Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and their in vitro characterization as inhibitors of protein-protein interaction

Article abstract of DOI:10.1021/jm401733v

The 4EGI-1 is the prototypic inhibitor of eIF4E/eIF4G interaction, a potent inhibitor of translation initiation in vitro and in vivo and an efficacious anticancer agent in animal models of human cancers. We report on the design, synthesis, and in vitro ch

Full text of DOI:10.1021/jm401733v

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Article
Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and
their in vitro characterization as inhibitors of protein-protein interaction
Poornachandran Mahalingam, Khuloud Takrouri, Ting Chen, Rupam Sahoo, Limo
Chen, Gerhard Wagner, Bertal H. Aktas, Jose A Halperin, and Michael Chorev
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401733v • Publication Date (Web): 14 May 2014
Downloaded from http://pubs.acs.org on June 1, 2014
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Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and their in
vitro characterization as inhibitors of protein-protein interaction
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Poornachandran Mahalingam , Khuloud Takrouri , Ting Chen , Rupam Sahoo , Limo Chen ,
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1,2
1,2
1,2*
Gerhard Wagner , Bertal H. Aktas , Jose A. Halperin , and Michael Chorev
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Laboratory for Translational Research, Harvard Medical School, One Kendall Square,
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Cambridge, MA, 02139; Hematology Laboratory for Translational Research, Brigham and
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Women’s Hospital, 20 Shattuck Street, Thorn 7, Boston, MA 02115; Department of
Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave,
Boston, MA 02115
*
To whom correspondence should be addressed:
Prof. Michael Chorev,
Hematology Laboratory for Translational Research
Brigham and Women’s Hospital
2
0 Shattuck Street, Thorn 7, Boston, MA 02115
Phone: 617 525-3146. Fax: 617 582-6069.
E-mail: michael_chorev@hms.harvard.edu
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ABSTRACT:
The 4EGI-1 is the prototypic inhibitor of eIF4E/eIF4G interaction, a potent inhibitor of
translation initiation in vitro and in vivo, and an efficacious anti-cancer agent in animal models of
human cancers. We report on the design, synthesis, and in vitro characterization of a series of
rigidified mimetic of this prototypic inhibitor in which the phenyl in the 2-(4-(3,4-
dichlorophenyl)thiazol-2-yl) moiety was bridged into a tricyclic system. The bridge consisted
one of the following: ethylene, methyleneoxide, methylenesulfide, methylenesulfoxide and
methylenesulfone. Numerous analogues in this series were found to be markedly more potent
than the parent prototypic inhibitor in the inhibition of eIF4E/eIF4G interaction thus preventing
the eIF4F complex formation, a rate limiting step in the translation initiation cascade in
eukaryotes, and in inhibition of human cancer cell proliferation.
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KEYWORDS: translation initiation, eukaryotic initiation factor, protein-protein interaction,
inhibition of translation initiation, eIF4E, eIF4G, eIF4F, rigidification, fluorescence
polarization.
INTRODUCTION
Translation initiation in eukaryotic cells is a highly regulated process and plays an
1
important role in cell proliferation, differentiation, survival, and maintenance of homeostasis .
Disruption and/or perturbation of cap-dependent translation is associated with many
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pathophysiological processes such as Wolcott-Rallison syndrome, fragile X syndrome ,
neurodegenerative disorders such as Alzheimer’s disease and proliferative disorders such as
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, 4
malignant transformation . Translation initiation commences with the binding of the eukaryotic
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initiation factor 4E (eIF4E) to the mRNA 5’-end cap (m GpppN, where N is any nucleotide and
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m is a methyl group) structure, which is present in all mRNAs. Protein-protein interaction
between eIF4E and eIF4G, the scaffolding protein, enables the recruitment of eIF4A, a DEAD-
box RNA helicase, and formation of the eIF4F complex that unwinds the secondary structure of
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mRNAs and allows the docking and assembly of the 40S ribosome . The 40S ribosome complex
then traverses the 5’ untranslated region (UTR) until it recognizes the initiation codon AUG,
followed by the 60S large ribosomal subunit binding to form the 80S initiation complex, which is
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competent to enter the elongation cycle .
Under normal cellular conditions, eIF4F complex is limited as eIF4E is secluded from
eIF4G by binding to hypophosphorylated eIF4E binding proteins (4E-BP). Stimulation of the
phosphatidylinositol 3-kinase/AKT/mTOR pathway leads to hierarchical 4E-BP
phosphorylation, dislodging hyperphosphorylated 4E-BP from eIF4E and enabling assembly of
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eIF4E complex. Since, both the 4E-BPs and eIF4G share the same binding motif on eIF4E
,
the former can function as an endogenous inhibitor of cap-dependent translation initiation. As
such ectopic overexpression of 4E-BP can inhibit cap-dependent protein synthesis, inhibit tumor
growth and revert the malignant phenotype of eIF4E-overexpressing cancer cells.
eIF4F complex assembly is rate limiting for translation initiation and is predominantly
dependent on the availability of eIF4E. Although eIF4F complex formation increases the
translation of all cap-dependent mRNAs and thereby increases global protein synthesis rate,
mRNAs vary widely in their inherent “translatability”, that is primarily dictated by the length
and structure of their 5’-UTRs. mRNAs with long and structurally complex 5’-UTRs (i.e.,
“
weak” mRNAs) are most sensitive to restrictive abundance of eIF4E and therefore to the limited
availability of the eIF4F complex. These “weak” mRNAs, which encode proteins that play
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important roles in cell growth, proliferation, and apoptosis
, are poorly translated when eIF4F
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complex is scarce leading to inefficient unwinding of “weak” mRNA and subsequently
preventing ribosome loading. In contrast, most mRNAs that are characterized by relatively short,
unstructured 5’UTRs, the so called “strong” mRNAs, express housekeeping proteins such as -
actin, continue to be efficiently scanned to achieve robust initiation codon recognition, effective
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ribosome loading and efficient translation even when eIF4F complex levels are limiting .
Dysregulated eIF4E-dependent translational control is implicated in the pathobiology of
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human disorders including autism , fragile X syndrome , tuberous sclerosis , and some
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cancers . eIF4E function is particularly critical for the expression of a wide array of proteins that
contribute to all aspects of malignancy, including growth factors such as c-myc and cyclin D1,
angiogenesis factors such as VEGF and FGF-2, and anti-apoptotic proteins such as survivin and
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Bcl-2 . Inhibition of either eIF4E expression by antisense RNA or the eIF4E/eIF4G interaction
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by overexpression of 4E-BPs reverses the malignant phenotype in vitro and in vivo . Hence,
disrupting the formation of eIF4F complex will retard translation initiation in general and in
particular translation initiation of “weak” mRNAs that encode a wide array of proteins involved
in pathophysiological processes including pro-oncogenes, growth factors, cell cycle regulators
and transcription factors will yield powerful molecular probes and may lead to novel drug
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candidates
.
We have previously reported the discovery of (2-(2-(4-(3,4-dichlorophenyl)thiazol-2-
yl)hydrazono)-3-(2-nitrophenyl)propanoic acid) (1) the eIF4E/eIF4G inhibitor-1 (4EGI-1), which
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inhibits protein-protein interaction . The high throughput screening (HTS) campaign of small
molecule libraries employed a cell free fluorescence polarization assay (FP). In vitro, 1 inhibits
expression of regulatory proteins such as cyclins D1 and E, C-myc, and Bcl-2 without affecting
the expression of housekeeping proteins such as actin and -tubulin and enhances the
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dissociation of eIF4G from eIF4E. Moreover, we reported significantly lower IC50 for the
inhibition of proliferation of transformed malignant Ph+, which is transformed by the bcr-abl,
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cells than that for non-malignant non-transformed Ph- cells by 1 . Similarly, Tamburini et al.
