Development of Mithramycin Analogues with Increased Selectivity toward ETS Transcription Factor Expressing Cancers

Article abstract of DOI:10.1021/acs.jmedchem.8b01107

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

Full text of DOI:10.1021/acs.jmedchem.8b01107

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Article
Development of Mithramycin Analogues with Increased
Selectivity towards ETS Transcription Factor Expressing Cancers
Prithiba Mitra, Joseph M. Eckenrode, Abhisek Mandal, Amit
K. Jha, Shaimaa M. Salem, Markos Leggas, and Jurgen Rohr
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01107 • Publication Date (Web): 16 Aug 2018
Downloaded from http://pubs.acs.org on August 17, 2018
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Journal of Medicinal Chemistry
Manuscript Title: Development of Mithramycin Analogues with Increased Selectivity towards ETS
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Transcription Factor Expressing Cancers
Prithiba Mitra,† Joseph M. Eckenrode,† Abhisek Mandal, Amit K. Jha, Shaimaa M. Salem, Markos
Leggas* and Jürgen Rohr*
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Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lee T. Todd, Jr.
Building, 789 South Limestone Street, Lexington, KY 40536ꢀ0596, USA
ABSTRACT: Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS tranꢀ
scription factor EWSꢀFLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic
window compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to
generate analogues via peptide coupling and fragmentꢀbased drug development strategies. Cytotoxicity
assays in ETS and nonꢀETS dependent cell lines identified two dipeptide analogues, 60 and 61, with
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9.1ꢀ and 15.6ꢀfold selectivity, respectively, compared to 1.5ꢀfold for 1. Importantly,
the cytotoxicityof 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory caꢀ
pacity of these analogues against EWSꢀFLI1 mediated transcription in Ewing sarcoma. Structural analyꢀ
sis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably
via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target
transcription factors for clinical use.
INTRODUCTION
Mithramycin (MTM; 1, Figure 1) is an aureolic acidꢀtype polyketide drug produced by various soil
bacteria of the genus Streptomyces and was found to possess activity against a wide variety of human
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cancers. MTM (1) was clinically evaluated in the 1960s and 70s as an agent for the chemotherapy of
various cancers. Despite some remarkable success using MTM (1) as a single agent, the results were
mixed due to its narrow therapeutic index and considerable variation in patients’ ability to tolerate the
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drug. Another concern was the lack of understanding of MTM’s (1) modeꢀofꢀaction. Taken together
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these limitations limited clinical use of MTM (1) as a chemotherapeutic agent and it has now been
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largely abandoned. Interest in MTM (1) was renewed recently, after the drug was identified as the top
inhibitor of the ETS transcription factor fusion, EWSꢀFLI1, in a screen of more than 50,000 natural
products and synthetic compounds. FLI1 and ERG are ETS transcription factors that are expressed as
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fusions with EWS and are the primary cause of Ewing sarcoma. Aside from Ewing sarcoma, aberrant
ETS transcription factors contribute significantly to the malignancy of prostate cancer, leukemia and
lymphoma. With respect to prostate cancer, approximately 50% of patients express a truncated form of
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ERG as a result of the TMPRSS2ꢀERG gene fusion. Interestingly, the DNA binding domain of ERG
and FLI1 is conserved and thus molecules that interfere with the activity of one should also inhibit the
other. Given the importance of these aberrant transcription factors in driving malignancy, the clinical use
of MTM (1) gave investigators hope for a “targeted” therapy. This was tested in a recent national cancer
institute (NCI) conducted clinical study where Ewing sarcoma patients were enrolled to assess the utility
of MTM (1) in a population of patients, all of whom express ETS fusions. Unfortunately, the results
were inconclusive because the trial was terminated early, due to toxicities. As such, the development of
less toxic and more selective analogues of MTM (1) is highly desirable.
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Figure 1. Structure of Mithramycin (MTM; 1), Mithramycin SA (MTMSA; 2), Mithramycin SAꢀ
Tryptophan (MTMSAꢀTrp; 3) and Mithramycin SA Phenylalanine (MTMSAꢀPhe; 4)
Our effort to develop such analogues is largely based on mechanistic studies that focused on underꢀ
standing MTM’s (1) mechanism of action. At the molecular level, it is known that MTM (1) binds to
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GCꢀrich DNA as a Mg coordinated dimer and modulates the activity of the transcription factor Sp1 and
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presumably others. We previously found that mithramycin SA (MTMSA; 2, Figure 1), which is a comꢀ
binatorial biosynthetic analogue of MTM (1) produced by S. argillaceus, upon inactivation of the mtmW
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gene, has no cytotoxicity (Table 1, Entry 19). Using the free carboxylic acid group in the 3ꢀside chain
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of MTMSA, we coupled natural amino acids and small molecules to generate a series of analogues,
out of which MTMSAꢀTrp (3, Figure 1) and MTMSAꢀPhe (4, Figure 1) were found to have cytotoxicity,
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comparable to MTM (1). Most importantly, we demonstrated with crystallography studies that the 3ꢀ
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side chain of the MTMSA analogues can interact with FLI1. In such complexes, aromatic 3ꢀside chain
MTMSA derivatives have sufficient length to directly interact with the FLI1 DNA binding domain of
EWSꢀFLI1, reflecting the in vitro potency of MTMSAꢀTrp (3) and MTMSAꢀPhe (4) against Ewing sarꢀ
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coma. Moreover, these studies pose a new modeꢀofꢀaction hypothesis, which requires a ternary MTM
1)ꢀDNAꢀFLI1 (or MTM (1)ꢀDNAꢀERG) complex. In this article, we describe our efforts to find more
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(
selective MTM (1) analogues for the treatment of cancers expressing aberrant ETS fusions or ETS facꢀ
tors. Our approach combined fragmentꢀbased drug development (FBDD) with structureꢀactivity relaꢀ
tionship (SAR) studies starting from of MTMSAꢀTrp (3).
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RESULTS AND DISCUSSION
Initial pharmacological studies showed that both, MTMSAꢀTrp (3) and MTMSAꢀPhe (4), appeared to
have the potential to overcome the limitation of MTM (1), with MTMSAꢀTrp (3) being the more cytoꢀ
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toxic of the two derivatives ( Table 3, Entry 10). However, their selectivity towards Ewing sarcoma
cell lines were only slightly improved compared to MTM (1) (Table 3). We chose MTMSAꢀTrp (3) as a
starting point to better understand and improve its potential binding properties to EWSꢀFLI1, by varying
its electronic, steric and hydrogen bonding properties of tryptophan residue, with the major objective to
increase the selectivity of it towards Ewing sarcoma cell lines while maintaining a cytotoxic activity
comparable to MTM (1).
Reagents and conditions: a) Phthalic anhydride, Et N, toluene, reflux, 15 h, 80%; b) NaH, Alkyl broꢀ
3
.
mide, DMF, 0 °C to rt, 10 h; c) NH NH H O, MeOHꢀDCM, rt.
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Scheme 1. Selective indole Nꢀalkylation of tryptophan
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To evaluate a potential hydrogen bonding of the indoleꢀNH, it was protected by Nꢀalkylation. After inꢀ
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itial protection of the primary amine group of
Lꢀtryptophan hydrochloride (5) with phthalimide using
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phthalic anhydride in the presence of excess triethylamine in refluxing toluene, which gave the protectꢀ
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ed tryptophan 6 in 80% yield, the indole Nꢀmethylation of 6 with MeIꢀNaH in DMF provided 7 in
0% yield, and benzylation with benzyl bromide under similar conditions furnished Nꢀbenzylated trypꢀ
tophan 8 in 70% yield (Scheme 1). Likewise, treatment with allyl bromide and prenyl bromide gave Nꢀ
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allyl tryptophan 9 and Nꢀprenyl tryptophan 10, respectively. Treatment with hydrazine hydrate in
MeOHꢀDCM at room temperature ensured the removal of the phthalimide protection group in all cases
(7 ꢀ 10), to provide the corresponding amines (11 ꢀ 14) in 50 ꢀ 65% yield (Scheme 1).
