Synthesis of 1,2,5,6-tetrahydro-3-pyridinylalanine: A key intermediate in the preparation of thrombin inhibitor UK-239,326

Article abstract of DOI:10.1080/00397910701839210

A short synthesis of N-protected 1,2,5,6-tetrahydro-3-pyridinylalanine derivatives (8-10) was accomplished using a simple procedure of quaternization of (3-pyridyl)alanine 6 with benzyl bromide followed by reduction with NaBH4. Amino acid 10 was then converted to thrombin inhibitor UK-239,326 (2), which required the sequential functionalization of four amines to give four different functional groups (viz., sulfonamide, carboxamide, guanidine, and methylamine). Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910701839210

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Synthesis of 1,2,5,6-
Tetrahydro-3-pyridinylalanine:
A Key Intermediate in the
Preparation of Thrombin
Inhibitor UK-239,326
Paul V. Fish a , Alan D. Brown a , John C. Danilewicz
a , David Ellis a , J. David Hardstone a & Keith James
a
a Discovery Chemistry , Pfizer Global Research and
Development , Sandwich, Kent, U.K.
Published online: 25 Aug 2010.
To cite this article: Paul V. Fish , Alan D. Brown , John C. Danilewicz , David
Ellis , J. David Hardstone & Keith James (2008) Synthesis of 1,2,5,6-Tetrahydro-3-
pyridinylalanine: A Key Intermediate in the Preparation of Thrombin Inhibitor
UK-239,326, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 38:16, 2787-2798, DOI:
10.1080/00397910701839210
To link to this article: http://dx.doi.org/10.1080/00397910701839210
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Synthetic Communications1, 38: 2787–2798, 2008
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701839210
Synthesis of 1,2,5,6-Tetrahydro-3-
pyridinylalanine: A Key Intermediate in the
Preparation of Thrombin Inhibitor UK-239,326
Paul V. Fish, Alan D. Brown, John C. Danilewicz, David Ellis,
J. David Hardstone, and Keith James
Discovery Chemistry, Pfizer Global Research and Development,
Sandwich, Kent, U.K.
Abstract: A short synthesis of N-protected 1,2,5,6-tetrahydro-3-pyridinylalanine
derivatives (8–10) was accomplished using a simple procedure of quaternization
of (3-pyridyl)alanine 6 with benzyl bromide followed by reduction with NaBH4.
Amino acid 10 was then converted to thrombin inhibitor UK-239,326 (2), which
required the sequential functionalization of four amines to give four different
functional groups (viz., sulfonamide, carboxamide, guanidine, and methylamine).
Keywords: Tetrahydropyridinylalanine, thrombin inhibitors, UK-239,326
INTRODUCTION
There has been considerable interest in discovering potent orally active
thrombin inhibitors.[1,2] Our own efforts identified phenylamidine inhibi-
tor 1 (UK-156,406), which was selected for clinical evaluation as a poten-
tial anticoagulant.[3] Further exploration of this template identified
guanidine inhibitor 2 (UK-239,326), which offered the advantage of
improved thrombin inhibitory potency (Ki 0.016 nM) and selectivity over
related enzymes (>2000-fold vs. FVIIa, FXa, FXIa, trypsin, plasmin,
tPA, uPA).[4] Evaluation of 2 in preclinical models of anticoagulant
activity[5] required the synthesis of multigram quantities of 1,2,5,6-tetra-
hydro-3-pyridinylalanine derivatives 4. In this article, we report a short
Received October 19, 2007
Address correspondence to Paul V. Fish, Discovery Chemistry, Pfizer Global
Research and Development, Sandwich, Kent CT13 9NJ, U.K. E-mail: paul.
fish@pfizer.com
2787

2788
P. V. Fish et al.
synthesis of protected 1,2,5,6-tetrahydro-3-pyridinylalanine derivatives 8
and 10 and their conversion to thrombin inhibitor 2.
