Design, synthesis, and biological evaluation of semicarbazide-sensitive amine oxidase (SSAO) inhibitors with anti-inflammatory activity

Article abstract of DOI:10.1021/jm050538l

In an attempt to examine the effect of inhibition of semicarbazide- sensitive amine oxidase (SSAO; EC 1.4.3.6, also known as VAP-1) as a novel anti-inflammatory target, the structure/mechanism based design and synthesis of a series of novel hydrazino-cont

Full text of DOI:10.1021/jm050538l

2166
J. Med. Chem. 2006, 49, 2166-2173
Design, Synthesis, and Biological Evaluation of Semicarbazide-Sensitive Amine Oxidase (SSAO)
Inhibitors with Anti-inflammatory Activity
Eric Y. Wang,* Hongfeng Gao, Luisa Salter-Cid, Jun Zhang, Li Huang, Erika M. Podar, Andrew Miller, Jingjing Zhao,
Anne O’Rourke, and Matthew D. Linnik
La Jolla Pharmaceutical Company, 6455 Nancy Ridge DriVe, San Diego, California 92121
ReceiVed June 7, 2005
In an attempt to examine the effect of inhibition of semicarbazide-sensitive amine oxidase (SSAO; EC
1.4.3.6, also known as VAP-1) as a novel anti-inflammatory target, the structure/mechanism based design
and synthesis of a series of novel hydrazino-containing small molecules are described. The in vitro biological
results show that compounds 4a,c are highly potent SSAO inhibitors with notable selectivity toward SSAO
over monoamine oxidases A and B (MAO-A and MAO-B). SAR studies based on compound 4c were
performed, and the results are discussed. The most potent and selective compound, 4a (IC50 ) 2 nM), is an
orally active, competitive, and apparently irreversible inhibitor of SSAO that is effective at reducing disease
incidence and severity in an in vivo animal disease model of multiple sclerosis.
Introduction
further supporting a role for this enzyme in inflammation. The
fact that SSAO has both enzymatic and adhesion activities
combined with the strong correlation between its upregulation
in many inflammatory conditions makes it an interesting
therapeutic target for drug discovery.
Semicarbazide-sensitive amine oxidase (SSAO) is a common
name for a group of enzymes, containing copper and quinone
cofactor1 and sensitive to semicarbazide, that converts primary
amines into the corresponding aldehydes, while releasing
ammonia and hydrogen peroxide:2
Several series of compounds have been reported in the
literature as SSAO inhibitors (Chart 1). The majority of reported
SSAO inhibitors were either originally developed for other
therapeutic uses or are simple chemical reagents with highly
reactive structural element(s).13 Most of these compounds lack
the combination of high potency and selectivity for SSAO. For
example, phenelzine is equally potent on SSAO and MAO-A.14
MDL 72161, a fluoroallylamine derivative that was originally
designed as MAO-B inhibitor for the treatment of Parkinson’s
disease, exhibits high inhibitory potency on rat aorta SSAO (IC50
) 2.5 nM) with no apparent selectivity over MAOs.15 On the
other hand, MDL 72274, a chloroallylamine derivative, was
reported to be a potent and selective rat aorta SSAO (IC50 ) 8
nM) inhibitor.15
RCH2NH2 + H2O + O2 SSAO8 RCHO + H2O2 + NH3
SSAO is found in a great variety of species from prokaryotes
to eukaryotes. In mammals, two forms of the SSAO protein
have been identified: a tissue-bound form and a soluble plasma
form.3 The tissue-bound SSAO contains a short intracellular
domain, a single transmembrane domain, and a long extracellular
domain that contains the active site.4 Plasma SSAO appears to
be the result of proteolytic cleavage of membrane-bound SSAO.
Cloning of the adhesion molecule vascular adhesion protein-1
(VAP-1) revealed that it is identical to membrane-bound SSAO.5
In humans, cell-associated SSAO is widely expressed in
endothelial, smooth muscle, and adipose cells.6 Cell-surface
expression of SSAO is tightly regulated and is significantly
upregulated at sites of inflammation.6a,7 SSAO is unique among
other endothelial cell-expressed adhesion molecules because of
its amine oxidase enzymatic activity. Studies have shown that
SSAO-mediated adhesion and transendothelial migration of
leukocytes through endothelial cell layers is mediated by its
enzymatic activity and can be blocked by small-molecule
inhibitors.8 It has been reported that increases in the levels of
plasma and/or membrane-associated SSAO occur in many
inflammation-associated diseases, including rheumatoid arthritis,
inflammatory bowel disease, type 1 and type 2 diabetes,
atherosclerosis, and chronic heart failure.9 The possible involve-
ment of SSAO in the inflammatory processes associated with
Alzheimer’s disease has also been reported.10
As part of a continuing effort to explore novel approaches
for treating acute and chronic inflammatory diseases, we became
interested in examining SSAO as a therapeutic target. We herein
report the structure/mechanism based inhibitor design and
synthesis of a series of hydrazino-containing small-molecule
inhibitors of SSAO. The synthesized compounds were evaluated
in an in vitro inhibition assay against SSAO in rat lung
homogenates. Structure-activity relationship (SAR) studies
based on compound 4c were conducted, and the results are
discussed. Enzyme kinetic and pharmacokinetic studies were
also performed for the most potent compound 4a. In addition,
compound 4a was further evaluated in an in vivo animal disease
model of human multiple sclerosis. In vivo SSAO inhibition
with compound 4a produced a reduction in disease incidence
and severity in a murine model of acute experimental autoim-
mune encephalomyelitis. These results suggest that SSAO
inhibition may represent a novel approach for the treatment of
inflammatory diseases.
Recent results with transgenic mice overexpressing human
SSAO support these disease associations.11,12 SSAO-deficient
mice are symptom-free in the resting state but display impaired
leukocyte adhesion to endothelial cells of inflamed vessels,12
Chemistry
The general synthetic routes for the preparation of compounds
4, 7, 9, and 11 are depicted in Scheme 1.
