Phenolic modification as an approach to improve the pharmacology of the 3-acyloxy-2-benzylpropyl homovanillic amides and thioureas, a promising class of vanilloid receptor agonists and analgesics

Article abstract of DOI:10.1016/S0968-0896(01)00387-X

In order to improve the analgesic activity and pharmacokinetics of thioureas 2 and 3, which we previously developed as potent vanilloid receptor (VR) agonists, we prepared and characterized phenolic modifications of them and of their amide surro-gates (7, 8). The aminoethyl analogue of the amide template 13 was a potent analgesic with an EC₅₀ =0.96 μg/kg in the AA-induced writhing test and with better in vivo stability than the parent phenol. Copyright

Full text of DOI:10.1016/S0968-0896(01)00387-X

Bioorganic & Medicinal Chemistry 10 (2002) 1171–1179
Phenolic Modification as an Approach to Improve the
Pharmacology of the 3-Acyloxy-2-benzylpropyl Homovanillic
Amides and Thioureas, a Promising Class of Vanilloid Receptor
Agonists and Analgesics
Jeewoo Lee,a,* Jiyoun Lee,a Myung-Sim Kang,a Kang-Pil Kim,b Suk-Jae Chung,b
Peter M. Blumberg,c Jung-Bum Yid and Young Ho Parkd
aLaboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku,
Seoul 151-742, South Korea
bLaboratory of Pharmaceutics, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku,
Seoul 151-742, South Korea
cLaboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences, National Cancer Institute,
NIH, Bethesda, MD 20892, USA
dPacific R&D Center, 314-1, Bora-Ri, Kiheung-Eup, Yougin-Si, Kyounggi-Do 449-900, South Korea
Received 13 August 2001; accepted 27 October 2001
Abstract—In order to improve the analgesic activity and pharmacokinetics of thioureas 2 and 3, which we previously developed as
potent vanilloid receptor (VR) agonists, we prepared and characterized phenolic modifications of them and of their amide surro-
gates (7, 8). The aminoethyl analogue of the amide template 13 was a potent analgesic with an EC50=0.96 mg/kg in the AA-induced
writhing test and with better in vivo stability than the parent phenol. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
tification of RTX as a novel vanilloid with a potency
The vanilloid receptor (VR) is a nociceptor that
responds to multiple pain-producing stimuli such as
heat and protons and is activated by ligands such as
capsaicin (CAP),1,2 resiniferatoxin (RTX),3 and natural
aldehydes4 such as scutigeral. The vanilloid receptor has
been shown to be expressed on a subset of primary
afferent neurons of the C-fiber ‘pain’ pathway. The
activation of this receptor triggers cation influx and fir-
ing of action potentials, leading centrally to the percep-
tion of pain.5 Desensitization of the receptor in response
to specific ligands has long been considered to be a
promising therapeutic approach for the treatment of
neuropathic pain and other pathological conditions
involving C-fiber neurons.6,7
three to four orders of magnitude greater than that of
capsaicin suggested exciting opportunities for exploita-
tion through medicinal chemistry.9ꢀ14 Structurally,
RTX represents a typical vanilloid in the A and B
regions (correspond to 4-hydroxy-3-methoxyphenyl and
amide bond in CAP), but with a tricyclic diterpene in
place of the typical C region substituents (hydrophobic
side chain) found in capsaicin and its congeners.2 Over
the past few years, we have explored simplified RTX
analogues as VR ligands based on our hypothetical
pharmacophore model and have disclosed the first lead
compound 1 (Fig. 1).15 Using this approach, we recently
reported ultrapotent VR agonists, thioureas 2 and 3,
with Ki values of 19 and 11 nM, respectively, in an
[3H]RTX binding assay on dorsal root ganglion neu-
rons. These two compounds are thus approximately
280- and 480-fold more potent than capsaicin for
receptor binding.16 The particular feature of these com-
pounds is a 3-acyloxy-2-benzylpropyl moiety as a C-
region which we designed to mimic key putative phar-
macophores of the diterpene moiety in RTX.
Among the VR agonists studied, only a few compounds
are currently marketed (e.g., capsaicin) or undergoing
clinical trial (e.g., resiniferatoxin, DA-5018).8 The iden-
*Corresponding author. E-mail: jeewoo@snu.ac.kr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00387-X

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J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
Figure 1.
One of the pharmacological problems for capsaicin
analogues as analgesics is that the pharmacophoric
3-methoxy-4-hydroxyphenyl A-region is metabolically
labile, mostly as the result of O-glucuronidation of the
phenolic hydroxyl, leading to low bioavailability.
Incorporation of an aminoethyl group into capsaicin
analogues, for example DA-5018 and SDZ-249,482, was
found to reduce their pungencies and improve their
pharmacokinetic profiles by blocking metabolic clear-
ance.17 In the present study, we have sought to combine
aminoethyl or acidic groups with our potent VR ago-
nists in order to improve their pharmacokinetic profiles
and analgesic activities.
stituted by azide to give 11 and 12, respectively. Reduc-
tion of the azides under catalytic hydrogenation finally
produced the 2-aminoethyl analogues 13 and 14.
Since thiourea groups are labile to base-mediated alkyl-
ation conditions, we synthesized aminoethyl analogues
(20, 21) of the thiourea templates (2, 3) by another route
shown in Scheme 2. Commercially available 4-hydroxy-
3-methoxybenzylamine hydrochloride (15) was readily
converted into 18 by following the previous scheme.
N-tert-Butoxycarbonylamine 18 was transformed into
the corresponding isothiocyanate 19 by acidic deprotec-
tion followed by condensation with 1,1-thiocarbonyl di-
2(1H)-pyridone.18 Coupling of 19 with amines prepared
from 4 and 5, followed by PPh3-mediated reduction
produced 2-aminoethyl analogues of the thiourea tem-
plates (20, 21), respectively.
In this paper, we describe the syntheses, in vitro receptor
activities, antinociceptive activities and in vivo stability of
O-aminoethyl, acetate and succinate analogues of 3-acyl-
oxy-2-benzylpropyl homovanillic thioureas and amides.
