Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase

Article abstract of DOI:10.1021/jm8015798

Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to L-homocysteine, yielding dimethylglycine and L-methionine. In this study, we prepared a new series of BHMT inhibitors. The inhibitors were designed to mimic the hypothetical transition state of BHMT substrates and consisted of analogues with NH, N(CH₃), or N(CH ₃)₂ groups separated from the homocysteine sulfur atom by a methylene, ethylene, or a propylene spacer. Only the inhibitor with the N(CH₃) moiety and ethylene spacer gave moderate inhibition. This result led us to prepare two inhibitors lacking a nitrogen atom in the S-linked alkyl chain: (RS,RS)-5-(3-amino-3-carboxypropylthio)-3-methylpentanoic acid and (RS)-5-(3-amino-3-carboxypropylthio)-3,3-dimethylpentanoic acid. Both of these compounds were highly potent inhibitors of BHMT. The finding that BHMT does not tolerate a true betaine mimic within these inhibitors, especially the nitrogen atom, is surprising and evokes questions about putative conformational changes of BHMT upon the binding of the substrates/products and inhibitors.

Full text of DOI:10.1021/jm8015798

3652
J. Med. Chem. 2009, 52, 3652–3665
Structure-Activity Study of New Inhibitors of Human Betaine-Homocysteine
S-Methyltransferase
†
†
†
†
†,‡
Va´clav Vaneˇk, Milosˇ Budeˇsˇ´ınsky´, Petra Kabeleova´, Miloslav Sanda, Milan Kozˇ´ısˇek, Ivona Hancˇlova´,† Jana Mla´dkova´,†
ˇ
Jirˇ´ı Brynda,† Ivan Rosenberg,† Markos Koutmos,§ Timothy A. Garrow,| and Jirˇ´ı Jira´cˇek*,†
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, V.V.i., FlemingoVo na´m. 2, 166 10 Prague 6,
Czech Republic, Department of Biochemistry, Faculty of Science, Charles UniVersity, HlaVoVa 8, 128 00, Prague 2, Czech Republic, Life
Sciences Institute, UniVersity of Michigan, 210 Washtenaw AVenue, Ann Arbor, Michigan 48109, Department of Food Science and Human
Nutrition, UniVersity of Illinois, 905 South Goodwin AVenue, Urbana, Illinois 61801
ReceiVed December 15, 2008
Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine
to L-homocysteine, yielding dimethylglycine and L-methionine. In this study, we prepared a new series of
BHMT inhibitors. The inhibitors were designed to mimic the hypothetical transition state of BHMT substrates
and consisted of analogues with NH, N(CH3), or N(CH3)2 groups separated from the homocysteine sulfur
atom by a methylene, ethylene, or a propylene spacer. Only the inhibitor with the N(CH3) moiety and
ethylene spacer gave moderate inhibition. This result led us to prepare two inhibitors lacking a nitrogen
atom in the S-linked alkyl chain: (RS,RS)-5-(3-amino-3-carboxypropylthio)-3-methylpentanoic acid and (RS)-
5-(3-amino-3-carboxypropylthio)-3,3-dimethylpentanoic acid. Both of these compounds were highly potent
inhibitors of BHMT. The finding that BHMT does not tolerate a true betaine mimic within these inhibitors,
especially the nitrogen atom, is surprising and evokes questions about putative conformational changes of
BHMT upon the binding of the substrates/products and inhibitors.
Introduction
toward betaine of 12 nM)15,16 was cocrystallized with BHMT,
and the structure of the complex was determined by X-ray
crystallography.7 Inhibitor 1 was also used in a study of the
intrinsic fluorescence of BHMT.17 This study helped to provide
a better understanding of the catalytic mechanism of the enzyme
and confirmed the ordered Bi-Bi reaction. Recently, inhibitor
1 was used in mice to study the role of BHMT in sulfur
metabolism in vivo.18 This pioneering pharmacological study
confirmed the role of BHMT in the synthesis of methionine
from homocysteine in vivo.
Other identified and possible physiological functions of
BHMT require further characterization. Betaine has been shown
to be an osmolyte in rat liver cells.19 It is likely that BHMT
helps to maintain the cellular osmolytic equilibrium by main-
taining the correct betaine concentration.20 Potent, stable, and
selective BHMT inhibitors would be useful in studies of the
role of the enzyme in liver and kidney osmolytic balance.
BHMT activity was shown to be increased in type 2 diabetes.21
A role for BHMT in lipid and phospholipid metabolism is highly
probable, but it remains unclear and demands further investiga-
tion.22,23 It is clear that many questions about the physiological
role of BHMT in mammalian organisms exist and that potent,
selective, and stable inhibitors of this enzyme will have an
important role in future studies, especially if BHMT-deficient
mice remain unavailable.24
We are particularly interested in the structural requirements
for ligand binding to the active site of BHMT. The design and
synthesis of compounds that closely resemble the structure of
substrates upon binding to BHMT, especially the structure of
the hypothetical transition state (4), should yield highly potent
inhibitors.25–27 The structures of inhibitors 1-3, which we
developed previously,15 are considerably different from the
structure of the transition state (4). The main difference is in
the “betaine” part of their molecules. Therefore, we decided to
design and synthesize new compounds that would better mimic
the structure of the transition state (4), which should yield highly
Betaine-homocysteine S-methyltransferase1 (BHMT; EC 2.1.
1.5) catalyzes a methyl transfer from betaine to L-homocysteine
to form dimethylglycine and L-methionine, respectively. This
enzyme is abundant in mammals but only in the liver and
kidney.2 It has been predicted that BHMT is responsible for up
to half of the homocysteine remethylation capacity in liver.3
BHMT follows an ordered Bi-Bi reaction mechanism with
homocysteine being the first substrate to bind and methionine
being the last product off.4 Cloning of human BHMT cDNA,
overexpression of the enzyme in E. coli, as well as a purification
protocol for preparation of homogeneous enzyme have been
previously described.5 BHMT was shown to be a Zn-metal-
loenzyme. The metal ion is essential for its catalytic activity.
Site-directed mutagenesis studies have shown that the Zn2+ ion
is coordinated by three cysteine ligands.6 BHMT is a member
of a family (Pfam 02574) of zinc and thiol/selenol-dependent
methyltransferases.7
Our laboratory is particularly interested in the synthesis and
biological evaluation of inhibitors of BHMT. During the past
several years, we developed potent and selective inhibitors of
human recombinant BHMT: phosphinic pseudopetides8–14 and
S-alkylated derivatives of homocysteine.15 Among the series of
S-alkylated homocysteines, we identified compounds 1-3 as
highly potent inhibitors of BHMT. They were believed to mimic
the structure of the hypothetical transition state 4 of BHMT
substrates, betaine and L-homocysteine, upon binding to the
active site of the enzyme. The competitive inhibitor 1 (Kiapp
* To whom correspondence should be addressed. Phone: +420220183441.
Fax: +420220183571. E-mail: jiracek@uochb.cas.cz.
†
Institute of Organic Chemistry and Biochemistry, Academy of Sciences
of the Czech Republic.
‡
Department of Biochemistry, Faculty of Science, Charles University.
Life Sciences Institute, University of Michigan.
Department of Food Science and Human Nutrition, University of
§
|
Illinois.
10.1021/jm8015798 CCC: $40.75
2009 American Chemical Society
Published on Web 05/20/2009