RESULTS AND DISCUSSION
Our synthesis of 2 was based on the derivitization of the amino and carboxy
groups of the central amino acid 4 and required the sequential functionali-
zation of four amines to give four different functional groups (viz., sulfona-
mide, carboxamide, guanidine, and methylamine) (Fig. 1). Hence, 4 would
need to be suitably protected so that the desired reacting groups could be
unmasked selectively at the appropriate point. Raucher and Lawence have
shown that 3-ethylpyridine can be efficiently converted to 3-ethyl-1,2,5,6-
tetrahydropyridine with N-alkylation of the pyridine.[6] Application of
this method was attractive for our synthesis of amino acid 4 because chiral
pyridinyl amino acids are commercially available, the chemistry seemed
to be simple and efficient, and the amino acid product 4 (R1 ¼ BOC,
R3 ¼ Bn) would have the two N atoms orthogonally protected.
Commercially available N-t-butoxycarbonyl-3-(3-pyridyl)-(S)-ala-
nine (6) was converted to 1,2,5,6-tetrahydro-3-pyridylalanines 8 and 10
using the sequence outlined in Scheme 1. Quaternization of 6 with benzyl
bromide followed by a simple trituration with ethyl acetate gave pyridi-
nium salt 7 as a solid in excellent yield. Reduction of 7 with sodium
Figure 1. Key building blocks for the synthesis of 2.

Synthesis of 1,2,5,6-Tetrahydro-3-pyridinylalanine
2789
Scheme 1. Synthesis of 1,2,5,6-tetrahydropyridinylalanine 10. (a) PhCH2Br,
MeCN, 94%; (b) NaBH4, EtOH, 8a:8b 9:1, 87%; (c) Me2NC(OBut)2, PhMe,
110 ꢀC; (d) separation by chromatography, 9a 63%, 9b 7%; (e) HCO2H, 70%.
borohydride yielded predominantly the desired 1,2,5,6-tetrahydropyri-
dine 8a (shown) along with the 1,2,3,6-regioisomer 8b (ratio ca. 9:1) in
a combined yield of 87%; it proved easier to separate these isomers after
the next reaction, and so they were carried forward as this mixture. The
esterification of 8 proved tricky because standard activated coupling
reagents caused partial racemization of the chiral center. After some
effort, N,N-dimethylformamide di-t-butylacetal[7] was identified as a
reagent suitable for the conversion of 8 to 9 in acceptable yield and
without significant loss of chiral integrity (>99% ee). All chiral intermedi-
ates were assessed by HPLC. The two regioisomers 9a and 9b were easily
separated by column chromatography at this point. The N-BOC carba-
mate of 9a was then selectively deprotected in the presence of the t-butyl
ester using neat formic acid at room temperature to give diamine 10.
Amino acid 10 was suitably protected for conversion to 2 (Schemes 2
and 3). Reaction of 10 with sulfonyl chloride 3[8] under standard con-
ditions gave sulfonamide 11 in good yield. Deprotection of the t-butyl
ester with HCl gave acid 12, which was then converted to the corres-
ponding acid chloride in situ and coupled with amine 5[9] to furnish

2790
P. V. Fish et al.
Scheme 2. Synthesis of 15. (a) 3, DIPEA, CH2Cl2, 88%; (b) HCl, CH2Cl2, quant.;
(c) (COCl)2, DMF, CH2Cl2, then 5, DIPEA, CH2Cl2, 80%; (d) CH3CH(Cl)
OC(O)Cl, Proton-sponge1, CH2Cl2, then MeOH, 80 ꢀC, quant.; (e) BOCNHC
(¼NBOC)SMe, HgCl2, TEA, CH2Cl2, 85%.
carboxamide 13. Debenzylation of 13 by treatment with 1-chloroethyl
chloroformate and Proton-sponge1 followed by decomposition of the
intermediate carbamate with MeOH gave amine 14. Installation of the
protected guanidine group was achieved by the reaction of 14 with
bis-BOC-S-methylisothiourea[10] in the presence of HgCl2 to give 15.
Scheme 3. Synthesis of thrombin inhibitor 2. (a) Na2CO3, MeOH-H2O, 85%; (b)
aq. HCHO, NaBH(OAc)3, CH2Cl2, 66%; (c) HCl, CH2Cl2, 95%; (d) NaOH,
H2O, then cHCl to pH 8.5, 59%.