To diversify the tryptophan residue, iridiumꢀcatalyzed borylation and palladium catalyzed crossꢀ
coupling reactions were applied. Borylation allowed the introduction of various functionalities into the
indole core of tryptophan, since the carbonꢀboron bond can be easily modified. For the C7 diversificaꢀ
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tion of tryptophan, the required borylated tryptophan 15 was prepared following the method develꢀ
oped by Movagasshi and coꢀworkers starting from protected tryptophan 16 (Scheme 2). To incorporate
an allyl residue into tryptophan, which could serve as a handle for further modifications through Grubbs
chemistry, 7ꢀallyltryptophan 17 was chosen.
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Reagents and conditions: a) CuI, 1,10ꢀPhen, KI, MeOHꢀH O, 40%; b) Allyl(nꢀBu) Sn, Pd(PPh ) ,
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PhMe, 120 °C, 60%; c) MeOH, Cu(OAc) H O, Et N, O , rt 12 h; d) CuTC, Togni reagent, 1,10ꢀPhen,
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DCM, LiOH, H O, rt; e) Pd (dba) , SPhos, K PO , PhI, PhMe, 80 °C, 70%.
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Scheme 2. C7 Functionalization of tryptophan using iridium catalyzed borylation chemistry
Borylated tryptophan 15 was treated with potassium iodide in the presence of CuI as the catalyst and
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,10ꢀphenanthroline as the ligand to avail 7ꢀiodotryptophan 18, which upon Stille coupling reaction
with allyltributylstannane furnished the allyltryptophan derivative 17 in 60% yield (Scheme 2). Chenꢀ
Lam coupling of 15 failed to provide an electron enriched methoxytryptophan 19. Trifluoromethylation
of 15 using the Togni reagent produced a complex mixture, possibly because of the interference of the
free indoleꢀNH. However, Suzuki coupling reaction of 15 with iodobenzene in the presence of Pd (dba)3
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as the catalyst resulted in 7ꢀphenyltryptophan 20 in 70% yield (Scheme 2).
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Reagents and conditions: a) Ir[(cod)OMe] (5 mol%), phenanthroline (10 mol%), HBPin (0.25 equiv),
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B Pin (4.0 equiv), hexane, 80 °C, 70%; b) NaN , Cu(OAc) H O, MeOH, 70%; c) Phenyl acetylene,
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CuI, DIPA, HOAc, DCM, 72%; d) 4 N aq HCl, EtOAc, rt, 4 h; TBAF, THF, rt, 2 h, 85% (2 Steps); e)
CuTC, Togni reagent, 1,10ꢀPhen, DCM, LiOH, H O, rt, 60%; f) Cu(OTf) , KF; g) AgOTf, NaOH, Seꢀ
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lectflour.
Scheme 3. C6 Functionalization of tryptophan using iridium catalyzed borylation chemistry
Functionalization of C6 of tryptophan was achieved by following a recently developed borylation of
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tryptophan 21 by Baran et al., which provided an inseparable 4:1 mixture of C6and C5 borylated Trp
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2 (Scheme 3). Treatment of the borylated tryptophan 22 with sodium azide in the presence of Cu(II)
acetate as the catalyst produced 6ꢀazido tryptophan 24 in good yield, which under Cu(I) catalyzed click
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chemistry conditions provided the triazolylꢀphenyltryptophan 25 in 72% yield. After removal of the
tertꢀbutyl carbamate and triisopropyl silyl protection, the triazolylꢀphenyltryptophan free amine 23 was
obtained in excellent yield. Trifluoromethylation of 22 using the Togni reagent successfully yielded an
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H N
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32: X = 4,5-benzo
I
N(Boc)2
Pd(OAc)2,
Ref 25
DABCO,DMF,
85 °C, 12 h
CO Me
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steps
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HO C
CO H
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OHC
CO Me
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NHBoc
50%
N
N(Boc)2
H
S-Glutamic acid
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CO Me
CO Me
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CO Me
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N(Boc)2
N(Boc)
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N(Boc)2
O N
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MeO
N
N
N
H
H
H
33 (50%)
34 (38%)
35 (42%)
Scheme 4. Palladium catalyzed tryptophan synthesis
inseparable mixture of C5 and C6 trifluoromethlytated tryptophan 26 in 60% combined yield, compleꢀ
menting the requirement of the indoleꢀNH protection. However, fluorination of 22 remained inaccessiꢀ
ble under both nucleophilic and electrophilic conditions, owing to the labile nature of the triisopropyl
silyl group (Scheme 3).
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The palladium catalyzed tryptophan synthesis methodology was utilized to access electron rich and
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electron deficient tryptophan residues. The required aldehyde 28 was prepared from Sꢀglutamic acid in
4 steps, subjected to the palladium catalyzed intramolecular crossꢀcoupling reaction with three 2ꢀ
iodoaniline derivatives(30 to 32) in the presence of palladium acetate as the catalyst and DABCO as the
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base. The reaction yielded 4ꢀnitrotryptophan 33 (50%), 4ꢀmethoxytryptophan 34 (38%) and benꢀ
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zotryptophan 35 (42%). NMR data of all the tryptophan derivatives (33 ꢀ 35) are in good agreement
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with previously reported data (Scheme 4).
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Ar1
HCl H N
Ar2
Ar2
HOBt, DCC, NMM,
THF, 0 °C to rt
O
H
N
OMe
OH
.
2
BocHN
BocHN
OMe
Ar1
+
O
O
O
O
HN
HN
H
N
O
H
O
BocHN
OMe
NH
H
N
N
O
BocHN
OMe
BocHN
OMe
NH
O
O
36 (85%)
37 (80%)
38 (80%)
N
O
H
O
N
H
BocHN
OMe
N
N
BocHN
OMe
O
O
39 (70%)
40 (75%)
Scheme 5. Synthesis of select dipeptides by HOBtꢀDCC coupling
20ꢀ22
Five dipeptides (36 - 40)
were prepared following an FBDD approach to combine the two potent
Phe and Trp structural elements. Using DCCꢀHOBt coupling reaction of corresponding NBoc protected
amino acids and methyl ester hydrochlorides in the presence of Nꢀmethylmorpholine (NMM) as the
base, the desired dipeptides were obtained in good yields (Scheme 5).
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Treatment of 41 with (±)ꢀepibromohydrin in the presence of cesium carbonate as base afforded the
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6
7
8
9
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
1
:1 diastereomeric mixture of epoxyꢀtethered tryptophan 42. Deprotection of 42 by TFAꢀDCM afforded
the trifluoroacetate salt 43 in 80% yield. Similarly, reaction with allyl bromide yielded 5ꢀOꢀallyl tryptoꢀ
phan derivative 44 in 82% yield. Both the Oꢀallyl group and the epoxy residue could serve as reactive
handles for further derivatization (Scheme 6).
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
Reagents and conditions: a) Cs CO , DMF, (±)ꢀEpibromohydrin, 80 °C, 12 h, 68%; b) TFA, DCM, rt, 6
2
3
h, 80%; c) Cs CO , DMF, Allyl bromide, 80 °C, 12 h, 82%.
2
3
Scheme 6. Alkylation of 5ꢀhydroxy tryptophan
After deprotection of the tertꢀbutyl carbamate of tryptophan derivatives by 4 N aq HCl in ethyl acetate,
all of the free amines of the tryptophan derivatives were coupled with MTMSA (2) using PyBop as reaꢀ
1
0
gent and DIPEA as the base. In each occasion, the corresponding MTMSA (2) analogues (45 ꢀ 63)
were obtained in 10 ꢀ 26% yields (Scheme 7, vide SI for HPLC profile, HRMS data and individual
yields of the reaction). The reaction of 43 with MTMSA (2) produced an inseparable mixture of diaꢀ
stereomeric MTMSA (2) coupled 1,2ꢀdiols, due to the opening of the epoxide ring under the reaction
conditions.