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reported that 1 dramatically reduces the clonogenic growth of AML progenitors with a moderate
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impairment of normal CD34+ hematopoietic progenitor cologenicity . Together, these studies
support the proposition that inhibition of cap-dependent translation initiation will affect
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predominantly cells whose growth is fast or unregulated rather than quiescent cells . In vivo, it
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effectively inhibits xenograft tumor growth in mouse model of human cancer . Moreover, 1
proved to be an important molecular probe in understanding the role of eIF4F in memory
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formation and consolidation autism spectrum disorders and viral infection .
Titration of GB1-eIF4E, a fusion protein tagged with a solubility enhancing domain, with
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and measuring the N-HSQC spectrum suggests that it is binding to residues present on the
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eIF4G-binding surface of eIF4E . In the absence of high resolution structure of the complex of 1
with eIF4E we carried out extensive structure-activity relationship studies to identify the
essential pharmacophore and understand the structural latitude presented by this prototypical
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inhibitor of eIF4E/eIF4G interaction
. Structural rigidification that introduces conformational
constraint around rotatable bonds was practiced advantageously in many systems and was
reported to contribute to higher specificity and potency, greater metabolic stability, and improved
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bioavailability
.
In light of the importance of the phenyl substitution on position 4 of the thiazolidine ring
we sought to delineate the spatial relationship between these two aromatic moieties by restricting
the rotation around the bond connecting the two rings. Ring closure between the 1- or 6-position
of the 3,4-dichlorophenyl ring (C) and the 5-position of the thiazolidine ring (B) in 1 will
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generate nearly coplanar, condensed and rigid tricyclic systems that are formally formed by
rotamers representing dihedral angles of 0˚ and 180˚ around the bond connecting the 4-thiazolyl
and the phenyl ring (Scheme 1, structures Type P1 and Type P2). Specifically, the resultant
rigid tricyclic scaffolds generated by bridging position 5 of the thiazolidine with the ortho
position of the phenyl ring (substituting position 4 of the same thiazolidine ring) via one of the
following linkers - ethylene, methyleneoxide, methylenesulfide, methylenesulfoxide and
methylenesulfone will fuse the thiazolidine ring to 3,4-dihydrotetralin, chromene, thiachromene,
oxothiachromene, and thiodioxochromene, respectively. Evidently, these fused tricyclic scaffolds
are found in many biological active compounds. For example: 4,5-dihydronaphtho[1,2-d]thiazole
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scaffold is found in a ligand that has significant 5-HT3 receptor affinity , and in ligands that acts
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as allosteric enhancers of A1 adenosine receptors ; 4H-chromeno[4,3-d]thiazole scaffold is
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present in some antibacterial agents ; 4H-thiochromeno[4,3-d]thiazole scaffold has been
reported to constitute agents with antimicrobial, analgesic, anti-inflammatory, and anesthetic
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activities
and 4H-sulfoxo- and 4H-sulfonochromeno[4,3-d]thiazole scaffolds constitute some
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antifungal agents .
With the intention to characterize the impact of partial molecular rigidification we
connected rings B and C forming either the 3,4-dihydrotetralin or chromene ring systems. We
have focused our study on the previously reported prototypic 1 and kept the o-NO -substitution
2
on ring A and the dichloro-substitution on ring C. In addition, we substituted ring C with the
more polar dimethoxy-substitutions. Our study reported herein explored the role of
conformational rigidification of the 4-[3’,4’-dichlorophenyl]thiazolyl part of 1 on its potency to
inhibit eIF4E/eIF4G interaction employing a fluorescence polarization assay and proliferation of
human cancer cells.
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RESULTS AND DISCUSSION
Chemistry.
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Conceptually, there are two possible modes for connecting rings B and C of 1 by forming
a new 6-membered ring that is part of the fused tricyclic system. In the first mode (P1), position
C5 of the 2-thiazolyl, (ring B) and the position C2’ of 3’,4’-dichlorphenyl (ring C) are connected
to form the new six-membered ring (Scheme 1, system I). In the second mode (P2), the same
position of the 2-thiazolyl, (ring B) is connected to C6’ of 3’,4’-dichlorphenyl (ring C) again
forming another variant of a fused tricyclic system (Scheme 1, system II). In addition, the
availability of (E)- and (Z)-configurations around the carbimino bond of the hydrazono function,
enables formation of two geometrical isomers for each of the two cyclization modes described
above. While the mode of cyclization is dictated by the choice of starting material, isomers (E)
and (Z) are generated simultaneously as a mixture that requires chromatographic separation. The
purity of the final rigidified 1 mimetic was established by analytical RP-HPLC and exceeded
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95%, their structural integrity and identity was established by H- and C-NMR, and HR-MS.
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Cl
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NO2
(
E/Z)-4EGI-1 (1)
Rigidification
C5-Thiazolyl.......C2'-dichlorophenyl
Rigidification
C5-Thiazolyl.......C6'-dichlorophenyl
Cl
O
Cl
2'
HO
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C
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'
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Cl
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N
N
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B
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'
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NO2
H
NO2
Type P1
Type P2
Cl
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Cl
O
HO
Cl
C
HO
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N
H
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B
C
N
Rigid tricyclic
structures
I and II
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B
S
A
Cl
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N
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H
NO2
NO2
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II
Rigidified analogs (E/Z)
Rigidified analogs (E/Z)
X = CH , O, S, SO, SO
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2
Scheme 1. Strategy for the rigidification of 1
Our stepwise synthetic strategy followed the general pathway used in the synthesis of
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3
(
E/Z)-1 (Scheme 2) . Accordingly, -bromination of the bicyclic ketone 2 generated the
corresponding phenacyl bromide 3. Reaction of the bromide 3 with the thiosemicarbazide led to
the formation of the 2-hydrazinyl-thiazole 4 that was then condensed with the o-nitro-
phenylpyruvic acid to generate the partially rigidified (E/Z)-1 mimetic 5 (Scheme 2).
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X
NO2
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X = CH , O, S, SO, SO
2
2
Scheme 2. Synthetic strategy of partially rigidified 1 mimetic
Synthesis of the 4,5-dihydronaphtho[1,2-d]thiazolyl-containing 1 mimetic 13a and 13b is
outlined in Scheme 3. Friedel Crafts acylation of o-dichlorobenzene, 6 with succinic anhydride
4
4
in the presence of AlCl
3
or n-BuLi led to the formation of the respective isomers 4-(3,4-
dichlorophenyl)- and 4-(2,3-dichlorophenyl)-4-oxobutanoic acid (7a and 7b, respectively).
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Clemmensen reduction of the ketones
was followed by intramolecular polyphosphoric acid-
4
7
45
46
mediated acylation generating the corresponding -tetralone derivatives 9a and 9b that
were then -brominated to the corresponding 2-bromo-6,7-dichloro-3,4-dihydronaphthalen-
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1(2H)-one, 10a
and 2-bromo-5,6-dichloro-3,4-dihydronaphthalen-1(2H)-one, 10b. Hantzsch-
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type reaction between thiosemicabazide and these -bromoketones generated predominantly
the corresponding hydrazines 11a and 11b in moderate yields side-by-side with lesser amounts
of the side-product amines 12a and 12b. Finally, condensation between 4,5-dihydronaphtho[1,2-
d]thiazol-2-yl)hydrazines, 11a and 11b, with 2-(o-nitrophenyl)pyruvic acid in the presence of
5% acetic acid afforded the isomeric mixture of the desired rigidified 1 mimetic, (E/Z)-13a
(
Type P1) and (E/Z)-13b (Type P2) in 35-40% yield. The individual (E)- and (Z)-isomers were
separated by reverse phase column chromatography. In a separate reaction, when a mixture of
1a and 12a treated with 2-(o-nitrophenyl)pyruvic acid under similar reaction conditions only
1
the formation (E/Z)-13a was observed with the unreacted diazine 12a presumably due to its
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weak nucleophilicity relative to 11a. And also considering the tedious chromatographic process
required for the isolation of the hydrazine (11a) and the thiadiazine (12a) it was decided to use
the crude amine mixtures in the syntheses of other analogical derivatives of 1 (Compounds
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(
E/Z)-23a-b, (E/Z)-29a-b, (E/Z)-32a-b, (E/Z)-35a-b and (E/Z)-46a-c, Schemes 4-7). The
respective yields of the hydrazines were based on the LCMS analyses of the mixtures.
