Synthesis of quinoline thioethers as novel small molecule enhancers of monoclonal antibody production in mammalian cell culture

Article abstract of DOI:10.1016/j.tet.2010.09.020

A small library of novel quinoline derivatives containing different thioether substituents at position 4 have been synthesized and screened in murine hybridoma cell culture for their ability to enhance monoclonal antibody (mAb) production. From this set of compounds, four compounds have been discovered that enhanced immunoglobulin G (IgG) titer over negative control cultures due to an increase in specific productivity. These results demonstrate the utility of using organic synthesis and parallel ligand screening methods to discover novel cell culture additives that may be useful for increasing mAb yield in industrial biomanufacturing processes.

Full text of DOI:10.1016/j.tet.2010.09.020

Tetrahedron 66 (2010) 9461e9467
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of quinoline thioethers as novel small molecule enhancers
of monoclonal antibody production in mammalian cell culture
*
Shahid A. Kazi, Geoffrey F. Kelso, Simon Harris, Reinhard I. Boysen, Jamil Chowdhury, Milton Hearn
Centre for Green Chemistry, Monash University, Melbourne 3800, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2010
Received in revised form 16 August 2010
Accepted 6 September 2010
Available online 21 September 2010
A small library of novel quinoline derivatives containing different thioether substituents at position 4
have been synthesized and screened in murine hybridoma cell culture for their ability to enhance
monoclonal antibody (mAb) production. From this set of compounds, four compounds have been dis-
covered that enhanced immunoglobulin G (IgG) titer over negative control cultures due to an increase in
specific productivity. These results demonstrate the utility of using organic synthesis and parallel ligand
screening methods to discover novel cell culture additives that may be useful for increasing mAb yield in
industrial biomanufacturing processes.
Keywords:
Small molecule enhancer
Monoclonal antibody
Hybridoma
Ó 2010 Elsevier Ltd. All rights reserved.
IgG
Quinoline thioether
1. Introduction
the best of our knowledge, not been reported. Such ligand discov-
ery and screening methods could provide an avenue to identify
The ability of inexpensive low molecular weight compounds
(LMWCs) to enhance monoclonal antibody (mAb) yield in mam-
malian cell culture would be immensely beneficial in industrial
biomanufacturing operations and other end-user applications. In
particular, the discovery and deployment of suitable LMWCs could
lead to improved large-scale production efficiencies, enhancements
to product development, and better cost effectiveness. Several
different low molecular weight compounds that enhance mAb titer
in mammalian cell culture are already known, including histone
deacetylase inhibitors, such as sodium butyrate,1 valproic acid,
and other aliphatic carboxylate analogues,2 the mTOR inhibitor,
rapamycin,3 the chemical chaperones 4-phenyl-butyric acid and
other aromatic carboxylic acids,4 DNA methyltransferase inhibitors,
such as hexano-hydroxamic acid; as well as dimethylsulfoxide and
retinoic acid.5
Combinatorial or parallel synthesis using solid- and liquid-
phase approaches to prepare low molecular weight organic mole-
cules based on known pharmacophores has evolved in recent years
to become a powerful tool for the development of chemical
libraries of novel lead compounds in drug discovery.6 However, the
screening of such synthetic libraries for the discovery of novel cell
culture additives for the production of recombinant proteins has, to
new classes of compounds having the desired and more potent
functionalities, and which may act synergistically with known
chemical additives.
Among the broad range of pharmacophores available for drug
development are nitrogen heterocycle scaffolds, containing differ-
ent functional groups that can be selectively modified by organic
synthesis to generate libraries consisting of a diverse range of
derivatives.7 One such nitrogen heterocycle scaffold is quinoline,
a rigid planar molecule, that is, a pharmacophore present in the core
of numerous physiologically active agents that display interesting
therapeutic properties by acting as either enzyme inhibitors or as
ligands for cell surface receptors.8
Structurally, quinoline can be readily modified with a broad
range of substituent groups to provide the molecular diversity
necessary to achieve a library of compounds whereby different
members can show different biological effects.9 As part of our effort
to discover novel low molecular weight compounds, which enhance
the yield of expressed recombinant proteins, including genetically
engineered monoclonal antibodies (mAbs), using mammalian cell
culture, we have used quinoline as a scaffold to synthesize a small
library of derivatives containing different thioether substituents at
the 4-position, and a N-(2-(2-(2-amino-ethoxy)ethoxy)ethyl)car-
boxamide group at the 2-position to improve water solubility
(Fig. 1). Each member of this chemical library was then screened to
evaluate their effect on immunoglobulin G (IgG) mAb titer and cell
viability using two murine hybridoma cell lines of different clonal
* Corresponding author. Tel.: þ61 03 99054547; fax: þ61 03 99058501; e-mail
address: milton.hearn@monash.edu (M. Hearn).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.020

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S.A. Kazi et al. / Tetrahedron 66 (2010) 9461e9467
uniform heat distribution affecting product formation during the
microwave process. Finally, polyphosphoric acid11 (PPA) was cho-
sen as both a catalytic cyclization reagent and solvent, and gave the
4(1H)-quinolone 4 at lower temperature and in greater yield (78%).
The desired product was easily purified on multigram scales by
simply washing with water. The intermediate 4 was then chlori-
nated with phosphorous oxychloride to give the desired quinoline
analogue as the diversity scaffold 5 in excellent yield.
Since the quinoline core is poorly water soluble, we introduced
a hydrophilic N-(2-(2-(2-amino-ethoxy)ethoxy)ethyl)-carboxamide
group at position 2 to improve water solubility and biocompatibility.
