Synthesis and solid-phase purification of anthranilic sulfonamides as CCK-2 ligands

Article abstract of DOI:10.1016/j.bmcl.2007.09.087

A novel strategy for the synthesis of cholecystokinin-2 receptor ligands was developed. The route employs a solution-phase synthesis of a series of anthranilic sulfonamides followed by a resin capture purification strategy to produce multi-milligram quant

Full text of DOI:10.1016/j.bmcl.2007.09.087

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Bioorganic & Medicinal Chemistry Letters 17 (2007) 6905–6909
Synthesis and solid-phase purification of anthranilic
sulfonamides as CCK-2 ligands
Craig R. Woods,a,* Michael D. Hack,a Brett D. Allison,a Victor K. Phuong,a
Mark D. Rosen,a Magda F. Morton,a Clodagh E. Prendergast,b Terrance D. Barrett,a
Nigel P. Shankleya and Michael H. Rabinowitza
aJohnson & Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121, USA
bDepartment of Physiology, University of Liverpool, Liverpool L69 3BX, UK
Received 2 August 2007; revised 25 September 2007; accepted 26 September 2007
Available online 29 September 2007
Abstract—A novel strategy for the synthesis of cholecystokinin-2 receptor ligands was developed. The route employs a solution-
phase synthesis of a series of anthranilic sulfonamides followed by a resin capture purification strategy to produce multi-milligram
quantities of compounds for bioassay. The synthesis was used to produce >100 compounds containing various functional groups,
highlighting the general applicability of this strategy and to address specific metabolism issues in our CCK-2 program.
Ó 2007 Elsevier Ltd. All rights reserved.
O
The cholecystokinin-2 receptor (CCK-2) remains an
important target for the potential treatment of gastroin-
testinal disorders such as acid reflux, gastroesphogeal re-
flux disease, peptic ulcers1 as well as GI adenocarcinoma.2
In the 20 years since the disclosure of CCK-2,3 no com-
pounds targeting this receptor have become approved
pharmaceuticals owing primarily to the variable pharma-
cokinetics and poor physiochemical properties of the clin-
ical candidates.3,4 Recently, we have re-examined CCK-2
as a target in our medicinal chemistry efforts in order to
develop a small molecule antagonist with increased drug
like properties.5
Cl
N
HN
O
S
O
N
N
S
1
To assess its moderate in-vivo half-life, an identification
of the metabolites formed in the presence of human liver
microsomes (HLM) was performed on 2 and analogs.
Rapid oxidation of the benzothiadiazole and piperidine
rings was found and hence could contribute significantly
to the high in-vivo clearance (Fig. 1).5
Testing of Johnson & Johnson compound library collec-
tion via high throughput screening identified a number
of novel structural types showing good affinity for the
CCK-2 receptor. Compound 1 exhibited good affinity
(pKI = 6.4) and selectivity over the related CCK-1 recep-
tor (pKI < 5). Pharmacokinetic (PK) analysis showed a
moderate half-life (0.35 0.03 h) and clearance (0.42
0.01 L/kg/h) upon iv administration to the rat.
To improve metabolic stability with respect to the ben-
zothiadiazole moiety specifically,6 a synthetic effort
O
O
OH
O
N
N
N
HLM
HN
Br
HN
Br
HN
Br
+
O
O
O
S
S
S
O
O
O
N
S
N
S
O
N
S
2
Keywords: Cholecystokinin; CCK; CCK-2; Anthranilic sulfonamides;
Sulfonamide synthesis; Resin capture purification; Solid-phase purifi-
cation; Trisamine-PS; TBD-Me; TBD-PS.
N
N
N
*
Corresponding author. Tel.: +1 858 784 3115; fax: +1 858 784
3267; e-mail: cwoods4@prdus.jnj.com
Figure 1. Metabolites of 2 formed in the presence of human liver
microsomes.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.087

6906
C. R. Woods et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6905–6909
O
NH2
NH2
5 equiv.
N
O
N
O
2 equiv.
RSO2Cl
N
O
H2N
Br
O2N
Br
trisamine-PS
N
N
HN
Br
3
O
HN
Br
pyridine,
DCM
S
+
H2N
Br
Scavenge
RSO2Cl
O
O
S
O
Cl
R
R1
O
3
S
O
5 equiv.
