3-Amino-4-sulfonylpyridinone acetamide and related pyridothiadiazine thrombin inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00154-9

We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-substituent, and

Full text of DOI:10.1016/S0960-894X(03)00154-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1441–1444
3-Amino-4-sulfonylpyridinone Acetamide and Related
Pyridothiadiazine Thrombin Inhibitors
Philip E. J. Sanderson,a,* Kellie J. Cutrona,a Kelly L. Savage,a Adel M. Naylor-Olsen,b
Denise J. Bickel,c Dennis L. Bohn,c Franklin C. Clayton,c Julie A. Krueger,d
S. Dale Lewis,d Bobby J. Lucas,d Elizabeth A. Lyle,c Audrey A. Wallace,c
Denice C. Welshc and Youwei Yane
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Molecular Design and Diversity, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
eDepartment of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA
Received 13 September 2002; revised 4 February 2003; accepted 8 February 2003
Abstract—We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone
acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-sub-
stituent, and forms a hydrogen bond to the insertion loop tyrosine OH. We also describe a related series of fused bicyclic dihy-
drothiadiazinedioxide derivatives, of which one had improved pharmacokinetics in dogs after oral dosing.
# 2003 Elsevier Science Ltd. All rights reserved.
The number of patents and papers describing the use of
the 3-aminopyridinone acetamide template for the
synthesis of thrombin inhibitors is growing rapidly.1 We
have noted that simple 3-aminopyridinones are
unstable2 and have described three methods for their
stabilization. The first is simply to derivatize the 3-
amino group as a sulfonamide or amide.2 The second
method is to incorporate a nitrogen in the ring at the 4-
position to give a pyrazinone.2 A third is to substitute
an electron withdrawing group such as trifluoromethyl
at the 4-position (structure 1).3 This last method gave
compounds with low nanomolar potency against
thrombin despite the lack of a group to fill the ‘distal
while using the same stabilization method, we reasoned
that it should be possible to stabilize the pyridinone and
fill the ‘distal binding pocket’ using a 4-sulfonyl sub-
stituent. Modelling of a 3-amino-4-sulfonylpyridinone
template 2 in the active site of thrombin showed that
one of the sulfonyl oxygens could form a hydrogen
bond to either the tryptophan indole NH or the tyrosine
phenol of thrombin’s insertion loop. Simultaneously,
the other oxygen would form an intramolecular hydro-
gen bond to the 3-amino group, locking the sulfone in a
limited number of conformations, one of which points
the sulfone substituent directly into the ‘distal binding
pocket’.
binding
pocket’.
However,
these
4-trifluoro-
methylpyridinones were not sufficiently efficacious to
warrant further development. To improve upon them,
In this communication we describe the synthesis and
properties of a variety of 3-amino-4-sulfonylpyridinones
2 in which the 4-sulfonyl group serves as a con-
formationally constraining electron withdrawing lin-
ker.4,5 We also describe
a related series of 4-
aminosulfonylpyridinones 3 and their bicyclic thiadiazine
derivatives 4.6 The latter offer an achiral alternative
to the chiral bicyclic lactam template 5 reported
previously.7,8
*Corresponding author. Tel.:+1-215-652-4957; fax:+1-215-652-3971;
e-mail: phil_sanderson@merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00154-9

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P. E. J. Sanderson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1441–1444
venient because the 2-sulfonylbenzothiazole is prepared
under mild conditions and the reduction and subsequent
nucleophilic addition is done in one pot. Reduction of
the nitro group followed by ester hydrolysis and coupl-
ing to 2-amino-5-aminomethyl-6-methylpyridine2 gave
thrombin inhibitor 2g.
The route to the 4-aminosulfonylpyridinones and
derived pyridothiadiazine dioxides is exemplified by the
synthesis of compounds 3d and 4d (Scheme 2).6 Addi-
tion of 2-mercaptobenzothiazole to 4-chloropyridinone
6 gave thioether 9. The highly deactivated sulfur in 9
was inert to oxidation under standard conditions. On
the other hand, oxidation of the amine 10 was
uncontrollable, so protection as its bis-BOC derivative
11 was necessary. Oxidation was then accomplished
using potassium permanganate to give sulfone 12.11
Zinc in ethanolic acetic acid was used to reduce the
2-sulfonylbenzothiazole to the sulfinate.12 Reaction of
zinc sulfinate 13 with NCS then gave sulfonyl chloride
14.13 Coupling of 14 with cyclobutylmethylamine gave
sulfonamide 15 which was deprotected, hydrolyzed and
coupled to the P1 group to give thrombin inhibitor 3d.