Inhibitors of Human BHMT
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3653
potent inhibitors of BHMT that could be useful in future in
vivo pharmacological studies.
selenide 34. Deprotection of the latter compound yielded the
desired selenide 9.
Amino acid 10 was prepared according to the method of
Barraclough et al.30 from DEAM and ethyl 8-bromooctanoate,
followed by standard acidic decarboxylation and deprotection.
For the synthesis of the transition state analogues bearing
the N-CH2-S motif (Scheme 4), a protected homocysteine
derivative 36 was prepared in high yield following the synthetic
method described by Zhu et al.34 Alkylating agent 37 was
obtained by a phase-catalyzed reaction35 of 36 with bromo-
chloromethane in the presence of crushed solid KOH. We did
not succeed in the isolation of this compound due to its high
reactivity (e.g., rapid decomposition on a silica column);
however, the CH2Cl2/CH2BrCl solution of compound 37 proved
to be sufficiently stable for subsequent synthetic use. During
the preparation of 37, formation of the djenkolic acid36
homologue 38 was invariably observed. After standard acidic
deprotection, free amino acid 39 was obtained.
Results and Discussion
We prepared two series of BHMT inhibitors (Table 1).
Compounds 5-10 in series 1 were designed using the scaffold
of the previously published potent inhibitor 1 to investigate the
role of the number and position of sulfur atom(s) in the
inhibitors. Compounds 11-21 from series 2 were designed to
mimic the hypothetical transition state (4) of betaine and
homocysteine substrates upon binding to the active site of
BHMT.
Chemistry. Compounds 5 and 6 were synthesized directly
by recombination of free unprotected acids (Scheme 1). Acidic
ring-opening of γ-aminobutyrolactone in an aqueous solution
containing DL-homocysteine followed by oxidation with hydro-
gen peroxide afforded disulfide 5 (accompanied by disulfide 22
and DL-homocystine as byproduct). Similarly, compound 6
bearing the thioformyl moiety was obtained by the phase-transfer
catalyzed reaction28 of 3-mercaptopropionic acid and DL-ho-
mocysteine in dichloromethane, followed by HPLC resolution
of the resulting mixture.
The alkylation of dimethylglycine tert-butyl ester with
chloromethylsulfide 37 in the presence of one equivalent of DBU
(1,8-diazabicyclo[5.4.0]undec-7-ene) at room temperature re-
sulted in the formation of quaternary compound 40, which gave
the desired amino acid 11 after deprotection.
While alkylation of the tertiary amines proceeded smoothly
at room temperature, alkylation of primary and secondary
amines demanded prolonged heating of the reaction mixture.
Surprisingly, when 2 equiv of DBU were used in the reaction
of 37 with tert-butyl-2-(methylamino)acetate hydrochloride,
formation of an unknown compound was observed and 43 was
not detected in the reaction mixture. Analysis of the product
revealed N-alkylated DBU (41), which gave the corresponding
compound 42 after deprotection. In addition, whereas the
alkylation of the above amine in the presence of 1 equiv of
DBU gave the expected compound 43, an attempt to prepare
the respective secondary amine from ꢀ-alanine tert-butyl ester
(as well as from glycine and alanine tert-butyl esters) failed,
giving only bis-alkylated product 44 and, despite our best efforts,
no trace of the expected secondary amine. This observation
indicates a strong preference of 37 for secondary and tertiary
amines over primary amines. Moreover, the acidic deprotection
of 43 and 44 caused their complete decomposition, and no traces
of the desired free compounds were detected.
The series of compounds bearing the ethylene linker N-CH2-
CH2-S and propylene linker N-(CH2)3-S was prepared from
halogenides 45 and 53, respectively (Schemes 5 and 6). Unlike
37, both 45 and 53 are stable compounds, with much weaker
reactivity toward amines than 37; therefore, harsh reaction
conditions37,38 were required. The alkylation of dimethylglycine
tert-butyl ester to quaternary amines was not feasible. This
problem was successfully bypassed by the alkylation of 46 and
54 by methyl iodide. During alkylation of ꢀ-alanine tert-butyl
ester, a bis-alkylated compound 52 was isolated as a byproduct.
Finally, inhibitors 20 and 21, with branched alkyl side chains,
were synthesized starting from 3-methyl- and 3,3-dimethylglu-
taranhydride, respectively, which were converted in two steps39–41
into bromide esters 58 and 59, respectively. After coupling with
thiol 36, protected sulfides 60 and 61 were obtained in good
yields, which, after deprotection, afforded the target compounds
20 and 21 (Scheme 7).
The treatment of diethyl acetamidomalonate (DEAMa) with
NaH followed by addition of 1-bromo-3-chloropropane yielded
a product that is considered in the literature29,30 to be chloride
23a. However, our analysis revealed that the product was a
mixture of chloride 23a and bromide 23b at a ratio of 59:41
(as determined by NMR). The coupling of a mixture of the
compounds 23a and 23b with sodium 4-mercaptoethylbutanoate
produced the corresponding protected sulfide 24 in good yield.
The target compound 7 was obtained after treatment with 3 N
HCl under reflux.
Synthesis of intermediate 29 (including its precursors 27 and
28), which was proposed as a precursor for the synthesis of
compounds 8 and 9, is described in detail in Scheme S1,
Supporting Information.
Attempts to prepare the oxygen-containing analogue (8) of
sulfide 1 by standard Williamson ether synthesis employing
alcohol 28, bromide 29, or several similar precursors were
unsuccessful. Therefore, an alternative approach (Scheme 2) was
used starting from DEAM, which was alkylated with 2,2′-
dichloro-diethylether.31 The resulting chloride 30 was converted
into iodide 31 and reacted with methylacrylate in the presence
of the Zn(Cu) pair,32 which gave the desired structure 32 in
high yield. The final analogue 8 was obtained after acidic
deprotection.
The selenium analogue (9) of sulfide 1 was synthesized from
29 in three steps (Scheme 3). The reaction with in situ generated
disodium diselenide33 afforded diselenide 33, which was then
treated with NaBH4 and ethyl 5-bromovalerate to generate
Inhibition Experiments. First, we determined the percent
inhibition of BHMT using the test compounds at 20 µM. For
the most potent compounds, we then determined the percent
inhibition at 1 µM and their IC50 values. The percentages of
inhibition were measured at relatively low concentrations of
a Abbreviations: ACES, N-(2-acetamido)-2-aminoethanesulfonic acid;
DEAM, diethyl acetamidomalonate; HEPES, 4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid; ITC, isothermal titration calorimetry; TEBAC,
benzyl triethylammonium chloride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-
ene; TFA, trifluoroacetic acid; Tris, tris(hydroxymethyl)aminomethane.

3654 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Vaneˇk et al.
Table 1. Relative Inhibition of Human BHMT by Compounds 1 and 5-21a
a The percent inhibition of each compound was determined at 20 and 1 µM. See the Experimental Section for details. b All assays were done in triplicate,
and the obtained data were reproducible within (15%. c All data points for IC50 values were derived from assays performed in duplicate, and the values were
obtained from 3 different assays. Nd means not determined.
substrates, 0.25 mM betaine and 100 µM DL-homocysteine (Km
values of BHMT for betaine and DL-homocysteine are 2 mM
and 8 µM, respectively), in order to maximize our ability to
detect inhibition. In contrast, we determined IC50 values of the
most potent inhibitors at higher substrate concentrations (2 mM
betaine and 1 mM DL-homocysteine) so that we could determine
IC50 values in measurable concentrations more accurately. The
results of the inhibition experiments are summarized in Table
1. For a clearer description of inhibitor structures, we will
use the numbering of atoms as shown in a simple inhibitor
scaffold (62, below).
Among the inhibitors 5-10, only compound 9 with a selenium
atom at position 1 in scaffold 62 and with four CH2 groups in the
alkyl chain strongly inhibited BHMT. Nevertheless, replacement
of sulfur with a selenium atom yields an inhibitor that is 5-fold
weaker than compound 1. If position 1 is occupied by an oxygen
or CH2, the respective compounds 8 and 10 are almost inactive.
The low inhibitory potency of compound 7, having a sulfur atom
at position 2, reveals the high specificity of BHMT for the
“homocysteine” moiety in inhibitors. Previously reported15 inhibitor
3, which has a second sulfur atom at position 4, displayed very
strong potency toward BHMT. In this study, we prepared and tested
compounds 5 and 6, which have a second sulfur atom at positions
2 and 3, respectively. Disulfide compound 5 is a very weak inhibitor
of BHMT, and compound 6, which has a thioformyl moiety,
displays only moderate inhibitory potency with an IC50 of 3.26
µM. The data on compounds 5-10 reveal the crucial role of a
sulfur atom at position 1, which provides a good balance of
nucleophilicity, electronegativity, and atom size for the interaction
between inhibitors and the catalytic Zn2+ in the active site of
BHMT. Hence, the potential replacement of the “homocysteine”
moiety in inhibitors with a suitable mimetic remains a challenge.
The inhibitory potencies of all tested compounds were
compared to the “reference” inhibitor 1. In our previous study,15
we determined a Kiapp of 12 nM with respect to betaine for this
compound. In the present series of experiments, we determined
an IC50 of 0.138 µM for this compound.

Inhibitors of Human BHMT
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3655
Scheme 1a
a Reagents and conditions: (i) (a) HCl (20%), 60 h, rt, Ar; (b) H2O2 (30%), rt, 1 d. (ii) NaOH, CH2Cl2, H2O, benzyl triethylammonium chloride (TEBAC),
rt, Ar, 1 d. (iii) (a) NaH, DMF, rt; (b) Br(CH2)3Cl, 30 °C, 1 d. (iv) Sodium 4-mercaptoethylbutanoate, EtOH, rt, 1 d. (v) 3 M HCl, reflux, 3 h.
Scheme 2a
a Reagents and conditions: (i) (a) NaH, DMF, rt; (b) (ClCH2CH2)2O. (ii) NaI, Me2CO, 65 °C. (iii) Methyl acrylate, Zn(Cu), EtOH-H2O, ultrasound, rt. (iv)
3 M HCl, reflux.
Scheme 3a
a Reagents and conditions: (i) Se, NaOH, N2H4 ·H2O, DMF, Ar, 60 °C. (ii) NaBH4, ethyl 5-bromovalerate, EtOH, Ar, 0 °C. (iii) (a) aq NaOH, rt, 1 h; (b)
TFA/CH2Cl2/thioanisole, rt, 0.5 h. (iv) (a) EtONa, DMF, rt; (b) Br(CH2)7COOEt, reflux, 10 h. (v) 3 M HCl, reflux, 3 h.
Concerning the inhibitors of series 2, we first prepared
compounds 11 and 12, which have a dimethylamino moiety in
positions 3 and 4, respectively. These structures should ideally
mimic the transition state 4; however, compound 11 was
completely inactive at 20 µM, and inhibitor 12 gave only very
weak inhibition at this concentration. Therefore, we prepared
compounds 13-17, all of which have a nitrogen atom at position
4, not as quaternary amines, but as tertiary or secondary amines.
We found that all secondary amines 14-17 were very poor
inhibitors of BHMT. Only tertiary amine 13 gave promising,
but still moderate, inhibition of BHMT, with an IC50 value of
about 2.5 µM. Given this finding, we prepared compounds 18
and 19, both with a nitrogen atoms at position 5, with a
quaternary amine in 18, and a tertiary amine in 19. The idea
was to investigate if the propylene spacer placed between sulfur
and nitrogen is better suited for optimal inhibition of BHMT
than the ethylene spacer. The propylene spacer should provide
sufficient flexibility to the inhibitors for better adaptation to

