Benzisothiazolones as modulators of macrophage migration inhibitory factor

Article abstract of DOI:10.1016/j.bmcl.2011.05.127

Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds were found to possess antagonist activity in the low micromolar range with the most potent being the 6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.

Full text of DOI:10.1016/j.bmcl.2011.05.127

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4545–4549
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzisothiazolones as modulators of macrophage migration inhibitory factor
William L. Jorgensen a, , Alexander Trofimov a, Xin Du b, Alissa A. Hare a, Lin Leng b, Richard Bucala b,
⇑
⇑
a Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA
b Department of Medicine, Yale University School of Medicine, New Haven, CT 06520-8031, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity
of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds
were found to possess antagonist activity in the low micromolar range with the most potent being the
6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant
inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were
also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 March 2011
Revised 27 May 2011
Accepted 31 May 2011
Available online 12 June 2011
Keywords:
MIF inhibitors
Benzisothiazolone
Cysteine modification
Macrophage migration inhibitory factor
MIF antagonists
Structure-based drug design
benzoxazolone core, was further pursued and yielded antagonists
The cytokine MIF (macrophage migration inhibitory factor) is
emerging as an important therapeutic target for inflammatory,
autoimmune, and hyperproliferative diseases.1 MIF possesses mul-
tiple biological functions and it participates in many cellular pro-
cesses. In addition to activating mitogen-activated protein kinases
(MAPK),2 MIF directly down-regulates p53 expression and func-
tion,3 and it counter-regulates the expression of glucocorticoids.4
Via binding to its cellular receptor CD74, MIF can promote cell sur-
vival through activation of the PI3K/Akt pathway. The roles of MIF
in cardiovascular diseases, inflammation, and innate immune re-
sponses are well documented.1,5,6 MIF is implicated in multiple dis-
orders including rheumatoid arthritis,7 glomerulonephritis,8
diabetes,9 atherosclerosis,10 sepsis,11 asthma,12 and acute respira-
tory distress syndrome.13 Moreover, it has been shown that inhibi-
tion of MIF or CD74 attenuates prostate cancer cell growth.14 In
addition to its activities as a cytokine, MIF is an enzyme that cata-
lyzes keto–enol tautomerization. It appears that the tautomerase
active site is located in proximity to the MIF-CD74 binding site.15
Thus, although evidence has not emerged that the enzymatic activ-
ity of MIF is significant in mammals, substantial effort has been di-
rected at the discovery of small-molecule inhibitors of both MIF’s
enzymatic and signaling activities.16 Our joint efforts began with
virtual screening that yielded 11 structurally diverse inhibitors of
MIF-CD74 binding with micromolar activities.17 One series, with a
with IC50 values as low as 7.5 nM in the tautomerase assay and
80 nM in the receptor binding assay.18 In parallel de novo design ef-
forts, triazole derivatives emerged as viable alternatives. This led to
the report of additional antagonists, and to the first agonists of MIF-
CD74 signaling.19 Another promising hit from the virtual screening
was 2-(4-chlorophenyl)-6-fluoro-benzo[d]isothiazol-3(2H)-one 1a
with an IC50 of 4.2 l
M in the tautomerase assay.17 Results of further
explorations of benzisothiazolones are reported here.
O
N
Cl
S
F
1a
Our computational studies indicated that 1a should bind to MIF
in the manner illustrated in Figure 1. Notably, results of Monte Car-
lo free-energy perturbation calculations20 found that (a) the orien-
tation with the carbonyl group of 1a pointing towards Ile64 is
preferred by ca. 6 kcal/mol over the 180°-rotated alternative with
the carbonyl group pointed towards Tyr95, and (b) substitutions
at C5 or C6 with F, Cl, OH, OCH3, and CN should be comparably via-
ble and preferred over CH3. This model received subsequent sup-
port from crystallographic studies of complexes of 2 and 3 with
MIF.21 Compounds 1a, 2, and 3 align well with the bicyclic ring sys-
tems inserted into the active site and with the fluorine of 1a and
bicyclic OH groups of 2 and 3 proximal to Asn97, and the C@O
⇑
Corresponding authors.
E-mail
addresses:
william.jorgensen@yale.edu
(W.L.