Cyclization to the bicyclic thiadiazine dioxide 4d was
then accomplished with aqueous ethanolic formalde-
hyde with ammonia,14 followed by treatment with warm
dilute hydrochloric acid to break down the dimer which
formed under the cyclization conditions.
Scheme 1. Synthesis of compound 2g: (a) 8, NaBH4, EtOH, 2 h, then
add acetic acid to pH 4–5 and 6, 2 h; (b) H2, 10% Pd/C, EtOH, 3 h; (c)
LiOH, THF, MeOH, H2O, 2 h, 73% over 3 steps; (d) P1.2HCl,2 EDC,
HOBT, NMM, DMF, 16 h, 65%.
The biological results for the sulfones 2a–i are given in
Table 1. They are significantly more potent than the 4-
trifluoromethylpyridinones 1.3 However, they are
slightly less potent than the fused bicyclic lactams 5,
although this is offset by lower protein binding (e.g.,
sulfone 2h is 45% free in human plasma whereas the
corresponding lactam 5 with the same cyclobutylmethyl
side chain is 19% free).7 Compound 2h is clearly the
most potent sulfone derivative (Ki=0.2 nM,
2ÂAPTT=0.23 mM) and it has good selectivity against
trypsin (Ki=0.55 mM) and activated protein C, t-PA,
plasma kallikrein and plasmin (Ki>100 mM). Cyclo-
propylmethylsulfone 2g is 9-fold less potent than 2h in
the enzyme assay but only 2-fold in the APTT assay,
presumably a result of its lower protein binding. Both
2g and 2h are fully efficacious in the rat ferric chloride
arterial thrombosis model.16
The synthetic route to the 4-sulfones starts from 4-
chloropyridinone 69,10 and is exemplified by the syn-
thesis of compound 2g (Scheme 1).4 This direct route
involves nucleophilic addition of the preformed side
chain as its sulfinate salt to give the 3-nitro-4-sulfo-
nylpyridinone 7. In general, the sulfinate salts were pre-
pared either by reaction of the corresponding organo-
lithium with sulfur dioxide, by reduction of the sulfonyl
chloride with sodium sulfite, or as illustrated in Scheme
1, by sodium borohydride reduction of the benzothi-
azole (Bt) derivative (8).11 This last method is con-
Scheme 2. Synthesis of 3d and 4d: (a) BtSH, Et3N, EtOH, reflux, 15 min, 91%; (b) H2, 10% Pd/C, EtOAc, 16 h, 86%; (c) (BOC)2O, DMAP,
CH2Cl2, 16 h, 73%; (d) KMnO4, AcOH, H2O, 16 h, 56%; (e) Zn, AcOH, EtOH, 16 h; (f) NCS, CH2Cl2, À5 ꢀC- RT, 45 min; (g) (c-C4H7)CH2NH2,
CH2Cl2, NMM, 16 h; (h) HCl, EtOAc, 0 ꢀC–rt, 3 h, 44% over 4 steps; (i) LiOH, THF, MeOH, H2O, 1 h, 78%; (j) P1.2HCl,2 EDC, HOAT, NMM,
DMF, 16 h, 40%; (k) formaldehyde, H2O, EtOH, NH4OH, reflux, 6 h; (l) warm 1 M HCl, 40% over 2 steps.