* To whom correspondence should be addressed. Phone: (858) 646-
6616. Fax: (858) 626-2845. E-mail: eric.wang@ljpc.com.
10.1021/jm050538l CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/14/2006

SSAO Inhibitors with Anti-inflammatory ActiVity
Chart 1. Some Known SSAO Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2167
while having dramatic impact on selectivity. The aqueous
solubility tests show that compounds 4a,c are highly soluble at
pH 7.4 while compounds 4b,d are less soluble. The vinylic
fluorine substitution, compound 15, also provides potent SSAO
inhibition with less specificity toward SSAO.
SAR studies on additional alkyl groups attached to either of
the hydrazine nitrogen atoms were performed on the basis of
compound 4c. The in vitro results, as shown in Table 1, reveal
that the introduction of an additional alkyl group attached to
either of the hydrazine nitrogen atoms results in progressively
lower potency compared with the parent compound 4c. The
potency of compounds decreases as the size of alkyl group
attached to either of the hydrazine nitrogen atoms increases.
The compounds lose potency completely when a bulky alkyl
group (i.e. benzyl group) is attached to either hydrazine nitrogen
atom. Moreover, the terminal nitrogen atom is more sensitive
to the reduction of potency when an additional alkyl group is
attached, which can be attributed to the increased size of the
molecule that makes it unable to bind to TPQ.
Compounds 2 were prepared from commercially available
compounds 1 according to the literature procedures.16 The
alkylations of compounds 2 with tert-butyl carbazate or di-tert-
butylhydrazodiformate were conducted in the presence of Et3N
or NaH, respectively, which provided compounds 3 and 5.
Compounds 4 were obtained as hydrochloride salts in good
yields upon removing the Boc group from compounds 3 under
acidic conditions. Further SAR studies were performed on the
basis of compound 4c. Thus, compounds 6 were prepared by
alkylation of compound 5c with a variety of alkyl halides in
the presence of NaH. Compounds 7 were then obtained as
hydrochloride salts by removing the Boc protecting groups from
compounds 6 under acidic conditions. Compounds 8 were made
by alkylation of compound 3c with a variety of alkyl halides in
the presence of diisopropylethylamine. Upon stirring compounds
8 under acidic conditions, compounds 9 were obtained as
hydrochloride salts. Compound 11 was obtained from the
deprotection of compound 10 that was prepared by dimethyla-
tion of compound 3b.
Experiments were performed to determine the kinetic char-
acteristics of the inhibition of SSAO produced by compound
4a. Analysis of kinetic data reveals that the kinetics is consistent
with a first-order inhibition process. The apparent Km increased,
while Vmax stays unchanged as the concentration of compound
4a increases.21 These data demonstrate that compound 4a is a
competitive inhibitor of SSAO and suggest that compound 4a
occupies the substrate-binding pocket of SSAO.
Dialysis experiments were performed using enzyme plus
inhibitor to determine whether 4a is a reversible or irreversible
SSAO inhibitor. The results revealed that enzyme inhibition
induced by 4a is minimally modified by 24 h dialysis at 4 °C,
indicating that compound 4a acts as an apparent irreversible
inhibitor of SSAO.21 Compound 4a was further assessed as a
time-dependent inhibitor of SSAO by using the method de-
scribed by Kitz and Wilson.22 Increasing concentration of 4a
or the duration of preincubation time with 4a resulted in greater
enzyme inhibition (Figure 1). These results indicated that
compound 4a is a time-dependent inhibitor of SSAO.
In an attempt to examine the effect of vinylic fluorine
substitution on SSAO potency, we synthesized compound 15
as shown in Scheme 2. Thus, compound 14 was obtained
through a coupling reaction of compound 12 and compound
13, which were prepared according to literature procedures.17
Upon two consecutive deprotections, compound 15 was obtained
as the hydrochloride salt.
The in vivo pharmacokinetic (PK) profile of compound 4a
was established in rats after per os (po) administration of 5.0
mg/kg. As shown in Figure 2, a single oral dose of compound
4a was administered to female Sprague Dawley rats.23 The PK
parameters of compound 4a are summarized in Table 2. The
results indicate that compound 4a is rapidly absorbed after oral
administration as indicated by short Tmax with a mean Cmax value
of 366 ng/mL. Compound 4a has oral bioavailability and a
relatively long elimination half-life in rat plasma, presumably
due to high plasma protein binding, as has been shown in
ADMET studies of compound 4a.21
Results and Discussion
The inhibitory properties of all desired final compounds
toward SSAO were initially evaluated in vitro with a radio-
chemical assay18 using benzylamine-14C as substrate. A crude
rat lung homogenate was used as the source of SSAO for the
routine potency measurements.19 The inhibitory properties of
compounds that showed potent SSAO inhibition (IC50 < 100
nM) were further evaluated against recombinant human MAO-A
and MAO-B in vitro with a coupled colorimetric assay.20 The
ratios of IC50 values for SSAO inhibition and MAO inhibition
were taken as an approximate index of selectivity for the tested
compounds. Table 1 summarizes the in vitro biological results
for the synthesized compounds. The approximate IC50 values,
presented in Table 1, were calculated using GraphPad Prism
software from the inhibition curves obtained with an inhibitor
concentration between 10-3 and 10-10 M.
Inhibition of SSAO with compound 4a has shown in vivo
efficacy in several animal disease models.24 To further examine
the effect of SSAO inhibition on a chronic neurological
inflammatory disease, we tested compound 4a in an animal
model of human multiple sclerosis. Multiple sclerosis is a
chronic immune-mediated disease of the CNS characterized by
patchy perivenular inflammatory infiltrates in areas of demy-
elination and axonal loss. Experimental autoimmune encephalitis
(EAE) in rodents is a well-characterized and reproducible animal
model of human multiple sclerosis.25 In general, EAE animal
models can be induced in mice by immunization with encepha-
litogenic myelin antigens in the presence of adjuvant. The
pathogenesis of EAE comprises presentation of myelin antigens
to T cells in the periphery, migration of activated T cells to the
The in vitro data show that compounds 4a-d are highly
potent and selective SSAO inhibitors. Comparing these four
compounds on the basis of the substitution on the phenyl ring,
the order of potency is H > 4-Cl > 2-Me > 4-F, while the
order of selectivity is H > 4-F > 2-Me > 4-Cl. The result
indicates that the ring substitution has limited effect on potency

2168 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Wang et al.