Jorgensen),
richard.bucala@yale.edu (R. Bucala).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.127

4546
W. L. Jorgensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4545–4549
Table 1
Inhibitory activities for benzisothiazolones in the MIF tautomerase assay
Rm
O
R5
R6
N
Rp
S
R7
Compd
R5
R6
R7
Rm
Rp
IC50 (lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
H
H
H
H
H
H
H
H
F
H
H
H
OMe
Cl
CH2OAc
CH2OMe
H
OMe
H
H
H
H
H
H
OMe
H
H
H
Cl
OMe
CN
H
H
H
H
Cl
H
Cl
Cl
Cl
Cl
OMe
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
4.2
6.2
6.4
25
5.0
2.3
2.4
1.9
8.0
2.8
6.6
5.6
5.6
6.5
4.9
2.8
6.2
8.5
11
OMe
H
H
H
F
H
H
F
H
H
H
Br
CN
H
H
OH
H
F
F
Cl
F
Figure 1. Computed structure of the complex of 1a bound in the MIF tautomerase
site after energy-minimization with MCPRO20 starting with protein coordinates
from the 1GCZ crystal structure.16a
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
groups of 1a and 2 hydrogen-bonded to the NH of Ile64. The syn-
thetic efforts were focused accordingly with particular interest in
preparing the 6-OH analog in view of precedents indicating poten-
tial value of hydrogen-bonding with Asn97.17 The structures of the
synthesized analogs, 1b–1y, are recorded in Table 1.
NO2
CF3
H
H
Br
CN
H
H
H
1s
1t
H
H
H
H
3.1
7.9
19
1.0
5.9
4.8
1u
1v
1w
1x
1y
OH
OH
OH
CH2OH
O
H
H
N
SO2NH2
S
HO
HO
O
sulfuryl chloride.27 Benzisothiazolones 1u–1v were prepared as de-
picted in method D starting from readily available 5-bromo-2-fluo-
robenzamide 13.28 Cu-catalyzed N-arylation of 13 with 1-choro-4-
iodobenzene (14) allowed preparation of arylamides 15 en route
to 1u and 1v via the protocol in method C. Finally, preparation of hy-
droxyl-substituted analogs 1w–1y required use of protecting
groups, as detailed in Scheme 2. Precursors 1f, 1h, and 17 were pre-
pared by the methods in Scheme 1 and deprotected in a final step.
The ability of the N-arylbenzisothiazolones 1 to inhibit the tau-
tomerase activity of MIF was evaluated with 4-hydroxyphenylpyru-
vate (4-HPP) as the substrate, as previously presented.17,29 Human
MIF, prepared recombinantly, was used throughout. All compounds
3
2
Synthetic routes used for the preparation of N-aryl-
benzisothiazolones 1b–1v are summarized in Scheme 1. Com-
pounds 1b–1l were prepared starting from the easily available
secondary arylamides22 4 via method A (Scheme 1). Ortho-lithiation
of 4 with sec-butyllithium in the presence of tetramethylethylenedi-
amine (TMEDA),23 followed by quenching of the dianion with di-
methyl disulfide afforded the corresponding arylmethylthioethers
5 in good yields. Sulfides 5 were heated with NaIO4 in methanol to
yield the sulfoxides 6, which were cyclized with thionyl chloride
to afford the desired N-arylbenzisothiazolones 1b–1l in moderate
to good yields.24 Benzisothiazolones 1m–1p were prepared via
method B in Scheme 1. Introduction of sulfide functionality into
arylamide 7 en route to 9 required protection of the bisactivated
ortho-site of arylamides 7 with a trimethylsilyl group.25 Thus, 7
was subjected to standard directed lithiation conditions followed
by trapping of the generated dianion by TMS chloride to provide
amides 8. Then 8 was subjected to the second ortho-lithiation, di-
methyl disulfide quench, and desilylation with tetrabutylamonium
fluoride (TBAF) to yield the sulfides 9. These products were subse-
quently oxidized and cyclized as in method A to afford the desired
1m–1p. Analogs 1q–1t, which possess functional groups that are
incompatible with ortho-lithiation methodology, were prepared
starting from the corresponding 2-fluorobenzoic acids 10 via meth-
od C. Benzoyl chlorides, formed by refluxing 10 in thionyl chloride,
were reacted with anilines to afford amides 11 in good to excellent
yields. For 1t, the cyano group was introduced via Pd-catalyzed cya-
nation of the aryl bromide.26 Amides 11 were subjected to SnAr reac-
tion with sodium thiomethoxide in DMSO to afford thioethers 12 in
good yields. Finally, 12 was cyclized to 1q–1t upon treatment with
show good activities ranging from 1 to 25 lM (Table 1). As expected
from the structural model, there was little effect on the activities
from varying the solvent-exposed substituents at the para (Rp) posi-
tion (1a, 1b, 1c). Some substitutions at the meta position provided
improvement, for example, 1f and 1g, while variation at the 6-posi-
tion did lead to the most potent compounds, 1h and 1w. Indeed, the
hydroxyl analog 1w is the most active at 1 lM. We have found few
compounds more active than this in the 4-HPP tautomerase assay
including the covalent inhibitor 4-IPP (4-iodo-6-phenylpyrimidine),
which yields an IC50 of 4.5 l
M.17–19
Many of the compounds were also tested in the MIF-CD74
binding assay. As previously detailed, this assay features biotin-
ylated MIF and immobilized CD74 ectodomain (CD7473–232) with
streptavidin conjugated alkaline phosphatase processing p-nitro-
phenylphosphate as the reporter.17,30 Most of the present ben-
zisothiazolones showed weak inhibitory activity that did not
reach the 50% level in the micromolar range. The two strongest
antagonists are 1c and 1w, which showed 49% inhibition of
binding at 78 nM and 23% inhibition at 4 nM, respectively. A
few of the compounds emerged as agonists19; the strongest is
1i, which enhances the MIF-CD74 binding by 2.7-fold at 4 lM.

