Critical modifications of the ISO-1 scaffold improve its potent inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity

Article abstract of DOI:10.1016/j.bmcl.2006.04.038

Based on the scaffold of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of the proinflammatory cytokine MIF, two critical modifications and chiral resolution have significantly improved the potency of the

Full text of DOI:10.1016/j.bmcl.2006.04.038

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3376–3379
Critical modifications of the ISO-1 scaffold improve
its potent inhibition of macrophage migration inhibitory
factor (MIF) tautomerase activity
Kai Fan Chenga and Yousef Al-Abeda,b,
*
aDepartment of Medicinal Chemistry, The Feinstein Institute for Medical Research, North Shore-LIJ Health System,
350 Community Drive, Manhasset, NY 11030, USA
bNew York School of Medicine, New York, NY, USA
Received 16 February 2006; revised 5 April 2006; accepted 6 April 2006
Available online 6 May 2006
Abstract—Based on the scaffold of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor
of the proinflammatory cytokine MIF, two critical modifications and chiral resolution have significantly improved the potency of
the inhibition. Compound (R)-17 is 20-fold more potent than ISO-1 and inhibits MIF tautomerase activity with an IC50 of 550 nM.
Ó 2006 Elsevier Ltd. All rights reserved.
Macrophage migration inhibitory factor (MIF) is a pro-
inflammatory cytokine, critically involved in the patho-
genesis of inflammatory disorders.1,2 Our recent studies
have clearly defined MIF as a critical factor in the path-
ophysiology of sepsis.3 We showed that abolition of
MIF activity during sepsis by antibodies or ISO-1 im-
proves cardio-circulatory efficiency and prevents the
lethality associated with sepsis.3,4 We designed our spe-
cific inhibitor ISO-1 to fit into the hydrophobic active
site of MIF, an interaction confirmed by the crystal
structure of the MIF complex with ISO-1 (Scheme 1).5
Administration of ISO-1 in a clinically relevant model
of sepsis confers moderate protection (80% vs 40% con-
trol). These results identify ISO-1 as the first small mol-
ecule inhibitor of MIF proinflammatory activities with
therapeutic implications and indicate the potential of
the MIF active site as a novel target for therapeutic
interventions in human sepsis. To improve the potency
of ISO-1, herein, we explored the SAR of ISO-1.
hol.5 Herein, we discovered that mono-fluorination onto
the ortho position of the phenolic group of ISO-1 im-
proved the inhibition of MIF activity up to 41%
(Scheme 5). Hence, we investigated the alkyl tail of
ISO-1 with various ester and amide analogues. The
new synthetic route provides the precursor (ISO-1-acid)
in large scale (Scheme 2).9 Esterification and amide for-
mation between the ISO-1-acid and alcohol (or amine)
were accomplished using a standard DCC coupling pro-
tocol (DCC, DMAP or HOBt) (Scheme 3).9
As summarized in Schemes 3 and 4, we found that the
ester analogues are more potent inhibitors than the
amide counterpart (e.g., compounds 14 (IC50 = 2.5 lM)
vs
6
(IC50 = 24 lM) and
3
(IC50 = 8.5 lM) vs
Previously, we have identified critical functional groups
within the ISO-1 scaffold as evidenced by the loss of its
MIF inhibitory effect upon methylation of the para-
hydroxyl functional group, oxidation of 4,5-dihydrois-
oxazole to isoxazole or reduction of methyl ester to alco-
Keywords: MIF; ISO-1; Tautomerase activity; Enzyme.
*
Scheme 1. MIF tautomerizes dopachrome methyl esters and the
structure of MIF inhibitor dubbed ‘ISO-1.’
Corresponding author. Tel.: +1 516 562 3406; fax: +1 516 562
1022; e-mail: yalabed@nshs.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.038

K. F. Cheng, Y. Al-Abed / Bioorg. Med. Chem. Lett. 16 (2006) 3376–3379
3377
Scheme 2. Synthesis of the ISO-1-acid.
Scheme 4. Synthesis of compounds 10–16.