Scheme 1a
a Conditions. (a) Method A: NBS, CCl4, reflux (for the preparation of 2a-c), Method B: Yb(OTf)3-TMSCl, NBS, CH2Cl2-THF (4:1), rt (for the
preparation of 2d). (b) H2NNHBoc, NEt3, MeOH, reflux. (c) BocNHNHBoc, NaH, DMF, rt. (d) RX (X ) I or Br), DIEA, DMF, rt. (e) MeI, NaH, DMF,
rt. (f) RX (X ) I or Br), NaH, DMF, rt. (g) HCl, Et2O, MeOH, rt.
Scheme 2a
a Conditions. (a) PPh3, DEAD, THF, rt. (b) (i) NH2NHMe, THF, rt; (ii) MeOH, 4.0 M HCl in 1,4-dioxane, rt.
Table 1. Inhibitory Potencies at SSAO, MAO-A, and MAO-B
CNS, and development of inflammation and demyelination upon
recognition of the same antigens in the CNS.
compd
SSAO IC50 a (nM)
MAO-A IC50 (nM) MAO-B IC50 (nM)
4a
4b
4c
4d
15
7a
7b
7c
7d
9a
9b
9c
9d
11
2.0 ( 0.21
3.0 ( 0.15
6.1 ( 0.51
4.0 ( 0.26
5.0 ( 0.32
1.7 ( 0.12
2.3 ( 0.25
>1000
1300 ( 180
1000 ( 160
6000 ( 250
2000 ( 150
2800 ( 400
NDb
71000 ( 5200
As shown in Figure 3, the vehicle-treated group showed an
80% disease incidence and moderate clinical severity during
the study period. In contrast, compound 4a-treated mice
exhibited a statistically significant reduction of disease severity
relative to vehicle, with 50% of mice affected. Statistically
significant differences in disease severity between the 4a and
vehicle-treated groups continued after stopping compound 4a
administration and were observed until the end of the study (day
30). In addition, there is no statistically significant difference
in disease severity between 4a and the positive control group
(p ) 0.18).
24500 ( 2300
81000 ( 3600
90000 ( 8900
650 ( 100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>1000
240 ( 40
630 ( 55
420 ( 30
>1000
2700 ( 360
Compound 4a treatment also prevented body weight loss in
the mice during the dosing period relative to vehicle-treated
animals (p ) 0.04). As expected, the loss of body weight
correlated with the clinical severity in vehicle-treated mice. In
addition, the inhibitory effect of compound 4a on the acute EAE
development persisted for 1 week after the last treatment (day
25).
a The values of IC50 (µM) are the means obtained from five concentra-
tions of each inhibitor studied at a substrate (benzylamine-14C) concentration
of 20 mM, while MAO-A and MAO-B activities were blocked with
clorgyline and pargyline at 1.0 µM, respectively. b ND ) not determined.
It is well-recognized that certain hydrazine-containing com-
pounds are capable of inducing organ damage, including liver

SSAO Inhibitors with Anti-inflammatory ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2169
Table 2. Plasma Pharmacokinetic Profile of 4aa
Dose (mg/kg), po 5
max (min) 30 ( 0
max (ng/mL)
366 ( 116 AUCINF (min × ng/mL) 19 8447 ( 40 977
T
1/2 (min)
677 ( 99
T
C
AUCall (min × ng/mL) 80 415 ( 17 815
a Mean ( SD (n ) 3). Pharmacokinetic parameters were derived from
noncompartmental model using WinNonlin 4.0.1.
Figure 3. (A) Compound 4a reduces clinical severity of acute EAE
in mice. (B) Compound 4a reduces the disease incidence of acute EAE
in mice. Groups of 8-10 mice received either 4a at 20 (mg/kg)/dose
or vehicle control, daily for 25 consecutive days while methotrexate
(MTX) in saline (3 mg/kg, 3× per week) was used as positive control.
Dosing was initiated 1 day after the immunization, and all doses were
administered ip. Animals were monitored for disease incidence and
clinical severity based on signs of neurological impairment, paralysis,
and death according to a prospectively defined scoring system based
on a 0-5 scale, where 0 indicates normal locomotion and motor
function and 5 indicates death.
Figure 1. (A) Preincubation time-dependent inhibition of rat lung
SSAO by 10 nM (9), 25 nM (O), 50 nM (0), and 100 nM ([) 4a.
Each point is the mean of duplicate determinations expressed as a
percentage of corresponding control samples preincubated without
inhibitor. (B) Kitz and Wilson replot of the data.
SSAO in adhesion, acute, and chronic inflammation has been
demonstrated by a variety of interventions, including antibodies,
enzyme inhibitors, gene knockout, and topaquinone mu-
tant.12,13,24,26 The inhibitory antibodies and genetic mutants were
not reported to be hepatotoxic, and the acute antiinflammatory
effects are observed within the first few hours of administration.
These well-controlled studies provide strong support for the
hypothesis that SSAO inhibition reduces leukocyte trafficking
at sites of inflammation, with a resultant reduction in local
inflammation. They are also consistent with the hypothesis that
the benefit observed with 4a in the acute EAE model is in part
due to SSAO inhibition.