3656 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Vaneˇk et al.
Scheme 4a
a Reagents and conditions: (i) NaOH, CH2Cl2, H2O, TEBAC, rt, Ar, 1 d. (ii) TFA/CH2Cl2/thioanisole, rt, 0.5 h. (iii) DBU, CH2Cl2, rt, 1 h. (iv) DBU,
CH2Cl2, 50 °C, 6 h.
the active site. However, compound 18 did not inhibit BHMT
at all, and compound 19, with a tertiary amine at position 5,
was only a modest inhibitor of BHMT, as it was significantly
weaker than its homologue 13, which has a tertiary amine at
position 4.
The above-described results indicate the following: (i) position
4 of scaffold 62 is the best suited for substitutions in the alkyl
chain of inhibitors, (ii) a tertiary amine is better accommodated
than a quaternary amine or secondary amine at position 4, but
(iii) the BHMT enzyme does not accept a nitrogen atom at
positions 3-5 of the scaffold 62 very well.
Given these data, we decided to synthesize and test com-
pounds 20 and 21, for which position 4 of the scaffold is
branched with one or two methyl groups, respectively. We found
that both compounds are highly potent inhibitors of BHMT;
inhibitor 20 is equipotent to the “reference” compound 1, and
we found that compound 21 has an IC50 value of 84 nM toward
BHMT. This value is lower than the IC50 that we determined
for inhibitor 1 of about 138 nM. In a previous study,15 we
determined the Kiapp of inhibitor 1 for betaine to be about 12
nM. We can competently assume that inhibitor 21 will also have
a significantly stronger Kiapp than inhibitor 1. However, in this
study, we did not repeat the extremely time-consuming enzyme
activity assays required to determine the Kiapp, which also require
high levels of radioactive betaine. Instead, we investigated the
interaction of BHMT with inhibitor 21 in more detail using
isothermal titration calorimetry. This technique allows for the
precise determination of Kd together with the determination of
changes in the total Gibbs free energy upon binding (decom-
posed to enthalpic and entropic contributions).
Isothermal Titration Calorimetry (ITC). Isothermal titra-
tion calorimetry (ITC) was used to monitor the binding of
inhibitor 21 to human recombinant BHMT. The experiments
were performed without the presence of BHMT substrates. Three
independent titrations of compound 21 in a solution of BHMT
were done separately in three buffers with different enthalpies
of ionization (HEPES, ∆Hion ) 5.03 kcal·mol-1; ACES, ∆Hion
) 7.51 kcal·mol-1; Tris, ∆Hion ) 11.3 kcal·mol-1). These
experiments indicated the same binding enthalpies, as can be
seen from the superposition of the individual titration curves
(Figure S1, Supporting Information), which suggests that there
is no net proton transfer associated with ligand binding.42,43 The
titration performed in HEPES buffer is shown in Figure 1.
Compound 21 is a mixture of two enantiomers. The crystal
structure of inhibitor 1 complexed with BHMT revealed that
only the S-enantiomer binds to the active site of the enzyme.7
The Glu159 of BHMT forms a hydrogen bond with the amino
group of the L-homocysteine moiety of inhibitor 1. Models of
D-homocysteine in the active site place the amino group more
than 4.0 Å away from the carboxylate of Glu159. Glu159
therefore establishes stereospecificity for the L-form of ho-
mocysteine. From these results, we presumed that only the
S-enantiomer of compound 21 will interact with BHMT. Later,
during the manuscript revision process, we confirmed this

Inhibitors of Human BHMT
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3657
Scheme 5a
a Reagents and conditions: (i) ClCH2CH2Cl, DBU 1 equiv, rt, Ar, 3 h. (ii) NaI, Na2CO3, Bu4NBr, dioxane, Ar, reflux, 40 h. (iii) CH3I, dioxane, rt, 1 d.
(iv) TFA/CH2Cl2/thioanisole, rt, 0.5 h.
Scheme 6a
a Reagents and conditions: (i) Br(CH2)3Br, DBU 1 equiv, rt, 1 h. (ii) NaI, Na2CO3, Bu4NBr, dioxane, Ar, reflux, 16 h. (iii) CH3I, dioxane, rt, 1 d. (iv)
TFA/CH2Cl2/thioanisole, rt, 0.5 h.

3658 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Vaneˇk et al.
Scheme 7a
a Reagents and conditions: (i) (a) NaBH4, THF, rt, 3 d (b) H+. (ii) HBr/EtOH, rt, 3 d. (iii) NaH, THF, rt, 1 d. (iv) (a) 1 M NaOH, H2O-Dx, 60 °C, 5 h;
(b) TFA/CH2Cl2/thioanisole, rt, 0.5 h.
possible to separate the interaction step from the unknown
exothermic process. The stoichiometry of the main interaction
was estimated to be 1.1 ( 0.1, which is in a good agreement
with the previous finding that one molecule of the similar
inhibitor 1 binds to one BHMT monomer subunit (four inhibitor
molecules per BHMT tetramer).7 Inhibitor 21 (S-enantiomer)
binds to BHMT with a dissociation constant (Kd) of about 51
nM (calculated from an association constant Ka ) (2.0 ( 0.6)
× 107 M-1), which corresponds to a total Gibbs free energy
change ∆G ) -10.0 ( 0.2 kcal ·mol-1. It is decomposed into
enthalpic (∆H ) -29.5 ( 1.2 kcal ·mol-1) and entropic (-T∆S
) 19.6 ( 1.3 kcal·mol-1) contributions. This considerably large
and favorable enthalpic contribution suggests a strong and direct
interaction of the inhibitor with the enzyme via hydrogen bonds
or ionic interactions. On the other hand, a large and positive
entropic contribution is unfavorable and may reflect possible
conformational changes of the enzyme upon binding of the
inhibitor. Recently, we studied the binding mechanism of BHMT
using its intrinsic fluorescence.17 This study confirmed the
previously proposed ordered Bi-Bi mechanism of BHMT. It
was shown that homocysteine is the first substrate to bind and
that this binding probably induces a conformational change of
the enzyme, which allows the binding of betaine, the second
substrate. In 2004, it was shown that dimerization of BHMT
might be required for substrate binding.44 His338 of the BHMT
“dimerization arm” (residues 319-371) contributes to betaine
binding at the active site of the other monomer, indicating that
the complete active site is formed upon dimerization. A more
Figure 1. Isothermal titration of inhibitor 21 with BHMT. Titration
recent study45 suggested that loop L2 (residues 74-79) is
was performed at 25 °C in 50 mM HEPES/NaOH, pH 7.5, containing
involved in the conformational change associated with oc-
5 mM 2-mercaptoethanol. Upper graph: experimental data. Lower
cupancy at the betaine-binding site. Gonzalez et al.46 proposed
graph: fit (line) to the integrated heats (full circles) from each injection
of compound 21, corrected for the heat of dilution of this inhibitor.
For details, see Experimental Section.
that, in ligand-free BHMT, L2 is open, which allows ligands
access to the active site, but L2 closes after formation of the
ternary complex (binding of betaine). Phe76 and Tyr77 are
important betaine-binding determinants in this process. It is
probable that inhibitor 21 interacts with BHMT in a similar
manner; first, the “homocysteine” part of the inhibitor binds,
and after an induced conformational change of BHMT, the
S-linked alkyl chain of the inhibitor binds. These conformational
changes could explain the high and positive entropic contribution
that we observed. In the above-mentioned fluorescence study,17
we determined the Kd of compound 1 (mixture of R- and
S-enantiomers) for the enzyme to be about 280 nM. The Kd of
presumption by the synthesis and characterization of pure S-
and R- enantiomers of compound 21 (compounds 63 and 64,
Supporting Information). Therefore, in our calculation of
enthalpy change, we considered the concentration of compound
21 to be half of the total concentration of the R,S-compound.
After addition of a small amount of ligand to the protein, an
unknown exothermic process was detected from the titration
curve (Figure 1). The other main exothermic response was
considered to be due to the interaction of the reactants. Using
a model for two sets of sites using the Origin software, it was

Inhibitors of Human BHMT
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3659
inhibitor 21 for BHMT as determined in this study using ITC
is significantly tighter (51 nM for S-enantiomer) and confirms
the status of compound 21 as the most potent inhibitor of BMHT
ever reported.
Considerations Regarding the Mode of Interaction between
Inhibitors and BHMT. The higher inhibitory potency of
inhibitor 21 suggests that this compound might represent a better
mimic of transition state 4 than the previously reported inhibitor
1. Nevertheless, a number of questions arise from the inhibitory
activities of our new compounds.
Molecular modeling of the BHMT-inhibitor 21 complex (see
Supporting Information, Figure S2) suggests that compound 21
might bind BHMT in a similar manner as compound 1.
However, the calculation of binding energies for compounds 1
and 21 (see Supporting Information) indicated that inhibitor 21
should be a weaker binder than inhibitor 1 in this type of
interaction. We predicted binding energies of -8.5 and -5.9
kcal·mol-1 for compounds 1 and 21, respectively. This observa-
tion contradicts our inhibition experiments, which revealed that
compound 21 was a tighter inhibitor than 1 (IC50 values of 84
and 138 nM, respectively), and it also contradicts an experi-
mental value of ∆G of about -10.0 kcal.mol-1 for compound
21 as determined by ITC. These findings also suggest that
inhibitor 21 is a stronger binder than 1. We suppose that inhibitor
21 binds to BHMT in a similar but not identical manner as
inhibitor 1. This slightly different mode of interaction results
in a higher binding affinity of compound 21 compared to 1.
Figure 2. Schematic representation of proposed structural changes in
BHMT during binding of substrates and products. Blue ovals designated
as S1-S6 represent different structural states of the BMHT monomer.
Hcy is homocysteine, Bet is betaine, Met is methionine, Dmg is
dimethylglycine, and TS is the putative transition state of the substrates.
For details, see the text.
site of BHMT,7 and it was recently shown that the binding of
ligands (substrates or inhibitor) replaces Tyr160, which is the
fourth, displaceable ligand of zinc in BHMT.46,47 These facts
do not support the hypothesis that our inhibitors bind outside
of the active site of BHMT, but rather evoke questions about
putative conformational changes of BHMT upon binding of
substrates/products or our inhibitors to the active site. Interest-
ingly, Awad et al.16,48 proposed a similar hypothesis 25 years
ago. They suggested that the binding site for betaine might exist
in two states, one permitting the binding of a positively charged
group and the other a neutral group. It is possible that our potent
inhibitors (e.g., 1, 20, and 21) bind to some transient structure
of BHMT, which may be similar to a structure of the enzyme
that exists when it binds methionine (Met) or dimethylglycine
(Dmg), for example, but relatively different from the struc-
ture of BHMT in a complex with homocysteine (Hcy) or betaine
(Bet). Along this line of reasoning, it is possible that the 3D
structure of the hypothetical transition state 4 is different from
the structures presented by inhibitors 1, 20, and 21. We
attempted to express this hypothesis in Figure 2. We suppose
that the structure of the active site of BHMT may exist in several
different conformations (S1-S6 in Figure 2). Therefore, due
to local rearrangements of its active site, BHMT can adapt its
structure to different binders, substrates, and products (Hcy, Bet,
Met, and Dmg). Structure S1 is the structure of BHMT without
substrates. After binding of the first substrate, Hcy, the BHMT
structure is modified such that it opens the binding site for the
second substrate, Bet, and then adopts the structure S2. The
transfer of a methyl group from Bet to Hcy proceeds via
the structure of transition state when BHMT passes through
structure S4. This process results in the formation of products
Met, Dmg, and the respective BHMT structure S5. Finally, Dmg
and then Met leave the active site and the BHMT structure
changes from S5 to S6 and then back to the basic structure S1.
It is not excluded that, for example, structures S2 and S6 are
the same or that S3 does not exist because S4 is formed
immediately after Bet binds. Such relatively complicated
structural rearrangements of BHMT during a catalytic cycle
would be in agreement with a very slow turnover rate of the
enzyme.5,6,49 We propose that our strong inhibitors 1, 20, and
21 fit well into the active site of structure S5. We suppose that
Dmg bound to BMHT (in S5) has a deprotonated tertiary amine
The finding that BHMT does not tolerate betaine mimics in
our inhibitors, especially the presence of the nitrogen atom, is
surprising. However, the structure7 of the complex of human
BHMT with inhibitor 1 reveals that the inhibitor butyl chain is
surrounded by a series of aromatic residues (Phe76, Tyr77,
Tyr160, Phe261, and Phe 267) and that the carboxyl group
interacts with Trp44 and Tyr77. The positive charge of the
quaternary amine in compounds 11, 12, and 18 would be rather
disadvantageous for binding to the hydrophobic pocket of
BHMT surrounding the butyl chain of inhibitor 1. It is probable
that compounds with secondary (14-17) and tertiary (13 and
19) amines have protonated nitrogen atoms, which are respon-
sible for their low inhibitory potency compared to inhibitors 1,
20, and 21. We evaluated this hypothesis by NMR spectroscopy
of compounds 13 and 16 over a broad pH range. The NMR
spectra of quaternary amine 12 were measured for comparison.
1
The H and 13C NMR titration experiments (see Supporting
Information, Figure S3) revealed a pK2 ∼ 9.5 for the secondary
and tertiary amino groups in both compounds 13 and 16. These
values indicate that the equilibrium between protonated and
deprotonated forms of the nitrogen atoms at position 4 at pH
7.5, the pH used in our enzymatic assay, is shifted strongly to
the protonated form, which may explain why they were much
weaker inhibitors compared to compounds 20 and 21. This
observation implies that the deprotonated forms of compounds
13 and 16 likely bind to BHMT and are responsible for the
observed moderate inhibition. The higher inhibitory potency of
inhibitor 13 compared to 16 could be attributed to a positive
contribution of the N-linked methyl group.
All of the above reasoning leads us to the conclusion that
compound 1, and probably also compounds 20 and 21, do not
correctly mimic the 3D structure of transition state 4 and that
the substrates betaine and homocysteine bind BHMT differently
than our inhibitors. The inhibitor-binding cavity of BHMT is
relatively narrow and is buried deeply in the structure of the
BMHT monomer.7 It was clearly shown that the sulfur atom of
inhibitor 1 directly interacts with the Zn2+ atom in the active

