Synthesis and biological activity of novel MIF antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.12.127

Two series of novel furan and indole compounds were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds from both series inhibited the enzymatic activity of MIF at levels equal to or significan

Full text of DOI:10.1016/j.bmcl.2010.12.127

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1508–1511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of novel MIF antagonists
Sarala Balachandran a, , Pradip K. Gadekar a, Santosh Parkale a, Vitthal N. Yadav a, Divya Kamath b,
⇑
Sneha Ramaswamy b, Somesh Sharma a,b, Ram A. Vishwakarma a, Nilesh M. Dagia b
a Department of Medicinal Chemistry, Piramal Life Sciences, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400 063, India
b Department of Pharmacology, Piramal Life Sciences, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400 063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel furan and indole compounds were synthesized and probed for inhibition of macro-
phage migration inhibitory factor (MIF) activity. Several compounds from both series inhibited the enzy-
matic activity of MIF at levels equal to or significantly better than ISO-1 (an early MIF inhibitor). The
majority of the compounds that robustly inhibited the spontaneous secretion/release/recognition of
MIF from freshly isolated human peripheral blood mononuclear cells were from the furan series (com-
pounds 5, 9, 13, 15, and 16). In contrast, compounds that markedly inhibited the MIF-induced production
of pro-inflammatory cytokines were predominantly from the indole series (compounds 26, 29, and 32).
Ó 2011 Elsevier Ltd. All rights reserved.
Received 21 October 2010
Revised 20 December 2010
Accepted 28 December 2010
Available online 1 January 2011
Keywords:
Inflammation
Cytokines
MIF
Furan
Indole
Macrophage migration inhibitory factor (MIF), a pro-inflamma-
tory cytokine, plays a critical role in the pathogenesis of a variety of
inflammatory and autoimmune diseases.1,2 Therefore, a promising
therapeutic approach to diminish pathological inflammation is to
inhibit the production and/or biological activity of MIF. Given that
MIF possesses enzymatic properties,1,2 an earliest pharmacological
strategy adopted to combat inflammation was to target the cata-
lytic pocket of MIF.1,2 Indeed, this approach has been extensively
utilized by academia as well as industry to discover small molecule
inhibitors of MIF enzymatic activity.2–6 Interestingly, a recent
study demonstrated that tautomerase null MIF protein is capable
of binding to its receptor(s) and initiating MIF signal transduction
pathway(s).7 These results suggest that enzymatic activity of MIF is
dispensable for its biological activity.7 Accordingly, it is imperative
to discover compounds that inhibit not only the enzymatic activity
of MIF but also (more importantly) the biological function of MIF.5
Previously, we have synthesized novel 1,2,4-oxadiazole, phthali-
mide, amide and other derivatives of ISO-1 (an early MIF inhibitor;
Fig. 1) which not only suppress the enzymatic activity of MIF but
also (more importantly) inhibit the MIF-induced production of
interleukin-6 (IL-6) and/or interleukin-1b (IL-1b) significantly bet-
ter than ISO-1.6 In the aforementioned study,6 we explored the
structure–activity relationship of isoxazoline compounds wherein
the main structural element was that of 4-hydroxy phenyl
unit. With the objective of diversifying our chemistry, in this
study, we replaced the 4-hydroxy phenyl unit with heterocycles
such as furan and indole. The aforementioned heterocycles were
chosen—in part—because of industrial interest.8 Subsequently, we
ascertained the potential of these novel compounds to inhibit
MIF biological function.
The key intermediate (E,Z)-furan-3-carbaldehyde oxime (2), re-
quired for synthesis of different dihydro-isoxazoles, was obtained
from furan-3-carbaldehyde (1). Compound 1 was treated with
hydroxylamine hydrochloride in the presence of pyridine and eth-
anol (1:1) to afford 2 in 90% yield. Oxime 2 was treated with 1-flu-
oro-4-vinylbenzene in the presence of sodium hypochlorite in THF
to yield compound 3. With the objective of synthesizing various
derivatives with aliphatic and cyclic substitutions, intermediate 2
was reacted with different alkenes [1-(trifluoromethyl)-4-vinyl-
benzene, 1-methoxy-4-vinylbenzene, styrene, vinylcyclohexane,
but-3-en-1-ol, vinyl pivalate, dimethyl 2-methylenesuccinate,
methyl methacrylate, 4-allylmorpholine, 1-vinylpyrrolidin-2-one,
2-vinylisoindoline-1,3-dione, 1-allyl-1H-indole] in the presence
of sodium hypochlorite in THF to yield the desired products
(4–11 and 13–16, respectively; Fig. 2). 3-(Furan-3-yl)-5-(methyl)-
5-(1-methoxy-1-oxomethyl)-4,5-dihydroisoxazole (11) was treated
O
O
N
O
HO
Figure 1. ISO-1: An early MIF inhibitor.