Comparable results on the effects of percentage disease
incidence and clinical severity were also observed when MDL
72974a, a known potent SSAO inhibitor, was used in a separate
acute EAE study.21
Figure 2. Plasma concentration profile of 4a in cannulated rats, po
5.0 mg/kg.
damage. The possibility therefore exists that part or all of the
benefit attributed to compound 4a in the acute EAE model could
be due to corticosteroid induction secondary to liver toxicity,
rather than the effect of SSAO inhibition. Corticosteroids were
not measured in this experiment, which can be seen as a
limitation of the experiment. However, there is good reason to
believe that the effect of 4a in the acute EAE model is in part
due to SSAO inhibition. The beneficial effect of inhibiting of
Conclusion
A series of hydrazino-containing compounds have been
synthesized and evaluated as SSAO inhibitors. Compounds
4a,4c are highly potent and selective SSAO inhibitors. The SAR
studies based on 4c show that potency decreases when an
additional alkyl group is attached to either hydrazine nitrogen
atom. The compounds lose potency completely when an

2170 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Wang et al.
temperature under N2 for 7 h when TLC showed the reaction was
completed. The reaction mixture was concentrated in vacuo. The
residue was purified by flash column chromatography (silica gel,
0-5% EtOAc/hexane) to afford the desired product.
additional larger alkyl group (i.e., benzyl group) is attached to
either hydrazine nitrogen atom. Terminal hydrazine nitrogen
atom is more sensitive to the reduction of potency when an
additional alkyl group is attached. Substitution on the phenyl
ring did not change compound potency significantly but had a
profound effect on the selectivity of the compounds. In addition,
the vinylic fluorine substitution, compound 15, has less influence
on potency but has significant effect on selectivity. The most
potent and selective compound, 4a, is an orally active, competi-
tive, and apparently irreversible inhibitor of SSAO. Inhibition
of SSAO with compound 4a in vivo produced a reduction in
disease incidence and severity in a murine model of human
multiple sclerosis. These results suggest that inhibition of SSAO
may be an effective approach for inhibiting inflammation.
The biological results obtained with compound 4a in an in
vivo model of multiple sclerosis illustrate the potential utility
of inhibiting SSAO activity in a chronic inflammatory disease.24
While the hydrazine moiety may limit the clinical utility of 4a
for interventions requiring chronic administration, the SAR
information gained from this series may be relevant to the design
of small-molecule SSAO inhibitors in related series.
(a) N,N′-Di(tert-butoxycarbonyl)-N′-[2-(4-fluorophenyl)allyl]-
hydrazine (5c). The title compound was obtained as a white solid
1
(7.1 g, 89%). mp: 56-57.5 °C. H NMR (CDCl3, 300 MHz): δ
1.43 (s, 9H), 4.52 (br s, 2H), 5.14 (s, 1H), 5.42 (s, 1H), 6.95-7.02
(m, 2H), 7.29-7.51 (m, 2H). ESMS: m/z 389.1 (M + Na)+.
(b) N,N′-Di(tert-butoxycarbonyl)-N′-[2-(2-methylphenyl)allyl]-
hydrazine (5d). The title compound was obtained as a white solid
(6.87 g, 76%). mp: 104-105 °C. 1H NMR (CDCl3, 300 MHz): δ
1.46 (s, 9H), 2.31 (s, 3H), 4.30 (br s, 2H), 5.04 (br s, 1H), 5.28 (d,
J ) 11.9 Hz, 1H), 6.98-7.19 (m, 4H). ESMS: m/z 385.1 (M +
Na)+.
General Procedure for the Preparation of Compounds 6. A
mixture of 5c (1.0 equiv) and NaH (1.6 equiv) in DMF (30 mL)
was stirred at room temperature for 20 min. To this stirred solution
were added alkyl halides (1.6 equiv). The resulting mixture was
stirred at room temperature under N2 overnight. The TLC showed
the reaction was completed. The solvent was removed in vacuo.
The residue was purified by flash column chromatography (silica
gel, 0-5% EtOAc/hexane) to provide the desired product.
(a) N,N′-Di(tert-butoxycarbonyl)-N′-[2-(4-fluorophenyl)allyl]-
N-methylhydrazine (6a). The title compound was obtained as a
Experimental Section
1
colorless oil (1.02 g, 98%). H NMR (CDCl3, 300 MHz): δ 1.40
Melting points were recorded on a Thomas-Hoover capillary
melting point apparatus and are uncorrected. Elemental analyses
were performed by NuMega Resonance Labs, San Diego, CA. 1H
NMR spectra were recorded on Bruker NMR spectrometers
operating at 300.1 and at 500 MHz. MS spectra (ESI) were
performed on a LCQ Finnigan mass spectrometer in positive mode.
High-resolution mass spectra (HRMS) were recorded on IonSpec
Ultima FTMS instrument at the Scripps Center for Mass Spec-
trometer in The Scripps Research Institute, La Jolla, CA. Analytical
chromatography was performed on Whatman PE Sil G/UV silica
gel plate (250 µM). Flash column chromatography was performed
using J. T. Baker silica gel (40 µM). All commercially obtained
reagents were used as received unless otherwise noted.
General Procedure for the Preparation of Compounds 3. A
mixture of H2NNHBoc (2.0 equiv) and NEt3 (2.0 equiv) in MeOH
(30 mL) was stirred at room temperature for 20 min. To this stirred
solution was added R-bromomethylstyrene16a (1.0 equiv). The
resulting mixture was kept at gentle reflux and monitored with thin-
layer chromatography (TLC). After refluxing for 3 h, the TLC
showed that the reaction was completed. The reaction mixture was
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, 10% EtOAc/hexane) to give the desired
product.
(s, 9H), 1.42, 1.45 (two s, total 9H), 2.75 (s, 3H), 3.95-4.03 (m,
2H), 5.20 (s, 1H), 5.49 (s, 1H), 6.95-7.08 (m, 2H), 7.40-7.55(m,
2H). ESMS: m/z 403.1 (M + Na)+.
