Solid-phase synthesis of a library based on biphenyl-containing trypsin-like serine protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.100

The solid-phase synthesis of a library based on an unusual biphenyl-containing trypsin-like serine protease inhibitor is described. Key to this effort was the synthesis of a highly functionalized aryl boronic acid reagent which required the development of

Full text of DOI:10.1016/j.bmcl.2009.08.100

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6477–6480
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Solid-phase synthesis of a library based on biphenyl-containing trypsin-like
serine protease inhibitors
a
a
a
b
b
Shuhao Shi a, , Shirong Zhu , Samuel W. Gerritz , Bogumila Rachwal , Zheming Ruan , Robert Hutchins ,
*
Ramesh Kakarla a, Michael J. Sofia a, James Sutton b, Daniel Cheney b
a Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, CT 06492, United States
b Bristol-Myers Squibb Company, PO Box 4000, Princeton, NJ 08543-4000, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The solid-phase synthesis of a library based on an unusual biphenyl-containing trypsin-like serine prote-
ase inhibitor is described. Key to this effort was the synthesis of a highly functionalized aryl boronic acid
reagent which required the development of a novel and efficient method to convert a triflate to a pina-
colboronate in large scale.
Received 4 March 2009
Revised 20 August 2009
Accepted 24 August 2009
Available online 17 September 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Solid phase library
Biphenyl
Trypsin-like serine protease inhibitor
Pinacolboronate
Nanokan
The trypsin-like serine protease superfamily contains a number
of drug discovery targets, including thrombin, elastase, urokinase,
and coagulation factors VIIa, Xa, and XIa.1 The therapeutic impor-
tance of this superfamily has prompted the discovery of a number
of common pharmacophores, as shown in Figure 1. Chemotypes
1–3 are peptidomimetic inhibitors,2–4 but structure 4 (IC50 8 nM
for factors VIIa, first disclosed by Ono Pharmaceuticals in 19995)
represents a structurally unique class of trypsin-like serine prote-
ase inhibitors due to its biphenyl scaffold, a ‘privileged’ structure
found in 4.3% of all known drugs.6 Our ongoing interest in mem-
bers of the trypsin-like serine protease superfamily prompted us
to embark on the solid-phase synthesis of a library of potential pro-
tease inhibitors related to chemotype 4.
Boronate 6 was synthesized as shown in Scheme 2. Compound
148 was converted to the orthogonally protected diester 8 in excel-
lent yield according Forsch and Rosowsky’s procedure.9 Miyaura
et al. reported an efficient one-step procedure10 for preparation of
arylboronates from aryl triflates and bis(pinacolato)-diboron 15 uti-
lizing a palladium-catalyzed coupling in the presence of a base.
However, when we tried to convert triflate 8 to arylboronate 6
according Miyaura’s standard conditions (dioxane, 80 °C, 1.1 equiv
of diboron 15, 1 equiv of triflate 8, 3% equiv of Pd(dppf), 3% equiv
of dppf, 3 equiv of KOAc, 20 h), only trace quantities of arylboronate
6 were obtained along with large amounts of unreacted triflate 8. In
order to make sufficient quantities of 6 for use in a large solid-phase
library synthesis, further optimization was required.
As reported by Kohrt et al.,7 the synthesis of 4 was not described
in detail in the original Ono disclosure. Accordingly, as shown in
Scheme 1, we developed a solid-phase retrosynthesis of 5 with
three points of diversity (R1, R2, R3R4). A Suzuki coupling was iden-
tified as the critical bond-forming step to permit disconnection of 5
to two components, pinacolboronate 6 and resin-bound amide 7.
Boronate 6 was envisioned to arise from triflate 8, which in turn
could arise from commercial available salicylic acid 9. Resin-bound
amide 7 could be disconnected into 2-halobenzoyl chloride 10 and
resin-bound amine 11 which could arise from PL-FMP resin 12 and
primary amine 13 via reductive amination.
After extensive experimentation, we found that by increasing
the Pd(dppf) loading to 8% of the triflate, adding no external dppf,
the reaction was complete after 2 h at 60–65 °C and product 6 was
obtained in more than 90% yield. However, the purification of the
product 6 was problematic- it could not be isolated by crystalliza-
tion and had to be purified by flash chromatography. Although the
Rf of 6 is 0.31 in DCM, the pinacolboronate rapidly hydrolyzed
upon exposure to silica gel even when the silica was pre-treated
with triethylamine. To prevent hydrolysis, a significantly more po-
lar eluent (DCM/EtOAc = 7:3) was used to quickly flush the product
from a column loaded with silica pre-treated with triethylamine.
This purification protocol provided 6 contaminated with approxi-
mately 5% of diboronate 15. This impure material worked very well
for Suzuki couplings in solution, but the presence of diboronate 15
* Corresponding author. Tel.: +1 203 677 7239; fax: +1 203 677 6984.
E-mail address: shij@bms.com (S. Shi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.100

