibitors of tumor necrosis factor converting enzyme (TACE) and matrix
metalloproteases (MMPs) have been synthesized via the Claisen-Ireland rearrangement. Aryl
residues have been introduced to fill the enzyme’s P1′ specificity pocket. The best compound
inhibits MMPs and TACE with nanomolar potency and inhibits the release of TNFR from cells
with an IC50 of 48 nM. Oral administration to rats inhibits the LPS-induced plasma TNFR
levels with an ED50 of 1 mg/kg.
In tr od u ction
leading to sub-micromolar inhibition of cellular TNFR
release.13 The vast majority of variations in the â-posi-
tion, due to synthetic restrictions, focused on alkyl and
aryl-alkyl residues that did not improve the TACE
inhibitory activity over that of marimastat.14,15
Rheumatoid arthritis (RA) is a chronic, inflammatory
joint disease which affects about 1% of the population
worldwide. Clinical signs include pain and joint swell-
ing, and over the long term, the erosion of cartilage and
bone leads to irreversible destruction of the affected
joints. The cytokines tumor necrosis factor R (TNFR) and
interleukin-1â (IL-1â) are well-established mediators of
inflammation and joint destruction.1 Clinical studies
and preclinical models suggest a central early role of
TNFR which seems to orchestrate the network of pro-
inflammatory cytokines.2 Antagonizing the TNFR by the
soluble TNF receptor (Enbrel) has been proven to be
effective for the treatment of RA.3
In an attempt to improve the potency on TNFR
inhibition and to overcome the poor in vivo properties
of succinate-based hydroxamate MMP/TACE inhibitors,
we have introduced novel modifications in R- and
â-positions of the succinate hydroxamate template via
a Claisen-Ireland rearrangement.16 This methodology
was used to place aryl substituents in the position â to
the hydroxamate, which resulted in a series (Figure 1)
of novel and very potent dual inhibitors of MMPs and
TACE as well as cellular TNFR release.
The degradation of cartilage and bone collagen in RA
is accomplished by matrix metalloproteinases (MMPs),
produced by synovial fibroblasts, chondrocytes, and
infiltrating leukocytes.4 Several different MMPs have
been shown to be present in the joints of RA patients.5
Attacking the inflammatory as well as the erosive
component of RA with one therapeutic agent might be
possible by designing dual inhibitors of MMPs and
TNFR convertase (TACE), a metalloproteinase respon-
sible for the release of TNFR from its precursor,
proTNFR.6,7 MMPs and TACE are Zn-dependent neutral
metalloproteinases; the enzymes share considerable
structural homology in the active-site region as shown
by X-ray crystallography.8 There is a vast amount of
literature on inhibitors of MMPs containing hydroxam-
ates, thiols, carboxylates, or phosphinates as the Zn-
chelating group,9 and dual inhibition of MMPs and
TNFR release was described for some MMP inhibitors.10
Very recently, the reversed hydroxamate dual MMP/
TACE inhibitor GW3333 has been described.11
Many of the MMP/TACE inhibitors reported to date
are derived from marimastat (1), the first MMP inhibi-
tor which entered clinical trials for cancer treatment.12
Despite their nanomolar potency on MMPs, many of the
succinate-based hydroxamate MMP inhibitors only mod-
estly inhibit cellular TNFR release. TACE inhibitory
activity in the marimastat template was improved by
derivatization of the position R to the hydroxamate,
Ch em istr y
The succinate inhibitors 2-5 presented in this study
were synthesized by applying a Claisen-Ireland rear-
rangement17 to build the succinate template and to
introduce novel substituents R1 and R2. Scheme 1
describes the synthesis of compound 5 in detail. Com-
pounds 2-4 were prepared in analogy to the described
synthesis; however, the syntheses were carried through
without separating the diastereomeric mixtures ob-
tained after the Claisen-Ireland rearrangement. Sepa-
ration of the diastereomers was done at the end of the
synthesis via reverse-phase HPLC.
