Design and synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa)

Article abstract of DOI:10.1016/j.bmc.2003.12.039

A series of 3-mercapto-propionic acid derivatives that function as reversible inhibitors of carboxypeptidase U have been prepared. We present a successful design strategy using cyclic, low basicity guanidine mimetics resulting in potent, selective and bioavailable inhibitors of carboxypeptidase U (TAFIa).

Full text of DOI:10.1016/j.bmc.2003.12.039

Bioorganic & Medicinal Chemistry 12 (2004) 1151–1175
Design and synthesis of potent, orally active, inhibitors of
carboxypeptidase U (TAFIa)
Magnus O. Polla,a,* Louise Tottie,a Carita Norden,a,y Marcel Linschoten,a,{
Djordje Musil,a,x Susanne Trumpp-Kallmeyer,a,k Inger R. Aukrust,b,{ Rune Ringom,b,**
Kjetil H. Holm,b Siren M. Neset,b Marcel Sandberg,b,{ John Thurmond,c Peng Yu,c
Georgeta Hateganc and Herb Andersonc
aDepartment of Medicinal Chemistry, AstraZeneca R&D Molndal, S-431 83 Molndal, Sweden
¨
¨
bSINTEF Applied Chemistry, Organic Synthesis, PO Box 124 Blindern, N-0314 Oslo, Norway
cdeCODE genetics, 2501 DaveyRoad, Woodridge, IL 60517, USA
Received 7 November 2003; accepted 22 November 2003
Abstract—A series of 3-mercapto-propionic acid derivatives that function as reversible inhibitors of carboxypeptidase U have been
prepared. We present a successful design strategy using cyclic, low basicity guanidine mimetics resulting in potent, selective and
bioavailable inhibitors of carboxypeptidase U (TAFIa).
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
available. The antiplatelets are also associated with
bleeding complications. Clearly, there is a need for an
orally active antithrombotic or thrombolytic drug that
is clinically safe and requires less monitoring.
Venous and arterial thromboembolism is the largest
cause of disease and death in the Western world and
more than 30% of the population will once experience a
thromboembolic episode. Therapy available today
includes thrombolytics, anticoagulants and antiplatelets.
However, none of these therapies seems to be optimal. The
only thrombolytics on the market are the plasminogen
activators, which are parenterally administered and
associated with more or less severe bleeding compli-
cations. Among the anticoagulants, Warfarin requires
closely monitored therapy because of the high risk of
bleeding. Heparin on the other hand is not orally
Procarboxypeptidase U1 (proCPU, EC 3.4.17.20,
thrombin activatable fibrinolysis inhibitor, TAFI,2 pro-
carboxypeptidase R3 and plasma procarboxypeptidase
B
4,5) is a metallopeptidase, circulating in plasma. The
function of this enzyme in fibrinolysis was elucidated in
the mid 1990s.6 After activation, the enzymatic activity
of CPU is to cleave off C-terminal basic amino acids,
that is, lysine and arginine residues, from peptides and
proteins (Chart 1).
During fibrinolysis, C-terminal lysine and arginine residues
are formed by the action of plasmin on the fibrin network
and thereby high-affinity binding sites for plasminogen
and tPA (tissue-type plasminogen activator) are created.
Upon binding to the clot, plasminogen is efficiently
converted to plasmin and an acceleration in the rate of
fibrinolysis is achieved. Through the action of CPU on
partly degraded fibrin, the C-terminal lysine residues are
cleaved off. This diminishes the amount of plasminogen
and tPA able to bind to fibrin and thereby the amount of
plasmin formed. This proposed mechanism for suppression
of fibrinolysis provides the rationale for inhibiting CPU
in order to enhance clot lysis. Furthermore, since the
* Corresponding author. Tel.: +46-31-7762269; fax: +46-31-
7763724; e-mail: magnus.polla@astrazeneca.com
y
Current adress: AstraZeneca R&D Lund, Scheelevagen 2, S-221 87
Lund, Sweden.
Current adress: Atomos AB, Brusewitz vag 18, SE-13649Haninge,
Sweden.
Current adress: Merck KGaA –– Germany, Frankfurter Strasse
250-64293 Darmstadt, Germany.
Current adress: Untere Apotheke, Oberstadtstr.16, Haigerloch,
Germany.
{
x
k
{
Current adress: Synthetica AS, Oslo Research Park, Gaustadalleen
21, N-0349 Oslo, Norway.
**Current adress: Biovitrum AB, Rapsgatan 7, Box 6443, SE-751 37,
Uppsala, Sweden.
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.039

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M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
Chart 1. Binding of a substrate (black) in the CPU active site (red).
coagulation is unaffected, CPU inhibition may result in
less bleeding complications than conventional therapy.7
This fact may contribute to the interest in the pharmaceu-
tical industry for CPU as a potential drug target.8 The
concept of CPU inhibition for stimulation of fibrinolysis
is supported by in vitro studies in human blood and
plasma as well as in vivo studies in animals.9 A high
molecular weight peptide CPU inhibitor, potato carboxy-
peptidase inhibitor (PTCI), has been shown to be active
in rabbit venous and arterial thrombosis models.10À12
Figure 1. X-ray structure of compound 2 in complex with CPB. Zink
atom in magenta, water molecules in red and hydrogen bonds in
dotted yellow lines.
interactions (CPB numbering20). The guanidine forms
a bidentate charge–charge interaction with Asp256 in
the specificity pocket. Although racemic 2 was used for
the crystallisation, the configuration of the inhibitor
found in the X-ray structure correspond to the unnatural
form of arginine.
So far, only a few low molecular weight inhibitors of
CPU, for example, compound 113 and 214 with IC50=20
mM and 270 mM respectively, have been described
(Chart 2). Both compounds where originally reported as
carboxypeptidase N (CPN) inhibitors (CPN IC50=200
nM and 17 mM respectively) and both compounds have
also been reported to inhibit pancreatic carboxy-
peptidase B (CPB) with IC50=8 mM.15,16
Compound 1 and 2 have two major drawbacks from a
drug discovery point of view. Firstly, both compounds
are potent inhibitors of CPN. CPN is believed to have an
important function in plasma as an inactivator of ana-
phylatoxins and in the processing of peptide hormones.21
Consequently, selectivity towards CPN was an important
parameter to consider during optimisation. Secondly,
both compounds are extremely polar resulting in low
oral bioavailability. Our approach was to improve
bioavailability simultaneously with potency and selec-
tivity towards CPN. The strongly basic guanidine group
was identified as the major obstacle for achieving a high
bioavailability and we reasoned that a low basicity
guanidine replacement should solve that problem. Fur-
thermore, if cyclic guanidine replacements where used,
increased potency and selectivity may be an additional
bonus. It was decided to use the neutral thiol moiety as zinc
binding group since charged alternatives, for example, imi-
dazole, carboxylate or phosphinate groups would reduce
the possibility to obtain compounds with good drug
metabolism and pharmacokinetic (DMPK) properties.
In this paper, we report the design and synthesis of the
first potent, selective and orally active inhibitors of
CPU.17
2. Results and discussion
CPU has a very low solubility and is also a very
unstable enzyme in vitro, factors probably contributing
to the fact that no X-ray crystallographic structure has
been published. The X-ray structure of several complexes
between CPA and different inhibitors has been reported18
while for CPB only the X-ray coordinates for the back-
bone atoms has been reported.19 In order to obtain a
more relevant starting point for drug design, the structure
of compound 2 bound to porcine CPB was solved using
X-ray crystallography to a resolution of 1.55 A (Fig. 1).
As expected, one of the carboxylates in compound 2
interacts with the zinc atom while the other carboxylate
interacts in a bidentate fashion with Arg145 and in a
monodentate fashion with Arg124 through charge–charge
A homology model of CPU was considered to be sufficient
for the first design steps and such a model was con-
structed using the protein structure analysis program
WHAT IF22 based on the X-ray structure of porcine CPB
(49% sequence identity) in complex with compound 2.
The CPU homology model indicated only small back-
bone deviations from the CPB structure.
In the first design cycle six different cyclic basic
groups was incorporated using the CPA inhibitor 2-
mercaptomethyl-3-phenyl-propionic acid as a template.23
Although no crystal structure has been reported for
2-mercaptomethyl-3-phenyl-propionic acid we reasoned
that the binding conformation should be similar to the
Chart 2. Commercial carboxypeptidase inhibitors 1 and 2.

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1153
conformation reported for benzylsuccinic acid in com-
plex with CPA.24 Superimposing the X-ray structures of
compound 2 in complex with CPB with benzylsuccinic
acid in complex with CPA indicate that basic groups
could be merged with the 2-mercaptomethyl-3-phenyl-
propionic acid template (Fig. 2).
X-ray structure of compound 8 complexed to bovine CPB
shows that this compound binds very much as expected.
The thiol interacts with the zinc atom in a tetragonal
fashion while the carboxylate interact in a bidentate
fashion with Arg143 and in a monodentate fashion with
Arg125. The aminopyridine forms a bidentate interaction
with Asp257 in the specificity pocket. The positions of
the aminopyridine nitrogens are identical to the guanidine
nitrogens of compound 2 in the X-ray structure.
The resulting inhibitors 3–8 were energy minimised in
the active site of the CPU homology model using the
docking programs DOCK25 and GOLD.26 The pre-
dicted binding conformations for all compounds looked
reasonable with the thiol interacting with the zinc atom,
the carboxylate interacting with Arg124 and Arg145
and the basic group interacting with Asp256 (CPB
numbering27).
With the potent and fairly selective compound 8 as a
starting point, we tried to increase selectivity towards
CPN further. The CPU homology model indicated that
there is some space around the pyridine ring, especially
around the 2-and 3p- osition, and our next step was to
investigate this area. Compounds 9–11 was first
designed to evaluate this (Chart 4).
Compounds 3–5 (Chart 3), containing five-membered
ring basic groups, with calculated pKa values of 11.3,
10.8 and 5.4 respectively, gave valuable information
(Table 1). Compounds 3–5 where equally potent, with
CPU IC50 values around 1 mM but the selectivity
towards CPN differed significantly with CPN IC50
values of 0.5 mM, 8 mM and 40 mM, respectively. Inter-
estingly, the basicity does not seem to influence CPU
binding affinity indicating that the interaction with
Asp256 may be of hydrogen bond character and not a
charge–charge interaction.28 However, compound 5 has
the possibility to interact in a bidentate fashion with
Asp256, possibly compensating for the effect of lower
basicity. Compound 6 had a similar profile as the pyr-
rolidine 3, with a CPU IC50 of 3 mM and a CPN IC50 of
0.5 mM, while the 2-aminopyridine 7, showed reversed
selectivity with a CPU IC50 of 3 mM and a CPN IC50 of
6 mM. The regioisomer, aminopyridine 8 proved to be
the best of the six compounds in the first design cycle.
This compound was potent with an IC50 of 0.2 mM
(Ki=22 nM) with 15 times selectivity toward CPN.
Furthermore, the weakly basic 2-aminopyridine (calculated
pKa=7) should result in a higher bioavailability than
the guanidine-containing starting points, 1 and 2. The
Chart 3. Inhibitors 3–8.
Table 1. Enzyme inhibition data on compounds 1–19
b
Compd
CPUa
IC50 (mM)
CPNa
IC50 (mM)
CPBa
IC50 (mM)
pKa
calcd
1
2
3
4
5
6
7
8
20
270
1.6
1
1.3
3.2
3.2
0.2
7.9
6.3
1
10
13
250
83
0.9
0.5
0.6
1.6
0.2
17
0.5
8
40
0.5
6.3
3.2
5
1.3
8
8
12.7
12.7
11.3
10.2
5.9
11.3
7.3
6.8
7.4
7.0
7.1
4.0
6.2
6.8
7.0
6.9
6.6
3.2
3.2
0.2
7.9
10
0.13
5
4.0
0.13
0.8
4
40
41
0.8
0.05
0.5
0.2
9
10
11
12
13
14
15
16
17
18
19
50
>2000
398
500
>1000
72
20
16
200
6.8
7.2
a The assays was performed as described earlier30 at pH 7.4 (CPU and
CPN) and at pH 6.0 (CPB).
b pKa was calculated with ACD/Labs 6.00 from Advanced Chemistry
Development Inc., Canada.
Figure 2. Superimposition of the X-ray structures of compound 2 in
complex with CPB (magenta), and benzylsuccinic acid (orange) in
complex with CPA (yellow).

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M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
good bioavailability (74%) and a medium clearance
(27 mL/min/kg), indicating that our design strategy
was indeed successful. In a disseminated intravascular
coagulation model in the rat, the compound increases
the rate of thrombus dissolution with an ED50
value of around 1 mmol/kg after intravenous or oral
administration.29
3. Inhibitor synthesis
The presence of the 3-mercaptopropanoic acid fragment
in the proposed inhibitors led us to consider a synthetic
strategy based on malonate alkylation chemistry. This
would provide the possibility to investigate different
basic groups using one synthetic approach. One of the
key intermediates, bromide 23, was synthesized from 6-
amino-nicotinic acid (20) in four steps. Esterification
and BOC protection followed by reduction to the alcohol
and finally bromination using Ph3P and CBr4 in methyl-
ene chloride gave bromide 23 in 30% overall yield.
While the 5-chloro analogue (27) could be synthesized
using the same route, the methyl-substituted analogues
33, 37 and 41 had to be synthesized using another route.
These bromides was conveniently accessed from the
corresponding aryl bromides via a palladium catalyzed
coupling with a hydroxymethyl equivalent, TBSOCH2-
SnBu3, followed by deprotection of the TBS group and
bromination (Scheme 1).
Chart 4. Inhibitors 9–13.
Methyl substitution at the 4-and 6-position, compound
9 and 10 respectively, resulted in decreased CPU affi-
nity. On the other hand, methyl substitution at the 3-
position resulted in an increased selectivity towards
CPN with only a slight decrease in CPU affinity. Chloro
substitution at the 3-position, compound 12, resulted in
significantly decreased CPU affinity. This could be an
effect of the low basicity (calculated pKa=4.3).
In the X-ray structure of compound 2 complexed to
CPB, two water molecules can be seen close to the gua-
nidine nitrogens (Fig. 1). The well-tolerated methyl
group in compound 11 served as a handle and com-
pound 13 was designed with the intention to replace or
interact with these water molecules. This attempt resul-
ted however, in a decrease in binding affinity of more
than two orders of magnitude. In the CPB crystal
structures as well as in the CPU homology model, there
was a small pocket below the carboxylate in the inhibi-
tors, close to the zinc atom. Attempts to reach this
pocket from the a-position showed that only small sub-
stituents where tolerated, with ethyl (compound 14) and
hydroxymethyl (compound 15) being detrimental for
activity (Chart 5). The smaller substituents hydroxy,
fluoro and methyl (compounds 16, 17 and 18, respec-
tively) resulted in only slightly decreased binding affi-
nity. Compound 18 showed an increased selectivity
towards CPN and the combination of a-methyl and 2-
methyl substitution (compound 19) resulted in more
than two orders of magnitude selectivity towards CPN.
As expected from the homology model, the compounds
show no selectivity towards CPB.
With the key bromides at hand, alkylation with diethyl
malonate/NaH followed by careful ester hydrolysis to
form the monoacid could be accomplished. This was
followed by a consecutive decarboxylation/Mannich
reaction to give the alkenoic esters 43, 46, 49, 52 and 55.
Michael addition of thioacetic acid followed by exhaustive
deprotection using boiling concentrated hydrochloric
acid gave compounds 8–12 as the hydrochloride salts
(Scheme 2).
Compound 7 was also synthesized using this method,
starting from 2-acetylamino-isonicotinic acid. Ester-
ification and deacetylation was accomplished using
BF3*OEt2 in EtOH followed by Boc protection of the
amine function and reduction of the ester using LiAlH4.
The obtained alcohol was then brominated (Ph3P/CBr4/
CHCl2) and subsequently converted to compound 7
using the same procedure as for compound 8–12
(Scheme 3).
The most potent inhibitor, compound 8, was selected
for further studies. DMPK studies in the rat show a
Compound 4 was synthesized starting from cis-(4-
hydroxymethyl-cyclopent-2-enyl)-carbamic acid tert-
butyl ester (62), via mesylation and bromination using
LiBr in acetone. The bromide was then converted to
compound 4 using the standard route (Scheme 4).
The synthesis of compound 3 started from 1-benzyl-5-
oxo-pyrrolidine-3-carboxylic acid (65), with concom-
itant reduction of the acid and the lactam carbonyl
group. After debenzylation and reprotection as the BOC
derivative, the alcohol was converted to the bromide (67)
via the triflate. The synthesis was completed using the
standard procedures (Scheme 5).
Chart 5. Inhibitors 14–19.

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1155
Scheme 1. Conditions: (i) SOCl2, EtOH; (ii) Boc2O, DMAP, tBuOH/acetone; (iii) LiAlH4, THF; (iv) Ph3P, CBr4, CHCl2; (v) TBSOCH2SnBu3,
(Ph3P)2PdCl2, 1,2-dichloroethane; (vi) Bu4NF, THF.
Compounds 17 and 18 were synthesised from bromide
23 and the fluoro-and methyls-ubstituted malonates,
respectively. In this case, ester hydrolysis was followed by
reduction of the acid to give the corresponding alcohols
(72 and 73) via the mixed anhydride (MeOCOCl, Et3N
then NaBH4). The alcohol was then converted to the
thiol using the Mitsunobu reaction followed by exhaustive
deprotection (Scheme 6).
A similar route was used for compounds 14 and 19, but
ethyl-or methyls-ubstituted Meldrums acid was used
instead of the corresponding malonate. Alkylation of
the Meldrums acid derivatives using triethyl amine/
DMSO and bromide 23 or 41, gave compound 74 and
75, respectively. Treatment with NaOEt in EtOH resulted
in ring opening giving the monoacids in good yield. The
acid was then reduced to the alcohol via the mixed
anhydride method. The alcohol was then converted to
thiols 14 and 19 using the Mitsunobu reaction followed
by removal of the protecting groups using concentrated
aqueous HCl (Scheme 7).
The synthesis of compound 15 was accomplished using
this route as well, but starting from the malonate.
Double alkylation, first with bromide 41 as earlier, and
then with BnOCH2Cl gave the disubstituted malonate,
Scheme 2. Conditions: (i) (EtOCO)2CH2, NaH, THF; (ii) KOH,
EtOH/CH2Cl2; (iii) Et2NH, CH2O(aq), CH2Cl2; (iv) Et3N, AcSH; (v)
HCl(aq)
.
Scheme 3. Conditions: (i) BF3*OEt2, EtOH; (ii) Boc2O, DMAP, tBuOH/acetone; (iii) LiAlH4, THF; (iv) Ph3P, CBr4, CHCl2; (v) (EtOCO)2CH2,
NaH, THF; (vi) KOH, EtOH/CH2Cl2; (vii) Et2NH, CH2O(aq), CH2Cl2; (viii) Et3N, AcSH; (ix) HCl(aq)
.

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M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
Scheme 4. Conditions: (i) MsCl, Et3N, CH2Cl2; (ii) LiBr, acetone;
(iii) (EtOCO)2CH2, NaH, THF; (iv) KOH, EtOH/CH2Cl2; (v) Et2NH,
CH2O(aq), CH2Cl2; (vi) Et3N, AcSH; (vii) HCl(aq)
.
Scheme 6. Conditions: (i) tBuOCOCHMeCOOEt, NaH, DMF or
(EtOCO)2CHF, NaH, DMF; (ii) NaOH, EtOH/THF; (iii) MeOCOCl,
Et3N, THF; (iv) NaBH4, THF; (v) AcSH, DEAD, Ph3P, THF;
which was then hydrolysed to the monoacid. Reduction
of the carboxylic acid to the corresponding alcohol
could not be accomplished in good yields using our
standard procedure on this compound. After some
experimentation, it was found that activation of the acid
using Bop/Et(iPr)2N in THF reproducibly gave the
active ester. It was then necessary to exchange the solvent
to ethanol and to use LiBH4 instead of NaBH4 in order
to obtain the alcohol in good yield. The benzyl ether was
then deprotected using catalytic hydrogenation to give the
symmetric diol (79). One of the hydroxy groups was then
converted to the thiol using the Mitsunobu reaction.
This procedure gave the mono-thiol in 45% yield after
standard deprotection (Scheme 8).
(vi) HCl(aq)
.
acetylated in order to facilitate purification. Standard
deprotection then gave compound 13 (Scheme 9).
The tertiary alcohol 16 was synthesized starting from
intermediate 43. Osmiumtetroxide catalyzed dihydroxyl-
ation followed by introduction of the thioacetate using
Mitsunobu conditions and subsequent deprotection
gave compound 16 in good yield (Scheme 10).
Aldehyde 83 was used as starting material for the
synthesis of compound 5. A Knoevenagel reaction with
diethylmalonate followed by reduction of the double
bond and BOC protection of the amine gave the
required intermediate. The standard procedure via
Mannich and Michael sequences gave compound 5 after
deprotection (Scheme 11).
The synthesis of compound 13 was accomplished using
the same method as for compounds 8. However, the
hydroxymethyl group could not be introduced using
TBSOCH2SnBu3 and instead Bu3SnCHCH2 was used
as a protected hydroxymethyl equivalent and coupling
partner for aryl bromide 29. After conversion to the
thioacetate 81, the vinyl group was cleaved using ozono-
lysis with reductive workup. This procedure resulted in a
mixture of alcohols containing unchanged thioacetate as
well as some free thiol. This material was immediately
Compound 6 was synthesized using another strategy
starting from pyridine 86. Concomitant hydrogenation
of the olefin and the pyridine ring gave a piperidine that
was subsequently BOC protected. Carboxylic acid 87 was
then alpha-alkylated using BrCH2SBn as a thiomethyl
Scheme 5. Conditions: (i) Red-Al, THF; (ii) NH4O2CH, Pd/C, MeOH; (iii) Boc2O, K2CO3, THF/H2O; (iv) TfCl, Et3N, CH2Cl2; (v) LiBr, acetone;
(vi) (EtOCO)2CH2, NaH, THF; (vii) KOH, EtOH/CH2Cl2; (viii) Et2NH, CH2O(aq), CH2Cl2; (ix) Et3N, AcSH; (x) HCl(aq)
.

