synthesis and experimental validation of new pdi...

Research ArticleSynthesis and Experimental Validation of New PDI Inhibitorswith Antiproliferative Activity

Mariateresa Badolato Gabriele Carullo Francesca Aiello and Antonio Garofalo

Department of Pharmacy Health and Nutritional Sciences University of Calabria Via Pietro Bucci87036 Arcavacata di Rende Italy

Correspondence should be addressed to Francesca Aiello francescaaiellounicalit

Received 2 April 2017 Revised 3 May 2017 Accepted 9 May 2017 Published 15 June 2017

Academic Editor Joaquin Campos

Copyright copy 2017 Mariateresa Badolato et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily of redox enzymes PDI is a multifunctional proteinthat catalyzes disulfide bond formation cleavage and rearrangement in unfolded or misfolded proteins and functions as achaperone in the endoplasmic reticulum Besides acting as a protein folding catalyst several evidences have suggested that PDI canbind small molecules containing for example a phenolic structure which includes the estrogenic one Increasing studies indicatethat PDI is involved in both physiology and pathophysiology of cells and tissues and is involved in the survival and proliferation ofdifferent cancers Propionic acid carbamoyl methyl amides (PACMAs) showed anticancer activity in human ovarian cancer bothin vitro and in vivo by inhibiting PDI The inhibition of PDIrsquos activity may have a therapeutic role in various diseases includingcancer In the present study we designed and synthesized a diversified small library of compounds with the aim of identifying a newclass of PDI inhibitors Most of synthesized compounds showed a good inhibitory potency against PDI and particularly 4-methylsubstituted 26-di-tert-butylphenol derivatives (8ndash10) presented an antiproliferative activity in a wide panel of human cancer celllines including ovarian ones

1 Introduction

Protein disulfide isomerase (PDI) belongs to the thioredoxinsuperfamily of oxidoreductases and is the founding memberof the PDI family consisting of 20 related mammalianproteins All members of this family share the thioredoxin-like domain structure characterized by the 120573120572120573120572120573120572120573120573120572 fold[1] PDI (Figure 1) is a soluble 55-kDa protein with fourtandem thioredoxin-like domains namely a b b1015840 and a1015840The homologous a and a1015840 domains contain the catalyticallyactive site of PDI which consists of a CGHC (Cys-Gly-His-Cys)motifThe cysteines can exist either in an intramoleculardisulfide (oxidized PDI) or in the dithiol form (reducedPDI) and interact with newly synthesized proteins mediatingthiol-disulfide exchanges The b and b1015840 domains link theactive site domains and assist in the binding of proteinsubstrates The b1015840 domain was identified as the chaperonedomain by NMR and X-ray crystallography Between theb1015840 and the a1015840 domains there is a short x-linker interdomain

region responsible for the U-shape structure of PDI Theacidic C-terminus of PDI is followed by an endoplasmicreticulum (ER) retrieval signal KDEL [1ndash3] A recent crystalstructure of yeast PDI reveals that this protein has a highflexibility essential for its enzymatic activity in vitro andin vivo [4] Several studies have suggested that PDI canfunction as an intracellular binding protein In human breastcancer cells it has been shown that PDI can modulate theintracellular level of 17120573-estradiol (E2) increase its hormonalactivity and reduce its metabolic availability [5] Althoughthe E2-binding site structure of human PDI is still not knownit is located to a hydrophobic pocket between the b and the b1015840domains [6]

PDI is a multifunctional protein acting as a foldingenzyme by catalyzing the formation (oxidation) cleavage(reduction) and rearrangement (isomerization) of the disul-fide bonds in unfolded or misfolded proteins [7] It has alsobeen proposed to function as a molecular chaperone in therefolding of denatured protein in vitro [8 9] Depending on

HindawiJournal of ChemistryVolume 2017 Article ID 2370359 9 pageshttpsdoiorg10115520172370359

2 Journal of Chemistry

CGHC CGHC KDEL

a b x CChaperone

E2-binding site

b㰀 a㰀

Figure 1 Domain structure of PDI

its initial concentration PDI exhibits both chaperone andantichaperone activities [10] PDI is ubiquitously expressedbut is primarily localized in the ER of eukaryotic cellswhere it maintains an oxidant environment contributingto ER homeostasis [11ndash14] Despite the presence of theER retention sequence PDI has been localized in diversesubcellular places such as cell surface cytosol mitochondriaand extracellular matrix [15] The mechanism by which PDIgets away from the ER is still unclear Originally PDI wasconsidered necessary to maintain healthy cells and tissueswhile recent studies indicate that this protein is involved inboth their physiology and pathophysiology In particular PDIhas a protective effect in neurodegenerative conditions andcardiovascular diseases PDI is also implicated in mediatingthe entry of pathogens during infectious diseases [3] Inmanydifferent cancer types including brain lymphoma kidneyovarian prostate lung and male germ cell tumors PDIis highly expressed and upregulated [16] Although morethan four decades of studies exist on PDI its role in cancerprogression is not well understood yet Acting as an ERchaperone PDI might play a role in the survival of metastaticbreast cancers [17] Suppression of apoptosis by PDI hasbeen proposed as possible mechanism for tumor growth andmetastasis In fact knockdown of PDI induces cytotoxicityin human breast cancer and activates apoptotic signalingin MCF-7 cells [18] Cell surface PDI is also associatedwith cancer progression and migration of glioma cells [19]Since PDI is involved in many important cell functionsamong which is supporting the growth and invasion ofvarious cancer types in the past years PDI has receivedconsiderable attention as a potential drug target especiallyfor cancer therapy [16] The modulation of PDIrsquos activitywith function-specific inhibitors may have a therapeuticrole in various diseases including cancer A wide rangeof drugs including antibiotics estrogen polyphenols andheterocyclic compounds have been found to inhibit PDI [20ndash23] Propionic acid carbamoyl methyl amides (PACMAs) caninhibit irreversibly PDI by forming a covalent bond (C-S)with the cysteines in the active site In particular PACMA31 showed anticancer activity in human ovarian cancer bothin vitro and in vivo [24] As an important considerabledrug target PDI has attracted our interest With the aimof identifying new PDI inhibitors considered an attractingapproach for the treatment of cancer we screened a smalllibrary of compounds using the insulin turbidimetry assay[16] Lactone frame was identified as a potential inhibitorof PDI activity so we modified and optimized the identifiedscaffold to improve upon its potency (Figure 2)