3660 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Vaneˇk et al.
color of esters). For flash column chromatography, silica gel 40-60
µm (Fluka) was used. TLC and preparative silica gel chromatog-
raphy were carried out in the following solvent systems (v/v):
chloroform-ethanol 9:1 (C1), chloroform-ethanol 19:1 (C2), ethyl
acetate-acetone-ethanol-water 4:1:1:1 (H1), ethyl acetate-
acetone-ethanol-water 6:1:1:0.5 (H3), 2-propanol-conc aqueous
ammonia-water 7:1:2 (IPAV), 50% EtOAc-toluene (T1), 20%
ethyl acetate-toluene (T2). For gradient RP-HPLC purification and
analysis, a Waters LC 625 System (Milford, MA) was used.
Different gradients of acetonitrile (1-80%) in water containing
0.1% (v/v) of TFA were used for the elution of compounds.
Preparative RP-HPLC of the target compounds was performed using
a Phenomenex (Luna C-18, 5 µm, 25 cm × 2.12 cm, Torrance,
CA) column. Purified compounds were coevaporated several times
with water at 75 °C to remove TFA and then lyophilized from the
water. Analytical RP-HPLC was performed using a Watrex
(Nucleosil 120, 5 µm, C18, 25 cm × 0.46 cm; Prague, Czech
Republic) column. Anion-exchange analytical HPLC was performed
using an AminoPac PA10 (0.4 cm × 25 cm, Dionex Corporation,
Sunnyvale, CA) column with a BioLC system (GP50 gradient
pump, ED50 electrochemical detector) from Dionex Corporation
(Sunnyvale, CA). Details of the HPLC conditions are given in the
Supporting Information. HRMS mass spectra were obtained on a
FTMS mass spectrometer LTQ-orbitrap XL (Thermo Fisher,
nitrogen atom, probably because it is formed in the active site.
On the other hand, our inhibitors that have a nitrogen atom at
position 4 exist as mainly protonated molecules. For this reason,
they bind weakly but their carbon-containing counterparts (20
and 21) bind strongly.
Conclusions
We prepared two series of new inhibitors of human recom-
binant BHMT and performed a systematic structure-activity
study with the aim of investigating the structural requirements
of the active site of the enzyme. The first series of inhibitors
varies with regard to the position and number of sulfur atoms
in the compounds, which are derived from the structure of the
previously published inhibitor 1. We found that the presence
of a well-positioned sulfur atom in this type of compound is
necessary for potent inhibition of BHMT. The second series of
inhibitors was designed to mimic the hypothetical transition state
(4) of the BHMT substrates betaine and homocysteine upon
binding to the active site. We synthesized and tested analogues
with NH, N(CH3), or N(CH3)2 groups separated from the
homocysteine sulfur atom by methylene, ethylene, or propylene
spacers. Only the inhibitor with the N(CH3) moiety and an
ethylene spacer between nitrogen and sulfur gave moderate
inhibition. These results led us to prepare two inhibitors lacking
a nitrogen atom in the S-linked alkyl chain, inhibitor 20 and
inhibitor 21, which are both highly potent inhibitors of BHMT.
We also investigated the interaction of inhibitor 21 with BHMT
using isothermal titration microcalorimetry and found a highly
enthalpic interaction and Kd of about 51 nM for the S-enantiomer
of this inhibitor. Compound 21 is the most potent inhibitor of
BHMT reported to date. The finding that BHMT does not
tolerate betaine mimics in inhibitors, especially the presence of
the nitrogen atom, is surprising and evokes questions about the
catalytic mechanism of the reaction and about putative confor-
mational changes of BHMT upon the binding of substrates/
products in the active site. It seems reasonable to assume that
the predominant effect driving the binding of inhibitors or
substrates to BHMT is an electrostatic effect. However, the work
reported here shows that potent inhibitors of BHMT require
that the central part of the compounds be without a positive
charge. We interpret these results to indicate that our inhibitors
bind to the enzyme in a mode similar to the products Met and
Dmg. On the other hand, good mimics of the 3D structure of
transition state 4 should demand the presence of a positive
charge in the central part of the molecule, however, the structure
and composition of true transition state 4 mimics have thus far
eluded us. Additional information about the structure of BHMT
complexed with varying substrate(s)-product(s) combinations,
or complexed with the new inhibitory ligands described here
(e.g., inhibitors 20 and 21), will lead our future synthetic efforts
to prepare BHMT ligands that deepen our understanding of the
chemical events and structural changes that occur during BHMT
catalysis.
1
Bremen, Germany) in electrospray ionization mode. H and 13C
NMR spectra were recorded on a Bruker AVANCE-500 (1H at
500.13 MHz, 13C at 125.7 MHz) and an AVANCE-600 spectrom-
eter (1H at 600.13 MHz, 13C at 150.9 MHz) in CDCl3, DMSO-d6
or D2O solution. The 2D-H,H-COSY, 2D-H,C-HSQC, and 2D-H,C-
HMBC spectra were used for the structural assignment of proton
and carbon signals.
Deprotection: General Procedure A. A solution of the protected
compound in aqueous HCl (4 M, 10 mL/mmol) was heated under
reflux for 3 h and then evaporated in vacuo. The residue was
dissolved in water and purified by the standard procedure (see
General part).
Deprotection: General Procedure B. A solution of the protected
compound in a mixture of dioxane (5 mL/mmol) and EtOH (2 mL/
mmol) was treated with aqueous NaOH (5 M, 2 mL/mmol), and
the reaction mixture was stirred for 4 h at 80 °C (TLC in T1). The
solution was acidified with saturated aq citric acid to pH 3 before
being extracted with EtOAc (2 × 50 mL). The organic layer was
washed with water (50 mL) and evaporated in vacuo. The residue
was dissolved in a mixture of TFA-CH2Cl2-thioanisole-H2O (47:
47:3:3 v/v, 5 mL/mmol), and the solution was stirred at rt for 30
min. Then, H2O (10 mL/mmol) was added, and the aqueous phase
was washed with Et2O (5 × 10 mL). The final product was obtained
after standard purification (see General part).
Deprotection: General Procedure C. The protected compound
was dissolved in a mixture of TFA-CH2Cl2-thioanisole-H2O (47:
47:3:3 v/v, 5 mL/mmol), and the solution was stirred at rt for 30
min. Then, H2O (10 mL/mmol) was added, and the aqueous phase
was washed with Et2O (5 × 10 mL). The final product was obtained
after standard purification (see General part).
Synthesis of compounds 27-29, 63, and 64 and spectral data
for nontarget compounds 23a, 23b, 24, 30-35, 38-55, and 58-61
are given in Supporting Information.
(RS)-2-Amino-4-[(3-carboxypropyl)disulfanyl]butanoic acid (5)
and 4,4′-disulfanediyldibutanoic acid (22). A solution of DL-homo-
cysteine (0.300 g, 2.22 mmol) and γ-thiobutyrolactone (0.576 mL,
6.66 mmol, 3 equiv) in 5 M hydrochloric acid (20 mL) was stirred
at rt for 60 h under an argon atmosphere, and then hydrogen
peroxide (30% aq solution, 0.454 mL, 2 equiv) was added.
Formation of a white precipitate was observed after 10 min, and
the reaction was allowed to proceed overnight (TLC in IPAV). The
reaction mixture was then coevaporated three times with water.
The residue was treated with water (20 mL), and the insoluble solid
was filtered off and dried over P2O5 to give 22 as a white solid
Experimental Section
Chemistry: General. Unless otherwise stated, materials were
obtained from commercial suppliers (Sigma-Aldrich, Fluka, Merck)
and used without purification. The solvents were evaporated at 40
°C and 2 kPa, and the products were dried over phosphorus
pentoxide at rt and 13 Pa. The course of the reactions was checked
on TLC plates (Fluka, Merck). More specifically, the products were
detected by UV monitoring after spraying with ninhydrin (dark-
blue color of amines), a 1% aqueous solution of KMnO4 (sulfur
compounds), and a 1% ethanolic solution of 4-(4-nitrobenzyl)py-
ridine, followed by heating and treating with gaseous ammonia (blue
1
(0.523 g, 66% from the total γ-thiobutyrolactone). H NMR (500
MHz, D2O) 1.80 (m, 4H), 2.15 (m, 4H), 2.61 (m, 4H). 13C NMR
(125.7 MHz, D2O) 25.45 (2 × C), 36.20 (2 × C), 37.86 (2 × C),