To evaluate the antiꢀproliferative properties and selectivity of MTMSA (2) derivatives towards aberrant
ETS transcription factors, such as EWSꢀFLI1, the following screening was performed: In the initial
screen, MTM (1) analogues were tested for 72 h growth inhibition (GI ) in TCꢀ32 cells, a commonly
50
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Journal of Medicinal Chemistry
NHBoc
NH2
NHMTMSA
1
2
3
4
5
6
7
8
9
2
R O C
2
MTMSA (2)
2
R O C
2
4
N aq HCl,
EtOAc
2
R O C
2
PyBOP, TEA, DMF
N
H
10 - 26%
R1
N
N
H
R1
R1
H
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
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
NHMTMSA
CO Me
NHMTMSA
2
NHMTMSA
MeO C
MeO C
MeO C
2
2
2
NHMTMSA
R
N
N
N
N
N
H
N
H
N
R
H
R
45: R = Me
Ph
4
9: R = OMe
50: R = NO2
51: R = O-allyl
52: R = Phenyl
53: R = Allyl
54
4
4
4
6: R = Benzyl
7: R = Allyl
8: R = Prenyl
CO Me
2
CO Me
2
HN
O
H
NHMTMSA
NHMTMSA
CO Me
2
N
MTMSAHN
OMe
NHMTMSA
N
R
N
O
H
H
55: R = CF3
56: R = F
57
58
5
9
O
R N
H
O
N
O
H
MTMSAHN
OMe
NH
H
N
N
MTMSAHN
OMe
O
MTMSAHN
OMe
O
O
N R
61: R = H
62: R = Me
63
6
0
Scheme 7. Synthesis of MTMSA (2) analogues
Table 1. Initial cytotoxicity (GI ) screen in TCꢀ32 (Ewing sarcoma) and PCꢀ3 (nonꢀEwing sarcoma)
5
0
cell line of MTMSAꢀTrp (3) analogues
1
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Page 12 of 43
TCꢀ32
PCꢀ3
1
2
3
4
5
6
7
8
9
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
Ewing sarcoma
Prostate cancer
GI ratio
5
0
PCꢀ3 :
TCꢀ32
Entry MTMSA (2) analogues
EWSꢀFLI1 Type 1
No ETS Translocation
GI (nM)
CI (95%)
GI (nM)
CI (95%)
50
50
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
2
3
1
2
45, R = Me, R = R =
139
2297
113 ꢀ 174
1557
> 10000
3139
1165 ꢀ 2252
11.2
NE*
1.2
4
R = H
1
2
3
46, R = Bn, R = R =
1011 ꢀ 5884
1509 ꢀ 5216
NE*
NE*
2553 ꢀ 9763
NE*
4
R = H
1
2
3
3
47, R = Allyl, R = R
2715
4
=
1
R = H
2
4
48a, R = Prenyl, R =
> 10000
> 10000
2402
> 10000
> 10000
> 10000
2989
NE*
NE*
NE*
3.8
3
4
R = R = H
1
2
5
48b, R = Prenyl, R =
NE*
NE*
3
4
R = R = H
2
1
6
52, R =Phenyl, R =
2160 ꢀ 2652
535 ꢀ 1201
441 ꢀ 2496
1958 ꢀ 3017
NE*
3
4
R = R = H
2
1
3
7
53, R =Allyl, R = R
794
2662 ꢀ 3687
> 2652
> 2046
4
= R = H
3
1
8
54, R = Trizolyl, R =
1030
5878
5.7
2
4
R = R = H
3
1
2
9
55, R = CF , R = R =
2339
2395
1.0
3
4
R = H
3
1
2
4
10
56, R = F, R = R = R
27
25 ꢀ 30
187
617
145 ꢀ 242
571 ꢀ 667
6.9
0.9
= H
4
1
2
11
49, R = OMe, R = R
675
577 ꢀ 790
3
=
R = H
4
1
2
12
50, R = NO , R = R =
353
53
313 ꢀ 408
37 ꢀ 75
646
454
174 ꢀ 2051
275 ꢀ 763
1.8
8.6
2
3
R = H
4
1
13
51, R = OꢀAllyl, R =
2
3
R = R = H
57
14
3505
541
2974ꢀ5602
353 – 834
13 ꢀ 20
25 ꢀ 42
26 ꢀ 38
NE*
> 10000
1605
76
NE*
1106 ꢀ 2714
66 ꢀ 88
NE*
3.0
15
58
1
1
6
7
MTMSAꢀTrp (3)
MTMSAꢀPhe (4)
MTM (1)
16
4.8
32
910
524 ꢀ 1606
62 ꢀ 112
NE*
28.4
2.6
18
32
83
19
MTMSA (2)
> 10000
> 10000
NE*
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Journal of Medicinal Chemistry
*
Regression not estimable
1
2
3
4
5
6
7
8
9
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
used Ewing sarcoma cell line expressing EWSꢀFLI1 sensitive to MTM (1). Analogues with a GI < 250
5
0
nM in the TCꢀ32 cell line were then further tested against PCꢀ3 cells, a prostate cancer cell line lacking
EWSꢀFLI1 expression, also for 72 h growth inhibition (Table 1 and 2). In this assay, MTM (1) disꢀ
played 2.6ꢀfold lower GI in TCꢀ32 cells as compared to PCꢀ3 cells (Table 1). Thus, analogues with
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
>
3ꢀfold selectivity towards TCꢀ32 cell line were considered selective and were further investigated in a
broader panel of cell lines to confirm and validate the selectivity in the context of multiple ETS fusion
or aberrant ETS expression and across multiple cancer types that do not depend on the ETS fusions. In
this secondary screen, analogues were tested in panel of seven additional Ewing sarcoma cell lines that
express the majority of the EWSꢀETS fusions and on the only available prostate cancer cell line (VCaP)
Table 2. Initial cytotoxicity (GI ) screen against TCꢀ32 (Ewing sarcoma) and PCꢀ3 (nonꢀ Ewing sarꢀ
5
0
coma) cell line of MTMSA (2)ꢀdipeptide analogues
TCꢀ32
PCꢀ3
GI ratio
50
CH
3
CH
3
OMe
O HO
O
H
HO
HO
O
N
OMe
AA
O
Ewing sarcoma
Prostate cancer
PCꢀ3 : TCꢀ32
H
O
O
H
3
C
OH OH
CH
O
EWSꢀFLI1 Type 1
No ETS Translocation
HO
O
H
3
C
CH
3
3
HO
O
O HO
O
O
3
H C
OH
Entry
Analogues
GI (nM)
CI (95%)
26 ꢀ 55
GI (nM)
CI (95%)
51 ꢀ 108
578 ꢀ 2225
376 ꢀ 862
NE*
5
0
50
1
2
3
4
59, AA = TrpꢀPhe
60, AA = PheꢀTrp
61, AA = TrpꢀTrp
37
75
1128
2.0
47
41
39 ꢀ 56
25 ꢀ 68
NE*
24.0
13.9
NE*
568
62, AA =
7834
> 10000
NMeTrpNMeTrp
5
6
7
8
63, AA = PheꢀPhe
MTMSAꢀTrp (3)
MTMSAꢀPhe (4)
MTM (1)
232
16
121 ꢀ 448
13 ꢀ 20
25 ꢀ 42
26 ꢀ 38
1132
76
504 ꢀ 2586
66 ꢀ 88
4.9
4.8
32
910
83
524 ꢀ 1606
62 ꢀ 112
28.4
2.6
32
Regression not estimable
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Page 14 of 43
that expresses aberrant ERG. Results were compared to a panel of an additional 8 cancer cell lines that
lack aberrant expression of ETS fusions (Table 3, Figure 2) to identify the most desired analogue. MTM
(1), MTMSAꢀTrp (3) and MTMSAꢀPhe (4) were used as controls.