Based on the structure of knownPDImodulators bearingspecific substituents in critical positions we also designedand synthesized a new class of compounds 4-methyl sub-stituted 26-di-tert-butylphenol derivatives We kept some

Table 1 Inhibition of PDI reductase activity (expressed in ) by thesynthesized compounds and IC50 values of the most potent ones

Compound PDI reductase activityinhibition () IC50 (120583M)

PACMA 31 805 100 plusmn 0101 5722 3593 807 gt304 933 gt305 876 gt306 6467 2268 985 151 plusmn 0099 975 169 plusmn 02210 823 417 plusmn 040Each value represents the average of three independent experiments plusmn SD

structural similarities with the known ligands with the aimof enriching and diversifying our small library (Figure 3)

In this study we investigated and reported the inhibitoryeffects of all synthesized compounds against the reductaseactivity of PDI Compounds 3ndash5 and 8ndash10 highly inhibitedthe PDI reductase activity (see Table 1) In addition thecytotoxicity of all synthesized compounds was also evaluatedagainst different cancer cell lines including ovarian tumor(seeTable 2) Compounds8ndash10 showed a goodpotency againThese results suggest that the new 4-methyl substituted 26-di-tert-butylphenol derivatives are potential antiproliferativeagents targeting PDI

2 Material and Methods

21 Chemistry All reagents were purchased from Sigma-Aldrich and used without further purification Solvents weredried and distilled according to conventional proceduresReactions were carried out under nitrogen (N2) atmospheremonitored by thin layer chromatography (TLC) on silicagel plates (Merck 60F254 02mm) and visualized by UVlight at 254 and 366 nm of wavelength Organic solutionswere dried over anhydrous NaSO4 and evaporated on arotary evaporator under reduced pressure Final compoundswere purified by flash chromatography columns on silica gel(Merck 60 230ndash400mesh 0040ndash0063mm)Melting pointswere obtained by a GallenKamp21374 apparatus and wereuncorrected 1H-NMR spectra were recorded on a Bruker300MHz spectrometer Chemical shift (120575) was reported inppm using tetramethylsilane (TMS) as the internal referencestandard Multiplicities coupling constants reported as a Jvalue inHertz (Hz) and number of protons are indicated par-entheticallyMass spectra datawere determined after electronimpact ionization at 70 eV with HP 5973MS spectrometerYields refer to purified products and are not optimized

211 Synthetic Procedures As known in literature [25]the indole and pyrrole lactones (1ndash5) were synthesized as

Journal of Chemistry 3

N

O O

R

R1 H2 OMe3 Br

N

O

O

4 I H5 H

N

X

X6 O7 S

N

O

ORퟏ Rퟐ

CO-NH-Ph-Ph

R1

R2

Figure 2 Chemical modifications made to the identified scaffold

HO

HO

H H

H

OH N

OHN

O

S

O

O

O

O

PACMA 31

S

R

R

8

9

10

N

NO

N

Eퟐ

Figure 3 Design of a new class of PDI inhibitors

depicted in Scheme 1 The further formation of amide 5 wasobtained following a classical synthetic method

Scheme 2 represents the synthetic pathway for 11-phenyl-5H11H-benzo[f]pyrrolo[21-c][14]oxazepine (6) The start-ing (1-(2-fluorobenzyl)-1H-pyrrol-2-yl)(phenyl)methanolwassynthesized following the procedure described by Garofaloet al [26] Finally the cyclization of compound 6 wasobtained according to the reaction conditions described byEffland and Davis [27]

The synthesis of 11-phenyl-5H11H-benzo[f]pyrrolo[21-c][14]thiazepine (7) was realized following a similar proce-dure [26]

The 4-methyl substituted 26-di-tert-butylphenol deriva-tives (8ndash10) were synthesized by a catalyst-free reactionbetween thiophene-2-carbaldehyde a cyclic amine and 26-di-tert-butylphenol (Scheme 3)

212 General Procedure for the Synthesis of 5H-Benzo[4513]oxazino[32-a]indol-5-one Derivatives (1ndash3) and 7-iodo-5H-benzo[d]pyrrolo[21-b][13]oxazin-5-one (4) The synthe-sis of the aforementioned compounds was obtained followingthe same procedure Characterization data are in agreementwith published data [25]

213 5H-Benzo[4513]oxazino[32-a]indol-5-one (1) Brownsolid (49 yield) mp 123-124∘C 1H NMR known com-pound [25]

214 9-Methoxy-5H-benzo[45][13]oxazino[32-a]indol-5-one (2) Amorphous yellow solid (32 yield) 1H NMR(CDCl3) 120575 828 (dd 1H J = 14 51 Hz) 809 (d 1H J =83Hz) 791ndash778 (m 1H) 737ndash731 (m 2H) 711 (t 1H) 694(dd 1H J = 26 42Hz) 616 (s 1H) 339 (s 3H) MS (EI70 eV)mz 266 [M + H]+

215 9-Bromo-5H-benzo[45][13]oxazino[32-a]indol-5-one(3) Beige solid (54 yield) mp 162ndash163∘C 1HNMR knowncompound [25]