Synthesis of 1,2,5,6-Tetrahydro-3-pyridinylalanine
2791
Selective basic hydrolysis of the trifloroacetamide group of 15 with
Na2CO3 gave tetrahydroisoquinoline 16, which was N-methylated by
reductive amination using aqueous formaldehyde and NaBH(OAc)3 to
give 17. Finally, unmasking of the bis-BOC-guanidine with HCl furn-
ished 18, and mild basic hydrolysis of the methyl ester with NaOH fol-
lowed by acidification to pH 8.5 gave 2, which precipitated from the
reaction mixture. Recrystallization from hot acetone–water gave 2 as a
crystalline zwitterion trihydrate with excellent purity (>99%) and good
recovery.
CONCLUSION
A short synthesis of N-protected 1,2,5,6-tetrahydro-3-pyridinylalanine
derivatives (8–10) was accomplished using a simple procedure of quater-
nization of (3-pyridyl)alanine 6 with benzyl bromide followed by
reduction with NaBH4. New synthetic amino acids 8–10 may prove to
be useful bioisosteres for lysine or arginine in the synthesis of novel pep-
tidomimetics. Amino acid 10 was converted to thrombin inhibitor UK-
239,326 (2) on a multigram scale.
EXPERIMENTAL SECTION
General
Proton nuclear magnetic resonance (1H NMR) data were obtained using a
Nicolet QE-300 or a Bruker AC-300. Low-resolution mass spectral (LRMS)
data were recorded on a Fisons Instruments Trio 1000 (thermaspray); the
observed ions quoted refer to the isotopic composition of lowest mass. Col-
umn chromatography was performed using Merck silica gel 60 (230–
400 mesh) or MCI reverse-phase gel (CHP20p, 75–150m) manufactured
by Mitsubishi-Kasei corporation. The following abbreviations are used: t-
butyloxycarbonyl (BOC), diisopropylethylamine (DIPEA), N,N-dimethyl-
formamide (DMF), dimethylsulphoxide (DMSO), and triethylamine (TEA).
N-t-Butoxycarbonyl-3-(1-benzyl-3-pyridinium)-(S)-alanine Bromide (7)
Benzyl bromide (30.83 g, 21.44 mL, 180.3 mmol) was added to a solution of
N-t-butoxycarbonyl-3-(3-pyridyl)-(S)-alanine (6) (16.00 g, 60.08 mmol) in
acetonitrile (204mL) at room temperature, and the resultant solution was
stirred for 18 h. The mixture was then evaporated, and the resultant white
residue was triturated with ethyl acetate (250 mL), filtered, and washed with
more ethyl acetate (250 mL) and pentane (250 mL) to give the product as a

2792
P. V. Fish et al.
solid. This was dried in vacuo to give 7 (24.8 g, 94%) as a white powder. 1H
NMR (DMSO-d6) d 1.23 (9H, s), 2.98–3.40 (2H, m), 4.31 (1H, br s), 5.82
(2H, s), 7.10 (1H, br s), 7.38–7.55 (5H, m), 8.10 (1H, dd), 8.48 (1H, d),
9.03 (1H, d), 9.20 (1H, s). Anal. calcd. for C20H25N2O4Br: C, 54.93; H,
5.76; N, 6.41. Found: C, 54.77; H, 5.71; N, 6.23%.