N
N
Capture
N
R1
sulfonamide,
remove impurities
TBD-PS
O
Figure 2. Retrosynthetic analysis of the anthranilic sulfonamides.
O
N
10%TFA
in DCM
HO2C
O2N
ClOC
O2N
N
b
a
N
S
R
Br
N
HN
S
R
Br
O
O
O2N
Br
Br
Br
Release
sulfonamide
O
O
N
c
N
H
O
O
N
Scheme 2. Synthetic and purification protocols for the production of
anthranilic sulfonamides.
d
N
H2N
Br
O2N
Br
3
solid support through an ionic interaction. Components
in the reaction mixture not containing acidic moieties
could then be removed by filtration and the sulfonamide
product later isolated by release from the solid support
by treatment with an acidic solution. Similar ‘catch
and release’ methods9 using both ionic10 and covalent11
interactions have been used efficiently in library synthe-
ses12 of ethers,13 amines,14 sulfonates,15 other moieties,16
and in the alkylation of N-Boc sulfonamides,17 however,
to our knowledge, not for the purification of
sulfonamides.18
Scheme 1. Reagents and conditions: (a) KMnO4, H2O or n-Bu4NM-
nO4, pyridine, rt, 5–18 h; (b) SOCl2, D; (c) piperidine, DCM, TEA; (d)
SnCl2Æ2H2O, EtOAc, CH2Cl2, rt.
was undertaken to find a suitable replacement and pro-
vide compounds which maintain the same potency to-
ward CCK-2, but offer improved resistance toward
CYP 450 oxidation.
Retrosynthetic analysis of the anthranilic sulfonamides
shows the sulfonamide linkage to be the logical discon-
nection in order to rapidly access arene-based replace-
ments for the benzothiadiazole (Fig. 2).
To optimize the reaction and purification strategies
(Scheme 2), we began by determining that addition of
two equivalents of sulfonyl chloride in the presence of
a mild base (pyridine) was sufficient to efficiently drive
the reaction to completion in a reasonable time frame
(approximately 1 h) and produce minimal amounts of
bis-sulfonamide addition product. Minor formation of
the latter was not expected to be an issue, however, as
it would lack the requisite acidic NH sulfonamide pro-
ton, and hence be removed in our ‘catch and release’
purification strategy during washing.
The intermediate anthranilic amine (3) can be readily
accessed in four steps from 4-bromo-2-nitro-toluene5,7
(Scheme 1). The sulfonyl chloride components were
obtained commercially and chosen to contain similar
aromatic cores to the benzothiadiazole, but range in ste-
ric size, polar functionality, conformational preference,
and overall polarity.
In order to streamline synthetic throughput, we
decided to investigate and optimize a purification
methodology for isolation of the proposed sulfonamide
library. Automated liquid chromatographic purification
systems of normal or reverse phase offer a viable, high
throughput method for purification, however, these
methods require a large volume of solvent and can
be time consuming. Alternatively, purification schemes
involving solid supported reagents or scavengers8 offer
the advantage of speed and ease of use, as reagents can
simply be filtered from solution upon completion of
reaction. Their usage, however, must be tailored to
appropriately fit the chemistry at hand and are not
suitable to all targets.