This modification was achieved by reacting the ester 5 with a small
excess of 2, 20-(ethylene-dioxy)-bis-ethyl-amine. Although mono-
acylation of diamines is usually difficult to accomplish, we obtained
an excellent transformation (98%) at room temperature by employ-
ing p-toluenesulfonic acid as a catalyst. This procedure eliminated
the need for a less efficient deprotection and activation of the
masked carboxylic acid, and is both cost-effective and scalable.
For our initial screening studies for novel cell culture additives
we chose to introduce diversity at position 4 by nucleophilic sub-
stitution of the chloride with different commercially available
thiols to generate a small library 8aei (Fig. 2). Alternatively, 5 could
also be thiolated and the ester subsequently substituted with 2,20-
(ethylenedioxy)-bis-ethylamine to give the final product. However,
this route was found to be more chromatographically intensive.
Therefore most of the library members were prepared from 7
(Scheme 1). It can be noted that the quantitative 19F NMR spec-
troscopy of the various purified synthetic molecules revealed that
several compounds, e.g., 8b, 8c, 8d, 8f, and 8h, occurred as the
corresponding stoichiometric TFA salts despite extensive lyophili-
sation procedures being employed following their reversed phase
chromatographic purification. The occurrence of these compounds
as their TFA salts is consistent with the chemical shift differences of
Fig. 1. General structure of the low molecular weight quinoline thioether compounds,
whereby R could be alkyl, aryl, alkylamino, carboxylmethyl, etc.
ancestry. Four of the compounds investigated significantly enhanced
the IgG titer, mainly by enhancing specific productivity. These find-
ings clearly demonstrate the potential of using molecules derived
from such approaches to enhance the yield of mAbs and other
recombinant proteins in research and industrial biomanufacturing
processes.
2. Results and discussion
2.1. Chemistry
As part of our earlier SAR design approaches, 6-bromo-4-
chloroquinolinic acid methyl ester (5) was identified as a suitable,
synthetically accessible, diversity scaffold that could be used to
generate a library of structures for bioactivity screening (Scheme 1).
Tosynthesizethisscaffold,4-bromoaniline1was firstreactedwith
dimethyl acetylenedicarboxylate (DMAD) 2 to give the Michael ad-
duct, dimethyl anilinofumarate, 3 in excellent yield on multigram
scales. We initially attempted to convert the Michael adduct 3 to the
4-(1H)-quinolone
4 by a
thermal ConradeLimpach method10
involving refluxing 3 in diphenyl ether. However, under a variety of
conditions involving both conventional heating and microwave
techniques with different solvents, contact times and reaction tem-
peratures, these methods invariably gave viscous tars containing an
unsatisfactory yield of the desired product that was difficult to purify.
To overcome these problems, advantage was taken of solventless
microwave techniques since the starting material was a liquid. Under
these conditions, ring closure to the enamine 3 readily occurred.
The yield from this reaction was moderate (40%), leaving behind
mainly starting material, presumably as a consequence of non-
the terminal methylene at
d
¼3.08 ppm for the TFA salt compared to
the free base at
d
¼2.80 ppm.
2.2. Cell culture procedures
The effect of the quinoline compounds on IgG titer and cell vi-
ability was measured at four different concentrations from 80 nM
to 100
mM. Of the compounds tested, 8b, 8c, and 8d (as their tri-
fluoracetate salts) at 80 nM increased the IgG titer from clone A by
Scheme 1. Reagents and conditions: (a) MeOH, reflux, 2 h, 97%; (b) PPA, 130 ꢀC, 1 h, 78%; (c) POCl3, 100 ꢀC, 1.5 h, 95%; (d) NaSR, DMF, rt, 2e12 h; (e) 2,20ethylene dioxy-biss-
ethylamine, PTSA, MeOH, 24 h, 84%.

S.A. Kazi et al. / Tetrahedron 66 (2010) 9461e9467
9463
Fig. 2. List of quinoline compounds discussed in the test.
1.2e1.4-fold, relative to the control culture (Fig. 3A), but did not
enhance IgG production from clone B, whilst compound 8h
increased IgG titer from clone B by 1.2-fold at 80 nM concentration
(Fig. 3B). The remaining compounds, tested at the same concen-
tration, did not enhance IgG production from either clone. In gen-
eral, the test compounds either had no significant effect (p>0.05)
on cell viability or slightly reduced cell viability at both concen-
trations (Fig. 3).
These findings demonstrate that the significant increases in IgG
titer elicited by 8b, 8c, 8d, and 8h can be attributed to enhanced
specific productivity (in terms of nmol IgG/cell/min) by both clones.
Further increasing the concentration of the other test compounds
Low molecular weight cell culture additives that increase the
yield of recombinant proteins, including mAbs, from cultured
mammalian cells would have significant utility in the bio-
manufacturing industry. As noted above, previous studies on the
effect of low molecular weight compounds on mAb production
have largely focused on compounds with known bioactivity, e.g.,
histone deacetylase, DNA methyltransferase and mTOR inhibitors,
or their analogues, where productivity enhancements in similar
ranges to those determined in the current investigations have been
observed. An alternative approach to discover novel compounds
that enhance mAb production is to screen synthetic compound li-
braries built around known pharmacophores. Here, we have syn-
thesized and screened members of a small library of quinoline
derivatives, a pharmacophore with a range of known bioactivities,
to discover several novel compounds that enhance mAb production
at the 80 nM level.