N-t-Butoxycarbonyl-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanine
(8a)
Sodium borohydride (6.42 g, 170 mmol) was added in several portions to
a solution of 7 (24.75 g, 56.59 mmol) in ethanol (250 mL) at 0 ꢀC. The
reaction mixture was then warmed to room temperature and stirred for
a further 1.5 h. The solvent was removed by evaporation, the residue
taken up in water (200 mL), and the resultant solution was acidified to
pH 6 by the cautious addition of aqueous 2N HCl solution. The solution
thus obtained was loaded directly onto reverse-phase MCI gel and puri-
fied by column chromatography using a gradient elution of water–meth-
anol (85:15 to 10:90). The appropriate fractions were combined,
evaporated in vacuo, and then azeotroped with ethyl acetate to give 8
as a white powder (17.68 g, 87%). The desired product 8a was contami-
nated with ca. 10% of the 1,2,3,6-tetrahydropyridine regioisomer 8b,
which could not be separated at this point. 1H NMR (DMSO-d6; 8a only)
d 1.27 (9H, s), 2.00 (2H, br s), 2.08–2.26 (2H, m), 2.41 (2H, br s), 2.82
(2H, br s), 3.50 (2H, s), 3.96 (1H, br s), 5.48 (1H, br s), 6.71 (1H, br s),
7.18–7.33 (6H, m). Anal. calcd. for C20H28N2O4.0.3EtOAc: C, 65.74;
H, 7.93; N, 7.19. Found: C, 65.74; H, 7.84; N, 7.53%.
N-t-Butoxycarbonyl-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanine
t-Butyl Ester (9a) and N-t-Butoxycarbonyl-3-(1-benzyl-1,2,3,6-tetrahydro-
3-(R,S)-pyridyl)-(S)-alanine t-Butyl Ester (9b)
N,N-Dimethylformamide di-t-butyl acetal (14.16 g, 16.64 mL, 69.60 mmol)
was added to a slurry of 8 in toluene (500 mL), and the resulting mixture
was heated at relux for 5.5 h. The reaction mixture was cooled to room tem-
perature and diluted with ethyl acetate (1000 mL). The solution was washed
with saturated sodium bicarbonate (500 mL) and water (500 mL), dried
(MgSO4), and evaporated to give a pale yellow oil. Purification by chromato-
graphy on silica gel (2 Kg) using hexane–EtOAc (90:10 to 0:100) as eluant gave
1
9b (2.10 g, 7%) followed by 9a (18.10 g, 62.5%). Compound 9a: H NMR
(CDCl3) d 1.42 (18H, s), 2.18 (2H, br s), 2.30 (1H, dd), 2.42 (1H, dd), 2.55
(2H, m), 2.97 (2H, s), 3.60 (2H, s), 4.19 (1H, br s), 4.92 (1H, br s), 5.56 (1H,
s), 7.20–7.58 (m, 5H); LRMS m=z 417.5 (MHþ ). Anal. calcd. for

Synthesis of 1,2,5,6-Tetrahydro-3-pyridinylalanine
2793
C24H36N2O.0.15H2O: C, 68.76; H, 8.73; N, 6.68. Found: C, 68.49; H, 8.68; N,
6.74%. Compound 9b: 1H NMR (CDCl3) d 1.42 (9H, s), 1.45 (9H, s), 1.60 (1H,
m), 1.83 (1H, m), 2.21–2.48 (2H, m), 2.62–2.90 (1H, m), 2.98 (2H, dd), 3.60
(2H, dd), 4.18 (1H, br s), 5.03 (1H, br s), 5.60–5.78 (2H, m), 7.20–7.58 (5H,
m); LRMS m=z 417.6 (MHþ). Anal. calcd. for C24H36N2O.0.1H2O: C,
68.90; H, 8.72; N, 6.70. Found: C, 68.70; H, 8.71; N, 6.66%.
3-(1-Benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanine t-Butyl Ester (10)
N-t-Butoxycarbonyl-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanine
t-butyl ester (9a) (11.54 g, 27.7mmol) was dissolved in aqueous formic acid
(88%, 187 mL), and the mixture was stirred at room temperature for 22h.