The syntheses of a-substituted acetic acid analogues (29,
30) of the thiourea template as outlined in Scheme 3
were started from commercially available 4-hydroxy-3-
methoxybenzonitrile (22). Nitrile 22 was converted into
the corresponding carbobenzyloxyamino (Cbz) group to
afford 25 in the conventional way in three steps. The
phenol of 25 was alkylated with methyl bromoacetate to
produce 26 which was then hydrolyzed and depro-
tected to amine 28. Condensation of 28 with 2-(3,4-
dimethyl(or 4-tert-butyl)benzyl)-3-pivaloyloxypropyl iso-
thiocyanate, previously reported,19 afforded the final
compounds 29 and 30, respectively.
Chemistry
The syntheses of the amide templates (7, 8) and their
2-aminoethy analogues (13, 14) are described in Scheme
1. Azides (4, 5), previously reported,16 were reduced to
amines by hydrogenation under Lindler’s catalyst,
which in situ reacted with the pentafluorophenyl ester of
homovanillate (6) to afford amides 7 and 8, respectively.
Phenolic hydroxyls of 7 and 8 were alkylated with 1,2-
dibromoethane and then their bromides were sub-
Scheme 1. Reagents: (a) (i) H2, Lindler’s cat, EtOH; (ii) 6, EtOAc; (b) BrCH2CH2Br, KOH, Bu4NOH; (c) NaN3, DMF; (d) H2, Pd/C, MeOH.

J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
1173
Scheme 2. Reagents: (a) Boc2O, NEt3, dioxane–H2O; (b) BrCH2CH2Br, KOH, Bu4NOH; (c) NaN3, DMF; (d) CF3CO2H , CHCl2; (e) 1,1-thio-
2
carbonyl di-2(1H)-pyridone, NEt3, DMF; (f) (i) 4 (or 5), H2, 5% Pd/C, MeOH; (ii) CH2Cl2; (g) PPh3, H2O–THF.
Scheme 3. Reagents: (a) ClCH2OCH3, NEt(i-Pr)2, CH2Cl2; (b) (i) LiAlH4, THF; (ii) BnOCOCl, NEt3, CH2Cl2; (c) CF3CO2H , CHCl2; (d)
2
BrCH2CO2CH3, K2CO3, acetone; (e) NaOH, THF–H2O; (f) H2, Pd/C MeOH; (g) 2-(3,4-dimethyl(or 4-t-butyl)benzyl)-3-pivaloyloxy-
propylisothiocyanate, NEt3, DMF.
Scheme 4. Reagents: (a) BnOH, NaH, THF; (b) 25, DCC, DMAP, CH2Cl2; (c) H2, Pd/C, MeOH; (d) 2-(3,4-dimethyl(or 4-t-butyl)benzyl)-3-piva-
loyloxypropylisothiocyanate, DMF.
The synthesis of succinate analogues (35, 36) was shown
in Scheme 4. Refluxing of succinic anhydride (31) with
sodium salt of benzyl alcohol gave monobenzyl succin-
ate 32 which was then esterified with phenol 25 to pro-
duce 33. Hydrogenation of 33 followed by condensation
with isothiocyanates used in Scheme 3 afforded 35 and
36, respectively.
Maximal response was compared to that induced by
capsaicin at a concentration of 3 mM. The analgesic
activities of the compounds were evaluated in the
phenyl-p-benzoquinone (PBQ)-induced or acetic acid
(AA)-induced writhing test in mice; the ED50 values
indicate the concentrations required to cause a 50%
reduction in the number of writhes.16 Both the PBQ-
and AA-induced writhing tests usually showed similar
values, as indicated for ovanil and DA-5018, which were
used as reference compounds. The in vitro and in vivo
results are presented in Table 1.
Results and Discussion
The activities of the synthesized vanilloid analogues as
agonists were assessed in vitro using a 45Ca influx assay
and their antinociceptive activities were measured using
the writhing test. The calcium influx assay was carried
out using cultured rat dorsal root ganglion (DRG)
neurons as previously described and the potencies of the
compounds were expressed as the EC50 ꢁ SEM.17
In the calcium influx assay, the parent phenolic ana-
logues (thioureas: 2, 3; amides: 7, 8) demonstrated
potent agonistic activities with EC50 values of 0.058,
0.361, 0.162, 0.182 mM, which were 7.5, 1.2, 2.7 and 2.4-
fold (2, 3, 7 and 8) more potent than capsaicin (EC50
=0.435 mM), respectively. Their analgesic activities in

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J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
the PBQ-induced writhing test improved much more
than in the calcium influx assay. Amides 7 and 8 with
ED50 values of 12 and 3.5 mg/kg were 25- and 86-fold
more potent than capsaicin (ED50=300 mg/kg) in this
assay for analgesia, as previously reported in a series of
thiourea analogues (2, 3).16
In order to examine the in vivo stability of the two
potent analgesics 2 and 13, the compounds were injected
intravenously into the rat and then measured in plasma
collected at regular intervals (Fig. 2). Whereas the phenolic
compound 2 showed a very short half life with t1/2=3 min
and was completely cleared in ca. 10 min, the
aminoethyl analogue 13 showed enhanced stability with
t1/2=30 min, indicating that the aminoethyl surrogate
was more metabolically stable than the parent phenol.
The higher analgesic potency of 13 compared to the
parent compound presumably reflects its better phar-
macokinetics.
Incorporation of the 2-aminoethyl group into the phe-
nols led to a series of compounds (13, 14, 20 and 21) in
which agonistic and analgesic activities were compar-
able or a little reduced compared to the parent com-
pounds, with the exception of 13. Compound 13
possessed potent analgesic activity (ED50=0.96 mg/kg)
in the AA-induced writhing assay, being ca. 18- and 2.4-
fold more potent than olvanil and DA-5018. Although
the in vitro activity of 13 was less than that of the parent
compound 7, presumably its enhanced in vivo stability
more than compensated. In a series of capsaicin ana-
logues, it has been noted previously that only 2-ami-
noethyl analogues among blocking groups of the phenolic
OHgroup retained the antinociceptive properties of the
parent compounds, showed reduced irritancy, and, by
avoiding rapid phenolic glucuronidation in vivo, dis-
played better pharmacokinetic profiles.17 This conclu-
sion appears to hold likewise for the present series.
