Novel diphenylamine 2,4′-dicarboxamide based azoles as potential epidermal growth factor receptor inhibitors: Synthesis and biological activity

Article abstract of DOI:10.1248/cpb.59.1124

Several hybrid molecules of diphenylamine-2,4′-dicarboxamide with various azolidinones and related heterocyclic rings have been synthesized and explored as epidermal growth factor receptor (EGFR) kinase inhibitors. Most of them displayed promising in vitro tyrosine kinase inhibition as well as potent cellular antiproliferative activity in the EGFR over-expressing breast cancer cell line (MCF-7). Compounds 12b and 13b that exhibited the highest inhibition in the kinase assay (89, 81% inhibition at 10 μM, respectively), showed potent antiproliferative effect against MCF-7 tumor cell line (IC₅₀ 1.04, 0.91 μM respectively). Molecular docking studies revealed that these compounds can bind to ATP binding site of the EGFR kinase domain and were involved in H-bonding with Met 793, in analogy to the known EGFR tyrosine kinase inhibitors. Moreover, compounds 15a-c possessed profound antitumor activity (IC₅₀ 0.59-0.73 μM) and significant EGFR-TK inhibition, making them of particular interest. In summary, the newly synthesized compounds provide promising new lead for the future design and development of anticancer agents of potential EGFR-TK inhibitory activity.

Full text of DOI:10.1248/cpb.59.1124

1124
Regular Article
Chem. Pharm. Bull. 59(9) 1124—1132 (2011)
Vol. 59, No. 9
ꢀ
Novel Diphenylamine 2,4 -Dicarboxamide Based Azoles as Potential
Epidermal Growth Factor Receptor Inhibitors: Synthesis and Biological
Activity
a
Sahar Mahmoud ABOU-SERI, ,a Nahla Ahmed FARAG,b and Ghaneya Sayed HASSAN
*
a Pharmaceutical Chemistry Department, Faculty of Pharmacy Cairo University; Kasr El-Aini Street, Cairo, P.O. Box
11562, Egypt: and b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University; Cairo,
P.O. Box 1, Heliopolis, Egypt. Received April 28, 2011; accepted June 22, 2011
Several hybrid molecules of diphenylamine-2,4ꢀ-dicarboxamide with various azolidinones and related hete-
rocyclic rings have been synthesized and explored as epidermal growth factor receptor (EGFR) kinase inhibitors.
Most of them displayed promising in vitro tyrosine kinase inhibition as well as potent cellular antiproliferative
activity in the EGFR over-expressing breast cancer cell line (MCF-7). Compounds 12b and 13b that exhibited the
highest inhibition in the kinase assay (89, 81% inhibition at 10 mM, respectively), showed potent antiproliferative
effect against MCF-7 tumor cell line (IC50 1.04, 0.91 mM respectively). Molecular docking studies revealed that
these compounds can bind to ATP binding site of the EGFR kinase domain and were involved in H-bonding with
Met 793, in analogy to the known EGFR tyrosine kinase inhibitors. Moreover, compounds 15a—c possessed pro-
found antitumor activity (IC50 0.59—0.73 mM) and significant EGFR-TK inhibition, making them of particular
interest. In summary, the newly synthesized compounds provide promising new lead for the future design and
development of anticancer agents of potential EGFR-TK inhibitory activity.
Key words antiproliferative agent; azolidinone; diphenylamine; tyrosine kinase inhibitor
Breast cancer is the second leading cause of cancer deaths poted.20) Furthermore, dianilinophthalimide 6 that can bind
in women today and is the most common cancer among competitively with the ATP to EGFR showed good selectiv-
women, excluding nonmelanoma skin cancers.1) Despite the ity between different tyrosine kinases.21) Therefore, azolidi-
progress achieved in anticancer therapy about 1.3 million nones are considered good scaffolds for future design of ty-
women will be diagnosed with breast cancer annually world- rosine kinase inhibitors. In addition, benzamides and beza-
wide and about 465000 will die from the disease.1) Accord- midines were synthesized as mimics of the classical EGFR
ingly, novel therapeutic strategies to improve prognosis are inhibitors 4-anilino-quinazolines.22) Also, we have recently
urgently needed.
reported preliminary results on antiproliferative action of
It is increasingly apparent that a number of protein tyro- 2,4ꢀ-bis-heterocyclic diphenylamine 7 against MCF-7 cell
sine kinases, specifically epidermal growth factor receptor line, that might be mediated through the inhibition EGFR ki-
(EGFR) tyrosine kinase and/or its family members play a nase activity.23)
critical role in the development and progression of many
Promoted by these observations and in continuation to the
solid tumors, including breast cancer.2,3) Over-expression of previous work,23) we designed and synthesized hybrid mole-
EGFR has been observed in about 70% of breast cancer4) and cules of diphenylamine-2,4ꢀ-dicarboxamide (as dibenzamide
is associated with later stages of carcinogenesis5,6) and is im- template) and different azolidinones or related heterocyclic
plicated in the development of drug resistance with poor rings 11, 13—17 aiming to obtain highly active antitumor
prognosis.7—9) Therefore inhibitors of EGFR kinase activity agents, with probable EGFR tyrosine kinase inhibitory activ-
have emerged as promising new approach to cancer therapy.
ity. The new hybrids were inspired by compound 7 through
During the last decade azolidinones heterocycles, particu- replacing the diphenylamine scaffold with diphenylamine-
larly imidazolidinones, thiazolidinones and pyrrolidinone 2,4ꢀ-dicarboxamide and replacement of the thiadiazole ring
have gained special attention as potential lead compounds for with various azolidinones. Such substitution pattern could
novel anticancer agents.10) The antineoplastic properties of target different regions of the ATP binding site of the recep-
azolidinones and related heterocycles are most probably tor. Molecular modeling showed that the chosen azolidinone
caused by their affinity to different anticancer biotargets, ring systems at the 4ꢀ-position of diphenylamine-2,4ꢀ-dicar-
such as extracellular signal-regulated kinases (ERK boxamide core could mimic the interaction of the adenine
kinases),11,12) JNK-stimulating phosphatase-1 (JSP-1) 1,13) portion of ATP with the hinge region of the EGFR ATP bind-
tumor necrosis factor-alpha (TNF-a),14,15) antiapoptic com- ing site. Moreover, the 2-subtituent was expected to orient to-
plex Bcl-XL-BH3 2,16) integrin avb3 receptor 3,17) etc. (Fig. ward the solvent front so that, the produced interaction might
1). Recent reports showed that imidazolidinones, and thiazo- gain an additional potency.
lidinones were found to inhibit the kinase activity of EGFR.