216 7-Iodo-5H-benzo[d]pyrrolo[21-b][13]oxazin-5-one (4)Yellow solid (28 yield) mp 128∘C 1H NMR (CDCl3) 120575 852(s 1H) 803 (dd 1H J = 21 86Hz) 725 (dd 1H J = 2386Hz) 697 (s 1H) 642 (d 1H J = 23Hz) 571 (s 1H) MS(EI 70 eV)mz 312 [M + H]+


Table 2 Cytotoxicity of the synthesized compounds against a panel of human cancer cell lines

Compound IC50 (120583M)MCF-7 MDA-MB 231 U87G OVCAR-8 Mia PaCa-2 HCT116 p53++ SKOV3WT

1 gt50 gt50 gt50 nt nt nt nt2 gt50 gt50 gt50 nt nt nt nt3 gt50 gt50 gt50 nt nt nt nt4 gt50 gt50 gt50 nt nt nt nt5 gt50 424 plusmn 06 451 plusmn 09 nt nt nt nt6 gt50 gt50 gt50 nt nt nt nt7 gt50 gt50 gt50 nt nt nt nt8 58 plusmn 02 87 plusmn 01 85 plusmn 03 34 plusmn 04 50 plusmn 03 111 plusmn 05 292 plusmn 059 61 plusmn 04 97 plusmn 03 88 plusmn 03 38 plusmn 06 51 plusmn 02 133 plusmn 06 281 plusmn 0210 79 plusmn 09 97 plusmn 03 183 plusmn 08 69 plusmn 09 70 plusmn 09 193 plusmn 06 409 plusmn 06Each value represents the average of three independent experiments plusmn SD

N

HO Oa N

O O

Reagent and conditions

R R

a

O

OH

N N

O

OR1

R2

R1

R2

R1 H2 OMe3 Br

4 I H5 H

Rퟏ Rퟐ

(a)MnO2 toluene reflux

CO-NH-Ph-Ph

Scheme 1

F

NOH b

N

O

6

Reagent and conditions(b) NaH DMFC6H6

Scheme 2

SH

OcAmine

HOHO

S

R

(c) Dry toluene reflux

R

8

9

10

N

NO

N

+ +


Scheme 3


217 General Procedure for the Synthesis of N-([111015840-Biphenyl]-4-methyl)-5-oxo-5H-benzo[d]pyrrolo[2-b][13]oxazine-8-car-boxamine (5) The starting 5-oxo-5H-benzo[d]pyrrolo[21-b][13]oxazine-8-carboxylic acid for the synthesis of com-pound 5 was obtained following the procedure mentionedabove [25] A solution of 5-oxo-5H-benzo[d]pyrrolo[21-b][13]oxazine-8-carboxylic acid (0044mmol 1 eq) andPCl5 (0053mmol 12 eq) in dry toluene (01mL) wasstirred at rt for 3 h To the obtained acyl chloride solution(004mmol 1 eq) Et3N (008mmol 2 eq) and [111015840-biphenyl]-4-ylmethanamine (006mmol 15 eq) were addedand the reaction mixture was kept at rt for 24 h Thenthe solvent was evaporated under vacuum and the residuewas solubilized in chloroform and washed with brine Theorganic layer was dried over anhydrous NaSO4 filteredand concentrated under vacuum The pure compound wascrystalized from chloroform

218 N-([111015840-Biphenyl]-4-methyl)-5-oxo-5H-benzo[d]pyrro-lo[2-b][13]oxazine-8-carboxamine (5) Yellow solid (8yield) mp 165∘C 1HNMR (CDCl3) 120575 770 (s 1H) 760ndash750(m 3H) 750ndash730 (m 9H) 715 (dd 1H J = 08 26Hz) 684(dd 1H J = 08 37Hz) 637 (dd 1H J = 25 37Hz) 471 (d2H J = 56Hz) MS (EI 70 eV)mz 386 [M + H]+

219 General Procedure for the Synthesis of 11-Phenyl-5H11H-benzo[f]pyrrolo[21-c][14]oxazepine (6) The synthesis of theaforementioned compound was realized following the pro-cedure described by Garofalo et al [26] and by Effland andDavis [27]

2110 11-Phenyl-5H11H-benzo[f]pyrrolo[21-c][14]oxazepine(6) Brown solid (41 yield) mp 124ndash126∘C 1HNMR(DMSO) 120575 760ndash745 (m 5H) 722 (s 1H) 697ndash683 (m 4H)680 (s 1H) 590ndash578 (m 1H) 545 (s 1H) 508ndash498 (d 1HJ = 156Hz) MS (EI 70 eV)mz 262 [M + H]+

2111 General Procedure for the Synthesis of 11-phenyl-5H11H-benzo[f]pyrrolo[21-c][14]thiazepine (7) The title com-pound was synthesized following the procedure describedby Garofalo et al [26]

2112 11-Phenyl-5H11H-benzo[f]pyrrolo[21-c][14]thiazepine(7) Beige solid (46 yield) mp 126ndash128∘C 1HNMR knowncompound [26]

2113 General Procedure for the Synthesis of 4-Methyl Sub-stituted 26-di-tert-butylphenol Derivatives (8ndash10) A stirredsolution of thiophene-2-carbaldehyde (105 eq) and sec-ondary heterocyclic amine (231 eq) in dry toluene wasrefluxed (110∘C) under N2 atmosphere overnight Then thereaction mixture was cooled down to 90∘C and a solutionof 26-di-tert-butylphenol in dry toluene was added dropby drop The reaction mixture was refluxed again under N2atmosphere overnight The solvent was evaporated undervacuum methanol was added to the reaction residue andwas stirred and heated at 60∘C for 30 minutes The obtained

precipitate was collected by filtration and purified by crys-tallization frommethanol or by chromatographic column onsilica gel (n-hexaneethyl acetate different ratios as eluent)