1
2
3
4
5
6
7
8
9
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
Analogue 45 was designed to evaluate the potential role of the indoleꢀNH. Nꢀmethylation will cut off
the potential hydrogenꢀbonding donation and will increase the hydrophobicity of the indole ring. The Nꢀ
methyl analogue 45 was found to be less active than MTMSAꢀTrp (3) against the TCꢀ32 and PCꢀ3 cell
lines (Table 1; Entry 1), but more selective towards TCꢀ32. The increasing hydrophobicity of 45 probaꢀ
bly makes it more targetꢀspecific than MTMSAꢀTrp (3). Inspired by these results, we synthesized Nꢀ
benzylated analogue 46 to evaluate the effect of an additional aromatic ring. Surprisingly, Nꢀbenzylated
analogue 46 completely lost its activity, against both the TCꢀ32 and the PCꢀ3 cell lines, probably due to
steric hindrance at the binding site (Table 1;
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
Table 3. Ewing sarcoma selectivity index of select MTMSA (2) analogues determined by median cytoꢀ
toxicity (GI ) in Ewing sarcoma cell lines compared to nonꢀEwing sarcoma cell lines
5
0
Entry
MTMSA (2)
Median
Median GI50
in nonꢀEwing
sarcoma cell
lines
Ratio of Median
GI50
Selectivity
Index ratio
GI in
Analogues
50
Ewing
sarcoma
cell lines
nonꢀEwing sarcoma
Analogues :
MTM (1)
:
Ewing sarcoma
(nM)
(
Selectivity Index)
(nM)
1
2
60
52
991
856
152
1644
532
419
1956
71
19.1
15.6
2.4
2.4
1.1
0.9
3.5
1.5
4.7
2.3
12.7
10.4
1.6
1.6
0.7
0.6
2.3
1
61
55
3
59
64
4
45
684
485
466
561
46
5
51
56
6
7
63
8
MTM (1)
MTMSAꢀPhe (4)
MTMSAꢀTrp (3)
9
117
47
545
109
3.1
1.5
10
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Journal of Medicinal Chemistry
1
2
Entry 2). Further modification by Nꢀallylation (cf 47) and Nꢀprenylation (cf 48a ꢀ b) to achieve a secꢀ
ondary interaction with the transcription factor also failed, resulting in complete loss of activity against
both cell lines (Table 1; Entry 3, 4, and 5).
3
4
5
6
7
8
9
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
Analogues 52 and 53 were synthesized to understand the steric requirement of the C7 position of the
tryptophan ring. Both, the C7 phenyl analogue 52 and C7 allyl analogue 53 lost their activity, suggesting
that the C7 position has to remain nonꢀsubstituted to avoid steric hindrance (Table 1; Entry 6, 7). To exꢀ
plore a click chemistry handle and to add a more distant (from the indole) phenyl ring, we designed the
C6 functionalized analogue 54, but it was found to be inactive (Table 1; Entry 8), which discouraged us
to further follow this approach for FBDD expansion.
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
It is well documented that incorporation of CF or F in a drug molecule can strongly affect the binding
3
affinity, pharmacokinetic properties, and bioavailability, mostly by inserting strong Hꢀbond acceptor
sites. The presence of a CF or F substituent in a drug also increases the hydrophilicity and the electronic
3
environment of the molecule, and significantly slows down the oxidative metabolism of the molecule
without altering its size drastically. We found that analogue 55 with Cꢀ6ꢀCF lost its activity completely
3
in both cell lines (Table 1; Entry 9). However, the sterically less demanding Cꢀ6ꢀF analogue 56 was
found to be active in TCꢀ32 cells with a selectivity of 6.9 and thus stood out as one of the potential canꢀ
didates to be further studied and improved (Table 1; Entry 10). The increase in hydrophilicity while
maintaining similar size as the unsubstituted tryptophan (cf 3) could be the reason behind its activity
with improved selectivity, in comparison to 55. Electron rich or deficient tryptophans, 49 and 50, reꢀ
spectively, were both found to be less active than the parent MTMSAꢀTrp (3), suggesting the imꢀ
portance of the steric factors over the electronic nature of the tryptophan ring (Table 1; Entry 11 and 12).
Incorporation of an allylic residue at indoleꢀN (cf. 47) and C7 of the tryptophan ring (cf. 53) resulted in
loss of activity in both the TCꢀ32 and PCꢀ3 cell lines, hence we synthesized analogue 51 with a 5ꢀOꢀ
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Page 16 of 43
allyl tryptophan residue to explore substitution at the 5ꢀposition. Analogue 51 was found to be active in
the TCꢀ32 cell line with a selectivity of 8.6 (Table 1; Entry 13). These results indicate the accessibility
of the C5 over the C7 position to incorporate an additional residue and to further expand the Trp residue
by FBDD, with the ultimate goal to avail a secondary interaction with EWSꢀFLI1. Analogue 57, with an
additional fused benzene ring added to tryptophan, documented a ~ 34 fold decrease in activity in the
TCꢀ32 cell line, once more restricting the steric requirements for interaction with EWSꢀFLI1, and thus
wiped out the possibility of adding πꢀdonation directly to the tryptophan ring (Table 1; Entry 14). Simiꢀ
larly, analogue 58, where the indole ring was replaced by a naphthalene ring, lost activity in the TCꢀ32
cell line, which again showed the importance of the indole ring for interactions with EWSꢀFLI1 (Table
1
2
3
4
5
6
7
8
9
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
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
; Entry 15).
Dipeptide analogues 59 - 63 were tested in TCꢀ32 cell lines to validate the FBDD concept in the most
straightforward way, combining Phe and Trp. The fact that the Nꢀmethyl tryptophan analogue 45
showed a ~ 4 fold increase in selectivity (Table 1; Entry 1) impelled us to include TrpꢀNꢀmethylation
into this series of analogues. Analogue MTMSAꢀTrpꢀPhe (59) was found to have similar activity as
MTMSAꢀTrp (3), but was less selective, while MTMSAꢀPheꢀTrp (60), with the opposite arrangement
of the amino acid residues, showed significantly increased selectivity compared to 3 (Table 2; Entry 1
and 2). Likewise, analogue MTMSAꢀTrpꢀTrp (61), which contains two consecutive tryptophan moieties,
was found to have much better selectivity than MTMSAꢀTrp (3) (Table 2; Entry 3). Both 60 and 61 are
about equally active in the TCꢀ32 cell line (as 3). This shows the importance of the Trp residue to be in a
more distant position from the DNAꢀinteracting MTM (1) core, to interact with the transcription factor
and thus may account for the observed improved selectivity towards the EWSꢀFLI1 expressing TCꢀ32
cell line over the PCꢀ3 cell line. It is noteworthy that the diꢀNꢀmethyl analogue of 61 (cf 62) lost its acꢀ
tivity completely, which indicates the importance of the beta indoleꢀNH for the secondary interaction
with EWSꢀFLI1 (Table 2; Entry 4). The loss of activity against TCꢀ32 of analogue 63 indicates that a
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Journal of Medicinal Chemistry
second phenylalanine residue is not advantageous (Table 2; Entry 5). These studies concluded that a
tryptophan moiety in the more distant, second position of the analogues is crucial, and increases drastiꢀ
cally the selectivity towards EWSꢀFLI1 while maintaining reasonable cytotoxic activity.
1
2
3
4
5
6
7
8
9
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
Overall, the above described initial screen identified 5 novel MTMSA (2) analogues with cytotoxicity
(
GI ) in TCꢀ32 cells of less than 250 nM, and selectivity against TCꢀ32 cells greater than 3 times of that
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
of PCꢀ3, determined by PCꢀ3 : TCꢀ32 GI ratio (Table 1 and 2, highlighted in red). To deepen these
5
0
findings and to further investigate the most promising analogues, we expanded the array of cancer cell
lines to include a panel of 8 Ewing sarcoma cell lines (expressing aberrant ETS transcription factors)
versus 9 nonꢀEwing sarcoma cell lines (lacking aberrant ETS transcription factors) (Figure 2, vide SI).