Schiff bases in the inhibitory effect of MIF.5 Hence, we
needed to resolve the optically active ISO-1 and deter-
mine the inhibitory activity for each isomer. Chiral res-
olution was accomplished as previously described
through fractional crystallization of the diastereomeric
cinchonidine salts of methylated ISO-1-acid (Scheme
5).7,9 The (S)-configuration salts were found to be less
soluble and were crystallized out from the chiral mix-
ture. The (S)- and (R)-configuration salts were then
acidified by 1 N HCl, demethylated with BBr3, and
esterified with TMSCl in methanol to yield (S)-ISO-1
(90% ee) and (R)-ISO-1 (90% ee). Both isomers were
tested for activity in the MIF dopachrome tautomerase
Scheme 3. Synthesis of compounds 2–9.
7(IC50 = 26 lM)). We further investigated the influence
of the bulkiness of the ester group on the potency of
inhibiting MIF activity. The esterification process of
ISO-1-acid was accomplished with TMSCl using the
following alcohols: ethanol, 10; 1-propanol, 11; 2-propa-
nol, 12; 1-butanol, 13; cyclohexanol, 14; cyclohexyl-
methanol, 15; and neopentylalcohol, 16 (Scheme 4).9
As shown in Scheme 4, the most bulky alcohol (com-
pound 16, neopentyl ester analogue) shows a superior
inhibition activity (IC50 = 1.5 lM) which is ten times
more potent than ISO-1.
The crystal structure analysis of MIF/ISO-1 complex
predicted that the (R)-isomer of ISO-1 binds in the
active site, suggesting that the (R)-isomer would bind
with higher affinity to the MIF active site. This is evident
from the hydrogen bond formation between the side-
chain nitrogen of Lys-32 and both the carbonyl oxygen
of the carboxymethyl group and the oxygen of the
isoxazoline ring.6 Previously, we have shown the impor-
tance of the absolute configuration of the amino acid
Scheme 5. Synthesis of stereoisomers of ISO-1 and compound 17.

3378
K. F. Cheng, Y. Al-Abed / Bioorg. Med. Chem. Lett. 16 (2006) 3376–3379
TLC plates were used. Spots on TLC plates were visualized
under a short wavelength UV lamp or stained with I2
vapor. NMR spectra were performed on a Jeol Eclipse 270
spectrometer at 270 MHz for 1H NMR spectra and
67.5 MHz for the 13C NMR spectra. Coupling constants
are reported in Hertz (Hz), and chemical shifts are reported
in parts per million (ppm) relative to deuterated solvent
peak. The coupling constants (J) are measured in Hertz
(Hz) and assigned as s (singlet), d (doublet), t (triplet), m
(multiplet), and br (broad). Low-resolution mass spectra
were acquired using Thermofinnigan LCQ DecaXPplus
quadrupole ion trap MS with negative-ion or positive-ion
mode. Preparation of 4-methoxybenzaldehyde oxime. To a
solution of 4-methoxybenzaldehyde (5.0 g, 36.8 mmol) in
methanol (300 mL) were added hydroxylamine hydrochlo-
ride (7.6 g, 110.4 mmol) and 2 N NaOH (37 mL,
73.6 mmol). The mixture was stirred at room temperature
for 6 h. The mixture was neutralized to pH 4 by using 1 N
HCl. Excess methanol was removed in vacuo to precipitate
out the oxime. The precipitations were filtered and washed
with water. The product was dried under vacuum and
yielded a white solid (4.9 g, 88%): 1H NMR (270 MHz,
acetone-d6) d 8.07 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 6.95 (d,
J = 8.2 Hz, 2H), 3.82 (s, 3H). Preparation of ISO-1-acid. To
a solution of 4-methoxybenzaldoxime (4 g, 26 mmol) in
anhydrous DMF (500 mL) was added NCS (5.2 g,
39 mmol). The reaction mixture was stirred for 5 h at rt
affording the chloro oxime. To this solution, vinylacetic
acid (6.6 mL, 78 mmol) was added, followed by the
dropwise addition of triethylamine (5.5 mL, 39 mmol) in
DMF (50 mL). The reaction mixture was stirred under N2
at rt for 48 h. The solvent was removed in vacuo and the
residue was taken up in EtOAc. The EtOAc solution was
washed with 0.5 N HCl, water, and brine, and dried with
anhydrous MgSO4. The final solution was concentrated in
vacuo and dried under vacuum pump to afford 1 in
quantitative yield. A solution of 1 (40–50 mM in dry
dichloromethane) was treated with an excess (8–10 equiv)
of boron tribromide (1 M solution in dichloromethane,
Aldrich cat No.: 211222) at 0 °C under N2. The reaction
mixture was allowed to reach room temperature over 5–6 h
and then quenched with aqueous saturated NaHCO3
(caution: BBr3 reacts violently with water). The mixture
was stirred for 1/2 h and then diluted with water and
CH2Cl2. The organic layer was separated from aqueous and
discarded. The aqueous portion was neutralized with 1N
HCl to pH 4 and extracted with EtOAc. The combined
EtOAc solution was washed with brine and dried with
anhydrous MgSO4 to afford ISO-1-acid as a pale yellow
assay. (R)-ISO-1 inhibited MIF tautomerase activity
with an IC50 of 7 lM, but (S)-ISO-1 was 50% less active
with an IC50 of 13 lM (Scheme 5).