The formic acid was removed by evaporation in vacuo (temperature
<30ꢀC), and the residue was partitioned between ethyl acetate (400 mL)
and aqueous citric acid (400 mL, 1 M). The organic phase was washed with
citric acid (2ꢁ 300 mL, 1 M), and the combined aqueous extracts were basi-
fied with solid sodium bicarbonate. The mixture was then extracted with
CH2Cl2 (3 ꢁ 500 mL), and the organic extracts were dried (MgSO4) and
evaporated to give the crude product as a yellow oil. Purification by chro-
matography on silica (240g) using CH2Cl2–MeOH (95:5 to 90:10) as eluant
gave 10 as a pale yellow oil (9.53g, 70%). 1H NMR (CDCl3) d 1.45 (9H, s),
1.65 (2H, br s), 2.10–2.17 (3H, m), 2.37 (1H, dd), 2.51–2.46 (2H, m), 2.92
(2H, br s), 3.41 (1H, dd), 3.60 (2H, s) 5.57 (1H, br s), 7.28–7.37 (5H, m);
LRMSm=z 317.2 (MHþ ). Anal. calcd. for C19H28N2O2.0.12CH2Cl2: C,
70.23; H, 8.71; N, 8.57. Found: C, 70.14; H, 8.73; N, 8.55%.
N-(2-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-
benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanine t-Butyl Ester (11)
A solution of 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulpho-
nyl chloride[8] (3) (11.57 g, 35.3 mmol) in CH2Cl2 (35 mL) was added
dropwise to a stirred solution of 10 (9.31 g, 29.4 mmol) and DIPEA
(7.7 mL, 44.1 mmol) in CH2Cl2 (25 mL) at 0 ꢀC. The mixture was allowed
to warm to room temperature and stirred for 18 h. The solvent was eva-
porated, and the residue partitioned between EtOAc (300 mL) and water
(150 mL). The organic phase was washed with saturated sodium bicar-
bonate (2 ꢁ 150 mL) and brine (150 mL), dried (MgSO4), and evaporated
to give an oil. Purification by chromatography on silica (500 g) using
hexanes–EtOAc (90:10 to 40:60) as eluant gave 11 as an off-white foam
1
(15.65 g, 88%). H NMR (CDCl3) (mixture of rotamers) d 1.27 (9H, s),
2.12 (2H, br s), 2.23–2.39 (2H, m), 2.44–2.53 (2H, m), 2.86–2.99 (4H,
m), 3.56 (2H, s), 3.85–3.88 (3H, m), 4.78–4.83 (2H, m), 5.08 (1H, br s),

2794
P. V. Fish et al.
5.54 (1H, s), 7.23–7.33 (6H, m), 7.63–7.69 (2H, m). LRMS m=z 608.6
(MHþ). Anal. calcd. for C30H36N3O5SF3.0.05CH2Cl2: C, 58.98; H,
5.95; N, 6.87. Found: C, 58.90; H, 6.03; N, 6.64%.
N-(2-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-
(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanine Hydrochloride (12)
HCl gas was bubbled through a solution of 11 (15.53 g, 25.55 mmol) in
CH2Cl2 (150 mL) at 0 ꢀC until saturated. The solution was allowed to
warm to room temperature over 30 min and then stirred for 6 h. The
solvent was evaporated, and the residue was azeotroped with CH2Cl2
1
(3 ꢁ 100 mL) to give 12 as a pale yellow foam (16.4 g, quant). H NMR
(DMSO-d6) (mixture of rotamers) d 2.20–2.38 (4H, m), 2.65–3.60 (6H,
m), 3.80 (3H, m), 4.20 (2H, m), 4.79 (2H, m), 5.60 (1H, s), 7.34–7.70
(8H, m), 8.03 (1H, d), 10.93 (1H, br s); LRMS (APCI) m=z 552.0
(Mþ). Anal. calcd. for C26H28N3O5SF3.0.6CH2Cl2: C, 50.00; H, 4.76;
N, 6.58. Found: C, 49.75; H, 4.84; N, 6.52%.