as their trifluoracetate salts to 1
by either clone. Compound 8d elicited a 2.2-fold enhancement of
IgG titer from clone A at 0.8 M (Fig. 3A) whilst 8h increased IgG
titer 1.4-fold from clone B at 0.8 M (Fig. 3B). Both of these increases
were also due to enhanced specific productivity. Higher concen-
trations of the library compounds (10 M and 100 M) were
cytotoxic in these bioassays (data not shown).
mM did not increase IgG production
m
m
The quinoline derivatives synthesized in this study fell into three
broad categories, namely aliphatic, aromatic, and carboxylic thio-
ether substituents at the 4 position. In general, the compounds that
m
m

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S.A. Kazi et al. / Tetrahedron 66 (2010) 9461e9467
Fig. 3. Effect of quinoline compounds on IgG titer (80 nM, gray and 0.8 mM, diagonal columns) and cell viability (80 nM, open and 0.8 mM, dotted columns). Data are presented as the
mean percent of control from three replicatesꢁS.D. *p<0.05; **p<0.01; (A). From clone A culture; (B) From clone B culture.
enhanced IgG titer were those bearing a hydrophobic aliphatic or
unsubstituted aromatic thiother substituent, with the propyl-con-
taining derivative 8d giving a 2.2-fold enhancement with clone A.
The exception to this trend was compound 8c, which contained
a carboxylic acid group at the end of the propyl chain. It is also clear
from this work that hybridoma cell lines of different clonal ancestry
are affected differently by cell culture additives. Therefore in an in-
dustrial setting it would be prudent to screen potential novel cell
culture additives against different cell lines to determine the best
producers and conditions for optimal and maximal mAb production.
productivity at levels below 100 nM with reduced cytotoxity at
higher concentrations.
4. Experimental section
4.1. General
All reagents and solvents were purchased from SigmaeAldrich,
Acros Organics or Alfa Aesar and used without further purification.
TLC was performed on Merck silica gel 60 F254 precoated aluminum
plates. The components were visualized by fluorescence under
254 nm ultraviolet irradiation or by exposure to a variety of
development/charring reagents where necessary.
3. Conclusion
In conclusion, we have designed a simple synthetic route toward
a new series of 4-thio-substituted quinoline derivatives with
different side chain substituents These compounds have been sub-
jected to cell-based assays to investigate their effects on the pro-
duction of immunoglobulin G (IgG) mAb by two murine hybridoma
cell lines of different clonal ancestry. From this study we have
identified several novel additives that enhance mAb production by
up to 2.2-fold with specific hybridoma cell lines. These results clearly
demonstrate the utility of using organic synthesis to generate
compound libraries to discover novel active, cell culture additives to
improve productivity and yields of expressed recombinant proteins
in biomanufacturing processes and value-add to the final production
technology. Implementation of such technologies would deliver
economic benefits through increased productivity in large-scale cell
culture procedures, in traditional bioreactor or wave-bag format,
with concomitant reduction of manufacturing costs in unit batch
operations. Based on the above results, future studies related to the
introduction of structural diversity around the A ring of 8 are
anticipated to lead to even greater enhancements in expression
Microwave-assisted reactions were carried out in a CEM-Ex-
plorer Automated Microwave Workstation with temperatures
measured by an online IR sensor. Flash chromatography was per-
formed with a FlashMaster II instrument using Merck silica gel 60,
0.040e0.063
mm (230e400 mesh) for normal phase and Grace
Vydac C18 silica gel (Davisil), 35e70
mm for reverse phase chro-
matography using the automated gradient elution mode.
The 1H, 13C, and 19F NMR spectra were recorded on a Bruker
DPX-300 spectrometer (300 MHz 1H, 75 MHz 13C) and Bruker DPX-
400 spectrometer (400 MHz 1H, 100.6 MHz 13C). The 1H NMR
spectra refer to solutions in deuterated solvents as indicated. The
solvent peaks were used as an internal reference, except CDCl3
where tetramethylsilane (TMS) was used as the internal standard (
d
0.00 ppm). The 19F NMR spectra were acquired as solutions in
deuterated solvents as indicated and CFCl3 (trichloro-fluoro-
methane) used as the internal standard (d 0.00 ppm). Since auto-
mated NMR techniques were used, the peak for residual H2O/
CD3OH was automatically referenced to 4.87 ppm. Because the
chemical shift of this signal is sensitive to the presence of acidic and

S.A. Kazi et al. / Tetrahedron 66 (2010) 9461e9467
9465
basic compounds the signal due to CHD2OD did not always appear
at w3.30 ppm. Accordingly, the 1H NMR data for all samples
recorded in CD3OD were corrected using the CHD2OD signal as
internal reference. Infrared spectra were recorded on a Per-
kineElmer Spectrum RXI Fourier Transform infrared spectrometer
as KBr disks of solids or thin films of liquid (neat) between sodium
chloride plates. Low resolution electrospray ionisation mass spectra
(ESI) were recorded on a Micro mass Platform II API QMS Electro-
spray mass spectrometer with cone voltage at 25 V as solutions in
MeOH unless otherwise indicated. HRMS were recorded with an
Agilent 6220 Accurate Mass TOF LC/MS spectrometer in the positive
(ESIþ) mode. During the course of these studies it was noted that
calculations based on the ChemDraw and the website http://www-
jmg.ch.cam.ac.uk/tools/magnus/MolWeight.html software give
different values for exact masses from the third decimal place.