trans-Dibromobut-2-ene 6 was reacted with benzyl
alcohol under phase-transfer catalysis to give the benzyl
ether 7. DBU-mediated coupling of 7 with (4-methoxy-
phenyl)acetic acid yielded the allyl ester 8. The Claisen-
Ireland rearrangement of 8 was initiated with Li-HMDS
and TMS-Cl in the presence of catalytic amounts of
TiCl4 and resulted in the succinate precursor 9, which
was isolated as a mixture of 4 diastereomers. The anti-
isomer 10 was obtained enantiomerically pure via
crystallization of 9 as its (-)-1-(S)-phenylethylamine
salt and subsequent acidification. Reaction of 10 with
tert-leucine methylamide under EDCI/HOBT conditions
and oxidation of the resulting coupling product 11 in a
three-step process gave the succinate derivative 12.
Conversion of the acid 12 to the hydroxamic acid
inhibitor 5 was achieved by coupling with o-benzylhy-
* Corresponding author. Phone: ++41 61 324 4440. Fax: ++41 61
324 4774. E-mail: ulf.neumann@pharma.novartis.com.
10.1021/jm0110993 CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/27/2002

2290 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Kottirsch et al.
were challenged by an iv LPS injection and bled 1 h
later. Concentrations of TNFR in rat plasma were
determined, and a dose-dependent response was ob-
served with all compounds tested. The starting com-
pound marimastat (1) only showed statistically signifi-
cant inhibition (87%) at the 30 mg/kg dose. In contrast,
compounds 3-5 already had significant effects at much
lower doses (3, 57% at 10 mg/kg; 5, 62 and 79% at 3
and 10 mg/kg, respectively), with 5 being the most
potent compound in the test with an ED50 of 1 mg/kg.
Con clu sion
The application of the Claisen-Ireland rearrange-
ment for the synthesis of succinate-based hydroxamate
TACE/MMP inhibitors provides a convenient route for
the introduction of aryl substituents in the position â
to the hydroxamate. Compared to marimastat, the
replacement of the isobutyl residue by para-substituted
phenyl groups has led to improved potency against both
TACE and cellular TNFR release. The para-methoxy
derivative 5 was found to be a sub-nanomolar inhibitor
of TACE with nanomolar activity in the cellular TNFR
release assay as well as a sub-nanomolar inhibitor
against MMP-1, MMP-2, and MMP-3. The in vivo
activities of 5 in the LPS rat model justified proposing
5 for testing in chronic models and, possibly, as a
candidate for further development.
F igu r e 1. Structures of novel MMP/TACE inhibitors.
droxylamine and EDCI to 13 and removal of the
protection groups via Pd-BaSO4-catalyzed hydrogena-
tion. Proof of the absolute stereochemistry of the suc-
cinate inhibitor 5 was established via X-ray crystallog-
raphy.
Discu ssion
The inhibitory potency of the succinate inhibitors was
determined against recombinant TACE, MMP-1, MMP-
2, and MMP-3 using a fluorogenic substrate. TNFR
inhibition in cells was measured in LPS-stimulated
human mononuclear cells (hPBMCs). The results are
summarized in Table 1.
Exp er im en ta l Section
MMP Assa ys. In vitro inhibition of MMP-1 and -2 was
measured using the fluorescence-quenched peptide substrate
Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (5 µM) and a
MMP concentration of 0.1 nM in 0.1 M Tris-HCl, containing
0.1 M NaCl, 10 mM CaCl2, 0.05% Brij-35, and 0.1% PEG6000,
at 25 °C and pH 7.5. Inhibitors (11 concentrations), enzyme,
and buffer were incubated in a black 96-well plate for 1 h. The
reaction was started by the addition of substrate, and fluo-
rescence (325/400 nm) was measured for 20 min in a Spectra-
max Gemini plate reader (Molecular Devices). The rates were
fitted to the equation v ) vo/(1 + [I]/Ki) where v and vo are the
rates in the presence and absence of inhibitor, respectively,
and Ki is the dissociation constant. The substrate concentration
of 5 µM is ,KM for all MMPs, and no correction for substrate
competition is needed. MMP-3 was assayed using Ac-Pro-Leu-
Ala(S)-Nvl-Trp-NH2 and DTNB in 50 mM MES and 10 mM
CaCl2 at pH 6.5.18 Inhibitors and the enzyme were preincu-
bated for 3 h, and the reaction was followed at 410 nm in a
BioTec Elx808 plate reader for 40 min.