2114 26-Di-tert-butyl-4-(piperidin-1-yl(thiophen-2-yl)meth-yl)phenol (8) Yellow solid (33 yield) mp 133ndash34∘C1HNMR (DMSO) 120575 739ndash736 (m 1H) 714 (s 2H) 692ndash687(m 2H) 685 (bs 1H) 455 (s 1H) 225 (s 4H) 152 (s 6H)135 (s 18H) 13CNMR (CDCl3) 120575 2466 2626 (x2C) 3028(x6C) 3434 (x2C) 5226 (x2C) 7131 12440 12493 12506(x2C) 12595 13140 13515 14850 14852 15257HRMSmz384 [M minusH]minus 301 [M - piperidinyl]minus

2115 26-Di-tert-butyl-4-(morpholino(thiophen-2-yl)meth-yl)phenol (9) Reddish orange oil (46 yield) 1HNMR(DMSO) 120575 799 (d 1H J = 30Hz) 785 (s 1H) 768 (s 1H)758 (d 1H J = 30Hz) 728ndash724 (m 1H) 718ndash715 (m 1H)687 (s 1H) 401ndash398 (m 4H) 210ndash198 (m 4H) 130 (s9H) 124 (s 9H) 13CNMR (CDCl3) 120575 3160 (x6C) 3472(x2C) 5213 (x2C) 6699 (x2C) 7165 12501 (x2C) 1255112681 12704 12796 13613 14850 14852 15257 HRMSmz 301 [M - morpholino]-

2116 26-Di-tert-butyl-4-(pyrrolidin-1-yl(thiophen-2-yl)meth-yl)phenol (10) Brown oil (46 yield) 1HNMR (DMSO) 12057580 (d 1H J = 30Hz) 784 (s 1H) 767 (s 1H) 757 (d 1H J =30Hz) 729ndash724 (m 1H) 720ndash716 (m 1H) 713 (s 1H) 148(s 8H) 130 (s 9H) 124 (s 9H) HRMSmz 372 [M + H]+

22 Biology

221 Cell Culture All used tumor cell lines (MCF-7 MDA-MB 231 U87G OVCAR-8 Mia PaCa-2 HCT116 p53++ andSKOV3 WT) were grown at 37∘C in a humidified incubatorwith 5CO2 in RPMI-1640medium supplementedwith 10fetal bovine serum (FBS) The culture medium was changedtwice a week

222 Treatment with Synthesized Compounds and MTTColorimetric Assay Cells were seeded in 180 120583L mediumin 96-well plates and incubated at 37∘C After overnightattachment cells were treated with the compounds (finalconcentration from 30 120583M to 012 120583M in duplicate) DMSOwas used as negative control Plates were incubated in thepresence of compounds for 72 h and then cytotoxicity wasmeasured with colorimetric assay based on the use ofMTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide) 20120583L of MTT solution in DPBS (Dulbeccorsquosphosphate-buffered saline) (3mgmL) was added and plateswere incubated for 3-4 h Mitochondrial reductase enzymesin viable cells reduce the yellow tetrazolium MTT in itsformazan which has a purple colorwhen dissolved inDMSOMedia were finally replaced with 125 120583L of DMSO and cellviability was determined by measuring absorbance on amultiwell scanning spectrophotometer using a wavelength of570 nm The IC50 (half-maximum inhibitory concentration)valueswere determined usingGraphPadPrism and expressed


as average of three independent experiments plusmn standarddeviation (SD) value

223 Colony Formation Assay Cells were seeded in 180 120583Lmedium in 96-well plates and allowed to attach overnight at37∘CThen cells were treated with the compounds (final con-centrations 10 120583M 5 120583M and 1 120583M in duplicate) DMSO wasused as negative control After 24 h treatment compound-containingmediumwas carefully removed and replaced with200120583L of fresh medium supplemented with 10 FBS Cellswere kept in culture at 37∘Cuntil visible colonies were formedin control wells Then medium was removed and colonieswere stained with 50120583L of 05 crystal violet solution for30min at rt Finally colonies were washed 3 times with100 120583L of water to remove excessive stain and allowed to dryovernight under hood Colonies were imaged with OdysseyImaging System (LI-COR Biosciences Lincoln NE)

224 ScratchWound Healing Assay Cells were seeded in 96-well plates at a high density to have 70ndash80 confluency afterovernight incubation at 37∘C and then starved for 24 hUsing200120583L tip every well was gently and slowly scratched andwashed twice with DPBS Fresh medium supplemented with5 FBS and containing compounds (final concentrations10 120583M and 1 120583M) was added and cells were grown at 37∘Cuntil the wound in control wells was closed Finally mediumwas removed and cells were stainedwith 50120583L of 05 crystalviolet solution for 30min at rt Finally cells were washed3 times with 100 120583L of water to remove excessive stain andallowed to dry overnight under hoodWells were imagedwithOdyssey Imaging System (LI-CORBiosciences LincolnNE)

225 Measurement of PDI Reductase Activity Reductaseactivity was assayed by measuring the PDI-catalyzed reduc-tion of insulin in the presence of compounds (final concen-trations from 30 120583M to 033 120583M in duplicate) in a 100 120583Lreaction volume containing 05mM DTT 100mM sodiumphosphate buffer pH70 05mMDTT 2mMEDTA 013mMbovine insulin (Sigma) and 13 120583M purified human PDIThen the aggregation of reduced insulin chains was mea-sured spectrophotometrically at 650 nmThe IC50 valuesweredetermined using GraphPad Prism and expressed as averageof three independent experimentsplusmn standard deviation value