Calculated exact masses of the various compounds reported below
were derived with the ChemDraw software.
p-toluenesulfonic acid (10 mg). The resulting solution was stirred at
60 ꢀC for 24 h before concentrated in vacuo. The residue was diluted
with saturated NaHCO3 (50 mL) and extracted with ethyl acetate
(3ꢂ50 mL). The combined organic extracts were washed with brine
(2ꢂ50 mL), dried over MgSO4, and concentrated invacuo. The residue
was purified by flash column chromatography (MeOH/EtOAc, 1:1
with 1% Et3N) to give N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-
bromo-4-(methyl-thio)quinoline-2-carboxamide (83 mg, 76%) as
a pale yellow semi-solid; IR (KBr) nmax: 3388, 2868, 1676, 1526, 1484,
1427,1392,1306,1119, 826 cmꢃ1; 1H NMR (MeOD):
d 2.64 (s, 3H), 2.77
(t, J¼5.3 Hz, 2H), 3.53 (t, J¼5.3 Hz, 2H), 3.63e3.75 (m, 8H), 7.72 (dd,
J¼9.0,1.9 Hz,1H), 7.79 (d, J¼9.0 Hz,1H), 7.84 (s,1H), 8.03 (d, J¼1.7 Hz,
1H); 13C NMR (MeOD):
d 14.4, 40.8, 42.3, 70.8, 71.5, 71.6, 73.5, 114.1,
123.0, 126.7, 129.1, 133.1, 134.9, 145.5, 150.4, 151.5, 166.5; HRMS (ES):
m/z [MþH]þ calcd for C17H23BrN3O3S: 428.0643, found 428.0640.
4.1.4. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-(phenylthio)quinoline-2-carboxamide trifluoroacetate salt (8b). To
a stirred solution of methyl 6-bromo-4-(phenylthio) quinoline-2-
carboxylate 6b (187 mg, 0.50 mmol) in dry MeOH (5 mL) were
added 2,20-ethylene dioxy-bis-ethylamine (222 mg, 1.498 mmol)
and a catalytic amount of p-toluenesulfonic acid (20 mg). The
resulting solution was stirred at 60 ꢀC for 24 h and then concen-
trated in vacuo. The residue was diluted with saturated NaHCO3
(50 mL) and extracted with ethyl acetate (3ꢂ50 mL). The combined
organic extracts were washed with brine (2ꢂ50 mL), dried over
MgSO4, and concentrated in vacuo. The residue was purified by
reverse phase flash chromatography (acetonitrile/water, 4:1 with
1% TFA) as eluent to give N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-6-
bromo-4-(phenylthio)-quinoline-2-carboxamide (176 mg, 59%, as
the TFA salt)12 as an off-white semi-solid; IR (KBr) nmax: 3387, 2927,
Melting points were determined using a Stuart melting point
apparatus (SMP3) with digital thermometer and are uncorrected.
4.1.1. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-chloroquinoline-2-carboxamide (7). To a stirred solution of methyl
6-bromo-4-chloroquinoline-2-carboxylate 5 (208 mg, 0.692 mmol)
in dry methanol/chloroform, 2:1 (5 mL) were added 2,20-ethylene
dioxy-bis-ethylamine (321 mg, 2.166 mmol) and a catalytic amount
of p-toluenesulfonic acid (10 mg). The resulting solution was stirred
at room temperature for 24 h and then concentrated in vacuo. The
residue was diluted with saturated NaHCO3 (50 mL) and extracted
with ethyl acetate (3ꢂ50 mL). The combined organic extracts were
washed with brine (2ꢂ50 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified by reverse phase flash chroma-
tography (acetonitrile/water, 1:1) to give N-(2-(2-(2-aminoethoxy)
ethoxy) ethyl)-6-bromo-4-chloroquinoline-2-carboxamide (245 mg,
84%) as a yellow solid. MP: 97.5e98.8 ꢀC; IR (KBr) nmax: 3370, 2869,
1678, 1528, 1483, 1431, 1205, 1131, 834, 800, 722 cmꢃ1
(MeOD):
;
1H NMR
d
3.07 (t, J¼5.0 Hz, 2H), 3.59 (t, J¼5.1 Hz, 2H), 3.63e3.73
(m, 8H), 7.54e7.61 (m, 3H), 7.60 (s, 1H), 7.62e7.66 (m, 2H), 7.94 (dd,
J¼8.9, 1.7 Hz, 1H), 8.03 (d, J¼8.9 Hz, 1H), 8.42 (d, J¼1.8 Hz, 1H); 13C
1663, 1527, 1482, 1305, 1136, 983, 927, 889, 830 cmꢃ1
;
1H NMR
(MeOD):
d
2.75 (t, J¼5.3 Hz, 2H), 3.52 (t, J¼5.3 Hz, 2H), 3.63e3.75 (m,
NMR (MeOD):
d
40.5, 40.8, 68.0, 70.7, 71.5(ꢂ2), 116.8, 123.5, 126.9,
8H), 7.98 (dd, J¼8.9, 2.1 Hz, 1H), 8.06 (d, J¼8.9 Hz, 1H), 8.25 (s, 1H),
128.8, 129.7, 131.6, 131.7(ꢂ2), 133.3, 135.4, 136.7(ꢂ2), 146.0, 150.5,
8.41 (d, J¼2.1 Hz, 1H). 13C NMR (MeOD):
d
40.8, 42.3, 70.7, 71.5, 71.6,
151.3, 166.2; 19F NMR (DMSO-d6):
d
ꢃ73.12 (s); HRMS (ES): m/z
73.6, 120.9, 125.0, 127.5, 129.5, 133.2, 136.1, 144.1, 147.5, 151.7, 165.4;
HRMS (ES): m/z [MþH]þ calcd for C16H20BrClN3O3: 416.0371, found
416.0369.