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1157
Scheme 9. Conditions: (i) Bu3SnCHCH2, (Ph3P)4Pd, 1,2-dichloro-
ethane; (ii) DIBAL, THF; (iii) Ph3P, CBr4, CHCl2; (iv) (EtOCO)2CH2,
NaH, DMF; (v) KOH, EtOH/CH2Cl2; (vi) Et2NH, CH2O(aq), CH2Cl2;
(vii) Et3N, AcSH; (viii) O3, EtOH, then NaBH4, water; (ix) Ac2O,
KHCO3; (x) HCl(aq)
.
Scheme 7. Conditions: (i) 5-alkyl-2,2-dimethyl-1,3-dioxane-4,6-dione,
Et3N, DMSO; (ii) NaOEt, EtOH; (iii) MeOCOCl, Et3N, THF; (iv)
NaBH4, THF; (v) AcSH, DEAD, Ph3P, THF; (vi) HCl(aq)
.
Scheme 10. Conditions: (i) OsO4, N-methyl morpholine N-oxide,
acetone/water; (ii) DEAD, Ph3P, AcSH; (iii) HCl(aq)
.
Scheme 8. Conditions: (i) tBuOCOCH2COOEt, NaH, THF; (ii)
BnOCH2Cl, LDA, THF; (iii) KOH, EtOH; (iv) Bop, Et(iPr)2N, THF;
(v) LiBH4, EtOH; (vi) H2, Pd/C, EtOH/HCl; (vii) AcSH, DEAD,
Ph3P, THF; (viii) HCl(aq)
.
equivalent. Benzyl deprotection using sodium in liquid
ammonia followed by BOC deprotection gave com-
pound 6 (Scheme 12).
4. Conclusion
The data shown in this paper suggest that the 3-mer-
capto-propionic acid scaffold is useful for preparing
potent inhibitors of carboxypeptidase U (TAFIa). The
introduction of cyclic, low basicity guanidine mimetics,
for example, 2-amino-pyridine, resulted in good oral
bioavailability and several compounds show a good
selectivity towards CPN.
Scheme 11. Conditions: (i) (EtOCO)2CH2, piperidine, MS 4A, DMF/
CH2Cl2; (ii) NaCNBH3, HCl(aq), EtOH; (iii) Boc2O, Et3N, DMAP,
CH2Cl2; (iv) KOH, EtOH/THF; (v) Et2NH, CH2O(aq), CH2Cl2;
(vi) Et3N, AcSH; (vii) HCl(aq)
.

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M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
di-tert-butyl dicarbonate (6.95 g, 32 mmol) were added.
The reaction mixture was stirred at room temperature
overnight. The THF was removed under reduced pressure,
water added and the aqueous phase was extracted with
EtOAc. The combined organic phases were dried and
concentrated under reduced pressure to give 4.64 g of
the crude product. Flash chromatography (Heptane/
EtOAc: 1/0!68/32) of the crude product afforded 3-
hydroxymethyl-pyrrolidine-1-carboxylic acid tert-butyl
ester (3.82 g, 60%) as a colorless oil. 1H NMR
(300 MHz, CDCl3): d 1.45 (br s, 9H), 1.55–2.05 (m, 3H),
2.3–2.5 (m, 1H), 3.0–3.2 (m, 1H), 3.2–3.7 (m, 5H). 13C
NMR (75 MHz, CDCl3): d 154.6, 79.2, 64.2, 48.7, 48.2,
45.4, 45.0, 41.3, 40.5, 28.1, 27.4.
Scheme 12. Conditions: (i) H2 (60 bar), Ru (5% on alumina), NH3/
H2O; (ii) Boc2O, NaHCO3, DMAP, THF/H2O; (iii) LDA, BrCH2SBn,
THF; (iv) Na, NH3(l), THF; (v) TFA, Et3SiH, CH2Cl2.
5.2.3. 3-Trifluoromethanesulfonyloxymethyl-pyrrolidine-
1-carboxylic acid tert-butyl ester. Methanesulfonyl chlo-
ride (0.4 mL, 5.17 mmol) was added dropwise to a
solution of 3-hydroxymethyl-pyrrolidine-1-carboxylic
acid tert-butyl ester (1 g, 4.97 mmol) and triethyl amine
(1.04 mL, 7.46 mmol) in CH2Cl2 (15 mL) at 0 ꢀC. The
reaction mixture was stirred at room temperature over-
night. After filtration CH2Cl2 was added, and the
organic phase was washed with 1 M HCl, dried and
concentrated under reduced pressure to yield 3-tri-
fluoromethanesulfonyloxymethyl-pyrrolidine-1-car-
boxylic acid tert-butyl ester (1.4 g, 97%). 1H NMR
(400 MHz, CDCl3): d 1.45 (br s, 9H), 1.65–1.78 (m, 1H),
1.98–2.1 (m, 1H), 2.55–2.68 (m, 1H), 3.0 (s, 3H), 3.1–3.2
(m, 1H), 3.3–3.6 (m, 3H), 4.1–4.25 (m, 2H).
5. Experimental
5.1. General
Mass spectra were recorded on a VG Platform II mass
spectrometer equipped with an electrospray interface
1
(LC-MS). H NMR measurements were performed on
Varian UNITY plus 400, 500 and 600 spectrometers,
1
operating at H frequencies of 400, 500 and 600 MHz
respectively. NMR spectra were recorded in DMSO,
D2O, CD3CN or mixtures thereof. Chemical shifts are
given in ppm with the solvent as internal standard.
Chromatography separations were performed using
Merck Silica gel 60 (0.063–0.200 mm). The compounds
named below were named using ACD/Name version
6.06/11 June 2002 available from advanced chemistry
development inc., Canada.
5.2.4. 3-Bromomethyl-pyrrolidine-1-carboxylic acid tert-
butyl ester (67). A mixture of 3-trifluoromethane-
sulfonyloxymethyl-pyrrolidine-1-carboxylic acid tert-butyl
ester (3.85 g, 13.8 mmol) and LiBr (3.61 g, 42 mmol) in
dry acetone (30 mL) was refluxed overnight. The reaction
mixture was allowed to cool to room temperature,
filtered and concentrated. The residue was dissolved in
CH2Cl2 and washed with water, dried and concentrated
under reduced pressure to give 6 g of the crude product.
Purification by flash chromatography (Heptane/EtOAc:
1/0!68/32) afforded compound 67 (2.84 g, 78%) as a
5.2. 2-Mercaptomethyl-3-pyrrolidin-3-yl-propionic acid (3)
5.2.1. (1-Benzyl-pyrrolidin-3-yl)-methanol (66). Red-Al
(160 mL of a 3.5 M solution in toluene, 560 mmol) was
added to a solution of 1-benzyl-5-oxo-pyrrolidine-3-
carboxylic acid (20 g, 91 mmol) in dry THF (650 mL)
under argon. The reaction mixture was refluxed for 2.5
h and then poured onto a mixture of crushed ice and
NaOH (20%). The phases were separated, the aqueous
phase was extracted with toluene and the combined
organic phases were dried and concentrated under
reduced pressure to give crude 66 as a yellow oil 17.9 g.
1H NMR (400 MHz, CDCl3): d 1.6–1.8 (m, 1H), 1.9–2.1
(m, 1H), 2.2–2.4 (m, 2H), 2.4–2.55 (m, 1H), 2.55–2.7 (m,
1H), 2.75–2.9 (m, 1H), 3.48–3.55 (m, 1H), 3.58 (br s,
2H), 3.63–3.7 (m, 1H), 7.14–7.35 (m, 5H).
1
colorless oil. H NMR (400 MHz, CDCl3): d 1.46 (br s,
9H), 1.65–1.8 (m, 1H), 2.0–2.1 (m, 1H), 2.5–2.65 (m,
1H), 3.05–3.15 (m, 1H), 3.26–3.65 (m, 5H).
5.2.5. 2-(1-tert-Butoxycarbonyl-pyrrolidin-3-ylmethyl)-
malonic acid diethyl ester (68). Diethyl malonate (1.93
mL, 12.7 mmol) was added dropwise to a solution of
NaH (60%; 0.51 g, 12.8 mmol) in dry THF (15 mL) at
0 ꢀC. The mixture was stirred at room temperature for 1
h after which it was added to a refluxed mixture of
compound 67 (2.8 g, 10.6 mmol) in dry THF (30 mL).
The reaction mixture was further refluxed for 19 h, and
then concentrated to almost dryness. Water (1 L) was
added, and the product was extracted with CH2Cl2. The
combined organic phases were dried and concentrated
under reduced pressure to yield 3.3 g of the crude pro-
duct. Purification by flash chromatography (CH2Cl2/
EtOAc: 1/0!68/32) afforded compound 68 (1.64 g,
5.2.2. 3-Hydroxymethyl-pyrrolidine-1-carboxylic acid tert-
butyl ester. Pd (10% on carbon, 6.1 g) and ammonium
formate (10g, 158 mmol) were added to a solution of
crude 66 (6.1 g, 32 mmol) in methanol (220 mL) under
argon. After reflux for 15 min the reaction mixture was
filtered while warm through a pad of Celite, the Celite
was further washed with methanol, and the combined
organic phases were concentrated. The residue was
dissolved in THF (35 mL) and water (35 mL), the solution
was cooled to 0 ꢀC and K2CO3 (22 g, 159 mmol) and
1
45%). H NMR (400 MHz, CDCl3): d 1.26 (t, J=7 Hz,
6H), 1.44 (br s, 9H), 1.46–1.55 (m, 1H), 1.95–2.05 (m,
3H), 2.05–2.2 (m, 1H), 2.8–2.95 (m, 1H), 3.16–3.3 (m,

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1159
1H), 3.3–3.4 (m, 1H), 3.4–3.6 (m, 2H), 4.2 (q, J=7 Hz,
4H). 13C NMR (100 MHz, CDCl3): d 169.1, 169.0,
154.4, 79.1, 61.5, 51, 50.8, 45.3, 36.6, 31.9, 31.1, 28.5, 14.
2H). 13C NMR (100 MHz, CDCl3): d 195.1, 173.9,
173.8, 154.4, 79.0, 60.8, 51.1, 45.4, 44.6, 35.3, 35.2, 30.7,
30.6, 30.5, 28.5, 14.2.
5.2.6. 2-(1-tert-Butoxycarbonyl-pyrrolidin-3-ylmethyl)-
malonic acid monoethyl ester. A solution of KOH (0.26
g; 4.6 mmol) in ethanol (7 mL) was added to a solution
of compound 68 (1.52 g; 4.4 mmol) in ethanol (7 mL) at
0 ꢀC. The reaction mixture was stirred at room temper-
ature overnight, concentrated under reduced pressure
and the residue was dissolved in water (500 mL). The
aqueous layer was washed with diethyl ether, acidified
to pH 3 by 0.5 M HCl, and extracted with diethyl ether.
The organic phase was dried and concentrated under
reduced pressure to yield 2-(1-tert-butoxycarbonyl-pyr-
rolidin-3-ylmethyl)-malonic acid monoethyl ester (1.13
5.2.9. 2-Mercaptomethyl-3-pyrrolidin-3-yl-propionic acid
(3). A solution of 3-(3-acetylsulfanyl-2-ethoxycarbonyl-
propyl)-pyrrolidine-1-carboxylic acid tert-butyl ester
(0.52 g; 1.45 mmol) in concentrated HCl (15 mL) was
refluxed under argon for 1 h. The reaction mixture
was allowed to cool to room temperature and concentrated
under reduced pressure to afford a diastereomeric mix-
ture of compound 3 as the hydrochloride salt (0.33 g;
1
100%). H NMR (400 MHz, D2O): d 1.6–1.95 (m, 3H),
2.2–2.5 (m, 2H), 2.65–3.0 (m, 4H), 3.25–3.35 (m, 1H),
3.4–3.6 (m, 2H). 13C NMR (100 MHz, D2O): d 178.8,
50.2, 50.1, 48.1, 45.5, 36.0, 33.8, 30.2, 29.9, 25.8, 25.6.
MS (+) 190 (M+1). HRMS (ESI) calcd for
C8H15NO2S 189.0824, found 189.0850.
1
g, 81%). H NMR (400 MHz, CDCl3): d 1.28 (t, J=7
Hz, 3H), 1.45 (br s, 9H), 1.47–1.6 (m, 1H), 1.95–2.1 (m,
1H), 2.1–2.25 (m, 3H), 2.85–2.95 (m, 1H), 3.2–3.3 (m,
1H), 3.35–3.6 (m, 3H), 4.22 (q, J=7 Hz, 2H). 13C NMR
(100 MHz, CDCl3): d 172.7, 172.5, 169.2, 169.1, 154.7,
79.6, 61.8, 51, 50.5, 45.5, 36.5, 32.0, 31.0, 28.5, 14.0.
5.3. 3-(cis-4-Amino-cyclopent-2-enyl)-2-mercaptomethyl-
propionic acid (4)
5.3.1. cis-Methanesulfonic acid 4-tert-butoxycarbonyl-
amino-cyclopent-2-enylmethyl ester. Methanesulfonyl
chloride (0.76 mL, 9.8 mmol) was added to a solution of
compound 62 (2 g, 9.4 mmol) and triethyl amine (1.96
mL, 14.1 mmol) in CH2Cl2 (30 mL) at 0 ꢀC. The reaction
mixture was stirred at room temperature overnight.
After filtration CH2Cl2 was added, and the organic phase
was washed with 1 M HCl, dried and concentrated under
reduced pressure to yield cis- methanesulfonic acid 4-
tert-butoxycarbonylamino-cyclopent-2-enylmethyl ester
5.2.7. tert-Butyl 3-[2-(ethoxycarbonyl)prop-2-en-1-yl]-
pyrrolidine-1-carboxylate (69). Diethyl amine (0.34 mL;
3.3 mmol) was added to a mixture of 2-(1-tert-butoxy-
carbonyl-pyrrolidin-3-ylmethyl)-malonic acid mono-
ethyl ester (0.69 g, 2.19 mmol) in 36% aqueous solution
of formaldehyde (0.27 mL, 3.5 mmol), CH2Cl2 (1.6 mL)
and water (1.6 mL) at 0 ꢀC. The reaction mixture was
stirred at room temperature overnight, poured onto ice-
water (500 mL) and extracted with CH2Cl2. The com-
bined organic phases were washed with 5% NaHCO3,
dried and concentrated under reduced pressure to yield
1
(2.64 g, 96%). H NMR (400 MHz, CDCl3): d 1.3–1.4
(m, 1H), 1.44 (s, 9H), 2.53–2.65 (m, 1H), 3.0 (br s, 4H),
4.12–4.22 (m, 2H), 4.55–4.8 (m, 2H), 5.75–5.85 (m, 2H).
13C NMR (100 MHz, CDCl3): d 155.1, 134.7, 132.4,
79.4, 72.0, 56.0, 44.0, 37.4, 34.7, 28.4.
1
compound 69 (0.55 g, 87%) as a colorless oil. H NMR
(400 MHz, CDCl3): d 1.3 (t, J=7 Hz, 3H), 1.45 (br s,
9H), 1.47–1.65 (m, 1H), 1.9–2.0 (m, 1H), 2.37 (m, 3H),
2.85–3.0 (m, 1H), 3.2–3.3 (m, 1H), 3.35–3.6 (m, 2H), 4.2
(q, J=7 Hz, 2H), 5.55 (s, 1H), 6.18 (s, 1H). 13C NMR
(100 MHz, CDCl3): d 166.9, 154.5, 139.0, 125.9, 79,
60.7, 51.1, 50.8, 45.5, 45.1, 37.7, 36.9, 35.5, 31.4, 30.6,
28.5, 14.2.
5.3.2.
cis-(4-Bromomethyl-cyclopent-2-enyl)-carbamic
acid tert-butyl ester. A mixture of cis-methanesulfonic acid
4-tert-butoxycarbonylamino-cyclopent-2-enylmethyl ester
(2.51 g, 8.6 mmol) and LiBr (2.24 g, 25.8 mmol) in dry
acetone (20 mL) was refluxed overnight. The reaction
mixture was allowed to cool to room temperature,
filtered and concentrated. The residue was dissolved in
CH2Cl2 and washed with water, dried and concentrated
under reduced pressure to give cis-(4-bromomethyl-
cyclopent-2-enyl)-carbamic acid tert-butyl ester (2.23 g,
94%). 1H NMR (400 MHz, CDCl3): d 1.26–1.36 (m,
1H), 1.45 (s, 9H), 2.55–2.68 (m, 1H), 3.02–3.13 (m, 1H),
3.36–3.48 (m, 2H), 4.5–4.8 (m, 2H), 5.74–5.83 (m, 2H).
13C NMR (100 MHz, CDCl3): d 155.1, 134.6, 133.9,
79.3, 56.1, 46.3, 38.2, 37.1, 28.4.
5.2.8. 3-(3-Acetylsulfanyl-2-ethoxycarbonyl-propyl)-pyr-
rolidine-1-carboxylic acid tert-butyl ester. Thioacetic
acid (5 mL), which had been cooled to 0 ꢀC, was added
to a mixture of 3-(2-ethoxycarbonyl-allyl)-pyrrolidine-1-
carboxylic acid tert-butyl ester (0.72 g; 2.54 mmol) and
triethyl amine (0.37 mL; 2.67 mmol) at 0 ꢀC. The reaction
mixture was stirred at 0 ꢀC for 30 min, at room temper-
ature for 23 h and then poured onto ice-water (400 mL).
The aqueous layer was extracted with CH2Cl2. The
combined organic phases were washed with saturated
NaHCO3, dried and concentrated under reduced pressure.
The crude product was purified by flash chromatography
(CH2Cl2/EtOAc: 1/0!68/32) to yield 3-(3-acetylsulfanyl-
2-ethoxycarbonyl-propyl)-pyrrolidine-1-carboxylic acid
tert-butyl ester (0.72 g, 79%) as an oil. 1H NMR
(400 MHz, CDCl3): d 1.27 (t, J=7 Hz, 3H), 1.44 (br s,
9H), 1.53–1.7 (m, 2H), 1.73–1.85 (m, 1H), 1.95–2.1 (m,
1H), 2.1–2.25 (m, 1H), 2.32 (s, 3H), 2.53–2.65 (m, 1H),
2.8–2.9 (m, 1H), 2.95–3.15 (m, 2H), 3.2–3.3 (m, 1H),
3.4–3.5 (m, 1H), 3.5–3.6 (m, 1H), 4.16 (q, J=7 Hz,
5.3.3. 2-(cis-4-tert-Butoxycarbonylamino-cyclopent-2-en-
ylmethyl)-malonic acid diethyl ester (63). Diethyl mal-
onate (1.29 mL, 8.5 mmol) was added to a mixture of
NaH (60%, 0.34 g; 8.5 mmol) in DMF (10 mL). After
stirring at room temperature for 15 min a solution of
cis-(4-bromomethyl-cyclopent-2-enyl)-carbamic
acid
tert-butyl ester (1.95 g, 7.1 mmol) in DMF (12 mL) was
added, and the reaction mixture was stirred at 60 ꢀC for
19 h. EtOAc was added and the organic phase was