Chemistry The general methods for the synthesis of tar-
Where, some 1-phenethyl-5-(E)-benzylidine hydantoins 4 in- get compounds 11—17 are depicted in Charts 1—4. The key
hibited EGFR autophosphorylation and polyglutamic acid/ intermediate compounds 9 and 10 were synthesized through
tyrosine (polyGAT) phosphorylation.18,19) Moreover, a series the reaction of diacid hyrazide 8 with the appropriate iso-
of hydrazono thiazolidin-4-one 5 that own high antineoplas- cyanates or isothiocynates in absolute ethanol, respectively,
tic activity against breast cancer cells MCF-7 and were po- Chart 1. Cyclization of bis-semicarbazides 9 with oxalyl
tent inhibitors of EGFR autophosphorylation has been re- chloride in benzene produced a mixture of bis-2,4,5-trioxo-
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: saharshaarawy_69@yahoo.com

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Fig. 1. Some Examples of Azolidinones with Anticancer Activity and EGFR Tyrosine Kinase Inhibition
Reagents and conditions: (a) isocyanates, absolute ethanol, reflux, (b) isothiocyantes, absolute ethanol, reflux.
Chart 1
imidazolidines 11 and bis-3,5,6-trioxotriazinanes 12, which tected. Its EI mass spectrum showed ion peak at m/z 644 des-
were separated by fractional solubility from ethanol, Chart 2. ignated as [Mꢁ1]ꢁ and different fragmentation pattern as-
According to literature,24) the obtained products could be dif- signed to different degradation products. It fragmented to
ferentiated based on their fragmentation in the mass spectra. yield product ions at m/z 223 corresponding to [C14H9NO2]+,
The detailed mass spectrometry analysis of compounds 11 m/z 211 and 210 corresponding to [C9H12N3O3]ꢁ, suggesting
and 12 provided diagnostic fragment ions that enabled dis- the presence of 3,5,6-trioxotriazinane ring system (Fig. 3).
1
crimination among alternative structures. The electron im- Moreover, in the H-NMR spectra of 12a, b singlet signals
pact (EI) mass spectrum of compound 11b had a measured derived from the cyclic NH were detected at 11.79—
elemental composition of C32H33N7O8 at m/z 643 designated 11.81 ppm. On the other hand, treatment of bis-thiosemicar-
as molecular ion peak [M] ꢁ. It fragmented to yield product bazides 10 with oxalyl chloride under the same condition
·
ions at m/z 253 corresponding to [C14H11N3O2]ꢁ and m/z 195 gave the substituted bis-4,5-dioxo-2-thioxoimidazolidines 13
[C9H11N2O3]ꢁ, suggesting the presence of 2,4,5-trioxoimida- as sole product. The EI mass spectrum of 13c revealed ion
zolidine ring system (Fig. 2). In addition, 1H-NMR spectra of peak at m/z 664 assigned as [Mꢁ1]ꢁ and important frag-
compounds 11a, b displayed the presence of the more ments at m/z 254 and 253 corresponding to [C14H11N3O2]ꢁ
deshielded amide signals resonating at 12.25—11.82 ppm. and m/z 206 and 205 equivalent to [C9H5N2O2S]ꢁ indicating
Meanwhile, compound 12b, the molecular ion was not de- the formation of 4,5-dioxo-2-thioxoimidazolidine ring sys-

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Vol. 59, No. 9
Reagents and conditions: (a) oxalyl chloride, benzene, refluxes.
Chart 2
Fig. 4. The Proposed Fragmentation of Compound 13c to Its Main Ions in
EI Mass Spectrum
Fig. 2. The Proposed Fragmentation of Compound 11b to Its Main Ions in
EI Mass Spectrum
succinic anhydride or phthalic anhydride in refluxing glacial
acetic acid furnished the corresponding imides’ analogues 16
and 17, respectively, Chart 4. Both the analytical and spectral
1
data (IR, H-NMR, 13C-NMR and MS) of all the newly syn-
thesized compounds were in full agreement with the pro-
posed structures.
Results and Discussion
In Vitro EGFR Kinase Inhibitory Activity The kinase
inhibitory activity of the newly synthesized compounds was
evaluated using EGFR kinase activity assay by enzyme-
linked immunosorbent assay (ELISA).25,26) The assay was
based on the inhibition of phosphorylation of the tyrosine
kinase-specific peptide poly glutamic acid/tyrosine
[poly(Glu/Tyr), 4 : 1] by EGFR. Results are illustrated in
Table 1. The inhibitory activities are given as percentage in-
hibition at concentration of 10 mm of the inhibitor.22,26) Most
Fig. 3. The Proposed Fragmentation of Compound 12b to Its Main Ions in
EI Mass Spectrum
1
tem (Fig. 4). Also, in the H-NMR spectra of compounds of the tested compounds exhibited significant EGFR-TK in-
13a—c, downfield signals belonging to the amide proton hibitory activity with percentage inhibition ranging from 89
were observed between 12.72—12.20 ppm. Alternatively, cy- to 33%, where compounds 12b and 13b were the most active
clocondensation of bis-thiosemicarbazides 10 with chloro- (89, 81% inhibition, respectively).
acetic acid in presence of anhydrous sodium acetate in reflux-
The Enzyme Assay Results Elicited That: 1) The 3,5,6-tri-
ing glacial acetic acid afforded the bis-2-imino-4-oxothia- oxotriazinanes 12a, b were more potent enzyme inhibitors
zolidine derivatives 14, while their reaction with 4-bromo- than the corresponding 2,4,5-trioxoimidazolidines 11a, b and
phenacyl bromide yielded the bis-2-imino thiazolines 15, the compounds bearing cyclohexyl substituent 11b and 12b
Chart 3. Finally, condensation of diacid hydrazide 8 with showed higher inhibitory effect than those having p-

September 2011
1127
Reagents and conditions: (a) oxalyl chloride, benzene, reflux, (b) chloroacetic acid, anhydrous sodium
acetate, glacial acetic acid, reflux, (c) 4-bromophenacyl bromide, ethanol/chloroform, reflux.
