Development of a scalable synthesis of a nonbasic inhibitor of the serine protease tryptase

Article abstract of DOI:10.1016/j.tetlet.2006.04.149

A chromatography-free process for the synthesis of a bis(benzimidazole)difluoromethane inhibitor of the serine protease tryptase is described. This synthesis features the introduction of the gem-difluoro moiety using the electrophilic fluorinating reagent N-fluoro-bis(phenylsulfonimide) as well as the stepwise introduction of both benzimidazole rings. A protocol for the destruction of reactive, process-related substances produced in the synthesis is also presented.

Full text of DOI:10.1016/j.tetlet.2006.04.149

Tetrahedron Letters 47 (2006) 4591–4595
Development of a scalable synthesis of a nonbasic inhibitor of
the serine protease tryptase
Jeffrey M. Dener,a,* Colin O’Bryan,a Robert Yee,b Emma J. Shelton,b David Sperandio,b
Tania Mahajan,b James T. Palmer,b Jeffrey R. Spencerb and Zhiwei Tongb
aDepartment of Process Chemistry, Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, United States
bDepartment of Medicinal Chemistry, Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, United States
Received 23 February 2006; revised 14 April 2006; accepted 28 April 2006
Available online 22 May 2006
Abstract—A chromatography-free process for the synthesis of a bis(benzimidazole)difluoromethane inhibitor of the serine protease
tryptase is described. This synthesis features the introduction of the gem-difluoro moiety using the electrophilic fluorinating reagent
N-fluoro-bis(phenylsulfonimide) as well as the stepwise introduction of both benzimidazole rings. A protocol for the destruction of
reactive, process-related substances produced in the synthesis is also presented.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
itors and determined that the potency of these com-
pounds against trypsin-like proteases such as Factor
Xa, and tryptase result from the tetrahedral binding of
zinc(II) ions to both the hydroxyl group of Ser-195
and the imidazole moiety of His-57 in the active site of
the enzyme, along with two nitrogens of the bis(benz-
imidazole)methane moiety of the inhibitor.6 This novel
mechanism takes advantage of ambient levels of zinc
in vivo, especially in the granules of the mast cell. How-
ever, development of orally active BABIM derivatives as
serine protease inhibitors, including tryptase, were ham-
pered by the presence of the polar benzamidine and met-
abolically labile benzylamine P1 moieties, illustrated in
the BABIM class (5) as well as the unsymmetrical deriv-
ative, APD-10 (6). In order to identify an orally active
tryptase inhibitor based on the bis(benzimidazole)meth-
ane motif, efforts were undertaken to identify related
derivatives which did not rely on the benzamidine or
similar polar functionality for binding affinity in the P1
pocket of the enzyme. Ultimately, SAR efforts identified
CRA-9249 (7) as an orally active tryptase inhibitor de-
void of these polar functionalities at P1 (Fig. 2). In order
to provide multigram and ultimately larger quantities of
7 for pre-clinical evaluation, we set out to develop a scal-
able process for this compound.
Inhibition of mast cell tryptase has been recognized as a
viable therapeutic target for allergic and inflammatory
diseases such as allergic rhinitis, conjunctivitis, dermati-
tis, inflammatory bowel disease (IBD), and asthma.1 For
example, a Phase IIa clinical evaluation of the tryptase
inhibitor APC-366 (1) showed significance in the pri-
mary endpoint of reduction of the late asthmatic re-
sponse when administered by inhalation.2 In addition,
in a Phase II clinical trial with patients exhibiting mildly
to moderately active ulcerative colitis, intravenous
administration of APC-2059 (2) resulted in 58% of these
patients experiencing a clinically significant benefit in
symptoms associated with the disease.3 Other tryptase
inhibitors, either in pre-clinical or clinical development,
include RWJ-56423 (3) and BMS-354326 (4). Structures
for these compounds are shown in Figure 1.