10000
1000
100
10
Figure 2. Median GI of select MTMSA (2) analogues in a panel of 8 Ewing sarcoma (TCꢀ32, 5838,
5
0
RDꢀES, TCꢀ71, Aꢀ673, ESꢀ2, ESꢀ7, ESꢀ8) as well as 9 nonꢀEwing sarcoma (PCꢀ3, DU 145, A549,
LNCaP, Uꢀ118 MG, HeLa, HCT116, DMS 114, PANCꢀ1) cell lines.
17
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Despite of initial lack in selectivity and cytotoxicity, we included analogues 59 and 63, respectively, to
get a complete overview of dipeptide analogues. MTM (1), MTMSAꢀTrp (3) and MTMSAꢀPhe (4) were
used as controls. The final selectivity ratio was calculated by taking the median GI of the nonꢀEwing
1
2
3
4
5
6
7
8
9
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
5
0
sarcoma panel over the Ewing sarcoma panel (Table 3, Figure 2). The median is presented as we were
not able to estimate the GI (exceeded 10µM), which precludes the estimation of a mean value. Of the
5
0
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
7
novel MTMSA (2) analogues that were tested on this secondary screen, only 3 were found to maintain
selectivity greater than 3 against the panel of Ewing sarcoma cell lines versus nonꢀEwing sarcoma cell
lines, namely 60, 61 and 63. The resulting Ewing sarcoma selectivity index ranked as follows: 60 (19.1)
>
61 (15.6) > > MTMSAꢀPhe (4) (4.7) > 63 (3.5) > 59 (2.4) = 45 (2.4) > MTMSAꢀTrp (3) (2.3) > MTM
(1) (1.5) > 51 (1.1) > 56 (0.9) (Table 3, Figure 2).
Taking both Ewing sarcoma cytotoxicity (GI ) and selectivity index into account, a potency order of
5
0
MTMSA (2) analogues was determined to be 60 > 61 > > MTMSAꢀTrp (3) = MTMSAꢀPhe (4) > MTM
(1). Several initially promising MTMSA (2) analogues (cf 45, 51, 56, and 63) were eliminated in the
secondary screen when tested against the panel of Ewing sarcoma cell lines because of their poor cytoꢀ
toxicity (GI > 250 nM). In contrast, analogue 59 was cytotoxic (median GI of 64), but eliminated
5
0
50
due to poor selectivity (selectivity index < 3) (Table 3, Figure 2). Interestingly and somewhat surprisingꢀ
ly, MTMSAꢀPhe (4) had a three times better ETS selectivity index of 4.7 than MTMSAꢀTrp (3, selectivꢀ
ity index 1.5), which initially was the lead molecule in this study (Table 3, Figure 2). Based on these
cytotoxicity screens, the overall best candidates for further development are analogues 60 and 61, with a
median ETS cytotoxicity of (GI ) 52 nM and 55 nM, respectively, and a drastically improved selectiviꢀ
5
0
ty of > 10ꢀfold towards ETS depended cell lines, in comparison to MTM (1) (Table 3, Figure 2).
Previous modification of MTM (1) leading to the identification of ECꢀ8105 with improved suppresꢀ
sion of EWSꢀFLI1 by almost 10ꢀfold, focused on the introduction of an allyl carbonate residue in the 3B
24
position of the disaccharide residue of MTM (1) (Figure 1). However, from our previous study of
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Journal of Medicinal Chemistry
DNAꢀMTMSAꢀTrp (3) and DNAꢀMTMSAꢀPhe (4) crystal structures, it was anticipated that the 3ꢀside
1
2
3
4
5
6
7
8
9
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
1
2
chain can interact with FLI1 DNA binding domain. Therefore, the identification of MTMSAꢀPheꢀTrp
(60) and MTMSAꢀTrpꢀTrp (61) with improved selectivity of 19.1 and 15.6, respectively, cemented our
initial hypothesis that adding an additional tryptophan residue to MTMSAꢀPhe (4) and MTMSAꢀTrp (3)
at the 3ꢀside chain position would enhance their interaction with EWSꢀFLI1.
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
Additionally, we tested all 7 MTMSA (2) analogues in VCaP cells, which overexpress the TMPRSS2ꢀ
ERG gene fusion, a common genomic alteration harbored by prostate cancer cells. We initially expected
that they would follow a similar selectivity trend to 5838 cells, which overexpress the more rare EWSꢀ
ERG gene alteration in Ewing sarcoma. Our results indeed show that analogues 60 and 61 are the most
selective for VCaP cells over other prostate cancer cell lines, with a selectivity ratio of 25.4 and 18.3,
respectively (Table 4), consistent with their Ewing sarcoma selectivity index (cf Table 3, Figure 2).
Moreover, these analogues were more selective than MTM (1) for 5838 cells, however, not in the exact
same rank order (Table 4).
Table 4. GI and selectivity index of select MTMSA (2) analogues in cancer cells expressing aberrant
5
0
ERG transcription factors.
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Entry MTMSA
(2)
VCaP
Median
Ratio of
5838
Ratio of
median GI50
NonꢀEwing
sarcoma cell
lines (cf
1
2
3
4
5
6
7
8
9
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
GI in median GI50
Prostate Cancer
50
Ewing Sarcoma
Analogues
prostate
cell
lines,
lacking
TMPR
SS2ꢀ
Prostate
cells lacking
TMPRSS2ꢀ
ERG :
TMPRSS2ꢀERG
EWSꢀERG
GI50 CI (95%)
(nM)
GI (nM)
CI
(95%)
50
Table 3) :
5838
VCaP
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
ERG
1
60
39
34 ꢀ 45
991
25.4
149
121 ꢀ
6.7
186
2
3
4
61
59
45
31
21
25 ꢀ 37
12 ꢀ 34
568
75
18.3
3.6
53
39
40 ꢀ 70
25 ꢀ 60
98 ꢀ
16.2
3.9
307 87 ꢀ 1430
1557
5.1
121
13.3
151
5
6
51
56
485 371 ꢀ 645
319 235 ꢀ 457
454
187
0.9
0.6
24
20 ꢀ 29
229 ꢀ
22.2
0.9
466
9
29
350 ꢀ
66
7
63
442 377 ꢀ 513
1132
2.6
579
3.4
9
8
9
MTM (1)
41
32 ꢀ 52
48
1.2
4.9
43
38
30 ꢀ 61
30 ꢀ 49
1.7
MTMSAꢀ 150
70 ꢀ 321
732
14.3
Phe (4)
10
MTMSAꢀ
60
45 ꢀ 80
76
1.3
6
4 ꢀ 10
18.2
Trp (3)
Since it is recognized that MTM displaces Sp1 from DNA and likely affects the expression of Sp1 tarꢀ
get genes (e.g., BCLꢀ2), we performed qRTꢀPCR to determine the effect of MTM (1) and MTMSA anaꢀ
logue treatment on the expression of those genes as well as on EWSꢀFLI1 and its target gene NR0B1.
The expression of EWSꢀFLI1 was reduced to approximately 70 ꢀ 75% after 6 h and then further reduced
after 12 h treatment (Figure 3A). The NR0B1 promoter contains a microsatellite region of GGAA reꢀ
peats of DNA that binds to EWSꢀFLI1 for transcriptional regulation, through the conserved ETS binding
25
domain of FLI1. NR0B1 mRNA expression was not affected by MTM (1) at the GI at 6 and 12 h, but
5
0
was reduced to ~75% after 6 h of treatment with MTMSA analogues, and it was further reduced after 12
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Journal of Medicinal Chemistry
h (Figure 3B). In contrast both Sp1 and BCLꢀ2 mRNA expression was reduced with MTM (1) and
1
2
3
4
5
6
7
8
9
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
MTMSA analogues treatments after 6 and 12 h (Figure 3C and 3D). Previously it was reported that 6 h
2
6
treatment with 100 nM MTM (1) had no effect on mRNA expression of CCK. Therefore, we used this
gene as a negative control. Our results also showed no change in mRNA expression of CCK after 6 h
treatment at the GI (32 nM, ref. Table 1) for MTM (1), and minimal effect with MTMSA analogues.