P. E. J. Sanderson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1441–1444
1443
Table 1. Inhibition constants,a in vitro anticoagulant potency (2ÂAPTT),b efficacy in the rat ferric chloride thrombosis modelc and pharmaco-
kinetic parameters in dogs after oral administration at 1 mg/kg
Compd
R
Thrombin
inhibition
Ki, nM
Trypsin
inhibition
Ki, nM
2ÂAPTT
mM
Protein
binding
% freed
Rat
FeCl3
Final plasma
concn (mM)f
Dog Cmax
(mM)
t1/2
(min)
AUC
(mM h)
e
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
4a
4b
4c
4d
PhCH2
Ph
4-MePh
2-Thienyl
c-C6H11
c-C3H5
(c-C3H5)CH2
(c-C4H7)CH2
(c-C5H9)CH2
c-C3H5
(c-C3H5)CH2
c-C4H7
(c-C4H7)CH2
c-C3H5
(c-C3H5)CH2
c-C4H7
(c-C4H7)CH2
1.6
3.8
94
1.7
3
1600
500
800
340
0.47
0.66
—
0.39
0.75
1.64
0.4
22
19
—
40
38
—
70
45
29
—
53
45
23
—
25
—
—
1/6
4/6
—
3/6
1/6
—
0/5
0/6
2/6
—
2/6
1/6g
0/6
5/6
1/6g
—
0.25
0.57
—
0.43
0.65
—
0.55
0.68
0.53
—
0.54
0.86
0.64
—
0.45
—
—
—
—
—
0.37
0.57
—
—
—
—
—
—
0.59
—
63
—
—
—
—
—
66
47
—
—
—
—
—
—
136
—
—
0.62
—
—
—
—
—
0.8
2.58
—
—
—
—
—
—
2.14
—
260
17
2800
1900
550
1.8
0.2
1.2
7.4
4.5
1.7
0.75
19
0.23
0.4
630
2900
1000
1100
740
10000
6100
6300
1600
0.85
0.75
0.35
0.47
0.75
0.79
1.11
0.92
2.1
10
3.9
0.65
—
—
—
—
—
aSee ref 15.
bConcentration of compound required to double the human activated partial thromboplastin time.
cSee ref 16.
dHuman plasma.
eOcclusions at 10 mg/kg/min iv infusion.
fArithmetic average at the end of the infusion.
gOne reflow.
The X-ray crystal structure 2g bound in thrombin’s
active site is shown in Figure 1 and it confirmed the
essential features of the modelling.17 The aminopyridine
and the pyridinone bind in the expected manner.2 There
is a long hydrogen bond (3.5 A) between one of the
sulfone oxygens and the OH of the insertion loop tyro-
sine, a hydrogen bond between the other sulfone oxygen
and the 3-amino group, and the cyclopropyl group
occupies the ‘distal binding pocket’. The energetic con-
tribution of the sulfone hydrogen bonds is impossible to
determine independent of the other factors which affect
binding. However, it is interesting to note that there
is a large difference in potency between benzylsulfone
2a and its benzylthioether analogue (1.6 vs 79 nM,
respectively).4
The 4-aminosulfonylpyridinones 3a and 3c are less
potent in the enzyme assay than their alkylsulfone
counterparts 2g and 2h. However 3c (2ÂAPTT=0.35
mM) is efficacious in the rat, as is its homologue 3d.
The constraint imposed on the side chain of derived
thiadiazines 4a and 4c significantly affects their
potency. Insertion of a methylene to compensate for
this constraint (4b and 4d) improves the activity
against the enzyme in each case. Compound 4b was
studied in further depth and it retains the selectivity of
Figure 1. The crystal structure of 2g in the thrombin active site.

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P. E. J. Sanderson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1441–1444
2h and is efficacious in the rat. Furthermore, while the
half-lives of sulfones 2a, 2g and 2h were between 47 and
66 min after oral administration to dogs, 4b had a
longer half-life (136 min) with an AUC of 2.14 mM h.
6. Sanderson, P. E. J.; Cutrona, K. J. US Patent 6,117,888,
2000.
7. Coburn, C. A.; Rush, D. M.; Williams, P. D.; Homnick, C.;
Lyle, E. A.; Lewis, S. D.; Lucas, B. J.; Di Muzio-Mower, J. M.;
Juliano, M.; Krueger, J. A.; Vastag, K.; Chen, I.-W.; Vacca,
J. P. Bioorg. Med. Chem. Lett. 2000, 10, 1069. See also ref 1.
8. For covalent, arginine aldehyde derivatives with 4-alkyl-
pyridinone acetamide groups see: Reiner, J. E.; Lim-Wilby, S.;
Brunck, T. K.; Ha-Uong, T.; Goldman, E. A.; Abelman,
M. A.; Nutt, R. F.; Semple, J. E.; Tamura, S. Y. Bioorg. Med.
Chem. Lett. 1999, 9, 895.