O
Cl
(i)
Cl
Cl
R1
HO
R1
R1
O
Cl
Cl
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a
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(iv)
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R2
HO
O
Cl
O
O
6
9
a, R = H, R = Cl
1
2
1
2
8
8
a, R = H, R = Cl
(ii)
1
2
9
b, R = Cl, R = H
1
2
b, R = Cl, R = H
HO
O
(
v)
7b
R1
R1
Cl
R2
Cl
(vi)
Cl
S
S
NH2
R2
N
Br
R2
N
NH
N
O
H N
2
1
2
1
2
10a, R = H, R = Cl
1
2a, R = H, R = Cl
1
2
1
1a, R = H, R = Cl
1
2
1
2
10b, R = Cl, R = H
1
2b, R = Cl, R = H
1
2
1
1b, R = Cl, R = H
(vii)
NO2
H
S
N
R1
N
N
HO
Cl
O
R2
1
2
(
E/Z)-13a, R = H, R = Cl
1
2
(E/Z)-13b, R = Cl, R = H
1
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3
Scheme 3. Synthesis of 4,5-dihydronaphtho[1,2-d]thiazolyl-containing 1 mimetic: (i) succinic anhydride, AlCl , 65
˚
C, 80%; (ii) succinic anhydride, n-BuLi, -78 C, 30%; (iii) Zn(Hg), Con. HCl, toluene, reflux, 36 h, 30-60%; (iv)
polyphosphoric acid, 130 ˚C, 12 h, 30%. (v) Br , ether, 30 min, 88-90%; (vi) thiosemicarbazide, dioxane, 48 h, 40-
2
5
0%; (vii) 2-(o-nitrophenyl)pyruvic acid, 5% AcOH in EtOH (1:2, v/v), reflux, 1 h, 38-45%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of rigidified oxymethylene-bridged 1 mimetic (E/Z)-23a (Type P1) and (E/Z)-
2
3b (Type P2) started with O-alkylation of 2,3-dichloro- and 3,4-dichlorophenol, respectively,
with -propiolacone in the presence of sodium hydroxide or sodium hydride. This was followed
by acid catalyzed cyclization of the 3-phenoxypropionic acids 15 and 18 into the corresponding
5
0
chroman-4-one derivatives 16, 19, and 20 . While the cyclization of 3-(2,3-
5
1
dichlorophenoxy)propanoic acid (15) in the presence of either HFliq or Eaton’s reagent afforded
,8-dichlorochroman-4-one (16) in good yield, cyclization of 3-(3,4-dichlorophenoxy)propanoic
7
acid (18) in the presence of HFliq afforded 7,8-dichlorochroman-4-one (19) but in the presence of
Eaton’s reagent it yielded 5,6-dichlorochroman-4-one (20). Due to excessive steric hindrance
considerations we shelved ketone 20 and carried -bromination of 16 and 19 successfully by
5
2
2
employing pyridinium bromide perbromide (PBPB) . In contrast, attempt to use Br under
conditions similar to those used for the preparation of 10a and 10b generated multiple products
and mixtures difficult to resolve. Conversion of the -bromo ketones 21a and 21b followed to
the hydrazines 22a and 22b and the subsequent condensation with 2-(o-nitrophenyl)pyruvic acid
followed the same procedures as described above for the 4,5-dihydronaphtho[1,2-d]thiazolyl-
containing 1 mimetic 13a and 13b and generated the anticipated oxymethylene-bridged 1
mimetic (E/Z)-23a and (E/Z)-23b (Scheme 4).
1
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
Cl
OH
Cl
O
O
Cl
O
O
Cl
Cl
(
v)
(
iii)/(iv)
(
i)
R1
HO
15
Cl
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
16
R2
Br
Cl
Cl
O
O
HO
Cl
Cl
Cl
O
(iii)
(ii)
(v)
1
2
2
1a, R = H, R = Cl
1
2
21b, R = Cl, R = H
HO
Cl
1
7
O
O
1
9
1
8
(iv)
(vi)
Cl
Cl
O
20
NO2
R1
H
S
N
O
O
N
R1
Cl
Cl
(vii)
S
N
HO
N
R2
NH
O
R2
H N
2
1
2
1
2
22a, R = H, R = Cl
(
E/Z)-23a, R = H, R = Cl
1
2
2
2b, R = Cl, R = H
1
2
(
E/Z)-23b, R = Cl, R = H
Scheme 4. Synthesis of chromene derived 1 mimetic: (i) -propiolactone, NaH, DMF, 100 °C, 42%; (ii) -
propiolactone, NaOH, H O, 100 °C, 50%; (iii) HFliq, -78 °C, 12 h, 76%; (iv) Eaton's Reagent, 70 °C, 75%; (v)
2
pyridinium bromide perbromide, CHCl - EtOH, 50 °C, 74-76%; (vi) thiosemicarbazide, dioxane, 40-50%; (vii) 2-
3
(o-nitrophenyl)pyruvic acid, 5% AcOH-ethanol (1:2, v/v), reflux, 1 h, 35-40%.
As a logical extension of oxymethylene to thiomethylene rigidified analogs of 1, the
synthesis of a set of rigidified mimetic of 1, 29a and 29b was performed. The synthetic protocol
started from the S-alkylation of 2,3- and 3,4-dichlorothiophenol with 3-bromopropionic acid in
the presence of sodium hydroxide at elevated temperature yielding respective thioaryl propionic
acids, 25a and 25b which on H SO cyclization afforded 4-thiochroman-1-ones, 26a and 26b in
2
4
5
3
54
excellent yield . The 2-bromo-4-thiochroman-4-ones , 27a and 27b obtained from the
1
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thiachromanones 25a and 25b respectively gave thiomethylene rigidified mimetic of 1, (E/Z)-
2
9a and (E/Z)-29b in 35-40% yields (Scheme 5) by following the general synthetic steps
described in scheme 2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R1
R1
SH
R1
R1
Cl
S
Cl
S
Cl
S
(
ii)
(
i)
(iii)
R2
R2
Cl
R2
Br
R2
HO
O
2
O
O
1
26a, R = H, R = Cl
1
2
1
2
1
2
27a, R = H, R = Cl
24a, R = H, R = Cl
25a, R = H, R = Cl
1
2
26b, R = Cl, R = H
1
2
1
2
1
2
27b, R = Cl, R = H
2
4b, R = Cl, R = H
25b, R = Cl, R = H
(iv)
NO2
R1
H
N
S
N
S
S
R1
Cl
Cl
(v)
S
N
HO
R2
N
NH
O
R2
H
N
2
1
2
1
2
28a, R = H, R = Cl
(
E/Z)-29a, R = H, R = Cl
1
2
2
8b, R = Cl, R = H
1
2
(
E/Z)-29b, R = Cl, R = H
Scheme 5. Synthesis of thio-chromene derived 1 mimetic: (i) 3-bromopropionic acid, NaOH, 100 °C, 88-94%; (ii)
Con. H SO , -10 °C-RT, 90%; (iii) Br , 60-69%; (iv) thiosemicarbazide, dioxane, 40-50%; (vi) 2-(o-
2
4
2
nitrophenyl)pyruvic acid, 5% AcOH-ethanol (1:2, v/v), reflux, 1 h, 35-40%.