3 Results and Discussion

Using an insulin-based turbidimetric assay [16] we screeneda small library of various chemical scaffolds to identify smallmolecules suitable to inhibit PDI The assay identified thelactone as an inhibitory compound of PDI Heterocyclesare widely studied because of their well-known biologicalactivities among these indole and pyrrole frames Dueto their attractive importance we designed and synthe-sized some indole lactones variously substituted with bothelectron-donor and attractor groups We also synthesizedpyrrole lactones bearing different substituents such as theamide moiety Finally we wanted to enlarge the central

six-membered ring of the lactone structure into a seven-membered heterocycle containing either oxygen or sulfuratom and bearing an appended phenyl ring All the chem-ical modifications were investigated with the aim of betterunderstanding whether the new synthesized molecules couldimprove the potency of the lead compound against PDI

Recent studies have suggested that PDI can bind smallmolecules containing for example a phenolic structurewhich includes endogenous hormones like estrogen Inparticular it has been demonstrated that estradiol fits intoa hydrophobic pocket placed between the b and the b1015840domains of PDI and its hydroxyl group on C3 interactswith the histidine residue present in position 256 throughformation of an H-bond (Figure 4) [6] A class of PACMAshas exhibited cytotoxic effect in a panel of human cancer celllines especially against the resistant ovarian cancer cell lineNCIADR-RES [28] A series of PACMAs electron-deficientcompounds were identified as potent PDI inhibitors ableto react irreversibly with the thiol groups of cysteines inthe catalytically active site of PDI forming covalent adductsIn particular PACMA 31 showed an anticancer activity inhuman ovarian cancer both in vitro and in vivo It has beensuggested that the electrophilic alkyne group of PACMA 31 isessential for its cytotoxicity [24]

Based on the structure of E2 and PACMA 31 we designeda new class of 4-methyl substituted 26-di-tert-butylphenolderivatives simplifying the structure of the known mod-ulators The phenolic frame the hydroxyl group and theappended thiophene ringwere kept in the same position of E2and PACMA 31 substituting the propionyl group with a tert-butyl and the amide moiety with a cyclic amine (Figure 5)

PDI catalyzes the reduction of insulin in the presence ofdithiothreitol (DTT) the reduced insulin chains aggregateand turbidimetry is monitored using a spectrophotometerReductase activity of PDIwas then assayed bymeasuring thereduction of insulin in the presence of compounds Firstlyall synthesized compounds were screened in the insulin tur-bidimetry assay at a single concentration (100120583M) As shownin Table 1 three lactone derivatives 3ndash5 and all 4-methylsubstituted 26-di-tert-butylphenol derivatives 8ndash10 showedthe best inhibition of PDI reductase activity (inhibition gt80)

Dose-response curves and IC50 values were calculatedfor the most potent compounds Compounds 8ndash10 inhibitedPDI in a dose-dependent manner in particular piperidineand morpholine derivatives showed an IC50 lower than 2120583M(Figure 6)

These results suggest that although lactone frame couldmoderately inhibit the reductase activity of PDI 4-methylsubstituted 26-di-tert-butylphenol structure is better suitedas potential PDI inhibitor

Increased levels of PDI have been found in a varietyof human cancer cell lines including ovarian ones [16]Increasing evidence suggests that PDI supports the survivaland proliferation of several cancer types In particular it hasbeen showed that PDI activity is essential for human ovariancancer cells [24] To demonstrate the PDI inhibitory activ-ity of the synthesized compounds and verify a correlationbetween the inhibition of both PDI and cancer cell growth we


(a) (b) (c)

O

H

O H

HNN

194

H256

K254

L287 G284

K283L234

I248

I238

P235L236

N232

K207E211

F228

N214

H231

(d)

Figure 4 Docking analysis of the binding interaction of E2 inside human PDI b-b1015840 fragment (Figure by Fu et al [6])

OH H

H

OH

N

O HN

O

S

O

O

O

O

O

S

NHO

S

N

HH

Figure 5 Superimposition of new 4-methyl substituted 26-di-tert-butylphenol derivatives and known modulators

determined the cytotoxicity of all derivatives against a panelof human cancer cell lines (Table 2)

We chose seven different human cancer cell lines whichinclude breast ovarian brain colorectal and pancreatic can-cer and investigated a selectivity of synthesized compoundsagainst a specific cancer type The synthesized heterocycliccompounds did not show a potent antiproliferative effect inall tested cell lines only compound 5 exhibited a moderatecytotoxicity against breast and brain cancer cells On theother hand the new derivatives 8ndash10 were cytotoxic in all thechosen cell lines

It is important to note that they showed the best antipro-liferative activity in the human ovarian cancer cells with IC50values lower than 10 120583M Additionally the same compoundsshowed the capability to inhibit the formation of colonies andthe migration of human ovarian cancer cells (Figure 7)

These results confirm PDI as preferred target of thenew4-methyl substituted 26-di-tert-butylphenol derivativesAlthough they could bind PDI in a similar way of E2 byforming an H-bond with a specific residue in the proteinstructure additional experiments are ongoing to verify thesite of binding and themode of action of 4-methyl substituted26-di-tert-butylphenol derivatives

4 Conclusions

Recently PDI has been considered an attracting drug targetespecially for cancer therapy With this purpose we designedand synthesized a diversified small library of compounds andevaluated their inhibitory effects against the reductase activ-ity of PDI The 4-methyl substituted 26-di-tert-butylphenolderivatives with an aliphatic cyclic amine exhibited thebest inhibition with IC50 values lower than 2 120583M (8 and 9)and 5 120583M (10) The possible involvement of PDI inhibition


8910

0500 10 15minus05minus10log (휇M)

0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion

Figure 6 Dose-response curves of the most potent compounds

10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)