(b) N,N′-Di(tert-butoxycarbonyl)-N′-[2-(4-fluorophenyl)allyl]-
N-ethylhydrazine (6b). The title compound was obtained as a
1
colorless oil (1.00 g, 93%). H NMR (CDCl3, 300 MHz): δ 0.92
(t, J ) 7.2 Hz, 3H), 1.40 (s, 9H), 1.42, 1.44 (two s, total 9H), 3.06
(q, J ) 7.2 Hz, 2H), 3.90-3.99 (m, 2H), 5.21 (S, 1H), 5.48 (s,
1H), 6.95-7.08 (m, 2H), 7.35-7.55 (m, 2H). ESMS: m/z 417.1
(M + Na)+.
(c) N,N′-Di(tert-butoxycarbonyl)-N′-[2-(4-fluorophenyl)allyl]-
N-isopropylhydrazine (6c). The title compound was obtained as
a colorless oil (1.00 g, 90%). 1H NMR (CDCl3, 300 MHz): δ 1.02
(d, J ) 6.9 Hz, 3H), 1.13 (d, J ) 6.9 Hz, 3H), 1.39 (s, 9H), 1.45,
1.47 (two s, total 9H), 3.39-3.63 (m, 1H), 3.78-3.93 (m, 1H),
5.22 (s, 1H), 5.49 (s, 1H), 6.94-7.06 (m, 2H), 7.36-7.54 (m, 2H).
ESMS: m/z 431.2 (M + Na)+.
(d) N,N′-Di(tert-butoxycarbonyl)-N′-[2-(4-fluorophenyl)allyl]-
N-benzylhydrazine (6d). The title compound was obtained as a
1
colorless oil (0.87 g, 70%). H NMR (CDCl3, 300 MHz): δ 1.38
(s, 9H), 1.43, 1.47 (two s, total 9H), 4.08 (d, J ) 15.0 Hz, 2H),
4.54 (d, J ) 14.7 Hz, 1H), 4.80 (d, J ) 14.7 Hz, 1H), 5.14 (s, 1H),
5.45 (s, 1H), 6.86-7.07 (m, 2H), 7.12-7.28 (m, 5H), 7.42-7.58
(m, 2H). ESMS: m/z 479.1 (M + Na)+.
(a) N-tert-Butoxycarbonyl-N′-(2-phenylallyl)hydrazine (3a).
The title compound was obtained as a white solid (1.34 g, 44%).
mp: 79-80 °C. 1H NMR (CDCl3, 300 MHz): δ 1.50 (s, 9H), 3.90
(s, 2H), 5.30 (s, 1H), 5.51 (s, 1H), 7.26-7.38 (m, 3H), 7.45-7.52
(m, 2H). ESMS: m/z 271.1 (M + Na)+.
General Procedure for the Preparation of Compounds 8. A
mixture of 3c (1.0 equiv) and N,N-diisopropylethylamine (2.0 equiv)
in DMF (10 mL) was stirred at room temperature for 20 min. To
this solution were added alkyl halides (2.0 equiv). The resulting
mixture was stirred at room temperature overnight. The TLC
showed the reaction was completed. The solvent was removed in
vacuo. The residue was purified by flash column chromatography
(silica gel, 5% EtOAc/hexane) to provide the product.
(a) N-tert-Butoxycarbonyl-N′-[2-(4-fluorophenyl)allyl]-N′-
methylhydrazine (8a). The title compound was obtained as a white
solid (0.62 g, 98%). mp: 95-96 °C. 1H NMR (CDCl3, 300 MHz):
δ 1.42 (s, 9H), 2.61 (s, 3H), 3.73 (br s, 2H), 5.21 (s, 1H), 5.42 (s,
1H), 6.95-7.08 (m, 2H), 7.46-7.59 (m, 2H). ESMS: m/z 303.1
(M + Na)+.
(b) N-tert-Butoxycarbonyl-N′-[2-(4-chlorophenyl)allyl]hydra-
zine (3b). The title compound was obtained as a white solid (2.7
g, 32%). mp: 94-96 °C. 1H NMR (CDCl3, 300 MHz): δ 1.45 (s,
9H), 3.85 (s, 2H), 5.27 (s, 1H), 5.45 (s, 1H), 7.29 (d, J ) 8.4 Hz,
2H), 7.42 (d, J ) 8.4 Hz, 2H). ESMS: m/z 305.1 (M + Na)+.
(c) N-tert-Butoxycarbonyl-N′-[2-(4-fluorophenyl)allyl]hydra-
zine (3c). The title compound was obtained as a white solid (1.9
1
g, 24%). mp: 83-84.5 °C. H NMR (CDCl3, 300 MHz): δ 1.48
(S, 9H), 3.87 (d, J ) 4.5 Hz, 2H), 5.26 (d, J ) 0.6 Hz, 1H), 5.43
(d, J ) 0.6 Hz, 1H), 7.02 (t, J ) 9 Hz, 2H), 7.47 (dd, J ) 5.4, 7.8
Hz, 2H). ESMS: m/z 289.1 (M+Na)+.
General Procedure for the Preparation of Compounds 5. A
mixture of di-tert-butylhydrazodiformate (1.0 equiv) and NaH (1.0
equiv) in dimethylformade (DMF; 40.0 mL) was stirred at room
temperature under N2 for 20 min. To this solution was added a
solution of substituted-R-bromomethylstyrene16 (0.75 equiv) in
DMF (5.0 mL). The resulting mixture was stirred at room
(b) N-tert-Butoxycarbonyl-N′-[2-(4-fluorophenyl)allyl]-N′-eth-
ylhydrazine (8b). The title compound was obtained as a white solid
1
(0.23 g, 42%). mp: 54-55 °C. H NMR (CDCl3, 300 MHz): δ
1.06 (t, J ) 4.3 Hz, 3H), 1.41 (s, 9H), 2.86 (br s, 2H), 3.81 (br s,
2H), 5.24 (br s, 1H), 5.43 (br s 1H), 7.01 (t, J ) 5.3 Hz, 2H), 7.55
(br s, 2H). ESMS: m/z 317.1 (M + Na)+.