5
0
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
However, after 12 h, CCK mRNA expression is reduced to ~60% for both MTM (1) and MTMSA anaꢀ
logues (Figure 3E).
Figure 3. mRNA expression of (A) EWSꢀFLI1 and associated gene, (B) NR0B1, are decreased after 6
and 12 h treatments with MTMSA analogues at respective GI , analyzed by qRTꢀPCR. mRNA expresꢀ
50
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sion of (C) Sp1, a wellꢀknown downstream target of MTM (1), and associated gene, (D) BCLꢀ2, were
also analyzed. mRNA expression of (E) CCK was analyzed as a negative control, previously reported as
1
2
3
4
5
6
7
8
9
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
2
6
unaffected after 6 h of treatment with MTM at 100 nM. Relative expressions were calculated using
GAPDH expression.
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
To further investigate the interference of MTM (1) and MTMSA analogues with the binding of ETS
transcription factors, we developed cell lines that express luciferase under the control of EWSꢀFLI1.
TCꢀ32 cells, which express EWSꢀFLI1, were stably transfected with a luciferase reporter vector und the
control of the full length NR0B1 promoter. Cells were treated for 12 h with multiple concentrations beꢀ
tween 0 and 10 µM. All MTMSA analogues decrease luciferase expression to 50% (Figure 4A). Addiꢀ
tionally, a luciferase reporter vector under the control of a CMV promoter was tested as a nonꢀspecific
control. None of the analogues reduced CMV driven luciferase signal at the range of their GI concenꢀ
5
0
trations. MTM (1) and analogue 59 decrease CMV driven luciferase expression down to ~50% and
75%, respectively, at concentrations between 1 and 10 µM, while analogues 60 and 61 had no effect at
~
all, even when treated up to 10 µM (Figure 4B).
Figure 4. (A) MTMSA analogues decrease the expression of luciferase controlled by NR0B1 promotꢀ
er, a validated binding promoter of EWSꢀFLI1. TCꢀ32 cells, expressing EWSꢀFLI1, were stably transꢀ
fected with luciferase reporter vectors and treated for 12 h. (B) MTMSA analogues 60 and 61 did not
decrease luciferase expression controlled by a nonꢀspecific CMV promoter.
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Journal of Medicinal Chemistry
1
2
3
4
5
Lack of effect of analogue MTMSAꢀPheꢀTrp (60) and MTMSAꢀTrpꢀTrp (61) on CMV promoter drivꢀ
en transcription while maintaining activities against EWSꢀFLI1 mediated transcription support the obꢀ
served selectivity in our cytotoxicity assays (cf. Table 3, Figure 2). Furthermore, MTMSAꢀTrpꢀPhe (59),
which reduces CMV promoter driven transcription, lacks selectivity in our cytotoxicity assays (cf. Table
3, Figure 2). This supports the conclusions made after the initial screening (Table 2), namely that a Trp
residue in a second, more distant position from the MTM (1)ꢀDNA binding core, is crucial for an interꢀ
action with the EWSꢀFLI1 transcription factor. The importance of the Phe or the Trp moiety in the first
position of the MTMSAꢀ3ꢀside chain and the impact of an additional Trp residue (cf MTMSA (2) tripepꢀ
tide analogues) remains to be investigated.
6
7
8
9
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
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
CONCLUSIONS
This study was aimed on refinement of two amino acid derivatives of MTMSA (2), namely, MTMSAꢀ
Trp (3) and MTMSAꢀPhe (4), which both showed promising activity and increased selectivity in a preꢀ
liminary cytotoxicity assay looking at effects on cell lines overexpressing aberrant ETS transcription
factors. This was expected, since crystallographic studies investigating DNAꢀFLI1 interactions and the
DNA binding modalities of the MTMSAꢀPhe (4) and MTMSAꢀTrp (3) analogues resulted in a new
modeꢀofꢀaction hypothesis of MTM (1) and these derivatives: a ternary complex of MTM (1)ꢀDNAꢀ
FLI1 (similarly MTM (1)ꢀDNAꢀERG in the context of prostate cancer), in which the MTM (1) analogue
binds to the minor groove of certain DNA microsatellites, with its core and trisaccharide side chain, and
simultaneously to the FLI1 portion of EWSꢀFLI1 within the major groove of DNA. Aromatic residues
of MTMSA (2) analogues are well poised to increase protein binding. Initially, the focus was on refining
the Trp residue, since MTMSAꢀTrp (3) showed better cytotoxicity than MTMSAꢀPhe (4) (Table 3). The
array of MTMSAꢀTrp (3) derivatives was achieved through iridiumꢀcatalyzed borylation and palladium
catalyzed tryptophan syntheses. The borylated tryptophans were further used to diversify the indol ring
2
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of tryptophan, before the PyBop coupling reaction with MTMSA (2) to generate sterically and electronꢀ
ically different MTMSAꢀTrp (3) analogues.
1
2
3
4
5
6
7
8
9
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
Later, the studies were expanded to a fragment based drug development (FBDD) approach, to comꢀ
bine the protein interactive aromatic rings of Trp and Phe, which both appeared to be advantageous eleꢀ
ments to increase selectivity towards ETS fusion expressing cell lines. These studies started with pheꢀ
nylꢀexpanded Trp derivatives, in which the indole of Trp was expanded by an aromatic ring, or through
phenylꢀTrp derivatives, and ended with dipeptide analogues of MTMSA (2), with all combinations of
dipeptide side chains, namely MTMSAꢀTrpꢀPhe (59), MTMSAꢀPheꢀTrp (60), MTMSAꢀPheꢀPhe (63)
and MTMSAꢀTrpꢀTrp (61).
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
Initially all analogues were tested in the Ewing sarcoma TCꢀ32 cell line that expresses EWSꢀFLI1
type I, the genotype in the majority of Ewing sarcoma patients. For target specificity analysis, these anaꢀ
logues were also tested in prostate PCꢀ3 cell line that lacks dependence on aberrant ETS transcription
factors. These comparative tests resulted in the identification of 7 MTMSA analogues with increased
specificity towards the Ewing sarcoma cell line (Table 1 and 2). As a result they were further evaluated
against multiple Ewing sarcoma cell lines, as well as VCaP prostate cancer cell line, all of whom exꢀ
press various aberrant ETS transcription factors. This was compared to a panel of cell lines lacking exꢀ
pression of aberrant ETS transcription factors. Analogues 60 and 61 were found to have cytotoxic activiꢀ
ties comparable to MTM (1), but showed > 10ꢀfold increased selectivity towards aberrant ETS dependꢀ
ent cell lines (Table 3 and 4, Figure 2). To further investigate if this selectivity is a result of activity
against aberrant ETS transcription factors we looked at the effects on EWSꢀFLI1 and target gene
NR0B1 mRNA expression with analogue treatment. Furthermore, we tested a luciferase reporter vector
controlled by NR0B1 promoter, which binds to EWSꢀFLI1. We found that analogues 60 and 61 deꢀ
creased EWSꢀFLI1 and NR0B1 mRNA expression, as well as NR0B1 promoter driven luciferase signal
(Figure 3A and 3B, Figure 4A). However, analogue 60 and 61 did not decrease nonꢀspecific CMV proꢀ
moter driven luciferase signal (Figure 4B), correlating well with the observed selectivity in cytotoxicity
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Journal of Medicinal Chemistry
assays (Table 3, Figure 2). This is in comparison to MTM (1) and analogue 59, which decreased nonꢀ
specific CMV promoter driven luciferase signal (Figure 4B), and therefore had no observed selectivity
in cytotoxicity assays (Table 3, Figure 2).