Figure 7 Inhibition of colony formation (a) and migration (b) ofOVAR-8 cells

in the mechanism of action of the mentioned compoundswas confirmed with the antiproliferative activity in vitroin particular against human ovarian cancer cell line Theseresults encourage further investigation about the bindingand mode of action of 4-methyl substituted 26-di-tert-butylphenol derivatives and a biological evaluation in vivoas anticancer PDI inhibitors

Additional Points

SupplementaryData Supplementary data associatedwith thisarticle can be found at httpsdoiorg10115520172370359

Conflicts of Interest

The authors declare no conflicts of interest regarding thepublication of this paper

Acknowledgments

The authors thank Professor Nouri Neamati at the Depart-ment of Medicinal Chemistry College of Pharmacy andTranslational Oncology Program University of MichiganNorth Campus Research Complex 2800 Plymouth Road

Building 520 Ann Arbor MI 48109 USA for allowing themto perform the biological assays in his facilities

References

[1] G Kozlov P Maattanen D Y Thomas and K Gehring ldquoAstructural overview of the PDI family of proteinsrdquoFEBS Journalvol 277 no 19 pp 3924ndash3936 2010

[2] H A Khan and B Mutus ldquoProtein disulfide isomerase a multi-functional protein with multiple physiological rolesrdquo Frontiersin Chemistry vol 2 no 70 pp 1ndash9 2014

[3] S Parakh and J D Atkin ldquoNovel roles for protein disulphideisomerase in disease states a double edged swordrdquo Frontiers inCell and Developmental Biology vol 3 no 30 pp 1ndash11 2015

[4] G Tian F-X Kober U Lewandrowski A Sickmann WJ Lennarz and H Schindelin ldquoThe catalytic activity ofprotein-disulfide isomerase requires a conformationally flexiblemoleculerdquo Journal of Biological Chemistry vol 283 no 48 pp33630ndash33640 2008

[5] X Fu P Wang and B T Zhu ldquoProtein disulfide isomerase isa multifunctional regulator of estrogenic status in target cellsrdquoJournal of Steroid Biochemisty amp Molecular Biology vol 112 pp127ndash137 2008

[6] X-M Fu P Wang and B T Zhu ldquoCharacterization of theestradiol-binding site structure of human protein disulfideisomerase (PDI)rdquo PLoS ONE vol 6 1 no 11 article e27185 p10 2011

[7] F Hatahet and L W Ruddock ldquoProtein disulfide isomerase acritical evaluation of its function in disulfide bond formationrdquoAntioxidants and Redox Signaling vol 11 no 11 pp 2807ndash28502009

[8] O Serve Y Kamiya and K Kato ldquoRedox-Dependent Chap-eroning following PDI Footstepsrdquo in Protein Folding E CWalters Ed pp 489ndash500NOVAScience PublishersNewYorkNY USA 2011

[9] A K Wallis and R B Freedman ldquoAssisting oxidative proteinfolding How do protein disulphide-isomerases couple confor-mational and chemical processes in protein foldingrdquo Topics inCurrent Chemistry vol 328 pp 1ndash34 2013

[10] P Klappa H C Hawkins and R B Freedman ldquoInteractionsbetween protein disulphide isomerase and peptidesrdquo EuropeanJournal of Biochemistry vol 248 no 1 pp 37ndash42 1997

[11] K R Hasan S M Khursheed and P Salahuddin ldquoProteindisulfide isomerase structure mechanism of oxidative proteinfolding and multiple functional rolesrdquo Journal of Biochemistryand Molecular Biology Research vol 2 no 3 pp 173ndash179 2016

[12] H F Gilbert ldquoProtein disulfide isomerase and assisted proteinfoldingrdquo Journal of Biological Chemistry vol 272 no 47 pp29399ndash29402 1997

[13] M Molinari C Galli V Piccaluga M Pieren and P PaganettildquoSequential assistance of molecular chaperones and transientformation of covalent complexes during protein degradationfrom the ERrdquo Journal of Cell Biology vol 158 no 2 pp 247ndash2572002

[14] S-O Lee K Cho S Cho I Kim C Oh and K Ahn ldquoProteindisulphide isomerase is required for signal peptide peptidase-mediated protein degradationrdquo EMBO Journal vol 29 no 2pp 363ndash375 2010

[15] C Turano S Coppari F Altieri and A Ferraro ldquoProteins ofthe PDI family Unpredicted non-ER locations and functionsrdquoJournal of Cellular Physiology vol 193 no 2 pp 154ndash163 2002


[16] S Xu S Sankar andN Neamati ldquoProtein disulfide isomerase apromising target for cancer therapyrdquoDrug Discovery Today vol19 no 3 pp 222ndash240 2014

[17] P Thongwatchara W Promwikorn C Srisomsap DChokchaichamnankit P Boonyaphiphat and P ThongsuksaildquoDifferential protein expression in primary breast cancer andmatched axillary node metastasisrdquo Oncology Reports vol 26no 1 pp 185ndash191 2011

[18] T Hashida Y Kotake and S Ohta ldquoProtein disulfide iso-merase knockdown-induced cell death is cell-line-dependentand involves apoptosis in MCF-7 cellsrdquo Journal of ToxicologicalSciences vol 36 no 1 pp 1ndash7 2011

[19] D Goplen J Wang P Oslash Enger et al ldquoProtein disulfideisomerase expression is related to the invasive properties ofmalignant gliomardquo Cancer Research vol 66 no 20 pp 9895ndash9902 2006

[20] P E Lovat M Corazzari J L Armstrong et al ldquoIncreasingmelanoma cell death using inhibitors of protein disulfideisomerases to abrogate survival responses to endoplasmic retic-ulum stressrdquo Cancer Research vol 68 no 13 pp 5363ndash53692008

[21] B G Hoffstrom A Kaplan R Letso et al ldquoInhibitors of proteindisulfide isomerase suppress apoptosis induced by misfoldedproteinsrdquo Nature Chemical Biology vol 6 no 12 pp 900ndash9062010