Another class of tryptase inhibitors is based on the 2,20-
bis(benzimidazole)methane scaffold, including the sym-
metrical bis(5-amidino-2-benzimidazolyl)methane (BA-
BIM; 5). The BABIM series afforded one of the first
potent inhibitors of mast cell tryptase, having been ini-
tially reported as a potent inhibitor of other serine
proteases in the late 1970s.4,5
Research at Celera (formerly Axys Pharmaceuticals) led
to the discovery of a novel binding mode of these inhib-
2. Results and discussion
The initial route to CRA-9249 was based on a stepwise,
‘one-pot’ coupling of 2,2-difluoromalonic acid (8) with
*
Corresponding author. E-mail: jdener@yahoo.com
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.04.149

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J. M. Dener et al. / Tetrahedron Letters 47 (2006) 4591–4595
O
O
H
NH2
O
H2N
N
H
N
N
O
H
H
N
NH
N
N
O
O
OH
O
O
O
N
N
NH
NH2
H
H
HN
O
N
N
H
HN
NH2
O
1
2
APC-366
APC-2059
HO
O
H
N
S
N
N
O
O
CO2H
HN
O
N
H
N
N
O
HN
HN
O
NH2
3
4
RWJ-56423
BMS-354326
Figure 1. Representative tryptase inhibitors.
to the bis(benzimidazole)methane fragment. Generation
of the gem-difluoromethane bridgehead would rely on
the reaction of the bridgehead methylene of the bis benz-
imidazole moiety with an electrophilic fluorinating re-
agent. This transformation is based upon a successful
strategy previously employed at Celera on a similar
bis(benzimidazole)methane derivative.9 The synthesis
of the key bis(benzimidazole)difluoromethane acid 20
is shown in Scheme 2. Both benzimidazole rings in 20
were constructed from two separate 1,2-diaminobenzene
derivatives according to this scheme. First, the right-
hand benzimidazole ring was constructed starting from
H
N
H
N
H2N
NH2
NH
N
N
NH
5
"BABIM"
O
CH3
N
H
N
N
H
H2N
N
N
6
APD-10
O
CH3
H
N
F
F
F
O
N
N
CH3
HN
O
H
a
O
N
N
F
HO2C
CO2H
N
H
+
F
F
F
H2N
F
7
CRA-9249
9
8
Figure 2. Bis(benzimidazole)methane derived tryptase inhibitors.
CH3
F
O
F
F
NH2
NH2
N
O
+
HO2C
N
two different 1,2-diaminobenzene derivatives 9 and 11 to
each carboxylate functionality of 8 with the expensive
coupling agent bromotripyrrolidinophosphonium hexa-
fluorophosphate (PyBroP) to give the bis(amide) 12.7,8
Without isolation, compound 12 was heated in acetic
acid to generate both benzimidazole rings of CRA-
9249. Although this approach was satisfactory in pro-
viding modest amounts of 7 needed for early discovery
efforts, the route suffers from low yield (20% from diacid
8 after column chromatography) and therefore was
unsuitable for the manufacture of multigram to kilo-
gram quantities of the active pharmaceutical ingredient
(API, compound 7).
H
O
H2N
F
F
10
11
CH3
F
O
F
H
F
N
N
O
b,c
N
H
O
NH2
O
H2N
F
F
12
d
7
In order to arrive at a scalable process to 7, which cir-
cumvented the liabilities evident in the route described
in Scheme 1, we pursued a stepwise condensation route
Scheme 1. Reagents and conditions: (a) PyBrOP, N-methylmorpholine
(NMM), DMF, ꢀ10 °C to rt; (b) PyBrOP, DMF, ꢀ10 °C to rt; (c) 11,
NMM, ꢀ10 °C to rt; (d) AcOH, 85 °C, 3 h.

J. M. Dener et al. / Tetrahedron Letters 47 (2006) 4591–4595
4593
H
H
N
CO2H
a,b
N
CO2H
CH3O
O2N
CO2H
c
H3C
H3C
H2N
O2N
15
14
13
H
N
F
NO2
NH2
• HCl
NH2
F
d
f
e
CO2Et
11 +
EtO
N
CO2Et
F
F
16
17
18
CH3
N
CH3
N
CH3
N
O
H
N
H
N
H
F
X
F
F
CO2H
F
CO2H
CO2H
+
g
N
N
N
N
N
N
N
F
F
F
22
19
20, X = F
21, X = H
CH3
N
H
N
F
CO2H
h
20
+
H
+
CO2CH3
N
N
F
23
24
Scheme 2. Reagents and conditions: (a) 40% aq CH3NH2, 100–105 °C; (b) 1 N aq HCl, 75% for both steps; (c) H2, 5% Pd–C, THF–MeOH (8:3),
96%; (d) H2, 10% Pd–C, EtOH, 89%; (e) EtOH, rt to 35–40 °C; (f) 15, DMPU, 150 °C, 90%; (g) (PhSO2)2N–F, 1,4-dioxane, reflux; 91%; (h) MeOH,
reflux.