To determine which solid supported base would be suit-
able for capturing the sulfonamide, we initially explored
use of the trisamine-PS (tris-(2-aminoethyl)aminoethyl
polystyrene resin) to both quench the excess sulfonyl
chloride19 and capture the desired final product. These
efforts were only partly successful as the sulfonyl chlo-
ride was efficiently removed, however, capture of the fi-
nal sulfonamide was inefficient. pKa calculations show
approximately two orders of magnitude difference be-
tween the acidic sulfonamide and basic trisamine-PS
components,20 which should have been suitable to
our ends. Fortunately, the use of a more basic resin,
TBD-PS (1,5,7-triazabicyclo[4.4.0]dec-5-ene polystyrene
resin),21 proved highly effective, giving quantitative
recovery of the desired sulfonamide after capture and
release under acidic conditions with trifluoroacetic
acid in dichloromethane. Thus after these optimizations,
we found that the overall transformation could be
The anthranilic sulfonamides (such as 1 or 2) bear an
acidic sulfonamide NH proton, which can be deproto-
nated by a solid supported base and thus could in theory
cause the anthranilic sulfonamide to be captured on a

C. R. Woods et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6905–6909
6907
completed in several hours and provide the desired sul-
fonamides in an average yield of 70% with an average
purity of >95%.22
ans (4q, 4r), and other heterocycles (4s, 4t). Many
functional groups are represented which had the poten-
tial for acid instability such as esters (4i, n, o, q, r),
amides (4m), and nitriles (4e), as well as basic amines
in the core or periphery. Several moieties were found
to be incompatible with this methodology, however,
including compounds containing carboxylic acid, isox-
azole, [1,2,4]-oxadiazole, and [1,2,4]-thiadiazole moie-
ties, presumably due to either insolubility under
reaction conditions or acid instability during the cleav-
age step.
Using this methodology, we were able to produce >100
compounds for our CCK-2 program and rapidly inves-
tigate a variety of benzothiadiazole replacements pos-
sessing varied functionality. A representative cross
section of the compounds is shown in Figure 3. Several
different types of aryl and heteroaryl cores were pro-
duced including phenyls (4a–g), thiophenes (4h–p), fur-
R1=
O
F
F
Cl
N
HN
Br
O
NO2
O
S
O
R1
CF3
O
4a
4b
4c
4d
(81%, >98) (64%, >98) (96%, >98) (54%, >98)
S
N
CN
N
S
N
O
N
N
S
4e
4g
4f
4h
(23%,a >98)
(81%, >98)
(96%, 97)
(52%, >98)
Br
S
O
S
S
S
Cl
N
O
MeO2C
N
N
CF3
S
4i
4j
4k
4l
(70%, >98)
(96%, 94)
(81%, >98)
(80%, >98)
CO2Me
S
CO2Me
S
S
S
F3C
HN
O
4m
4n
4o
4p
(85%, >98)
(89%, >98)
(55%, >98)
(55%, >98)
MeO2C
O
Cl
N
O
N
N N
S
MeO2C
4s
4q
4r
4t
(82%, >98)
(81%, >98)
(86%, >98)
(85%, 95)
Figure 3. A cross-section of the compounds synthesized, showing functionality amenable to the solid-phase purification methodology described
a
herein. Percent yield and purity are in parentheses below the compound number, respectively. Purity of starting sulfonyl chloride was poor in this
example contributing to low yield.

6908
C. R. Woods et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6905–6909
Table 1. Microsomal stabilitya and potencyb for aryl sulfonamides 4a–t
Supplementary data
HLMa
RLMa
pKIb
Characterization data for compounds 4a–t. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2007.
09.087.
2
0c
77
44
3
29
78
49
25
9
7.6
6.3
6.2
5.7
5.6
5.3
5.3
5.1
5.4
5.3
5.5
6.1
5.3
5.2
5.8
5.1
5.5
5.3
6.3
5.8
5.1
4a
4b
4c
4d
4e
4f
41
7
100
23
66
47
0c
13
0c
5
0c
63
2
References and notes
4g
4h
4i
1. Vakil, N. Aliment. Pharmacol. Ther. 2004, 19, 1041;
Lehmann, F. S.; Hildebrand, P.; Beglinger, C. Drugs
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4j
39
68
87
0c
4k
4l
2. Reubi, J. C.; Schaer, J. C.; Waser, B. Cancer Res. 1997, 57,
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Drug Dispos. 1996, 17, 1; McDonald, I. M. Expert Opin.
Ther. Pat. 2001, 11, 445.
89
0c
3
0c
23
10
0c
7
4m
4n
4o
4p
4q
4r
4s
4t
37
0c
19
100
11
100
30
85
4. Allison, B. D.; Phuong, V. K.; McAtee, L. C.; Rosen, M.;
Morton, M.; Prendergast, C.; Barrett, T.; Lagaud, G.;
Freedman, J.; Li, L.; Wu, X.; Venkatesan, H.; Pippel, M.;
Woods, C.; Rizzolio, M. C.; Hack, M.; Hoey, K.; Deng,
X.; King, C.; Shankley, N. P.; Rabinowitz, M. H. J. Med.