Inhibitors of Human BHMT
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3661
182.72 (2 × C). HRMS (ESI) calcd for C8H15O4S2 [M + H]+
239.0412; found 239.0404.
(elution with a linear gradient of EtOAc in toluene) to give
compound 30 as a pale-yellow viscous oil (4.24 g, 57%).
Diethyl 2-[2-(2-Iodoethoxy)ethyl]-2-acetamidomalonate (31). An-
hydrous sodium iodide (2.78 g, 18.5 mmol, 3 equiv) was added to
a solution of chloride 30 (2.0 g, 6.2 mmol) in dry acetone (20 mL),
and the reaction mixture was heated to 65 °C in a sealed flask for
24 h with stirring. The mixture was then diluted with Et2O (50
mL), and the precipitated inorganic salts were removed by filtration.
Evaporation of the filtrate under reduced pressure gave a crude
product, which was purified by flash chromatography on silica
(elution with a linear gradient of EtOAc in toluene) to give
compound 31 as a colorless viscous oil (2.43 g, 95%).
The aqueous filtrate was concentrated in vacuo, and the crude
product was purified by the standard procedure (see General part),
giving 5 as a colorless hygroscopic semisolid (0.134 g, 24% from
1
DL-homocysteine). H NMR (500 MHz, D2O) 1.89 (m, 2H), 2.20
(m, 1H), 2.28 (m, 1H), 2.40 (m, 2H), 2.67 (t, 2H), 2.74 (t, 2H),
4.03 (dd, 1H). 13C NMR (125.7 MHz, D2O) 23.61, 29.29, 32.22,
32.53, 36.76, 51.95, 178.05 (2 × C). HRMS (ESI) calcd for
C8H16NO4S2 [M + H]+ 254.0521; found 254.0514.
(RS)-2-Amino-4-[(2-carboxyethylthio)methylthio]butanoic Acid
(6). 3-Mercaptopropionic acid (386 µL, 4.44 mmol, 3 equiv) and
TEBAC (0.017 g, 0.075 mmol 0.05 equiv) were added to a stirred
solution of DL-homocysteine (0.200 g, 1.48 mmol) and NaOH (1.2
g, 30 mmol, 20 equiv) in H2O (10 mL). Then, dichloromethane
(10 mL) was added and the reaction mixture was vigorously stirred
at rt for 1 d (TLC in IPAV). The aqueous layer was passed through
a column of Dowex 50 (Et3N-cycle) and evaporated to dryness.
The residue was dissolved in water and purified by the standard
procedure to give 6 as a white solid (0.102 g, 27%). 1H NMR (500
MHz, D2O + NaOD) 1.81 (m, 1H), 1.93 (m, 1H), 2.49 (t, 2H),
2.70 (m, 2H), 2.85 (t, 2H), 3.33 (dd, 1H), 3.80 (s, 2H). 13C NMR
(125.7 MHz, D2O + NaOD) 29.83, 29.97, 36.60, 36.93, 39.77,
57.95, 183.50, 185.25. HRMS (ESI) calcd for C8H14NO4S2 [M +
H]+ 252.0364; found 252.0360.
Diethyl 2-(3-Chloropropyl)-2-acetamidomalonate (23a) and Di-
ethyl 2-(3-Bromopropyl)-2-acetamidomalonate (23b). Diethyl ac-
etamidomalonate (4.88 g, 22.5 mmol, 1.00 equiv) was added in
five portions to an ice-cooled suspension of NaH (60% suspension
in oil, 1.04 g, 26.0 mmol, 1.17 equiv) in anhydrous DMF (12.5
mL) in a flask equipped with a calcium dichloride tube, and the
mixture was left to stand at rt for 1 h. Then, 1-bromo-3-
chloropropane (2.46 mL, 25.0 mmol, 1.12 equiv) was added
dropwise over 10 min, and the reaction was allowed to proceed at
30 °C for 24 h (TLC in T1). After removal of the solvent in vacuo,
the crude product was suspended in Et2O, and the inorganic solids
were filtered off. The organic phase was washed with H2O twice
before being dried over Na2SO4 and evaporated in vacuo. The
resulting residue was purified by flash chromatography on silica
(elution with a linear gradient of EtOAc in toluene) to yield 3.70 g
of a mixture of 23a and 23b as colorless needles. The mixture was
used directly in the next step without further resolution.
Diethyl 2-{2-[4-(Methoxycarbonyl)butoxy]ethyl}-2-acetamidoma-
lonate (32). The zinc-copper couple was prepared according to a
previously described procedure32 by the sonication of zinc dust (0.50
g, 8 mmol) and CuI (0.356 g, 2.4 mmol) in water (2 mL) under
argon; after 2 min, the mixture turned to a black heavy suspension.
Then, EtOH (2 mL) and methyl acrylate (0.90 mL, 10 mmol, 10
equiv) were added through the septum via syringe, and finally a
solution of 31 (0.415 g, 1 mmol) in iPrOH (1.5 mL) was added
over a period of 1 h under sonication; the temperature was not
allowed to rise above 20 °C. Sonication was continued for 1 h,
and then saturated aq NaCl (5 mL) was added. The resulting
precipitate was filtered over celite. The solid was extracted with
Et2O (100 mL), and the filtrate was partitioned between water and
Et2O, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by flash chromatography on silica (elution with a linear
gradient of EtOAc in toluene) to give compound 32 as a colorless
viscous oil (0.195 g, 52%).
(RS)-5-(3-Amino-3-carboxypropoxy)pentanoic Acid (8). Com-
pound 32 (0.180 g, 0.82 mmol) was deprotected according to
general procedure A, yielding 8 as a white solid (0.098 g, 93%).
1H NMR (600 MHz, D2O) 1.62 (m, 4H), 2.23 (m, 2H), 2.41 (t,
2H), 2.52 (m, 2H), 3.68 (m, 2H), 4.16 (dd, 1H). 13C NMR (150.9
MHz, D2O) 23.64, 30.65, 32.10, 36.10, 54.16, 68.97, 73.22, 174.63,
181.56. HRMS (ESI) calcd for C9H18NO5 [M + H]+ 220.1185;
found 220.1183.
(RS,RS)-Di-tert-butyl-4,4′-diselanediylbis[2-(tert-butoxycarbony-
lamino)butanoate] (33). N2H4 ·H2O (0.062 mL, 1.27 mmol, 1.1
equiv) was added to a suspension of Se (0.100 g, 1.27 mmol, 1.1
equiv) and NaOH (0.138 g, 3.45 mmol, 3 equiv) in DMF (5 mL),
and the mixture was stirred at 60 °C under argon for 3 h. Then, a
solution of bromide 29 (0.390 g, 1.15 mmol, 1 equiv) in DMF (3
mL) was added to the flask through the septum by syringe, and
stirring was continued for a further 3 h at 60 °C (TLC in T2). The
reaction mixture was concentrated in vacuo, and the residue was
partitioned between Et2O and H2O. The organic phase was
separated, washed with brine, and dried over Na2SO4. After
evaporation, the crude product was purified by flash chromatography
on silica (elution with a linear gradient of EtOAc in toluene) to
give compound 33 as a yellow viscous oil (0.295 g, 76%).
Diethyl 2-[3-(3-Ethoxycarbonylpropylthio)propyl]-2-acetami-
domalonate (24). Sodium 4-mercaptoethylbutanoate50 (0.40 g, 2.20
mmol, 1.5 equiv) was added to a mixture of 23a and 23b (0.50 g,
ca. 1.57 mmol in total) in anhydrous EtOH (10 mL), and the reaction
mixture was stirred overnight (TLC in T1). The resultant white
precipitate was filtered off, and the solution was evaporated in
vacuo. The crude product was purified by flash chromatography
on silica (elution with a linear gradient of EtOAc in toluene) to
give compound 24 as a colorless solid (0.317 g, 50%).
(RS)-2-Amino-5-(3-carboxypropylthio)pentanoic Acid (7). Com-
pound 24 (0.310 g, 0.76 mmol) was deprotected according to
general procedure A, affording 7 (0.147 g, 82%) as a white solid.
1H NMR (600 MHz, D2O) 1.70 (m, 1H), 1.77 (m, 1H), 1.90 (m,
2H), 2.02 (m, 1H), 2.06 (m, 1H), 2.50 (m, 2H), 2.62 (m, 2H), 2.64
(m, 2H), 4.04 (dd, 1H). 13C NMR (150.9 MHz, D2O) 26.77, 26.97,
31.68, 32.83, 32.90, 35.39, 55.68, 172.66, 181.00. HRMS (ESI)
calcd for C9H19N2O4S [M + H]+ 251.1066; found 251.1062.
Diethyl 2-[2-(2-Chloroethoxy)ethyl]-2-acetamidomalonate (30).
Diethyl acetamidomalonate (5 g, 23 mmol, 1 equiv) was slowly
added to an intensively stirred suspension of NaH (60% dispersion
in oil; 0.92 g, 23 mmol, 1 equiv) in DMF (40 mL) at 0 °C. After
20 min, sodium iodide (0.345 g, 2.3 mmol, 0.1 equiv) and bis(2-
chlorethyl)ether (13.5 mL, 115 mmol, 5 equiv) were added, and
the reaction mixture was stirred for 24 h at 60 °C (TLC in T1).
The mixture was then concentrated in vacuo, and the dark-orange
residue was partitioned between water (250 mL) and Et2O (2 ×
250 mL). The organic layer was separated, washed with water (2
× 100 mL), dried over Na2SO4, and concentrated in vacuo. The
crude product was purified by flash chromatography on silica
(RS)-Ethyl 5-[3-tert-butoxycarbonyl-3-(tert-butoxycarbonylami-
no)propylselanyl]pentanoate (34). NaBH4 (0.048 g, 1.26 mmol, 3
equiv) was added to a solution of diselenide 33 (0.285 g, 0.42 mmol)
and ethyl 5-bromovalerate (0.150 mL, 0.93 mmol, 2.2 equiv) in
abs EtOH (5 mL) under an argon atmosphere at 0 °C, and the
mixture was stirred until the yellow color of the diselenide
disappeared (ca. 5 min). The reaction was allowed to proceed at rt
overnight (TLC in T2). The mixture was then partitioned between
Et2O and H2O. The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified by flash chromatography on silica (elution with a linear
gradient of EtOAc in toluene) to give compound 34 as a colorless
viscous oil (0.344 g, 87%).
(RS)-5-(3-Amino-3-carboxypropylselanyl)pentanoic Acid (9). Com-
pound 34 (0.320 g, 0.69 mmol) was deprotected according to
general procedure B, yielding 9 as a white solid (0.122 g, 63%).
1H NMR (600 MHz, D2O + NaOD) 1.63 (m, 2H), 1.67 (m, 2H),
1.87 (m, 1H), 1.97 (m, 1H), 2.19 (t, 2H), 2.60 (m, 2H), 2.64 (m,
2H), 3.30 (dd, 1H). 13C NMR (150.9 MHz, D2O + NaOD) 22.02,