1
2
3
4
5
6
7
8
9
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
This expanded study confirmed that the tryptophan ring in second position of the 3ꢀside chains of anaꢀ
logue 60 and 61 may play a significant role to afford better selectivity against ETS dependent cell lines.
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
EXPERIMENTAL SECTION
Chemistry
General
All commercial reagents were used without further purification. The required amine for the synthesis
of 56 and 58 were prepared by protecting the commercially available corresponding tryptophan in the
presence of thionyl chloride in methanol. Solvents were dried and distilled following the standard proꢀ
cedures. TLC was carried out on preꢀcoated plates (Merck silica gel 60, GF254), and the spots were visꢀ
ualized with UV, fluorescent light or by charring with phosphomolybdic acid hydrate (PMA). Column
1
13
chromatography was performed on silica gel (230ꢀ400 mesh). H and C NMR spectra for the comꢀ
1
13
pounds were recorded with Varian 400 or 600 MHz spectrometers. H and C chemical shifts are reꢀ
ported in ppm downfield of tetramethylsilane and referenced to residual solvent peaks (CHCl ; δ =
3
H
7
.26 and δ = 77.23, d ꢀMeOH; δ = 3.31 and δ = 49.1). Multiplicities are reported using the following
C 4 H C
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad resonance, ap =
apparent. The phrase ‘usual work up’ or ‘worked up in usual manner’ refers to washing of the organic
phase with water (2 × 1/4 the volume of organic phase) and brine (1 × 1/4 the volume of organic phase),
and drying (anhydrous Na SO ), filtration, and concentration under reduced pressure. Yields referred to
2
4
isolated yields after purification. Analytical LC/MS was performed on a Waters 2965 (Kinetex 5U EVO
C18 100A , 250 × 4.6 mm, a linear gradient from A/B 75:25 to 30:70 (20 min), 30:70 to 0:100 (2 min),
0
:100 (2 min, ); 0:100 to 75:25 (2 min), 75:25 (4 min) (A = H O + 0.1% formic acid, B = MeCN + 0.1%
2
formic acid), flow rate 0.5 mL/min) equipped with an Waters ZQ 2000 mass spectrometer and Waters
2
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2
996 photodiode array detector. The purity of all analogues used in the bioassays was determined by
1
2
3
4
5
6
7
8
9
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
this method to be >95%. Mass spectra were taken on ABSciex QTOF mass spectrometer.
General Procedure A: Nꢀalkylation of 6
To a stirred solution of 6 (1 equiv) in dry DMF was added 60% NaH (1.5 equiv) in portion at 0 °C and
stirred for 30 min after which respective halide (1.5 equiv) was added. The reaction mixture was stirred
at rt for 12 h, cooled to 0 °C and quenched with methanol. It was diluted with ethyl acetate, worked up
in usual manner, and subjected to column chromatography with silica gel using 20% ethyl acetate in
hexane as eluent.
0
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4
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6
7
8
9
0
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9
0
General Procedure for the deprotection of phthalimide:
To a stirred solution of phtalimide (cf. 7 to 10) (1 equiv) in MeOHꢀDCM (1 : 1; 10 mL) was added
hydrazine hydrate (1.5 equiv) and the reaction mixture was stirred at rt for 24 h. It was then filtered
through celite, washed with ethyl acetate and concentrated under reduced vacuum. It was purified in reꢀ
verse phase silica using 20% acetonitrile in water as mobile phase to obtain the free amine (cf. 11 to 14)
in 45 ꢀ 60% yield. The free amine was only characterized by LCMS and directly used without further
purification in the PyBop coupling reaction with MTMSA (2).
General Procedure B: Procedure of synthesis of tryptophan from aldehyde 28
A mixture of oꢀiodoaniline (1.1 equiv), aldehyde 28 (1.0 equiv), and DABCO (3 equiv) in dry DMF
was degassed for 30 min using argon. Pd(OAc) (5 mol%) was added to the reaction, and the resulting
2
reaction mixture was heated at 85 °C in a pressure tube for 24 h. The reaction mixture was cooled to
room temperature, diluted with water, extracted with ethyl acetate and worked up in usual manner. The
crude product was purified by flash column chromatography to obtain the corresponding tryptophan deꢀ
rivative.
2
0
General Procedure C: Procedure for HOBtꢀDCC coupling reaction
To a solution of NBocAAꢀOH (1 equiv) in dry THF were added HOBt (1.2 equiv) and DCC (1.2
.
equiv) and stirred at 0 °C for 1 h. Then HCl AAꢀOMe (1.1 equiv) was added to the reaction mixture and
2
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Journal of Medicinal Chemistry
pH was adjusted in between 8 ꢀ 9 by adding excess NMM. The reaction mixture was stirred for 12 h at
room temperature, filtered and concentrated under reduced pressure. The residue was dissolved in ethyl
1
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7
8
9
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6
acetate, washed with saturated aq NaHCO and worked up in usual manner. The crude product was puriꢀ
3
fied by flash column chromatography to obtain the corresponding protected dipeptide.
General Procedure D: Procedure for PyBop coupling reaction
0
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2
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7
8
9
0
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9
0
To a stirred solution of MTMSA (2) (1 equiv) in dry DMF were added PyBop (1.5 equiv), free amine
(3.0 equiv) and excess triethyl amine (adjusted to pH 8). The reaction mixture was stirred at room temꢀ
perature under argon atmosphere until disappearance of MTMSA (2). It was quenched by adding satuꢀ
rated NaCl solution and extracted with nꢀBuOH. The organic fraction was collected and concentrated
under reduced pressure and purified by HPLC to obtain pure MTMSA (2) analogues.
Methyl (S)ꢀ3ꢀ(1ꢀbenzylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)propanoate (8)
Compound 8 (512 mg, 70%) was prepared as a yellow semisolid by Nꢀbenzylation of 6 (581 mg, 1.67
mmol) with benzyl bromide (0.3 ml, 2.5 mmol) using 60% NaH (100 mg, 2.5 mmol) as base, following
1
the general procedure A. H NMR (400 MHz, CDCl ) δ 7.75 – 7.73 (m, 2H), 7.67 – 7.62 (m, 3H), 7.17 –
3
7
2
1
4
.05 (m, 6H), 6.93 (s, 1H), 6.89 – 6.86 (m, 2H), 5.30 (dd, J = 9.9, 6.4 Hz, 1H), 5.16 (ABq, J = 14.8 Hz,
1
3
H), 3.81 (s, 3H), 3.79 – 3.75 (m, 2H).; C NMR (100 MHz, CDCl ) δ 169.8, 167.6, 137.6, 136.6,
3
34.1, 131.8, 128.7, 128.0, 127.5, 127.0, 126.6, 123.5, 122.1, 119.5, 118.9, 110.3, 109.8, 53.0, 52.8,
+
9.9, 24.9.; HRMS (TOF MS ES+) m/z calcd for C H N O [M+H] 439.1659, found 439.1638.
27
23
2
4
Methyl (S)ꢀ3ꢀ(1ꢀallylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)propanoate (9)
Compound 9 (400 mg, 85%) was prepared as a yellow semisolid by Nꢀallylation of 6 (425 mg, 1.22
mmol) with allyl bromide (0.16 ml, 1.83 mmol) using 60% NaH (75 mg, 1.83 mmol) as base, following
1
the general procedure A. H NMR (400 MHz, CDCl ) δ 7.76 – 7.73 (m, 2H), 7.66 – 7.60 (m, 2H), 7.61
3
(d, J = 7.9 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.13 (t, J = 8.0, 1H), 7.05 (t, J = 8.4, 1H), 6.88 (s, 1H),
5
.83 – 5.74 (m, 1H), 5.27 (t, J = 7.6 Hz, 1H), 4.97 – 4.93 (m, 1H), 4.78 – 4.73 (m, 1H), 4.62 – 4.50 (m,
1
3
2H), 3.80 (s, 3H), 3.75 (d, J = 8.1 Hz, 2H).; C NMR (100 MHz, CDCl ) δ 169.8, 167.6, 136.4, 134.1,
3
2
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1
33.5, 131.9, 127.9, 126.6, 123.5, 121.9, 119.3, 118.8, 116.7, 110.1, 109.7, 53.0, 52.8, 48.5, 24.9.;
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
+
HRMS (TOF MS ES+) m/z calcd for C H N O [M+H] 389.1501, found 389.1488.