W. L. Jorgensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4545–4549
4547
Method A
O
O
O
O
1. sBuLi, TMEDA, THF
-78oC, 5h
SOCl2
CH2Cl2
NaIO4, MeOH
550C
N
H
R1
R2
R2
N
N
H
N
H
R2
R1
R1
64 93%
R1
2. MeSSMe, rt, 12h
R2
S
Me
4
7
5
8
6
SMe
S
O
1b -1l
48 73%
-
64 80%
-
-
Method B
O
Me3Si
R1
O
O
1. sBuLi, TMEDA, THF
-78oC, 5h
1. sBuLi, TMEDA, THF
-78oC, 5h
O
R1
R1
R1
R2
N
H
N
H
SMe
R2
as above
N
H
R2
N
5
1
from to
R2
2. Me3SiCl, rt, 12h
2. MeSSMe, rt, 12h
S
9
from
1m 1p
-
23-47%
7
Method C
Cl
Cl
O
O
O
CO2H
F
SO2Cl2
CH2Cl2
1. SOCl2, reflux, 12h
N
NaSMe
R1
N
R1
R1
R1
N
Cl
H
H
DMSO
2. Cl-C6H4-NH2, THF, Et3N
11
S
F
SMe
12
40 72%
-
1q 1t
40 73%
-
-
89 100%
-
10
ZnCN2, Pd2dba3
R
1 = Br
R1 = CN
dppf, Zn, DMA
100oC, 12h
93%
Method D
Cl
Cl
CuI/K2CO3/DMF
MeHN
O
O
N
O
N
O
R1
Br
R1
NHMe
Br
SO2Cl2
CH2Cl2
NaSMe
DMSO
NH2
N
H
Cl
H
S
15
35%
F
I
Cl
14
F
SMe
16
86-98%
1u 1v
22-27%
-
13
ZnCN2, Pd2dba3
dppf, Zn, DMA
R1 = Br
R1 = CN
100oC, 12h
92%
Scheme 1. Synthesis of N-arylbenzisothiazolones 1b–1v.
O
O
BBr3
CH2Cl2
20%
N
Cl
N
Cl
S
S
MeO
HO
1h
1w
O
O
TBAF
N
N
THF
S
S
52%
OTBS
OAc
OH
F
F
17
1x
O
O
N
K2CO3
MeOH
78%
N
S
S
F
F
Figure 2. Relative potency of MIF antagonists in reducing MIF-dependent ERK1/2
phosphorylation in human synovial fibroblasts. The bottom numerals show the
ratio of phosphorylated to total ERK protein relative to MIF alone from densito-
metric scanning. Values are expressed in relative units from two experiments.
⁄P <0.05, ⁄⁄P <0.01 by t-test.
OH
1f
1y
Scheme 2. Final deprotection step in the syntheses of 1w–1y.