3662 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Vaneˇk et al.
25.97, 28.89, 32.37, 38.53, 39.76, 58.96, 185.56, 186.44. HRMS
(ESI) calcd for C9H18O4NSe [M + H]+ 284.0396; found 284.0395.
Diethyl 2-[(7-Ethoxycarbonyl)heptyl]-2-acetamidomalonate (35).
Sodium ethanolate was generated in situ by dissolving sodium
(0.072 g, 3.13 mmol, 1.2 equiv) in abs EtOH (2.5 mL) at rt in a
flask equipped with a reflux condenser fitted with a calcium chloride
tube. DEAM (0.571 g, 2.63 mmol, 1.1 equiv) was added, and after
15 min, a solution of ethyl 8-bromooctanoate (0.600 g, 2.39 mmol,
1 equiv) in EtOH (3 mL) was introduced, and the reaction was
heated to reflux for 10 h. The mixture was evaporated, partitioned
between Et2O and H2O, and the organic layer was dried over
Na2SO4 and concentrated in vacuo. The crude product was purified
by flash chromatography on silica (elution with a linear gradient
of EtOAc in toluene) to give compound 35 as a colorless viscous
oil (0.547 g, 51%).
(RS)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-{[(tert-butoxy-
carbonylmethyl)(methyl)amino]methylthio}butanoate (43). The
solution of 37, prepared as above, was added to a solution of tert-
butyl 2-(methylamino)aminoacetate hydrochloride (0.216 g, 1.19
mmol, 1 equiv) and DBU (0.181 mL, 1.19 mmol, 1 equiv) in
CH2Cl2 (4 mL), and the reaction was allowed to proceed in a sealed
flask under argon at 50 °C for 6 h. Then second portion of DBU
(0.181 mL, 1.19 mmol, 1 equiv) was added, the mixture evaporated
in vacuo, and the residue was purified by flash chromatography on
silica. Elution with a linear gradient of EtOAc in toluene gave 43
as a colorless glassy solid (0.250 g, 31%).
Di-tert-butyl (RS,RS)-4,4′-(tert-Butoxycarbonylmethylamino)bis-
(methylene)bis(sulfanediyl)bis[(2-tert-butoxycarbonylamino)bu-
tanoate] (44). The solution of 37, prepared as above, was added to
a solution of tert-butyl 2-aminoacetate hydrochloride (0.200 g, 1.19
mmol, 1 equiv) and DBU (0.181 mL, 1.19 mmol, 1 equiv) in
CH2Cl2 (4 mL), and the reaction was allowed to proceed in a sealed
flask under argon at 50 °C for 24 h. Then second portion of DBU
(0.181 mL, 1.19 mmol, 1 equiv) was added, the mixture was
evaporated in vacuo, and the residue was purified by flash
chromatography on silica. Elution with a linear gradient of EtOAc
in toluene gave 44 as a colorless glassy solid (0.143 g, 11%).
(RS)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-(2-chloroethylth-
io)butanoate (45). DBU (1.353 mL, 8.89 mmol, 1.1 equiv) was
added to a solution of thiol 36 (2.355 g, 8.08 mmol) in 1,2-
dichloroethane (20 mL), and the reaction mixture was heated under
argon at 80 °C for 3 h (TLC in T2). Excessive solvent was
evaporated in vacuo, and the crude product was purified by flash
chromatography on silica (elution with a linear gradient of EtOAc
in toluene) to give compound 45 as a white crystalline solid (2.456
g, 86%).
(RS)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-{2-[(tert-buto-
xycarbonylmethyl)(methyl)amino]ethylthio}butanoate (46). Chlo-
ride 45 (0.366 g, 1.03 mmol, 1 equiv), tert-butyl 2-(methylami-
no)acetate (0.150 g, 1.03 mmol, 1 equiv), sodium carbonate (0.165
g, 1.55 mmol, 1.5 equiv), sodium iodide (0.155 g, 1.03 mmol., 1
equiv), and tetrabutylammonium bromide (0.166 g, 0.52 mmol, 0.5
equiv) were placed in a flask, and the mixture was codistilled twice
with anhydrous CH2Cl2 (10 mL) to remove traces of water. Dioxane
(15 mL) was added, and the flask was equipped with a reflux
condenser and heated to reflux under argon for 40 h (TLC in T2).
After cooling, the mixture was filtered over celite, the solid extracted
with EtOAc (50 mL), and the filtrate evaporated in vacuo. The crude
product was purified by flash chromatography on silica (elution
with a linear gradient of EtOAc in toluene) to give compound 46
as a colorless solid (0.298 g, 62%).
(RS)-2-Aminodecanedioic Acid (10). Compound 35 (0.320 g, 0.69
mmol) was deprotected according to general procedure A, yielding
1
10 as a white solid (0.156 g, 87%). H NMR (600 MHz, D2O +
NaOD) 1.30 (m, 2H), 1.32 (m, 4H), 1.34 (m, 2H), 1.54 (m, 2H),
1.74 (m, 1H), 1.80 (m, 1H), 2.16 (t, 2H), 3.59 (dd, 1H). 13C NMR
(150.9 MHz, D2O + NaOD) 27.09, 28.54, 30.89, 31.04, 31.25,
34.16, 40.34, 57.86, 180.04, 187.08. HRMS (ESI) calcd for
C10H20NO4 [M + H]+ 218,1392; found 218,1382.
(RS,RS)-Di-tert-butyl 4,4′-(methylenedithio)-2,2′-di(tert-butoxy-
carbonylamino)dibutanoate (38) and (RS)-1-[3-(tert-butoxycarbo-
nylamino)-3-(tert-butoxycarbonyl)propylthio]-N-(tert-butoxycar-
bonylmethyl)-N,N-dimethylmethanammonium chloride (40). A
solution of chloromethylsulfide 37, which is necessary for the amine
alkylation, was obtained by intensive stirring of thiol 36 (0.345 g,
1.18 mmol),34 crushed solid KOH (0.067 g, mmol, 1 equiv), and
TEBAC (0.027 g, 0.12 mmol, 0.1 equiv) in a mixture of CH2BrCl
(2.5 mL) and CH2Cl2 (2.5 mL) for 1 h at rt. The solid precipitate
was filtered off, and the solution was directly used in subsequent
reaction. A solution of 37 was added to a solution of tert-butyl
2-(dimethylamino)acetate hydrochloride (0.160 g, 0.82 mmol, 1
equiv) and DBU (0.135 mL, 0.90 mmol, 1.1 equiv) in CH2Cl2 (4
mL), and the reaction was allowed to proceed at rt for 1 h. The
mixture was evaporated in vacuo, and the residue was purified by
flash chromatography on silica. Elution with a linear gradient of
EtOAc in toluene gave byproduct 38 as a colorless viscous oil
(0.065 g, 19% from thiol 36). Further elution with a linear gradient
of H1 in EtOAc yielded the desired ammonium salt 40 (0.125 g,
31% from the amine hydrochloride). Isolation of the byproduct 38
in the following procedures that employ a solution of 37 was not
performed.
(RS,RS)-4,4′-(Methylenedithio)-2,2′-(diamino)dibutanoic Acid
(39). Compound 38 (0.110 g, 0.18 mmol) was deprotected according
to the general procedure C, affording 39 as a white solid (0.045 g,
86%).
(RS)-2-[3-(tert-Butoxycarbonylamino)-3-(tert-butoxycarbonyl)-
propylthio]-N-(tert-butoxycarbonylmethyl)-N,N-dimethylethanam-
monium Iodide (47). A solution of 46 (0.196 g, 0.42 mmol) in
dioxane (5 mL) was treated with methyl iodide (0.066 mL, 0.84
mmol, 2.5 equiv), and the reaction mixture was left to react at rt
for 4 d (TLC in C1). The solution was then concentrated in vacuo,
and the residue was purified by flash chromatography on silica
(elution with a linear gradient of H3 in EtOAc) to give compound
47 as a dark-yellow solid (0.196 g, 89%).
(RS)-2-{[(3-Amino-3-carboxypropylthio)methyl]dimethylammoni-
um}acetate (11). Compound 40 (0.065 g, 0.13 mmol) was depro-
tected according to the general procedure C, affording 11 as a white
1
solid (0.020 g, 63%). H NMR (500 MHz, D2O) 2.22 (m, 1H),
2.33 (m, 1H), 2.97 (m, 1H), 3.02 (m, 1H), 3.26 (s, 6H), 4.14 (dd,
1H), 4.17 (s, 2H), 4.91 (s, 2H). 13C NMR (125.7 MHz, D2O) 32.59,
32.66, 53.20 (2 × C), 54.42, 63.54, 70.92, 170.74, 174.09. HRMS
(ESI) calcd for C9H19N2O4S [M]+ 251.1066; found 251.1077.
(RS)-1-{[3-(tert-Butoxycarbonyl)-3-(tert-butoxycarbonylamino)-
propylthio]methyl}-2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepin-
1-ium Chloride (41). The solution of 37, prepared as above, was
added to a solution of tert-butyl 2-aminoacetate hydrochloride
(0.200 g, 1.19 mmol, 1 equiv) and DBU (0.381 mL, 2.50 mmol,
2.1 equiv) in CH2Cl2 (4 mL), and the reaction was allowed to
proceed in a sealed flask under argon at 50 °C for 6 h. The mixture
was evaporated in vacuo, and the residue was purified by flash
chromatography on silica. Elution with a linear gradient of H1 in
EtOAc gave 41 as a white foam (0.180 g, 0.37 mmol).
(RS)-2-{[2-(3-Amino-3-carboxypropylthio)ethyl]dimethylammoni-
um}acetate (12). Compound 47 (0.188 g, 0.31 mmol) was depro-
tected according to the general procedure C, affording 12 as a white
1
solid (0.035 g, 42%). H NMR (500 MHz, D2O) 2.18 (m, 1H),
2.28 (m, 1H), 2.78 (m, 2H), 2.99 (m, 2H), 3.27 (s, 6H), 3.83 (m,
2H), 4.10 (s, 2H), 4.15 (t, 1H). 13C NMR (125.7 MHz, D2O) 26.03,
29.44, 32.36, 54.54 (2xC), 54.68, 65.11, 65.82, 170.67, 174.80.
HRMS (ESI) calcd for C10H21N2O4S [M]+ 265.1222; found
265.1236.
(RS)-2-Amino-4-{2-[(carboxymethyl)(methyl)amino]ethylthio}-
butanoic Acid (13). Compound 46 (0.181 g, 0.39 mmol) was
deprotected according to the general procedure C, affording 13 as
(RS)-2-Amino-4-[(2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepin-
1-ium-1-yl)methylthio]butanoate (42). Compound 41 (0.172 g, 0.35
mmol) was deprotected according to the general procedure C,
affording 42 as a white solid (0.062 g, 59%).
1
a white solid (0.71 g, 73%). H NMR (600 MHz, D2O) 2.20 (m,
1H), 2.31 (m, 1H), 2.80 (m, 2H), 2.99 (s, 3H), 3.00 (m, 2H), 3.50
(m, 2H), 4.08 (m, 2H), 4.21 (t, 1H). 13C NMR (150.9 MHz, D2O)