In order to obtain the most potent, specific, small mol-
ecule, MIF inhibitor, three critical steps need to be inte-
grated into the ISO-1 scaffold: (1) mono-fluorination
onto the ortho position of the phenolic group of ISO-
1; (2) a neopentyl ester functional group replacing the
methyl ester of ISO-1; (3) chiral resolution to obtain
an (R)-isomer. Compound 17 emerged after all as a po-
tent inhibitor of MIF activity with an IC50 of 750 nM.
After the classical chiral resolution, (R)-17 inhibited
MIF tautomerase activity with an IC50 of 550 nM, while
the (S)-17 was 50% less active with an IC50 of 1.1 lM
(Scheme 5). Compound (R)-17 is 20 times more potent
than the parent compound ISO-1.
After two critical modifications on ISO-1 scaffold, we
have improved the inhibition of MIF tautomerase activ-
ity to a nanomolar concentration (550 nM), which is 20
times potent than ISO-1. The in vivo and in vitro studies
of the new inhibitor are currently under investigation.
References and notes
1. Calandra, T.; Roger, T. Nat. Rev. Immunol. 2003, 3, 791.
2. Riedemann, N. C.; Guo, R. F.; Ward, P. A. Nat. Med.
2003, 9, 517.
3. Al-Abed, Y.; Dabideen, D.; Aljabari, B.; Valster, A.;
Messmer, D.; Ochani, M.; Tanovic, M.; Ochani, K.;
Bacher, M.; Nicoletti, F.; Metz, C.; Pavlov, V. A.; Miller,
E. J.; Tracey, K. J. J. Biol. Chem. 2005, 280, 36541.
4. Lin, X.; Sakuragi, S.; Metz, C.; Ojamaa, K.; Skopicki, H.;
Wang, P.; Al-Abed, Y.; Miller, E. J. Shock 2005, 24, 556.
5. Lubetsky, J. B.; Dios, A.; Han, J.; Aljabari, B.; Ruzsicska,
B.; Mitchell, R.; Lolis, E.; Al-Abed, Y. J. Biol. Chem. 2002,
277, 24976.
6. Dios, A.; Mitchell, R. A.; Aljabari, B.; Lubetsky, J.;
O’Connor, K.; Liao, H.; Senter, P. D.; Manogue, K. R.;
Lolis, E.; Metz, C.; Bucala, R.; Callaway, D. J. E.;
Al-Abed, Y. J. Med. Chem. 2002, 45, 2410.
7. Wityak, J.; Sielecki, T. M.; Pinto, D. J.; Emmett, G.; Sze, J.
Y.; Liu, J.; Tobin, A. E.; Wang, S.; Jiang, B.; Ma, P.;
Mousa, S. A.; Wexler, R. U.; Olson, R. E. J. Med. Chem.
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1
powder in good yield (75%). H NMR (300 MHz, acetone-
d6) d 10.65 (br, 1H), 8.75 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H),
6.85 (d, J = 8.7 Hz, 2H), 5.01 (m, 1H), 3.50 (m, 1H), 3.05
(m, 1H), 2.68 (m, 2H); ESI-MS m/z 220 (MÀ). General DCC
coupling procedure for formation of esters or amides. A
solution of ISO-1-acid (100 mM in dry dichloromethane)
was treated with 1.1 equiv DCC, 0.2 equiv DMAP, and
1.5 equiv alcohols (or 0.2 equiv HOBt and 1.5 equiv
amines). The mixture was stirred for 8 h at rt. The formed
white precipitate was filtered off and washed with CH2Cl2
and the filtrate was evaporated to dryness. The residue was
purified on silica gel (hexane/EtOAc/MeOH 4:3:1) to give
the esters or amides as a white solid. Compound 2 (65%
yield): 1H NMR (300 MHz, acetone-d6) d 8.75 (br, 1H),
7.54 (d, J = 8.7 Hz, 2H), 7.38 (m, 2H), 7.22 (m, 1H), 7.11
(m, 2H), 6.86 (d, J = 8.7 Hz, 2H), 5.10 (m, 1H), 3.54 (m,
1H), 3.27 (m, 1H), 2.96 (m, 2H); ESI-MS m/z 296 (MÀ).