TACE Assa y. The cDNA for human TACE (Met 1 to Asp
670) was cloned from a HeLa cell library into a baculovirus
transfer vector, and protein was expressed in Sf21 insect cells.
Protein was purified by a combination of anion-exchange, size-
exclusion, and affinity-chromatography steps, and was ap-
proximately 90% pure as shown by SDS-PAGE and N-termi-
nal sequencing. TACE was assayed using the same substrate
used for MMP-1 and -2 in 10 mM Hepes, containing 0.0075%
Brij35, pH 7.5. Inhibitor testing and calculation of Ki values
were done as described for the MMPs.
The use of the Claisen-Ireland rearrangement re-
sulted in a hydroxymethylene substituent in the position
R to the hydroxamate instead of a hydroxy group like
in marimastat (1). As seen with compound 2 in Table
1, this modification retained the marimastat-like po-
tency. While the activity of 2 on the MMPs was similar
to that of marimastat, a 2-fold loss in activity on TACE
as well as in the cellular TNFR release assay was found.
Replacement of the isobutyl side chain in 2 by a phenyl
group restored the inhibitory potency on TACE and
TNFR release as illustrated by 3 but was less suitable
for MMP activity. A drop in activity on all three MMPs
was observed. Further improvements in potency on
TACE and TNFR release were achieved by introduction
of para-substituents in 3. The para-methyl inhibitor 4
had similar activity on TACE compared to the phenyl
analogue 3, but the IC50 for cellular TNFR release
decreased by more than a factor of 3. While the potency
on MMP-2 increased 10-fold, the activity on MMP-1 and
MMP-3 was hardly influenced by this modification.
Changing the para-methyl group in 4 to a methoxy
group (e.g., 5) gave an additional increase in potency
on TACE and cellular TNFR release and also improved
the activity on MMP-1 and, to a greater extent, on
MMP-3. Compared to marimastat, the para-methoxy
compound 5 is a very potent inhibitor of TACE and
cellular TNFR release as well as a broad spectrum MMP
inhibitor with sub-nanomolar activity on all three
enzymes tested. The solubility of compounds 2-5 in
water is >10 mg/mL, and they show little or no binding
to plasma proteins (data not shown).
Cellu la r TNF r Relea se Assa y. Mononuclear cells (hPB-
MCs) were isolated from the blood of healthy donors by
centrifugation over Ficoll. Cells were washed in PBS, counted,
and cultured in RPMI/5% FCS at 37 °C and 5% CO2 in 96-
well plates. Final cell density was 105 cells/100 µL. Cells were
incubated with an inhibitor (7 concentrations) for 30 min and
then stimulated by LPS (5 µg/mL) and interferon-γ (100 units/
mL) for 3 h. Plates were centrifuged, and TNFR concentrations
in the supernatants were determined by ELISA.
The in vivo activity of compounds 1 and 3-5 was
demonstrated in an LPS-induced systemic TNFR release
model in rats. Four hours after oral dosing, the animals
Ra t LP S Ch a llen ge Mod el. Conscious male Sprague-
Dawley rats, kept under standard conditions, were orally dosed
by gavage with aqueous solutions of compounds 1 and 3-5 at

â-Aryl-Succinic Acid Hydroxamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2291
Sch em e 1a
aConditions: (a) BnOH, NaOH, CH2Cl2, Bu4NHSO4, 48%; (b) 4-MeO-phenylacetic acid, DBU, CH2Cl2, 69%; (c) Li-HMDS, TMS-Cl,
TiCl4, THF, -78 f 20 °C, 99%; (d) (-)-1-(S)-phenethylamine, 31%;. (e) L-tert-leucine methylamide, EDCI, HOBT, NEt3, DMF, 93%; (f)
NMM-N-oxid, OsO4, tert-butyl alcohol/water, 100%; (g) NaIO4, acetone/water, 100%; (h) NaClO2, 2-methyl-2-butene, tert-butyl alcohol/
water, 46%; (i) BnO-NH2, EDCI, HOBT, DMF, 46%; (j) H2, Pd-BaSO4, MeOH, 93%.