[22] MGaoQ YangMWang K DMiller GW Sledge andQ-HZheng ldquoSynthesis of radiolabeled protein disulfide isomerase(PDI) inhibitors as new potential PET agents for imaging ofthe enzyme PDI in neurological disorders and cancerrdquo AppliedRadiation and Isotopes vol 74 pp 61ndash69 2013

[23] R Jasuja F H Passam D R Kennedy et al ldquoProtein disulfideisomerase inhibitors constitute a new class of antithromboticagentsrdquo The Journal of Clinical Investigation vol 122 no 6 pp2104ndash2113 2012

[24] S Xu A N Butkevich R Yamada et al ldquoDiscovery of anorally active small-molecule irreversible inhibitor of proteindisulfide isomerase for ovarian cancer treatmentrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 109 no 40 pp 16348ndash16353 2012

[25] F Grande A Brizzi A Garofalo and F Aiello ldquoActivemanganese dioxide promoted cyclization of ortho-(1H-pyrrol-1-yl)aryl and heteroaryl carboxylic acids to 5H-pyrrolo[12-a][31]benzoxazin-5-one derivativesrdquo Tetrahedron vol 69 no47 pp 9951ndash9956 2013

[26] A Garofalo G Campiani V Nacci and I Fiorini ldquoPol-ycondensed heterocycles VIII Synthesis of 11-aryl-5H11H-pyrrolo[21-c][14]benzothiazepines by pummerer rearrange-ment-cyclization reactionrdquo Heterocycles vol 34 no 1 pp 51ndash60 1992

[27] R C Effland and L Davis ldquoSynthesis of pyrrolo[21-c][14]ben-zoxazepines 1 A novel heterocyclic ring systemrdquo Journal ofHeterocyclic Chemistry vol 22 no 4 pp 1071ndash1075 1985

[28] R Yamada X Cao A N Butkevich et al ldquoDiscovery andpreclinical evaluation of a novel class of cytotoxic propynoicacid carbamoylmethyl amides (PACMAs)rdquo Journal ofMedicinalChemistry vol 54 no 8 pp 2902ndash2914 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy


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International Journal ofInternational Journal of


Journal of

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Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


CGHC CGHC KDEL

a b x CChaperone

E2-binding site

b㰀 a㰀

Figure 1 Domain structure of PDI

its initial concentration PDI exhibits both chaperone andantichaperone activities [10] PDI is ubiquitously expressedbut is primarily localized in the ER of eukaryotic cellswhere it maintains an oxidant environment contributingto ER homeostasis [11ndash14] Despite the presence of theER retention sequence PDI has been localized in diversesubcellular places such as cell surface cytosol mitochondriaand extracellular matrix [15] The mechanism by which PDIgets away from the ER is still unclear Originally PDI wasconsidered necessary to maintain healthy cells and tissueswhile recent studies indicate that this protein is involved inboth their physiology and pathophysiology In particular PDIhas a protective effect in neurodegenerative conditions andcardiovascular diseases PDI is also implicated in mediatingthe entry of pathogens during infectious diseases [3] Inmanydifferent cancer types including brain lymphoma kidneyovarian prostate lung and male germ cell tumors PDIis highly expressed and upregulated [16] Although morethan four decades of studies exist on PDI its role in cancerprogression is not well understood yet Acting as an ERchaperone PDI might play a role in the survival of metastaticbreast cancers [17] Suppression of apoptosis by PDI hasbeen proposed as possible mechanism for tumor growth andmetastasis In fact knockdown of PDI induces cytotoxicityin human breast cancer and activates apoptotic signalingin MCF-7 cells [18] Cell surface PDI is also associatedwith cancer progression and migration of glioma cells [19]Since PDI is involved in many important cell functionsamong which is supporting the growth and invasion ofvarious cancer types in the past years PDI has receivedconsiderable attention as a potential drug target especiallyfor cancer therapy [16] The modulation of PDIrsquos activitywith function-specific inhibitors may have a therapeuticrole in various diseases including cancer A wide rangeof drugs including antibiotics estrogen polyphenols andheterocyclic compounds have been found to inhibit PDI [20ndash23] Propionic acid carbamoyl methyl amides (PACMAs) caninhibit irreversibly PDI by forming a covalent bond (C-S)with the cysteines in the active site In particular PACMA31 showed anticancer activity in human ovarian cancer bothin vitro and in vivo [24] As an important considerabledrug target PDI has attracted our interest With the aimof identifying new PDI inhibitors considered an attractingapproach for the treatment of cancer we screened a smalllibrary of compounds using the insulin turbidimetry assay[16] Lactone frame was identified as a potential inhibitorof PDI activity so we modified and optimized the identifiedscaffold to improve upon its potency (Figure 2)

Based on the structure of knownPDImodulators bearingspecific substituents in critical positions we also designedand synthesized a new class of compounds 4-methyl sub-stituted 26-di-tert-butylphenol derivatives We kept some

Table 1 Inhibition of PDI reductase activity (expressed in ) by thesynthesized compounds and IC50 values of the most potent ones

Compound PDI reductase activityinhibition () IC50 (120583M)

PACMA 31 805 100 plusmn 0101 5722 3593 807 gt304 933 gt305 876 gt306 6467 2268 985 151 plusmn 0099 975 169 plusmn 02210 823 417 plusmn 040Each value represents the average of three independent experiments plusmn SD

structural similarities with the known ligands with the aimof enriching and diversifying our small library (Figure 3)