Unlike the aminoethyl analogues, both the a-substituted
acetic acid (29, 30) and succinate analogues (35, 36)
possessed dramatically reduced agonistic and analgesic
activities even though previous report described these
analogues of synthetic capsanoid showed a little better
antinociceptive effect than parent one.20 These findings
indicate that a hydrogen bonding acceptor such as car-
boxylic acid is not an appropriate substituent, unlike the
amino group as a hydrogen bonding donor in the phe-
nolic modification for improving VR mediated calcium
influx and analgesic activity, and are consistent with
those of capsainoid analogues previously reported17 in
Table 1. Calcium influx assay and writhing test
45Ca Influx (EC50=mM)
PBQ-induced writhing test (ED50=mg/kg)
AA-induced writhing test (ED50=mg/kg)
Amides
7
8
13
14
Thioureas
2
3
20
21
29
30
35
36
Capsaicin
RTX
Olvanil
DA-5018
Indomethacinn
Aspirin
Morphine
0.162 (ꢁ0.33)
0.182 (ꢁ0.45)
1.57 (ꢁ0.23)
0.878 (ꢁ0.17)
12
3.5
0.960
8.12
0.058 (ꢁ0.008)
0.361 (ꢁ0.054)
0.083 (ꢁ0.012)
0.377 (ꢁ0.067)
7.77 (ꢁ0.80)
12.3 (ꢁ1.58)
7.04 (ꢁ1.3)
7.00 (ꢁ1.2)
0.435
0.516
1.016
14.0
48.2
431
365
53.1
60
300
0.01
30
3
400
17.4
2.27
0.234
3500
1000
Figure 2. In vivo stability of compounds 2 and 13.

J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
1175
which substitution of basic nitrogen acting as a hydro-
gen bonding acceptor for free amine reduced its in vitro
and in vivo activities.
(M+). Anal. calcd for C28H39NO5: C, 71.61; H, 8.37; N,
2.98. Found: C, 71.84; H, 8.39; N, 2.97.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-
bromoethoxy)-3-methoxyphenyl]acetamide (9) and N-[2-
(4-tert-butylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-bro-
moethoxy)-3-methoxyphenyl]acetamide (10). A mixture
of 6 (or 7) (1 mmol), 40% KOH(2 mL), tetra-
butylammonium hydroxide (40% in H2O, 0.2 mL) and
1,2-dibromoethane (1.5 mL) was heated at 50 ꢃC for 2 h.
The reaction mixture was diluted with CH2Cl2, washed
with H2O and brine, dried over MgSO4 and con-
centrated in vacuo. The residue was purified by flash
column chromatography over silica gel with EtOAc/
hexanes (1:1) as eluant to afford 9 (or 10).
In conclusion, we investigated amide surrogate and
phenolic modifications of our previously discovered
potent vanilloid receptor agonists 2 and 3 and found the
aminoethyl analogue of amide template 13 to be a
potent analgesic due to improved pharmacokinetics.
Compound 13 was selected as a candidate for further
development of novel analgesics.
Experimental
All chemical reagents were commercially available.
Melting points were determined on a Melting Point
Buchi B-540 apparatus and are uncorrected. Silica gel
column chromatography was performed on silica gel 60,
230–400 mesh, Merck. Proton NMR spectra were
recorded on a JEOL JNM-LA 300 at 300 MHz. Chem-
ical shifts are reported in ppm units with Me4Si as a
reference standard. Mass spectra were recorded on a
VG Trio-2 GC–MS. Elemental analyses were performed
with an EA 1110 Automatic Elemental Analyzer, CE
Instruments.
1
9: 78% yield, yellow oil; HNMR (CDCl ) d 6.7–7.05
3
(m, 6H, Ar), 5.76 (t, 1H, NH), 4.32 (t, 2H, J=6.6 Hz,
OCH2CH2Br), 4.00 (m, 1H, CH2OCO), 3.86 (s, 3H,
OCH3), 3.80 (m, 1H, CH2OCO), 3.64 (t, 2H,
J=6.6 Hz, OCH2CH2Br), 3.47 (s, 2H, COCH2Ar), 3.30
(m, 1H, CH2NH), 3.10 (m, 1H, CH2NH), 2.60 (dd, 1H,
J=7.3, 12.0 Hz, CH2Ph), 2.49 (d, 1H, J=7.3 Hz,
CH2Ph), 2.0–2.3 (m, 7H, 2 ꢂ CH3 and CH), 1.20 (s, 9H,
CO(CH3)3).
1
10: 88% yield, yellow oil; HNMR (CDCl ) d 7.28 (d,
3
2H, J=8.3 Hz, Ar), 7.02 (d, 2H, J=8.3 Hz), 6.88 (d, 1H,
J=7.8 Hz), 6.80 (d, 1H, J=2.8 Hz), 6.74 (dd, 1H,
J=2.8, 7.8 Hz), 5.80 (t, 1H, NH), 4.32 (t, 2H, J=5.4 Hz,
OCH2CH2Br), 4.02 (dd, 1H, J=4.6, 11.2 Hz,
CH2OCO), 3.89 (s, 3H, OCH3), 3.83 (dd, 1H, J=5.2,
11.2 Hz, CH2OCO), 3.64 (t, 2H, J=5.4 Hz,
OCH2CH2Br), 3.49 (s, 2H, COCH2Ar), 3.32 (m, 1H,
CH2NH), 3.10 (m, 1H, CH2NH), 2.56 (d, 2H,
J=7.6 Hz, CH2Ph), 2.12 (m, 1H, CH), 1.30 (s, 9H,
C(CH3)3), 1.20 (s, 9H, CO(CH3)3).