4-Methyl-1-[N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-
sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl]-1,2,3,6-
tetrahydropyridine-2(R)-carboxylic Acid Methyl Ester (13)
DMF (3 drops) was added to a stirred solution of acid 12 (16.4 g,
25.5 mmol) and oxalyl chloride (12.95 g, 8.90 mL, 102 mmol) in CH2Cl2
(150 mL) at 0 ꢀC. The reaction was warmed to room temperature and stir-
red for 2.5 h. The solvent was evaporated, and the residue was azeotroped
with CH2Cl2 (3 ꢁ 100 mL). The acid chloride was dissolved in CH2Cl2
(150 mL), and the solution was cooled to 0 ꢀC. A solution of 4-methyl-
1,2,3,6-tetrahydropyridine-2(R)-carboxylic acid methyl ester p-toluene
sulphonate[9] (5) (9.20 g, 28.11 mmol) and DIPEA (14.5 mL, 89.4 mmol)
in CH2Cl2 (30 mL) was added to this solution. The reaction was then
warmed to room temperature and stirred for 18 h. The solvent was then
evaporated, and the residue partitioned between EtOAc (300 mL) and
water (150 mL). The organic phase was washed with saturated sodium
bicarbonate (100 mL) and brine (100 mL), dried (MgSO4), and evapo-
rated in vacuo to give the crude product as a yellow gum. Purification
by chromatography on silica (450 g) using a gradient elution of hex-
ane–EtOAc (100:0 to 0:100) gave 13 as an pale yellow foam (14.06 g,
1
80%). H NMR (CDCl3) (mixture of rotamers) d 1.56 (3H, s), 2.10–
2.51 (8H, m), 2.80–3.06 (4H, m), 3.36–4.10 (9H, m), 4.18–4.28 (1H, m),
4.75–4.88 (2H, m), 5.18–5.30 (2H, m), 5.58 (2H, m), 7.18–7.20 (6H, m),
7.52–7.70 (2H, m); LRMS m=z 689.7 (MHþ). Anal. calcd. for

Synthesis of 1,2,5,6-Tetrahydro-3-pyridinylalanine
2795
C34H39N4O6SF3.0.15CH2Cl2: C, 58.47; H, 5.65; N, 7.99. Found: C,
58.21; H, 5.59; N, 7.92%.
4-Methyl-1-[N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-
sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl]-1,2,3,6-
tetrahydropyridine-2(R)-carboxylic Acid Methyl Ester (14)
A solution of 1-chloroethyl chloroformate (4.37 g, 30.6 mmol) in CH2Cl2
(30 mL) was added dropwise to a stirred solution of 13 (14.06 g,
20.41 mmol) and 1,8-bis(dimethylamino)naphthalene (873mg, 4.07mmol)
in CH2Cl2 (70 mL) at 0 ꢀC. The mixture was warmed to room temperature
and stirred for 2 h. The mixture was washed with aqueous citric acid sol-
ution (2 ꢁ 100 mL, 0.1 N) and brine (2 ꢁ 100 mL) and dried (MgSO4). The
solvents evaporated to give the intermediate carbamate as a pale yellow
gum. This material was dissolved in MeOH (120 mL), and the solution
was heated at reflux for 45 min. The solvent was then evaporated, and
the residue triturated with pentane (3 ꢁ 100 mL) to give 14 as a pale yel-
low foam (12.96 g, quant); this material was used in the next step without
further purification. A sample of 14 (100 mg) was purified by chromato-
graphy on silica (2 g) using CH2Cl2–MeOH–0.880NH3 (90:10:0.2) as
1
eluant to give 14 as a white foam (80 mg). H NMR (CDCl3) (mixture
of rotamers) d 1.68 (3H, s), 2.08–2.46 (6H, m), 2.89–3.05 (4H, m),
3.25–4.12 (10H, m), 4.25 (1H, m), 4.76–4.89 (2H, m), 5.18–5.30 (2H,
m), 5.63 (1H, s), 7.20 (1H, m), 7.58–7.70 (2H, m); LRMS m=z 599.6
(MHþ). Anal. calcd. for C27H33N4O6SF3.0.5CH2Cl2: C, 51.52; H, 5.35;
N, 8.74. Found: C, 51.53; H, 5.39; N, 8.51%.