commercially available 3-methoxy-4-nitrobenzoic acid
(13) which was converted to the 3-methylamino deriva-
tive 14 via a nucleophilic aromatic displacement of the
methoxy group with 40% aqueous methylamine. Reduc-
tion of the nitro group in 14 with hydrogen and 5%
palladium on carbon provided 4-amino-3-methylamino-
benzoic acid 15.7
anol also supported our concerns that these ketones
could also be toxic.
Hence, stringent specifications (<0.05%) for their pres-
ence were put in place for the final API. The treatment
of crude 20 in refluxing methanol served to degrade
the reactive monofluoro- and keto-derivatives 21 and
22 and reduce their levels below these stringent specifica-
tions prior to the introduction of the 20-(4-fluorophen-
oxy)ethylamine fragment. The resulting degradants 23
and 24 were very soluble and easily removed during
the methanol treatment.
The 3,5-difluoro-1,2-diaminobenzene required for the
left-hand benzimidazole ring was produced by catalytic
reduction of commercially available 3,5-difluoro-6-nit-
roaniline (16) with 10% Pd–C to afford the 3,5-diflu-
oro-1,2-diaminobenzene (11). Reaction of the diamine
11 with the imidate of ethyl cyanoacetate (17)10 in etha-
nol at 35–40 °C provided the left-hand benzimidazole
fragment 18 in 65% yield. Finally, thermal condensation
of ester 18 with a degassed suspension of 3-methyl-
amino-4-methyl benzoic acid 15 in DMPU at 150 °C
afforded the bis(benzimidazole)methanecarboxylic acid
19.11 Fluorination of this acid with an excess (250
mol %) of N-fluorobenzenesulfonimide afforded the de-
sired difluoro bridgehead intermediate 20 in 74% yield
after heating the crude product in refluxing methanol.
The hot methanol treatment served to degrade small
amounts of the monofluoro- and keto-derivatives 21
and 22 derived from incomplete fluorination and com-
petitive air oxidation of the methylene group in 19.
Although we had determined that bis(benzimid-
azole)methyl derivatives similar to 19 were toxic, we sus-
pected that the keto derivative 22, as well as the
corresponding amide related to 7, could also be toxic.
Furthermore, the chemical reactivity of 22 to hot meth-
Several routes were evaluated to prepare 4-fluorophen-
oxyethylamine derivative 29 required for the synthesis
of CRA-9249 (7), which are described in Scheme 3.
The first route started from the Mitsunobu reaction of
commercially available N-(tert-butoxycarbonyl)-amino-
ethanol 25 and 4-fluorophenol (26) using diisopropyl-
azodicarboxylate (DIAD) and triphenylphosphine to
provide Boc-protected 2-phenoxyethylamine 27. Unfor-
tunately this approach was ultimately abandoned due
the formation of modest amounts (15–20% as deter-
mined by proton NMR analysis) of the N-Boc aziridine
(28), which required a tedious chromatographic separa-
tion to remove this potentially troublesome impurity
from the desired N-Boc-aminophenoxyether.12,13
Although this route could be employed to afford the
target amine hydrochloride 29 after deprotection with
a solution of anhydrous HCl, a more scalable approach
was developed, based on the Delepine reaction.14 The

4594
J. M. Dener et al. / Tetrahedron Letters 47 (2006) 4591–4595
F
a-e
a,b
BOC
BOC
OH
20
7
+
N
H
HO
F
25
26
RO2C
CH3
N
F
F
O
O
N
c
N
H
+
HN
O
N
BOC
N
N
F
F
F
27
28
33
F
O
H
H2N
HCl
N
RO2C
O
F
34
29
N
Scheme 4. Reagents and conditions: (a) DMF, NMM, 0–5 °C; (b)
ROCOCl, (R = i-Pr or i-Bu), <10 °C; (c) 29, DMF; (d) NMM, <10 °C
to rt; (e) recrystallization from ethanol.
d
O
Br
+
N
N
N
F
30
31
isopropyl chloroformate in toluene was the preferred
reagent to generate the mixed anhydride, ensuring
>95% conversion and excellent yields of CRA-9249
(Scheme 4).