2
3
21
2
4
Methyl (R)ꢀ2ꢀ(tertꢀbutoxycarbonyl)ꢀ3ꢀ(7ꢀiodoꢀ1Hꢀindolꢀ3ꢀyl)propanoate (18)
To a solution of 15 (740 mg, 1.67 mmol) in methanol (15 mL) in a pressure tube, were added CuI (35
mg, 0.183 mmol, 10.0 mol%), 1,10ꢀphen (60.0 mg, 0.34 mmol, 20.0 mol%) and KI (420 mg, 2.53
mmol, 1.50 equiv). The mixture was stirred at room temperature, water (3.5 mL) was added and it was
sealed under air. The mixture was heated at 80 °C for 2 h, cooled to room temperature and diluted with
0
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2
3
4
5
6
7
8
9
0
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2
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7
8
9
0
1
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8
9
0
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7
8
9
0
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6
7
8
9
0
water (40 ml). It was extracted with Et O (3 × 35 mL) and worked up in usual manner. The crude comꢀ
2
pound was subjected to column chromatography with silica gel using 30% ethyl acetate in hexane as
1
eluent to obtain 18 as a white solid. H NMR (400 MHz, CDCl ) δ 8.19 (s, 1H), 7.54 (apt, J = 8.4 Hz,
3
2
H), 7.06 (s, 1H), 6.88 (t, J = 7.7 Hz, 1H), 5.08 (d, J = 8.2 Hz, 1H), 4.64 (dd, J = 12.8, 6.0 Hz, 1H), 3.67
1
3
(s, 3H), 3.30 – 3.20 (m, 2H), 1.42 (s, 9H).; C NMR (100 MHz, CDCl ) δ 172.7, 155.3, 138.1, 131.0,
3
1
28.0, 123.3, 121.5, 119.1, 112.1, 80.1, 76.1, 54.3, 52.5, 28.52, 28.49.; HRMS (TOF MS ES+) m/z calcd
+
for C H IN O [M+H] 445.0625, found 445.0620.
1
7
22
2
4
Methyl (R)ꢀ3ꢀ(7ꢀallylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(tertꢀbutoxycarbonyl)propanoate (17)
To a stirred solution of 18 (115 mg, 0.29 mmol) in dry toluene (5 ml), were added allyl tributyltin
(0.13 ml, 0.4 mmol) and tetrakis(triphenylphosphine)palladium(0) (70 mg, 0.06 mmol, 20 mol%). The
reaction mixture was refluxed under argon atmosphere for 24 h, cooled to room temperature and diluted
with ethyl acetate (50 ml). After usual work up followed by column chromatography with silica gel usꢀ
1
ing 25% ethyl acetate in hexane as eluent obtained 17 (56 mg, 60%) as a yellow liquid. H NMR (400
MHz, CDCl ) δ 8.17 (s, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.03 – 6.97 (m, 2H), 6.11
3
–
6.01 (m, 1H), 5.28 – 5.13 (m, 2H), 5.07 (d, J = 8.2 Hz, 1H), 4.64 (q, J = 6.3 Hz, 1H), 3.68 (s, 3H),
1
3
3.62 (d, J = 6.5 Hz, 2H), 3.28 (dd, J = 5.6, 2.6 Hz, 2H), 1.42 (s, 9H).; C NMR (100 MHz, CDCl ) δ
3
1
2
73.0, 155.4, 136.9, 135.7, 122.7, 122.5, 122.3, 120.1, 117.4, 116.6, 110.7, 80.0, 54.4, 52.4, 37.0, 28.5,
+
8.2.; HRMS (TOF MS ES+) m/z calcd for C H N O [M+H] 359.1971, found 359.1965.
2
0
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Methyl (R)ꢀ2ꢀ(tertꢀbutoxycarbonyl)ꢀ3ꢀ(7ꢀphenylꢀ1Hꢀindolꢀ3ꢀyl)propanoate (20)
1
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5
To a stirred solution of 18 (160 mg, 0.36 mmol) in dry toluene (5 ml), were added iodobenzene (70
µL, 0.6 mmol), K PO (180 mg, 0.84 mmol), Sphos (18 mg, 0.044 mmol, 12 mol%), and
3
4
6
7
8
9
1
1
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1
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5
5
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5
6
tris(dibenzylideneacetone)dipalladium(0) (20 mg, 0.02 mmol, 5 mol%). The reaction mixture was heatꢀ
ed under argon atmosphere at 80 °C for 16 h, cooled to room temperature and diluted with ethyl acetate
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
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3
4
5
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7
8
9
0
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3
4
5
6
7
8
9
0
(50 ml). Work up in usual manner followed by column chromatography with silica gel using 20% ethyl
1
acetate in hexane as eluent obtained 17 (100 mg, 70%) as a yellow liquid. H NMR (400 MHz, CDCl )
3
δ 8.34 (s, 1H), 7.62 (d, J = 7.2 Hz, 2H), 7.56 – 7.48 (m, 3H), 7.39 (t, J = 7.2 Hz, 1H), 7.22 – 7.19 (m,
2H), 7.02 (s, 1H), 5.11 (d, J = 8.2 Hz, 1H), 4.66 (q, J = 6.0 Hz, 1H), 3.70 (s, 3H), 3.37 – 3.27 (m, 2H),
1
3
1
1
.44 (s, 9H).; C NMR (100 MHz, CDCl ) δ 172.9, 155.4, 139.2, 134.2, 129.3, 128.4, 128.3 127.7,
3
25.9, 123.2, 122.3, 120.4, 118.3, 110.9, 80.0, 54.4, 52.5, 28.5, 28.3.; HRMS (TOF MS ES+) m/z calcd
+
for C H N O [M+H] 395.1971, found 395.1965.
2
5
26
2
4
Methyl (R)ꢀ3ꢀ(6ꢀazidoꢀ1ꢀ(triisopropylsilyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(tertꢀbutoxycarbonyl)propanoate (24,
mixture of C6 and C5 isomers, major C6 isomer)
To a solution of 22 (244 mg, 0.41 mmol) in methanol (5 ml), were added sodium azide (40 mg, 0.61
mmol) and copper(II)acetate monohydrate (8.2 mg, 0.041 mmol, 10 mol%). The reaction mixture was
heated at 55 °C under air for 12 h, diluted with ethyl acetate and worked up in usual manner. Purificaꢀ
tion by column chromatography with silica gel using 25% ethyl acetate in hexane as eluent obtained 24
1
(
147 mg, 70%) as a yellow liquid as an inseparable mixture of two isomers. H NMR (400 MHz, CDCl ,
3
Major C6ꢀazido isomer ) δ 7.49 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 1.9 Hz, 1H), 7.00 (s, 1H), 6.86 (dd, J =
8.5, 1.9 Hz, 1H), 5.09 (d, J = 8.2 Hz, 1H), 4.64 (q, J = 5.6 Hz, 1H), 3.63 (s, 3H), 3.26ꢀ 3.22 (m, 2H),
1
.68 ꢀ 161 (m, 3H), 1.43 (s, 9H), 1.13 (d, J = 6.0 Hz, 18H).; HRMS (TOF MS ES+) m/z calcd for
+
C H N O Si [M+H] 516.3006, found 516.3001.
26
42
5
4
Methyl
(2R)ꢀ2ꢀ(tertꢀbutoxycarbonyl)ꢀ3ꢀ(6ꢀ(4ꢀphenylꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)ꢀ(triisopropylsilyl)ꢀ1Hꢀ
indolꢀ3ꢀyl)propanoate (25, mixture of C6 and C5 isomers, major C6 isomer)
2
9
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