In this study we investigated and reported the inhibitoryeffects of all synthesized compounds against the reductaseactivity of PDI Compounds 3ndash5 and 8ndash10 highly inhibitedthe PDI reductase activity (see Table 1) In addition thecytotoxicity of all synthesized compounds was also evaluatedagainst different cancer cell lines including ovarian tumor(seeTable 2) Compounds8ndash10 showed a goodpotency againThese results suggest that the new 4-methyl substituted 26-di-tert-butylphenol derivatives are potential antiproliferativeagents targeting PDI

2 Material and Methods

21 Chemistry All reagents were purchased from Sigma-Aldrich and used without further purification Solvents weredried and distilled according to conventional proceduresReactions were carried out under nitrogen (N2) atmospheremonitored by thin layer chromatography (TLC) on silicagel plates (Merck 60F254 02mm) and visualized by UVlight at 254 and 366 nm of wavelength Organic solutionswere dried over anhydrous NaSO4 and evaporated on arotary evaporator under reduced pressure Final compoundswere purified by flash chromatography columns on silica gel(Merck 60 230ndash400mesh 0040ndash0063mm)Melting pointswere obtained by a GallenKamp21374 apparatus and wereuncorrected 1H-NMR spectra were recorded on a Bruker300MHz spectrometer Chemical shift (120575) was reported inppm using tetramethylsilane (TMS) as the internal referencestandard Multiplicities coupling constants reported as a Jvalue inHertz (Hz) and number of protons are indicated par-entheticallyMass spectra datawere determined after electronimpact ionization at 70 eV with HP 5973MS spectrometerYields refer to purified products and are not optimized

211 Synthetic Procedures As known in literature [25]the indole and pyrrole lactones (1ndash5) were synthesized as


N

O O

R

R1 H2 OMe3 Br

N

O

O

4 I H5 H

N

X

X6 O7 S

N

O

ORퟏ Rퟐ

CO-NH-Ph-Ph

R1

R2


HO

HO

H H

H

OH N

OHN

O

S

O

O

O

O

PACMA 31

S

R

R

8

9

10

N

NO

N

Eퟐ















N

HO Oa N

O O


R R

a

O

OH

N N

O

OR1

R2

R1

R2

R1 H2 OMe3 Br

4 I H5 H

Rퟏ Rퟐ


CO-NH-Ph-Ph

Scheme 1

F

NOH b

N

O

6


Scheme 2

SH

OcAmine

HOHO

S

R


R

8

9

10

N

NO

N

+ +


Scheme 3













22 Biology


















(a) (b) (c)

O

H

O H

HNN

194

H256

K254

L287 G284

K283L234

I248

I238

P235L236

N232

K207E211

F228

N214

H231

(d)


OH H

H

OH

N

O HN

O

S

O

O

O

O

O

S

NHO

S

N

HH






4 Conclusions



8910


0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion


10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)



Additional Points




Acknowledgments



References







































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


N

O O

R

R1 H2 OMe3 Br

N

O

O

4 I H5 H

N

X

X6 O7 S

N

O

ORퟏ Rퟐ

CO-NH-Ph-Ph

R1

R2


HO

HO

H H

H

OH N

OHN

O

S

O

O

O

O

PACMA 31

S

R

R

8

9

10

N

NO

N

Eퟐ















N

HO Oa N

O O


R R

a

O

OH

N N

O

OR1

R2

R1

R2

R1 H2 OMe3 Br

4 I H5 H

Rퟏ Rퟐ


CO-NH-Ph-Ph

Scheme 1

F

NOH b

N

O

6


Scheme 2

SH

OcAmine

HOHO

S

R


R

8

9

10

N

NO

N

+ +


Scheme 3













22 Biology


















(a) (b) (c)

O

H

O H

HNN

194

H256

K254

L287 G284

K283L234

I248

I238

P235L236

N232

K207E211

F228

N214

H231

(d)


OH H

H

OH

N

O HN

O

S

O

O

O

O

O

S

NHO

S

N

HH






4 Conclusions



8910


0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion


10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)



Additional Points




Acknowledgments



References







































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





N

HO Oa N

O O


R R

a

O

OH

N N

O

OR1

R2

R1

R2

R1 H2 OMe3 Br

4 I H5 H

Rퟏ Rퟐ


CO-NH-Ph-Ph

Scheme 1

F

NOH b

N

O

6


Scheme 2

SH

OcAmine

HOHO

S

R


R

8

9

10

N

NO

N

+ +


Scheme 3













22 Biology


















(a) (b) (c)

O

H

O H

HNN

194

H256

K254

L287 G284

K283L234

I248

I238

P235L236

N232

K207E211

F228

N214

H231

(d)


OH H

H

OH

N

O HN

O

S

O

O

O

O

O

S

NHO

S

N

HH






4 Conclusions



8910


0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion


10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)



Additional Points




Acknowledgments



References







































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of













22 Biology


















(a) (b) (c)

O

H

O H

HNN

194

H256

K254

L287 G284

K283L234

I248

I238

P235L236

N232

K207E211

F228

N214

H231

(d)


OH H

H

OH

N

O HN

O

S

O

O

O

O

O

S

NHO

S

N

HH






4 Conclusions



8910


0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion


10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)



Additional Points




Acknowledgments



References







































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















(a) (b) (c)

O

H

O H

HNN

194

H256

K254

L287 G284

K283L234

I248

I238

P235L236

N232

K207E211

F228

N214

H231

(d)


OH H

H

OH

N

O HN

O

S

O

O

O

O

O

S

NHO

S

N

HH






4 Conclusions



8910


0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion


10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)



Additional Points




Acknowledgments



References







































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


8910


0

50

100

150

P

DI r

educ

tase

activ

ity in

hibi

tion


10 휇M 5 휇M 1 휇M 10 휇M 1 휇M

1

2

3

Ctrl

(a) (b)



Additional Points




Acknowledgments



References







































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

synthesis and experimental validation of new pdi...

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