With a view to functionalize the thiochromene derived mimetic on the sulfur atom, -
55
bromo thiochromenes, 27a and 27b were subjected to oxidation at different conditions to afford
3
3a and 33b which on treatment with thiosemicarbazide followed by 2-(o-nitrophenyl)pyruvic
acid gave the methylene sulfoxide and methylene sulfone bridged 1 mimetic (E/Z)-32a and
E/Z)-32b and (E/Z)-35a and (E/Z)-35b, respectively (Scheme 6).
(
1
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O N
2
OH
O
R2
H
N
Cl
N
R1
O
S
(iv)
N
(iii)
Cl
N
S
R1
R2
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(i)
HN
H N
2
S
R2
O
Br
Cl
S
O
R1
O
1
2
1
2
31a, R = H, R = Cl
3
3
0a, R = H, R = Cl
1
2
1
2
31b, R = Cl, R = H
1
2
0b, R = Cl, R = H
(E/Z)-32a, R = H, R = Cl
R1
1
2
(E/Z)-32b, R = Cl, R = H
Cl
S
O N
2
R2
Br
OH
O
H
N
R2
1
2
2
7a, R = H, R = Cl
N
O
R1
1
2
Cl
2
7b, R = Cl, R = H
ii)
O O
S
N
Cl
R2
R2
S
(iii)
(v)
(
N
R1
HN
H N
Br
S O
S
Cl
S
2
O
O
R1
O O
1
2
1
2
1
2
(E/Z)-35a, R = H, R = Cl
3
3a, R = H, R = Cl
34a, R = H, R = Cl
1
2
1
2
1
2
3
3b, R = Cl, R = H
34b, R = Cl, R = H
(E/Z)-35b, R = Cl, R = H
Scheme 6. Synthesis of methylene sulfoxide and methylene sulfone derived 1 mimetic: (i) m-chloroperbenzoic acid
1 equiv.), CHCl , RT, 70-76%; (ii) m-chloroperbenzoic acid (4 equiv.), CHCl , Reflux, 72-83%; (iii)
(
3
3
thiosemicarbazide, dioxane, 40-50%; (iv) 2-(o-nitrophenyl)pyruvic acid, acetic acid, 2 h, RT, 40-42%; (v) 2-(o-
nitrophenyl)pyruvic acid, acetic acid, 30 min, RT, 35-45%.
In order to examine the effect of dimethoxy substituent on the ring A replacing dichloro
substituent, we synthesized three dimethoxy substituted rigidified analogs of 1 starting from the
5
6-58
respective tetralone and chromanones. Thus, 6,7-dimethoxy-4-chromanone (43a)
afforded
5
9,60
(
E/Z)-46a while 7,8-dimethoxy-4-chromanone (43b)
obtained from 4-oxo-4-(3,4-dimethoxyphenyl)butanoic acid (39) yielded (E/Z)-46b and (E/Z)-
6c respectively in analogy to the scheme 2 (Scheme 7).
, and 6,7-dimethoxy tetralone (43c)
6
1
4
1
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MeO
MeO
OH
MeO
MeO
OH Cl
(
ii)
(
i)
O
3
6
37
R1
O
O
MeO
MeO
MeO
R2
X
OMe
OMe
MeO
MeO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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45
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47
48
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(iii)
(iv)
(v)
HO
O
0
HO
O
3
8
4
1
2
3
9
43a, R = H, R = OMe, X=O
1
2
OMe
43b, R = OMe, R = H, X=O
OH
MeO
1
2
4
3c, R = OMe, R = H, X=CH
2
O
O
OMe
OMe
(vi)
(vi)
HO
(viii)
42
4
1
NO2
R1
R1
H
N
S
N
X
X
MeO
R2
X
R1
OMe
(x) MeO
(ix)
S
N
HO
R2
N
Br
NH
O
O
R2
H N
2
1
2
4
4
4
5a, R = H, R = OMe, X=O
1
2
4
4a, R = H, R = OMe, X=O
1
2
(
(
E/Z)-46a, R = H, R = OMe, X=O
1
2
5b, R = OMe, R = H, X=O
1
2
4
4b, R = OMe, R = H, X=O
1
2
E/Z)-46b, R = OMe, R = H, X=O
1
2
5c, R = OMe, R = H, X=CH
1
2
2
44c, R = OMe, R = H, X=CH
2
1
2
(E/Z)-46c, R = OMe, R = H, X=CH
2
Scheme 7. Synthesis of dimethoxy derivatives of tetralin and chromene based 1 mimetic: (i) 3-chloro propanoyl
chloride, BF .etherate, 60 °C, 3h; (ii) K CO , RT, 20 h; (iii) succinic anhydride, AlCl , nitrobenzene; (iv) Zn-Hg,
Con. HCl, reflux, 40%; (v) P O , benzene, reflux, 4h, 57-79%; (vi) oxetan-2-one, NaH, DMF (vii) P O , benzene, 90
3
2
3
3
2
5
2
5
°
C, 6h, 57%; (viii) pyridinium bromide perbromide, CHCl –EtOH, 50 °C, 30 min, 48-75%; (ix) thiosemicarbazide,
3
dioxane; (x) 2-(o-nitrophenyl)pyruvic acid, 5% AcOH-Ethanol (1:2, v/v), 35-41%;
Biology.
Our previously described fluorescence polarization (FP) assay was used to measure the
binding efficiency of new 1 mimetic to eIF4E by competing with a fluorogenic eIF4G derived
2
2
peptide containing the conserved eIF4E-binding motif (KRYDREFLLGF) . Displacement of the
N-fluorescein tagged eIF4G-drived peptide by a competing ligand causes a measureable
decrease in FP. The non-tagged version of the same eIF4G-derived peptide and DMSO were
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
used as positive and negative controls, respectively. The binding affinity of new rigidified
mimetic of 1 in which the 3,4-dichlorphenylthiazol-2-yl system was replaced with either 4,5-
dihydronaphthol[1,2-d]thiazol-3-yl or 4H-chromeno[4,3-d]thiazol-3-yl moieties or 4H-
thiochromeno[4,3-d]thiazol-3-yl or 4H-sulfoxo- and sulfonochromeno[4,3-d]thiazol-3-yl
moieties was tested in the FP assay and were compared to that of (Z)-isomer of parent 1. The
results were presented as a ratio between the IC of (Z)-1; the concentration of (Z)-1 needed to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
displace 50% of the fluorescent-tagged peptide, and the IC50 of the new 1 analog; the
concentration of the new analog needed to displace 50% of the fluorescent-tagged peptide when
measured in the same 98-well plate (Figure 1).