6478
S. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6477–6480
H
N
O
O
O
NH
CO2H
O
N
N
NH HN
NH HN
NH2
O
NH
OH
CF3
O
O
H
N
O
HN
S
N
H2N
O
CO2H
HN
H
COOH
N
H
CF3
NH
HN
H2N
Figure 1. Representative trypsin-like serine protease inhibitors.
1 (Inogatran)
2 (Argatroban)
H2N
NH
4
3
O
O
O
O
R4
N
O
O
CHO
OH
O
R3
TMS
2tBu
TMS
2tBu
CO
CO2H
CO
B
OTf
6
8
9
O
CO2H
R2
R1
+
OMe
CHO
N
H
O
X
O
X
R1
R1
+
R1-NH2
O
Cl
N
H
R2
N
+
5
R2
13
12
10
Scheme 1.
11
7
O
O
O
O
O
O
TMS
O
O
O
OH
B
B
TMS
TMS
O
15
O
(1 eq)
HO
t
CO2 Bu
DCC/DMAP
DCM, 0 ºC, 18h.
91%
t
t
PdCl2 (dppf) (8%)
CO2 Bu
CO2 Bu
B
OTf
6
79%
OTf
KOAc (3 eq)
Dioxane, 60-65 ºC
8
14
Scheme 2.
adversely affected the reaction on solid-phase because it was more
reactive than 6 in the Suzuki reaction with the solid-supported aryl
iodide. To overcome this issue, we reduced the diboron 15 to
1 equiv of the triflate and prolonged the reaction time to 20 h. Un-
der these conditions, boronate 6 was obtained in 79% yield with no
diboronate 15 contamination. A dimer byproduct (Suzuki coupling
between starting material triflate 8 and product boronate 6) was
clearly seen by UV (16%), but the presence of the dimer had no ef-
fect on the solid-phase reaction because it did not react with the
solid-supported aryl iodide. The optimized protocol11 is very effi-
cient and 180 g of the key intermediate 6 were prepared in several
batches.
The solid-phase synthesis of 5 is shown in Scheme 3, starting
with PL-FMP resin 12 loaded into Nanokans.12 The first diversity
TMS
(X =, I, Br)
X
t
CO2 Bu
O
OMe
CHO
OMe
1) R1-NH2
O
O
O
O
X
B
Cl
DMF/TMOF (2:1)
R2
O
R1
O
O
10
N
2) NaBH(OAc)
6
N
H
R2
HOAc
R1
DIEA/DCM
Pd(PPh3)4/K3PO4
DMF/H2O
80-90ºC, 20 h
RT, 2days
11
12
RT
7
Overnight
R4
N
R4
N
O
O
O
OH
O
R3
TMS
O
R3
1) Ghosez reagent
DCM, RT, 2h
t
CO2tBu
O
CO2 Bu
t
O
O
CO2H
O
CO2 Bu
TBAF
THF
1h
N
N
2) Amine, DCE
RT, 20h
HN
R1
N
TFA
DCM
R2
R2
R2
R2
R1
R1
R1
17
18
16
5
Scheme 3.

S. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6477–6480
6479
Table 1
Purity and yields of representative library products
O
H
N
R1
O
R2
N
R4
R3
HO2C
5
Entry
1
R1NH2
R2
NR3R4
Purity (%)
100
Yield (%)
NH2
66
52
50
53
79
65
N
N
BocN
2
3
4
5
6
100
100
100
97
NH2
OH
H
N
H
Me2N
NH2
BocHN
NH2
F
NH2
H
95
7
8
9
5-MeO
4-Cl
4,5-Di-MeO
97
97
97
93
43
34
45
32
BocN
H2N
H2N
NH2
OH
10
5-iPrO
11
12
13
14
15
99
98
87
82
81
81
66
55
56
74
OH
OH
N
H
NH2
F
4,5-Di-MeO
O
H2N
H2N
H2N
N
Et
N
14
The Boc-group in these reagents falls off in cleavage.