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-
hydroxy-3-methoxyphenyl]acetamide (7) and N-[2-(4-tert-
butylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-hydroxy-3-meth-
oxyphenyl]acetamide (8). A suspension of 4 (or 5)
(2.0 mmol) and 5% palladium on carbon (100 mg) in
MeOH(10 mL) was hydrogenated under a balloon of
hydrogen for 2 h. The reaction mixture was filtered and
the filtrate was concentrated in vacuo. The residue was
dissolved in CH2Cl2 (10 mL) and treated with homo-
vanillic pentafluorophenyl ester (6) (696 mg, 2 mmol)
prepared from homovanillic acid and pentafluorophenol
by DCC coupling. After being stirred overnight at room
temperature, the reaction mixture was concentrated in
vacuo and the residue was purified by flash column
chromatography over silica gel with EtOAc/hexanes
(1:2) as eluant to afford 7 (or 8).
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-
azidoethoxy)-3-methoxyphenyl]acetamide (11) and N-[2-
(4-tert-butylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-azido-
ethoxy)-3-methoxyphenyl]acetamide (12). A mixture of
9 (or 10) (1 mmol) and sodium azide (195 mg, 3 mmol)
in DMF (2 mL) was heated at 100 ꢃC for 8 h. The reac-
tion mixture was diluted with H2O and extracted with
EtOAc several times. The combined organic layers were
washed with H2O and brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash
column chromatography over silica gel with EtOAc/
hexanes (1:1) as eluant to afford 11 (or 12).
1
7: 96% yield, colorless oil; HNMR (CDCl ) d 6.65–
3
7.05 (m, 6H, Ar), 5.74 (t, 1H, NH), 4.00 (m, 1H,
CH2OCO), 3.88 (s, 3H, OCH3), 3.80 (m, 1H,
CH2OCO), 3.46 (s, 2H, COCH2Ar), 3.30 (m, 1H,
CH2NH), 3.08 (m, 1H, CH2NH), 2.59 (dd, 1H, J=7.3,
12 Hz, CH2Ph), 2.48 (d, 1H, J=7.3 Hz, CH2Ph), 2.0–2.3
(m, 7H, 2 ꢂ CH3 and CH), 1.20 (s, 9H, CO(CH3)3); MS
m/e 441 (M+). Anal. calcd for C26H35NO5: C, 70.72; H,
7.99; N, 3.17. Found: C, 70.99; H, 8.01; N, 3.15.
1
11: 95% yield, colorless oil; HNMR (CDCl ) d 6.7–
3
7.05 (m, 6H, Ar), 5.76 (t, 1H, NH), 4.17 (t, 2H,
J=5.3 Hz, OCH2CH2N3), 4.00 (m, 1H, CH2OCO), 3.86
(s, 3H, OCH3), 3.80 (m, 1H, CH2OCO), 3.62 (t, 2H,
J=5.3 Hz, OCH2CH2N3), 3.48 (s, 2H, COCH2Ar), 3.30
(m, 1H, CH2NH), 3.10 (m, 1H, CH2NH), 2.60 (dd, 1H,
J=7.3, 12.0 Hz, CH2Ph), 2.49 (d, 1H, J=7.3 Hz,
CH2Ph), 2.0–2.3 (m, 7H, 2 ꢂ CH3 and CH), 1.20 (s, 9H,
CO(CH3)3).
1
8: 94% yield, colorless oil; HNMR (CDCl ) d 7.28 (d,
3
2H, J=8.3 Hz, Ar), 7.02 (d,2H, J=8.3 Hz), 6.88 (d, 1H,
J=7.8 Hz), 6.77 (d, 1H, J=2.8 Hz), 6.72 (dd, 1H,
J=2.8, 7.8H z), 5.73 (t, 1H , NH ), 4.00 (dd, 1H , C2HOCO),
3.89 (s, 3H, OCH3), 3.82 (dd, 1H, CH2OCO), 3.46 (s, 2H,
COCH2Ar), 3.32 (m, 1H, CH2NH), 3.08 (m, 1H, CH2NH),
2.53 (d, 2H, J=7.8 Hz, CH2Ph), 2.10 (m, 1H, CH), 1.30
(s, 9H, C(CH3)3), 1.20 (s, 9H, CO(CH3)3); MS m/e 469
12: 92% yield, colorless oil; 1HNMR (CDCl 3) d 7.28 (d,
2H, J=8.3 Hz, Ar), 7.03 (d, 2H, J=8.3 Hz), 6.88 (d, 1H,

1176
J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
J=7.8 Hz), 6.80 (d, 1H, J=2.8 Hz), 6.74 (dd, 1H,
J=2.8, 7.8 Hz), 5.76 (t, 1H, NH), 4.17 (t, 2H, J=5.2 Hz,
OCH2CH2N3), 4.01 (dd, 1H, J=4.5, 11.2 Hz,
CH2OCO), 3.86 (s, 3H, OCH3), 3.80 (dd, 1H, J=5.0,
11.2 Hz, CH2OCO), 3.63 (t, 2H, J=5.2 Hz,
OCH2CH2N3), 3.48 (s, 2H, COCH2Ar), 3.32 (m, 1H,
CH2NH), 3.06 (m, 1H, CH2NH), 2.54 (d, 2H,
J=7.6 Hz, CH2Ph), 2.10 (m, 1H, CH), 1.30 (s, 9H,
C(CH3)3), 1.20 (s, 9H, CO(CH3)3).
tert-Butyl N-[4-(2-bromoethoxy)-3-methoxybenzyl]carba-
mate (17). The compound was obtained from 16 by fol-
lowing the method for 9 in 84% yield. 1HNMR
(CDCl3) d 6.75–6.90 (m, 3H, Ar), 4.79 (bs, 1H, NH),
4.31 (t, 2H, J=6.6 Hz, OCH2CH2Br), 4.24 (d, 2H,
J=5.8 Hz, CH2NHBoc), 3.86 (s, 3H, OCH3), 3.64 (t,
2H, J=5.8 Hz, OCH2CH2Br), 1.46 (s, 9H, C(CH3)3).
tert-Butyl N-[4-(2-azidoethoxy)-3-methoxybenzyl]carba-
mate (18). The compound was obtained from 17 by fol-
lowing the method for 11 in 98% yield. 1HNMR
(CDCl3) d 6.75–6.90 (m, 3H, Ar), 4.79 (bs, 1H, NH),
4.24 (d, 2H, J=5.8 Hz, CH2NHBoc), 4.17 (t, 2 H,
J=4.9 Hz, OCH2CH2N3), 3.86 (s, 3H, OCH3), 3.62 (t,
2H, J=4.9 Hz, OCH2CH2N3), 1.46 (s, 9H, C(CH3)3).