SSAO Inhibitors with Anti-inflammatory ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2171
(c) N-tert-Butoxycarbonyl-N′-[2-(4-fluorophenyl)allyl]-N′-iso-
propylhydrazine (8c). The title compound was obtained as a white
semisolid (0.38 g, 66%). 1H NMR (CDCl3, 300 MHz): δ 1.05 (d,
J ) 3.3 Hz, 6H), 1.40 (s, 9H), 3.21 (br s, 1H), 3.73 (br s, 2H),
5.28 (br s, 1H), 5.42 (br s, 1H), 7.01 (t, J ) 5.2 Hz, 2H), 7.58 (br
s, 2H). ESMS: m/z 331.1 (M + Na)+.
J ) 7.2 Hz, 3H), 3.04 (q, J ) 6.9 Hz, 2H), 3.93 (s, 2H), 5.30 (s,
1H), 5.50 (s, 1H), 7.04 (t, J ) 9.0 Hz, 2H), 7.42 (t, J ) 9.0 Hz,
2H). ESMS: m/z 195 (M + H)+. Anal. (C11H16ClFN2) C, H, N.
(g) N′-[2-(4-Fluorophenyl)allyl]-N-isopropylhydrazine Hy-
drochloride (7c). The title compound was obtained as a white solid
1
(0.42 g, 82%). mp: 140-141 °C. H NMR (D2O, 300 MHz): δ
(d) N-tert-Butoxycarbonyl-N′-[2-(4-fluorophenyl)allyl]-N′-
benzylhydrazine (8d). The title compound was obtained as a white
1.12 (d, J ) 6.6 Hz, 6H), 3.25-3.44 (m, 1H), 3.92 (s, 2H), 5.29
(s, 1H), 5.50 (s, 1H), 7.04 (t, J ) 8.7 Hz, 2H), 7.40-7.47 (m, 2H).
ESMS: m/z 209 (M + H)+. Anal. (C12H18ClFN2) C, H, N.
(h) N′-[2-(4-Fluorophenyl)allyl]-N-benzylhydrazine Hydro-
chloride (7d). The title compound was obtained as a light yellow
solid (0.44 g, 90%). mp: 149-150 °C. 1H NMR (D2O, 300
MHz): δ 4.01 (s, 2H), 4.16 (s, 2H), 5.32 (s, 1H), 5.53 (s, 1H),
7.03 (t, J ) 8.7 Hz, 2H), 7.28-7.60 (m, 2H). ESMS: m/z 257 (M
+ H)+. Anal. (C16H18ClFN2) C, H, N.
1
semisolid (0.40 g, 60%). H NMR (CDCl3, 300 MHz): δ 1.38 (s,
9H), 3.96 (br s, 2H), 4.10 (br s, 2H), 5.28 (br s, 1H), 5.45 (br s,
2H), 7.00 (t, J ) 5.2 Hz, 2H), 7.19-7.22 (m, 5H), 7.48 (br s, 2H).
ESMS: m/z 379.1 (M + Na)+.
N,N′-Di(tert-butoxycarbonyl)-N′-[2-(4-chlorophenyl)allyl]-
N,N′-dimethylhydrazine (10). A mixture of 3b (0.42 g, 1.49 mmol)
and NaH (0.11 g, 4.58 mmol) in DMF (10 mL) was stirred at room
temperature for 20 min. To this stirred solution was added MeI
(0.64 g, 4.51 mmol). The resulting reaction mixture was stirring
under N2 at room temperature overnight. The TLC showed the
reaction was completed. The solvent was removed in vacuo. The
residue was purified by flash column chromatography (silica gel,
5% EtOAc/Hexane) to give a colorless oil (0.29 g, 63%). 1H NMR
(CDCl3, 300 MHz): δ 1.43 (s, 9H), 2.59 (s, 3H), 2.73 (s, 3H),
3.73 (br s, 2H), 5.22 (s, 1H), 5.43 (s, 1H), 7.33 (d, J ) 8.7 Hz,
2H), 7.41 (d, J ) 8.7 Hz, 2H). ESMS: m/z 333.1 (M + Na)+.
General Procedure for the Preparation of Compounds 4, 7,
9, and 11. To a solution of Boc protected hydrazine compounds
(1.0 equiv.) in anhydrous MeOH (3.0 mL) was added a solution of
HCl in ether (5.0 equiv.). The resulting mixture was stirred under
N2 at room temperature and monitored by TLC. When TLC showed
that the reaction was completed, it was concentrated in vacuo. The
residue was dissolved in H2O (20 mL). The resulting aqueous
solution was washed with ether (2 × 10 mL) and then was basified
to pH 10 by adding 1.0 N NaOH solution. The resulting basic
solution was saturated with solid NaCl and extracted with ether (3
× 30 mL). The combined organic layers were dried (MgSO4),
filtered and concentrated in vacuo. The residue was dissolved in
ether (5.0 mL). To this solution was added a solution of HCl in
ether (5 equiv.). The solid formed was collected by filtration,
washed with ether, and then dried in vacuo to give the final
compounds as hydrochloride salts.
(i) N′-[2-(4-Fluorophenyl)allyl]-N′-methylhydrazine Hydro-
chloride (9a). The title compound was obtained as a white solid
1
(0.40 g, 95%). mp: 138-140 °C. H NMR (D2O, 500 MHz): δ
2.72 (s, 3H), 4.05 (s, 2H), 5.43 (s, 1H), 5.62 (s, 1H), 7.06 (t, J )
8.7 Hz, 2H), 7.39-7.48 (m, 2H). ESMS: m/z 181 (M + H)+. Anal.
(C10H14ClFN2) C, H, N.