Chart 3
Reagents and conditions: (a) succinic anhydride, glacial acetic acid, reflux (b) phthalic anhydride, glacial acetic acid, reflux.
Chart 4
chlorophenyl substituent 11a and 12a. 2) Among the azolidi-
Regarding the effect of substituent on different series, the
none containing compounds, the kinase inhibitory effect of results showed that: 1) The cyclohexyl substituted com-
the imidazolidinone derivatives 11a, b and 13a—c was pounds 11b, 12b and 14d were more potent EGFR-TK in-
higher than that of the 2-imino-4-oxothiazolidines 14a—d hibitors than the corresponding phenyl containing com-
and the pyrrolidinone derivatives 16 and 17. 3) Replacement pounds 11a, 12a and 14c. 2) For the series of 2-imino-4-bro-
of the 2-imino-4-oxothiazolidine moiety in 14a—d by 2- mophenyl thiazolines 15a—d, it was found that the kinase
imino-4-bromophenyl thiazoline in 15a—d produced com- inhibitory activity decreased as the size of the substituent on
pounds with enhanced kinase inhibition activity, except for the imino increased from ethyl 15a to cyclohexyl 15d. This
the cyclohexyl analogue 15d.
proved poor bulk tolerance for this class of compounds and

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Vol. 59, No. 9
Table 1. EGFR Tyrosine Kinase Inhibitory Activity of the Synthesized
Compounds, and Their in Vitro Cytotoxicity against Human Breast Cancer
Cell (MCF-7)
EFGR-TK
Inhibition [%] at 10 mM
In vitro cytotoxicity
IC50 (mM)
Compound
723)
11a
11b
12a
12b
13a
13b
13c
14a
14b
14c
14d
15a
15b
15c
97.7ꢃ2.3***
45.0ꢃ5.0*
70.0ꢃ4.2**
50.0ꢃ4.5*
89.0ꢃ8.0***
70.0ꢃ6.4**
81.0ꢃ7.1***
49.0ꢃ3.5*
52.0ꢃ5.3*
NA
33.0ꢃ3.1
63.0ꢃ5.2**
70.0ꢃ6**
52.0ꢃ4.5*
50.0ꢃ4.2*
NA
0.94ꢃ0.05
0.96ꢃ0.08**
0.84ꢃ0.06**
1.24ꢃ0.11*
1.04ꢃ0.13*
0.71ꢃ0.05***
0.91ꢃ0.11**
0.71ꢃ0.05***
1.11ꢃ0.12*
ND
4.44ꢃ0.30
0.62ꢃ0.05***
0.73ꢃ0.06***
0.64ꢃ0.05***
0.59ꢃ0.04***
ND
Fig. 6. Compound 13b Docked in EGFR Kinase Domain in 3D Diagram
the binding pocket and is considered crucial for the activity
of the known EGFR inhibitors.29,30) Meanwhile the cyclo-
hexyl substituent made a predominant hydrophobic interac-
tion with the adjacent lipophilic pocket lined with Ala 743,
Met 790, Leu 844 and Thr 854. The additional binding en-
ergy provided by the interaction of the cyclohexyl with this
pocket may be responsible for the high binding affinity of
this molecule. Also the 2-[(4-cyclohexyl-3,5,6-trioxo-1,2,4-
triazinane-1-yl)carbonyl]phenyl moiety was direct to the en-
trance of the active site, with phenyl extended to the solvent
exposed region. Where, the triazinane ring nitrogen N-2 and
the 6-oxo group together with carbonyl bridge were involved
in H-bonding with Leu 718 and Lys 716 (2.9, 3.1, 2.4 Å).
Docking of compound 13b—as an example of the azolidi-
none derivatives—is depicted in Fig. 6. The model revealed a
similar orientation to 12b. The 4ꢀ-[(4,5-dioxo-2-thioxoimida-
zolidin-1-yl)carbamoyl] moiety bound in the adenine binding
pocket, where the 5-oxo and the carbamoyl NH were en-
gaged in H-bonds with the backbone amide and carbonyl
atoms of Met 793 (2.6, 3.0 Å, respectively). The allyl group
in 13b was inserted in the adjacent lipophilic pocket defined
by Val 726, Met 790, Leu 844 and Thr 854, in analogy to the
cyclohexyl substituent in 12b. Moreover, the 2-[(3-allyl-4,5-
dioxo-2-thioxoimidazolidin-1-yl)carbamoyl]phenyl moiety
was oriented toward the entrance of binding site, so that the
oxygen atoms of the carbamoyl and the 5-oxo functionalities
15d
16
17
42.0ꢃ3.9*
NA
0.89ꢃ0.07**
ND
PD153035
99.6ꢃ2.0***
ND
NAꢂnot active (ꢄ10%), ND, not determined. Significant at: ∗ pꢅ0.05 ∗∗ pꢅ0.01
∗∗∗ pꢅ0.001.
Fig. 5. Molecular Docking of Compound 12b in EGFR Kinase Domain in
3D Diagram
could justify the inactivity of the cyclohexyl congener 15d were involved in three H-bonds with the side chain NH of
compared to the other cyclohexyl bearing compounds. Lys 728 and the backbone NH of Glu 1004 (Fig. 6). The mo-
Molecular Modeling To rationalize the obtained biolog- lecular docking results, along with the enzyme assay data
ical results and to describe the binding mode of the active suggesting that compounds 12b and 13b are potential in-
compounds with their predicted intracellular target, docking hibitors of EGFR.
analysis was carried out using Molecular Operating Environ-
Also, in order to clarify the reason for the lack of activity
ment MOE version 2008.10.27) Molecular docking of the of cyclohexyl substituted compound 15d. Comparison of the
most potent inhibitors 12b and 13b into the ATP binding site docked poses of 14d and 15d was performed (Figs. 7a, b). In
of the EGFR kinase domain was performed on the X-ray 14d the 2-[2-cyclohexylimino-4-oxothiazolidine] ring occu-
crystal structure of EGFR complex (3IKA.pdb).28) The bind- pied the adenine binding pocket with the 4-oxo group H-
ing model of compound 12b showed that the 4ꢀ-[(4-cyclo- bonded to Met 793 (2.6 Å) and 2-cyclohexylimino projecting
hexyl-3,5,6-trioxo-1,2,4-triazinane-1-yl)carbonyl] moiety away from the pocket (Fig. 7a). On the other hand, 15d re-
bound to the narrow hydrophobic pocket in the N-terminal of vealed flipping of the 2-cyclohexylimino-4-bromophenyl
EGFR, which is the binding site of adenine base of ATP (Fig. thiazoline moiety, locating the 2-cyclohexylimino in the nar-
5). A bidentate H-bonding interaction was observed between row adenine binding pocket. Therefore, compound 15d was
the 3,5,6-trioxo-1,2,4-triazinane ring and the main chain NH too sterically hindered to effectively occupy this pocket and
and CꢂO of Met 793 (2.9, 2.7 Å, respectively), hydrogen to make the necessary interaction with Met 793 (Fig. 7b).