The simplest of the rigidified 1 mimetic, (E/Z)-13a containing ethylene bridge between
C-6’-(2’,3’-dichlorophenyl) ring (C) and C-5 of thiazolyl ring (B) and (E/Z)-13b containing
ethylene bridge between C-2’-(4’,5’-dichlorophenyl) ring (C) and C-5 of thiazolyl ring (B)
display a 1.5- to 2-fold increase in binding affinity as compared to parent (Z)-1 (Entries 2-5,
Table 1). Linking thiazolyl ring B with the phenyl ring C via an oxymethylene (Entries 6-9,
Table 1) contribute to further increase in binding affinity reaching in (Z)-23a a 3-fold increase
(IC50=15.5 M) relative to the parent (Z)-1. However, rigidification via bridges containing
thiomethylene (Entries 10-13, Table 1), methylenesulfoxide (Entries 14-17, Table 1), or
methylenesulfone (Entries 18-21, Table 1) reverse the trend and do not contribute to binding
affinity enhancement relative to the non-rigidified parent 1. Interestingly, the most potent binders
to eIF4E in this series are 1 mimics containing the dihydronaphtho- and the chromeno-ring
systems carrying dimethoxy substituents (IC50=10.5 M for both (E)-46c and (E)-46a,
respectively). A plausible explanation for the decrease in binding affinity of the polar
methylenesulfoxide- and methylenesulfone-containing 1 mimetic relative to the less polar
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thiomethylene- and oxymethylene-containing analogs is the markedly decrease in the calculated
partition coefficient (CLogP) associated with the increased polarity. Comparison of CLogPs of
(
Z)-isomers of 13a (5.21), 23a (4.65), 29a (4.96), 32a (3.15), and 35a (2.88) that are constrained
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
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43
44
45
46
47
48
49
50
51
52
53
54
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56
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58
59
60
by the respective ethylene-, oxymethylene-, thiomethylene-, methylenesolfoxide-, and
methylenesulfone-bridges supports the above mentioned proposition.
Evidently, regardless of the substitution on ring C (dichloro or dimethoxy) we do not
observe a significant difference in binding affinity trends between Type P1 (a series) and Type
P2 (b series) for the rigidified 1 mimetic (e.g. cf. IC in the FP assay of (E)-13a and (Z)-13a
5
0
with (E)-13b and (Z)-13b, 21.5 and 34 with 32 and 43.5 M, respectively, or (E)-46a and (Z)-
6a with (E)-46b and (Z)-46b, 10.5, 30 and 51.5 with 16.5 and 55.5 M, respectively).
4
Figure 1. Dose Dependent Inhibition of eIF4E/eIF4G interaction by rigidified 1 mimetic.
The inhibition of cell proliferation data obtained in the SRB (sulforhodamine B) cell
proliferation assay with human cancer cell lines CRL-2351 breast and CRL-2813 melanoma
indicated that the rigidified 1-derived mimetic inhibited cell proliferation with IC50 values around
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1
2
2
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2
2
2
2
2
2
2
3
3
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5
5
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5
5
5
5
5
6
1
-20 µM. With few exceptions that might relate to solubility and cell penetration issues, almost
all compounds displaying higher affinity to eIF4E than (Z)-1 in the FP assay were also more
potent inhibitors in the SRB cell proliferation assay, suggesting that the compounds inhibit cell
proliferation through inhibition of translation initiation. In comparison with the parent non-
constrained 1 that inhibits equally the breast cancer and melanoma cells, the rigidified 1 mimetic
compounds were, in general, more potent in inhibiting proliferation of CRL-2813 melanoma
cells vs. the CRL-2351 breast cancer cells. While (Z)-13a, (Z)-23a, (E)-32a, and (E)-32b (IC =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
12.0, 12.3, 7.5, and 11.2 M, respectively) were slightly more potent than (Z)-1 (IC50= 15.3 M)
in inhibiting proliferation of the melanoma cells, they did not inhibit the proliferation of breast
cancer cells up to 20 M. Interestingly, the more polar rigidified 1 mimetic compounds, those
that are comprised of 4H-[1]benzothiopyrano[4,3-d]thiazole, 5,5-dioxide (35a and 35b,
CLogP=2.88), and dimethoxy-substituted dihydronaphtho[1,2-d]thiazole ((Z)-46c, CLogP=3.63)
and 4H-chromeno[4,3-d]thiazole (46a and 46b, CLogP=2.98), and in spite of having inhibitory
binding affinity comparable to that of (Z)-1 (CLogP=4.76) were devoid of cell proliferation
inhibitory activity up to concentrations of 20 M.
Evidently, we think of the cancer cell proliferation inhibitory activities as reporting of
global activity that combine on- and off-target effects. The high sensitivity of the adherent
human melanoma CRL-2813 relative to the human breast cancer CRL-2351 to inhibition of
proliferation by the rigidified 1 mimetic compounds may be attributed to the presence of a BRAF
mutation that makes them significantly more dependent on highly efficient cap-dependent
6
2
translation initiation .
1
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Table 1. Binding of the 1 rigidified mimetic to eIF4E as measured by fluorescence polarization assay
(
FP), and their potency to inhibit cancer cells proliferation as measured by sulforhodamine B (SRB)
assay.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
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50
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60
SRB Assay - IC50
(µM)
FP Assay
Entry
Compound
(Z)-1
X
-
R
1
R
2
R
3
IC50 (µM) CRL-2813 CRL-2351
1
2
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
H
43.5±1.52 15.3±2.5
11.6±0.2
4.1±0.7
(E)- 13a
(Z)- 13a
(E)-13b
(Z)-13b
(E)-23a
(Z)-23a
(E)-23b
(Z)-23b
(E)-29a
(Z)-29a
(E)-29b
(Z)-29b
CH
CH
CH
CH
O
2
2
2
2
21.5±0.70
34±4.24
32±1.41
3.8±0.3
3
H
12.0±0.6 >20 (NA)
4
Cl
Cl
Cl
Cl
H
3.6±0.3
4.4±0.6
11.2±0.6
16.6±4.2
5
H
43.5±2.12 10.6±1.4
25±1.41 5.8±0.0
6
H
7
O
H
15.5±0.70 12.3±0.4 >20 (NA)
8
O
Cl
Cl
Cl
Cl
H
18.5±0.70
27±1.41
15.5±0.70
14±0.0
3.1±0.1
8.7±4.1
5.1±0.1
10.5±0.1
4.5±0.1
7.0±2.2
5.2±0.6
15.4±2.8
17.6±2.8
17.7±2.7
9.1±1.3
9
O
H
10
S
H
1
1
S
H
12
S
Cl
Cl
28±1.41
11.5±0.70
1
3
S
H
16.7±0.1
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
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5
5
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1
4
(E)-32a
(Z)-32a
(E)-32b
(Z)-32b
(E)-35a
(Z)-35a
(E)-35b
(Z)-35b
(E)-46c
(Z)-46c
(E)-46b
(Z)-46b
(E)-46a
(Z)-46a
SO
SO
SO
SO
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
30.5±2.12
30±1.41
7.5±0.7
4.1±0.1
>20 (NA)
10.3±0.4
15
1
1
1
1
6
7
8
9
Cl
Cl
H
48.5±6.36 11.2±1.7 >20 (NA)
32.5±2.12 11.4±0.3 19.8±0.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
SO
SO
SO
SO
2
Cl
Cl
H
40.5±6.36 >20 (NA) >20 (NA)
39±5.65 >20 (NA) >20 (NA)
49±2.82 >20 (NA) >20 (NA)
49±2.82 >20 (NA) >20 (NA)
2
2
2
H
20
Cl
Cl
H
2
2
2
2
1
2
3
4
H
CH
CH
O
2
OMe OMe 10.5±0.70 12.1±0.7 >20 (NA)
OMe OMe 45±2.82 >20 (NA) >20 (NA)
2
H
OMe OMe
OMe OMe
H
H
16.5±3.53 >20 (NA) >20 (NA)
55.5±4.94 >20 (NA) >20 (NA)
25
O
2
2
6
O
H
H
OMe OMe 10.5±0.70 >20 (NA) >20 (NA)
OMe OMe 51.5±4.94 >20 (NA) >20 (NA)
7ꢀ
O
*
NA: Not active.
Disruption of eIF4E/eIF4G interaction.