Compound 3 (60% yield): 1H NMR (300 MHz, acetone-d6)
d 8.78 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.7 Hz,
2H), 6.90 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 5.10
8. MIF tautomerase activity was measured by UV–vis record-
ing spectrophotometry (SHIMADZU, UV1600U). A fresh
stock solution of L-dopachrome methyl ester was prepared
at 2.4 mM through oxidation of L-3,4-dihydroxyphenylal-
anine methyl ester with sodiumperiodate. 1 lL of MIF
solution (800–900 ng/mL) and 1 lL of a DMSO solution
with various concentrations of the enzymatic inhibitor were
added into a plastic cuvette (10 mm, 1.5 mL) containing
0.7 mL assay buffer (50 mM potassium phosphate, pH 7.2).
Then L-dopachrome methyl ester solution (0.3 mL) was
added to the assay buffer mixture. Activity was determined
at room temperature and the spectrometric measurements
were made at k = 475 nm for 20 seconds by monitoring the
rate of decolorization of L-dopachrome methyl ester in
comparison to a standard solution.
9. All solvents were HPLC-grade from Fisher Scientific. Silica
gel (Selecto Scientific, 32–63 lm average particle size) was
used for flash column chromatography (FCC). Aluminum-
backed Silica Gel 60 with a 254 nm fluorescent indicator

K. F. Cheng, Y. Al-Abed / Bioorg. Med. Chem. Lett. 16 (2006) 3376–3379
3379
(m, 1H), 3.76 (s, 3H), 3.53 (m, 1H), 3.25 (m, 1H), 2.76 (m,
(m, 1H), 4.10 (q, 2H), 3.51 (m, 1H), 3.12 (m, 1H), 2.66 (m,
2H) 1.19 (t, 3H); ESI-MS m/z 248 (MÀ). Compound 11: 1H
NMR (300 MHz, acetone-d6) d 8.75 (s, 1H), 7.51 (d,
J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.98 (m, 1H), 4.01
(t, 2H), 3.51 (m, 1H), 3.15 (m, 1H), 2.66 (m, 2H) 1.60 (m,
2H), 0.89 (t, 3H); ESI-MS m/z 262 (MÀ). Compound 12: 1H
NMR (300 MHz, acetone-d6) d 8.74 (s, 1H), 7.51 (d,
J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.97 (m, 2H), 4.72
(m, 1H), 3.51 (m, 1H), 3.12 (m, 1H), 2.63 (m, 2H) 1.18 (d,
1
2H); ESI-MS m/z 326 (MÀ). Compound 4 (40% yield): H
NMR (300 MHz, acetone-d6) d 8.75 (br, 1H), 7.55 (d,
J = 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.03 (d,
J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.11 (m, 1H),
3.55 (m, 1H), 3.23 (m, 1H), 2.95 (m, 2H), 1.30 (s, 9H); ESI-
MS m/z 352 (MÀ). Compound 5 (30% yield): 1H NMR
(300 MHz, acetone-d6) d 8.75 (s, 1H), 7.72 (d, J = 8.7 Hz,
2H), 7.17 (d, J = 8.7 Hz, 2H), 6.86 (s, 2H), 5.10 (m, 1H),
3.55 (m, 1H), 3.25 (m, 1H), 2.96 (m, 2H), 2.05 (s, 9H); ESI-
MS m/z 338 (MÀ). Compound 6 (80% yield): 1H NMR
(300 MHz, acetone-d6) d 8.79 (s, 1H), 7.52 (d, J = 8.7 Hz,
2H), 7.00 (br, 1H), 6.85 (d, J = 8.7 Hz, 2H), 4.94 (m, 1H),
3.64 (m, 1H), 3.42 (m, 1H), 3.10 (m, 1H), 2.52 (m, 1H), 2.37
(m, 1H) 1.90–1.00 (m, 10H); ESI-MS m/z 301 (MÀ).