Ta ble 1. Inhibition of TACE and TNFR Production in hPBMCs
2-4 were synthesized in a similar fashion; however, the
syntheses were carried through without separating the dia-
stereomeric mixtures obtained after the Claisen-Ireland
rearrangement. Separation of the diastereomers was done at
the end of the synthesis by preparative HPLC on RP-18 silica
using methanol/water as eluent. The stereochemistry of com-
pounds 2-4 was assigned in analogy to compound 5 by the
NMR shifts of the tert-butyl groups, the order of elution from
the RP-HPLC, and their biological activity.
and MMPs by Succinate-Type Hydroxamate Inhibitorsa
TACE
TNFR
MMP-1 MMP-2 MMP-3
compound Ki (nM) IC50 (nM) Ki (nM) Ki (nM) Ki (nM)
1
2
3
4
5
6.3
12.9
4.9
3.8
0.6
1001
1606
928
269
48
0.7
0.6
3.5
2.0
1.1
0.6
0.3
4.8
0.3
0.5
8.9
9.8
13.2
15.2
0.9
(4-Br om obu t-2-en yloxym eth yl)ben zen e (7). To a solu-
tion of trans-1,4-dibromobut-2-ene (50.00 g, 233.8 mmol),
benzyl alcohol (26.6 mL, 257.2 mmol), and tert-butylammoni-
umhydrogensulfate (7.94 g, 23.4 mmol) in CH2Cl2 (200 mL)
was added an aqueous solution of NaOH (113.7 mL, 2.1 mol).
The mixture was stirred for 24 h at room temperature, diluted
with water (400 mL), and extracted with Et2O (2 × 500 mL).
The combined organic extracts were dried over Na2SO4 and
filtered, and the solvents were removed. The residue was
purified by flash chromatography on silica using hexanes/
EtOAc (9/1) as eluant to give 7 (27.04 g, 48%). 1H-NMR
(CDCl3): 3.98 (d, 2H, J ) 7.3 Hz), 4.05 (d, 2H, J ) 4.4 Hz),
4.54 (s, 2H), 5.82-6.08 (m, 2H), 7.25-7.45 (m, 5H).
(4-Meth oxyp h en yl)a cetic Acid 4-Ben zyloxybu t-2-en yl
Ester (8). A solution of DBU (3.47 g, 22.8 mmol) in CH2Cl2
(10 mL) was added to a solution of 7 (5.0 g, 20.7 mmol) and
(4-methoxyphenyl)acetic acid (3.44 g, 20.7 mmol) in CH2Cl2
(60 mL) within 15 min at room temperature. The reaction
mixture was stirred for 24 h at room temperature; the CH2-
Cl2 was evaporated, and the residue was dissolved in EtOAc/
water (1/1, 100 mL). The solution was extracted with EtOAc
and dried over Na2SO4, and the solvent was removed. The
residue was purified by flash chromatography on silica using
hexanes/EtOAc (9/1) as eluant to give 8 (4.70 g, 69%). 1H-NMR
(CDCl3): 3.58 (s, 2H), 3.79 (s, 3H), 4.03 (d, 2H, J ) 4.1 Hz),
4.52 (s, 2H), 4.61 (d, 2H, J ) 4.1 Hz), 5.80-5.90 (m, 2H), 6.85
(d, 2H, J ) 8.7 Hz), 7.21 (d, 2H, J ) 8.7 Hz), 7.30-7.40 (m,
5H). MS (DCI, CH4) m/e: 326 (M+).