Chem. 2006, 49, 6371.
a Human liver microsome (HLM) and rat liver microsome (RLM)
stabilities reported as percent of compound remaining after 30 min.
b Negative logarithm of the antagonist equilibrium dissociation con-
stant calculated from the concentration required to displace 50% 125I-
CCK-8S (pIC50) by the method of Cheng and Prussoff. All values are
0.3 log units unless otherwise stated.
5. For chemical changes related to the modification of the
piperidine amide region, see Ref. 5.
c Compound concentration was below detection limits.
6. Deng, X.; Stefanick, S.; Pippel, M. C. W.; Mani, N. S.
Org. Process Res. Dev. 2006, 10, 1287.
7. Ley, S. V.; Ladlow, M.; Vickerstaffe, E. Exploiting Chem.
Divers. Drug Discov. 2006, 3; Booth, R. J.; Hodges, J. C.
Acc. Chem. Res. 1999, 32, 18.
The relative stability toward CYP 450 oxidation of the
compounds was examined in the presence of human
and rat liver microsomes (Table 1).23 Several of the com-
pounds showed good stability after 30 min. This was a
vast improvement over compound 2 under the same
conditions which showed rapid degradation.
8. Keating, T. A.; Armstrong, R. W. J. Am. Chem. Soc. 1996,
118, 2574.
9. Organ, M. G.; Dixon, C. E.; Mayhew, D.; Parks, D. J.;
Arvanitis, E. A. Comb. Chem. High Throughput Screening
2002, 5, 211; Flynn, D. L.; Devraj, R. V.; Naing, W.;
Parlow, J. J.; Weidner, J. J.; Yang, S. Med. Chem. Res.
1998, 8, 219.
10. Cheng, S.; Comer, D. D.; Williams, J. P.; Myers, P. L.;
Boger, D. L. J. Am. Chem. Soc. 1996, 118, 2567; Cheng,
S.; Tarby, C. M.; Comer, D. D.; Williams, J. P.; Caporale,
L. H.; Myers, P. L.; Boger, D. L. Bioorg. Med. Chem.
1996, 4, 727; Armstrong, R. W.; Brown, S. D.; Keating, T.
A.; Tempest, P. A. J. Comb. Chem. 1997, 153.
11. Bapna, A.; Vickerstaffe, E.; Warrington, B. H.; Ladlow,
M.; Fan, T.-P. D.; Ley, S. V. Org. Biomol. Chem. 2004, 2,
611.
Although we were able to achieve improved stabilities
by this measure, receptor affinity values of the analogs
(Table 1) were lower than for compound 2. The best
of the analogs from the library synthesis, 4a, was over
a full log unit lower relative to the direct benzothiadiaz-
ole analog 2. Subsequently, in a second stage of this
work, we undertook a chemical strategy to build struc-
turally similar benzothiadiazole-like replacements to
determine the requisite pharmacophore and modifiable
regions of the system using X-ray crystallography,
NMR, and computational studies and are subjects of fu-
ture disclosures from our laboratories.24
12. Xu, W.; Mohan, R.; Morrissey, M. M. Tetrahedron Lett.
1997, 38, 7337.
13. Liu, Y.-S.; Zhao, C.; Bergbreiter, D. E.; Romo, D. J. Org.
Chem. 1998, 63, 3471.
14. Seeberger, S.; Griffin, R. J.; Hardcastle, I. R.; Golding, B.
T. Org. Biomol. Chem. 2007, 5, 132.
15. Boisnard, S.; Chastanet, J.; Zhu, J. Tetrahedron Lett.
1999, 40, 7469; Hardcastle, I. R.; Moreno Barber, A.;
Marriott, J. H.; Jarman, M. Tetrahedron Lett. 2001, 42,
1363; Kulkarni, B. A.; Ganesan, A. Angew. Chem., Int.
Ed. Engl. 1997, 36, 2454; Parlow, J. J.; Case, B. L.; South,
M. S. Tetrahedron 1999, 55, 6785; Shuker, A. J.; Siegel, M.
G.; Matthews, D. P.; Weigel, L. O. Tetrahedron Lett. 1997,
38, 6149.