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-
aminoethoxy)-3-methoxyphenyl]acetamide (13) and N-[2-
(4-tert-butylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-ami-
noethoxy) - 3 - methoxyphenyl]acetamide (14). A suspen-
sion of 11 (or 12) (1 mmol) and 5% palladium on
carbon (50 mg) in MeOH(5 mL) was hydrogenated
under a balloon of hydrogen for 1 h. The reaction mix-
ture was filtrated and the filtrate was concentrated in
vacuo. The residue was purified by flash column chro-
matography over silica gel with CH2Cl2/MeOH(10:1)
as eluant to afford 13 (or 14).
4-(2-Azidoethoxy)-3-methoxybenzylisothiocyanate (19).
A solution of 18 (322 mg, 1 mmol) in CH2Cl2 (3 mL)
was treated with trifluoroacetic acid (0.9 mL) stirred at
room temperature for 1 h. The reaction mixture was
concentrated in vacuo by the aid of toluene to afford the
corresponding amine salt as a brown solid.
13: 83% yield, white solid, mp 38 ꢃC; 1HNMR (CDCl 3)
d 6.7–7.05 (m, 6H, Ar), 5.78 (t, 1H, NH), 4.07 (t, 2H,
J=4.9 Hz, OCH2CH2NH2), 4.00 (m, 1H, CH2OCO),
3.85 (s, 3H, OCH3), 3.80 (m, 1H, CH2OCO), 3.47 (s,
2H, COCH2Ar), 3.30 (m, 1H, CH2NH), 3.15 (t, 2H,
J=4.9 Hz, OCH2CH2NH2), 3.10 (m, 1H, CH2NH), 2.60
(dd, 1H, J=7.5, 12 Hz, CH2Ph), 2.50 (d, 1H, J=7.5 Hz,
CH2Ph), 2.15–2.3 (m, 6H, 2 ꢂ CH3), 2.08 (m, 1H, CH),
1.21 (s, 9H, CO(CH3)3); MS m/e 484 (M+). Anal. calcd
for C28H40N2O5: C, 69.39; H, 8.32; N, 5.78. Found: C,
69.61; H, 8.36; N, 5.76.
The amine salt was dissolved in DMF (3 mL), treated
with triethylamine (110 mg, 1.1 mmol) and stirred for
1 h. The mixture was treated with 1,1-thiocarbonyl di-
2(1H)-pyridone (244 mg, 1.1 mmol) and stirred for 3 h at
room temperature. The mixture was diluted with H2O
and extracted with diethyl ether several times. The
combined organic layers were washed with H2O and
brine, dried over MgSO4 and concentrated in vacuo.
The residue was purified by flash column chromato-
graphy over silica gel with EtOAc/hexanes (1:2) as
1
eluant to afford 19 as an oil (270 mg, 74%). HNMR
14: 89% yield, white solid, mp 36 ꢃC; 1HNMR (CDCl 3)
d 7.28 (d, 2H, J=8.3 Hz, Ar), 7.03 (d, 2H, J=8.3 Hz),
6.88 (d, 1H, J=7.8 Hz), 6.80 (d, 1H, J=2.8 Hz), 6.74
(dd, 1H, J=2.8, 7.8 Hz), 5.84 (t, 1H, NH), 4.07 (t, 2H,
J=4.9 Hz, OCH2CH2NH2), 4.02 (dd, 1H, J=4.5,
11.2 Hz, CH2OCO), 3.85 (s, 3H, OCH3), 3.82 (dd, 1H,
J=5.2, 11.2 Hz, CH2OCO), 3.47 (s, 2H, COCH2Ar),
3.32 (m, 1H, CH2NH), 3.15 (t, 2H, J=4.9 Hz,
OCH2CH2NH2), 3.08 (m, 1H, CH2NH), 2.53 (d, 2H,
J=7.6 Hz, CH2Ph), 2.12 (m, 1H, CH), 1.29 (s, 9H,
C(CH3)3), 1.20 (s, 9H, CO(CH3)3); MS m/e 512 (M+).
Anal. calcd for C30H44N2O5: C, 70.28; H, 8.65; N, 5.46.
Found: C, 70.50; H, 8.68; N, 5.45.
(CDCl3) d 6.8–6.95 (m, 3H, Ar), 4.65 (s, 2H, CH2NCS),
4.18 (t, 2H, J=5.1 Hz, OCH2CH2N3), 3.89 (s, 3H,
OCH3), 3.64 (t, 2H, J=5.1 Hz, OCH2CH2N3).