1160
M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
extracted with water and brine, dried and concentrated
under reduced pressure to give 2.44 g of the crude pro-
duct. Purification by flash chromatography (CH2Cl2/
EtOAc: 1/0!68/32) afforded of compound 63 (1.47 g,
58%). 1H NMR (400 MHz, CDCl3): d 1.05–1.15 (m,
1H), 1.24–1.30 (m, 6H), 1.44 (s, 9H), 1.85–1.95 (m, 1H),
2.02–2.12 (m, 1H), 2.53–2.65 (m, 2H), 3.34–3.40 (m,
1H), 4.15–4.25 (m, 4H), 4.5–4.75 (m, 2H), 5.65–5.70 (m,
1H), 5.75–5.80 (m, 1H). 13C NMR (100 MHz, CDCl3): d
169.3, 155.1, 136.3, 132.4, 79.2, 61.4, 56.3, 50.4, 42.2,
38.4, 35.1, 28.4, 14.0.
then poured onto ice-water (400 mL). The aqueous
layer was extracted with CH2Cl2. The organic phase was
washed with saturated NaHCO3, dried and con-
centrated under reduced pressure to give 1.7 g of the
crude product. Purification by flash chromatography
(CH2Cl2/EtOAc: 1/0!68/32) afforded acetylsulfanyl-
methyl-3-(cis-4-tert-butoxycarbonylamino-cyclopent-2-
enyl)-propionic acid ethyl ester (0.46 g, 65%). 1H NMR
(400 MHz, CDCl3): d 1.04–1.15 (m, 1H), 1.24–1.31 (m,
3H), 1.46 (s, 9H), 1.60–1.95 (m, 2H), 2.34 (s, 3H), 2.55–
2.72 (m, 3H), 3.01 (dd, JAB=13.5 Hz, JAX=9 Hz, 1H),
3.14 (dd, JAB=13.5 Hz, JAX=5 Hz, 1H), 4.10–4.22 (m,
2H), 4.5–4.8 (m, 2H), 5.65–5.71 (m, 1H), 5.75–5.83 (m,
1H). 13C NMR (100 MHz, CDCl3): d 195.2, 174.1,
155.1, 136.8, 136.5, 132.3, 132.1, 79.2, 60.8, 56.4, 44.3,
44.2, 42.2, 38.8, 38.7, 30.8, 30.7, 30.5, 28.4, 14.2, 14.1.
5.3.4. 2-(cis-4-tert-Butoxycarbonylamino-cyclopent-2-en-
ylmethyl)-malonic acid monoethyl ester. A solution of
KOH (0.19 g; 3.4 mmol) in ethanol (6 mL) was added to
a solution of compound 63 (1.15 g; 3.2 mmol) in ethanol
(6 mL) at 0 ꢀC. The reaction mixture was stirred at
room temperature overnight, concentrated and ice-
water (400 mL) was added. The aqueous phase was
washed with diethyl ether (the emulsion formed during
the extraction was treated with brine in order to get
good phase separation), acidified to pH 3 with 0.5 M
HCl, and extracted with diethyl ether. The organic
phase was dried and concentrated under reduced pressure
to afford 2-(cis-4-tert-butoxycarbonylamino-cyclopent-
2-enylmethyl)-malonic acid monoethyl ester (0.86 g,
81%) as white crystals. 1H NMR (500MHz, dioxane-d8):
d 1.0–1.1 (m, 1H), 1.22–1.27 (m, 3H), 1.41 (s, 9H), 2.4–
2.65 (m, 3H), 3.39–3.45 (m, 1H), 4.12–4.20 (m, 2H),
4.55–4.65 (m, 1H), 5.64–5.70 (m, 1H), 5.74–5.80 (m,
1H), 5.80–5.88 (m, 1H). 13C NMR (100 MHz, dioxane-
d8): d 170.5, 169.7, 155.4, 136.3, 133.5, 78.4, 61.4, 56.9,
50.5, 42.9, 38.9, 35.9, 28.5, 14.1.
5.3.7. 3-(cis-4-Amino-cyclopent-2-enyl)-2-mercaptomethyl-
propionic acid (4). A solution of 2-acetylsulfanylmethyl-
3-(cis-4-tert-butoxycarbonylamino-cyclopent-2-enyl)-
propionic acid ethyl ester (86 mg, 0.23 mmol) in con-
centrated HCl (5 mL) was refluxed under argon for 1 h.
The reaction mixture was allowed to cool to room
temperature and concentrated under reduced pressure
to afford a diasteromeric mixture of compound 4 as the
1
hydrochloride salt (65 mg, 100%). H NMR (400 MHz,
D2O): d 1.35–1.47 (m, 2H), 1.60–1.72 (m, 1H), 1.74–1.92
(m, 2H), 1.92–2.04 (m, 1H), 2.67–2.92 (m, 10H), 4.34–
4.43 (m, 2H), 5.79–5.85 (br s, 2H), 6.12 (d, 1H), 6.18 (d,
1H). 13C NMR (100 MHz, D2O): d 179.3, 141.6, 141.4,
127.0, 126.9, 56.9, 48.2, 48.0, 43.0, 42.9, 37.4, 37.2, 35.0,
25.9, 25.6. HRMS (ESI) calcd for C9H15NO2S 201.0824,
found 201.0854.
5.3.5. 2-(cis-4-tert-Butoxycarbonylamino-cyclopent-2-en-
ylmethyl)-acrylic acid ethyl ester (64). Diethyl amine
(0.31 mL; 3.0 mmol) was added to a mixture of 2-(cis-4
-tert-butoxy-carbonylamino-cyclopent-2-enylmethyl)-
malonic acid monoethyl ester (0.66 g, 2.0 mmol) in 36%
aqueous solution of formaldehyde (0.25 mL, 3.2 mmol),
CH2Cl2 (1.6 mL) and water (1.6 mL) at 0 ꢀC. The reac-
tion mixture was stirred at room temperature overnight,
poured onto ice-water (400 mL) and extracted with
CH2Cl2. During the extraction, phase separation was
improved by the addition of brine. The combined
organic phases were washed with 5% NaHCO3, dried
and concentrated under reduced pressure to yield com-
5.4. 3-(2-Amino-thiazol-5-yl)-2-mercaptomethyl-propionic
acid (5)
5.4.1. 2-(2-Amino-thiazol-5-ylmethylene)-malonic acid
diethyl ester (84). To a solution of 2-amino-thiazole-5-
carbaldehyde (6 g, 23 mmol) in CH2Cl2 (30 mL) and
DMF (30 mL), was added 4A molecular sieves, diethyl
malonate (3.5 mL, 23 mmol), piperidine (1.1 mL, 11.5
mmol) and acetic acid (0.7 mL, 11.5 mmol). The reac-
tion mixture was stirred at room temperature for 96 h.
Then EtOAc was added, and the reaction mixture was
filtered through Celite in order to remove the precipitate
formed. EtOAc (500 mL) was added to the filtrate, and
the organic phase was washed with NaHCO3 and brine.
The organic phase was dried and concentrated to yield
4.9 g of the crude product. Addition of petroleum ether
to a solution of the crude product in ethanol followed
1
pound 64 (0.56 g, 94%) as an oil. H NMR (400 MHz,
CDCl3): d 1.08–1.17 (m, 1H), 1.26–1.32 (m, 3H), 1.43 (s,
9H), 2.28–2.60 (m, 3H), 2.75–2.88 (m, 1H), 4.15–4.24
(m, 2H), 4.45–4.75 (m, 2H), 5.52 (br s, 1H), 5.62–5.70
(m, 1H), 5.75–5.82 (m, 1H), 6.17 (br s, 1H). 13C NMR
(100 MHz, CDCl3): d 167.2, 155.2, 139.1, 137.0, 131.8,
125.7, 79.2, 60.7, 56.3, 43.0, 38.6, 38.2, 28.4, 14.2.
1
by filtration afforded compound 84 (1.13 g, 18%). H
NMR (400 MHz, CDCl3): d 1.25–1.38 (m, 6H), 4.2–4.35
(m, 4H), 7.43 (s, 1H), 7.73 (s, 1H).
5.3.6. 2-Acetylsulfanylmethyl-3-(cis-4-tert-butoxycarbo-
nylamino-cyclopent-2-enyl)-propionic acid ethyl ester.
Thioacetic acid (4 mL), which had been cooled to 0 ꢀC,
5.4.2. 2-(2-Amino-thiazol-5-ylmethyl)-malonic acid diethyl
ester. NaCNBH3 (1.88 g; 29.9 mmol) was added to a
stirred solution of compound 84 (1.13 g, 4.2 mmol) in
ethanol at 0 ꢀC. The pH of the solution was monitored
by addition of a small amount of bromocresol Green to
the solution. Concentrated HCl was added dropwise
until the solution turned yellow. The ice bath was
removed, and the reaction mixture was stirred at room
was added to
carbonylamino-cyclopent-2-enylmethyl)-acrylic
a
mixture of 2-(cis-4-tert-butoxy-
acid
ethyl ester (0.56 g, 1.9 mmol) and triethyl amine (0.28
mL, 2.0 mmol) at 0 ꢀC. The reaction mixture was stirred
at 0 ꢀC for 30 min, at room temperature for 19 h and

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1161
temperature for 5 h. Water was added and the product
was extracted with CH2Cl2. The combined organic
phases were dried and concentrated to yield 2-(2-amino-
thiazol-5-ylmethyl)-malonic acid diethyl ester (1 g,
stirred at room temperature overnight, poured onto ice-
water and extracted with CH2Cl2. The combined
organic phases were washed with 5% NaHCO3, dried
and concentrated to yield compound 85 (69 mg, 80.9%).
1H NMR (400 MHz, CDCl3): d 1.29 (t, J=7 Hz, 3H),
1.43 (s, 9H), 3.72 (s, 2H), 4.21 (q, J=7 Hz, 2H), 5.63 (s,
1H), 6.26 (s, 1H), 7.08 (s, 1H).
1
87.8%). H NMR (400 MHz, CDCl3): d 1.23–1.30 (m,
6H), 3.24 (d, J=7 Hz, 2H), 3.56 (t, J=7 Hz, 1H), 4.14–
4.26 (m, 4H), 6.81 (s, 1H). 13C NMR (100 MHz, CDCl3):
d 168.3, 167.5, 136.6, 123.6, 61.7, 53.5, 26.3, 14.0.
5.4.6. 2-Acetylsulfanylmethyl-3-(2-tert-butoxycarbonyl-
amino-thiazol-5-yl)-propionic acid ethyl ester. Triethyl
amine (32 mL, 0.23 mmol) was added to a solution of
compound 85 (67 mg; 0.21 mmol) in thioacetic acid (0.4
mL) at 0 ꢀC. The reaction mixture was stirred at room
temperature for 48 h, and then poured onto ice-water.
The aqueous layer was extracted with CH2Cl2. The
combined organic phases were washed with saturated
NaHCO3, dried and concentrated to yield 190 mg of the
crude product. The crude product was purified by flash
chromatography (Heptane/EtOAc, 1:0!68:32) to yield
2-acetylsulfanylmethyl-3-(2-tert-butoxycarbonyl-amino-
thiazol-5-yl)-propionic acid ethyl ester (43 mg, 51.6%).
1H NMR (400 MHz, CDCl3): d 1.23 (t, J=7 Hz, 3H),
1.56 (s, 9H), 2.33 (s, 3H), 2.82–2.92 (m, 1H), 2.98–3.20
(m, 4H), 4.14 (q, J=7 Hz, 2H), 7.05 (s, 1H).
5.4.3. 2-(2-tert-Butoxycarbonylamino-thiazol-5-ylmethyl)-
malonic acid diethyl ester. Di-tert-butyl-dicarbonate
(0.6 g, 27.5 mmol) was added to a solution of triethyl
amine (0.4 mL; 30.1 mmol), 4-(dimethylamino)pyridine
(0.34 g, 27.8 mmol) and 2-(2-amino-thiazol-5-ylmethyl)-
malonic acid diethyl ester (0.75 g, 27.5 mmol) in CH2Cl2
(5 mL) at 0 ꢀC. The reaction mixture was stirred at
room temperature overnight. Additional di-tert-butyl-
dicarbonate (0.15 g, 0.7 mmol) was added at 0 ꢀC, and
the reaction mixture was stirred at room temperature
for 1 h. CH2Cl2 was added, and the organic phase was
extracted with 0.3 M KHSO4 and brine. The organic
phase was dried and concentrated to yield 0.78 g of the
crude product. NMR indicated that approximately 40%
of 2-(2-amino-thiazol-5-ylmethyl)-malonic acid diethyl ester
remained. Di-tert-butyl-dicarbonate (0.6 g, 27.5 mmol) was
added to a solution of the crude product (0.78 g), triethyl
amine (0.4 mL, 30.1 mmol), 4-(dimethylamino)pyridine
(0.34 g, 27.8 mmol) in CH2Cl2 (5 mL) at 0 ꢀC. The
reaction mixture was stirred at room temperature over-
night and the work up procedure was repeated. The
crude product (1 g) was purified by flash chromato-
graphy (Heptane/EtOAc, 1:1) and HPLC to afford 2-(2-
tert-butoxycarbonylamino-thiazol-5-ylmethyl)-malonic
acid diethyl ester (184 mg, 17.9%). 1H NMR (400 MHz,
CDCl3): d 1.20–1.28 (m, 6H), 1.56 (s, 9H), 3.31 (d, J=7
Hz, 2H), 3.59 (t, J=7 Hz, 1H), 4.13–4.26 (m, 4H), 7.07
(s, 1H). 13C NMR (100 MHz, CDCl3): d 168.1, 161.1,
152.8, 134.7, 126.4, 81.7, 61.6, 53.4, 28.2, 25.9, 13.9.
5.4.7. 3-(2-Amino-thiazol-5-yl)-2-mercaptomethyl-propionic
acid (5). A solution of 2-acetylsulfanylmethyl-3-(2-tert-
butoxycarbonylamino-thiazol-5-yl)-propionic acid ethyl
ester (43 mg, 0.11 mmol) in concentrated HCl (1.5 mL)
was refluxed under argon for 1.5 h. The reaction mixture
was allowed to cool to room temperature and con-
centrated under reduced pressure to yield 30 mg of the
crude product. The crude product was purified by pre-
parative HPLC to afford the title compound 5 (8 mg,
1
21%) as the hydrochloride salt. H NMR (500 MHz,
D2O): d 2.75–3.1 (m, 5H), 7.0 (br s, 1H). MS (+) 219
(M+1). HRMS (ESI) calcd for C7H10N2O2S2 218.0184,
found 218.0237.
5.4.4. 2-(2-tert-Butoxycarbonylamino-thiazol-5-ylmethyl)-
malonic acid monoethyl ester. 2-(2-tert-Butoxy-
carbonylamino-thiazol-5-ylmethyl)-malonic acid diethyl
ester (158 mg, 0.43 mmol) was dissolved in ethanol (1
mL) and THF (0.5 mL), and a solution of KOH (24 mg,
0.43 mmol) in ethanol (0.14 mL) was added at 0 ꢀC. The
reaction mixture was stirred at room temperature for 96
h, and then poured onto ice-water. The aqueous phase
was extracted with diethyl ether, acidified to pH 3 by
addition of 0.5 M HCl, and extracted with diethyl ether.
The combined organic phases were dried and concentrated
to yield 2-(2-tert- butoxycarbonylamino-thiazol-5-yl-
methyl)-malonic acid monoethyl ester (97 mg, 66.4%).
1H NMR (400 MHz, CDCl3): d 1.27 (t, J=7 Hz, 3H),
1.54 (s, 9H), 3.23–3.42 (m, 2H), 3.57–3.68 (m, 1H), 4.23
(q, J=7 Hz, 2H), 7.02 (s, 1H).
5.5. 2-Mercaptomethyl-3-piperidin-4-yl-propionic acid (6)
5.5.1. 3-Piperidin-4-yl-propionic acid. A solution of 3-
pyridin-4-yl-acrylic acid (86) (4.20 g, 28.0 mmol) in
water (50 mL) and ammonia (aq, 25%, 4 mL) was
hydrogenated at 60 bar in a high-pressure steel autoclave
in presence of ruthenium (5% on alumina, 439 mg). When
hydrogen pressure remained constant (3 days) the catalyst
was removed from the reaction mixture by filtration.
The catalyst was washed with ethanol and water, and the
ethanol was removed from the solution on a rotavapor
and the aqueous solution was freeze dried to give 3-
1
piperidin-4-yl-propionic acid (4.30 g, 100%). H NMR
(300 MHz, D2O): d 1.25 (m, 2H), 1.47 (m, 3H), 1.82 (m,
2H), 2.13(m, 2H), 2.80 (m, 2H), 3.25 (m, 2H).
5.5.2. 4-(2-Carboxy-ethyl)-piperidine-1-carboxylic acid tert-
butyl ester (87). A solution of 3-piperidin-4-yl-propionic
acid (4.79 g, 30.5 mmol), di-tert-butyl-dicarbonate (6.98
g, 32.0 mmol) and NaHCO3 (2.69 g, 32.0 mmol) in THF/
water (1:1, 50 mL) were stirred at room temperature for
22 h. Another portion of di-tert-butyl-dicarbonate (2.00
g, 9.10 mmol) was added together with a catalytic
amount of DMAP, the resulting mixture was stirred for
5.4.5. 2-(2-tert-Butoxycarbonylamino-thiazol-5-ylmethyl)-
acrylic acid ethyl ester (85). To a mixture of 2-(2-tert-
butoxycarbonylamino-thiazol-5-ylmethyl)-malonic acid
monoethyl ester (94 mg, 0.27 mmol), a 36% aqueous
solution of formaldehyde (36 mL, 1.2 mmol), CH2Cl2
(0.2 mL) and water (0.2 mL) was added at 0 ꢀC diethyl
amine (30 mL, 0.40 mmol). The reaction mixture was

1162
M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
another four days. THF was removed under reduced
pressure and the aqueous phase was extracted with
CH2Cl2. The aqueous was then acidified to pH 2
with 1M HCl and the acid extracted with CH2Cl2. The
combined organic extracts were washed with brine,
dried and concentrated in vacuo to yield compound 87 as
a white solid (6.36 g, 81%). 1H NMR (300MHz, CDCl3):
d 1.0–1.2 (m, 2H), 1.45 (s+m, 10H), 1.6 (m, 4H), 2.39
(m, 2H), 2.65 (m, 2H), 4.10 (m, 2H).
1H), 2.9–3.05 (m, 5H), 3.38–3.48 (m, 2H). MS (+) 204
(M+1).
5.6. 2-(2-Amino-pyridin-4-ylmethyl)-3-mercapto-propionic
acid (7)
5.6.1. 2-Amino-isonicotinic acid ethyl ester. 2-Acetyl-
amino-isonicotinic acid (56) (10.8 g, 60.0 mmol) was
.
suspended in ethanol (150 mL) and BF3 OEt2 (22 mL,
138 mmol) was added. The mixture was refluxed over-
night, and after cooling to room temperature 10%
NaHCO3 (250 mL) was added. The product was
extracted with chloroform and the combined organic
extracts were washed with water and dried. Filtering
and concentration afforded 2-amino-isonicotinic acid
ethyl ester (7.46 g, 79%) as pale yellow crystals. 1H
NMR (300 MHz, CDCl3): d 1.40 (t, 3H), 4.35 (q, 2H),
4.60 (bs, 2H), 7.05 (m, 1H), 7.15 (m, 1H), 8.15 (m, 1H).
5.5.3. 4-(3-Benzylsulfanyl-2-carboxy-propyl)-piperidine-
1-carboxylic acid tert-butyl ester (88). BuLi (1.6 M, 15.3
mL, 24.4 mmol) was added to a solution of diisopropyl-
amine (3.43 mL, 24.4 mmol) in THF (3 mL) at À78 ꢀC
under argon. After a few min the solution was allowed
to warm up to room temperature over a period of 15
min. The resulting LDA solution was slowly added to a
solution of compound 87 (3.07 g, 11.9 mmol) in THF (7
mL) at À78 ꢀC. The resulting solution was stirred at
À78 ꢀC for 10 min, THF (20 mL) was added during that
time in order to dissolve the anion, which had solidified.
The dianion was cooled to À7 8 ꢀC and bromomethyl
thiobenzylether (2.72 g, 12.5 mmol) was added as a
solution in THF (3 mL), the solution was stirred at
À78 ꢀC for 30 min, at 0 ꢀC for 30 min and then allowed
to warm up to room temperature and stirred overnight.
The reaction mixture was acidified with 1 M HCl, diluted
with EtOAc and the organic phase was washed with
water and dried. The crude product was purified by
flash chromatography (MeOH/CHCl3, 1:9) to yield
5.6.2. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-isonicotinic
acid ethyl ester (57). To a solution of 2-amino-iso-
nicotinic acid ethyl ester (5.00 g, 30 mmol) in t-BuOH
(45 mL) and acetone (15 mL) was added DMAP (50
mg, 0.41 mmol) and di-t-butyl dicarbonate (16.4 g, 75.0
mmol). The reaction was stirred at room temperature
overnight and hexane (60 mL) was added. The reaction
mixture was cooled in a refrigerator for 3 h and filtered
to give compound 57 (8.71 g, 79%). 1H NMR
(300 MHz, CDCl3): d 1.40 (t, 3H), 1.50 (s, 18H), 4.40 (q,
2H), 7.75 (m, 1H), 7.80 (m, 1H), 8.60 (m, 1H).
1
compound 88 as a pale yellow oil (3.12 g, 66%). H
NMR (300 MHz, CDCl3): d 0.9–1.1 (m, 2H), 1.40 (m,
1H), 1.45 (s, 9H), 1.60 (m, 3H), 2.45 (m, 1H), 2.65 (m,
4H), 3.70 (s, 2H), 4.05 (bs, 2H), 7.27 (m, 5H).
5.6.3. (4-Hydroxymethyl-pyridin-2-yl)-carbamic acid tert-
butyl ester (58). A solution of to compound 57 (35.0 g,
95.5 mmol) in THF (350 mL) was treated with LiAlH4
(7.25 g, 191 mmol) and refluxed for 1 h under nitrogen.
The reaction mixture was poured carefully onto crushed
ice and the product extracted several times with CHCl3
and CHCl3:MeOH (9:1). The combined organic extracts
were dried, filtered and concentrated under reduced
pressure to give compound 58 (18.5 g, 86%) as a pale
5.5.4. 4-(2-Carboxy-3-mercapto-propyl)-piperidine-1-car-
boxylic acid tert-butyl ester. Sodium metal (513 mg,
22.5 mmol) was added in portions during 5 min to a
solution of compound 88 (0.9 g, 2.29 mmol) in THF (45
mL) and liquid ammonia (50 mL) at À60 ꢀC under
argon. After stirring for 15 min ammonium chloride
(1.7 g, 31.5 mmol) was added in portions. The cooling
bath was removed and the ammonia was evaporated
using a stream of argon. 0.5 M NaOH was added and
the mixture was washed with heptane. The aqueous
phase was acidified with 2 M HCl and extracted with
methylene chloride. The organic phase was washed
with brine, dried and concentrated under reduced pressure
to give crude 4-(2-Carboxy-3-mercapto-propyl)-piper-
idine-1-carboxylic acid tert-butyl ester (0.7 g, 100%).
This material was used directly in the next step.
1
yellow solid. H NMR (300 MHz, CDCl3): d 1.50 (s,
9H), 4.60 (s, 2H), 7.00 (m, 1H), 7.90 (m, 1H), 8.25 (m,
1H), 8.60 (bs, 1H).
5.6.4. (4-Bromomethyl-pyridin-2-yl)-carbamic acid tert-
butyl ester (59). Compound 58 (8.00 g, 35.6 mmol) was
dissolved in CH2Cl2 (150 mL) and treated with PPh3
(11.2 g, 42.8 mmol) under nitrogen. The reaction flask
was cooled in an ice bath and CBr4 (14.2 g, 42.8 mmol)
was added in small portions. The ice bath was removed
after 30 min and the reaction mixture was stirred over-
night at room temperature. The reaction mixture was
concentrated under reduced pressure and acetonitrile
(50 mL) was added. The reaction flask was placed in a
refrigerator for 3 h and the precipitate filtered and
washed with cold acetonitrile. The white solid was dried
5.5.5. 2-Mercaptomethyl-3-piperidin-4-yl-propionic acid
(6). To a solution of crude 4-(2-Carboxy-3-mercapto-
propyl)-piperidine-1-carboxylic acid tert-butyl ester (0.7
g, 2.29 mmol) in methylene chloride (8 mL) under argon
was added triethylsilane (731 mL, 4.58 mmol) followed
by TFA (4 mL). The reaction mixture was stirred for
60 min and then concentrated under reduced pressure.
Purification by HPLC (10!30% acetonitrile, 0.1%
TFA in water) gave compound 6 as the TFA salt (447
1
in vacuo giving compound 59 (8.38 g, 82%). H NMR
(300 MHz, CDCl3): d 1.50 (s, 9H), 4.40 (s, 2H), 7.00 (m,
1H), 8.05 (m, 1H), 8.30 (m, 1H), 8.90 (bs, 1H).
5.6.5. 2-(2-tert-Butoxycarbonylamino-pyridin-4-ylmethyl)-
malonic acid diethyl ester (60). To a solution of NaH
(80%, 0.17 g, 4.00 mmol) in THF (5 mL) at 0 ꢀC under
1
mg, 61%). H NMR (400 MHz, D2O): d 1.34–1.50 (m,
2H), 1.54–1.76 (m, 3H), 1.90–1.99 (m, 1H), 2.0–2.1 (m,