[MþH]þ calcd for C22H25BrN3O3S: 490.0800, found 490.0801.
4.1.5. Synthesis of 3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethyl-carba-
moyl)-6-bromoquinolin-4-ylthio)propanoic acid trifluoroacetate salt
(8c). To a stirred solution of 3-mercaptopropionic acid (106 mg,
1.0 mmol) in dry THF (15 mL) at 0 ꢀC was slowly added sodium
hydride (72 mg, 3.0 mmol). The resulting suspension was stirred for
1 h at room temperature and then evaporated to dryness. The
resulting white solid was slowly added to a stirred solution of N-(2-
(2-(2-aminoethoxy)ethoxy) ethyl)-6-bromo-4-chloroquinoline-2-
carboxamide 7 (208 mg, 0.499 mmol) in dry DMF (10 mL) and the
mixture was stirred for 2 h at room temperature. The reaction was
quenched with distilled water (50 mL), concentrated in vacuo, and
the residue was purified by reverse phase flash chromatography
(acetonitrile/water, 1:1 with 1% TFA) to give 3-(2-(2-(2-(2-amino-
ethoxy)ethoxy)ethylcarbamoyl)-6-bromoquino- lin-4-ylthio)pro-
pionic acid (175 mg, 58%, as the TFA salt)12 as a pale yellow
semi-solid; IR (KBr) nmax: 3440, 2920, 1671, 1590, 1484, 1426, 1310,
4.1.2. Synthesis of methyl 6-bromo-4-(methylthio)-quinoline-2-car-
boxylate (6a). To a stirred solution of methyl 6-bromo-4-chloro-
quinoline-2-carboxylate 5 (1.0 g, 3.328 mmol) in dry DMF (10 mL)
at room temperature was slowly added sodium thiomethoxide
(231 mg 3.296 mmol). The resulting suspension was stirred for 3 h
at room temperature and the reaction was then quenched with
water (50 mL) and extracted with ethyl acetate (3ꢂ50 mL). The
combined organic extracts were washed with water (2ꢂ50 mL),
brine (2ꢂ50 mL), dried over MgSO4, and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/hexane,
1:1) to give methyl 6-bromo-4-(methylthio)-quinoline-2-carbox-
ylate (876 mg, 85%) as an off-white solid. MP: 172.8e173.5 ꢀC; IR
(KBr) nmax: 1714, 1560, 1452, 1331, 1243, 1049 cmꢃ1 1H NMR
;
(CDCl3):
d
2.70 (s, 3H), 4.08 (s, 3H), 7.84 (d, J¼8.7 Hz, 1H), 7.94 (s,
1H), 8.11 (d, J¼8.9 Hz, 1H), 8.29 (s, 1H); 13C NMR (CDCl3):
d
14.2,
1144, 1006, 826, 638 cmꢃ1; 1H NMR(MeOD):
d
2.65 (t, J¼7.3 Hz, 2H),
53.4, 115.3, 122.9, 125.8, 128.3, 132.8, 134.1, 145.1, 147.3, 149.9,
165.9; HRMS (ES): m/z [MþH]þ calcd for C12H11BrNO2S: 311.9688,
found 311.9691.
3.07 (t, J¼5.1 Hz, 2H), 3.45 (t, J¼7.3 Hz, 2H), 3.66e3.76 (m, 10H), 7.80
(dd, J¼9.0, 2.1 Hz, 1H), 7.90 (d, J¼9.0 Hz, 1H), 8.01 (s, 1H), 8.15 (d,
J¼2.0 Hz, 1H); 13C NMR (MeOD):
d 29.1, 37.5, 40.6, 40.8, 68.3, 71.1,
71.5, 71.6, 115.0, 123.0, 127.0, 129.5, 133.3, 135.1, 145.8, 150.4, 150.7,
4.1.3. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-(methylthio)quinoline-2-carboxamide (8a). To a stirred solution of
methyl 6-bromo-4-(methylthio)-quinoline-2-carboxylate 6a (80 mg,
0.256 mmol) in dry methanol (5 mL) were added 2,20-ethylene dioxy-
bis-ethylamine (114 mg, 0.769 mmol) and catalytic amount of
166.7, 179.0; 19F NMR (DMSO-d6):
d
ꢃ73.56 (s); HRMS (ES): m/z
[MþH]þ calcd for C19H25BrN3O5S: 486.0698, found 486.0697.