N
Br-
e-g
N+
O
N
29
N
F
32
CRA-9249 (7) was isolated in 72% yield after chlorofor-
mate-mediated amide bond formation and recrystalliza-
tion of the crude solid. Ultimately, this process was used
to generate over 95 g of 7 in >99.0% purity. The limit of
detection for the keto- and monofluoro impurities was
less than 0.03% as determined by spiking experiments
with authentic samples. The only impurity identified at
significant levels (>0.1%) was the acid precursor 20
(0.25%).16
Scheme 3. Preparation of the right-hand amine 29. Reagents and
conditions: (a) Ph3P, THF, rt to 0–5 °C; (b) i-PrO2CN@NCO2Pr-i, 0–
5 °C, then 0–5 °C to rt; (c) silica gel chromatography then 4 N HCl in
1,4-dioxane, CH2Cl2, 0–5 °C to rt (60% from 25); (d) CHCl3, reflux,
95%; (e) EtOH, 12 N HCl; (f) 4 N aq NaOH, H2O, 88% for two steps;
(g) anhyd HCl in 1,4-dioxane, then Et2O, 85%.
commercially available 4-fluorophenoxy-ethylbromide
(30) was reacted with hexamethylenetetramine (31) to
provide the ammonium salt 32. Hydrolysis of 32 to the
free amine followed by conversion to the HCl salt pro-
vided amine 29 on a multigram scale without chroma-
tography. Several other routes were explored but none
were as convenient as the Delepine reaction for large-
scale preparations of amine 29.15
Acknowledgments
We thank Joyce Mordenti, Mike Green, and Mike
Venuti for their commitment to and guidance during
the execution of this work. In addition Celera would
like to acknowledge the support and contributions of
Bayer A.G. in the identification of CRA-9249.
The coupling of amine 29 and acid 20 using mixed anhy-
drides afforded very good to excellent yields after addi-
tion of water to crystallize the product from N,N-
dimethylformamide (DMF). We noted that the nature
of the chloroformate employed in the reaction made a
significant difference in conversion and yield of the crude
product. Use of either isobutyl chloroformate (R = i-
Bu) or isopropyl chloroformate (R = i-Pr) afforded
CRA-9249 (7) in excellent purity. However isopropyl
chloroformate was found to be superior to isobutyl chlo-
roformate in the conversion of 20 to 7. The latter reagent
provided as much as 18% of unreacted 20 which fortu-
nately could be easily recovered through acidification
of the filtrate. Attempts to convert unreacted acid 20
through charging additional chloroformate to the reac-
tion mixture resulted in the formation of side-products,
such as compound 33 which is produced by N-acylation
of the free N–H of the left-hand benzimidazole nitrogen
and carbamate 34 which arises from competitive reac-
tion of excess chloroformate with amine 30. Thus, 1 M
References and notes
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Fleming, T. J.; Mordenti, J.; Stenkoski-Nix, L. C.; Kurth,
M. C. Alim. Pharmacol. Ther. 2002, 16, 407–413.
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J. M. Dener et al. / Tetrahedron Letters 47 (2006) 4591–4595
4595
6. (a) Janc, J. W.; Clark, J. M.; Warne, R. L.; Elrod, K. C.;
Katz, B. A.; Moore, W. R. Biochemistry 2000, 39, 4792–
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Jenkins, T. E.; Johnson, C. R.; Ross, M. J.; Luong, C.;
Moore, W. R.; Stroud, R. M. Nature 1998, 391, 608–612.
7. Mittendorf, J.; Henning, R.; Raddatz, S.; Schlemmer,
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Yoshino. T. PCT Int. Appl. WO 0020401, 2000. CRA-
9249 was developed by Bayer as part of an agreement
between Bayer and Arris Pharmaceutical Corporation,
now part of Celera. Celera re-acquired the tryptase
program from Bayer in 2002.