In view of these results, inhibition of MIF signal transduction
by 1c and 1w was further investigated in human target cells,
specifically, primary synovial fibroblasts. The expectation is that
MIF-CD74 inhibition should reduce ERK (extracellular-signal-reg-
ulated kinase) phosphorylation, which is relevant to the control
of hyperproliferative and autoimmune diseases.31 Thus, the test
compounds were added together with MIF for 30 min to
1 Â 106 synovial fibroblasts cultured in DMEM/0.1% fetal bovine
serum. The cells then were lysed and the intracellular contents
of phospho-ERK1/2 and total ERK1/2 were quantified by specific
antibodies and western blotting. The results in Figure 2 show
that addition of MIF enhances ERK phosphorylation, while co-
administration with 1c and 1w results in substantial reduction
of phosphorylation. The most profound effects are obtained with
the 6-hydoxy analog 1w, which reduces the phosphorylation by
35% at 1 lM and still by 20% at 100 nM. For comparison, results
are included for the well-studied MIF tautomerase inhibitor ISO-
132; the racemate yields a 19% reduction of ERK phosphorylation
at 2 lM.

4548
W. L. Jorgensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4545–4549
activity and receptor binding functions. However, the extent of
O
covalent modification appears small, and it is unlikely that it can ac-
count for the absolute activities in Table 1 or of the relative values.
For example, electron-withdrawing groups should make the ben-
zisothiazolones more reactive electrophiles; however, this is not
apparent in comparisons of the IC50 values such as for 1f and 1g ver-
sus 1a and 1c, and in comparing the isomeric 1t and 1v, where 1v
would be expected to be more reactive towards nucleophilic attack
at the conjugating para-sulfur site.
In summary, N-phenylbenzisothiazolones have been examined
for their ability to inhibit the tautomerase and signaling activities
of the cytokine MIF. Numerous analogs have been reported with
activities in the low micromolar range for inhibition of the tautom-
erase activity. Though the expectation is that they are reversible
inhibitors that bind near the tautomerase active site, mass spectro-
scopic investigation of the original virtual screening hit 1a demon-
strated that it yields a small amount of covalent modification of
Cys56 or Cys59 of MIF. The most potent MIF tautomerase inhibitor
O
HO
N
O
O
N
Se
ISO-1
ebselen
A final issue that was addressed was the possible covalent
modification of MIF by the benzisothiazolones. Many MIF inhibi-
tors have been reported to function in this manner by bonding to
the nucleophilic Pro1 or Cys residues.15b,33 Indeed, Ouertatani-
Sakouhi et al. recently reported 12 covalent modifiers of Pro1
from high-throughput screening and mass spectrometric analy-
sis33; these compounds feature reactive carbonyl functionality
that is normally avoided when seeking reversible inhibitors.34
However, they also demonstrated that the anti-inflammatory
agent ebselen inhibits MIF by covalent attachment specifically
to Cys80 among the three Cys options (56, 59, and 80).33 The
modification causes disruption of MIF trimer assembly and,
therefore, negates formation of the tautomerase active sites.33 Be-
sides the structural similarity of 1a and ebselen, it has also been
shown that some N-arylbenzisothiazolones can react with cyste-
ines yielding opening of the benzisothiazolone ring and disulfide
formation.35 The latter process should be facilitated by electron-
withdrawing substituents in the para position of the N-phenyl
ring as in the reported case with a p-sulfamoyl group.
reported here is the 6-hydroxy analog 1w at 1 lM. This compound
was also shown to cause striking retardation of ERK phosphoryla-
tion in synovial fibroblasts. Investigations continue in several veins
including evaluation of the anti-inflammatory and anti-prolifera-
tive activities of the compounds.
Acknowledgments
Gratitude is expressed to T. Lam and E. Voss of the Keck Biotech-
nology Facility for the mass spectrometric analyses, and to the
National Institutes of Health (AI042310, AR049610, AR050498,
GM032136) and the Treat B. Johnson Fund at Yale for support.
Thus, mass spectrometric studies were pursued to assess possi-
ble covalent attachment of 1a to MIF in a similar manner to the
prior investigation of 4-IPP and a benzoxazolone MIF inhibitor.18
Equimolar quantities of human MIF and 1a were mixed (30 lM of
each reactant in 0.01 mL) for 1 h at 25 °C and the reaction mixture
was analyzed (Bruker 9.4T FT-ICR). A small peak appeared at the
mass position expected for the covalent attachment of one mole-
cule of 1a with a MIF monomer (12,616 Da), as shown in Figure 3.
Analysis of the four highest peaks in the MIF alone and MIF + 1a iso-
topic distributions indicates that ca. 3% of the MIF is modified by 1a.
References and notes
1. (a) Lolis, E.; Bucala, R. Expert Opin. Ther. Targets 2003, 7, 153; (b) Greven, D.;
Leng, L.; Bucala, R. Expert Opin. Ther. Targets 2010, 14, 253.