bonding with this amino acid helps to fix the adenine base in
On the other hand, no conclusive reason could account for

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1129
Fig. 7. The Docking Poses of Compounds 14d (a) and 15d (b) in EGFR Kinase Domain, Showing the Different Positions of the 2-Cyclohexylimino Group
as Indicated by Green Arrows
the difference in activity between compound 13b and 14b. It levels. Compounds 12b and 13b exploited the most potent
could be referred to difference in substitution pattern and rel- kinase inhibitory activity (89, 81% at 10 mM, respectively).
ative position of the ally substituent that was reflected as bad Molecular docking models of these compounds indicated
docking scores of 14b (ꢆ7.26) compared to 13b (ꢆ15.07). that they bound to the ATP binding site of EGFR forming a
This indicated the formation of unstable receptor-ligand bidentate H-bond interaction with Met 793. Therefore, the
complex and low binding affinity of 14b.
molecular docking results, along with the enzyme assay data
In Vitro Cytotoxicity The compounds that showed suggested that compounds 12b and 13b are potential in-
EGFR inhibitory activity were evaluated for their antiprolif- hibitors of EGFR. In addition, the cytotoxicity assay results
erative activity on human breast cancer cell line (MCF-7) by indicated that all evaluated compounds own high antiprolifer-
Skehan’s method.31) MCF-7 is known to over-express EGFR ative activity against EGFR over-expressing breast cancer
and provides a good measure to determine the effectiveness cells (MCF-7) at micromolar and submicromolar levels. In
of EGFR-TK inhibition. As shown in Table 1, all of the particular, the 2-imino-4-bromophenyl thiazoline derivatives
tested compounds exhibited potent activity against MCF-7 15a—c which possessed moderate EGFR-TK inhibition
cell line, indicating that these compounds are potential in- showed unexpected high potencies as antitumor agents (IC50
hibitors of EFGR-TK. The majority of compounds (11a, b, 0.59—0.73 mM), making them of special interest. In sum-
13a—c, 14d, 15a—c, 16) showed significant antiproliferative mary, even if further structure–activity investigation is
effect at sub-micromolar level with IC50 ranging from 0.59 to needed for potency optimization of this class of compounds
0.97 mM. On the other hand, compounds 12a, b and 14a pos- with respect to the enzyme test, they seem potentially attrac-
sessed remarkable antitumor activity at micromolar level tive as antiproliferative agents.
with IC50 ranging from 1.04 to 1.24 mM, while compound 14c
Experimental
demonstrated the least activity with IC50 of 4.44 mM. Analysis
Chemistry Melting points were determined on Galle Kamp and Kofler
melting point apparatus and are uncorrected. Elemental analyses (C, H, N)
were performed by Micro Analytical Center, Faculty of Science, Cairo Uni-
of the data in Table 1 illustrated that the nature of the ring
system on the diphenylamine-2,4ꢀ-dicarboxamide core af-
fected the antitumor activity. In general, 4,5-dioxo-2-thioxo- versity. Infrared spectra were recorded on Shimadzu IR 435 Spectropho-
tometer or on a Genesis II FT-IR TM, Mattson, 5225, Verona Road, Madison
wi. 53711 U.S.A., using KBr discs. H-NMR spectra were scanned on Var-
ian XL-300 MHz, Varian XL-200 MHz or Joel FX 90Q-90 MHz (chemical
imidazolidines 13a—c exhibited better cytotoxic activity
than the 2,4,5-trioxoimidazolidine analogues 11a, b. With re-
1
spect to the thiazole containing compounds 14a—d and
15a—c, it can be observed that the 2-imino-4-bromophenyl
thiazolines 15a—c had superior anticancer effect compared
to the corresponding 2-imino-4-oxothiazolidine derivatives
14a—d [c.f. 14c, 15c]. Moreover, the potency of the 2-
imino-4-bromophenyl thiazoline derivatives 15a—c was
found to increase parallel to the increase in lipophilicity of
the 2-imino substituent. Finally, the pyrrolidinone derivative
16 revealed antiproliferative activity comparable to that of
the 2,4,5-trioxoimidazolidine derivatives 11a, b.
shifts are given in part per million ppm downfield from TMS), 13C-NMR
spectrum was scand on Varian XL-300 MHz . Mass spectra were made on a
Finnigan MAT, SSQ 7000 spectrophotometer at 70 eV. Analytical thin-layer
chromatography (TLC) was performed on precoated silica gel plates 60-F-
254 (Merck; 0.25 mm), developing with chloroform. Compounds 8 and
10a—c were prepared according to the reported procedure.23)
General Procedure for Preparation of 2,4ꢀ-Bis-(4-substituted semicar-
bazidocarbonyl)diphenylamine (9a, b) A mixture of the diacid hydrazide
8 (1.0 g, 3.5 mmol) and the appropriate isocyanate (10.5 mmol) in absolute
ethanol (20 ml) was refluxed for 8 h the reaction mixture was concentrated
and left to cool. The separated solid was filtered and dried. The product was
crystallized from ethanol.
2,4ꢀ-Bis-[4-(4-chlorophenyl)semicarbazidocarbonyl)]diphenylamine (9a):
1
Conclusion
Yield, 95%; mp 264—266 °C. H-NMR (200 MHz) (DMSO-d6) d: 6.97—
7.82 (16H, m, Ar-H), 9.61 (4H, s, 2ꢇp-chlorophenyl–NHCꢂONH, D2O
exch.), 9.88 (3H, s, NH and 2ꢇCONH, D2O exch.). IR (KBr) cmꢆ1: 3290.3,
3219.3 and 3183.7 (NH)s, 305.61 (CH aromatic), 1663.9 and 1623.1
(CꢂO)s. Anal. Calcd for C28H23Cl2N7O4.H2O: C, 55.09; H, 4.13; N, 16.06.