4.1.6. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-(propylthio)quinoline-2-carboxamide trifluoroacetate salt (8d). To

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S.A. Kazi et al. / Tetrahedron 66 (2010) 9461e9467
a stirred solution of propane thiol (76 mg, 1.0 mmol) in dry
methanol (5 mL) was slowly added sodium methoxide (1 mL, 25%
w/w). The resulting suspension was stirred for 1 h at room tem-
perature and then N-(2-(2-(2-aminoethoxy)ethoxy) ethyl)-6-
bromo-4-chloroquinoline-2-carboxamide 7 (209 mg, 0.502 mmol)
in dry methanol (2 mL) was slowly added to the suspension. The
mixture was stirred for 4 h at room temperature and the reaction
was then quenched with water (50 mL), concentrated in vacuo
and the residue extracted with ethyl acetate (3ꢂ50 mL). The
combined organic extracts were washed with brine (2ꢂ50 mL),
dried over MgSO4, and concentrated in vacuo. The residue was
purified by reverse phase flash chromatography (acetonitrile/wa-
ter, 1:1 with 1% TFA) to give N-(2-(2-(2-aminoethoxy)ethoxy)-
ethyl)-6-bromo-4-(propylthio) quinoline-2-carboxamide (142 mg,
50%, as the TFA salt)12 as a brown semi-solid; IR (KBr) nmax: 3399,
was then quenched with water (50 mL), concentrated in vacuo and
the residue extracted with ethyl acetate (3ꢂ50 mL). The combined
organic extracts were washed with brine (2ꢂ50 mL), dried over
MgSO4, and concentrated in vacuo. The residue was purified by
reverse phase flash chromatography (acetonitrile/water, 1:1 with 1%
TFA) to give N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-6-bromo-4-(4-
hydroxy-phenyl- thio) quinoline-2-carboxamide (87 mg, 57%, as the
TFA salt)12 as a yellow solid. MP: 133.8e135.2oC; IR (KBr) nmax: 3088
(br), 2929, 1675, 1599, 1581, 1534, 1484, 1431, 1282, 1203, 1132, 834,
722 cmꢃ1; 1H NMR (MeOD):
d
3.06 (t, J¼5.1 Hz, 2H), 3.59 (t, J¼5.0 Hz,
2H), 3.64e3.69 (m, 8H), 6.98 (d, J¼8.8 Hz, 2H), 7.46 (d, J¼8.8 Hz, 2H),
7.50 (s,1H), 7.88 (dd, J¼9.0, 2.1 Hz,1H), 7.96 (d, J¼9.0 Hz,1H), 8.34 (d,
J¼2.1 Hz, 1H); 13C NMR (MeOD):
d
40.5, 40.8, 68.0, 70.8, 71.5(ꢂ2),
115.8, 117.4, 118.8(ꢂ2), 123.3, 126.8, 128.7, 133.2, 135.3, 139.1(ꢂ2),
146.0, 150.6, 153.5, 161.5, 166.5; 19F NMR (DMSO-d6):
d
ꢃ73.16 (s);
2872, 1676, 1527, 1484, 1391, 1308, 1119, 828 cmꢃ1
(MeOD):
1.10 (t, J¼7.2 Hz, 3H), 1.76 (q, J¼7.3 Hz, 2H), 3.04 (t,
J¼7.2 Hz, 2H), 3.11 (t, J¼4.7 Hz, 2H), 3.61e3.77 (m, 10H), 7.65e7.75
(m, 2H), 7.76 (s, 1H), 7.94 (s, 1H); 13C NMR (MeOD):
14.0, 22.7,
34.1, 40.6, 40.7, 68.0, 70.8, 71.4, 71.5, 114.5, 122.9, 126.7, 129.1, 133.0,
;
1H NMR
HRMS (ES): m/z [MþH]þ calcd for C22H25BrN3O4S: 506.0749, found
d
506.0740.
d
4.1.9. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-(2-(tert-butylamino)ethylthio) quinoline-2-carboxamide (8g). To
a stirred solution of BOC-cystamine (88.5 mg, 0.50 mmol) in dry
THF (20 mL) at 0 ꢀC was slowly added 60% sodium hydride emul-
sion (70 mg, 1.75 mmol). The resulting suspension was warmed
to room temperature and then stirred for 20 min. N-(2-(2-(2-
134.8, 145.4, 149.9, 150.6, 166.3; 19F NMR (DMSO-d6):
d
ꢃ73.09 (s);
HRMS (ES): m/z [MþH]þ calcd for C19H27BrN3O3S: 456.0957, found
456.0959.
4.1.7. Synthesis of 2-(2-(2-(2-(2-aminoethoxy)ethoxy) ethylcar-
bamoyl)-6-bromoquinolin-4-yl)thiobenzoic acid (8e). To a stirred
solution of thiosalicylic acid (154 mg, 1.0 mmol) in dry DMF (5 mL)
at 0 ꢀC was slowly added sodium hydride (72 mg, 3.0 mmol). The
resulting suspension was warmed to room temperature and stir-
red for 1 h. N-(2-(2-(2-Aminoethoxy)ethoxy)-ethyl)-6-bromo-4-
chloroquin-oline-2-carboxamide 7 (208 mg, 0.499 mmol) in dry
DMF (2 mL) was then added and the mixture was stirred for 24 h
at room temperature. The reaction was slowly quenched with
water (50 mL) and extracted with ethyl acetate (2ꢂ50 mL) to
remove excess thiosalicylic acid. The aqueous extract was con-
centrated in vacuo and acidified with ethanolic hydrochloric acid
(20 mL) and then evaporated to dryness. The residue was purified
by flash chromatography (MeOH/EtOAc, 1:1 with 1% Et3N) to give
2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethylcarbamoyl)-6-bromo-qui-
nolin-4-yl)thio)benzoic acid (128 mg, 48%) as a brown liquid; IR
(KBr) nmax: 3387, 2928, 1691, 1593, 1545, 1486, 1421, 1397, 1293,
Aminoethoxy)
ethoxy)ethyl)-6-bromo-4-chloroquinoline-2-car-
boxamide 7 (105 mg, 0.252 m mol) in dry THF (2 mL) was then
added and the mixture was stirred for 4 h at room temperature. The
reaction was then quenched with water (50 mL) and extracted with
ethyl acetate (3ꢂ50 mL). The combined organic extracts were
washed with brine (2ꢂ50 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified by flash column chromatography
(MeOH/EtOAc, 1:1 with 1% Et3N) as eluent to give N-(2-(2-(2-
aminoethoxy)ethoxy)ethyl)-6-bromo-4-(2(tert-butyl-amino)ethyl-
thio)quinoline-2-carboxamide (133 mg, 95%) as an off-white solid.