8. Although bis(amide) 12 was not isolated, this intermediate
could be produced as a mixture of regioisomers. The
specific regioisomer shown here is based on the assump-
tion that acylation occurs on the more basic nitrogens
found on each 1,2-diaminobenzene derivative 9 and 11.
9. Sperandio, D.; Gangloff, A. R.; Litvak, J.; Goldsmith, R.;
Hataye, J. M.; Wang, V. R.; Shelton, E. J.; Elrod, K.;
Janc, J. W.; Clark, J. M.; Rice, K.; Weinheimer, S.;
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14. (a) Galat, A.; Elion, G. J. Am. Chem. Soc. 1939, 61, 3585–
3586. For a review of synthetic applications of hexameth-
´
ylenetetramine, including the Delepine reaction, please see
´
the following: (b) Blazˇevic, N.; Kolbah, D.; Belin, B.;
ˇ
´
Sunjic, V.; Kajfezˇ, F. Synthesis 1979, 161–176.
15. Other routes explored in the preparation of 29 included
reduction of nitrile 35 and reaction of bromide 30 with
ammonia. Reaction of 35 using either catalytic [e.g., H2/
PtO2; transformation (a)] or chemical reduction [e.g.,
BH3ÆTHF or LiAlH4, THF, reflux; transformation (b)]
were unsatisfactory, providing either C–O bond cleavage
as the primary reaction pathway or formation of small
amounts of N,N-bis[2-(40-fluorophenoxy)ethyl]amine (36).
The hydrogenolysis of the C–O bond of a-phenoxyaceto-
´
nitriles is a known process: Benarab, A.; Boye, S.; Savelon,
L.; Guillaumet, G. Tetrahedron Lett. 1993, 34, 7567–7568,
Displacement of the bromide in 30 with ammonia in
several solvent combinations and conditions (transforma-
tion c) was also plagued by formation of 36 and the
corresponding trialkylamine which could not be easily
separated from the desired product.
26
+
29
a
b
10. Imidate 17 was prepared in 85% yield on a 1.1 mol scale
from the reaction of ethyl cyanoacetate and HCl gas in a
mixture of toluene and ethanol (ꢁ6:1) at 0–5 °C.
F
NC
O
O
11. The conditions used to produce 19 also afforded small
amounts of the decarboxylated bis(benzimidazole)meth-
ane derivative shown below, whose identity was confirmed
by comparison to an authentic sample.
HN
29
35
30
+
F
2
36
CH3
H
F
c
H
N
N
29
+ 36
N
N
F
16. The 95-g lot of 7 also provided acceptable analysis results
including combustion (CHN), inductively coupled plasma
(ICP), residue on ignition (ROI) and moisture content
(Karl Fischer).
12. The preparation of compound 29 by alkylation of a-
chloroacetamide with 4-fluorophenol followed by LiAlH4
reduction has been described in the literature: Shtacher, G.;
Taub, W. J. Med. Chem. 1966, 9, 197–203. However, we
did not evaluate this process for the work described here.
13. Protection of commercially available 2-bromoethylamine
hydrobromide with di-tert-butyl dicarboxylate and N-
methylmorpholine (NMM), followed by bromide displace-
ment with 4-fluorophenol and cesium carbonate also
provided 27 contaminated with aziridine 28.
LC-MS: m/z 516.0 [M+1]+; 1H NMR (DMSO-d6; 400
MHz) d 8.8 (t, 1H), 8.3 (s, 1H), 7.85 (d, 1H), 7.8 (d, 1H),
7.3 (d, 1H), 7.2 (t, 1H), 7.1 (m, 2H), 7.0 (m, 2H), 4.1
(t, 2H), 4.0 (s, 3H), 3.7 (q, 2H); CHN: calcd: C, 58.26; H,
3.52; N, 13.59; F, 18.43. Found: C, 58.03; H, 3.28; N,
13.62; F, 18.55; ICP results: Zn: <1 ppm; Cu < 1 ppm.
ROI: <0.10%; % moisture (Karl Fischer): <0.10%.