6480
S. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6477–6480
element (R1) was introduced via reductive amination. Thus, PL-
FMP resin in Nanokans (1.2 mmol/g, 8 mg/Nanokan) was treated
with primary amine in DMF/TMOF for 4 h, followed by treatment
with NaBH(OAc)3 and HOAc for 2 days at room temperature to af-
ford resin-bound amine 11. The second diversity element (R2) was
introduced via coupling benzoyl chloride 10 to resin-bound amine
11 in the presence of DIEA in DCE. The biphenyl core 16 was con-
structed via Suzuki coupling between 7 and boronate 6 at 80–
90 °C for 20 h. It was critically important to use degassed DMF
and we found that aqueous potassium phosphate was optimal.
The 2-(trimethylsilyl)ethyl ester was removed by treating 16 with
TBAF in THF for 1 h to afford acid 17. The third diversity element
(NR3R4) was introduced via amide formation. Thus, dried resin-
bound acid 17 was treated with 1-chloro-N,N,2-trimethyl-prope-
nylamine (Ghosez reagent)13 in DCM for 2 h, then washed with
DCM and treated with amine in DCE for 20 h at rt. For amino alco-
hols, an alternative method was applied. Resin-bound acid 17 was
treated with EDC, HOBt, DIEA and amino alcohol in DMA for 20 h.
In either case, the final product 5 was cleaved from resin 18 with
TFA/DCM (1:1) at rt for 2 h, then the resin was filtered off and
solution was collected in 96-well plates via IRORI Nanokan sys-
tem. Concentration of the filtrate afforded product 5 without fur-
ther purification. The purity of library products was determined
by HPLC at 254 nm and mass spectroscopy. Product samples
which met our purity criteria (product peak P80% by HPLC at
254 nm, correct MS) were submitted. The quantity of products
was determined by weight (most of library products were ob-
tained in 1–3 mg per compound). The library size is more than
10,000 products and the more than 80% of library products were
submitted. The purity and yields of some representative products
are shown in Table 1.
In summary, we have discovered a very unique and easy meth-
od to convert a triflate to a pinacolboronate in large scale. We also
developed an efficient solid-phase chemistry for large library of
biphenyl core with multiple functionalities.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.08.100.
References and notes
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was validated with a wide variety of building blocks to determine
best reagents and conditions. The 1H and 13C NMR of one product
can be seen in Supplementary data. In the reductive amination,
anilines with halides, –OMe, –SMe, –NO2, –CN, amide, acetamide
and alkylamines gave generally high yields (80–100%), some
anilines with an tertiary amine gave lower yields (60–79%). In
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withdrawing groups gave high yields (80–100%), but aromatic bro-
mines with electrondonating groups gave lower yields (42–68%). In
the amide formation via Ghosez reagent pathway, general amines
gave high yields (80–100%), some amines with tertiary amine and
pyridine ring gave mediate yields (68–79%), and amino alcohols
gave low yields (42–70%). The yields from reagents amino alcohol
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and DIEA in DCE. By reagent rehearsal, low yielding candidates
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11. General experimental procedure: A mixture of triflate 8 (9.39 g,19.96 mmol),
bis(pinacolato)diboron 15 (5.068 g,19.96 mmol), PdCl2(dppf) (1.168 g,
1.596 mmol, 8%) and KOAc (6.855 g, 69.84 mmol) in degased 1,4-dioxane
(100 mL) was heated at 60–65 °C for 20 h. After cooled down, dioxane was
removed by Rotovap, then DCM and water were added to the crude mixture.
Layers were separated and the aqueous layer was extracted by DCM one more
time. The combined organic extract was washed once with water and dried over
potassium carbonate. The crude product was purified by flash chromatography
(DCM and ethyl acetate, 70:30) to give 8.37 g (93.5%, theoretical yield 8.949 g) of
compound 6. LCMS showed M+1 (449) peak with purity of 84% for the compound
and peak 665 (Mdimer+Na+) as the major impurity (16%). NMR (1H, 13C) can be
seen in Supplementary data.
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