2. (a) Santos, L. L.; Lacey, D.; Yang, Y.; Leech, M.; Morand, E. F. J. Rheumatol. 2004,
31, 1038; (b) Mitchell, R. A.; Metz, C. N.; Peng, T.; Bucala, R. J. Biol. Chem. 1999,
274, 18100.
3. Mitchell, R. A.; Liao, H.; Chesney, J.; Fingerle-Rowson, G.; Baugh, J.; David, J.;
Bucala, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 345.
4. (a) Calandra, T.; Bernhagen, J.; Metz, C. N.; Spiegel, L. A.; Bacher, M.; Donnelly,
T.; Cerami, A.; Bucala, R. Nature 1995, 377, 68; (b) Leech, M.; Metz, C.; Bucala,
R.; Morand, E. F. Arthritis Rheum. 2000, 43, 827.
5. (a) Zernecke, A.; Bernhagen, J.; Weber, C. Circulation 2008, 117, 1594; (b)
Herder, C.; Illig, T.; Baumert, J.; Mueller, M.; Klopp, N.; Khuseyinova, N.;
Meisinger, C.; Martin, S.; Thorand, B.; Koenig, W. Atherosclerosis 2008, 200, 380.
6. Calandra, T.; Roger, T. Nat. Rev. Immunol. 2003, 3, 791.
7. Kim, H.-R.; Park, M.-K.; Cho, M.-L.; Yoon, C.-H.; Lee, S.-H.; Park, S.-H.; Leng, L.;
Bucala, R.; Kang, I.; Choe, J.; Kim, H.-Y. J. Rheumatol. 2007, 34, 927.
8. Lan, H. Y. Nephron 2008, 109, 79.
9. Sanchez-Zamora, Y.; Terrazas, L. I.; Vilches-Flores, A.; Leal, E.; Juarez, I.;
Whitacre, C.; Kithcart, A.; Pruitt, J.; Sielecki, T.; Satoskar, A. R.; Rodriguez-Sosa,
M. FASEB J. 2010, 24, 2583.
10. Noels, H.; Bernhagen, J.; Weber, C. Trends Cardiovascul. Med. 2009, 19, 76.
11. Bozza, M.; Satoskar, A. R.; Lin, G.; Lu, B.; Humbles, A. A.; Gerard, C.; David, J. R. J.
Exp. Med. 1999, 189, 341.
With 4-IPP, which has a similar IC50 (4.5 lM) in the tautomerase as-
say, ca. 20% of the protein was modified.18 Further FT-ICR MS anal-
ysis of a trypsin-digested sample revealed a peptide of M = 6083 Da
that corresponds to the covalent adduct of the MIF12–66 fragment
and 1a (Fig. 3). These data are consistent with covalent attachment
of MIF and 1a at Cys56 or Cys59. The fact that Cys59 is well-buried
in the structure of the MIF trimer shifts the likelihood of the mod-
ified residue towards being Cys56. While the catalytic Pro1 is not
modified by 1a, cysteine modification could influence MIF confor-
mational integrity and account for alterations in tautomerization
MIF12-66 + 1a
M=6083.091 Da
10+
'1234.73292
12. Yamaguchi, E.; Nishihira, J.; Shimizu, T.; Takahashi, T.; Kitashiro, N.; Hizawa,
N.; Kamishima, K.; Kawakami, Y. Clin. Exp. Allergy 2000, 30, 1244.
13. Lai, K. N.; Leung, J. C. K.; Metz, C. N.; Lai, F. M.; Bucala, R.; Lan, H. Y. J. Pathol.
2003, 199, 496.
14. Meyer-Siegler, K. L.; Iczkowski, K. A.; Leng, L.; Bucala, R.; Vera, P. L. J. Immunol.
2006, 177, 8730.
5+
'1217.62544
MIF, M=12,337 Da
15. (a) Senter, P. D.; Al-Abed, Y.; Metz, C. N.; Benigni, F.; Mitchell, R. A.; Chesney, J.;
Han, J.; Gartner, C. G.; Nelson, S. D.; Todar, G. J.; Bucala, R. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 144; (b) Winner, M.; Meier, J.; Zierow, S.; Rendon, B. E.;
Crichlow, G. V.; Riggs, R.; Bucala, R.; Leng, L.; Smith, N.; Lolis, E.; Trent, J. O.;
Mitchell, R. A. Cancer Res. 2008, 68, 7253; (c) McLean, L. R.; Zhang, Y.; Li, H.;
Choi, Y.-M.; Han, Z.; Li, Y. Bioorg. Med. Chem. Lett. 2009, 20, 1821.