Found: C, 54.71; H, 4.51; N,15.70.
Several hybrid molecules of diphenylamine-2,4ꢀ-dicarbox-
amide with various azolidinones and related heterocyclic
rings have been synthesized and investigated for their ability
to inhibit EGFR tyrosine kinase activity. Most of them dis-
played promising EGFR-TK inhibitory activity at various
2,4ꢀ-Bis-(4-cyclohexylsemicarbazidocarbonyl)diphenylamine (9b): Yield,

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Vol. 59, No. 9
85%; mp 241—242 °C. 1H-NMR (200 MHz) (DMSO-d6) d: 1.01—1.24
(10H, m, cyclohexyl), 1.67—1.79 (10H, m, cyclohexyl), 3.35 (2H, br s,
2ꢇCH-1 cyclohexyl), 6.22 (2H, s, 2ꢇcycolhexyl-NHCꢂO, D2O exch.),
113.32, 116.27, 124.52, 128.59, 129.45, 133.97, 137.01, 139.01, 140.82,
151.8, 153.00, 157.92 (CꢂO), 160.86 (CꢂO), 166.41 (CꢂO), 178.91
(CꢂS). IR (KBr) cmꢆ1: 3243.1, br (NH)s, 2980.6 and 2935.0 (CH), 1797.9,
6.97—7.84 (8H, m, Ar-H), 9.48 (2H, s, 2ꢇNHCꢂO, D2O exch.), 9.93 (1H, 1740.0 and 1702.8 (CꢂO)s, 1240.6 (CꢂS). Anal. Calcd for C24H21N7O6S2:
s, NH, D2O exch.), 10.12 (2H, s, 2ꢇCONH, D2O exch.). IR (KBr) cmꢆ1
:
C, 50.79; H, 3.73; N, 17.27. Found: C, 50.53; H, 3.56; N,17.32.
3348.7 and 3247.7 (NH)s, 3064.3 (CH aromatic), 2927.7 and 2853.5 (CH
cyclohexyl), 1683 and 1634 (CꢂO)s. Anal. Calcd for C28H37N7O6S2: C,
62.78; H, 6.96; N, 18.30. Found: C, 62.70; H, 7.00; N, 18.00.
2,4ꢀ-Bis[N-(3-allyl-4,5-dioxo-2-thioxoimidazolidin-1-yl)carbamoyl]-
diphenylamine (13b): Yield 60%, mp 92—94 °C. 1H-NMR (200 MHz)
(DMSO-d6) d: 4.57 (4H, br s, 2ꢇCH2-CHꢂCH2), 5.17—5.26 (4H, t,
2ꢇCH2-CHꢂCH2), 5.83 (2H, m, 2ꢇCH2-CHꢂCH2), 6.65—8.22 (8 H, m,
Ar-H), 11.44 (1H, s, NH, D2O exch.), 12.22 (2H, s, 2ꢇCONH, D2O exch.).
IR (KBr) cmꢆ1: 3221.2, br (NH)s, 2926.4 (CH), 1798.2, 1740.9 and 1704.8
(CꢂO)s, 1271,4 (CꢂS). Anal. Calcd for C26H21N7O6S2: C, 52.78; H, 3.58;
N, 16.57. Found: C, 52.60; H, 3.40; N, 16.32.
2,4ꢀ-Bis-(4-cyclohexylthiosemicarbazidocarbonyl)diphenylamine
(10d) A mixture of the diacid hydrazide 8 (1.0 g, 3.5 mmol) and cyclo-
hexyl isothiocyanate (1.48 g, 1.5 ml, 10.5 mmol) in absolute ethanol (20 ml)
was refluxed for 8 h. The reaction mixture was concentrated and left to cool.
The separated solid was filtered and dried (yield 95%), the product was crys-
tallized from ethanol, mp 184—186 °C. 1H-NMR (200 MHz) (DMSO-d6) d:
2,4ꢀ-Bis[N-(4,5-dioxo-3-phenyl-2-thioxoimidazolidin-1-yl)carbamoyl]-
1.01—1.24 (10H, m, cyclohexyl), 1.67—1.79 (10H, m, cyclohexyl), 4.12 diphenylamine (13c): Yield 62%, mp 165—167 °C. 1H-NMR (200 MHz)
(2H, br s, 2ꢇCH-1 cyclohexyl), 6.97—7.86 (8H, m, Ar-H), 9.12 (2H, s, (DMSO-d6) d: 7.42—8.29 (18H, m, Ar-H), 11.97 (1H, s, NH, D2O exch.),
2ꢇcyclohexyl-NHCꢂS, D2O exch.), 9.48 (1H, s, NH, D2O exch.), 10.07 12.20 (2H, s, 2ꢇCONH, D2O exch.). IR (KBr) cmꢆ1: 3261.7, br (NH)s,
(2H, s, 2ꢇNHCꢂS, D2O exch.), 10.33 (2H, s, 2ꢇCONH, D2O exch.). IR
3064.1 (CH aromatic), 1796.5, 1738.2 and 1701.5 (CꢂO)s, 1340.4 (CꢂS).
(KBr) cmꢆ1: 3269.3, br (NH)s, 2928.4 and 2852.3 (CH cyclohexyl), 1649.9 MS m/z: 664 (Mꢁ1). Anal. Calcd for C26H21N7O6S2: C, 57.91; H, 3.19; N,
(CꢂO)s, 1253.9 (CꢂS)s Anal. Calcd for C28H37N7O2S2: C, 59.23; H, 6.57; 14.77. Found: C, 57.90; H, 3.59; N, 14.44.
N, 17.27. Found: C, 58.90; H, 6.30; N, 17.58.