In summary, we designed and synthesized a series of
highly potent and efficacious 3-amino-4-sulfonylpyri-
dinone acetamide thrombin inhibitors. The functionally
dense sulfone stabilizes the aminopyridinone, con-
formationally constrains the 4-substituent via a hydro-
gen bond to the adjacent amino group, and hydrogen
bonds to the insertion loop tyrosine. We prepared a
related series of bicyclic dihydrothiadiazinedioxides
from the corresponding 4-aminosulfonylpyridinones.
The cyclopropylmethyl derivative 4b is efficacious in
rats and has improved pharmacokinetics in dogs after
oral dosing compared to the 4-sulfonylpyridinones 2
and bicyclic lactams 5, making the pyridothiadiazine
template an attractive, achiral alternative to the bicyclic
lactam template.
9. Coburn, C. A., Kolatac, C., Rush, D. M., Vacca, J. P. US
Patent 5,869,487, 1999.
10. For the synthesis of
a related 4-(trifluoromethyl-
sulfonyloxy)pyridinone, see: Lassalle, G.; Bellevergue, P.;
Bourbier, J. C.; Galtier, D.; Martin, V. WO9831671, 1998; US
Patent 6,252,082, 2001. See also ref 5.
11. Ueno, Y.; Kojima, A.; Okawara, M. Chem. Lett. 1984,
2125.
12. Conditions developed for the reductive cleavage of ben-
zothiazole-2-sulfonamides: Vedejs, E.; Lin, S.; Klapars, A.;
Wang, J. J. Am. Chem. Soc. 1996, 118, 9796.
13. For the reaction of lithium sulfinates with NCS, see: Gra-
ham, S. L.; Hoffman, J. M.; Gautheron, P.; Michelson, S. R.;
Scholz, T. H.; Schwam, H.; Shepard, K. L.; Smith, A. M.;
Smith, R. L.; Sondey, J. M.; Sugrue, M. F. J. Med. Chem.
1990, 33, 749.
Acknowledgements
We thank Dan McMasters for his help with the figures.
14. Cragoe, E. J.; Nicholson, J. A.; Sprague, J. M. J. Med.
Pharm. Chem. 1961, 4, 369.
15. Lewis, S. D.; Ng, A. S.; Baldwin, J. J.; Fusetani, N.;
Naylor, A. M.; Shafer, J. A. Thromb. Res. 1993, 70, 173. All
the Ki data quoted in this report are for the human enzymes.
16. Kurz, K. D.; Main, B. W.; Sandusky, G. E. Thromb. Res.
1990, 60, 269. Lewis, S. D.; Ng, A. S.; Lyle, E. A.; Mellot,
M. J.; Appleby, S. D.; Brady, S. F.; Stauffer, K. J.; Sisko, J. T.;
Mao, S.-S.; Veber, D. F.; Nutt, R. F.; Lynch, J. J.; Cook, J. J.;
Gardell, S. J.; Shafer, J. A. Thromb. Haemost. 1995, 74, 1107.
17. The crystals of 2g bound to the a-thrombin-hirugen complex
were prepared and the diffraction data collected as previously
described: Lyle, T. A.; Chen, Z.; Appleby, S. D.; Freidinger,
R. M.; Gardell, S. J.; Lewis, S. D.; Li, Y.; Lyle, E. A.; Lynch, J. J.;
Mulichak, A. M.; Ng, A. S.; Naylor-Olsen, A. M.; Sanders,
W. M. Bioorg. Med. Chem. Lett. 1997, 7, 67. Chen, Z.; Li, Y.;
Mulichak, A. M.; Lewis, S. D.; Shafer, J. A. Arch. Biochem.
Biophys. 1995, 322, 198. The diffraction data was collected to
1.9 A resolution with Rsym of 0.057 and completeness of 98%.
The structure was determined using the difference Fourier
method and was refined to an R factor of 0.199.
References and Notes
1. Coburn, C. A. Exp. Opin. Ther. Pat. 2001, 11, 1.
2. Sanderson, P. E. J.; Lyle, T. A.; Cutrona, K. J.; Dyer, D. L.;
Dorsey, B. D.; McDonough, C. M.; Naylor-Olsen, A. M.;
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5. For covalent, boronate thrombin inhibitors containing
4-sulfonylpyridinone acetamide groups, see: Lassalle, G.; Bel-
levergue, P.; Mallart, S.; Bourbier, J. C.; Galtier, D.; Martin,
V. FR 2758329, 1998.