(j) N′-[2-(4-Fluorophenyl)allyl]-N′-ethylhydrazine Hydrochlo-
ride (9b). The title compound was obtained as a white solid (0.14
g, 90%). mp: 118-119 °C. 1H NMR (D2O, 500 MHz): δ 1.27 (t,
J ) 7.3 Hz, 3H), 3.24 (br s, 2H), 4.28 (br s, 2H), 5.61 (s, 1H),
5.78 (s, 1H), 7.18 (t, J ) 8.7 Hz, 2H), 7.54 (dd, J ) 5.3, 8.7 Hz,
2H). ESMS: m/ z 195 (M + H)+. Anal. (C11H16ClFN2) C, H, N.
(k) N′-[2-(4-Fluorophenyl)allyl]-N′-isopropylhydrazine Hy-
drochloride (9c). The title compound was obtained as a white solid
1
(0.22 g, 85%). mp: 146-147 °C. H NMR (D2O, 500 MHz): δ
1.31 (d, J ) 6.7 Hz, 3H), 3.56-3.71 (m, 1H), 4.10-4.30 (m, 2H),
5.63 (s, 1H), 5.78 (s, 1H), 7.19 (t, J ) 8.7 Hz, 2H), 7.54 (d, J )
5.3, 8.7 Hz, 2H). ESMS: m/z 209 (M + H)+. Anal. (C12H18ClFN2)
C, H, N.
(l) N′-[2-(4-Fluorophenyl)allyl]-N′-benzylhydrazine Hydro-
chloride (9d). The title compound was obtained as a white solid
1
(0.25 g, 90%). mp: 170-171 °C. H NMR (D2O, 500 MHz): δ
4.06 (s, 2H), 4.17 (s, 2H), 5.51 (s, 1H), 5.70 (s, 1H), 7.12 (t, J )
8.7 Hz, 2H), 7.28-7.37 (m, 2H), 7.38-7.50 (m, 5H). ESMS: m/z
257 (M + H)+. Anal. (C16H18ClFN2) C, H, N.
(a) N′-(2-Phenylallyl)hydrazine Hydrochloride (4a). The title
compound was obtained as a white solid (0.66 g, 90%). mp: 153-
154.5 °C. 1H NMR (D2O, 300 MHz): δ 4.05 (s, 2H), 5.40 (s, 1H),
5.60 (s, 1H), 7.25-7.42 (m, 5H). ESMS: m/z 149 (M + H)+. Anal.
(C9H13ClN2) C, H, N.
(b) N′-[2-(4-Chlorophenyl)allyl]hydrazine Hydrochloride (4b).
The title compound was obtained as a white solid (0.60 g, 100%).
mp: 124-126 °C. 1H NMR (D2O, 300 MHz): δ 4.06 (s, 2H), 5.43
(s, 1H), 5.65 (s, 1H), 7.35 (d, J ) 9.0 Hz, 2H), 7.40 (d, J ) 9.0
Hz, 2H). ESMS: m/z 183 (M + H)+. Anal. (C9H12Cl2N2) C, H, N.
(c) N′-[2-(4-Fluorophenyl)allyl]hydrazine Hydrochloride (4c).
The title compound was obtained as a off-white solid (0.55 g, 90%).
mp: 149-150 °C. 1H NMR (D2O, 300 MHz): δ 4.16 (s, 2H), 5.49
(s, 1H), 5.70 (s, 1H), 7.07 (t, J ) 8.7 Hz, 2H), 7.42 (dd, J ) 5.4,
8.7 Hz, 2H). ESMS: m/z 167 (M + H)+. Anal. (C9H12ClFN2) C,
H, N.
(m) N′-[2-(4-Chlorophenyl)allyl]-N,N′-dimethylhydrazine Hy-
drochloride (11). The title compound was obtained as a white solid
(0.17 g, 74%). mp: 108-110 °C. H NMR (D2O, 300 MHz): δ
2.58 (s, 3H), 2.69 (s, 3H), 3.93 (s, 2H), 5.42 (s, 1H), 5.60 (s, 1H).
HRMS (MALDI-FTMS). Calcd for C11H15ClN2H: 211.0995.
Found: 211.0995.
1
N′-[(E)-(2-Phenyl-3-fluoroallyl)]-N′-tert-butoxycarbonylami-
nophthalimide (14). To a cooled solution of (E)-2-phenyl-3-
fluoroallyl alcohol 12 17a (1.1 g, 7.47 mmol), PPh3 (2.94 g, 11.22
mmol), and N-tert-butoxycarbonylaminophthalimide 13 17b (1.95 g,
7.47 mmol) in tetrahydrofuran (THF; 120 mL) was added dropwise
a solution of diethyl azodicarboxylate (DEAD) (1.8 mL, 11.09
mmol) in THF (5.0 mL). The resulting mixture was stirred under
N2 at room temperature overnight and then concentrated in vacuo.
The residue was purified by flash column chromatography (silica
1
gel, 10% EtOAc/haxane) to give a colorless oil (1.3 g, 44%). H
(d) N′-[2-(2-Methylphenyl)allyl]hydrazine Hydrochloride (4d).
The title compound was obtained as a white powder (0.56 g, 95%).
mp: 107-108.5 °C. H NMR (D2O, 300 MHz): δ 2.30 (s, 3H),
NMR (CDCl3, 300 MHz): δ 1.30, 1.44 (two s, total 9H), 4.55,
4.63 (two s, 2H), 6.81 (two d, J ) 81.0 Hz, 1H), 7.23-7.45 (m,
5H), 7.71-7.95 (m, 4H). ESMS: m/z 419.1 (M + Na)+.
1
4.02 (s, 2H), 5.35 (s, 1H), 5.65 (s, 1H), 7.16-7.28 (m, 2H), 7.28-
7.34 (m, 2H). ESMS: m/z 163 (M + H)+. Anal. (C10H15ClN2) C,
H, N.
N′-[(E)-(2-Phenyl-3-fluoroallyl)]hydrazine Hydrochloride (15).
A mixture of 14 (1.3 g, 3.28 mmol), NH2NHMe (0.26 mL, 4.72
mmol) in THF (50 mL) was stirred under N2 at room temperature
for 24 h and then concentrated in vacuo. The residue was diluted
with EtOAc. The solid formed was filtered and washed with EtOAc.