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1163
argon was added diethyl malonate (0.64 g, 4.00 mmol).
After the mixture was stirred for 15 min the mixture was
added to a refluxed mixture of compound 59 (1.00 g,
3.48 mmol) in THF (10 mL), and the mixture was
refluxed for 2 h. The mixture was concentrated under
reduced pressure and the residue was partitioned
between water and chloroform. The organic layer was
washed with water and brine and dried. After filtration
and evaporation of the solvent, the crude product was
purified by flash chromatography (MeOH/ CH2Cl2,
(300 MHz, CDCl3): d 1.20 (t, 3H), 1.51 (s, 9H), 2.30 (s,
3H), 2.95 (m, 3H), 3.09 (m, 2H), 4.10 (q, 2H), 6.70 (d,
1H), 7.78 (s, 1H), 8.15 (d, 1H), 8.30 (s, 1H).
5.6.9. 2-Acetylsulfanylmethyl-3-(2-amino-pyridin-4-yl)-
propionic acid ethyl ester. TFA (0.5 mL) was added to
a solution of 2-acetylsulfanylmethyl-3-(2-tert-butoxy-
carbonylamino-pyridin-4-yl)-propionic acid ethyl ester
(50 mg, 0.13 mmol) in methylene chloride under argon.
The solution was stirred for 60 min and concentrated
under reduced pressure to give crude 2-acetylsulfanyl-
methyl-3-(2-amino-pyridin-4-yl)-propionic acid ethyl ester
1
1:100) to give compound 60 (0.80 g, 55%). H NMR
(300 MHz, CDCl3): d 1.25 (t, 6H), 1.55 (s, 9H), 3.22 (d,
2H), 3.70 (t, 1H), 4.20 (q, 4H), 6.90 (d, 1H), 7.88 (s, 1H),
8.19 (d, 1H), 8.42 (bs, 1H).
1
(52 mg, 100%). H NMR (500 MHz, CD3OD3): d 1.15
(t, 3H), 2.32 (s, 3H), 2.73–2.83 (m, 2H), 2.86–2.93 (m,
1H), 3.01–3.07 (dd, 1H), 3.12–3.18 (dd, 1H), 4.03–4.12
(m, 2H), 6.39 (s, 1H), 6.43 (d, 1H), 7.77 (d, 1H).
5.6.6. 2-(2-tert-Butoxycarbonylamino-pyridin-4-ylmethyl)-
malonic acid monoethyl ester. A solution of KOH (0.19
g, 3.43 mmol) in ethanol (5 mL) was added to a solution
of compound 60 (1.20 g, 3.27 mmol) in ethanol (5 mL)
and methylene chloride (5 mL) at 0 ꢀC. The mixture was
stirred for 18 h at room temperature. The mixture
was concentrated under reduced pressure and water was
added to the residue. The aqueous layer was washed
with diethyl ether, acidified to pH 4 by 1M HCl, and
extracted with methylene chloride. The organic layer
was washed with water, brine and dried. After filtration
and evaporation in vacuo, the crude product was pur-
ified by flash chromatography (CH3OH/CH2Cl2, 1:20)
to yield 2-(2-tert-butoxycarbonylamino-pyridin-4-ylme-
5.6.10. 2-(2-Amino-pyridin-4-ylmethyl)-3-mercapto-propio-
nic acid (7). 2-Acetylsulfanylmethyl-3-(2-amino-pyridin-
4-yl)-propionic acid ethyl ester (52 mg, 0.13 mmol) was
dissolved in concentrated HCl (2 mL) under argon. The
solution was heated to reflux for 1 h. Concentration
under reduced pressure gave compound 7 as the hydro-
chloride salt (32 mg, 100%). 1H NMR (500 MHz,
CD3OD): d 2.70 (bs, 2H), 2.85–3.0 (m, 3H), 6.76 (bs,
1H), 6.81 (bs, 1H), 7.67 (bs, 1H). MS (+) 213 (M+1).
5.7. 3-(6-Amino-pyridin-3-yl)-2-mercaptomethyl-propionic
acid (8)
1
thyl)-malonic acid monoethyl ester (0.90 g, 81%). H
NMR (300 MHz, CDCl3): d 1.25 (t, 3H), 1.55 (s, 9H),
3.22 (m, 2H), 3.78 (t, 1H), 4.21 (q, 2H), 6.95 (d, 1H),
8.05 (m, 2H), 9.00 (bs, 1H).
5.7.1. 6-Amino-nicotinic acid ethyl ester. 2-Amino-5-
pyridinecarboxylic acid (20) (25.0 g, 181 mmol) was
suspended in ethanol (190 mL) and SOCl2 (15 mL, 206
mmol) was added. The mixture was refluxed for 10 h
and more SOCl2 (16 mL) was added. After 3 days with
reflux (and more SOCl2 (10 mL) added each day), the
reaction mixture was cooled to room temperature and
diethyl ether was added. After 24 h at À20 ꢀC the
mixture was filtered. The crude salt was dissolved in
methanol (214 mL) and a solution of NaOH (40.0 g,
23.5 mmol) in methanol (90 mL) was added. The reac-
tion mixture was stirred for 1 h and THF (270 mL) was
added. The reaction mixture was filtered through a plug
of silica (THF/MeOH) and concentrated under reduced
pressure to give 6-amino-nicotinic acid ethyl ester (36.2
5.6.7. 2-(2-tert-Butoxycarbonylamino-pyridin-4-ylmethyl)-
acrylic acid ethyl ester (61). A solution of diethylamine
(0.26 g, 2.67 mmol) in methylene chloride (4 mL) was
added to a mixture of 2-(2-tert-butoxycarbonylamino-
pyridin-4-ylmethyl)-malonic acid monoethyl ester (0.90
g, 2.66 mmol) and 37% aq solution of formaldehyde
(0.24 g, 3.00 mmol) at 0 ꢀC. The mixture was stirred for
5 h at room temperature and the mixture was poured
onto ice-water and extracted with methylene chloride.
The organic layer was washed with 5% NaHCO3 and
dried. The crude product was purified by flash chroma-
tography (1% methanol in CH2Cl2) to yield compound
1
g, 97%). H NMR (300 MHz, CDCl3): d 1.40 (t, 3H),
4.35 (q, 2H), 4.60 (bs, 2H), 7.05 (m, 1H), 7.15 (m, 1H),
8.15 (m, 1H).
1
61 (0.58 g, 71%). H NMR (300 MHz, CDCl3): d 1.25
(t, 3H), 1.55 (s, 9H), 3.62 (s, 2H), 4.20 (q, 2H), 5.55 (s,
1H), 6.30 (s, 1H), 6.80 (d, 1H), 7.85 (s, 1H), 8.10 (d,
1H), 8.65 (bs, 1H).
5.7.2. 6-tert-Butoxycarbonylamino-nicotinic acid ethyl
ester (21). To a solution of 6-amino-nicotinic acid ethyl
ester (36.0 g, 217.0 mmol) in t-BuOH (308 mL) and
acetone (103 mL) was added DMAP (0.53 g, 4.34
mmol) and di-t-butyl dicarbonate (72.0 g, 330 mmol).
The reaction was stirred at room temperature for 10 h
followed by addition of more di-t-butyl dicarbonate
(2.60 g). After 10 h stirring at room temperature hexane
(470 mL) was added. The reaction mixture was cooled
to À20 ꢀC for 2 h and filtered. The filtrate was washed
with hexane/dichloromethane (3:1) and dried in vacuo to
5.6.8. 2-Acetylsulfanylmethyl-3-(2-tert-butoxycarbonyl-
amino-pyridin-4-yl)-propionic acid ethyl ester. A solution
of compound 61 (0.48 g, 1.57 mmol) and triethylamine
(0.17 g, 1.64 mmol) was added to thioacetic acid (3 mL)
at 0 ꢀC under nitrogen. The mixture was stirred at room
temperature for 4 h. The mixture was poured onto ice-
water and extracted with CH2Cl2. The organic phase
was washed with saturated NaHCO3 (aq) and dried.
The crude product was purified by flash chromato-
graphy (2.5% MeOH in CH2Cl2) to give 2-acetyl-
sulfanylmethyl-3-(2-tert-butoxycarbonylamino-pyridin-4-
1
give compound 21 (40.5 g, 70%). H NMR (300 MHz,
CDCl3): d 1.40 (t, 3H), 1.50 (s, 18H), 4.40 (q, 2H), 7.75
(m, 1H), 7.80 (m, 1H), 8.60 (m, 1H).
1
yl)-propionic acid ethyl ester (0.60 g, 100%). H NMR

1164
M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
5.7.3. (5-Hydroxymethyl-pyridin-2-yl)-carbamic acid tert-
butyl ester (22). To a stirred solution of compound 21
(3.50 g, 13.1 mmol) in THF (20 mL) under nitrogen was
added LiAlH4 (0.91 g, 24.0 mmol) in THF (20 mL) over
a period of 2 h. The reaction mixture was stirred for 6 h,
and then NH4Cl (satd) was added (carefully) until neu-
tral solution. The mixture was filtered and concentrated
under reduced pressure to give compound 22 (2.00 g,
68%). 1H NMR (300 MHz, CDCl3): d 1.52 (s, 9H), 4.62
(s, 2H), 7.68 (d, 1H), 7.98 (d, 1H), 8.19 (bs, 1H), 8.29 (s,
1H).
carbonylamino-pyridin-3-ylmethyl)-malonic acid mono-
ethyl ester (1.00 g, 2.96 mmol) and 37% aq solution of
formaldehyde (0.25 g, 3.05 mmol) at 0 ꢀC. The mixture
was stirred for 16 h at room temperature and then
poured onto ice-water and extracted with methylene
chloride. The organic layer was washed with 5%
NaHCO3 and dried. Filtration and concentration under
1
reduced pressure gave compound 43 (0.75 g, 83%). H
NMR (300 MHz, CDCl3): d 1.25 (t, 3H), 1.52 (s, 9H),
3.55 (s, 2H), 4.15 (q, 2H), 5.45 (s, 1H), 6.22 (s, 1H), 7.50
(d, 1H), 7.85 (d, 1H), 8.10 (s, 1H), 8.20 (bs, 1H).
5.7.4. (5-Bromomethyl-pyridin-2-yl)-carbamic acid tert-
butyl ester (23). Triphenylphosphine (8.70 g, 33.1
mmol) and carbontetrabromide (17.0 g, 51.2 mmol)
were added to a suspension of compound 22 (7.00 g,
31.2 mmol) in CH2Cl2 (200 mL) at room temperature.
Stirring was continued for 5 h followed by evaporation
of the solvent. Acetonitrile (200 mL) was added and the
mixture was cooled to À20 ꢀC for 2 h. The mixture was
then filtered and the crystalline residue washed with
cold acetonitrile (2ꢁ10 mL), to give compound 23 (5.96
5.7.8. 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarbonyl-
amino-pyridin-3-yl)-propionic acid ethyl ester. Compound
43 (0.49 g, 1.60 mmol) and triethylamine (0.17 g, 1.64
mmol) were added to thioacetic acid (3 mL) at 0 ꢀC.
The mixture was stirred at room temperature for 6 h.
The mixture was poured onto ice-water and extracted with
CH2Cl2. The organic phase was washed with saturated
NaHCO3 and dried. The crude product was purified by
flash chromatography (MeOH/CH2Cl2, 2.5:100) to give
2-acetylsulfanylmethyl-3-(6- tert-butoxycarbonylamino-
pyridin-3-yl)-propionic acid ethyl ester (0.36 g, 61%).
1H NMR (300 MHz, CDCl3): d 1.25 (t, 3H), 1.52 (s,
9H), 2.30 (s, 3H), 2.85 (m, 3H), 3.09 (m, 2H), 4.05 (q, 2H),
7.45 (d, 1H), 7.90 (d, 1H), 8.09 (s, 1H), 8.50 (s, 1H).
1
g, 67%). H NMR (300 MHz, CDCl3): d 1.54 (s, 9H),
4.41 (s, 2H), 7.68 (d, 1H), 7.98 (d, 1H), 8.32 (s, 1H), 9.00
(bs, 1H).
5.7.5. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
malonic acid diethyl ester (42). To a suspension of NaH
(0.49 g, 16.3 mmol, 80%) in THF (15 mL) at 0 ꢀC was
added diethyl malonate (2.61 g, 16.3 mmol). The mixture
was stirred for 15 min and was then added dropwise to a
refluxed mixture of compound 23 (3.90 g, 13.6 mmol) in
THF (25 mL), and the resulting solution was refluxed
for 15 min. After evaporation of the solvent, the crude
product was purified by flash chromatography (metha-
nol/CH2Cl2, 1:100!2.5:100) to give compound 42 (2.18
5.7.9. 3-(6-Amino-pyridin-3-yl)-2-mercaptomethyl-propionic
acid (8). 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarb-
onylamino-pyridin-3-yl)-propionic acid ethyl ester (38 mg,
0.096 mmol) was dissolved in concd HCl (2.0 mL) under
argon. The solution was stirred at room temperature for
1 h and then heated to reflux for 1 h. Concentration
under reduced pressure gave compound 8 (25.7 mg,
1
100%) as the hydrochloride salt. H NMR (500 MHz,
CD3OD): d 2.74–2.78 (m, 2H), 2.84–2.94 (m, 3H), 7.02
(d, 1H), 7.72 (s, 1H), 7.89 (d, 1H). MS (+) 213 (M+1).
HRMS (ESI) calcd for C9H12N2O2S 212.0619, found
212.0678.
1
g, 44%). H NMR (300 MHz, CDCl3): d 1.20 (t, 6H),
1.52 (s, 9H), 3.15 (d, 2H), 3.55 (t, 1H), 4.15 (q, 4H), 7.55
(d, 1H), 7.70 (bs, 1H), 7.90 (d, 1H), 8.10 (s, 1H).
5.7.6. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
malonic acid monoethyl ester. A solution of KOH (0.37
g, 6.54 mmol) in ethanol (5 mL) was added to a solution
of compound 42 (2.18 g, 5.95 mmol) in ethanol (25 mL)
and methylene chloride (10 mL) at 0 ꢀC. The mixture was
stirred for 18 h at room temperature. The mixture
was concentrated under reduced pressure and the residue
dissolved in water. The aqueous layer was washed with
ether, acidified to pH 4 by 1M HCl and extracted
with methylene chloride. The organic layer was washed
with water, brine and dried. After filtration and con-
centration under reduced pressure, the crude product
was purified by flash chromatography (methanol/
CH2Cl2, 1:20) to yield 2-(6-tert-butoxycarbonylamino-
pyridin-3-ylmethyl)-malonic acid monoethyl ester (1.00
5.8. 3-(6-Amino-4-methyl-pyridin-3-yl)-2-mercaptomethyl-
propionic acid (9)
5.8.1. 2-Amino-5-bromo-4-methylpyridine (34). 2-
Amino-4-methylpyridine (110 g, 1.02 mol) in hydro-
bromic acid (1 L, 48%) was stirred at 70 ꢀC and hydrogen
peroxide (300 mL, 15% in water) was added dropwise
over a one h at such a rate that the temperature of the
reaction mixture remained at 70–80 ꢀC. The mixture was
stirred for 90 min at 70 ꢀC and poured onto crushed ice.
The pH was adjusted to 4–5 with sodium carbonate and
the precipitated solid (containing mostly dibrominated
products) was filtered off and discarded. The pH was
subsequently raised to 9 and the precipitated product
collected by filtration. Recrystallization from toluene
1
1
g, 50%). H NMR (300 MHz, CDCl3): d 1.30 (t, 3H),
1.52 (s, 9H), 3.10 (m, 2H), 3.55 (t, 1H), 4.25 (q, 2H),
7.55 (d, 1H), 7.85 (m, 1H), 8.05 (m, 1H), 9.20 (bs, 1H).
gave compound 34 (76.3 g, 40%). H NMR (300 MHz,
CDCl3): d 2.27 (s, 3H), 4.35 (s, 2H), 6.38 (s, 1H), 8.07 (s,
1H).
5.7.7. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
acrylic acid ethyl ester (43). Diethylamine (0.29 g, 3.00
mmol), water (2 mL) and methylene chloride (2 mL)
5.8.2. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-5-bromo-
4-methylpyridin. Compound 34 (5.70 g, 30.5 mmol) in
chloroform was treated with di-tert-butyl dicarbonate
(20.0 g, 91.60 mmol) and DMAP (0.60 g, 4.91 mmol).
was added to
a
mixture of 2-(6-tert-butoxy-

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1165
The reaction mixture was left at ambient temperature
overnight and was then concentrated under reduced
pressure. Flash chromatography (hexane/EtOAc, 95:5)
gave 2-[N,N-bis(tert-butoxycarbonyl)amino]-5-bromo-4-
methylpyridin (8.02 g, 68%). 1H NMR (300 MHz, CDCl3):
d 1.45 (s, 18H), 2.40 (s, 3H), 7.25 (s, 1H), 8.49 (s, 1H).
was added to a solution compound 51 (1.1 g, 2.29
mmol) in ethanol (10 mL) and methylene chloride (4
mL) at 0 ꢀC. The mixture was stirred for 18 h at room
temperature. The mixture was concentrated under
reduced pressure and the residue dissolved in water.
Ethyl acetate was added and the organic layer was
washed with 0.5 M HCl, water, brine and dried. After
filtration and concentration under reduced pressure
gave crude 2-(6-[N,N-bis(tert-butoxycarbonyl)-amino]-
4-methyl-pyridin-3-ylmethyl)-malonic acid monoethyl
ester (1.0 g, 97%). 1H NMR (500 MHz, CD3OD): d
1.19, (t, 3H), 1.36, (s, 18H), 2.41 (s, 3H), 3.24, (d, 2H),
3.66 (t, 1H), 4.05–4.16 (m, 2H), 7.14 (s, 1H), 8.18 (s,
1H).
5.8.3. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-5-(tert-butyl-
dimethyl-silanyloxymethyl)-4-methylpyridin (35). A solu-
tion of 2-[N,N-bis(tert-butoxycarbonyl)amino]-5-bromo-
4-methylpyridin (15.0 g, 38.70 mmol), tert-butyl-di-
methyl-tributylstannanylmethoxy-silane (25.4 g, 58.3
mmol), and bis(triphenylphosphine)palladium(II) dichlor-
ide (0.90 g, 1.42 mmol) in 1,2-dichloro-ethane (50 mL)
was stirred at 90 ꢀC for two days. The mixture was cooled
to 0 ꢀC and diethyl ether (200 mL) was added followed by
saturated aqueous potassium fluoride (40 mL). The
mixture was stirred vigorously for 30 min and filtered.
The organic phase was washed with water, dried and
concentrated under reduced pressure. Flash chromato-
graphy (hexane/EtOAc, 95:5) gave compound 35 (10.0
5.8.7. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-4-methyl-
pyridin-3-ylmethyl)-acrylic acid ethyl ester (52). Diethyl-
amine (0.26 g, 2.67 mmol) was added a mixture of 2-(6-
[N,N-bis(tert-butoxycarbonyl)amino]-4-methyl-pyridin-3-
ylmethyl)-malonic acid monoethyl ester (1.0 g, 2.2
mmol) and 37% aq solution of formaldehyde (0.24 g,
3.00 mmol) in methylene chloride (2 mL) at 0 ꢀC. The
mixture was stirred for 16 h at room temperature and
ethyl acetate was added. The organic layer was washed
with water and 5% NaHCO3 and dried. Concentration
under reduced pressure followed by flash chromato-
graphy (toluene/ethyl acetate, 3:1!1:1) gave compound
1
g, 57%). H NMR (300 MHz, CDCl3): d 0.10 (s, 6H),
0.91 (s, 9H), 1.44 (s, 18H), 2.32 (s, 3H), 4.72 (s, 2H),
7.00 (s, 1H), 8.40 (s, 1H).
5.8.4. 5-Bromomethyl-2-[N,N-bis(tert-butoxycarbonyl)-
amino]- 4-methylpyridin (37). Tetrabutylammonium
fluoride (13.9 g, 44.1 mmol) was added to a solution of
compound 35 (10.0 g, 24.3 mmol) in THF (100 mL).
The reaction mixture was stirred for 3 h at room temp-
erature. Concentration under reduced pressure followed
by flash chromatography (hexane/EtOAc, 50:50) gave
compound 36 (5.0 g, 67%). Triphenylphosphine (4.69 g,
17.9 mmol) and CBr4 (4.89 g, 14.8 mmol) was added to
a solution of compound 36 (5.00 g, 22.0 mmol) in di-
chloromethane (130 mL) at 0 ꢀC. The reaction mixture
was stirred for 3 h and was then diluted with dichloro-
methane. The organic phase was washed with water,
dried and concentrated under reduced pressure. Flash
chromatography (hexane/EtOAc, 80:20) gave compound
37 (5.35 g, 90%). 1H NMR (300 MHz, CDCl3): d 1.46 (s,
18H), 2.42 (s, 3H), 4.47 (s, 2H), 7.12 (s, 1H), 8.34 (s, 1H).
1
52 (0.81 g, 88%). H NMR (500 MHz, CDCl3): d 1.30,
(t, 3H), 1.53, (s, 18H), 2.26 (s, 3H), 3.62 (s, 2H), 4.22
(q, 2H), 5.16 (s, 1H), 6.23 (s, 1H), 7.04 (s, 1H), 8.19 (s,
1H).
5.8.8. 2-Acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxy-
carbonyl)amino]-4-methyl-pyridin-3-yl)-propionic acid ethyl
ester. Triethylamine (0.279 mL, 2.0 mmol) was added
to a solution of 2-(6-[N,N-bis(tert-butoxycarbonyl)amino]-
4-methyl-pyridin-3-ylmethyl)-acrylic acid ethyl ester (0.8
g, 1.9 mmol) in thioacetic acid (3 mL) at 0 ꢀC. The
mixture was stirred at room temperature for 16 h. Ethyl
acetate was added and the organic phase was washed
with water, saturated NaHCO3 and brine and dried.
The crude product was purified by flash chromato-
graphy (toluene/ethyl acetate, 3:1!1:2) to give pure 2-
acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxycarbonyl)-
amino]-5-methyl-pyridin-3-yl)-propionic acid ethyl ester
(200 mg, 21%) and slightly impure 2-acetyl-
sulfanylmethyl-3-(6-[N,N-bis(tert-butoxycarbonyl)amino]-
4-methyl-pyridin-3-yl)-propionic acid ethyl ester (0.68
g). 1H NMR (500 MHz, CDCl3): d 1.17, (t, 3H), 1.44, (s,
18H), 2.35 (s, 3H), 2.85-3.17 (m, 5H), 4.02-4.12 (m, 2H),
7.04 (s, 1H), 8.17 (s, 1H).
5.8.5. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-4-methyl-
pyridin-3-ylmethyl)-malonic acid diethyl ester (51). To a
suspension of NaH (0.24 g, 6.0 mmol, 60%) in DMF (5
mL) was added diethyl malonate (0.91 mL, 6.0 mmol)
and the mixture was stirred for 15 min. A solution
compound 37 (2.0 g, 5.0 mmol) in DMF (5 mL) was
added and the resulting solution stirred for 120 min at
60 ꢀC. Ethyl acetate was added and the mixture was
washed with water and brine and dried. After evapora-
tion of the solvent, the crude product was purified by
flash chromatography (CH3OH/CH2Cl2, 1:100!1:20)
to give a pure fraction of compound 51 (1.15 g, 48%)
together with an impure fraction (1.1 g). 1H NMR
(500 MHz, CDCl3): d 1.23, (t, 6H), 1.44, (s, 18H), 2.36
(s, 3H), 3.23, (d, 2H), 3.61 (t, 1H), 4.10–4.25 (m, 4H),
7.03 (s, 1H), 8.22 (s, 1H).
5.8.9. 3-(6-Amino-4-methyl-pyridin-3-yl)-2-mercapto-
methyl-propionic acid (9). 2-Acetylsulfanylmethyl-3-(6-
[N,N-bis(tert-butoxycarbonyl)amino]-4-methyl-pyridin-3-
yl)-propionic acid ethyl ester (36 mg, 0.072 mmol) was
dissolved in concentrated HCl (3.0 mL). The solution
was heated to reflux for 1 h. Concentration under
reduced pressure gave compound 9 (18.7 mg, 98%) as
1
the hydrochloride salt. H NMR (500 MHz, CD3OD):
5.8.6. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-4-methyl-
pyridin-3-ylmethyl)-malonic acid monoethyl ester. A solu-
tion of KOH (141 mg, 2.52 mmol) in ethanol (2 mL)
d 2.42 (s, 3H), 2.72–2.95 (m, 5H), 6.81 (s, 1H), 7.58 (s,
1H). MS (+) 227 (M+1). HRMS (ESI) calcd for
C10H14N2O2S 226.0776, found 226.0801.