MP: 83.5e84.8 ꢀC; IR (KBr) nmax: 3312, 2865, 1717, 1655, 1529, 1482,
1171, 1132, 840 cmꢃ1 1H NMR (MeOD):
; d 1.41 (s, 9H), 2.80 (t,
J¼5.2 Hz, 2H), 3.31(t, J¼6.5 Hz, 2H), 3.45 (t, J¼6.5 Hz, 2H), 3.54 (t,
J¼5.3 Hz, 2H), 3.64e3.76 (m, 8H), 7.77 (dd, 1H, J¼9.0, 2.0 Hz, 1H),
7.84 (d, J¼9.0 Hz, 1H), 7.93 (s, 1H), 8.12 (d, J¼1.8 Hz, 1H); 13C NMR
(MeOD):
d
28.9(ꢂ3), 32.4, 40.1, 40.7, 42.1, 70.8, 71.5, 71.6, 73.0, 80.4,
115.1, 123.2, 127.0, 129.5, 133.2, 135.1, 145.8, 149.8, 150.2, 158.4,
166.4; HRMS (ES): m/z [MþH]þ calcd for C23H34BrN4O5S: 557.1433,
found 557.1426.
1183, 1130, 1012, 824, 763 cmꢃ1 1H NMR(DMSO-d6):
; d 2.76 (t,
J¼5.3 Hz, 2H), 3.45e3.58 (m, 10H), 7.12 (dd, J¼10.3, 1.4 Hz, 1H), 7.27
(dt, J¼10.3, 2.2 Hz, 1H), 7.37 (dt, J¼10.0, 1.8 Hz, 1H), 7.77 (s, 1H), 7.78
(dd, J¼10.0, 2.1 Hz, 1H), 7.94 (dd, J¼9.0, 2.1 Hz, 1H), 8.02 (d,
J¼9.0 Hz, 1H), 8.29 (d, J¼2.7 Hz, 1H), 8.80(t, J¼7.8 Hz, 1H); 13C NMR
4.1.10. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-(iso-propylthio)quinoline-2-carboxamide trifluoroacetate salt (8h).
To a stirred solution of isopropanethiol (38 mg, 0.50 mmol) in
dry methanol (5 mL) was slowly added sodium methoxide
(0.5 mL, 25% w/w). The resulting suspension was stirred for 1 h at
room temperature and then N-(2-(2-(2-aminoethoxy)ethoxy)-
(DMSO-d6):
d
38.4, 38.8, 66.7, 68.6, 69.5(ꢂ2), 119.4, 121.7, 126.0,
128.1, 128.4, 129.1, 129.5, 129.6, 132.1, 132.5, 133.9, 142.2, 144.5, 148.1,
149.6, 163.4, 169.7; 1H NMR(MeOD/DMSO-d6):
d
2.76 (t, J¼5.1 Hz,
2H), 3.45e3.60 (m, 10H), 7.26e7.30 (m, 2H), 7.34e7.40 (m, 1H), 7.60
(d, J¼8.1 Hz, 1H), 7.61 (s, 1H), 7.78 (dd, J¼9.0, 2.1 Hz, 1H), 7.90 (d,
J¼9.0 Hz, 1H), 8.31 (d, J¼2.1 Hz, 1H); 13C NMR (MeOD/DMSO-d6):
ethyl)-6-bromo-4-chloroquinoline-2-carboxamide
7 (105 mg,
0.252 mmol) in dry methanol (2 mL) was added. The mixture was
stirred for 24 h at room temperature and the reaction then
quenched with water (50 mL), concentrated in vacuo, and the
residue extracted with ethyl acetate (3ꢂ50 mL). The combined
organic extracts were washed with brine (2ꢂ50 mL), dried over
MgSO4, and concentrated in vacuo. The residue was purified by
reverse phase flash chromatography (acetonitrile/water, 0e80%
gradient elution with 1% TFA) to give N-(2-(2-(2-aminoethoxy)-
ethoxy)ethyl)-6-bromo-4-(iso-propylthio)quinoline-2-carbox-amide
d
40.4, 40.8, 68.3, 70.8, 71.4, 71.5, 119.1, 123.4, 127.6, 127.9, 129.8,
130.0, 130.7(ꢂ2), 133.4, 135.3, 136.1, 146.3, 150.9, 151.2, 166.4, 168.2;
HRMS (ES): m/z [MþH]þ calcd for C23H25BrN3O5S: 534.0698, found
534.0694.