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N0-[4-
(2-aminoethoxy)-3-methoxybenzyl]thiourea (20) and N-[2-
(4-tert-butylbenzyl)-3-(pivaloyloxy)propyl]-N0-[4-(2-ami-
noethoxy)-3-methoxybenzyl]thiourea (21). A suspension
of 4 (or 5) (0.6 mmol) and 5% palladium on carbon
(25 mg) in MeOH(5 mL) was hydrogenated under a
balloon of hydrogen for 2 h. The reaction mixture was
filtered and the filtrate was concentrated in vacuo. The
residue was dissolved in CH2Cl2 (5 mL) and treated with
isothiocyanate 19 (132 mg, 0.5 mmol). After being stir-
red for 15 h at room temperature, the reaction mixture
was concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel with
EtOAc/hexanes (1:10) as eluant to afford thiourea. The
above compound (0.3 mmol) was dissolved in THF
(3 mL) was treated with triphenylphosphine (157 mg,
0.6 mmol) and H2O (11 mg, 0.6 mmol), and stirred for
24 h at room temperature. The reaction mixture was
concentrated in vacuo and the residue was purified by
flash column chromatography over silica gel with
CH2Cl2/MeOH(10:1) as eluant to afford 20 (or 21).
tert - Butyl N - (4 - hydroxy - 3 - methoxybenzyl)carbamate
(16). A solution of 4-hydroxy-3-methoxy benzylamine
hydrochloride (15, 500 mg, 2.64 mmol) and triethyl-
amine (54 mg, 5.28 mmol) in H2O (10 mL) was treated
dropwise with a solution of di-tert-butyl dicarbonate
(1.15 g, 5.28 mmol) in dioxane (2 mL) for 20 min. After
being stirred for 24 h at room temperature, the reaction
mixture was extracted with CH2Cl2 several times. The
combined organic layers were dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash
column chromatography over silica gel with EtOAc/
hexanes (1:1) as eluant to give 16 as an oil (663 mg,
99%). 1HNMR (CDCl 3) d 6.7–7.0 (m, 3H, Ar), 5.65 (s,
1H, OH), 4.79 (bs, 1H, NH), 4.23 (d, 2H, J=5.6 Hz,
CH2NHBoc) 3.87 (s, 3H, OCH3), 1.43 (s, 9H, C(CH3)3).
1
20: 48% yield, sticky semisolid; HNMR (CDCl ) d
3
6.75–7.05 (m, 6H, Ar), 6.47 (bs, 2H, NH), 4.43 (bs, 2H,
NHCH2Ar), 4.13 (m, 1H, CH2OCO), 3.99 (t, 2H,

J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
1177
J=5.4 Hz, OCH2CH2NH2), 3.82 (s, 3H, OCH3), 3.7–3.8
(m, 2H, CH2OCO and CHCH2NH), 3.31 (m, 1H,
CHCH2NH), 3.07 (t, 2H, J=5.4 Hz, OCH2CH2NH2),
2.45–2.7 (m, 2H, CH2Ph), 2.15–2.3 (m, 7H, 2 ꢂ
CH3 and CH), 1.22 (s, 9H, C(CH3)3); MS m/e 515
(M+). Anal. calcd for C28H41N3O4S: C, 65.21; H,
8.01; N, 8.15; S, 6.22. Found: C, 65.46; H, 8.04; N, 8.13;
S, 6.19.
ture. After cooling in ice bath, the reaction mixture was
neutralized with saturated NaHCO3 solution, diluted
with H2O and extracted with CH2Cl2 several times. The
combined organic layers were washed with H2O and
brine, dried over MgSO4 and concentrated in vacuo.
The residue was purified by flash column chromato-
graphy over silica gel with EtOAc/hexanes (2:1) as
1
eluant to give 25 (5.46 g, 95%). HNMR (CDCl ) d
3
7.3–7.35 (m, 5H, phenyl), 6.86 (d, 1H, J=7.8 Hz), 6.76
(m, 2H), 5.14 (s, 2H, OCH2Ph), 4.30 (d, 2H, J=5.8 Hz,
NHCH2Ar), 3.86 (s, 3H, OCH3).
1
21: 54% yield, sticky semisolid; HNMR (CDCl ) d
3
7.30 (d, 2H, J=8.3 Hz), 7.10 (d, 2H, J=8.3 Hz), 6.75–
6.9 (m, 3H, Ar), 6.40 (bs, 2H, NH), 4.43 (bs, 2H,
NHCH2Ar), 4.12 (m, 1H, CH2OCO), 3.98 (t, 2H,
J=5.4 Hz, OCH2CH2NH2), 3.82 (s, 3H, OCH3), 3.7–3.8
(m, 2H, CH2OCO and CHCH2NH), 3.30 (m, 1H,
CHCH2NH), 3.06 (t, 2H, J=5.4 Hz, OCH2CH2NH2),
2.45–2.7 (m, 2H, CH2Ph), 2.20 (m, 1H, CH), 1.28 (s,
9H, C(CH3)3), 1.22 (s, 9H, C(CH3)3); MS m/e 543
(M+). Anal. calcd for C30H45N3O4S: C, 66.26; H, 8.34;
N, 7.73; S, 5.90. Found: C, 66.50; H, 8.33; N, 7.70; S,
5.88.
2-{[4-(Benzyloxycarbonylamino)methyl-2-methoxy]phe-
noxy}acetic acid (27). A solution of 25 (287 mg,
1 mmol) in acetone (20 mL) was treated with K2CO3
(552 mg, 4 mmol) and methyl bromoacetate (0.14 mL,
1.5 mmol) and refluxed for 3 h. After cooling, the reac-
tion mixture was filtered and the filtrate was con-
centrated in vacuo. The residue was purified by flash
column chromatography over silica gel with EtOAc/
1
hexanes (3:2) as eluant to afford 26 (323 mg, 90%). H
NMR (CDCl3) d 7.25–7.35 (m, 5H, phenyl), 6.75–6.85
(m, 3H, Ar), 5.11 (s, 2H, OCH2Ph), 4.65 (s, 2H, OCH2-
CO2Me), 4.28 (d, 2H, J=5.8 Hz, NHCH2Ar), 3.81 (s,
3H, OCH3), 3.76 (s, 3H, CO2CH3).
Benzyl N-(4-hydroxy-3-methoxybenzyl)carbamate (25).