1166
M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
5.9. 3-(6-Amino-2-methyl-pyridin-3-yl)-2-mercaptomethyl-
propionic acid (10)
mg, 7.97 mmol, 55% in mineral oil) in DMF (5 mL) at
0 ꢀC under argon. The reaction mixture was stirred for
45 min and a solution of compound 33 (2.0 g, 6.64
mmol) in DMF (5 mL) was added dropwise. The
mixture was stirred overnight (0 ꢀC!20 ꢀC). EtOAc was
added and the solution was washed with water and
brine, dried and concentrated under reduced pressure.
Flash chromatography (heptane/EtOAc, 4:1) gave
compound 48 (1.87 g, 74%). 1H NMR (500 MHz,
CDCl3): d 1.23, (t, 6H), 1.49, (s, 9H), 2.45 (s, 3H), 3.17,
(d, 2H), 3.59 (t, 1H), 4.11–4.24 (m, 4H), 7.42 (d, 1H),
7.56 (broad s, 1H), 7.66 (d, 1H).
5.9.1. (5-Bromo-6-methyl-pyridin-2-yl)-carbamic acid
tert-butyl ester. Compound 30 (25.0 g, 133.7 mmol) in
THF/tert-butanol (1:10, 550 mL) was treated with di-
tert-butyl dicarbonate (39.3 g, 180.0 mmol) and DMAP
(2.40 g, 19.6 mmol). The reaction mixture was stirred
for 4 h at 40ꢀC and concentrated under reduced pressure.
Flash chromatography (methylene chloride) gave (5-
bromo-6-methyl-pyridin-2-yl)-carbamic acid tert-butyl
ester (17.0 g, 44%). 1H NMR (300 MHz, CDCl3): d 1.40
:
(s, 9H), 2.50 (s, 3H), 7.40 (bs, 1H), 7.65 (d, 1H), 7.70 (d,
1H).
5.9.6. 2-(6-tert-Butoxycarbonylamino-2-methyl-pyridin-
3-ylmethyl)-malonic acid monoethyl ester. A solution of
KOH (300 mg, 5.35 mmol) in EtOH (4 mL) was added
to a solution of compound 48 (1.85 g, 4.86 mmol) in
EtOH/methylene chloride (2:1, 21 mL) at 0 ꢀC. The
mixture was stirred for 40 h at room temperature and
EtOAc was added. The mixture was washed with 0.5 M
HCl and brine, dried and concentrated under reduced
pressure to give crude 2-(6-tert-butoxycarbonylamino-2-
methyl-pyridin-3-ylmethyl)-malonic acid monoethyl
5.9.2. [5-(tert-Butyl-dimethyl-silanyloxymethyl)-6-methyl-
pyridin-2-yl]-carbamic acid tert-butyl ester (31). A solution
of (5-bromo-6-methyl-pyridin-2-yl)-carbamic acid tert-
butyl ester (27.5 g, 95.8 mmol), tert-butyl-dimethyl-tri-
butylstannanylmethoxy-silane (43.5 g, 100.2 mmol), and
bis(triphenylphosphine)palladium(II) dichloride (1.00 g,
1.40 mmol) in 1,2-dichloroethane (350 mL) was stirred at
reflux for 48 h. Additional bis(triphenylphosphine)-
palladium(II) dichloride (1.00 g, 1.40 mmol) was added
every 12 h. The mixture was cooled to 0 ꢀC and diethyl
ether (300 mL) was added followed by saturated aqueous
potassium fluoride (100 mL). The mixture was stirred
vigorously for 60 min and filtered. The organic phase
was washed with water, dried and concentrated under
reduced pressure. Flash chromatography (MeOH/
CH2Cl2, 1:99) compound 31 (15 g, 47%). 1H NMR
1
ester (1.45 g). H NMR (500 MHz, CDCl3): d 1.25, (t,
3H), 1.51, (s, 9H), 2.61 (s, 3H), 3.13–3.27 (m, 2H), 3.61
(t, 1H), 4.14–4.25 (m, 2H), 7.87 (d, 1H), 8.11 (d, 1H).
5.9.7. 2-(6-tert-Butoxycarbonylamino-2-methyl-pyridin-
3-ylmethyl)-acrylic acid ethyl ester (49). Diethylamine
(359 mg, 4.90 mmol) was added dropwise to a solution
of 2-(6-tert-butoxycarbonylamino-2-methyl-pyridin-3-
ylmethyl)-malonic acid monoethyl ester (1.44 g, 4.09
mmol) and formaldehyde (464 mg, 5.72 mmol, 37% in
water) in methylene chloride (35 mL) at 0 ꢀC under
argon. The mixture was stirred at room temperature over
night. Methylene chloride was added and the solution was
washed with Na2CO3 and brine, dried and concentrated
under reduced pressure to give compound 49 (1.03 g,
79%). 1H NMR (500 MHz, CDCl3): d 1.30, (t, 3H),
1.49, (s, 9H), 2.38 (s, 3H), 3.57 (s, 2H), 4.22 (q, 2H), 5.25
(d, 1H), 6.23 (d, 1H), 7.40 (d, 1H), 7.71 (d, 1H), 8.10
(broad s, 1H).
(300 MHz, CDCl3): ꢀ 0.07 (s, 6H), 0.90 (s, 9H), 2.35 (s,
:
3H), 4.62 (s, 2H), 7.25 (bs, 1H), 7.62 (d, 1H), 7.68 (d, 1H).
5.9.3. (5-Hydroxymethyl-6-methyl-pyridin-2-yl)-carbamic
acid tert-butyl ester (32). Tetrabutylammonium fluoride
(19.6 g, 62.4 mmol) was added to a solution of
compound 31 (10.5 g, 31.23 mmol) in THF (100 mL)
and stirred at room temperature overnight. Water was
added and the product extracted with chloroform. The
organic phase was dried and concentrated under
reduced pressure. Flash chromatography (MeOH/
1
CH2Cl2, 2.5:77.5) gave compound 32 (6.0 g, 81%). H
NMR (300 MHz, CDCl3): d 1.51 (s, 9H), 2.45 (s, 3H),
5.9.8. 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarbonyl-
amino-2-methyl-pyridin-3-yl)-propionic acid ethyl ester.
Triethylamine (0.556 mL, 3.99 mmol) was added to a
solution of 2-(6-tert-butoxycarbonylamino-2-methyl-
pyridin-3-ylmethyl)-acrylic acid ethyl ester (1.23 g, 3.84
mmol) in thioacetic acid (10 mL) at 0 ꢀC under argon.
The mixture was stirred at room temperature for 64 h.
EtOAc was added and the solution was washed with
Na2CO3 and brine, dried and concentrated under
reduced pressure. Flash chromatography (toluene/
EtOAc, 5:1!1:1) gave 2-acetylsulfanylmethyl-3-(6-tert-
butoxycarbonylamino-2-methyl-pyridin-3-yl)-propionic
acid ethyl ester (1.33 g, 87%). 1H NMR (500 MHz,
CDCl3): d 1.09, (t, 3H), 1.21, (s, 9H), 2.12 (s, 3H), 2.19
(s, 3H), 2.71–2.90 (m, 3H), 2.96–3.12 (m, 2H), 3.95–4.02
(m, 2H), 7.35 (d, 1H), 7.62 (d, 1H), 8.25 (broad s, 1H).
:
4.65 (s, 3H), 7.27 (bs, 1H), 7.62 (d, 1H), 7.70 (d, 1H).
5.9.4. 5-Bromomethyl-2-[N,N-bis(tert-butoxycarbonyl)-
amino]-pyrimidin (33). Triphenylphosphine (9.83 g, 37.5
mmol) and CBr4 (17.7 g, 53.5 mmol) was added to a
solution of compound 32 (8.50 g, 35.7 mmol) in di-
chloromethane (30 mL) at 0 ꢀC. The reaction mixture
was stirred for 3 h at room temperature and was then
diluted with dichloromethane. The organic phase was
washed with water, dried and concentrated under
reduced pressure. Flash chromatography (CH2Cl2) gave
compound 33 (4.05 g, 38%). 1H NMR (300 MHz,
CDCl3): d 1.49 (s, 9H), 2.48 (s, 3H), 4.45 (s, 2H), 7.53
:
(bs, 1H), 7.56 (d, 1H), 7.70 (d, 1H).
5.9.5. 2-(6-tert-Butoxycarbonylamino-2-methyl-pyridin-
3-ylmethyl)-malonic acid diethyl ester (48). A solution
of diethyl malonate (1.21 mL, 7.97 mmol) in DMF (2
mL) was added dropwise to a suspension of NaH (348
5.9.9. 3-(6-Amino-2-methyl-pyridin-3-yl)-2-mercapto-
methyl-propionic acid (10). Concentrated hydrochloric
acid (2 mL) was added to 2-acetylsulfanylmethyl-3-(6-

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1167
tert-butoxycarbonylamino-2-methyl-pyridin-3-yl)-pro-
pionic acid ethyl ester (77 mg, 0.19 mmol) under argon.
The reaction was heated to reflux for 110 min and
was then concentrated under reduced pressure to give
compound 10 (39 mg, 76%) as the hydrochloride salt.
1H NMR (500 MHz, D2O): d 2.49 (s, 3H), 2.73–2.92 (m,
5H), 6.81 (d, 1H), 7.77 (d, 1H). MS (+) 227 (M+1).
HRMS (ESI) calcd for C10H14N2O2S 226.0776, found
226.0835.
5.10.4. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-5-methyl-
pyridin-3-ylmethyl)-malonic acid diethyl ester (54). To a
suspension of NaH (0.24 g, 6.0 mmol, 60%) in DMF (5
mL) was added diethyl malonate (0.91 mL, 6.0 mmol)
and the mixture was stirred for 15 min. A solution
compound 41 (2.0 g, 5.0 mmol) in DMF (5 mL) was
added and the resulting solution stirred for 120 min at
60 ꢀC. Ethyl acetate was added and the mixture was
washed with water and brine and dried. After evapora-
tion of the solvent, the crude product was purified by
flash chromatography (CH3OH/CH2Cl2, 1:100!1:20)
to give compound 54 (1.2 g, 50%). 1H NMR (300 MHz,
CDCl3): d 1.23 (t, 6H), 1.38(s, 18H), 2.19 (s, 3H), 3.20
(d, 2H), 3.60 (t, 1H), 4.10–4.20 (m, 4H), 7.44 (s, 1H),
8.20 (s, 1H).
5.10. 3-(6-Amino-5-methyl-pyridin-3-yl)-2-mercaptomethyl-
2-methyl-propionic acid (11)
5.10.1. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-5-bromo-
3-methylpyridin. A solution of compound 38 (15.0 g,
80.2 mmol) in tert-butanol (500 mL) was treated with
di-tert-butyl dicarbonate (43.6 g, 200 mmol) and dime-
thylaminopyridine (0.60 g, 4.91 mmol). The reaction
mixture was stirred overnight and concentrated under
reduced pressure. Hexane was added and the product
precipitated as a solid. Filtering afforded 22.0 g (71%)
of 2-[N,N-bis(tert-butoxycarbonyl)amino]-5-bromo-3-
5.10.5. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-5-methyl-
pyridin-3-ylmethyl)-malonic acid monoethyl ester. A solu-
tion of KOH (154 mg, 2.75 mmol) in ethanol (2 mL)
was added to a solution compound 54 (1.2 g, 2.50
mmol) in ethanol (10 mL) and methylene chloride (4
mL) at 0 ꢀC. The mixture was stirred for 18 h at room
temperature. The mixture was concentrated under
reduced pressure and the residue dissolved in water.
Ethyl acetate was added and the organic layer was
washed with 0.5 M HCl, water, brine and dried. After
filtration and concentration under reduced pressure
gave crude 2-(6-[N,N-bis(tert-butoxy- carbonyl)amino]-
5-methyl-pyridin-3-ylmethyl)-malonic acid monoethyl
1
methylpyridin. H NMR (300 MHz, CDCl3): d 1.40 (s,
18H), 2.22 (s, 3H), 7.72 (s, 1H), 8.40 (s, 1H).
5.10.2. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-5-hydro-
xymethyl-3-methylpyridin (40). A solution of (tert-
butyldimethylsilyloxymethyl)tri-n-butyltin (47.6 g, 109
mmol), 2-[N,N-bis(tert-butoxycarbonyl)amino]-5-bromo-
3-methylpyridin (26.0 g, 67.1 mmol) and bis(triphenyl-
phosphine)palladium(II) dichloride (0.90 g, 1.42 mmol)
in 1,2-dichloroethane (80 mL) was stirred at 90ꢀC for two
days. The mixture was cooled to 0 ꢀC and ether (200 mL)
was added followed by saturated aqueous potassium
fluoride (40 mL). The mixture was stirred vigorously for
30 min, filtered and the organic phase separated and
washed with water. The organic phase was dried,
filtered and evaporated. The crude product was purified
by flash chromatography (hexane, then hexane:ethyl
acetate, 95:5) to afford compound 39 (18.0 g, 59%). A
solution of compound 39 (18.0 g, 39.8 mmol) in 150 mL
dry THF was treated with tetrabutylammonium fluoride
(25.1 g, 79.6 mmol) at room temperature overnight.
Concentration under reduced pressure followed by flash
chromatography (hexane:ethyl acetate, 1:1) afforded
1
ester (1.0 g, 88%). H NMR (300 MHz, CDCl3): d 1.23
(t, 3H), 1.37 (s, 18H), 2.08 (s, 2H), 2.20 (s, 3H), 3.24 (d,
2H), 3.64 (t, 1H), 4.12–4.22 (m, 2H), 7.53 (s, 1H), 8.25
(s, 1H).
5.10.6. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-5-methyl-
pyridin-3-ylmethyl)-acrylic acid ethyl ester (55). Diethyl-
amine (0.26 g, 2.67 mmol) was added a mixture of 2-(6-
[N,N-bis(tert-butoxycarbonyl)amino]-5-methyl-pyridin-3-
ylmethyl)-malonic acid monoethyl ester (1.0 g, 2.2
mmol) and 37% aq solution of formaldehyde (0.24 g,
3.00 mmol) in methylene chloride (2 mL) at 0 ꢀC. The
mixture was stirred for 16 h at room temperature and
ethyl acetate was added. The organic layer was washed
with water and 5% NaHCO3 and dried. Concentration
under reduced pressure followed by flash chromato-
graphy (toluene/ethyl acetate, 3:1!1:2) gave compound
1
compound 40 (8.00 g, 59%) as a white solid. H NMR
(300 MHz, CDCl3): d 1.39 (s, 18H), 2.22 (s, 3H), 4.70 (s,
2H), 7.60 (s, 1H), 8.27 (s, 1H).
1
55 (0.68 g, 73%). H NMR (300 MHz, CDCl3): d 1.31
(t, 3H), 1.44 (s, 18H), 2.24 (s, 3H), 3.66 (s, 2H), 4.22 (q,
2H), 5.50 (s, 1H), 6.31 (s, 1H), 7.46 (d, 1H), 8.24 (d, 1H).
5.10.3. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-5-bromo-
methyl-3-methyl-pyridin (41). Compound 40 (8.00 g,
23.6 mmol) and triphenylphosphine (7.43 g, 28.3 mmol)
were dissolved in dichloromethane (220 mL) and cooled
to 0 ꢀC. Carbon tetrabromide (9.49 g, 28.6 mmol) was
added and the reaction mixture allowed to reach room
temperature. Stirring was continued for 3 h. The solvent
was removed under reduced pressure and the residue
purified by flash chromatography (hexane:ethyl acetate,
80:20) to afford compound 41 (8.00 g, 77%) as a white
solid. 1H NMR (300 MHz, CDCl3): d 1.38 (s, 18H), 2.22
(s, 3H), 4.44 (s, 2H), 7.60 (s, 1H), 8.30 (s, 1H). 13C
NMR (75 MHz, CDCl3): d 17.1, 27.9, 28.9, 83.1, 131.2,
133.6, 139.9, 146.5, 150.7, 151.2.
5.10.7. 2-Acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxy-
carbonyl)amino]-5-methyl-pyridin-3-yl)-propionic
acid
ethyl ester. Triethylamine (0.234 mL, 1.68 mmol) was
added to a solution of compound 55 (0.68 g, 1.61 mmol)
in thioacetic acid (3 mL) at 0 ꢀC. The mixture was stirred
at room temperature for 16 h. Ethyl acetate was added
and the organic phase was washed with water, saturated
NaHCO3 and brine and dried. The crude product was
purified by flash chromatography (toluene/ethyl acetate,
3:1!1:2) to give pure 2-acetylsulfanylmethyl-3-(6-[N,N-
bis(tert-butoxycarbonyl)amino]-5-methyl-pyridin-3-yl)-
propionic acid ethyl ester (489 mg, 61%) and slightly
impure 2-acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxy-