We chose (E)-29a, a rigidified 1 mimetic that showed enhanced potency in the FP-assay
and the SRB-cell proliferation assay, and (E)-35b, another 1 mimetic with very low activity in
both assays, and tested their ability to disrupt the eIF4E/eIF4G complex formation in CRL-2813
melanoma cells (Figure 2). The state of association of eIF4E with eIF4G and 4E-BP1 was
7
determined by pull-down experiment on m GTP agarose resin. This demonstrated that full-length
eIF4G is displaced from eIF4E by (E)-29a that was not the case with the FP-less active
2
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derivative (E)-35b, in which no effect was observed. The disruption of the eIF4E/eIF4G
however, increased the 4E-BP1 level which is consistent with our previous finding that 1 inhibits
2
4
eIF4E/eIF4G interaction independently of 4E-BP1 binding to eIF4E . We speculated that this
increase in eIF4E/4E-BP1 level is likely due to the dissociation of eIF4G that exposes of a larger
4E-BP1 binding footprint that is present on eIF4E and is partially obscured by the bound
10
11
12
13
14
15
16
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44
45
46
47
48
49
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60
2
2
eIF4G .
a.
b.
RatioꢀofꢀeIF4G/eIF4E
Ratioꢀofꢀ4E‐BP1/eIF4E
5
4
3
2
1
0
DMSO
(E)‐29a
(E)‐35b
Figure 2. eIF4F Complex formation disruption by (E)-29a: a. (E)-29a displaces eIF4G from eIF4E and enhances
E-BP1 binding in reticulocyte lysate. After incubation of CRL-2813 cells with 30µM of each compound for 3 hrs,
4
the cells lysate was then used. A cap-affinity chromatography and SDS-PAGE immunoblotting were used to detect
eIF4E, eIF4G, and 4E-BP1. The eIF4E lanes shown come from the same gel and western blot. b. Quantitative
analysis of the effect of (E)-29a and (E)-35b on complex protein levels relative to eIF4E.
CONCLUSIONS
In an effort to optimize 1, the hit compound that was found to inhibit eIF4E/eIF4G
protein-protein interaction and translation initiation both in vitro and in-vivo, as a molecular
2
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1
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1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
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probe and potential drug candidate we focused on developing a rigidified 1 mimetic.
Rigidification of the 2-(4-(3,4-dichlorophenyl)thiazol-2-yl) moiety by introduction of a ethylene,
methyleneoxide, methylenesulfide, methylenesulfoxide or methylenesulfone bridges locking
condensed tricyclic systems yielded some very potent 1 mimics. One of these is (E)-29a, which
carries a methylenesulfide bridge, is 3-fold more potent than the parent 1 in competing for the
binding to eIF4E in the cell-free FP assay (Figure 1) with an IC =15.5 M, disrupts very
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
effectively eIF4E/eIF4G protein-protein interaction and concomitantly increases very effectively
binding of 4E-BP1 to eIF4E (Figure 2), and last but not least is a potent inhibitor of human
melanoma CRL-2813 cells proliferation IC =5.1 M (Table 1). Taken together, these results
5
0
suggest that the binding site on eIF4E for the 2-(4-(3,4-dichlorophenyl)thiazol-2-yl) moiety in 1
accommodates very nicely the fused nearly coplanar tricyclic system, which may mimic very
closely a rotamer population in 1 that binds to the macromolecular target. Recently, NMR
mapping of a solution of N-terminal fused eIF4E and 1, and high resolution x-ray analysis of co-
crystal structures of eIF4E and 1 or some of its analogs suggest that dissociation of eIF4E-eIF4G
complex in the presence of these inhibitors is through an allosteric mechanism (unpublished
data). It is very likely, that the similar activity profiles of 1 and the rigidified fused tricyclic 1
mimetic reported inhere strongly suggests that the latter also share a similar molecular
mechanism. For now, we conclude that the orientation of the phenyl in position 4 relative to the
thiazolidine ring depends on the nature of the bridge in the fused tricyclic system, the
substituents on that phenyl ring and interplay between the former two and the configuration
around the hydrazone function. Our on-going efforts will continue to look at optimization
through rigidification modes introducing conformational and configurational constraints to
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develop novel 1 mimetics as effective molecular probes. We are currently utilizing these
optimized 1 mimetics for the direct confirmation of their allosteric mechanism of action.
EXPERIMENTAL SECTION
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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38
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42
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44
45
46
47
48
49
50
51
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59
60
Chemistry.
General: All the starting materials were obtained from commercial sources and used as
purchased. Chromatography solvents were HPLC grade and were used without further
purification. Thin layer chromatography (TLC) analysis was performed using Merck silica gel 60
F-254 thin layer plates. LC-MS analyses were performed on Waters 2695 separator module with
(APCI mode) (XTerra C 30X100 mm column) micromass ZQ employing a flow rate of 0.5
8
ml/min and a solvent system that includes A – 0.1% v/v formic acid in water and B – 0.1% v/v
formic acid in acetonitrile. The scan range was m/z 100-1000. HRMS analyses were performed
on Agilent Technologies, 6120 Time-of-Flight-LC/MS instrument employing a linear gradient of
A-0.1% v/v FA in water and B-0.1% v/v TFA in acetonitrile. Melting points were measured in
open Pyrex capillaries in MEL TEMP “Electronthermal” apparatus and are uncorrected. The
purity of tested compounds was >95% as determined by RP-HPLC on a C18 Xbridge column
(
4.6 x 100 mm, 1 mL/min) with effluents monitored at 254 nm in Waters 2695 separator module.
Solvent system employed included a linear gradient of A-0.1% v/v TFA in water and B-0.1% v/v
TFA in acetonitrile. NMR spectra were recorded on a Varian 400MHz or 500MHz
spectrometers. The signal of the deuterated solvent was used as internal reference. Chemical
shifts () are given in ppm and are referenced to residual not fully deuterated solvent signal.
Coupling constants (J) are given in Hz.
Synthesis of 4-(3,4-dichlorophenyl-1-oxo-butyric acid (7a). AlCl
3
(1.99 g, 15 mmol) was
added to a solution of succinic anhydride (5 g, 50 mmol) in 1,2-dichlorobenzene (44.1 g, 3
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
°
mmol) at RT. The reaction mixture was heated to 60 C for 2.5 h and then inverse quenched onto
°
cold water (120 ml) maintaining the temperature below 50 C and stirred for 30 min. Then hexane
(
60 ml) was added and the stirring continued for 2 h to afford 7a as an off white solid, which was
filtered and dried under vacuum.
7a: White powder, Yield: 5.92 g (80%); mp: 181-182 C; H NMR (DMSO-d
ppm:  2.54-2.57 (t, 2H), 3.24 (t, 2H), 7.79 (d, J= 8 Hz, 1H), 7.90-7.93 (dd, J= 8 Hz and 4 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
°
1
6
, 400 MHz) in
1
3
1H), 8.12 (d, J= 4 Hz, 1H); C NMR (DMSO-d , 100 MHz) in ppm:  28.4, 33.9, 128.6, 130.4,
6
1
31.7, 136.6, 137.2, 174.3, 197.5; Purity of 100% as determined by RP-HPLC, t
R
= 13.55 min
(
linear gradient system of 0-100% B in A for 26 min); ESI-MS Calcd. M
W
for C10
H
8
Cl 247.07,
2 3
O
-
found m/z = 247.01 (M-H) .