Inhibitors of Human BHMT
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3663
27.62, 28.90, 32.05, 43.82, 54.41, 58.00, 59.37, 171.27, 174.54.
HRMS (ESI) calcd for C9H19N2O4S [M + H]+ 251.1066; found
251.1070.
(RS)-tert-Butyl 4-(3-Bromopropylthio)-2-(tert-butoxycarbony-
lamino)butanoate (53). DBU (0.598 mL, 4.32 mmol, 1.1 equiv)
was added to a solution of thiol 36 (1.145 g, 3.93 mmol) in 1,3-
dibromopropane (10 mL), and the reaction mixture was left to stand
overnight at rt (TLC in T2). Excessive solvent was evaporated in
vacuo, and the crude product was purified by flash chromatography
on silica (elution with a linear gradient of EtOAc in toluene) to
give compound 53 as a white crystalline solid (1.32 g, 81%).
(RS)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-{3-[(tert-butoxy-
carbonylmethyl)(methyl)amino]propylthio}butanoate (54). Using
the procedure outlined for 46, compound 54 was prepared from 53
(0.300 g, 0.73 mmol), tert-butyl 2-(methylamino)acetate hydro-
chloride (0.145 g, 0.80 mmol, 1.1 equiv), sodium carbonate (0.231
g, 2.18 mmol, 3.0 equiv), sodium iodide (0.109 g, 0.73 mmol, 1
equiv), and tetrabutylammonium bromide (0.117 g, 0.36 mmol, 0.5
equiv) as a colorless solid (0.290 g, 82%).
tert-Butyl 2-(RS)-(tert-Butoxycarbonylamino)-4-[2-(S)-(1-tert-bu-
toxycarbonylethylamino)ethylthio]butanoate (48). Using the pro-
cedure outlined for 46, compound 48 was prepared from 45 (0.377
g, 1.06 mmol), (S)-tert-butyl 2-aminopropanoate (0.170 g, 1.17
mmol, 1.1 equiv), sodium carbonate (0.372 g, 3.51 mmol, 3.0
equiv), sodium iodide (0.175 g, 1.17 mmol, 1 equiv), and tetrabu-
tylammonium bromide (0.190 g, 0.59 mmol, 0.5 equiv) as a
colorless solid (0.234 g, 47%).
2-(RS)-Amino-4-[2-(S)-(1-carboxyethylamino)ethylthio]bu-
tanoic Acid (14). Compound 48 (0.220 g, 0.48 mmol) was
deprotected according to the general procedure C, affording 14 as
a white solid (0.36 g, 30%).
tert-Butyl 2-(RS)-(tert-Butoxycarbonylamino)-4-[2-(R)-(1-tert-bu-
toxycarbonylethylamino)ethylthio]butanoate (49). Using the pro-
cedure outlined for 46, compound 49 was prepared from 45 (0.354
g, 1.00 mmol), (R)-tert-butyl 2-aminopropanoate (0.160 g, 1.10
mmol, 1.1 equiv), sodium carbonate (0.318 g, 3.00 mmol, 3.0
equiv), sodium iodide (0.150 g, 1.00 mmol, 1 equiv), and tetrabu-
tylammonium bromide (0.161 g, 0.50 mmol, 0.5 equiv) as a
colorless solid (0.171 g, 37%).
(RS)-2-Amino-4-{3-[(carboxymethyl)(methyl)amino]propylthio}bu-
tanoic Acid (19). Compound 54 (0.225 g, 0.47 mmol) was
deprotected according to the general procedure C, affording 19 as
1
a white solid (0.69 g, 55%). H NMR (600 MHz, D2O) 2.06 (m,
2H), 2.18 (m, 1H), 2.28 (m, 1H), 2.68 (m, 2H), 2.74 (t, 2H), 2.97
(s, 3H), 3.28 (m, 1H), 3.41 (m, 1H), 3.96 (bd, 1H), 4.05 (bd, 1H),
4.18 (t, 1H). 13C NMR (150.9 MHz, D2O) 26.16, 29.07, 30.13,
32.21, 44.10, 54.65, 58.67, 59.49, 171.57, 174.79. HRMS (ESI)
calcd for C10H21N2O4S [M + H]+ 265.1222; found 265.1219.
(RS)-3-[3-(tert-Butoxycarbonylamino)-3-(tert-butoxycarbonyl)-
propylthio]-N-(carboxymethyl)-N,N-dimethylpropan-1-ammo-
nium iodide (55). The procedure outlined for 47 was followed using
amine 54 (0.222 g, 0.47 mmol) and methyl iodide (0.072 mL, 1.16
mmol, 2.5 equiv). The reaction was complete after 2 d, and
compound 55 was isolated as a dark-yellow solid (0.153 g, 53%).
(RS)-2-{[3-(3-Amino-3-carboxypropylthio)propyl]dimethylammo-
nio}acetate (18). Compound 55 (0.140 g, 0.23 mmol) was depro-
tected according to the general procedure C, affording 18 as a white
2-(RS)-Amino-4-[2-(R)-(1-carboxyethylamino)ethylthio]bu-
tanoic Acid (15). Compound 49 (0.154 g, 0.33 mmol) was
deprotected according to the general procedure C, affording 15 as
a white solid (0.022 g, 27%). NMR: Mixture of diastereoisomers
ca. 1:1; doubling of carbon signals observed. 1H NMR (600 MHz,
D2O) 1.58 (d, 3H), 2.19 (m, 1H), 2.29 (m, 1H), 2.78 (m, 2H), 2.93
(m, 2H), 3.34 (m, 2H), 4.06 (q, 1H), 4.18 (t, 1H). 13C NMR (150.9
MHz, D2O) 17.01 + 17.05, 28.82 + 28.86, 29.53, 32.11, 47.41 +
47.45, 54.58 + 54.59, 58.78 + 58.82, 174.75, 175.22 + 175.24.
HRMS (ESI) calcd for C9H19N2O4S [M + H]+ 251.1066; found
251.1057.
1
solid (0.059 g, 94%). H NMR (600 MHz, D2O) 2.07 (m, 2H),
2.15 (m, 1H), 2.22 (m, 1H), 2.67 (m, 2H), 2.71 (t, 2H), 3.24 (s,
3H), 3.27 (s, 3H), 3.68 (m, 2H), 3.95 (s, 2H), 3.99 (t, 1H). 13C
NMR (150.9 MHz, D2O) 24.77, 29.17, 30.06, 32.65, 54.33 (2 ×
C), 55.76, 66.00, 66.20, 171.50, 176.00. HRMS (ESI) calcd for
C11H23N2O4S [M]+ 279.1379; found 279.1370.
(RS)-Ethyl 5-Bromo-3-methylpentanoate (58). (RS)-Tetrahydro-
4-methylpyran-2-one39 (0.410 g, 3.59 mmol) was treated with a
saturated solution of anhydrous HBr in EtOH (15 mL), and the
mixture was stirred at rt for 3 d (TLC in T2). The reaction was
quenched by pouring it into 200 mL of water and extracting it with
Et2O (3 × 50 mL). The combined organic extracts were sequentially
washed with water (100 mL), sat. aq NaHCO3 (50 mL), and water
(100 mL), dried over Na2SO4, and evaporated in vacuo. The crude
product was purified by flash chromatography on silica (isocratic
elution, toluene only) to give compound 58 as a colorless liquid
with a camphor odor (0.660 g, 82%).
Ethyl 5-Bromo-3,3-dimethylpentanoate (59). The procedure
outlined for 58 was followed using tetrahydro-4,4-dimethylpyran-
2-one39 (1.03 g, 8.00 mmol) and saturated solution of anhydrous
HBr in EtOH (20 mL), giving compound 59 as a colorless liquid
with a camphor odor (1.690 g, 89%).
Ethyl (RS,RS)-5-[3-(tert-Butoxycarbonylamino)-3-(tert-butoxy-
carbonyl)propylthio]-3-methylpentanoate (60). NaH (60% disper-
sion in oil; 0.061 g, 1.52 mmol, 1.2 equiv) was added to a solution
of thiol 36 (0.369 g, 1.27 mmol, 1 equiv) in anhydrous THF (5
mL) in a flask equipped with a calcium chloride tube. Bromide 58
(0.282 g, 1.27 mmol, 1 equiv) was added after 10 min, and the
reaction was allowed to proceed overnight (TLC in T2). The mixture
was then concentrated in vacuo, and the residue was purified by
flash chromatography on silica (elution with a linear gradient of
EtOAc in toluene) to give compound 60 as a colorless thick oil
(0.300 g, 55%). NMR: Mixture of diastereoisomersssome carbon
signals are doubled.
(RS)-tert-Butyl 2-(tert-Butoxycarbonylamino)-4-[2-(tert-butoxy-
carbonylmethylamino)ethylthio]butanoate (50). Using the proce-
dure outlined for 46, compound 50 was prepared from 45 (0.403
g, 1.14 mmol), tert-butyl 2-aminoacetate (0.164 g, 1.25 mmol, 1.1
equiv), sodium carbonate (0.397 g, 3.75 mmol, 3.0 equiv), sodium
iodide (0.171 g, 1.14 mmol, 1 equiv), and tetrabutylammonium
bromide (0.184 g, 0.57 mmol, 0.5 equiv) as a pale-yellow thick oil
(0.250 g, 45%).
(RS)-2-Amino-4-[2-(carboxymethylamino)ethylthio]butanoic Acid
(16). Compound 50 (0.220 g, 0.49 mmol) was deprotected according
to the general procedure C, affording 16 as a white solid (0.012 g,
10%). 1H NMR (600 MHz, D2O) 2.17 (m, 1H), 2.26 (m, 1H), 2.75
(m, 2H), 2.94 (m, 2H), 3.35 (t, 2H), 3.84 (s, 2H), 4.06 (t, 1H). 13
C
NMR (150.9 MHz, D2O) 28.88, 29.44, 32.36, 48.78, 50.75, 55.23,
172.63, 175.50. HRMS (ESI) calcd for C8H17N2O4S [M + H]+
237.0909; found 237.0906.
(RS)-tert-Butyl 2-(tert-Butoxycarbonylamino)-4-[2-(2-tert-butoxy-
carbonylethylamino)ethylthio]butanoate (51) and Di-tert-butyl
(RS,RS)-4,4′-[2,2′-(2-tert-Butoxycarbonylethylaminodiyl)bis(ethane-
2,1-diyl)bis(sulfanediyl)]bis[2-(tert-butoxycarbonylamino)bu-
tanoate] (52). The procedure outlined for 46 was followed using
45 (0.354 g, 1.00 mmol), tert-butyl 3-aminopropanoate (0.160 g,
1.10 mmol, 1.1 equiv), sodium carbonate (0.318 g, 3.00 mmol, 3.0
equiv), sodium iodide (0.150 g, 1.00 mmol, 1 equiv), and tetrabu-
tylammonium bromide (0.161 g, 0.50 mmol, 0.5 equiv). Elution
with a linear gradient of EtOAc in toluene gave 51 (0.377 g, 81%)
and 52 (0.120 g, 15%) as colorless thick oils.
(RS)-2-Amino-4-[2-(2-carboxyethylamino)ethylthio]butanoic Acid
(17). Compound 51 (0.370 g, 0.80 mmol) was deprotected according
to the general procedure C, affording 17 as a white solid (0.087 g,
43%). 1H NMR (600 MHz, D2O) 2.18 (m, 1H), 2.26 (m, 1H), 2.76
(m, 2H), 2.86 (t, 2H), 2.94 (m, 2H), 3.34 (t, 2H), 3.38 (t, 2H), 4.09
(t, 1H). 13C NMR (150.9 MHz, D2O) 28.96, 29.31, 32.38, 32.71,
45.66, 49.05, 55.14, 175.40, 177.10. HRMS (ESI) calcd for
C9H18NO4S [M + H]+ 236.0957; found 236.0945.
Ethyl (RS)-5-[3-(tert-Butoxycarbonylamino)-3-(tert-butoxycar-
bonyl)propylthio]-3,3-dimethylpentanoate (61). The procedure out-
lined for 60 was followed using thiol 36 (0.369 g, 1.27 mmol, 1