⇑
Corresponding author.
E-mail address: sarala.balachandran@piramal.com (S. Balachandran).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.127

S. Balachandran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1508–1511
1509
R1
3 = R1 = 4-FPh, R2 = H
10 = R1 = CH2 -COOCH3,R2 = COOCH3
11 = R1 = CH3 , R2 = COOCH3
OH
O
4 = R1 = 4-CF3Ph, R2 = H
R2
N
N
O
5 = R1 = 4-OCH3Ph, R2 = H
c
12 = R1 = CH3, R2 = COOH
H
H
6 = R1 = C6H5, R2 = H
R2
R1
13 = R1 = -CH2-morpholine, R2 = H
b
a
7 = R1 = C6H11, R2 = H
8 = R1 = CH2-CH2OH, R2 = H
14 = R1 = -N-pyrrolidin -2 -one, R2 = H
15 = R1 = -N-phthalimide, R2 = H
16 = R1 = indole- 1-yl -CH2 , R2 = H
O
O
O
9 = R1 = -OCOC(CH3)3, R2 =
H
2
1
Figure 2. Synthesis of compounds 2–16. Reagents and conditions: (a) NH2OHÁHCl/C5H5N/C2H5OH, rt, 15–16 h, 90%; (b) different alkenes/THF, bleach, rt, 24–26 h, 10–90%; (c)
compound 12 from 11, THF/MeOH/H2O, LiOH, rt, 3 h, 82%.
Table 1
with lithium hydroxide monohydrate in THF, methanol and water
to afford 12 (Fig. 2) in 82% yield.
Activity of synthesized furan and indole series compounds in MIF dopachrome
tautomerase assay
As a first step towards synthesizing indole derivatives, com-
pound 17 was treated with 1-bromo-3-(trifluoromethyl) benzene
in the presence of cesium carbonate and copper iodide in DMF at
165 °C to obtain 18. Subsequently, compound 18 was treated with
phosphoryl chloride and DMF at 165 °C to obtain 19. The key
intermediate 20 was obtained by treating 19 with hydroxylamine
hydrochloride in the presence of pyridine and ethanol (1:1). The
intermediate 20 was reacted with different alkenes [1-fluoro-
4-vinylbenzene, 1(trifluoromethyl)-4-vinylbenzene, 1-methoxy-
4-vinylbenzene, styrene, vinylcyclohexane, but-3-en-1-ol, vinyl
pivalate, dimethyl 2-methylenesuccinate, methyl methacrylate,
4-allylmorpholine, 1-vinylpyrrolidin-2-one, 2-vinylisoindoline-1,
3-dione, 1-allyl-1H-indole] in the presence of sodium hypochlo-
rite in THF to yield indole derivatives (21–29 and 31–34, respec-
tively) (Fig. 3). Compound 30 was obtained from 29 using a
hydrolysis procedure analogous to the one utilized to obtain 12
(Fig. 3). The structures of various synthesized compounds were
assigned on the basis of different spectral data (experimental
details are provided in the Supplementary data section).
R1
R2
Furans
Indoles
Compd
% Inh.
Compd
% Inh.
4-FPh
4-CF3Ph
4-OCH3Ph
C6H5
C6H11
CH2–CH2OH
OCOC(CH3)3
CH2–COOCH3
CH3
H
H
H
H
H
H
H
3
4
5
6
7
41**
41*
72**
59
21
22
23
24
25
26
27
28
29
30
31
32
33
34
100*
51
97*
71
54**
23*
30**
47
100*
91*
100*
76*
66
8
9
COOCH3
COOCH3
COOH
H
H
H
H
10
11
12
13
14
15
16
22*
29*
56
CH3
17*
100*
10*
–CH2-morpholine
–N-Pyrrolidin-2-one
-N-Phthalimide
Indol-1-yl-CH2
18*
0*
47*
100**
100*
% Inh. indicates % inhibition of MIF tautomerase activity at 100
59% inhibition at 100
experiments carried out at 1, 10, 30, and 100
Indicates p <0.05 compared to ISO-1.
Indicates p = 0.05–0.10 compared to ISO-1.
l
M. ISO-1 shows
l
M. Results presented are representative of three separate
lM.
*
**
We investigated the MIF inhibitory potential of the synthesized
compounds and, in parallel, compared their activity with ISO-1.