Synthesis of 4-(3,4-dichlorophenyl)butyric acid (8a). Pure Zn dust (98%) (2.6 g, 40 mmol) and
HgCl , (0.18 g, 0.66 mmol) were stirred with concentrated HCl (0.25 ml) and water (0.5 ml) for
0 min. The aqueous solution was then syringed out and the amalgamated zinc was suspended in
2
1
a mixture of water (4 ml) and con. HCl (8 ml). To this suspension were added 7a (1.0 g, 4 mmol)
followed by toluene (8 ml) and refluxed with stirring for 36 h with the addition of con.HCl (4
ml) in every 5 h. After cooling to RT reaction mixture was filtered and the filtrate was extracted
2 4
with ethyl acetate (100 ml), dried over anhydrous Na SO and concentrated under vacuum to
give the crude butyric acid (8a) as oil. It was then chromatographed on a silica gel column using
hexane–ethyl acetate mixture (9:1 v/v) as eluent.
°
8
a: White solid, Yield: 0.3 g (30%), R
f
: 0.4 (Methanol-dichloromethane, 1:9, v/v); mp: 61-63 C;
1
H NMR (CDCl , 400 MHz) in ppm:  1.91-1.97 (m, 2H), 2.35-2.39 (t, 2H), 2.61-2.64 (t, 2H),
3
1
3
7
.00-7.02 (dd, J= 8 Hz and 4 Hz, 1H), 7.26 (d, J= 4 Hz, 1H), 7.34 (d, J= 8 Hz, 1H); C NMR
(CDCl , 100 MHz) in ppm:  26.0, 33.3, 34.2, 123.2, 125.7, 128.1, 130.5, 131.8, 141.6, 179,9;
3
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Purity of 98.4% as determined by RP-HPLC, t
R
= 15.16 min (linear gradient system of 0-100% B
-
in A for 26 min); ESI-MS Calcd. M for C H Cl O 232.98, found, m/z = 231.02 (M-H) .
W
9
8
2
3,
Synthesis of 6,7-dichloro-3,4-dihydronaphthalen-1(2H)-one (9a). Polyphosphoric acid (35 g)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
34
35
36
37
38
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41
42
43
44
45
46
47
48
49
50
51
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59
60
°
was heated to melt at 120 C for 30 min. To this was added 8a (1.2 g, 5.1 mmol) and heated
°
further with stirring for 10h at 130 C. After cooling to RT, diluted with water (100 ml) and
extraction with ethyl acetate (100 ml) yielded an organic phase that was washed with a saturated
solution of NaHCO (50 ml), dried over anhydrous Na SO and evaporated under vacuum. The
3
2
4
oily residue was chromatographed on silica gel column with hexane–ethyl acetate (98:2, v/v) to
obtain 9a.
°
1
9
a: White solid, Yield: 0.32 g (30%); mp: 110-111 C; H NMR (CDCl
.09-2.14 (m, 2H), 2.62 (t, J= 6.0 Hz, 2H), 2.89 (t, J= 6.0 Hz, 2H), 7.34 (s, 1H), 8.03 (s, 1H); C
, 125 MHz) in ppm:  23.8, 29.0, 38.7, 129.1, 130.8, 131.5, 132.3, 137.7, 143.9,
196.3; Purity of 100% as determined by RP-HPLC, t = 16.79 min (linear gradient system of 0-
Cl O, 215.08, found: m/z =
3
, 500 MHz) in ppm: 
13
2
NMR (CDCl
3
R
1
2
00% of B in A for 26 min); ESI-MS Calcd. M
W
for C10
H
8
2
+
15.00228 [M+H] .
Synthesis of 2-bromo-6,7-dichloro-3,4-dihydronaphthalen-1(2H)-one (10a). To a solution of
9
a (0.1 g, 0.464 mmol) in dry diethyl ether (5 ml) was added a solution of bromine (0.074 g,
0
.464 mmol) in ether (1 ml) and stirred at RT for 30 min. The residue obtained after the removal
of the solvent under vacuum was treated with an aqueous solution of NaHCO
and extracted with dichloromethane (50 ml). The organic layer was dried over anhydrous
Na SO4, concentrated under vacuum and chromatographed on a silica gel column with ethyl
acetate-hexane mixture (5:95, v/v) to afford the pure bromide 10a.
3
(5% w/v, 10 ml)
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
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5
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6
°
1
1
0a: White powder, Yield: 0.120 g (88%); mp: 128-130 C; H NMR (CDCl
 2.83-2.88 (m, 2H), 3.22-3.29 (m, 2H), 4.69 (t, 1H), 7.40 (s, 1H), 8.12 (s, 1H); C NMR
CDCl , 100 MHz) in ppm:  25.4, 31.4, 49.2, 129.6, 130.5, 130.8, 132.1, 138.8, 142.3, 188.8;
Purity of 98.9% as determined by RP-HPLC, t
3
, 400 MHz) in ppm:
1
3
(
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
= 18.53 min (linear gradient system of 0-100% B
-
in A for 26 min); ESI-MS Calcd. M for C H BrCl O, 293.97, found: m/z = 292.75 (M-H) .
W
10
7
2
Synthesis 1-(7,8-dichloro-4,5-dihydronaphtho[1,2-d]thiazol-2-yl)hydrazine (11a) and 8,9-
dichloro-5,6-dihydro-4aH-naphtho[1,2-e][1,3,4]thiadiazin-3-amine (12a). A solution of 10a
(
0.4 g, 1.36 mmol) and thiosemicarbazide (0.124 g, 1.36 mmol) in anhydrous dioxane (20 ml)
°
was heated to 80 C for 1 h and then stirred at RT for 48 h. The resulting precipitate was filtered,
2 3
washed with dioxane (10 ml), and suspended in 2M Na CO (15 ml). The pale greenish yellow
solid of 11a and 12a was filtered, washed with water, and separated by preparative RP-HPLC
using linear gradient system of 10-50% B in A for 25 min.
°
1
11a: Off-white solid, Yield: 0.150 g (39%); mp: 160-162 C; H NMR (DMSO-d
6
, 400 MHz) in
ppm:  2.86-2.95 (m, 4H), 7.49 (s, 1H), 7.67 (s, 1H), 9.27 (bs, 2H); C NMR (DMSO-d , 100
MHz) in ppm:  21.3, 27.8, 123.9, 129.0, 129.6, 130.5, 131.9, 132.0, 135.9; Purity of 65.5% as
1
3
6
determined by RP-HPLC, t
ESI-MS Calcd. M
R
= 13.20 min (linear gradient system of 0-100% B in A for 26 min);
+
W
for C11
H
9
Cl
2
N
3
S, 286.18, Found: m/z = 285.98726 [M+H] .
°
1
12a: Off-white solid, Yield: 0.077 g (20%); mp: 214-216 C; H NMR (DMSO-d , 500 MHz) in
6
13
ppm:  1.73-1.84 (m, 2H), 2.77-2.95 (m, 2H), 4.31-4.35 (m, 1H), 7.66 (s, 1H), 8.05 (s, 1H); C
NMR (DMSO-d , 125 MHz) in ppm:  25.8, 27.1, 39.6, 127.0, 129.2, 129.7, 130.5, 131.4, 141.6,
48.0, 164.4; Purity of 96.8% as determined by RP-HPLC, t = 12.44 min (linear gradient
6
1
R
2
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W 9 2 3
system of 0-100% B in A for 26 min); ESI-MS Calcd. M for C11H Cl N S, 286.18, Found: m/z
+
=
285.98573 [M+H] .