1168
M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
carbonyl)amino]-5-methyl-pyridin-3-yl)-propionic acid
1
The mixture was then filtered and the crystalline residue
washed with cold acetonitrile. The filtrate was con-
centrated under reduced pressure and another crop of
bromide was obtained as described above. Compound
27 (1.86 g, 63%) was obtained as white crystals.%). H
NMR (300 MHz, CDCl3): d 1.54 (s, 9H), 4.41 (s, 2H),
7.30 (bs, 1H), 7.71 (d, J=1.8 Hz), 8.35 (d, J=1.8 Hz).
ethyl ester (0.34 g, 43%). H NMR (300 MHz, CDCl3):
d 1.22 (t, 3H), 1.41 (s, 18H), 2.22 (s, 3H), 2.36 (s, 3H),
2.88-3.17 (m, 5H), 4.06-4.16 (m, 2H), 7.43 (s, 1H), 8.17
(s, 1H).
1
5.10.8.
3-(6-Amino-5-methyl-pyridin-3-yl)-2-mercapto-
methyl-propionic acid (11). 2-Acetylsulfanylmethyl-3-(6-
[N,N-bis(tert-butoxycarbonyl)amino]-5-methyl-pyridin-3-
yl)-propionic acid ethyl ester (17 mg, 0.034 mmol) was
dissolved in concentrated HCl (3.0 mL). The solution
was heated to reflux for 1 h. Concentration under
reduced pressure gave compound 11 (8.9 mg, 100%) as
the hydrochloride salt. 1H NMR (500 MHz, CD3OD): d
2.26 (s, 3H), 2.72–2.75 (m, 2H), 2.83–2.91 (m, 3H), 7.60
(s, 1H), 7.77 (s, 1H). MS (+) 227 (M+1). HRMS (ESI)
calcd for C10H14N2O2S 226.0776, found 226.082.
5.11.4. 2-(6-tert-Butoxycarbonylamino-5-chloro-pyridin-
3-ylmethyl)-malonic acid diethyl ester (45). Diethyl mal-
onate (1.87 mL, 12.31 mmol) was added to a suspension
of NaH (0.54 g, 12.31 mmol, 55% in mineral oil) in dry
DMF (15 mL) at À8 ꢀC. This mixture was stirred for 15
min. before it was added dropwise to a solution of
compound 27 (3.30 g, 10.26 mmol) in dry DMF (50 mL)
at 0 ꢀC. The resulting solution was stirred for 40 min at
0 ꢀC, then saturated aqueous NH4Cl (5 mL) was added
carefully. Stirring at room temperature overnight and
concentration under reduced pressure gave a residue,
which was dissolved in water/CH2Cl2. The aqueous
layer was extracted with CH2Cl2 and the combined
organic extracts were dried, filtered and concentrated
under reduced pressure. Flash chromatography (1%
MeOH in CH2Cl2) gave compound 45 (2.45, 60%) as a
5.11. 3-(6-Amino-5-chloro-pyridin-3-yl)-2-mercaptomethyl-
propionic acid (12)
5.11.1. 6-Amino-5-chloro-nicotinic acid ethyl ester. N-
Chlorosuccinimide (21.7 g, 0.162 mol) was added to a
suspension of 6-amino-nicotinic acid ethyl ester (18.0 g,
0.108 mol) in acetonitrile (270 mL) and the mixture was
refluxed for 2 h. The reaction mixture was filtered and
concentrated under reduced pressure. The residue was
dissolved in dichloromethane, washed with water and
dried. Flash chromatography (2.5% MeOH in CH2Cl2)
gave pure 6-amino-5-chloro-nicotinic acid ethyl ester
1
sticky clear oil. H NMR (300 MHz, CDCl3): d 1.23 (t,
6H), 1.53 (s, 9H), 3.16 (d, 2H), 3.57 (t, 1H), 4.1–4.25 (m,
4H), 7.67 (s, 1H), 8.22 (s, 1H).
5.11.5. 2-(6-tert-Butoxycarbonylamino-5-chloro-pyridin-
3-ylmethyl)-malonic acid monoethyl ester. A solution of
KOH (0.44 g, 6.72 mmol, 85%) in ethanol (5 mL) was
added to a solution of compound 45 (2.45 g, 6.11 mmol)
in ethanol (25 mL) and methylene chloride (10 mL) at
0 ꢀC. The mixture was stirred for 18 h at room temper-
ature. The solvent was concentrated under reduced
pressure and the residue dissolved in water. The aqueous
layer was washed with ether, acidified to pH 4 by 1 M
HCl and extracted with methylene chloride and ethyl
acetate. The combined organic layers were washed with
water and brine and dried. Filtration and concentration
under reduced pressure gave the crude product which
was purified by flash chromatography (10% MeOH in
CH2Cl2) giving 2-(6-tert-butoxycarbonylamino-5-chloro-
pyridin-3-ylmethyl)-malonic acid monoethyl ester (1.41
g, 62%) as a yellow-white glassy foam. 1H NMR
(300 MHz, CDCl3): d 1.23 (t, 6H), 1.53 (s, 9H), 3.16 (d,
2H), 3.57 (t, 1H), 4.1–4.25 (m, 4H), 7.67 (s, 1H), 8.22 (s,
1H).
1
(17.23 g, 79%). H NMR (300 MHz, CDCl3): d 1.36 (t,
3H), 4.34 (q, 2H), 5.38 (br s, 2H, NH2), 8.08 (s, 1H),
8.62 (s, 1H).
5.11.2. (3-Chloro-5-hydroxymethyl-pyridin-2-yl)-carbamic
acid tert-butyl ester (26). DMAP (0.11 g, 0.9 mmol)
and di-tert-butyl dicarbonate (21.54 g, 99 mmol) was
added to a solution of 6-amino-5-chloro-nicotinic acid
ethyl ester (9.0 g, 45 mmol) in dichloromethane (250
mL). The reaction mixture was stirred for 24 h. DMAP
(0.02 equiv) and di-tert-butyl dicarbonate (3ꢁ0.5 equiv)
was added during the reaction time. The reaction mixture
was washed with water and dried. The crude product
was washed with hexane to give pure compound 25
(11.87 g, 66%). LiAlH4 (2.4 g, 63.2 mmol) was added in
portions over a period of 3.5 h to solution of compound
25 (11.5 g, 28.6 mmol) in THF (70 mL) at 0 ꢀC. The
reaction mixture was stirred in room temperature over
night, and then saturated aqueous NH4Cl was added
carefully followed by water. The solution was filtered,
dried and concentrated under reduced pressure to yield
5.11.6. 2-(6-tert-Butoxycarbonylamino-5-chloro-pyridin-
3-ylmethyl)-acrylic acid ethyl ester (46). Diethylamine
(3.67 mL, 3.67 mmol) was added dropwise followed by
water (2.5 mL) and CH2Cl2 (2.5 mL) to a mixture of 2-
(6-tert-butoxycarbonylamino-5-chloro-pyridin-3-ylmethyl)-
malonic acid monoethyl ester (1.40 g, 3.64 mmol) and
formaldehyde (0.29 mL, 3.75 mmol, 37% in water) in
CH2Cl2 (2 mL) at 0 ꢀC. The mixture was stirred for 20 h
at room temperature and then poured onto ice-water and
extracted with methylene chloride. The organic layer was
washed with 5% NaHCO3, dried and concentrated
under reduced pressure. Flash chromatography
(1!2.5% methanol in CH2Cl2) yielded compound 46
1
crude compound 26 (5.86 g, 79%) H NMR (300 MHz,
CDCl3): d 1.58 (s, 9H), 2.09 (s, 1H), 4.66 (2, 2H), 7.47 (s,
1H), 7.76 (d, J=2.0 Hz, 1H), 8.25 (d, J=2.0 Hz, 1H).
5.11.3. (5-Bromomethyl-3-chloro-pyridin-2-yl)-carbamic
acid tert-butyl ester (27). Triphenylphosphine (2.61 g,
9.7 mmol) followed by carbontetrabromide (4.58 g, 13.8
mmol) was added to a suspension of compound 26 (2.38
g, 9.2 mmol) in CH2Cl2 (60 mL) at 0 ꢀC. The mixture
was stirred at room temperature for 5 h and con-
centrated under reduced pressure. Acetonitrile (40 mL)
was added and the mixture was kept to À20ꢀC overnight.
1
(0.81 g, 65%). H NMR (300 MHz, CDCl3): d 1.26 (t,

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1169
3H), 1.53 (s, 9H), 3.56 (s, 2H), 4.17 (q, 2H), 5.53 (d,
1H), 6.26 (s, 1H), 7.19 (br s, NH), 7.53 (d, 1H), 8.28 (br
s, 1H).
NH4Cl was added carefully followed by water, and
the mixture was concentrated under reduced pressure. The
residue was suspended in MeOH/dichloromethane
(5:95) and filtered through silica gel. The filtrate was
dried and concentrated under reduced pressure to give
5.11.7. 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarbonyl-
amino-5-chloro-pyridin-3-ylmethyl)-propionic acid ethyl
ester. Thioacetic acid (4 mL) was added to a suspension
of compound 46 (0.732 g, 2.16 mmol) and triethylamine
(0.31 mL, 2.23 mmol) at 0 ꢀC. The mixture was stirred
at room temperature under argon overnight, poured
onto ice-water and extracted with CH2Cl2. The organic
phase was washed with saturated NaHCO3 until gas
evolution ceased and then dried. The crude product was
purified twice with flash chromatography (CH2Cl2, 1–
2.5% MeOH in CH2Cl2 and Hexane/EtOAc, 5:2!1:1)
to give pure 2-acetylsulfanylmethyl-3-(6-tert-butoxy-
carbonylamino-5-chloro-pyridin-3-ylmethyl)-propionic
acid ethyl ester (0.79 g, 87%). 1H NMR (300 MHz,
CDCl3): d 1.17 (t, 3H), 1.52 (s, 9H), 2.32 (s, 3H), 2.78–
2.98 (m, 3H), 3.0–3.15 (m, 2H), 4.08 (q, 2H), 7.21 (br s,
NH), 7.52 (d, 1H), 8.16 (d, 1H).
(5-hydroxymethyl-3-vinyl-pyridin-2-yl)carbamic
acid
tert-butyl ester (1.00 g, 78%). 1H NMR (300 MHz,
CDCl3): d 1.50 (s, 9H), 1.72, (bs, 1H), 4.70 (s, 3H), 5.45
(d, J=11.6 Hz, 1H), 5.78 (d, J=17.8 Hz, 1H), 6.78 (dd,
J=11.6, 17.8 Hz, 1H), 7.96 (d, J=1.8 Hz, 1H), 8.29 (d,
J=1.8 Hz, 1H).
5.12.3. (5-Bromomethyl-3-vinyl-pyridin-2-yl)carbamic acid
tert-butyl ester (80). To a stirred suspension of (5-
hydroxymethyl-3-vinyl-pyridin-2-yl)carbamic acid tert-
butyl ester (10.0 g, 40.0 mmol) in CH2Cl2 (150 mL) and
THF (60 mL) was added triphenylphosphine (11.5 g,
44.0 mmol) followed by carbontetrabromide (19.9 g,
60.0 mmol) at 0 ꢀC. The mixture was stirred at room
temperature for 1 h and then concentrated under
reduced pressure. Acetonitrile (100 mL) was added and
the mixture was kept at À20 ꢀC overnight. The mixture
was then filtered and the crystalline residue washed with
cold acetonitrile. The product was purified by flash
chromatography (MeOH/dichloromethane, 1:99) to
5.11.8. 3-(6-Amino-5-chloro-pyridin-3-yl)-2-mercapto-
methyl-propionic acid (12). A solution of 2-acetylsulf-
anylmethyl-3-(6-tert-butoxycarbonylamino-5-chloro-pyr-
idin-3-yl)-propionic acid ethyl ester (55mg, 0.132 mmol)
in concentrated aqueous HCl (4 mL) was refluxed for 90
min. The reaction was cooled and concentrated under
reduced pressure to give compound 12 as the HCl salt
1
give compound 80 (4.5 g, 36%). H NMR (300 MHz,
CDCl3): d 1.50 (s, 9H), 1.72, (bs, 1H), 4.42 (s, 2H), 5.49
(d, J=11.0 Hz, 1H), 5.78 (d, J=17.0 Hz, 1H), 6.78 (dd,
J=11.0, 17.0 Hz, 1H), 7.91 (d, J=1.8 Hz, 1H), 8.32 (d,
J=1.8 Hz, 1H).
1
(36mg, 96.4%). H NMR (400 MHz, D2O): d 2.70–2.97
(m, 5H), 7.73 (s, 1H), 8.09 (s, 1H). MS (+) 248 (M+1).
HRMS (ESI) calcd for C9H11N2O2ClS 246.0230, found
246.0286.
5.12.4. 2-(6-tert-Butoxycarbonylamino-5-vinyl-pyridin-3-
ylmethyl)-malonic acid diethyl ester. Diethyl malonate
(2.30 g, 14.3 mmol) was added to a suspension of NaH
(0.61 g, 14.3 mmol, 55%) in dry DMF (75 mL) at 0 ꢀC.
The mixture was stirred for 15 min and then added
dropwise to a solution of compound 80 (4.50 g, 14.3
mmol) in dry DMF (100 mL) at 0 ꢀC. The resulting
solution was stirred for 40 min at 0 ꢀC, and then satu-
rated aqueous NH4Cl (30 mL) was added carefully.
Concentration under reduced pressure gave a residue,
which was dissolved in water/CH2Cl2. The aqueous
layer was extracted with CH2Cl2 and the combined
extracts were dried, filtered and concentrated under
reduced pressure. Flash chromatography (MeOH/
CH2Cl2, 1:99) gave 2-(6-tert-Butoxycarbonylamino-5-
5.12. 3-(6-Amino-5-hydroxymethyl-pyridin-3-yl)-2-mer-
captomethyl-propionic acid (13)
5.12.1. 6-bis(tert-Butoxycarbonyl)amino-5-vinyl-nicotinic
acid ethyl ester. A mixture of compound 29 (4.50 g,
10.1 mmol), vinyltributyltin (3.52 g, 11.1 mmol) and
tetrakispalladium triphenyphosphine(0.50 g, 0.40 mmol)
in THF (15 mL) was stirred at reflux for 24 h. Tetra-
kispalladium triphenyphosphine(0.50 g) was added
and after 24 h at reflux the reaction mixture was cooled
and diluted with dichloromethane (100 mL). Saturated
aqueous KF (25 mL) was added and the solution stir-
red for 1 h. Water was added and the product extracted
with dichloromethane, the organic phase was dried
and concentrated under reduced pressure. Flash chroma-
tography (1% MeOH in dichloromethane) gave
6-bis(tert-butoxycarbonyl)-amino-5-vinyl-nicotinic acid
ethyl ester 3.20 g, (81%).1H NMR (300 MHz, CDCl3): d
1.30 (s, 18H), 1.36 (t, J=7.0 Hz, 3H), 4.38 (q, J=7.0
Hz, 2H), 5.46 (d, J=10.8 Hz, 1H), 5.83 (d, J=17.3 Hz,
1H), 6.65 (dd, J=10.8, 17.3 Hz, 1H), 8.40 (d, J=1.8 Hz,
1H), 8.92 (d, J=1.8 Hz, 1H).
vinyl-pyridin-3-ylmethyl)-malonic acid diethyl ester
1
(4.30 g, 77%). H NMR (300 MHz, CDCl3): d 1.21 (t,
:
J=7.1 Hz, 6H), 1.48 (s, 9H), 3.19 (d, J=7.7 Hz, 2H),
3.58 (t, J=7.7 Hz, 1H), 4.15 (q, J=7.1 Hz, 2H), 4.17 (q,
J=7.1 Hz, 2H), 5.42 (d, J=11.0 Hz, 1H), 5.72 (d,
J=17.5 Hz, 1H), 6.72 (dd, J=11.0, 17.5 Hz, 1H), 7.74
(d, J=2.4 Hz, 1H), 8.00 (s, 1H), 8.17 (d, J=2.4 Hz,
1H).
5.12.5. 2-(6-tert-Butoxycarbonylamino-5-vinyl-pyridin-3-
ylmethyl)-malonic acid monoethyl ester. A solution of
KOH (0.71 g, 12.6 mmol, 85%) in ethanol (10 mL) was
added to a solution of 2-(6-tert-butoxycarbonylamino-
5-vinyl-pyridin-3-ylmethyl)-malonic acid diethyl ester
(4.30 g, 11.0 mmol) in ethanol (25 mL) and dichloro-
methane (10 mL) at 0 ꢀC. The mixture was stirred for 6
h at room temperature. The solvent was concentrated
5.12.2. (5-Hydroxymethyl-3-vinyl-pyridin-2-yl)carbamic
acid tert-butyl ester. DIBAL (25 mL, 1M in hexane)
was added dropwise to a solution of 6-bis(tert-butoxy-
carbonyl)amino-5-vinyl-nicotinic acid ethyl ester (2.00
g, 5.1 mmol) in THF (40 mL) at 0 ꢀC. The mixture was
stirred at room temperature for 1 h. Saturated aqueous

1170
M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
under reduced pressure and the residue dissolved in
water. The aqueous layer was washed with ether, acid-
ified to pH 4 by 1 M HCl and extracted with dichloro-
methane. The organic layer was washed with water and
brine and dried. Filtration and concentration under
reduced pressure followed by flash chromatography
(MeOH/CH2Cl2, 1:9) gave 2-(6-tert-butoxycarbonylamino-
5-vinyl-pyridin-3-ylmethyl)-malonic acid monoethyl ester
initial volume. 50% aqueous NaCl was added and the
mixture was extracted with dichloromethane, dried and
concentrated under reduced pressure to give crude 3-(6-
tert-butoxy-carbonylamino-5-hydroxymethyl-pyridin-3-
yl)-mercaptomethyl-propionic acid ethyl ester (0.38 g,
63%).1H NMR (300 MHz, CDCl3): d 1.16 (t, J=7.1 Hz,
:
3H), 1.49 (s, 9H), 2.07 (s, 1H), 2.32 (s, 3H), 2.81–3.14 (b
m, 5H), 4.07 (q, J=7.1 Hz, 2H), 5.09 (s, 2H), 7.58 (d,
J=2.4 Hz, 1H), 8.16 (d, J=2.4 Hz, 1H).
1
(3.05 g, 76%). H NMR (300 MHz, CDCl3): d 1.25 (t,
J=7.1 Hz, 3H), 1.48 (s, 9H), 3.21 (m, 2H), 3.62 (t,
J=7.5 Hz, 1H), 4.20 (q, J=7.1 Hz, 2H), 5.38 (d,
J=11.1 Hz, 1H), 5.72 (d, J=17.4 Hz, 1H), 6.66 (dd,
J=11.1, 17.4 Hz, 1H), 7.83 (d, J=1.8 Hz, 1H), 8.00 (s,
1H), 8.05 (d, J=1.8 Hz, 1H).
5.12.9. Ethyl 3-{5-[(acetyloxy)methyl]-6-[(tert-butoxy-
carbonyl)amino]pyridin-3-yl}-2-[(acetylthio)methyl]pro-
panoate. A mixture of crude 3-(6-tert-butoxycarbonyl-
amino-5-hydroxymethyl-pyridin-3-yl)-mercaptomethyl-
propionic acid ethyl ester (0.38 g, 1.0 mmol) and
KHCO3 (0.11 g, 1.1 mmol) in acetic acid anhydride (1
mL) was stirred at room temperature for 5 h. Saturated
aqueous NH4Cl and water was then added. The mixture
was extracted with dichloromethane, dried and con-
centrated under reduced pressure. Flash chromatography
(CH2Cl2, 2.5% MeOH in CH2Cl2) gave ethyl 3-{5-[(acet-
yloxy)methyl]-6-[(tert-butoxycarbonyl)amino]pyridin-3-
yl}-2-[(acetylthio)methyl]propanoate (0.23 g, 60%).1H
NMR (300 MHz, CDCl3):? d?1.17 (t, J=7.1 Hz, 3H),
1.51 (s, 9H), 2.11 (s, 3H), 2.34 (s, 3H), 2.84–3.03 (m,
3H), 3.09 (dd, J=4.2, 6.0 Hz, 2H), 4.08 (q, J=7.1 Hz,
2H), 5.11 (s, 2H), 7.66 (s, 1H), 8.17 (s, 1H).
5.12.6. 2-(6-tert-Butoxycarbonylamino-5-vinyl-pyridin-3-
ylmethyl)- acrylic acid ethyl ester. Diethylamine (0.85
mL, 8.80 mmol) was added dropwise to a mixture of 2-
(6-tert-butoxycarbonylamino-5-vinyl-pyridin-3-ylmethyl)-
malonic acid monoethyl ester (3.05 g, 8.37 mmol) and
formaldehyde (0.71 g, 8.80 mmol, 37% in water) in
CH2Cl2 (2 mL) at 0 ꢀC. The mixture was stirred for 3 h
at room temperature and then poured onto ice-water and
extracted with dichloromethane. The organic layer was
washed with 5% NaHCO3 and dried. Flash chromato-
graphy (MeOH/CH2Cl2, 1:99) yielded 2-(6-tert-butoxy-
carbonylamino-5-vinyl-pyridin-3-ylmethyl)-acrylic
acid
1
ethyl ester (1.70 g, 61%). H NMR (300 MHz, CDCl3):
d 1.26 (t, J=7.4 Hz, 3H), 1.48 (s, 9H), 3.61 (s, 2H), 4.18
(q, J=7.4 Hz, 2H), 5.42 (d, J=11.4 Hz, 1H), 5.55 (s,
1H), 5.72 (d, J=18.0 Hz, 1H), 6.26 (s, 1H), 6.73 (dd,
J=11.4, 18.0 Hz, 1H), 7.72 (d, J=1.9 Hz, 1H), 8.16 (d,
J=1.9 Hz, 1H).
5.12.10. 3-[6-Amino-5-(hydroxymethyl)pyridin-3-yl]-2-
(mercaptomethyl)propanoic acid (13). A solution of
ethyl 3-{5-[(acetyloxy)methyl]-6-[(tert-butoxycarbonyl)-
amino]pyridin -3-yl}-2-[(acetylthio)methyl]propanoate
(50 mg, 135 mmol) in concentrated aqueous HCl (2 mL)
was refluxed for 60 min. The reaction was cooled and
concentrated under reduced pressure to give compound
5.12.7. 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarbonyl-
amino-5-vinyl-pyridin-3-yl)-propionic acid ethyl ester
(81). Thioacetic acid (4 mL) was added to a suspension
of 2-(6-tert-butoxycarbonylamino-5-vinyl-pyridin-3-yl-
methyl)-acrylic acid ethyl ester (0.73 g, 2.16 mmol) in
triethylamine (0.31 mL, 2.23 mmol) at 0 ꢀC. The mixture
was stirred at room temperature under argon overnight,
poured onto icewater and extracted with CH2Cl2. The
organic phase was washed with saturated NaHCO3
until gas evolution ceased and then dried. Flash chro-
1
13 as the HCl salt (37mg, 98.3%). H NMR (400 MHz,
D2O): d 2.70–2.95 (m, 5H), 4.65 (s, 2H), 7.68 (s, 1H),
7.88 (s, 1H). MS (+) 244 (M+1). HRMS (ESI) calcd
for C10H14N2O3S 242.0725, found 242.0785.
5.13. 2-(6-Amino-pyridin-3-ylmethyl)-2-mercaptomethyl-
butyric acid (14)
5.13.1. [5-(5-Ethyl-2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-
ylmethyl)-pyridin-2-yl]-carbamic acid tert-butyl ester
(74). Compound 23 (1.0 g, 3.48 mmol) was added to a
solution of 2,2-dimethyl-5-ethyl-1,3-dioxane-4,6-dione
(600 mg, 3.48 mmol) and triethylamine (0.51 mL, 3.66
mmol) in dimethyl sulfoxide (40 mL) under nitrogen.
The reaction mixture was stirred over night and water
(100 mL) was added. Filtration of the precipitate gave
compound 74 (1.15 g, 87%). 1H NMR (500 MHz,
CDCl3): d 0.96 (s, 3H), 1.02 (t, 3H), 1.53 (s, 9H),1.63 (s,
3H), 2.20 (q, 2H), 3.27 (s, 2H), 7.56 (d, 1H), 7.92 (m,
1H), 8.07 (m, 1H).
matography (MeOH/CH2Cl2, 1:99) gave compound 81
1
(0.51 g, 70%). H NMR (300 MHz, CDCl3): d 1.16 (t,
:
J=6.6 Hz, 3H), 1.48 (s, 9H), 2.34 (s, 3H), 2.80–3.15 (br
m, 5H), 4.09 (m, 2H), 5.42 (d, J=10.8 Hz, 1H), 5.75 (d,
J=17.8 Hz, 1H), 6.73 (dd, J=10.8, 17.8 Hz, 1H), 7.68
(d, J=2.2 Hz, 1H), 8.19 (d, J=2.2 Hz, 1H).
5.12.8. 3-(6-tert-Butoxycarbonylamino-5-hydroxymethyl-
pyridin-3-yl)-mercaptomethyl-propionic acid ethyl ester.
Ozone was bubbled through a solution of 2-acetyl-
sulfanylmethyl-3-(6-tert-butoxycarbonylamino-5-vinyl-
pyridin-3-yl)-propionic acid ethyl ester (0.65 g, 1.60
mmol) in ethanol (25 mL) at À78 ꢀC. O2 was then
bubbled through the mixture for 5 min followed by N2
bubbling for 15 min. A mixture of NaBH4 0.30 g, 8.00
mmol) in water was carefully added to the mixture at
À78 ꢀC, and the reaction mixture allowed to reach 0 ꢀC.
Stirring was continued for 3 h. Acetone (10 mL) was
added and the reaction mixture evaporated to 1/3 of the
5.13.2. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
2-ethyl-malonic acid monoethyl ester. A solution of
sodium metal (140 mg, 6.08 mmol) in ethanol (20 mL)
was added to a solution of compound 74 (1.15 g, 3.04
mmol) in ethanol (10 mL). The reaction was stirred for
90 min and methylene chloride was then added. The mix-
ture was washed with 0.5M HCl, dried and concentrated