In summary, we have developed an efficient and rapid
synthetic methodology for the preparation of >100
anthranilic sulfonamides for our ongoing medicinal
chemistry efforts. This methodology has produced a
small library of compounds allowing us to refine our
pharmacophore model for CCK-2 receptor binding of
small molecule antagonists.
Acknowledgment
16. Griffiths-Jones, C. M.; Hopkin, M. D.; Jonsson, D.; Ley,
S. V.; Tapolczay, D. J.; Vickerstaffe, E.; Ladlow, M.
J. Comb. Chem. 2007, 9, 422.
We thank Jiejun Wu for his assistance with the spectral
analysis of these compounds.

C. R. Woods et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6905–6909
6909
17. Sturino, C. F.; Labelle, M. Tetrahedron Lett. 1998, 39,
5891.
18. arrett, A. G. M.; Smith, M. L.; Zecri, F. J. J. Chem. Soc.,
Chem. Commun. 1998(21), 2317.
19. Booth, R. J.; Hodges, J. C. J. Am. Chem. Soc. 1997, 119,
4882.
by the addition of trisamine-PS resin (tris-(2-amino-
ethyl)aminoethyl polystyrene resin, 200 mg, 0.88 mmol,
Aldrich, 4.4 mmol/g loading), shaking for 1 h, followed
by filtration, and rinsing of the resin with dichlorometh-
ane. Purification of desired compound was carried out in
a two-step ‘catch and release’ method by addition of
TBD-PS resin (1,5,7-triazabicyclo[4.4.0]dec-5-ene polysty-
rene resin, 325 mg, 0.88 mmol, 5 equiv, Novabiochem,
2.7 mmol/g loading), shaking for 1 h, then collecting the
resin containing the sequestered product via filtration
and rinsing with dichloromethane. The desired product
was released by shaking the resin with a 10% trifluoro-
acetic acid solution in dichloromethane (10 mL) for
30 min followed by filtration and removal of solvent
under reduced pressure to yield the desired product as
the trifluoroacetate salt. In several cases, the product
obtained was an amber oil, but could easily be crystal-
lized by the addition and subsequent trituration with
diethyl ether.
20. pKa calculations were made using ACD/pKa DB, version
7.07, Advanced Chemistry Development, Inc., Toronto
ON, Canada, www.acdlabs.com, 2006 and showed the
pKa’s of the sulfonamide NH to be in the range of 6–8
depending on sulfonamide arene substitution and corre-
lates well with measured pKa values of analogs of 1 and 2
in Ref. 5. pKa of the primary amine of the trisamine-PS
resin was calculated to be 9.6 based on the N1-(2-Amino-
ethyl)-N1-[2-(4-methyl-benzyl-amino)-ethyl]-ethane-1,2-dia-
mine fragment of the resin.
21. pKa calculations were made using ACD/pKa DB, version
7.07, Advanced Chemistry Development, Inc., Toronto
ON, Canada, www.acdlabs.com, 2006 and showed the pKa
of TBD-PS to be 13.5 based on the 1-(4-methyl-benzyl)-
1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine frag-
ment of the resin.
22. Typical procedure for sulfonamide synthesis: To a 20 mL
reaction vial were added amine 3 (50.0 mg, 0.18 mmol,
1 equiv), dichloromethane (10 mL) and pyridine (100 lL,
0.88 mmol, 5 equiv). Sulfonyl chloride (0.36 mmol,
2 equiv) was then added and contents agitated on a
shaker plate for one hour or until reaction was complete
by HPLC. Excess sulfonyl chloride was then removed
23. Relative microsomal stability was assed by testing 2, 4a–t
with the same batch of human and rat liver microsomes
for thirty minutes under an identical protocol, followed by
determination of the amount of parent remaining via
LCMS analysis.
24. Rosen, M. D.; Hack, M. D.; Allison, B. D.; Phuong, V.
K.; Woods, C. R.; Morton, M. F.; Prendergast, C. E.;
Barrett, T. D.; Schubert, C.; Li, L.; Wu, X.; Freedman, J.
M.; Wu, J.; Shankley, N. P.; Rabinowitz, M. H., manu-
script in preparation.