16. For reviews, see: (a) Orita, M.; Yamamoto, S.; Katayama, N.; Fujita, S. Curr.
Pharm. Des. 2002, 8, 1297; (b) Garai, J.; Lorand, T. Curr. Med. Chem. 2009, 16,
1091.
17. Cournia, Z.; Leng, L.; Gandavadi, S.; Du, X.; Bucala, R.; Jorgensen, W. L. J. Med.
Chem. 2009, 52, 416.
18. Hare, A. A.; Leng, L.; Gandavadi, S.; Du, X.; Cournia, Z.; Bucala, R.; Jorgensen, W.
L. Bioorg. Med. Chem. Lett. 2010, 20, 5811.
m/z
1217.5 1218.5 1219.5
MIF + 1a,
M=12,616 Da
10+
'1262.52988
m/z
1235 1240 1245 1250 1255 1260 1265 1270
Figure 3. Mass spectrometric results demonstrating minor covalent modification of
MIF by 1a. The inset shows the product after trypsin digestion of the modified
protein.

W. L. Jorgensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4545–4549
4549
19. Jorgensen, W. L.; Gandavadi, S.; Du, X.; Hare, A. A.; Trofimov, A.; Leng, L.;
Bucala, R. Bioorg. Med. Chem. Lett. 2010, 20, 7033.
29. Bernhagen, J.; Mitchell, R. A.; Calandra, T.; Voelter, W.; Cerami, A.; Bucala, R.
Biochemistry 1994, 33, 14144.
20. Jorgensen, W. L.; Tirado-Rives, J. J. Comput. Chem. 2005, 26, 1689.
21. McClean, L. R.; Zhang, Y.; Li, H.; Choi, Y.-M.; Han, Z.; Vaz, R. J.; Li, Y. Bioorg. Med.
Chem. Lett. 2010, 20, 1821.
30. Shi, X.; Leng, L.; Wang, T.; Wang, W.; Du, X.; Li, J.; McDonald, C.; Chen, Z.;
Murphy, J. W.; Lolis, E.; Noble, P.; Knudson, W.; Bucala, R. Immunity 2006, 25,
595.
22. Shaw, A. Y.; Chen, Y.-R.; Tsai, C.-H. Synth. Comm. 2009, 39, 2647.
23. Metallinos, C.; Nerdinger, S.; Snieckus, V. Org. Lett. 1999, 1, 1183.
24. Uchida, Y.; Kozuka, S. Chem. Comm. 1981, 11, 510.
25. Mills, R. J.; Taylor, N. J.; Snieckus, V. J. Org. Chem. 1989, 54, 4372.
26. Didier, D.; Sergeyev, S. Tetrahedron 2007, 63, 3864.
27. Zlotin, S. G.; Kislitsin, P. G.; Podgursky, A. I.; Samet, A. V.; Semenov, V. V.;
Buchanan, A. C., III; Gakh, A. A. J. Org. Chem. 2000, 65, 8439.
28. Perez-Medrano, A.; Donnelly-Roberts, D. L.; Honore, P.; Hsieh, G. C.; Namovic,
M. T.; Peddi, S.; Shuai, Q.; Wang, Y.; Faltynek, C. R.; Jarvis, M. F.; Carroll, W. A. J.
Med. Chem. 2009, 52, 3366.
31. (a) Bucala, R.; Lolis, E. Drug News Perspect. 2005, 18, 417; (b) Ohori, M. Drug
News Perspect. 2008, 21, 245.
32. Lubetsky, J. B.; Dios, A.; Han, J.; Aljabari, B.; Ruzsicska, B.; Mitchell, R.; Lolis, E.;
Al Abed, Y. J. Biol. Chem. 2002, 277, 24976.
33. Ouertatani-Sakouhi, H.; El-Turk, F.; Fauvet, B.; Cho, M.-K.; Karpinar, D. P.; Le
Roy, D.; Dewor, M.; Roger, T.; Bernhagen, J.; Calandra, T.; Zweckstetter, M.;
Lashuel, H. A. J. Biol. Chem. 2010, 285, 26581.
34. Rishton, G. M. Drug Discovery Today 2003, 8, 86.
35. Sanchez, J. P. J. Heterocycl. Chem. 1997, 34, 1463.