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1171
under reduced pressure to give 2-(6-tert-butoxy-carbonyl-
amino-pyridin-3-ylmethyl)-2-ethyl-malonic acid mono-
ethyl ester (1.05 g, 95%). 1H NMR (400 MHz,
CD3SOCD3): d 0.85 (t, 3H), 1.15 (t, 3H), 1.62 (m, 2H),
3.03 (m, 2H), 4.04–4.14 (m, 2H), 7.49 (dd, 1H), 7.62 (d,
1H), 7.96 (d, 1H), 9.88 (s, 1H), 13.0 (bs, 1H).
À70 ꢀC. Tert-butyl ethyl malonate (154 mg, 0.82 mmol)
was added and the reaction mixture was stirred for 60
min. A solution of compound 41 (0.3 g, 0.75 mmol) in
tetrahydrofuran (10 mL) was then added dropwise. The
reaction mixture was allowed to warm to room temper-
ature and was stirred overnight. The reaction mixture was
cooled to 0 ꢀC and quenched with water. The aqueous
layer was extracted with ethyl acetate and the organic
phase was washed with brine and dried. Concentration
under reduced pressure followed by flash chromatography
(hexane/ethyl acetate, 3:1) yielded 2-(6-bis-tert-butoxy-
carbonylamino-5-methyl-pyridin-3-ylmethyl)-malonic
5.13.3. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
2-hydroxymethyl-butyric acid ethyl ester (76). Methyl
chloroformate (150 mL, 1.95 mmol) was added dropwise
to a solution of 2-(6-tert-butoxycarbonylamino-pyridin-
3-ylmethyl)-2-ethyl-malonic acid monoethyl ester (700
mg, 1.91 mmol) and Et3N (275 mL, 1.97 mmol) in THF
(15 mL) at-20 ꢀC under nitrogen. The reaction mixture
was stirred for 50 min., filtered and added dropwise to a
suspension of NaBH4 (80 mg, 2.1 mmol) in THF (15
mL) at-20 ꢀC. The reaction was stirred for 16 h at room
temperature. 0.2 M HCl was added followed by meth-
ylene chloride. The organic phase was washed with
brine and dried. Concentration under reduced pressure
followed by chromatography (toluene/EtOAc, 3:1!1:3)
gave compound 76 (300 mg, 45%). 1H NMR (500 MHz,
CDCl3): d 0.89 (t, 3H), 1.27 (t, 3H), 1.47–1.53 (m, 1H),
1.54 (s, 9H),1.61–1.73 (m, 1H), 2.83 (d, 1H), 3.03 (d,
1H), 3.46 (d, 1H), 3.73 (d, 1H), 7.56 (d, 1H), 7.92 (m,
1H), 8.07 (m, 1H).
1
acid tert-butyl ester ethyl ester (178 mg, 59% yield). H
NMR (300 MHz, CDCl3) d 1.26 (m, 3H), 1.39 (s, 9H),
1.43 (s, 3H), 2.19 (s, 3H), 3.16 (d, 2H), 3.52 (t, 1H), 4.16
(m, 2H), 7.44 (d, 1H), 8.20 (d, 1H).
5.14.2. 2-Benzyloxymethyl-2-(6-bis-tert-butoxycarbonyl-
amino-5-methyl-pyridin-3-ylmethyl)-malonic acid tert-
butyl ester ethyl ester. A solution of 2-(6-bis-tert-butoxy-
carbonylamino-5-methyl-pyridin-3-ylmethyl)-malonic
acid tert-butyl ester ethyl ester (200 mg, 393 mmol) in
tetrahydrofuran (5 mL) was cooled to 0 ꢀC and lithium
diisopropylamide (2.16 mL, 0.43 mmol, 2 M solution in
heptane/THF/ethylbenzene) was added. The mixture
was stirred for 0.5 h and benzyl chloromethyl ether
(66.90 mg, 0.393 mmol) was added. This mixture was
stirred for 1 h at 0 ꢀC and then allowed to warm to
room temperature overnight. Water was added and the
mixture was extracted with ethyl acetate. The combined
organic layer was washed with brine, dried and con-
centrated under reduced pressure to afford 0.23 g of
crude product. Purification by flash chromatography
(ethyl acetate/hexane, 1:5), yielded 2-benzyloxymethyl-2
-(6-bis- tert-butoxycarbonylamino-5-methyl-pyridin-3-
ylmethyl)-malonic acid tert-butyl ester ethyl ester (149 mg,
60% yield). 1H NMR (300MHz, CDCl3) d 1.25 (m, 3H),
1.37 (s, 9H), 1.43 (s, 9H), 2.12 (s, 3H), 3.33 (d, 2H), 3.62 (s,
2H), 4.15 (m, 2H), 4.51 (s, 2H), 7.30 (m, 6H), 8.11 (d, 1H).
5.13.4. 2-Acetylsulfanylmethyl-2-(6-tert-butoxycarbonyl-
amino-pyridin-3-ylmethyl)-butyric acid ethyl ester. Diiso-
propyl azodicarboxylate (296 mL, 1.53 mmol) was added
dropwise to a solution of triphenylphosphine (402 mg,
1.53 mmol) in THF (4 mL) at 0 ꢀC under argon and the
reaction was stirred for 30 min. A solution of thioacetic
acid (109 mL, 1.53 mmol) and compound 76 (0.27 g,
0.77 mmol) in THF (2 mL) was added dropwise during
10 min. The reaction was stirred for 60 min at 0 ꢀC and
then for 16 h at room temperature. Concentration under
reduced pressure followed by chromatography (heptane/
EtOAc, 10:1!1:1) gave 2-acetylsulfanylmethyl-2-(6-tert-
butoxycarbonylamino-pyridin-3-ylmethyl)-butyric acid
ethyl ester (193 mg, 61%). 1H NMR (500 MHz,
CD3OD): d 0.88 (t, 3H), 1.23 (t, 3H), 1.52 (s, 9H),1.56–
1.78 (m, 2H), 2.35 (s, 3H), 2.86 (dd, 2H), 3.15 (dd, 2H),
4.12 (q, 2H), 7.47 (dd, 1H), 7.74 (d, 1H), 7.96 (d, 1H).
5.14.3. 2-Benzyloxymethyl-2-(6-tert-butoxycarbonylamino-
5-methyl-pyridin-3-ylmethyl)-malonic acid mono-tert-
butyl ester (78). KOH (14 g, 250 mmol) was added
slowly to a solution of 2-benzyloxymethyl-2-(6-bis-tert-
butoxycarbonylamino-5-methyl -pyridin -3-ylmethyl)-
malonic acid tert-butyl ester ethyl ester (3.2 g, 5 mmol)
in CH2Cl2 (10 mL), EtOH (200 mL) and H2O (100 mL)
at 0 ꢀC. The reaction mixture was stirred at room
temperature for 4 days. The reaction was quenched by
slow addition of 1 M HCl at 0 ꢀC until pH=2–3 and
was then extracted with CH2Cl2. The combined organic
layer was dried and concentrated under reduced pressure.
Filtration through a plug of silica (EtOAc) yielded
compound 78 (1.9 g, 60%). 1H NMR (300 MHz,
CDCl3): d 1.46 (s, 9H), 1.50 (s, 9H), 2.13 (s, 3H), 3.25–
3.29 (m, 2H), 3.44 (d, J=9.6 Hz, 1H), 3.93 (d, J=9.3
Hz, 1H), 4.51 (d, J=12.3 Hz, 1H), 4.63 (d, J=12.3 Hz,
1H), 7.25–7.40 (m, 7H), 7.78 (s, 1H).
5.13.5. 2-(6-Amino-pyridin-3-ylmethyl)-2-mercaptomethyl-
butyric acid (14). 2-Acetylsulfanylmethyl-2-(6-tert-
butoxycarbonylamino-pyridin-3-ylmethyl)-butyric acid
ethyl ester (12.3 mg, 30 mmol) was dissolved in con-
centrated HCl (2 mL) under argon. The solution was
heated to reflux for 24 h. Concentration under reduced
pressure gave compound 14 as the hydrochloride salt
1
(8.3 mg, 100%). H NMR (500 MHz, CD3OD): d 0.91
(t, 3H), 1.71 (m, 2H), 2.68 (m, 2H), 2.92 (m, 2H), 6.96
(d, 1H), 7.65 (bs, 1H), 7.82 (dd, 1H). MS (À) 239 (M-1).
5.14. 3-(6-Amino-5-methylpyridin-3-yl)-2-(hydroxymethyl)-
2-(mercaptomethyl)-propanoic acid (15)
5.14.1. 2-(6-bis-tert-Butoxycarbonylamino-5-methyl-pyri-
din-3-ylmethyl)-malonic acid tert-butyl ester ethyl ester.
A mixture of sodium hydride (33 mg, 0.82 mmol, 60%
in mineral oil) in tetrahydrofuran (10 mL) was cooled to
5.14.4. 2-Benzyloxymethyl-3-(6-tert-butoxycarbonylamino-
5-methyl-pyridin-3-yl)-2-hydroxymethyl-propionic acid
tert-butyl ester. BOP (975 mg, 2.2 mmol) was added to
a solution of compound 78 (1g, 2 mmol) in THF (80

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M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
mL). DIPEA (470 mL, 2.4 mmol) was added and the
solution was stirred at room temperature for 5 h. The
solvent was removed under reduced pressure and EtOH
(60 mL) was added. To this mixture LiBH4 (175 mg) was
added in portions. The reaction mixture was stirred for 40
min and saturated aqueous NH4Cl was added followed
by extraction with EtOAc. The combined organic layer
was dried and concentrated under reduced pressure.
Flash chromatography (EtOAc/hexane, 1:9 to 3:7) yielded
2-benzyloxymethyl-3-(6- tert-butoxycarbonylamino-5-
pyridin-3-yl)-2-hydroxymethyl-propionic acid tert-butyl
ester (380 mg, 0.84 mmol) was dissolved in concd HCl
(4.0 mL) under argon. The solution was then heated to
reflux for 3 h. Concentration under reduced pressure
gave compound 15 (251 mg, 100%) as the hydrochloride
1
salt. H NMR (500 MHz, CD3OD): d 2.24 (s, 3H), 2.74
(d, 1H), 2.83 (d, 1H), 2.91 (s, 2H), 3.72 (d, 1H), 3.83 (d,
1H), 7.58 (s, 1H), 7.68 (s, 1H). HRMS (ESI) calcd for
C11H16N2O3S 256.0882, found 256.0942.
methyl-pyridin-3-yl)-2-hydroxymethyl-propionic
acid
5.15. 2-Mercaptomethyl-3-(6-amino-pyridin-3-yl)-2-hydr-
oxy-propionic acid (16)
tert-butyl ester (700 mg, 72%). 1H NMR (300 MHz,
CDCl3): d 1.42 (s, 9H), 1.50 (s, 9H), 2.22 (s, 3H), 2.89 (s,
2H), 3.54 (dd, 2H), 3.75 (q, 2H, J=13.2 Hz), 4.50 (s,
2H), 7.25–7.4 (m, 6H), 8.07 (s, 1H).
5.15.1. 2-Hydroxymethyl-3-(6-tert-butyoxycarbonylamino-
pyridin-3-yl)-2-hydroxy-propionic acid ethyl ester (82).
To a solution of compound 43 (2.5 g, 8.16 mol) in Ace-
tone/H2O (4/1, 200 mL) at 0 ꢀC was added N-methyl
morpholine N-oxide (1.0 g, 8.5 mmol) followed by OsO4
(12 mg). The reaction mixture was stirred at rt for 16 h.
Na2S2O5 (1.0 g) was then added and the mixture was
stirred at rt for 2 h. The mixture was extracted with
EtOAc, washed with brine and dried to afford crude
5.14.5. 3-(6-tert-Butoxycarbonylamino-5-methyl-pyridin-
3-yl)-2,2-bis-hydroxymethyl-propionic acid tert-butyl ester
(79).
A suspension of 2-benzyloxymethyl-3-(6-tert-
butoxycarbonylamino-5-methyl-pyridin-3-yl)-2-hydroxy-
methyl-propionic acid tert-butyl ester (0.6 g, 1.23 mmol),
Pd (0.15 g, 10% on carbon, 50% wet) and concentrated
HCl (0.4 mL) in ethanol (30 mL) was shaken under
hydrogen atmosphere (50 psi) for 18 h. The reaction
mixture was filtered through Celite and concentrated
under reduced pressure. Filtration through a plug of
silica (dichloromethane to MeOH/dichloromethane,
1:9) yielded compound 79 (398 mg, 81%). 1H NMR
(300 MHz, CDCl3): d 1.47 (s, 9H), 1.5 (s, 9H), 2.17 (s,
3H), 2.32 (s, 2H), 2.83 (s, 2H), 3.6–3.82 (m, 4H), 7.26–
7.47 (m, 2H), 8.12 (s, 1H). MS: (M+) 397.8 (M+1).
1
compound 82 (2.5 g, 90%) as a white solid . H NMR
(300 MHz, DMSO): d 1.52 (t, J=6.9 Hz, 3H), 1.47 (s,
9H), 2.73–2.87 (m, 2H), 3.39–3.60 (m, 2H), 4.01–4.08
(m, 2H), 4.91–4.95 (m, 1H), 5.11 (s, 1H), 7.54 (dd,
J=8.7, 2.1 Hz, 1H), 7.67 (d, J=8.7 Hz, 1H), 8.03 (s,
1H), 9.68 (s, 1H). 13C NMR (300 MHz, DMSO): d
14.09, 28.02, 37.01, 60.38, 66.87, 78.45, 79.44, 111.39,
126.24, 139.52, 148.72, 150.85, 152.76, 179.43. MS
(APCI): 341.0 (M+1).
5.14.6. 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarbonyl-
amino-5-methyl-pyridin-3-yl)-2-hydroxymethyl-propionic
acid tert-butyl ester. Triphenyl phosphine (215 mg, 0.82
mmol) and DEAD (130 mL, 0.82 mmol) was added to a
solution of compound 79 (200 mg, 0.41 mmol) in THF
(15 mL) After stirring for 30 min, thiolacetic acid (63
mL, 0.82 mmol) was added. After stirring for 5 h, the
solvent was removed under reduced pressure and satu-
rated aqueous NaHCO3 (1 mL) was added. The mixture
was extracted with EtOAc, dried and concentrated
under reduced pressure. Flash chromatography
(EtOAc/hexane, 1:9 to 3:7) yielded 60 mg pure product
and 100 mg of mixture product/reduced DEAD. This
fraction was repurified to provide 2-Acetylsulfanyl-
methyl-3-(6-tert-butoxycarbonylamino-5-methyl-pyridin-
3-yl)-2-hydroxymethyl-propionic acid tert-butyl ester in
a total yield of 100 mg (45%). FT–IR (cmÀ1): 3342,
1720, 1367, 1244 and 1156. 1H NMR (300 MHz,
CDCl3): d 1.42 (s, 9H), 1.5 (s, 9H), 2.24 (s, 3H), 2.40 (s,
3H), 2.85 (dd, 2H), 3.18 (q, 2H), 3.52–3.59 (m, 2H), 6.70
(s, 1H), 7.36 (s, 1H), 8.05 (s, 1H). 13C NMR
(75.45 MHz, CDCl3) d 17.80, 27.98, 28.21, 30.5, 31.48,
35.78, 53.57, 62.44, 80.64, 82.34, 125.94, 128.81, 141.19,
146.97, 148.53, 152.48, 172.12, 198.49. MS: (M+) 455.3
(M+1). Elemental analysis: Anal. calcd for C22H34-
N2O6S*H2O: C, 55.91; H, 7.68; N, 5.93. Found: C,
55.79; H, 7.21; N, 5.95.
5.15.2. 2-Acetylsulfanylmethyl-3-(6-tert-butyoxycarbonyl-
amino-pyridin-3-yl)-2-hydroxy-propionic acid ethyl ester.
To a solution of compound 82 (2g, 5.9 mmol) in THF
(120 mL) was added triphenylphosphine (3.1 g, 11.8
mmol) and diethylazodicarboxylate (2 g, 11.8 mmol).
After stirring for 0.5 h, thiolacetic acid (0.9 mL) was
added. The reaction mixture was stirred for 60 h. The
mixture was diluted with EtOAc, then quenched with
saturated aqueous NaHCO3 (10 mL). A white solid
formed, which was filtered and washed with EtOAc.
The organic layer was washed with water and brine, and
dried. Concentration under reduced pressure followed
by flash chromatography (EtOAc/hexane, 1:9 to 3:7)
gave 720 mg product with 90% purity and 480 mg
impure product. The 720 mg batch was purified by
recrystallization from diethyl ether to provide 580 mg pure
2-Acetylsulfanylmethyl-3-(6-tert-butyoxycarbonylamino-
pyridin-3-yl)-2-hydroxy-propionic acid ethyl ester. 1H
NMR (300 MHz, CDCl3): d 1.28 (t, 3H), 1.53 (s, 9H),
2.34 (s, 3H), 3.02 (m, 2H), 3.3 (d, 1H), 3.53 (d, 1H), 3.79
(s, 1H), 4.16–4.18 (m, 2H), 7.52 (d, J=8.7, 1H), 7.86 (d,
J=8.7, 1H), 8.11 (s, 1H), 8.38 (s, 1H). 13C NMR
(300 MHz, CDCl3): d 14.12, 28.29, 30.43, 37.56, 40.69,
62.56, 80.80, 111.63, 125.03, 139.76, 148.80, 151.27,
152.51, 173.53, 194.42. Elemental analysis: Anal. calcd
C 54.21%, H 6.52%, N 7.03%; found: C 54.34%, H
6.64%, N 7.01%. MS (CI): 399 (M+).
5.14.7. 3-(6-Amino-5-methylpyridin-3-yl)-2-(hydroxy-
methyl)-2-(mercaptomethyl)-propanoic acid (15). 2-Acetyl-
sulfanylmethyl-3-(6-tert-butoxycarbonylamino-5-methyl-
5.15.3. 2-Mercaptomethyl-3-(6-amino-pyridin-3-yl)-2-
hydroxy-propionic acid (16). 2-Acetylsulfanylmethyl-3-
(6-tert-butyoxycarbonylamino-pyridin-3-yl)-2-hydroxy-