General Procedure for Preparation of 2,4ꢀ-Bis-[N-(3-substituted-
General Procedure for Preparation of 2,4ꢀ-Bis-{N-[2-(substituted
imino)-4-oxothiazolidin-3-yl]carbamoyl}diphenylamine (14a—d) To a
2,4,5-trioxoimidazolidin-1-yl)carbamoyl]diphenylamine (11a, b) and suspension of the appropriate thiosemicarbazide 10a—d (1.0 mmol) in gla-
2,4ꢀ-Bis-[(4-substituted-3,5,6-trioxo-1,2,4-triazinane-1-yl)carbonyl]-
diphenylamine (12a, b) To a solution of 9a, b (1.0 mmol), in dry benzene
(20 ml), oxalyl chloride (0.17 ml, 2.0 mmol) was added drop wise while stir-
ring. The reaction mixture was refluxed at 60—65 °C for 2 h, the solvent was
distilled under vacuum. The obtained residue was treated with cold ethanol
then filtered. The filtrate was evaporated under vacuum, and the obtained
residue was crystallized from benzene–pet ether (9 : 1) to give 11a, b. The
residual product insoluble in cold ethanol was crystallized from ethanol to
yield 12a, b.
cial acetic acid (10 ml), anhydrous sodium acetate (0.33 g, 4.0 mmol) and
monochloroacetic acid (0.38 g, 4.0 mmol) were added. The reaction was re-
fluxed for 5 h. After cooling the mixture was diluted with ice water and al-
lowed to stand overnight in the fridge. The product was filtered, washed with
water and crystallized from the appropriate solvent.
2,4ꢀ-Bis-[N-[2-(ethylimino)-4-oxothiazolidin-3-yl]carbamoyl]diphenyl-
1
amine (14a): Yield 83%, crystallized from ethanol, mp 228—230 °C. H-
NMR (200 MHz) (DMSO-d6) d: 1.16—1.22 (6H, t, 2ꢇCH2CH3), 3.74—
3.77 (4H, q, 2ꢇCH2CH3), 4.04 (4H, s, 2ꢇCH2 of oxothiazolidine), 6.99—
7.81 (8H, m, Ar-H), 9.43 (1H, s, NH, D2O exch.), 10.67 (2H, s, 2ꢇCONH,
2,4ꢀ-Bis-{N-[3-(4-chlorophenyl)-2,4,5-trioxoimidazolidin-1-yl]car-
bamoyl}diphenylamine (11a): Yield 37%, mp 167—169 °C. 1H-NMR D2O exch.). IR (KBr) cmꢆ1: 3326.4 and 3164.7 (NH)s, 2980.9 (CH), 1717.5
(200 MHz) (DMSO-d6) d: 6.82—8.00 (16H, m, Ar-H), 11.19 (1H, s, NH, and 1638.4 (CꢂO)s. Anal. Calcd for C24H25N7O4S2: C, 53.42; H, 4.67; N,
D2O exch.), 12.25 (2H, s, 2ꢇNHCꢂO, D2O exch.). IR (KBr) cmꢆ1: 3506.7 18.17. Found: C, 53.70; H, 4.78; N, 18.30.
and 3450.0 (NH)s, 1702.0 and 1605.0 (CꢂO)s. MS m/z: 700 (Mꢁ). Anal.
2,4ꢀ-Bis-{N-[2-(allylimino)-4-oxothiazolidin-3-yl]carbamoyl}diphenyl-
1
Calcd for C32H19Cl2N7O8: C, 54.78; H, 2.73; N, 14.00. Found: C, 54.76; H, amine (14b): Yield 70%, crystallized from acetone, mp 218—220 °C. H-
2.73; N, 13.9.
NMR (200 MHz) (DMSO-d6) d: 3.77 (4H, br s, 2ꢇCH2-CHꢂCH2), 4.09
2,4ꢀ-Bis-[N-(3-cyclohexyl-2,4,5-trioxoimidazolidin-1-yl)carbamoyl]- (4H, s, 2ꢇCH2 of oxothiazolidine), 5.14—5.20 (4H, m, 2ꢇCH2-CHꢂCH2),
diphenylamine (11b): Yield 45%, mp 155—157 °C. 1H-NMR (200 MHz)
5.84 (2H, m, 2ꢇCH2-CHꢂCH2), 7.04—8.19 (8H, m, Ar-H), 9.38 (1H, s,
(DMSO-d6) d: 1.24—1.37 (10H, m, cyclohexyl), 1.62—1.86 (10H, m, cy- NH, D2O exch.), 10.68 (2H, s, 2ꢇCONH, D2O exch.). IR (KBr) cmꢆ1
:
clohexyl), 4.02 (2H, br s, 2ꢇCH-1 cyclohexyl), 6.63—8.24 (8H, m, Ar-H),
11.55 (1H, s, NH, D2O exch.), 11.82 (2H, s, 2ꢇCONH, D2O exch.). IR
3377.9 and 3170.8 (NH)s, 2972.2 and 2841.2 (CH), 1725.7 and 1636.0
(CꢂO)s. Anal. Calcd for C26H25N7O4S2.H2O: C, 53.69; H, 4.68; N, 16.86.
(KBr) cmꢆ1: 3507.5, br (NH)s, 2926.4 and 2845.6 (CH cyclohexyl), 1703.0 Found: C,53.70; H, 4.80; N, 17.00.
and 1606.7 (CꢂO)s. MS m/z: 643 (Mꢁ). Anal. Calcd for C32H33N7O8: C,
59.71; H, 5.17; N, 15.23. Found: C, 59.80; H, 5.20; N, 15.22.
2,4ꢀ-Bis-{N-[2-(phenylimino)-4-oxothiazolidin-3-yl]carbamoyl}diphenyl-
1
amine (14c): Yield 93%, crystallized from acetone, mp 176—178 °C. H-
2,4ꢀ-Bis-{[4-(4-Chlorophenyl)-3,5,6-trioxo-1,2,4-triazinane-1-yl]car-
NMR (300 MHz) (DMSO-d6) d: 4.18 (4H, s, 2ꢇCH2 of oxothiazolidine),
bonyl}diphenylamine (12a): Yield 32%, mp 237—239 °C. 1H-NMR
6.89—7.95 (18H, m, Ar-H), 9.39 (1H, s, NH, D2O exch.), 10.74 (2H, s,
(200 MHz) (DMSO-d6) d: 7.10—7.95 (16H, m, Ar-H), 11.58 (2H, s, 2ꢇNH 2ꢇCONH, D2O exch.). IR (KBr) cmꢆ1: 3285.1, br (NH)s, 3063.9 (CH aro-
of triazinanetrione, D2O exch.), 11.79 (1H, s, NH, D2O exch.). IR (KBr)
matic), 1727.2 and 1644.2 (CꢂO)s. Anal. Calcd for C32H25N7O4S2: C,
cmꢆ1: 3493.0 and 3307.3 (NH)s, 1753.9, 1702.2 and 1673.7 (CꢂO)s. Anal. 60.42; H, 3.96; N, 15.42. Found: C, 60.09; H, 4.33; N, 15.15.