3664 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Vaneˇk et al.
equiv), NaH (60% dispersion in oil; 0.061 g, 1.52 mmol, 1.2 equiv),
and bromide 59 (0.300 g, 1.27 mmol, 1 equiv), giving compound
61 as a colorless thick oil (0.430 g, 76%).
of 50 µM compound 21 (calculated for the S-enantiomer) were
stepwise injected into the sample cell containing 1.43 mL of 5.1
µM protein until saturation was achieved. The assay was ac-
companied by a control experiment in which compound 21 was
injected into buffer alone. The thermodynamic parameters and
association constants were estimated using MicroCal Origin
software.
(RS,RS)-5-(3-Amino-3-carboxypropylthio)-3-methylpentanoic Acid
(20). Compound 60 (0.290 g, 0.67 mmol) was deprotected according
to the general procedure B, affording 20 as a white solid (0.106 g,
64%). NMR: Mixture of diastereoisomers ca. 1:1; doubling of the
1
most of carbon signals observed. H NMR (600 MHz, DMSO-d6)
Acknowledgment. This work was supported by an NIH
Research Grant funded by the Fogarty International Center (R01
TW0052501, T.A.G. and J.J.), by a grant from the National
Institutes of Health (DK52501, T.A.G.), Research Project of
the Academy of Sciences of the Czech Republic (Z40550506,
J.J.), and by grants from the Ministry of Education, Youth and
Sports of the Czech Republic (LC060777, Research Centre for
Chemical Genetics, J.J., and 1M0508, Research Centre for New
Antivirals and Antineoplastics, M.K.).
0.89 (d, 3H), 1.40 (m, 1H), 1.53 (m, 1H), 1.93 (m, 1H), 1.97 (m,
1H), 2.02 (dd, 1H), 2.03 (m, 1H), 2.24 (dd, 1H), 2.48 (m, 1H),
2.53 (m, 1H), 2.56 (m, 1H), 2.62 (m, 1H), 3.96 (dd, 1H), 8.25 (b,
2H), 12.10 (b, 2H). 13C NMR (150.9 MHz, DMSO-d6) 19.36 +
19.39, 26.50 + 26.54, 28.39 + 28.43, 29.20 + 29.22, 30.36 +
30.38, 35.84 + 35.88, 41.09 + 41.11, 51.38, 170.98, 174.05. HRMS
(ESI) calcd for C10H20NO4S [M + H]+ 250.1113; found 250.1105.
(RS)-5-(3-Amino-3-carboxypropylthio)-3,3-dimethylpentanoic Acid
(21). Compound 61 (0.430 g, 0.96 mmol) was deprotected according
to the general procedure B, affording 21 as a white solid (0.154 g,
61%). 1H NMR (600 MHz, D2O) 1.02 (s, 6H), 1.63 (m, 2H), 2.18
(m, 1H), 2.27 (m, 1H), 2.28 (s, 2H), 2.61 (m, 2H), 2.72 (t, 2H),
4.14 (dd, 1H). 13C NMR (150.9 MHz, D2O) 28.51, 28.97, 29.09,
29.10, 32.32, 35.63, 43.88, 48.10, 54.69, 175.04, 180.04. HRMS
(ESI) calcd for C11H22NO4S [M + H]+ 264.1270; found 264.1276.
BHMT Inhibition Assays. Human recombinant BHMT was
prepared as described previously,6 and N-methyl-14C-betaine (57
mCi/mmol) was prepared and supplied by Moravek Biochemicals
(Brea, CA). Compounds were tested for their ability to inhibit
BHMT activity using an assay procedure that we have described
previously in detail.15,49 Briefly, DL-homocysteine was freshly
prepared by dissolving DL-homocysteine thiolactone hydrochloride
(15.4 mg) in 400 µL of 2 M NaOH. The solution was allowed to
stand for 5 min at rt. The solution was then neutralized by the
addition of 600 µL of a saturated solution of KH2PO4 and
immediately used in the BHMT assay.
The standard BHMT assay mixture (500 µL) used to determine
percent inhibition of activity contained 0.2 µM BHMT, different
concentrations of inhibitor (20 or 1 µM), 100 µM DL-homocysteine,
250 µM betaine (0.05 µCi), 10 mM ꢀ-mercaptoethanol, and 50 mM
K-phosphate buffer at pH 7.5. Human recombinant BHMT was
first mixed with the inhibitor(s), and then the substrates were added
and the mixture was incubated at 37 °C for 30 min. The reaction
was stopped by transfer of the reaction tubes to ice water and by
the addition of 2.5 mL ice-cold water. The samples were applied
to a Dowex 1 × 4 (200-400 mesh) column, and the nonreacted
betaine was washed from the column with water. Dimethylglycine
and methionine were eluted into scintillation vials with 1.5 mL of
1.5 M HCl, and then 10 mL of the scintillation mixture were added
to each vial and counted. Blanks contained all of the reaction
components except the enzyme; their values were subtracted from
the sample values. All samples were assayed in triplicate, and the
results (reproducible within ( 15%) are expressed relative (%) to
a sample containing no inhibitor.
Supporting Information Available: Synthesis of compounds
27-29, 63, and 64. Spectral data for nontarget compounds 23a,
23b, 24, 30-35, 38-55, and 58-61. HPLC purity data for target
compounds. Isothermal titration calorimetry (ITC). Molecular
modeling of inhibitor 21 into the active site of BHMT. 1H and 13
C
NMR spectra measurements for pH titration of compounds 12, 13,
and 16. Inhibition experiments with enantiomers 63 and 64. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
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measured using the conditions described above, except that the
concentrations of substrates used were 1 mM DL-homocysteine and
2 mM betaine (0.15 µCi). The inhibition at 10 different inhibitor
concentrations in duplicate was determined for each curve. The
data were analyzed by use of nonlinear regression fit using
the GraphPad Prism 5 program. The experiment was repeated three
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independent experiments ( SEM.
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21 to BHMT was monitored using a VP-ITC microcalorimeter
(MicroCal Inc., Northampton, MA) at 25 °C. The solutions of
reactants were prepared in 50 mM HEPES/NaOH, ACES/NaOH,
and Tris/HCl buffers, at pH 7.5, containing 5 mM ꢀ-mercaptoet-
hanol. The concentration of BHMT protein (calculated for a BHMT
monomer with a relative molecular weight of 45 kDa) was
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the respective buffer. The concentration of inhibitor 21 was
determined based on elemental analysis. Typically, 9 µL aliquots
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