Initially, dopachrome tautomerase assays were performed. Several
compounds from furan series (e.g., 5, 6, 7, 13, and 16) and indole
series (e.g.2, 1, 22, 23, 24, 25, 26, 27, 28, 29, 31, and 34) consistently
inhibited the dopachrome tautomerase activity of purified recom-
binant human MIF at levels equal to or better than that of ISO-1
(Table 1). Interestingly, compounds from the indole series inhib-
ited MIF enzymatic activity to a greater extent in comparison to
the furan series compounds (Table 1). Furthermore, the ester in in-
dole (29) series but not in furan series (11) elicited robust inhibi-
tion of MIF enzymatic activity as compared to its corresponding
acid (30 and 12, respectively). The findings with indole series com-
pounds are consistent with prior literature reports.9 Of note, com-
pounds in which R1 is indol-1-yl-CH2 (e.g., 16 and 34) were found
to be potent inhibitors of MIF enzymatic activity (Table 1).
Given our earlier observations5, we sought to find compounds
that inhibit not only the enzymatic activity of MIF but also (more
importantly) the biological function of MIF. Analogous to our re-
cent studies6, we investigated if these compounds inhibit the puri-
fied human MIF-induced expression of TNF-a, IL-6, and IL-1b from
human peripheral blood mononuclear cells (hPBMCs). Pre-treat-
ment of MIF with compounds from furan series (e.g., 4, 6, and
11) or indole series (e.g., 23, 25, 26, 29, 30, 31, 32, 33, and 34)
led to a marked suppression in the induced production of pro-
inflammatory cytokines which was at times comparable to-, and
at times significantly better than-, ISO-1 (Table 2). Collectively,
these results provide evidence that our novel MIF antagonists not
only inhibit the enzymatic activity of MIF, but also, more impor-
tantly, inhibit the biological function of MIF.
Based on these experiments, it was evident that there was no
one-to-one correlation between activity observed in MIF enzy-
matic assay and inhibition of pro-inflammatory cytokine produc-
tion seen in MIF biological assay. Indeed, compound 16 was
more potent than compound 4 in inhibiting the dopachrome tau-
tomerase activity of MIF (Table 1); however, compound 4 inhibited
MIF-induced production of pro-inflammatory cytokines to a great-
er extent than compound 16 (Table 2). Interestingly, the N-pyrroli-
R1
R2
21 = R1 = 4-FPh, R2 = H
28 = R1 = CH2 -COOCH3, R2 = COOCH3
OH
N
O
22 = R1 = 4-CF3Ph,R2 = H
23 = R1 = 4-OCH3Ph, R2 = H
24 = R1 = C6H5, R2 = H
29 = R1 = CH3, R2 = COOCH3
O
N
e
H
R1
H
30 = R1 = CH3, R2 = COOH
R2
31 = R1 = -CH2-morpholine, R2 = H
32 = R1 = -N-pyrrolidin -2 -one, R2 = H
33 = R1 = -N-phthalimide, R2 = H
34 = R1 = indole-1-yl -CH2, R2 = H
a
b
c
d
25 = R1 = C6H11, R2 = H
N
N
N
26 = R1 = CH2-CH2OH, R2 = H
N
N
H
27 = R1 = -OCOC(CH3)3, R2= H
F3C
18
F3C
F3C
F3C
17
19
20
Figure 3. Synthesis of compounds 18–34. Reagents and conditions: (a) 1-bromo-3-(trifluoromethyl) benzene, Cs2CO3, CuI, DMF, 165 °C, 3–4 h, 77%; (b) POCl3, DMF, 165 °C, 2–
3 h, 75%; (c) NH2OHÁHCl/C5H5N/C2H5OH, rt, 15–16 h, 90%; (d) different alkenes/THF, bleach, rt, 24–26 h, 10–90%; (e) compound 30 from 29, THF/MeOH/H2O, LiOH, rt, 3 h, 78%.

1510
S. Balachandran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1508–1511
Table 2
Activity of synthesized furan and indole series compounds in MIF-induced cytokine production assay in hPBMCs
R1
R2
Compd
Furans
% Inh.
Compd
Indoles
% Inh.