a
Inhibition constants, Ki, and IC50 values were determined as
described in the Experimental Section.
doses between 0.1 and 30 mg/kg (4-7 rats per group; controls
receiving vehicle only were included in the study). The rats
were anesthetized 3 h later for the remaining experimental
procedure. The carotid arteries (for blood sampling) and
jugular veins (for LPS injections) were cannulated, and 1 h
thereafter a single dose (500 µg/kg) of LPS was given. One
hour later, arterial blood was taken, and plasma was prepared
and, subsequently, was tested for TNFR concentrations (ELISA).
TNFR inhibition of compound-treated groups was calculated
as a percentage of controls treated with vehicle only. Addition-
ally, an ED50 was determined for compound 5.
Ch em istr y. Unless otherwise noted, materials were ob-
tained from commercial suppliers and used without purifica-
tion. Analytical thin-layer chromatography was performed
with precoated glass-backed plates (Merck Silicagel 60 F254).
Compounds were purified either by flash chromatography
using Merck Silicagel 60 (40-63 µm) or by preparative high-
pressure liquid chromatography on RP-18 silica gel. NMR
spectra were recorded on a Bruker MX-400 or DPX-400
spectrometer (400 MHz proton) in the indicated solvent.
Chemical shifts are expressed as parts per million (ppm)
downfield from tetramethylsilane, and J values are reported
in hertz (Hz). Fast atom bombardment mass spectroscopy (Xe,
8 keV) on a VG70-SE mass spectrometer was used for all
reported compounds. C,H,N analyses were carried out by
Novartis Services AG with all substances biologically tested.
3(R)-Ben zyloxym eth yl-2(S)-(4-m eth oxyp h en yl)p en t-4-
en oic Acid (10). To a solution of hexamethyldisilazane (22.62
g, 140.0 mmol) in THF (400 mL) was added 1.6 M nBuLi (87.60
mL, 140.0 mmol) in hexane at 0 °C. After being stirred for 10
min, the reaction mixture was cooled to -78 °C, and tri-
Gen er a l P r oced u r es for Com p ou n d s in F igu r e 1 a n d
Ta ble 1. The procedures used to synthesize the inhibitors in
Table 1 are described in detail for compound 5. Compounds

2292 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Kottirsch et al.
methylchlorosilane (15.23 g, 140.0 mmol) was added, followed
by a solution of 8 (30.50 g, 93.4 mmol) in THF (40 mL). After
the mixture had been stirred for 1 h at -78 °C, 1 M TiCl4 (1.89
mL, 1.9 mmol) in CH2Cl2 was added. The cooling bath was
removed, and the reaction mixture was allowed to warm to
room temperature; the mixture was stirred for one additional
hour, and the solvents were evaporated. The remaining residue
was taken up in ether (500 mL) and extracted with 0.1 N
NaOH (2 × 300 mL). The combined aqueous phases were
acidified with 1 N HCl and extracted with ether (2 × 300 mL),
and the combined organic layers were dried over Na2SO4. The
solvent was evaporated to yield the racemic mixture 9 (30.4
g, 93.1 mmol, 99.6%) as a raw product. Crude product 9 was
dissolved in ethanol (350 mL); (-)-1(S)-phenylethylamine
(12.42 g, 102.4 mmol) was added, and the resulting suspension
was heated to reflux while ethanol was added until a clear