Calcd for C32H19Cl2N7O8: C, 54.82; H, 2.73; N, 14.00. Found: C,55.03; H,
2.81; N, 14.11.
2,4ꢀ-Bis-{N-[2-(cyclohexylimino)-4-oxothiazolidin-3-yl]carbamoyl}-
diphenylamine (14d): Yield 82%, crystallized from acetone/water, mp
140—142 °C. H-NMR (200 MHz) (DMSO-d6) d: 1.10—1.31 (10H, m, cy-
1
2,4ꢀ-Bis-[(4-cyclohexyl-3,5,6-trioxo-1,2,4-triazinane-1-yl)carbonyl]-
diphenylamine (12b): Yield 40%, mp 224—226 °C. 1H-NMR (200 MHz) clohexyl), 1.60—1.82 (10H, m, cyclohexyl), 3.99 (4H, s, 2ꢇCH2 of oxothia-
(DMSO-d6) d: 1.24—1.37 (10H, m, cyclohexyl), 1.62—1.86 (10H, m, cy- zolidine), 4.20—4.50 (2H, m, 2ꢇCH-1 cyclohexyl), 7.28—7.85 (8H, m, Ar-
clohexyl), 4.03 (2H, br s, 2ꢇCH-1 cyclohexyl), 6.62—8.24 (8H, m, Ar-H),
11.81 (3H, s, 2ꢇNH of triazinanetrione and NH, D2O exch.). IR (KBr)
H), 9.36 (1H, s, NH, D2O exch.), 11.00 (2H, s, 2ꢇCONH, D2O exch.). IR
(KBr) cmꢆ1: 3278 (NH)s, 2930 and 2855 (CH), 1719 and 1630 (CꢂO)s,
cmꢆ1: 3491.1 and 3395.5 (NH)s, 2932.6 and 2858.5 (CH cyclohexyl), 1254 (CꢂS)s. Anal. Calcd for C32H37N7O4S2: C, 59.33; H, 5.76; N, 15.14.
1753.5, 1702.2 and 1675.4 (CꢂO)s. MS m/z: 644 (Mꢁ1). Anal. Calcd for
C32H33N7O8: C, 59.71; H, 5.17; N, 15.21. Found: C, 59.60; H, 5.00; N,
14.94.
Found: C, 59.20; H, 5.50; N, 14.80.
General Procedure for Preparation of 2,4ꢀ-Bis-{N-[4-(4-bromo-
phenyl)-2-(substituted imino)thiazol-3(2H)-yl]carbamoyl}diphenyl-
General Procedure for Preparation of 2,4ꢀ-Bis[N-(3-substituted-4,5- amine (15a—d) A mixture of the appropriate thiosemicarbazide 10a—d
dioxo-2-thioxoimidazolidin-1-yl)carbamoyl]diphenylamine (13a—c) To (1.0 mmol) and 4ꢀ-bromophenacyl bromide (0.56 g, 2.0 mmol) in ethanol–
a solution of 10a—c (1.0 mmol), in dry benzene (20 ml), oxalyl chloride
(0.17 ml, 2.0 mmol) was added drop wise while stirring. The reaction mix-
ture was refluxed at 60—65 °C for 2 h, the solvent was distilled under re-
duced pressure. The obtained residue was crystallized from benzene.
2,4ꢀ-Bis[N-(4,5-dioxo-3-ethyl-2-thioxoimidazolidin-1-yl)carbamoyl]-
diphenylamine (13a): Yield 55%, mp 130—132 °C. 1H-NMR (300 MHz)
chloroform (7 : 3) mixture (20 ml) was refluxed for 3 h. The mixture was
concentrated. After cooling the precipitate was filtered, washed several times
with ether and crystallized from the appropriate solvent.
2,4ꢀ-Bis-{N-[4-(4-bromophenyl)-2-(ethylimino)thiazol-3(2H)-yl]car-
bamoyl}diphenylamine (15a): Yield 65%, crystallized from acetone/water,
mp 210—212 °C. 1H-NMR (200 MHz) (DMSO-d6) d: 1.05—1.12 (6H, t,
(DMSO-d6) d: 1.10—1.28 (6H, t, 2ꢇCH2CH3), 3.77—3.99 (4H, q, 2ꢇCH2CH3), 3.32—3.43 (4H, q, 2ꢇCH2CH3), 6.91(2H, s, 2ꢇCH of thiazo-
2ꢇCH2CH3), 6.62—8.21 (8H, m, Ar-H), 11.35 (1H, s, NH), 12.72 (2H, s, line), 7.13—7.81 (16H, m, Ar-H), 9.46 (1H, s, NH, D2O exch.), 11.62 (1H,
2ꢇCONH). 13C-NMR (300 MHz) (DMSO-d6) d: 12.29 (CH3), 40.71 (CH2),
s, CONH, D2O exch.), 11.71 (1H, s, CONH, D2O exch.). IR (KBr) cmꢆ1
:

September 2011
1131
3182.3, br (NH)s, 3109.0 (CH aromatic), 2971.2 and 2929.6 (CH), 1654.1 purchased from Sigma-Aldrich. The cytotoxic activity was measured in vitro
(CꢂO)s. Anal. Calcd for C36H31Br2N7O2S2: C, 52.88; H, 3.82; N, 11.99. using the Sulfo-Rhodamine-B stain (SRB) assay using the method of Ske-
Found: C, 52.59; H, 3.82; N, 11.69.
han.31) Cells were inoculated in 96-well microtiter plate (104 cells/well) for
24 h before treatment with the compound(s) to allow attachment of cell to
the wall of the plate. Test compounds were dissolved in DMSO and diluted
with saline to the appropriate volume. Different concentrations of the com-
2,4ꢀ-Bis-{N-[4-(4-bromophenyl)-2-(allylimino)thiazol-3(2H)-yl]car-
bamoyl}diphenylamine (15b): Yield 60%, crystallized from acetone/water,
1
mp 196—198 °C. H-NMR (200 MHz) (DMSO-d6) d: 4.51 (4H, d, 2ꢇCH2-
CHꢂCH2), 4.91—5.24 (4H, m, 2ꢇCH2-CHꢂCH2), 5.75—5.84 (2H, m, pound under test (0.1, 2.5, 5, 10 mmol/ml) were added to the cell monolayer.