TNF-a
IL-1b
IL-6
TNF-a
IL-1b
IL-6
4-FPh
4-CF3Ph
4-OCH3Ph
C6H5
C6H11
CH2–CH2OH
–OCOC(CH3)3
CH2–COOCH3
CH3
H
H
H
H
H
H
H
3
4
5
6
7
0*
47*
67*
13
15
50*
0
21
22
23
24
25
26
27
28
29
30
31
32
33
34
0*
0*
4**
6**
13
76*
2.5
50
0*
2*
15*
37
23
31
14
0*
12**
43
9
15*
0*
0*
27
88*
0*
19
8
9
11**
0*
7**
1*
88*
15*
23
84*
12
6*
COOCH3
COOCH3
COOH
H
H
H
H
10
11
12
13
14
15
16
4*
57**
39
60*
11
7
0*
4**
65*
45*
36*
52*
24
30
32
0*
28
26
30
81*
39
42
68*
63*
30
89*
54*
51*
68*
43
CH3
15**
32
–CH2-morpholine
-N-Pyrrolidin-2-one
-N-Phthalimide
Indol-1-yl-CH2
49*
49*
13
6
22*
12**
19**
44
17
% Inh. indicates % inhibition of MIF-induced production of TNF-
a
, IL-1b or IL-6 at 100
l
M. ISO-1 shows 35%, 34%, and 12% inhibition for TNF-
M.
a, IL-1b, and IL-6, respectively, at
100 M. Results presented are representative of at least two experiments conducted at 10, 30, and 100
l
l
*
Indicates p <0.05 compared to ISO-1,
Indicates p = 0.05–0.10 compared to ISO-1.
**
Table 3
din-2-one substituted compound in the indole series (compound
32) inhibits MIF-induced production of pro-inflammatory cyto-
kines to a greater extent than the corresponding compound in fur-
an series (compound 14) (Table 2). Similarly, the indole alcohol 26
is more active than corresponding compound 8 from the furan ser-
ies (Table 2). In general, majority of the potent inhibitors of MIF-in-
Activity of synthesized furan and indole series compounds in MIF ELISA assays using
hPBMCs
R1
R2
Furans
Indoles
Compd
% Inh.
Compd
% Inh.
4-FPh
4-CF3Ph
4-OCH3Ph
C6H5
C6H11
CH2-CH2OH
–OCOC(CH3)3
CH2–COOCH3
CH3
H
H
H
H
H
H
H
3
4
5
6
7
51*
60*
82**
68*
35*
58*
86
21
22
23
24
25
26
27
28
29
30
31
32
33
34
0*
5*
duced production of TNF-a, IL-6, and IL-1b were from the indole
series (e.g., 26, 29, and 32). Within the indole series, N-substituted
cyclic compounds (32, 33) appeared to show better inhibition of
MIF biological function as compared to alkyls with cyclic substitu-
tion (31, 34) (Table 2).
1*
2*
22*
0*
8
9
5*
In an earlier study, we had observed that an isoxazole series
compound (a fluorinated analog of ISO-1, ISO-F) and a substituted
quinoline series compound containing the furan moiety (7E)
docked differentially in the active site of MIF.5 Thus, given our find-
ings in the MIF-induced cytokine production assay (Table 2), it
would be of interest to ascertain the docking pattern of furan
and indole series compounds. In particular, it would be interesting
to determine whether the docking pattern is critically dependent
on the heterocyclic core or on the aliphatic and cyclic substitutions.
It is well-established that MIF can bind to CD74, CXCR2, and
CXCR4.10 However, the identity of the MIF receptor(s) which is
(are) critically involved in MIF-induced production of pro-inflam-
matory cytokines is currently unknown. Preliminary investigations
in our laboratory suggest that CD74 is not the principal MIF recep-
tor mediating MIF-induced production of pro-inflammatory cyto-
kines in hPBMCs (Dagia et al., manuscript in preparation). It
would, thus, be of interest to ascertain if the furan and indole series
compounds affect the MIF binding to CXCR2 and/or CXCR4.
The synthesized compounds were also evaluated for their po-
tential to inhibit the secretion/release/recognition of MIF from
hPBMCs. Several compounds from the furan series (e.g., 5, 9, 13,
15, and 16) inhibited the secretion/release/recognition of MIF from
hPBMCs at levels equal to or better than ISO-1 (Table 3). Interest-
ingly, none of the synthesized compounds from indole series po-
tently inhibited the secretion/release/recognition of MIF from
hPBMCs.
COOCH3
COOCH3
COOH
H
H
H
H
10
11
12
13
14
15
16
0*
10*
0*
0*
CH3
21*
78**
36*
83
14*
0*
–CH2-morpholine
-N-Pyrrolidin-2-one
-N-Phthalimide
Indol-1-yl-CH2
0*
0*
95*
8*
% Inh. indicates % inhibition of secretion/release/recognition of MIF at 100
1 shows 87% inhibition at 100 M. Results presented are representative of at least
lM. ISO-
l
two experiments carried out at 3, 10, 30, and 100
lM.