solution was obtained. The solution was allowed to cool to room
temperature, and a crystalline product was isolated by filtra-
tion. It was recrystallized from ethanol (350 mL), and the
isolated crystals were suspended in EtOAc (300 mL); 2 N HCl
(200 mL) was added, and the resulting solution was extracted
with EtOAc (2 × 300 mL). The combined organic layers were
dried over Na2SO4, and the solvents were evaporated to yield
acid 10 (9.3 g, 31%) as a single enantiomer. HPLC analysis
showed a single peak tR ) 22.7 min (Chiralcel OD, n-hexanes/
i-PrOH/TFA ) 95/5/0.1) [tR (anti-isomer) ) 17.3/22.7 min/tR
and the solvents were removed in vacuo. The remaining
residue was taken up in ether (300 mL) and extracted with 2
N NaOH (2 × 200 mL). The aqueous layer was acidified with
2 N HCl and extracted with EtOAc (3 × 200 mL). The
combined organic layers were washed with brine and dried
over Na2SO4, and the solvents were removed in vacuo. The
remaining white solid was recrystallized from ether to obtain
acid 12 (1.85 g, 46%) in the form of white crystals. 1H-NMR
(DMSO): 0.85 (s, 9H), 2.46 (d, 3H, J ) 4.3 Hz), 3.25-3.32 (m,
1H), 3.45-3.50 (m, 1H), 3.59 (t, 1H, J ) 9.4 Hz), 3.71 (s, 3H),
3.88 (d, 1H, J ) 10.2 Hz), 4.13 (d, 1H, J ) 10.2 Hz), 4.44 (s,
2H), 6.83 (d, 2H, J ) 8.5 Hz), 7.28 (d, 2H, J ) 8.5 Hz), 7.28-
7.35 (m, 5H), 7.83 (q, 1H, J ) 4.3 Hz), 8.13 (d, 1H, J ) 8.7
Hz). MS (ESI) m/e: 493.3 [(M + Na)+].
N1-Ben zyloxy-2(R)-ben zyloxym eth yl-N4-(2,2-d im eth yl-
1(S)-m et h ylca r b a m oylp r op yl)-3(S)-(4-m et h oxyp h en yl)-
su ccin a m id e (13). To a solution of 12 (1.80 g, 3.8 mmol) in
DMF (20 mL) at 0 °C were added successively HOBT (0.64 g,
4.2 mmol), EDCI (0.73 g, 3.8 mmol), and triethylamine (1.16
g, 11.5 mmol). After 15 min, O-benzylhydroxylamine hydro-
chloride (0.67 g, 4.2 mmol) was added, and the mixture was
stirred for 18 h at room temperature. The solvent was
evaporated, and the residue was taken up in EtOAc (200 mL).
The organic layer was extracted with 2 N HCl (100 mL),
saturated NaHCO3 (100 mL), and brine (100 mL) and dried
over Na2SO4. The remaining solid was crystallized from EtOAc
to obtain compound 13 (0.93 g, 42%) as a white solid. From
the mother liquor, the solvents were removed in vacuo, and
the remaining oil was purified by flash chromatography on
silica gel using hexanes/EtOAc (3/7) as eluant to give an
1
(syn-isomer) ) 15.4/20.1 min]. H-NMR (DMSO): 2.98-3.05
(m, 1H), 3.41-3.55 (m, 2H), 3.59 (d, 1H, J ) 10.2 Hz), 3.72 (s,
3H), 4.47 (s, 2H), 4.82-4.91 (m, 2H), 5.41-5.51 (m, 1H), 6.83
(d, 2H, J ) 8.5 Hz), 7.18 (d, 2H, J ) 8.5 Hz), 7.25-7.35 (m,
5H), 12.25 (s, 1H). MS (DCI, CH4) m/e: 326 (M+).
1
additional batch of 13 (0.53 g, 24%). H-NMR (DMSO): 0.88
(s, 9H), 2.47 (d, 3H, J ) 4.8 Hz), 3.08-3.17 (m, 1H), 3.35-
3.41 (m, 1H), 3.62 (t, 1H, J ) 9.6 Hz), 3.69 (s, 3H), 3.86 (d,
1H, J ) 11.4 Hz), 4.12 (d, 1H, J ) 11.4 Hz), 4.13 (d, 1H, J )
11.4 Hz), 4.31 (d, 1H, J ) 11.4 Hz), 4.42 (s, 2H), 6.81 (d, 2H,
J ) 9.0 Hz), 7.12 (d, 2H, J ) 9.0 Hz), 7.24-7.37 (m, 10H),
7.80 (q, 1H, J ) 4.8 Hz), 8.12 (d, 1H, J ) 9.0 Hz), 10.97 (s,
1H). MS (ESI) m/e: 576.4 [(M + H)]+.