Synthesis of (E/Z)-2-(2-(7,8-dichloro-4,5-dihydronaphtho[1,2-d]thiazol-2-yl)hydrazono)-3-
10
11
12
13
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(
2-nitrophenyl)propanoic acid [(E/Z)-13a]. A suspension of 11a (0.186 g, 0.649 mmol) in 5%
(
v/v) acetic acid (7 ml) was added to 2-(o-nitrophenyl)pyruvic acid (0.135 g, 0.649 mmol) in
°
ethanol (14 ml) and was heated at 90-100 C for 1h. The yellow precipitate that formed upon
cooling to RT was filtered, washed with water and dried. The crude mixture containing the two
isomers, (E)-13a and (Z)-13a was purified on a RP-C18 FCC (100 g cartridge, flow-rate=40
ml/min) using a solvent system consisting of A - triethylammonium bicarbonate buffer (50 mM,
pH=8.5) and B - methanol. The purified isomers were precipitated from their respective fractions
following acidification with 10% HCl and separated by centrifugation. Repeated washes of the
pellets with 5% HCl followed by thorough washes with water and drying under vacuum yielded
the following:
°
1
(E)-13a: Yellow powder. Yield: 0.06 g (20%); mp: 255-256 C; H NMR (DMSO-d
6
, 400 MHz)
in ppm:  2.85-2.89 (m, 2H), 2.93-2.97 (m, 2H), 4.27 (s, 2H), 7.05 (d, J = 8.0 Hz, 1H), 7.47-7.51
1
3
(
m, 2H), 7.55 (s, 1H), 7.60-7.65 (m, 1H), 8.04-8.06 (m, 1H), 12.44 (bs, 1H); C NMR (DMSO-
d
6
, 100 MHz) in ppm:  21.3, 27.7, 29.8, 123.6, 125.7, 128.5, 129.2, 129.5, 129.7, 130.5, 131.7,
1
34.5, 136.0, 149.6, 166.0; Purity of 100% as determined by RP-HPLC, t = 8.37 min (linear
R
gradient system of 50-100% B in A for 20 min); HRMS(ESI) Calcd. M for C H Cl N O S,
W
20 14
2
4
4
+
4
77.32, Found: m/z= 477.01929 [M+H] .
°
1
(
Z)-13a: Yellow powder. Yield: 0.055 g (18%); Mp: 254-255 C; H NMR (DMSO-d , 400 MHz)
6
in ppm:  2.73-2.80 (m, 2H), 2.88-2.92 (m, 2H), 4.15 (s, 2H), 7.46-7.55 (m, 3H), 7.59 (s, 1H),
2
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13
7
.66-7.70 (m, 1H), 8.02-8.05 (m, 1H), 12.72 (bs, 1H); C NMR (DMSO-d
6
, 100 MHz) in ppm:
 21.2, 27.7, 36.7, 123.9, 125.2, 128.9, 129.3, 129.8, 130.4, 132.5, 133.60, 134.2, 135.9, 149.7,
1
5
64.7; Purity of 99.2% as determined by RP-HPLC, t
R
= 10.79 min (linear gradient system of
0
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9
0
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9
0
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2
3
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5
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7
8
9
0
0-100% B in A for 20 min); HRMS (ESI) Calcd. M for C H Cl N O S, 476.01128, Found:
W
20 14
2
4
4
+
m/z = 477.01923 [M+H] .
Synthesis of 4-(2,3-dichlorophenyl)butyric acid (7b). A solution of 1,2-dichlorobenzene (1.47
g, 10 mmol) in dry THF (10 ml) was added dropwise to n-butyl lithium (4 ml, 1.8 M in hexanes)
°
at -78 C under N and the mixture was stirred for half an hour. To the pale yellow reaction
2
mixture a solution of succinic anhydride (1.0 g, 10 mmol) in dry THF (10 ml) was added slowly
(
15 min) and stirred for 1 h at -78 °C. The reaction was quenched by water (20 ml) and acidified
with 5N HCl. The organic phase obtained following extraction with DCM (100 ml) was dried
over anhydrous Na SO and concentrated under vacuum to yield an oily residue which after
recrystallization in toluene afforded 7b.
2
4
°
1
7
b: White crystals, Yield: 0.74 g (30%); mp: 116-118 C; H NMR (CDCl
.81 (t, 2H), 3.20 (t, 2H), 7.25-7.29 (m, 1H), 7.34-7.36 (m, 1H), 7.53-7.55 (m, 1H); C NMR
, 100 MHz) in ppm:  28.4, 37.6, 126.9, 127.9, 132.5.134.3, 141.4, 178.7, 200.8; Purity
3
, 400 MHz) in ppm: 
1
3
2
(CDCl
3
of 99.4% as determined by RP-HPLC, t = 12.76 min (linear gradient system of 0-100% B in A
R
+
for 26 min); ESI-MS Calcd. M
W
for C10
H
8
Cl
2
O
3
, 247.07, Found: m/z = 268.97501 [M+Na] .
Synthesis of 5,6-dichloro-3,4-dihydronaphthalen-1(2H)-one (9b).
2
Step A: Pure Zn dust (98%) (4.0 g, 61 mmol) and HgCl , (0.4 g, 1.4 mmol) were stirred with
con. HCl (0.25 ml) and water (6.6 ml) for 10 min. The aqueous solution was removed and the
zinc amalgam was then suspended in water (2.5 ml) and con. HCl (6 ml). The stirred suspension
was treated with 7b (2.3 g, 9.3 mmol) and toluene (3.5 ml) and refluxed for 24 h with the
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addition of con. HCl (2 ml) in every 6 h. The reaction mixture was cooled to RT, filtered and the
filtrate was extracted with ethyl acetate (100 ml). The ethyl acetate fraction was dried over
anhydrous Na SO and concentrated under vacuum to give 8b as white solid.
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8
b: White solid, Yield: 1.2 g (57%); Purity of 99.2% as determined by RP-HPLC, t = 15.01 min
R
(linear gradient system of 0-100% B in A for 26 min); ESI-MS Calcd. M
W
for C10
2 2
H10Cl O ,
-
2
33.09, found: m/z = 230.74 (M-H) .
°
Step B: 8b (1.2 g, 5.1 mmol) was added to a polyphosphoric acid melt (35 g) at 130 C and
stirred for 10 h. The reaction mixture was cooled to RT and water (100 ml) was added. This
mixture was extracted with ethyl acetate (100 ml) and washed with saturated solution of
3 2 4
NaHCO (50 ml). The organic phase was dried over anhydrous Na SO and concentrated in
vacuum. The oily residue was subjected to silica gel column chromatography with hexane–ethyl
acetate mixture (98:2 v/v) to obtain pure 9b.
°
1
9b: Pale yellow solid, Yield: 0.32 g (30%); mp: 95-96 C; H NMR (CDCl
3
, 400 MHz) in ppm: 
2
.12-2.17 (m, 2H), 2.59-2.62 (t, 2H), 3.01-3.04 (t, 2H), 7.38 (d, J= 8.0 Hz, 1H), 7.86 (d, J= 8.0
1
3
Hz, 1H); C NMR (CDCl
3
, 100 MHz) in ppm:  22.3, 28.1, 38.0, 126.4, 128.4, 128.5, 132.6,
= 16.90 min (linear gradient
Cl O, 215.08, Found: m/z =
138.5, 143.9, 196.7; Purity of 98.3% as determined by RP-HPLC, t
R
system of 0-100% B in A for 26 min); ESI-MS Calcd. M
W
for C10
H
8
2
+
215.00095 [M+H] .
Synthesis of 2-bromo-5,6-dichloro-3,4-dihydronaphthalen-1(2H)-one (10b). Synthesis of 10b
followed identical procedure as described for 10a.
°
1
10b: White powder, Yield: 0.13 g (90%); mp: 102-103 C; H NMR (CDCl
3
, 400 MHz) in ppm:

2.49-2.59 (m, 2H), 3.16-3.19 (m, 2H), 4.67-4.69 (m, 1H), 7.47 (d, J= 8.0 Hz, 1H), 7.95 (d, J=
1
3
8.0 Hz, 1H); C NMR (CDCl , 100 MHz) in ppm:  24.9, 30.6, 48.6, 127.7, 129.0, 132.5, 139.5,
3
2
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