4-Methyl-1{[N-[2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-
sulphonyl]-3-[1-(N,N0-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-
pyridyl]-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylic Acid
Methyl Ester (15)
A stirred solution of amine 14 (12.96g, 20.41mmol) in CH2Cl2 (125 mL) at
0ꢀC was treated sequentially with TEA (8.24 g, 81.6 mmol), bis-t-butoxycar-
bonyl-S-methylisothiourea[10] (6.52 g, 22.41 mmol), and mercuric chloride
(6.09 g, 22.41 mmol), and the mixture was then warmed to room tempera-
ture and stirred for 22 h. The reaction mixture was filtered, and the filtrate
was washed sequentially with water (100 mL), aqueous citric acid (100 mL,
0.1 M), and brine (100mL) and then dried (MgSO4) and evaporated to give
the product as a yellow gum. Purification by chromatography on silica
(400 g) by gradient elution with hexane–EtOAc (80:20 to 0:100) gave 15
as an off-white foam (14.56 g, 85%). 1H NMR (CDCl3) (mixture of

2796
P. V. Fish et al.
rotamers) d 1.51 (18H, s), 1.68 (3H, s), 2.03–2.58 (6H, m), 2.99 (2H, m),
3.25–4.30 (12H, m), 5.70–5.89 (2H, m), 5.23 (1H, br s), 5.55–5.78 (2H,
m), 7.30 (1H, m), 7.55–7.85 (2H, m), 9.96–10.15 (1H, m); LRMS m=z
842.3 (MHþ ). Anal. calcd. for C38H51N6O10SF3.CH2Cl2: C, 50.59; H,
5.77; N, 9.08. Found: C, 50.28; H, 5.79; N, 9.06%.
4-Methyl-1-{N-[1,2,3,4-tetrahydroisoquinoline-7-sulphonyl]-3-[1-(N,N0-di-
t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl]-(S)-alanyl}-
1,2,3,6-tetrahydropyridine-2(R)-carboxylic Acid Methyl Ester (16)
A solution of sodium carbonate (6.65 g, 62.8 mmol) in water (82 mL) was
added dropwise to a stirred solution of 15 (13.3g, 15.8 mmol) in MeOH
(140mL) at room temperature. After 2 h, the bulk of the MeOHwas removed
by evaporation in vacuo, and the aqueous residue was diluted with water
(50 mL) and extracted with EtOAc (4ꢁ 200 mL). The combined organic
extracts were washed with brine (200 mL), dried (MgSO4), and evaporated
in vacuo to give the crude product as a yellow foam. Purification by chroma-
tography on silica (220 g) using a gradient elution with CH2Cl2–MeOH–
1
0.880NH3 (99:1:0 to 90:10:1) gave 16 as a white foam (10.30 g, 85%). H
NMR (CDCl3) (mixture of rotamers) d 1.50 (18H, s), 1.69 (3H, s), 2.05–
2.52 (6H, m), 2.78–2.88 (2H, m), 3.10–3.20 (2H, m), 3.39–4.35 (13H, m),
5.21–5.35 (2H, m), 5.63–5.72 (1H, m), 7.10–7.22 (1H, m), 7.40–7.65 (2H,
m); LRMS m=z 745.8 (MHþ). Anal. calcd. for C36H52N6O9S.0.4CH2Cl2:
C, 56.13; H, 6.83; N, 10.79. Found: C, 55.88; H, 6.94; N, 11.05%.
4-Methyl-1-{N-[2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl]-3-[1-
(N,N0-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl]-(S)-
alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylic Acid Methyl Ester (17)
A solution of aqueous formaldehyde (4.4 mL, 37% w=v solution) was
added in one portion to a stirred solution of 16 (10.3 g, 14.0 mmol) in
CH2Cl2 (100 mL) at room temperature for 30 min. Sodium triacetoxybor-
ohydride (4.15 g, 19.6 mmol) was then added in one portion, and the reac-
tion mixture was stirred for a further 4 h. A second portion of
formaldehyde (2.2 mL, 37%) was added after 1 h. The mixture was parti-
tioned between CH2Cl2 (600 mL) and saturated aqueous NaHCO3
(600 mL), and the organic phase was washed with sat. NaHCO3
(2 ꢁ 100 mL) and brine (2 ꢁ 100 mL), dried (MgSO4), and evaporated
to give the crude product as a pale white foam. Purification by chroma-
tography on silica (210 g) using a gradient elution of CH2Cl2–MeOH–
0.880NH3 (99:1:0 to 98:2:0.2) gave 17 as a white foam (7.00 g, 66%).