3(R)-Ben zyloxym eth yl-2(S)-(4-m eth oxyp h en yl)p en t-4-
en oic Acid (2,2-Dim eth yl-1(S)-m eth ylca r ba m oylp r op yl)-
a m id e (11). To a solution of 10 (3.00 g, 9.2 mmol) in DMF (25
mL) at 0 °C were added successively HOBT (1.55 g, 10.1
mmol), EDCI (1.76 g, 9.2 mmol), and triethylamine (2.79 g,
27.6 mmol). After 15 min, L-tert-leucine methylamide hydro-
chloride (2.0 g, 11.04 mmol) was added, and the mixture was
stirred for 18 h at room temperature. The solvent was
evaporated, and the residue was taken up in EtOAc (200 mL).
The organic layer was extracted with 2 N HCl (100 mL),
saturated NaHCO3 (100 mL), and brine (100 mL) and dried
over Na2SO4. The solvents were removed, and the residue was
dried in vacuo to obtain 11 (4.2.g, 93%), which was used in
N4-(2,2-Dim eth yl-1(S)-m eth ylca r ba m oylp r op yl)-N1-h y-
d r oxy-2(R)-h yd r oxym eth yl-3(S)-(4-m eth oxyp h en yl)su c-
cin a m id e (5). Compound 13 (0.90 g, 1.6 mmol) was hydro-
genated in MeOH (80 mL) with Pd-BaSO4 (0.80 g) under a
hydrogen atmosphere. After the solution was stirred for 6 h,
the catalyst was filtered off, and the solvent was removed in
vacuo. The remaining solid was crystallized from water to yield
the hydroxamate 5 (0.39 g, 63%) in the form of white crystals.
1H-NMR (DMSO): 0.92 (s, 9H), 2.46 (d, 3H, J ) 6.1 Hz), 2.87-
2.94 (m, 1H), 3.43-3.51 (m, 1H), 3.52-3.60 (m, 1H), 3.71 (s,
3H), 3.84 (d, 1H, J ) 12.7 Hz), 4.14 (d, 1H, J ) 9.4 Hz), 4.66
(t, 1H, J ) 4.3 Hz), 6.78 (d, 2H, J ) 8.7 Hz), 7.27 (d, 2H, J )
8.7 Hz), 7.78 (q, 1H, J ) 6.1 Hz), 8.03 (d, 1H, J ) 9.5 Hz),
8.49 (s, 1H), 10.19 (s, 1H). MS (ESI) m/e: 394.2 [(M - H)]-.
Anal. (C19H29N3‚0.66H2O) C, H, N.
1
the next step without further purification. H-NMR (DMSO):
0.98 (s, 9H), 2.47 (s, 3H), 2.98-3.05 (m, 1H), 3.41-3.51 (m,
2H), 3.72 (s, 3H), 3.79 (d, 1H, J ) 9.4 Hz), 4.16 (d, 1H, J ) 9.4
Hz), 4.42 (s, 2H), 4.78-4.85 (m, 2H), 5.35-5.50 (m, 1H), 6.80
(d, 2H, J ) 8.5 Hz), 7.24 (d, 2H, J ) 8.5 Hz), 7.25-7.35 (m,
5H), 7.80-7.85 (m, 1H), 7.96 (d, 1H, J ) 8.3 Hz). MS (FAB,
Thio) m/e: 453 [(M + H)]+.