The combined filtrate was concentrated in vacuo to give a semisolid
(0.98 g). It was used directly in the next step without any further
purification. It was dissolved in MeOH (5.0 mL). To this solution
was added HCl solution in 1,4-dioxane (4.0 M, 4.0 mL, 16 mmol).
The resulting mixture was stirred under N2 at room temperature
overnight and then concentrated in vacuo. The residue was diluted
with ether (30 mL) and filtered. The solid was washed with ether
(e) N′-[2-(4-Fluorophenyl)allyl]-N-methylhydrazine Hydro-
chloride (7a). The title compound was obtained as a white solid
1
(0.52 g, 90%). mp: 128-129 °C. H NMR (D2O, 300 MHz): δ
2.66 (s, 3H), 3.95 (s, 2H), 5.29 (s, 1H), 5.50 (s, 1H), 7.03 (t, J )
9.0 Hz, 2H), 7.42 (t, J ) 9.0 Hz, 2H). ESMS: m/z 181 (M + H)+.
Anal. (C10H14ClFN2) C, H, N.
(f) N′-[2-(4-Fluorophenyl)allyl]-N-ethylhydrazine Hydrochlo-
ride (7b). The title compound was obtained as a white solid (0.44
g, 90%). mp: 117-118 °C. 1H NMR (D2O, 300 MHz): δ 1.10 (t,

2172 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Wang et al.
(3 × 30 mL) and EtOAc (3 × 30 mL) and then dried in vacuo to
give a white solid (0.35 g, 53%). mp: 139-141 °C. 1H NMR (D2O,
300 MHz): δ 3.89 (s, 2H), 6.96 (d, J ) 81.0 Hz, 1H), 7.25-7.46
(m, 5H). ESMS: m/z 167 (M + H)+. Anal. (C9H12ClFN2) C, H,
N.
Acknowledgment. We thank Dr. Tenshang Joh, Dr. Shaw-
Ling Wang, and Mr. Dan Konkler for performing statistical
analyses on EAE studies.
Supporting Information Available: Results from elemental
analysis (pdf). This material is available free of charge via the
Internet at http://pubs.acs.org.
Preparation of Rat Lung Homogenates. Freshly excised rat
lungs were cut into small pieces, and then washed thoroughly with
PBS. The washed tissue was then homogenized at 1:20 (w/v) in
10 mM solution of potassium phosphate buffer (pH 7.8). The
homogenate was then centrifuged at 10000g for 10 min at 4 °C.
The supernatant was collected and kept frozen until ready to use.
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Radiochemical assays: SSAO activity was measured as described
by Fowler.18 Briefly, SSAO activity in 100 µL of lung homogenate
was determined radiochemically using 20 µM benzylamine-14C as
substrate and a determined amount of inhibitor. The reaction was
carried out at 37 °C in a final volume of 400 µL of 100 mM
potassium phosphate buffer (pH 7.2) and stopped with 100 µL of
2.0 M citric acid. Radioactively labeled products were extracted
into toluene/ethyl acetate (1:1, v/v) containing 0.6% (w/v) 2,5-
diphenyloxazole (PPO) before liquid scintillation counting. All
experiments were performed in the presence of clorgyline and
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and IC50 values were calculated from a dose-response curve using
GraphPad Prism software (version 3.03; San Diego, CA).
Enzyme Kinetic Study. Kinetic parameters were determined
using the radiochemical assay as described above. Briefly, rat lung
homogenates (25 µL) were incubated at 37 °C for 1 min with
various concentrations of benzylamine-14C (0.1-10 µM) in the
absence or presence of compound 4a (1-5 nM), then was kept in
ice, and quenched by adding 2 M citric acid solution (100 µL).
Following the workup procedures described above for the radio-
chemical assay, the Vmax for each reaction was calculated using
GraphPad Prism software.
Dialysis Experiments. To test whether reversible or irreversible
inhibition was produced by compound 4a, rat lung homogenates
were preincubated for 30 min with appropriate concentrations of
compound 4a to bring about complete or virtually complete
inhibition of SSAO activity. These samples were dialyzed at 4 °C
in an attempt to restore enzyme activity. Dialyzed samples including
uninhibited controls were assayed and compared with corresponding
nondialyzed samples otherwise treated identically.
Time-Dependent Inhibition Experiments. These experiments
were carried out by using the method described by Kitz and
Wilson.22 Briefly, mixtures containing rat lung homogenates (25
µL) and appropriate amount of 4a to give inhibitor concentration
from 10 to 100 nM were set up in ice-cold vessels and then
preincubated for various periods at 37 °C. After the appropriate
times, a 1:100 dilution was made for each mixture with ice-cold
0.2 M potassium phosphate buffer, pH 7.6. SSAO activity after
dilution was assayed by incubation of diluted sample (390 µL) with
400 nM benzylamine-14C (10 µL) at 37 °C for 30 min.
In Vivo Acute EAE Animal Study. To induce EAE on 7-8
week old C57BL/6 female mice, MOG35-55 in CFA containing 3.0
mg/mL Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI)
was injected at a dose of 300 µg over two sites at the back of the
mice. Pertussis toxin (500 ng) was given intraperitoneally (ip) on
days 0 and 2 postimmunization. Compound 4a (20 (mg/kg)/dose,
1 dose/day), Methotrexate (MTX) in saline (3 mg/kg, once a day
on M, W, F) and buffer were administered ip for 25 days starting
from day 1. Animals were monitored for body weight (M, W, F
from day 1), signs of paralysis, and death (M, W, and F from day
10). EAE clinical severity was scored according to a 0-5 scale
system.
Statistical Analysis. Statistical analysis was performed using a
repeated measure analysis to assess the treatment effect, and results
were considered to be significant if p < 0.05.

SSAO Inhibitors with Anti-inflammatory ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2173
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JM050538L