Compound 7 (88% yield): 1H NMR (300 MHz, acetone-
d6) d 9.10 (br,1H), 8.80 (br, 1H), 7.53 (m, 4H), 6.84 (m, 4H),
5.06 (m, 1H), 3.72 (s, 3H), 3.49 (m, 1H), 3.17 (m, 1H), 2.73
(m, 1H), 2.60 (m, 1H); ESI-MS m/z 325 (MÀ). Compound 8
(95% yield): 1H NMR (270 MHz, acetone-d6) d 7.52 (d,
J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 6.07 (br, 1H), 5.02
(m, 1H), 3.46 (m, 1H), 3.13 (m, 5H), 2.56 (m, 2H), 1.51 (m,
4H), 1.38 (s, 9H); ESI-MS m/z 414 (M+Na+). Compound 9
(90% yield): 1H NMR (270 MHz, acetone-d6) d 8.63 (br,
1H), 7.52 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.04
(m, 1H), 3.84 (m, 2H), 3.28 (m, 3H), 2.58 (m, 3H), 1.86 (m,
2H), 1.65 (m, 2H); ESI-MS m/z 292 (M+). General TMSCl
esterification procedure. To a solution of ISO-1-acid
(50 mg. 0.23 mmol) in a 3 mL alcohol (ethanol, 10; 1-
propanol, 11; 2-propanol, 12; 1-butanol, 13; cyclohexanol,
14; cyclohexylmethanol, 15; and neopentylalcohol, 16) was
added 0.1 mL TMSCl. The mixture was stirred for 2 hrs at
rt. (for 14, 15 and 16: 3 h at 50 °C). The mixture was
evaporated to dryness and the residue was subjected to
purification on silica gel (hexane/EtOAc 4:3) to afford a
white solid or pale yellow oil in quantitative yield.
1
j = 6.3 Hz, 6H); ESI-MS m/z 262 (MÀ). Compound 13: H
NMR (300 MHz, acetone-d6) d 8.78 (s, 1H), 7.52 (d,
J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.99 (m, 1H), 4.05
(t, 2H), 3.51 (m, 1H), 3.12 (m, 1H), 2.68 (m, 2H) 1.10–1.60
(m, 4H), 0.88 (t, 3H); ESI-MS m/z 276 (MÀ). Compound
1
14: H NMR (300 MHz, acetone-d6) d 8.84 (br, 1H), 7.52
(d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.98 (m, 1H),
4.72 (m, 1H),3.51 (m, 1H), 3.15 (m, 1H), 2.66 (m, 2H) 1.90–
1
1.20 (m, 10H); ESI-MS m/z 302 (MÀ). Compound 15: H
NMR (300 MHz, acetone-d6) d 8.78 (s, 1H), 7.55 (d,
J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 5.02 (m, 1H), 3.90
(d, J = 6.7 Hz, 2H), 3.51 (m, 1H), 3.15 (m, 1H), 2.72 (m,
2H) 1.80–0.90 (m, 11H); ESI-MS m/z 302 (MÀ). Compound
16: 1H NMR (300 MHz, acetone-d6) d 8.79 (s, 1H), 7.54 (d,
J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 5.05 (m, 1H), 3.82
(m, 2H), 3.52 (m, 1H), 3.18 (m, 1H), 2.75 (m, 2H) 0.96 (s,
9H); ESI-MS m/z 292 (M+). Classical resolution of acid 1
via crystallization of the cinchonidine salts. (R,S)-1 (1.3 g,
5.5 mmol) was dissolved in hot acetone (25 mL), cincho-
nidine (1.61 g, 5.5 mmol) was added, and the solution
was cooled to rt and allowed to stand at À20 °C
overnight. The resulting white solid was filtered to give
(S)-1 salts. The filtrate was concentrated in vacuo to
afford (R)-1 salts. To a solution of (R)-1 salts or (S)-1
salts (200 mg, 0.4 mmol) in chloroform (3 mL) was added
1 N HCl in ether (3 mL, 3 mmol). The resulting white
precipitate was filtered off and the filtrate was concen-
trated in vacuo to give (R)-1 or (S)-1 in quantitative
yield.
1
Compound 10: H NMR (300 MHz, acetone-d6) d 8.74 (s,
1H), 7.52 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.97