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1173
propionic acid ethyl ester (47 mg, 118 mmol) was dis-
solved in concentrated HCl (2 mL) under argon. The
solution was heated to reflux for 18 h. Concentration
under reduced pressure yielded compound 16 as the
reduced pressure. Flash chromatography (Hexane/
EtOAc, 9:1) gave compound 72 (30 mg, 32%) as a white
solid. 1H NMR (300 MHz, CDCl3): d 1.22 (t, J=7.2 Hz,
3H), 1.54 (s, 9H), 3.08 (s, 1H), 3.18 (s, 1H), 3.79–4.02
(m, 2H), 4.25 (q, J=7.2 Hz, 2H), 7.53 (dd, J=1.8, 8.7
Hz, 1H), 7.92 (d, J=8.4 Hz, 1H), 8.14 (d, J=1.8 Hz
1H), 8.44 (s, 1H). MS (CI): 342.8 (M+).
1
hydrochloride salt (31 mg, 99%). H NMR (500 MHz,
D2O): d 2.87 (d, 1H), 2.98 (d, 1H), 3.08 (d, 1H), 3.11 (d,
1H), 6.99 (d, 1H), 7.68 (s, 1H), 7.85 (d, 1H). HRMS
(ESI) calcd for C9H12N2O3S 228.0569, found 228.0616.
5.16.4. Ethyl-2-(acetylthiomethyl)-3-{6-[(tert-butoxy) car-
bonylamino](3-pyridyl]-2-fluoropropanoate. Diethyl azo-
dicarboxylate (74.8 mg, 0.43 mmol) was added to a
solution of compound 72 (70 mg, 0.21 mmol) and PPh3
(113 mg, 0.43 mmol) in THF (5 mL). The reaction
mixture was stirred for 10 min where after thiolacetic
acid (39 mg, 0.52 mmol) was added. The reaction mixture
was stirred for 16 h. Saturated aqueous NaHCO3 was
added and the mixture was extracted with EtOAc. The
organic layer was washed with brine and dried. Con-
centration under reduced pressure followed by flash
chromatography (Hexane/EtOAc, 9:1) gave ethyl-2-
(acetylthiomethyl)-3-{6-[(tert-butoxy) carbonylamino]
(3-pyridyl]-2-fluoropropanoate (30 mg, 37%) as a white
solid. m.p.; 113.5–114.5 ꢀC. 1H NMR (300 MHz,
CDCl3): d 1.21 (t, J=7.4 Hz, 3H), 1.52 (s, 9H), 2.38 (s,
3H), 3.12 (s, 1H), 3.19 (d, J=3.9 Hz, 1H), 3.43–3.57 (m,
2H), 4.14 (q, J=7.5 Hz, 2H), 7.46 (s, 1H), 7.53 (dd,
J=1.5, 8.4 Hz, 1H), 7.88 (d, J=8.7 Hz, 1H), 8.07 (d,
J=2.4 Hz, 1H). 13C NMR (300 MHz, CDCl3): d 193.50,
169.11, 168.78, 152.74, 151.95, 148.87, 140.01, 123.61,
112.00, 96.95, 94.38, 80.95, 62.09, 38.91, 38.62, 34.73,
34.42, 30.25, 28.21, 13.90. MS (CI): 400 (M+). FT-IR:
1159,1534, 1721, 2987, 3190 cmÀ1. Elemental analysis:
Anal. calcd C, 53.99; H, 6.29; N, 7.00; Found: C, 53.74;
H, 6.17; N, 6.89.
5.16. 2-Mercaptomethyl-3-(6-amino-pyridin-3-yl)-2-fluoro-
propionic acid (17)
5.16.1. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
2-fluoro-malonic acid diethyl ester. To a suspension of
NaH (1.2 g, 60% in mineral oil, 30.2 mmol) in DMF
(100 mL) was added diethyl fluoromalonate (4.3 g, 24.1
mmol) at 0 ꢀC. The reaction mixture was allowed to stir
at 0 ꢀC for 0.5 h. A slurry of compound 23 (6.9 g, 24.1
mmol) in DMF (100 mL) was then added dropwise at
0 ꢀC. The reaction mixture was then stirred at room
temperature for 16 h. Saturated aqueous ammonium
chloride (70 mL) was added slowly and the mixture was
extracted with EtOAc. The organic layers was washed
with brine and dried. Concentration under reduced
pressure followed by flash chromatography (Hexane/
EtOAc, 5:1) gave 2-(6-tert-butoxycarbonylamino-pyridin-
3-ylmethyl)-2-fluoro-malonic acid diethyl ester (7.0 g,
1
85%) as a white solid. H NMR (300 MHz, CDCl3): d
1.27 (t, J=6.9 Hz, 6H), 1.53 (s, 9H), 3.37 (s, 1H), 3.45
(s, 1H), 4.26 (q, J=7.2 Hz, 4H), 7.57 (d, J=8.7 Hz,
1H), 7.79 (s, 1H), 7.89 (d, J=8.7 Hz, 1H), 8.13 (s, 1H).
MS (CI): 384.8 (M+).
5.16.2. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
2-fluoro-malonic acid monoethyl ester (70). A solution of
KOH (300 mg, 5.7 mmol) in EtOH (30 mL) was added
dropwise to a suspension of 2-(6-tert-butoxycarbonyl-
amino-pyridin-3-ylmethyl)-2-fluoro-malonic acid diethyl
ester (2.0 g, 5.2 mmol) in EtOH (60 mL) at 0 ꢀC. The
reaction mixture was stirred for 30 min at 0 ꢀC and at
room temperature for 6 h. The reaction was quenched at
0 ꢀC using slow addition of 5% HCl (60 mL). The mix-
ture was extracted with EtOAc, washed with brine and
dried. Concentration under reduced pressure gave com-
5.16.5. 2-Mercaptomethyl-3-(6-amino-pyridin-3-yl)-2-
fluoro-propionic acid (17). Ethyl-2-(acetylthiomethyl)-3-
{6-[(tert-butoxy) carbonylamino](3-pyridyl]-2-fluoropro-
panoate (53 mg, 132 mmol) was dissolved in concentrated
HCl (2 mL) under argon. The solution was heated to
reflux for 1 h. Concentration under reduced pressure
yielded compound 17 as the hydrochloride salt (39 mg,
1
99%). H NMR (500 MHz, D2O): d 3.05-3.22 (m, 4H),
6.98 (d, 1H), 7.68 (s, 1H), 7.82 (d, 1H). HRMS (ESI)
calcd for C9H12N2O2SF 231.0604, found 231.0617.
1
pound 70 (1.5 g, 70%). H NMR (300 MHz, CDCl3): d
1.37 (t, J=7.2 Hz, 3H), 1.51 (s, 9H), 3.31 (t, J=7.2 Hz,
1H), 3.73 (dd, J=7.2 Hz, 1H), 4.39 (m, 2H), 7.99 (d,
J=8.7 Hz, 1H), 7.40–7.52 (m, 1H), 8.06 (s, 1H), 8.25 (d,
J=9.0 Hz, 1H). MS (CI): 356.9 (M+).
5.17. 3-(6-Amino-pyridin-3-yl)-2-mercaptomethyl-2-methyl-
propionic acid (18)
5.17.1. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
2-methyl-malonic acid tert-butyl ester ethyl ester. A solu-
tion of tert-butyl ethyl methylmalonate (457 mg, 2.26
mmol) in DMF (4 mL) was added dropwise to a sus-
pension of NaH (90 mg, 2.26 mmol, 60% in oil) in
DMF (4 mL). The reaction mixture was stirred for 20
min. A solution of compound 23 (500 mg, 1.74 mmol) in
DMF (2.5 mL) was added and the reaction was stirred
for 70 min. EtOAc was added and the mixture was
washed with water and brine, dried and concentrated
under reduced pressure. Chromatography (Heptane/
EtOAc, 3:1!1:3) gave 2-(6-tert-butoxycarbonylamino-
pyridin-3-ylmethyl)-2-methyl-malonic acid tert-butyl
ester ethyl ester (437 mg, 61% yield). 1H NMR
5.16.3. 3-(6-tert-Butoxycarbonylamino-pyridin-3-yl)-2-
fluoro-2-hydroxymethyl-propionic acid ethyl ester (72).
N-methyl morpholine (38.6 mg, 0.38 mmol) was added
to a solution of compound 70 (100 mg, 0.28 mmol) in
THF (10 mL) at 0 ꢀC. The reaction mixture was stirred
for 30 min. Isobutylchloroformate (40 mg, 0.31 mmol)
was then added dropwise at 0 ꢀC and the reaction mix-
ture was stirred for 1 h. NaBH4 (12 mg, 0.32 mmol) was
then added in portions and the mixture was stirred at
room temperature for 16 h. Saturated aqueous ammo-
nium chloride (10 mL) was added and the mixture was
extracted with EtOAc. The organic layer was washed
with brine (10 mL), dried and concentrated under

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M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
(500 MHz, CDCl3): d 1.30 (t, 3H), 1.35 (s, 3H), 1.48 (s,
9H), 1.56 (s, 9H), 3.15 (q, 2H), 4.22 (q, 2H), 7.60 (d,
1H), 7.97 (d, 1H), 8.06 (s, 1H).
5.18. 3-(6-Amino-5-methyl-pyridin-3-yl)-2-mercaptomethyl-
2-methyl-propionic acid (19)
5.18.1. 2-[N,N-bis(tert-Butoxycarbonyl)amino]-3-Methyl-
5-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-ylmethyl)-pyridin
(75). Compound 41 (1.6 g, 4.0 mmol) was added to a
solution of 2,2,5-trimethyl-1,3-dioxane-4,6-dione (630
mg, 4.0 mmol) and triethylamine (0.58 mL, 4.2 mmol) in
dimethyl sulfoxide (40 mL). The reaction mixture was
stirred overnight and water (100 mL) was added. The
mixture was extracted with EtOAc, the combined
organic phases washed with water and brine and dried.
Concentration under reduced pressure gave crude com-
5.17.2. 2-(6-tert-Butoxycarbonylamino-pyridin-3-ylmethyl)-
2-methyl-malonic acid mono-tert-butyl ester (71). 1M
NaOH (2 mL) was added to a solution of 2-(6-tert-
butoxycarbonylamino-pyridin-3-ylmethyl)-2-methyl-
malonic acid tert-butyl ester ethyl ester (0.42g, 1.03
mmol) in THF/EtOH (4 mL, 1:1). The reaction mixture
was stirred at 50 ꢀC for 16 h. CH2Cl2 was added and the
mixture was washed with 0.5 M HCl and brine and dried.
Concentration under reduced pressure gave compound
1
1
71 (348 mg, 89%). H NMR (500 MHz, CD3SOCD3): d
1.11 (s, 3H), 1.37 (s, 9H), 1.45 (s, 9H), 2.96 (dd, 2H),
7.50 (d, 1H), 7.66 (d, 1H), 8.01 (s, 1H), 9.63 (s, 1H).
pound 75 (2.06 g). H NMR (500 MHz, CDCl3): d 1.27
(s, 3H), 1.42 (s, 18H), 1.70 (s, 3H), 1.81 (s, 3H), 2.22 (s,
3H), 3.36 (s, 2H), 7.48 (s, 1H), 8.21 (s, 1H).
5.17.3. 3-(6-tert-Butoxycarbonylamino-pyridin-3-yl)-2-
hydroxymethyl-2-methyl-propionic acid tert-butyl ester
(73). Methyl chloroformate (75 mL, 0.92 mmol) was
added dropwise to a solution of compound 71 (348 mg,
0.915 mmol) and Et3N (123 mL, 0.92 mmol) in THF (6
mL). The reaction mixture was stirred for 20 min., fil-
tered and added dropwise to a suspension of NaBH4 (39
mg, 1.04 mmol) in THF (6 mL) at 0 ꢀC. The reaction
was stirred for 16 h at room temperature. 0.2 M HCl
was added followed by EtOAc. The organic phase was
washed with brine and dried. Concentration under
reduced pressure followed by chromatography (toluene/
EtOAc, 3:1!1:3) gave compound 73 (190 mg, 57%). 1H
NMR (500 MHz, CD3OD): d 1.09 (s, 3H), 1.43 (s, 9H),
1.53 (s, 9H), 2.69 (d, 1H), 2.88 (d, 1H), 3.50 (d, 1H),
3.59 (d, 1H) 7.58 (dd, 1H), 7.74 (d, 1H), 8.04 (d, 1H).
5.18.2. 2-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-5-methyl-
pyridin-3-ylmethyl)-2-methyl-malonic acid monoethyl es-
ter. A solution of sodium metal (184 mg, 8.0 mmol) in
ethanol (20 mL) was added to a solution of crude com-
pound 75 (2.06 g, ꢂ4.0 mmol) in ethanol (20 mL) under
argon. The reaction was stirred for 60 min. and meth-
ylene chloride was then added. The mixture was washed
with 0.5 M HCl and brine, dried and concentrated
under reduced pressure to give crude 2-(6-[N,N-bis(tert-
butoxycarbonyl)amino]-5-methyl-pyridin-3-ylmethyl)-2-
1
methyl-malonic acid monoethyl ester (1.9 g). H NMR
(500 MHz, CD3SOCD3): d 1.20 (t+s, 6H), 1.35 (s,
18H), 2.11 (s, 3H), 3.13 (q, 2H), 4.13 (m, 2H), 7.52 (d,
1H), 8.09 (d, 1H), 13.11 (bs, 1H).
5.18.3. 3-(6-[N,N-bis(tert-Butoxycarbonyl)amino]-5-methyl-
pyridin-3-yl)-2-hydroxymethyl-2-methyl-propionic acid
ethyl ester (77). Methyl chloroformate (338 mL, 4.4
mmol) was added dropwise to a solution of crude 2-(6-
[N,N-bis(tert-butoxycarbonyl)amino]-5-methyl-pyridin-3-
ylmethyl)-2-methyl-malonic acid monoethyl ester (1.9 g)
and Et3N (641 mL, 4.6 mmol) in THF (30 mL) at
À20 ꢀC. The reaction mixture was stirred for 50 min.,
filtered and added dropwise to a suspension of NaBH4
(182 mg, 4.8 mmol) in THF (30 mL) at À20 ꢀC. The
reaction was stirred for 16 h at room temperature. 0.5 M
HCl was added followed by methylene chloride. The
organic phase was washed with brine and dried. Con-
centration under reduced pressure followed by chroma-
tography (toluene/EtOAc, 10:1!1:3) gave compound
5.17.4. 2-Acetylsulfanylmethyl-3-(6-tert-butoxycarbonyl-
amino-pyridin-3-yl)-2-methyl-propionic acid tert-butyl ester.
Diethyl azodicarboxylate (160 mL, 1.01 mmol) was
added dropwise to a solution of 3-(6-tert-butoxycarbo-
nylamino-pyridin-3-yl)-2-hydroxymethyl-2-methyl-pro-
pionic acid tert-butyl ester (180 mg, 0.49 mmol) and
triphenylphosphine (266 mg, 1.01 mmol) in THF (6 mL)
and the reaction was stirred for 5 min. Thiolacetic acid (96
mL, 1.34 mmol) was added and the reaction was stirred for
16 h. Concentration under reduced pressure followed by
chromatography (toluene/EtOAc, 10:1!1:1) gave 2-acet-
ylsulfanylmethyl-3-(6-tert-butoxycarbonylamino-pyridin-3-
yl)-2-methyl-propionic acid tert-butyl ester (137 mg, 65%).
1H NMR (500 MHz, CD3OD): d 1.10 (s, 3H), 1.42 (s, 9H),
1.55 (s, 9H), 2.35 (s, 3H), 2.79 (d, 1H), 2.94 (d, 1H), 3.12 (d,
1H), 3.21 (d, 1H) 7.56 (d, 1H), 7.78 (d, 1H), 8.02 (s, 1H).
1
77 (885 mg, 49%). H NMR (400 MHz, CDCl3): d 1.08
(s, 3H), 1.24 (t, 3H), 1.39 (s, 18H), 2.09 (s, 3H), 2.70
(broad s, 1H), 2.95 (dd, 2H), 3.55 (dd, 2H), 4.18 (q, 2H),
7.42 (s, 1H), 8.13 (s, 1H).
5.17.5. 3-(6-Amino-pyridin-3-yl)-2-mercaptomethyl-2-
methyl-propionic acid (18). 2-Acetylsulfanylmethyl-3-(6-
tert-butoxycarbonylamino-pyridin-3-yl)-2-methyl-pro-
pionic acid tert-butyl ester (4 mg, 9.4? mmol) was dis-
solved in concentrated aqueous HCl under argon. The
solution was heated to reflux for 1 h. Concentration
under reduced pressure yielded compound 18 as the
hydrochloride salt (2.5 mg, 100%). 1H NMR (500 MHz,
CD3OD): d 1.20 (s, 3H), 2.62 (d, 1H), 2.76–2.83 (m,
2H), 2.95 (d, 1H), 6.94 (d, 1H), 7.64 (d, 1H), 7.80 (dd,
1H). MS (+) 227 (M+1). HRMS (ESI) calcd for
C10H14N2O2S 226.0776, found 226.0776.
5.18.4. 2-Acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxy-
carbonyl)amino]-5-methyl-pyridin-3-yl)-2-methyl-propionic
acid ethyl ester. Diisopropyl azodicarboxylate (755 mL,
3.91 mmol) was added dropwise to a solution of triphe-
nylphosphine (1.026 g, 3.91 mmol) in THF (10 mL) at
0 ꢀC and the reaction was stirred for 30 min. A solution
of thiolacetic acid (279 mL, 3.91 mmol) and compound
77 (885 mg, 1.96 mmol) in THF (5 mL) was added
dropwise during 10 min. The reaction was stirred for 60
min. at 0 ꢀC and then for 16 h at room temperature.
Concentration under reduced pressure followed by

M. O. Polla et al. / Bioorg. Med. Chem. 12 (2004) 1151–1175
1175
chromatography (heptane/EtOAc, 10:1 1:3) gave
impure 2-acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxy-
carbonyl)amino]-5-methyl-pyridin-3-yl)-2-methyl-pro-
pionic acid ethyl ester (1.46 g). 1H NMR (400 MHz,
CDCl3): d 0.85 (t, 3H), 1.15 (t, 3H), 1.62 (m, 2H), 3.03
(m, 2H), 4.04–4.14 (m, 2H), 7.49 (dd, 1H), 7.62 (d, 1H),
7.96 (d, 1H), 9.88 (s, 1H), 13.0 (bs, 1H).
8. WO 00/66550, WO 00/66557, WO 03/013526, WO 03/
027128, WO 01/19836, WO 02/14285, WO 03/061652 and
WO 03/061653.
9. Redlitz, A.; Nicolini, F. A.; Malycky, J. L.; Topol, E. J.;
Plow, E. F. Circulation 1996, 93, 1328.
10. Nagashima, M.; Werner, M.; Wang, M.; Zhao, L.; Light,
D. R.; Pagila, R.; Morser, J.; Verhallen, P. Thrombosis
Research 2000, 98, 333.
11. Klement, P.; Liao, P.; Bajzar, L. Blood 1999, 94, 2735.
12. Refino, C. J.; DeGuzman, L.; Schmitt, D.; Smyth, R.;
Jeet, S.; Lipari, M. T.; Eaton, D.; Bunting, S. Fibrinolysis
& Proteolysis 2000, 14, 305.
13. Hendriks, D.; Wang, W.; Scharpe, S.; Lommaert, M.-P.;
van Sande, M. Biochimica et Biophysica Acta 1990, 1034,
86.
14. Eaton, D. L.; Malloy, B. E.; Tsai, S. P.; Henzel, W.;
Drayna, D. The Journal of Biological Chemistry 1991, 266,
21833.
15. McKay, T. J.; Plummer, T. H., Jr. Biochemistry 1978, 17,
401.
16. Ryan, T. J.; Plummer, T. H., Jr. Biochemical and Biophy-
sical Research Communications 1981, 98, 448.
17. Barrow, J. C.; Nantermet, P. G.; Stauffer, S. R.; Ngo,
P. L.; Steinbeiser, M. A.; Mao, S.-S.; Carrol, S. S.; Bai-
ley, C.; Colussi, D.; Bosserman, M.; Burlein, C.; Cook,
J. J.; Sitko, G.; Tiller, P. R.; Miller-Stein, C. M.; Rose,
M.; McMasters, D. R.; Vacca, J. P.; Selnick, H. G.
TAFIa inhibitors based on the imidazole acetic acid
scaffold have recently been reported: J. Med. Chem.
2003, 46, 5294.
18. Pdb entries: 1bav, 1cbx, 1cps, 1dtd, 1f57, 1 hdq, 1 hdu, 1
hee, 2ctc, 3cpa, 4cpa, 5cpa, 6cpa, 7cpa, 8cpa.
19. Pdb entry: 1cpb.
20. Coll, M.; Guasch, A.; Aviles, F. X.; Huber, R. EMBO J.
1991, 10, 1.
5.18.5. 2-Acetylsulfanylmethyl-3-(6-amino-5-methyl-pyri-
din-3-yl)-2-methyl-propionic acid ethyl ester. Crude 2-
acetylsulfanylmethyl-3-(6-[N,N-bis(tert-butoxycarbonyl)-
amino]-5-methyl-pyridin-3-yl)-2-methyl-propionic acid
ethyl ester (1.46g) was dissolved in TFA (5 mL) and
stirred for 60 min. Concentration under reduced pressure
followed by chromatography (toluene/EtOAc, 1:1!
1:10!0:1) gave slightly impure 2-acetylsulfanylmethyl-3
-(6-amino-5-methyl-pyridin-3-yl)-2-methyl-propionic acid
ethyl ester (696 mg, 84%). 1H NMR (500 MHz,
CD3OD): d 1.16 (s, 3H), 1.23 (t, 3H), 1.58 (s, 3H), 2.23
(s, 3H), 2.76 (d, 1H), 2.94 (d, 1H), 3.19 (dd, 2H), 4.11
(m, 2H), 7.52 (s, 1H), 7.61 (s, 1H).
5.18.6.
methyl-2-methyl-propionic acid (19).
3-(6-Amino-5-methyl-pyridin-3-yl)-2-mercapto-
2-Acetylsulf-
anylmethyl-3-(6-amino-5-methyl-pyridin-3-yl)-2-methyl-
propionic acid ethyl ester (17 mg, 40 mmol) was
dissolved in concd HCl (2 mL) under argon. The solution
was heated to reflux for 150 min. Concentration under
reduced pressure gave compound 19 as the hydrochloride
salt (10.7 mg, 96%). 1H NMR (500MHz, CD3OD): d 1.20
(s, 3H), 2.23 (s, 3H), 2.61 (d, 1H), 2.79 (2d, 2H), 2.94 (d,
1H), 7.55 (m, 1H), 7.69 (m, 1H). MS (+) 241 (M+1).
21. Huey, R.; Bloor, C. M.; Kawahara, M. S.; Hugli, T. E.
Am. J. Pathol. 1983, 112, 48.
References and notes
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24. Pdb entry: 1cpx.
25. Ewing, T. J. A.; Makino, S.; Skillman, A. G.; Kuntz, I. D.
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30. Hendriks, D.; Scharpe, S.; van Sande, M. Clinical Chem-
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4. Eaton, D. L.; Malloy, B. E.; Tsai, S. P.; Henzel, W.;
Drayna, D. J. Biol. Chem. 1991, 266, 21833.
5. Plasma carboxypeptidase B should not be confused with
the related enzyme pancreatic carboxypeptidase B.
6. Bouma, B. N.; Marx, P. F.; Mosnier, L. O.; Meijers,
J. C. M. Thrombosis Research 2001, 101, 329.
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Lasky, N.; Halks-Miller, M.; Broze, G. J., Jr.; Fay, W. P.;
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