2(R)-Ben zyloxym et h yl-N-(2,2-d im et h yl-1(S)-m et h yl-
ca r b a m oylp r op yl)-3(S)-(4-m et h oxyp h en yl)su ccin a m ic
Acid (12). To a solution of 11 (4.05 g, 8.9 mmol) in acetone
(60 mL) were added a solution of NMM-N-oxide (1.57 g, 11.6
mmol) in water (70 mL) and a 0.2 M solution of OsO4 (1.34
mL, 0.27 mmol) in tert-butyl alcohol. The reaction mixture was
stirred for 18 h at room temperature; aluminum oxide (1.6 g)
and Na2SO3 (0.8 g) were added, and the mixture was filtered
through silica gel. The filtrate was diluted with EtOAc (200
mL); the organic layer was washed with 2 N HCl, saturated
NaHCO3, and brine and dried over Na2SO4. The solvent was
removed in vacuo, and the remaining bishydroxy compound
(4.4 g, 100%) was dissolved in acetone (50 mL). To this solution
was added NaIO4 (2.08 g, 9.7 mmol) dissolved in water (50
mL) within 15 min. The reaction mixture was stirred for 4 h
at room temperature, quenched with water, and extracted with
EtOAc (2 × 200 mL). The organic layer was washed with brine
and dried over Na2SO4, and the solvents were removed in
vacuo. The remaining aldehyde (4.0 g, 100%) was dissolved in
tert-butyl alcohol (70 mL) and 2-methyl-2-butene (20 mL). A
solution of NaClO2 (1.03 g, 11.4 mmol) and NaH2PO4 (1.37 g,
11.4 mmol) in water (20 mL) was added within 15 min. The
reaction mixture was stirred for 18 h at room temperature,
N4-(2,2-Dim eth yl-1(S)-m eth ylca r ba m oylp r op yl)-N1-h y-
d r oxy-2(R)-h yd r oxym et h yl-3(R)-(isobu t yl)su ccin a m id e
1
(2). H-NMR (DMSO): 0.74 (d, 3H, J ) 8.1 Hz), 0.79 (d, 3H,
J ) 8.1 Hz), 0.92 (s, 9H), 1.22-1.32 (m, 1H), 1.35-1.45 (m,
1H), 2.21-2.31 (m, 1H), 2.54 (d, 3H, J ) 4.3 Hz), 2.56-2.63
(m, 1H), 3.22-3.32 (m, 1H), 3.48-3.55 (m, 1H), 4.15 (d, 1H, J
) 10.2 Hz), 4.50 (t, 1H, J ) 4.3 Hz), 7.72-7.78 (m, 2H), 8.71
(s, 1H), 10.35 (s, 1H). MS (FAB, Thio) m/e: 352 [(M + Li)]+.
Anal. (C16H33N3‚H2O) C, H, N.
N4-(2,2-Dim eth yl-1(S)-m eth ylca r ba m oylp r op yl)-N1-h y-
d r oxy-2(R)-h yd r oxym eth yl-3(S)-p h en ylsu ccin a m id e (3).
1H-NMR (DMSO): 0.93 (s, 9H), 2.46 (d, 3H, J ) 4.3 Hz), 2.92-
3.01 (m, 1H), 3.43-3.51 (m, 1H), 3.52-3.60 (m, 1H), 3.93 (d,
1H, J ) 10.2 Hz), 4.14 (d, 1H, J ) 10.2 Hz), 4.68 (t, 1H, J )
4.3 Hz), 7.14-7.22 (m, 3H), 7.35-7.38 (m, 2H), 7.76 (q, 1H, J
) 4.3 Hz), 8.09 (d, 1H, J ) 9.4 Hz), 8.49 (s, 1H), 10.22 (s, 1H).
MS (FAB,Thio) m/e: 366 [(M + H)]+. Anal. (C19H29N3‚2.5H2O)
C, H, N.
N4-(2,2-Dim eth yl-1(S)-m eth ylca r ba m oylp r op yl)-N1-h y-
d r oxy-2(R)-h yd r oxym eth yl-3(S)-p-tolylsu ccin a m id e (4).
1H-NMR (DMSO): 0.90 (s, 9H), 2.23 (s, 3H), 2.43 (d, 3H, J )

â-Aryl-Succinic Acid Hydroxamates
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