1H NMR (CDCl3) (mixture of rotamers) d 1.43 (18H, s), 1.70 (3H, s),
2.07–2.50 (9H, m), 2.70 (2H, m), 2.96 (2H, m), 3.38–3.60 (4H, m), 3.62

Synthesis of 1,2,5,6-Tetrahydro-3-pyridinylalanine
2797
(3H, s), 3.65–4.28 (5H, m), 5.20–5.32 (2H, m), 5.53 (1H, m), 5.68 (1H, s),
7.13–7.21 (1H, d, d), 7.40–7.58 (2H, m), 10.02 (1H, br s); LRMS m=z
759.5 (MHþ ). Anal. calcd. for C37H54N6O9S.0.1CH2Cl2: C, 58.07; H,
7.12; N, 10.95. Found: C, 58.03; H, 7.23; N, 10.80%.
4-Methyl-1-[N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-
(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl]-1,2,3,6-
tetrahydropyridine-2(R)-carboxylic Acid Methyl Ester Dihydrochloride (18)
HCl gas was bubbled through a solution of 17 (6.3 g, 8.3 mmol) in
CH2Cl2 (50 mL) to give 18 (5.35 g, 95%) as a white powder.
[a]D þ 55.2ꢀ (c ¼ 1.05, MeOH); 1H NMR (DMSO-d6) (mixture of
rotamers) d 1.64–1.65 (3H, s,s), 1.92–2.55 (8H, m), 2.90 (3H, s), 3.00–
3.45 (2H, m), 3.50–3.98 (8H, m), 4.10 (1H, dd), 4.20–4.60 (3H, m), 5.21
(1H, m), 5.37 (1H, m), 5.63 (1H, br s), 7.36–7.70 (7H, m), 8.01–8.10
(1H, s, s), 11.53 (1H, br s); LRMS m=z 559.5 (MHþ ). Anal. calcd. for
C27H38N6O5S.2HCl.0.25CH2Cl2.1.5H2O: C, 48.14; H, 6.45; N, 12.36.
Found: C, 48.14; H, 6.34; N, 12.01%.
4-Methyl-1-[N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-
amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl]-1,2,3,6-
tetrahydropyridine-2(R)-carboxylic Acid (2)
Aqueous NaOH solution (24.0 mL, 1 M) was added with stirring to 18
(5.3 g, 8.39 mmol) at room temperature, and the resultant solution was
stirred for 1.25 h. The solution was then adjusted to pH 8.5 by the careful
addition of conc HCl. On standing at room temperature, the product
crystallized out of solution and was collected by filtration with water
washing. This material was recrystallized from hot acetone–water
(50 mL, 1:1) to give 2 as a white crystalline solid (2.69 g, 59%).
ꢀ
1
[a]D þ 77.1 (c ¼ 1, MeOH); H NMR (DMSO-d6) d 1.62 (3H, s), 1.92–
2.10 (4H, m), 2.53 (3H, s), 2.42–2.63 (4H, m), 2.80 (2H, m), 3.10–3.30
(2H, m), 3.39 (1H, d), 3.45 (2H, s), 3.60 (2H, s), 3.91 (1H, d), 4.22–4.38
(2H, m), 5.20 (1H, s), 5.57 (1H, s), 7.20 (1H, d), 7.39 (1H, s), 7.51 (1H,
d), 7.60 (1H, br d), 8.18 (4H, br s); LRMS m=z 545.5 (MHþ). Anal. calcd.
for C26H36N6O5S.3H2O: C, 52.16; H, 7.07; N, 14.04. Found: C, 52.04; H,
7.05; N, 13.90%.
ACKNOWLEDGMENT
We thank our colleagues for their expert and enthusiastic technical assis-
tance: Yvonne Ailwood, Leena Patel, and Anthony Pomeroy. We also

2798
P. V. Fish et al.
thank members of the Physical Sciences Group for spectroscopic and
analytical services.
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