A solution of 4-hydroxy-3-methoxy-benzonitrile (22,
10 g, 67 mmol) and diisopropylethylamine (17.52 mL,
100.5 mmol) in CH2Cl2 (100 mL) was treated with
chloromethylmethylether (6.11 mL, 80.4 mmol) and
stirred for 3 h at room temperature. The mixture was
diluted with CH2Cl2, washed with H2O, dried over
MgSO4 and concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel
A solution of 26 (323 mg, 0.9 mmol) in THF (1 mL) was
treated with 15% NaOHsolution (1 mL) and stirred for
30 min at room temperature. The reaction mixture was
neutralized with acetic acid, diluted with H2O and
extracted with CH2Cl2 several times. The combined
organic layers were washed with H2O, dried over
MgSO4 and concentrated in vacuo to afford 27 (276 mg,
using EtOAc/hexanes (1:2) as eluant to afford 23 (11.9 g,
1
92%). HNMR (CDCl ) d 7.05–7.3 (m, 3H, Ar), 5.29
3
1
(s, 2H, ArOCH2), 3.91 (s, 3H, ArOCH3), 3.51 (s, 3H,
CH2OCH3).
89%). HNMR (DMSO- d6) d 7.72 (t, 1H, NH), 7.25–
7.4 (m, 5 H, phenyl), 6.65–6.85 (m, 3H, Ar), 5.02 (s, 2H,
OCH2Ph), 4.16 (s, 2H, OCH2CO2H), 4.09 (d, 2H,
J=6.1 Hz, NHCH2Ar), 3.70 (s, 3H, OCH3).
A solution of 23 (9.66 g, 50 mmol) in THF (50 mL) was
added dropwise to a stirred suspension of lithiumalu-
minum hydride (7.6 g, 200 mmol) in THF (150 mL).
After being refluxed for 4 h, the reaction mixture was
cooled in ice bath and quenched carefully with 5 N
NaOH. Precipitated aluminum salts were removed by
filtration and the filtrate was evaporated. The residue
was partitioned with ether and H2O. The ether layer was
washed with H2O and brine, dried over MgSO4 and
concentrated in vacuo to give amine. The amine was
dissolved in CH2Cl2 (50 mL) and treated with triethyl-
amine (14 mL, 100 mmol) and benzylchloro formate
(10.7 mL, 75 mmol). After being stirred for 2 h at room
temperature, the reaction mixture was diluted with
CH2Cl2, washed with H2O and brine, dried over MgSO4
and concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel with
EtOAc/hexanes (1:2) as eluant to give 24 (8.28 g, 50%).
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N0-[4-
carboxymethoxy-3-methoxybenzyl]thiourea (29) and N-
[2-(4-tert-butylbenzyl)-3-(pivaloyloxy)propyl]-N0-[4-car-
boxymethoxy-3-methoxybenzyl]thiourea (30). A suspen-
sion of 27 (276 mg, 0.8 mmol) and 5% palladium on
carbon (30 mg) in MeOH(5 mL) was hydrogenated
under a balloon of hydrogen for 1 h. The reaction
mixture was filtrated and the filtrate was con-
centrated to afford amine 28. The amine was dissolved
in DMF (1 mL) was treated with triethylamine
(0.14 mL, 1.0 mmol) and stirred for 30 min. The solu-
tion was treated with a solution of 2-(3,4-dimethyl (or
4-tert-butyl)benzyl)-3-pivaloyloxypropyl isothiocyanate
(0.8 mmol).19 After stirring for 24 h at room tempera-
ture, the reaction mixture was diluted with H2O and
extracted with EtOAc several times. The combined
organic layers were washed H2O and brine, dried over
MgSO4 and concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel
with CH2Cl2/MeOH/AcOH (100:10:0.5) as eluant to
afford 29 (or 30).
1HNMR (CDCl ) d 7.3–7.35 (m, 5H, phenyl), 7.09 (d,
3
1H, J=8.1 Hz), 6.80 (m, 2H), 5.21 (s, 2H, ArOCH2),
5.14 (s, 2H, OCH2Ph), 4.32 (d, 2H, J=6.1 Hz,
NHCH2Ar), 3.85 (s, 3H, ArOCH3), 3.51 (s, 3H,
CH2OCH3).
A cooled solution of 24 (6.63 g, 20 mmol) in CH2Cl2
(20 mL) in ice bath was treated with trifluoroacetic acid
(2 mL) slowly and stirred for 10 min at room tempera-
29: 37% yield, white solid, mp 50 ꢃC; 1HNMR (CDCl 3)
d 6.75–7.05 (m, 6H, Ar), 6.54 (bs, 2H, NH), 4.51 (bs,
2H, NHCH2Ar), 4.46 (s, 2H, OCH2CO2H), 4.17 (m,

1178
J. Lee et al. / Bioorg. Med. Chem. 10 (2002) 1171–1179
1H, CH2OCO), 3.7–3.85 (m, 5H, OCH3, CH2OCO and
CH2NHCS), 3.22 (m, 1H, CH2NHCS), 2.5–2.7 (m, 2H,
CH2Ar), 2.2–2.4 (m, 7H, 2 ꢂ CH3 and CH), 1.23 (d, 9H,
C(CH3)3); MS m/e 530 (M+). Anal. calcd for
C28H38N2O6S: C, 63.37; H, 7.22; N, 5.28; S, 6.04.
Found: C, 63.61; H, 7.24; N, 5.25; S, 6.02.
(m, 7H, 2 ꢂ CH3 and CH), 1.23 (d, 9H, C(CH3)3); MS m/
e 441 (M+); MS m/e 572 (M+). Anal. calcd for
C30H40N2O7S: C, 62.91; H, 7.04; N, 4.89; S, 5.60.
Found: C, 63.16; H, 7.06; N, 4.87; S, 5.58.
36: 21% yield, white solid, mp 46 ꢃC; 1HNMR (CDCl 3)
d 7.30 (d, 2H, J=8.3 Hz), 7.06 (d, 2H, J=8.3 Hz), 6.7–
6.9 (m, 3H, Ar), 6.26 (bs, 2H, NH), 4.36 (d, 2H,
J=4.1 Hz, NHCH2Ar), 4.11 (dd, 1H, J=4.4, 11.4 Hz,
CH2OCO), 3.6–3.9 (m, 5H, OCH3, CH2OCO and
CHCH2NH), 3.27 (m, 1H, CHCH2NHCS), 2.4–2.5 (m,
4H, CH2Ar and COCH2CH2CO), 2.35 (t, 2H,
J=6.6 Hz, COCH2CH2CO), 2.31 (m, 1H, CH), 1.29 (s,
9H, C(CH3)3), 1.21 (s, 9H, C(CH3)3); MS m/e 600
(M+). Anal. calcd for C32H44N2O7S: C, 63.98; H, 7.38;
N, 4.66; S, 5.34. Found: C, 64.22; H, 7.41; N, 4.65; S,
5.32.