*
Indicates p <0.05 compared to ISO-1.
Indicates p = 0.05–0.10 compared to ISO-1.
**
cells is known to occur via a non-classical pathway.7 Reagents
modulating the ABC transporter, but not protein synthesis inhibi-
tors such as cycloheximide, are known to inhibit the secretion of
MIF.7 Whether ISO-1 and 5, 9, 13, 15, and 16 work in a similar man-
ner and inhibit the secretion of MIF by affecting the ABC trans-
porter is currently unknown and warrants further investigation.
However, the data generated from our recent study5 and earlier
studies11 are consistent with the hypothesis that the inhibitory
activity of these aforementioned compounds in cell-based ELISA
assays is, at least in part, due to these compounds binding directly
to MIF and preventing its recognition. Since several of these com-
pounds (e.g., 5, 9, 13, 15, and 16) showed equivalent or better
MIF inhibition potential in cell-based ELISA assays (in comparison
to ISO-1), it would be of interest to determine their ability to
interact with MIF in comparison to ISO-1. These studies are
currently ongoing in our laboratory. Most importantly, it would
be of interest to assess the efficacy of one or more of these MIF
inhibitors (e.g., 4, 26, 29, and 32) in a MIF-dependent model of
experimental inflammation.
In the aforementioned assay, it was found that the acid from the
indole (30) series showed relatively more (albeit marginal) MIF
inhibition than the corresponding ester (29). Of note, these obser-
vations are in direct contrast to our findings in the dopachrome
tautomerase assay (Table 1). The secretion of MIF from THP-1

S. Balachandran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1508–1511
1511
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.
In conclusion, we have synthesized novel furan and indole com-
pounds which inhibit the biological activity of MIF.
4. Dabideen, D. R.; Cheng, K. F.; Aljabari, B.; Miller, E. J.; Pavlov, V. A.; Al-Abed, Y. J.
Med. Chem. 2007, 50, 1993.
Acknowledgments
5. Dagia, N. M.; Kamath, D. V.; Bhatt, P.; Gupte, R. D.; Dadarkar, S. S.; Fonseca, L.;
Agarwal, G.; Chetrapal-Kunwar, A.; Balachandran, S.; Srinivasan, S.; Bose, J.;
Pari, K.; B-Rao, C.; Parkale, S. S.; Gadekar, P. K.; Rodge, A. H.; Mandrekar, N.;
Vishwakarma, R. A.; Sharma, S. Eur. J. Pharmacol. 2009, 607, 201.
6. Balachandran, S.; Rodge, A.; Gadekar, P. K.; Yadav, V. N.; Kamath, D.; Chetrapal-
Kunwar, A.; Bhatt, P.; Srinivasan, S.; Sharma, S.; Vishwakarma, R. A.; Dagia, N.
M. Bioorg. Med. Chem. Lett. 2009, 19, 4773.
We are thankful to department of analytical chemistry for pro-
viding NMR, mass, and HPLC for characterization of all molecules.
We thank Charmaine Dias and Prashant Vadnal for technical
assistance.
7. Fingerle-Rowson, G.; Kaleswarapu, D. R.; Schlander, C.; Kabgani, N.; Brocks, T.;
Reinart, N.; Busch, R.; Schütz, A.; Lue, H.; Du, X.; Liu, A.; Xiong, H.; Chen, Y.;
Nemajerova, A.; Hallek, M.; Bernhagen, J.; Leng, L.; Bucala, R. Mol. Cell. Biol.
2009, 29, 1922.
Supplementary data
8. Gaeta, F. C. A.; Baird, A.; Anchin, J.; Ying, W.; Florkiewicz, R.; Sircar, J.;
Sunilkumar, K. C. Avanir Pharmaceuticals, USA 2006, US7,129,236B2.
9. Zhang, X.; Bucala, R. Bioorg. Med. Chem. Lett. 1999, 9, 3193.
10. Schwartz, V.; Lue, H.; Kraemer, S.; Korbiel, J.; Krohn, R.; Ohl, K.; Bucala, R.;
Weber, C.; Bernhagen, J. FEBS Lett. 2009, 583, 2749.
11. Senter, P. D.; Al-Abed, Y.; Metz, C. N.; Benigni, F.; Mitchell, R. A.; Chesney, J.;
Han, J.; Gartner, C. G.; Nelson, S. D.; Todaro, G. J.; Bucala, R. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 144.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.127.
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