2ꢇCH2-CHꢂCH2), 6.40 (2H, s, 2ꢇCH of thiazoline), 7.10—7.95 (16H, m,
Ar-H), 9.41 (1H, s, NH), 11.34 (1H, s, CONH, D2O exch.), 11.44 (1H, s,
CONH, D2O exch.). IR (KBr) cmꢆ1: 3283.3, br (NH)s, 3096.7 (CH aro-
Triplicate wells were prepared for each individual dose. Monolayer cells
were incubated with the compound(s) for 48 h at 37 °C and in atmosphere of
5% CO2. After 48 h, cells were fixed, washed, and stained for 30 min with
matic), 2955.5, 2895.7 and 2820.3 (CH), 1659.0 (CꢂO)s. Anal. Calcd for 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by
C38H31Br2N7O2S2: C, 54.23; H, 3.71; N, 11.56. Found: C,54.33; H,3.54; four washes with 1% acetic acid, and attached stain was recovered with Tris-
N,11.66.
ethylenediaminetetraacetic acid (Tris–EDTA) buffer. Color intensity was
2,4ꢀ-Bis-{N-[4-(4-bromophenyl)-2-(phenylimino)thiazol-3(2H)-yl]-car- measured in an ELISA reader. The relation between surviving fraction and
bamoyl}diphenylamine (15c): Yield 73%, crystallized from ethanol/water, drug concentration is plotted to get the survival curve for breast tumor cell
mp 236—238 °C. 1H-NMR (200 MHz) (DMSO-d6) d: 6.58 (2H, s, 2ꢇCH of line after the specified time. The concentration required for 50% inhibition
thiazoline), 7.02—7.73 (26 H, m, Ar-H), 9.06 (1H, s, NH, D2O exch.), 11.36 of cell viability (IC50) was calculated and the results are given in Table 1.
(1H, s, CONH, D2O exch.), 11.54 (1H, s, CONH, D2O exch.). IR (KBr)
Molecular Modeling All molecular modeling calculation and docking
studies were carried out using Molecular Operating Environment MOE ver-
cmꢆ1: 3150.0, br (NH)s, 3056.9 (CH aromatic), 2964.8 and 2922.7 (CH),
1674.6 (CꢂO)s. Anal. Calcd for C44H31Br2N7O2S2: C, 57.84; H, 3.42; N, sion 2008.10.27) The target compounds were drawn on MOE. The structures
10.73. Found: C, 57.70; H, 3.30; N, 11.08.
were subjected to energy minimization using Hamiltonian-Force Field-
MMFF94x. The most stable conformers for each compound were retained
and partial charges were calculated. The X-ray crystal structure of the kinase
2,4ꢀ-Bis-{N-[4-(4-bromophenyl)-2-(cyclohexylimino)thiazol-3(2H)-yl]-
carbamoyl}diphenylamine (15d): Yield 68%, crystallized from acetone, mp
182—184 °C. 1H-NMR (200 MHz) (DMSO-d6) d: 1.06—1.71 (20H, m, domain of EGFR in complex with WZ4002 PDB ID code 3IKA was recov-
2ꢇcyclohexyl), 3.56 (2H, br s, overlapped, 2ꢇCH-1 of cyclohexyl), 6.24
(1H, s, CH of thiazoline), 6.26 (1H, s, CH of thiazoline), 7.02—7.73 (16 H,
m, Ar-H), 9.16 (1H, s, NH), 11.99 (1H, s, CONH), 12.05(1H, s, CONH). IR
ered RSCB protein data bank.28) The enzyme was prepared for docking as
follows: 1) The Co-crystallized ligand, and water molecules were removed.
2) The enzyme was 3D protonated, where hydrogen atoms were added at
(KBr) cmꢆ1: 3199.1 and 3111.3 (NH)s, 2930.8 and 2854.7 (CH), 1678.0 their standard geometry, the partial charges were computed and the system
(CꢂO)s. Anal. Calcd for C44H43Br2N7O2S2: C, 57.08; H, 4.68; N, 10.59. was optimized. Flexible ligand-rigid receptor docking of the most stable
Found: C, 57.30; H, 4.60; N, 10.40.
conformers was done with MOE-DOCK using triangle matcher as place-
General Procedure for the Preparation of 2,4ꢀ-Bis-(N-substituted car- ment method and London dG as a scoring function. The obtained poses were
bamoyl)-diphenylamine (16) and (17) A solution of the diacid hydrazide
8 (1.0 g, 3.5 mmol) and the appropriate acid anhydride (7.0 mmol) in glacial
acetic acid (10 ml) was refluxed for 3 h. The reaction mixture was concen-
trated and poured over crushed ice. The product was filtered, washed several
times with cold water then crystallized from the appropriate solvent.
subjected to force field refinement using the same scoring function. Ten con-
formers of the ligand were retained with the highest and best score. In order
to validate the docking procedure, WZ4002 was docked into the active site
of 1XKK. The docking results show that the compound exhibit similar inter-
action reported in literature.27)
2,4ꢀ-Bis-[N-(2,5-dioxopyrrolidin-1-yl)carbamoyl]diphenylamine (16):
Yield 60%, crystallized from ethanol/water, mp 178—180 °C. 1H-NMR
(90 MHz) (DMSO-d6) d: 2.84 (8H, s, 4ꢇCH2 of dioxopyrolidine), 7.03—
Acknowledgment The authors are grateful for Dr. Nadia Hamdy, De-
partment of Biochemistry, Faculty of Pharmacy, Ain Shams University, for
7.93 (8H, m, Ar-H), 9.46 (1H, s, NH), 10.90 (1H, s, CONH), 11.30 (1H, s, conducting the in vitro EGFR inhibitory activity assays. The authors thank
CONH). IR (KBr) cmꢆ1: 3276.1, br (NH)s, 2988.9 and 2943.7 (CH), 1730.3 the National Cancer Institute, Cairo University, for performing the in vitro
and 166.20 (CꢂO)s. Anal. Calcd for C22H19N5O6: C, 58.80; H, 4.26; N, cytotoxicity.
15.58. Found: C, 58.70; H, 4.52; N, 15.55.
2,4ꢀ-Bis(N-phthalimidocarbamoyl)diphenylamine (17): Yield 59%, crys-
tallized from ethanol/water, mp 166—8 °C. H-NMR (90 MHz) (DMSO-d6)
References
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