30: 52% yield, white solid, mp 48 ꢃC; 1HNMR (CDCl 3)
d 7.28 (d, 2H, J=8.3 Hz), 7.09 (d, 2H, J=8.3 Hz), 6.65–
6.9 (m, 3H, Ar), 6.54 (bs, 2H, NH), 4.58 (bs, 2H,
NHCH2Ar), 4.46 (s, 2H, OCH2CO2H), 4.11 (dd, 1H,
J=4.2, 11.5 Hz, CH2OCO), 3.6–3.9 (m, 5H, OCH3,
CH2OCO
and
CHCH2NH),
3.26
(m,
1H,
CHCH2NHCS), 2.64 (dd, 1H, J=6.3, 13.5 Hz, CH2Ar),
2.53 (dd, 1H, J=8.3, 13.5 Hz, CH2Ar), 2.31 (m, 1H,
CH), 1.29 (s, 9H, C(CH3)3), 1.21 (s, 9H, C(CH3)3); MS
m/e 558 (M+). Anal. calcd for C30H42N2O6S: C, 64.49;
H, 7.58; N, 5.01; S, 5.74. Found: C, 64.73; H, 7.61; N,
5.00; S, 5.73.
Characterization of pharmacokinetics for 2 and 13
4-{4-[(Benzyloxycarbonylamino)methyl]-2-methoxyphe-
noxy} - 4 - oxobutanoic acid (33). A solution of benzyl
alcohol (0.52 mL, 5 mmol) in THF (20 mL) was treated
with NaH(60%, 0.2 g, 5 mmol) and succinic anhydride
(31, 0.502 g, 5 mmol). After being refluxed for 4 h, the
reaction mixture was diluted with H2O and extracted
with EtOAc several times. The combined organic layers
were washed with H2O and brine, dried over MgSO4
and concentrated in vacuo. The residue was purified by
flash column chromatography over silica gel with
EtOAc/hexanes (1:1) as eluant to give 32 (0.866 g, 57%).
Pharmacokinetics of 2 and 13 were studied in rats. Male
Sprague–Dawley rats (body weight 250–300 g) were
anesthetized by ether, and the femoral artery and vein
were catheterized using polyethylene tubing (PE-50).
After a recovery period of approximately 2 h, 2 and 13
were intravenously injected (dose 0.5 mg/kg) via the
venous catheter. Arterial blood samples were collected
various times up to 1 h and analyzed for 2 and 13. Two
hundred mL of acetonitrile was added to 100 mL of
plasma for deproteination. The resulting mixture was
centrifuged, an aliquot (270 mL) of the supernatant col-
lected and evaporated under vacuum. The residue was
reconstituted by LC mobile phase (see below) and an
aliquot (20 mL) was injected on to an LC–MS system
(Finnigan LCQ Deca). The mobile phase was consisted
of acetonitrile and water (6:4 in volume), and the flow
rate was 0.5 mL/min. Selected ions monitored for 2 and
13 were 495.2 and 485.2, respectively. The peaks of the
drug were adequately separated from other peaks (i.e.,
retention times, 2: 7.5 min, 13: 5.3 min). The detection
limit for this assay was 10 ng/mL for 2 and 1 ng/mL for
13.
1HNMR (CDCl ) d 7.25–7.40 (m, 5H, phenyl), 5.15 (s,
3
2H, PhCH2), 2.70 (m, 4H, COCH2CH2CO).
A solution of 32 (0.866 g, 2.85 mmol) in CH2Cl2 (10 mL)
was treated with 25 (2.85 mmol), dicyclohexyl-
carbodiimide (4.2 mmol) and 4-dimethylaminopyridine
(0.42 mmol) successively and stirred for 24 h at room
temperature. The reaction mixture was diluted with
diethyl ether, precipitated solid was filtered and the fil-
trate was concentrated in vacuo. The residue was pur-
ified by flash column chromatography over silica gel
with EtOAc/hexanes (2:1) as eluant to give 33 as a white
solid (1.15 g, 85%); mp 64 ꢃC; 1HNMR (CDCl 3) d
7.25–7.40 (m, 10H, 2 ꢂ phenyl), 6.75–6.95 (m, 3H, Ar),
5.15 (s, 2H, CH2Ph), 5.14 (s, 2H, CH2Ph), 4.34 (d, 2H,
J=5.8 Hz, CH2NH), 3.75 (s, 3H, ArOCH3), 2.93 (t, 2H,
J=6.8 Hz, COCH2CH2CO), 2.79 (t, 2H, J=6.8 Hz,
COCH2CH2CO).
Acknowledgements
This research was supported by a Fund 2000 grant from
the Korea Research Foundation and BK21 project for
Medicine, Dentistry and Pharmacy.
N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-N0-{4-
[(3 - carboxypropanoyl)oxy] - 3 - methoxybenzyl}thiourea
(35) and N-[2-(4-tert-butylbenzyl)-3-(pivaloyloxy)propyl]-
N0 - {4 - [(3 - carboxypropanoyl)oxy] - 3 - methoxybenzyl}-
thiourea (36). The compounds were obtained from 33
by following the method for 29 (or 30) via 34.
References and Notes
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d 6.75–7.05 (m, 6H, Ar), 6.54 (bs, 2H, NH), 4.51 (bs,
2H, NHCH2Ar), 4.17 (m, 1H, CH2OCO), 3.7–3.85 (m,
5H, OCH3, CH2OCO and CH2NHCS), 3.22 (m, 1H,
CH2NHCS), 2.4–2.5 (m, 4H, CH2Ar and COCH2
CH2CO), 2.34 (t, 2H, J=6.8 Hz, COCH2CH2CO), 2.2–2.4
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