4.1.8. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-(4-hydroxyphenylthio)quinoline-2-carboxamide trifluoroacetate salt
(8f). To
a stirred solution of 4-hydroxy thiophenol (63 mg,
0.50 mmol) in dry methanol (5 mL) was slowly added sodium
methoxide (0.50 mL, 25% w/w). The resulting suspension was
stirred for 1 h at room temperature and then N-(2-(2-(2-amino-
ethoxy)ethoxy)ethyl)-6-bromo-4-chloroquin-oline-2-carbox-amide
7 (105 mg, 0.252 m mol) in dry methanol (2 mL) was added. The
mixture was stirred for 24 h at room temperature and the reaction
(78 mg, 55%, as the TFA salt)12 as a brown semi-solid; IR (KBr) nmax
3394, 2929, 1677, 1533, 1484, 1204, 1132, 834, 780, 722 cmꢃ1 1H
NMR(MeOD):
:
;
d
1.49 (d, J¼6.6 Hz, 6H), 3.10 (t, J¼5.1 Hz, 2H),
3.68e3.78 (m, 10H), 3.87 (dq, J¼6.7, 6.7 Hz, 1H), 7.85 (d, J¼9.1 Hz,
1H), 7.94 (d, J¼9.1 Hz, 1H), 8.08 (s, 1H), 8.26 (s, 1H); 13C NMR
(MeOD):
d
23.1(ꢂ2), 37.4, 40.6, 40.8, 68.0, 70.8, 71.5(ꢂ2), 116.4,

S.A. Kazi et al. / Tetrahedron 66 (2010) 9461e9467
9467
123.2, 127.3, 129.9, 133.4, 135.2, 146.2, 150.1, 150.3, 166.7; 19F NMR
120.5, 124.7, 125.7, 135.4, 137.1, 141.2, 161.8, 177.6; HRMS (ES): m/z
[MþH]þ calcd for C16H21BrN3O4: 398.0715, found 398.0714.
(DMSO-d6):
d
ꢃ73.08 (s); HRMS (ES): m/z [MþH]þ calcd for
C19H27BrN3O3S: 456.0957, found 456.0956.
Acknowledgements
4.1.11. Synthesis of 4-(2-(2-(2-(2-aminoethoxy)ethoxy) ethylcarba-
moyl)-6-bromoquinolin-4-ylthiobenzoic acid (8i). To a stirred solu-
tion of 4-mercaptobenzoic acid (28 mg, 0.180 mmol) in dry DMF
(4 mL) was slowly added sodium hydride (13 mg, 0.542 mmol). The
resulting suspension was stirred for 1 h at room temperature and
then N-(2-(2-(2-aminoethoxy)ethoxy ethyl)-6-bromo-4-chlor-
oquinoline-2-carboxamide 7 (50 mg, 0.120 mmol) in dry DMF
(2 mL) was added. The mixture was stirred for 2 h at room tem-
perature and the reaction then quenched with water, evaporated to
dryness, and purified by flash column chromatography (MeOH/
EtOAc, 1:1 with 1% Et3N) to give 4-(2-(2-(2-(2-aminoethoxy)eth-
These investigations were carried out with partial funding
support from the Australian Research Council and Cooperative
Research Centre for Polymers. We would like to thank MDS Pharma,
USA for assistance with the cell culture experiments.
References and notes
1. (a) Mimura, Y.; Lund, J.; Church, S.; Dong, S.; Li, J.; Goodall, M.; Jefferis, R. J.
Immunol. Methods 2001, 247, 205e216; (b) Sung, Y. H.; Song, Y. J.; Lim, S. W.;
Chung, J. Y.; Lee, G. M. J. Biotechnol. 2004, 112, 323e335.
2. (a) Gaurav, B.; Markus, H.; Ivan, K.; Fanny, D.; David, L. H.; Florian, M. W.
Biotechnol. Bioeng. 2008, 101, 182e189; (b) Liu, C.; Chu, I.; Hwang, S. J. Biosci.
Bioeng 2001, 91, 71e75.
oxy)ethylcarbamoyl)-6-bromo-quinol-in-4-ylthio)benzoic
acid
(59 mg, 92%) as an off-white solid; IR (KBr) nmax: 3433, 2999, 1691,
1593, 1545, 1420, 1293, 1184, 1116, 824, 764 cmꢃ1; 1H NMR (MeOD/
3. Balcarcel, R. R.; Stephanopoulos, G. Biotechnol. Bioeng. 2001, 76, 1e10.
4. Burrows, J. A. J.; Willis, L. K.; Perlmutter, D. H. Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 1796e1801.
5. (a) Allen, M. J.; Boyce, J. P.; Trentalange, M. T.; Treiber, D. L.; Rasmussen, B.;
Tillotson, B.; Davis, R.; Reddy, P. Biotechnol. Bioeng. 2008, 100, 1193e1204; (b)
Grunt, T. W.; Somay, C.; Pavelka, M.; Ellinger, A.; Dittrich, E.; Dittrich, C. J. Cell
Sci. 1991, 100, 657e666.
6. (a) Eric, M. G. Curr. Opin. Biotechnol. 1995, 6, 624e631; (b) Luc, V. H.; Gilbert, M.;
Nicolas, F. J. Chromatogr., B 1999, 725, 3e15; (c) Clemencia, P.; Jon, R. A.; Eva, B.;
Richard, A. H. Nat. Med. 2003, 9, 118e122; (d) Wess, G.; Urmann, M.;
Sickenberger, B. Angew. Chem., Int. Ed. 2001, 40, 3341e3350.
7. (a) An, H.; Dan, C. P. Chem. Rev. 2000, 100, 3311e3340; (b) Franzen, R. G. J. Comb.
Chem. 2000, 2, 195e214.
8. (a) Giardina, G. A.; Raveglia, L. F.; Grugni, M.; Sarau, H. M.; Farina, C.;
Medhurst, A. D.; Graziani, D.; Schmidt, D. B.; Rigolio, R.; Luttmann, M.;
Cavagnera, S.; Foley, J. J.; Vecchietti, V.; Hay, D. W. J. Med. Chem. 1999, 42,
1053e1065; (b) Deady, L. W.; Desneves, J.; Kaye, A. J.; Finlay, G. J.; Baguley, B.
C.; Denny, W. A. Bioorg. Med. Chem. 2000, 8, 977e984; (c) Alexandre, V. I.;
Vladimir, V. K.; Alexey, P. I.; Alexander, V. K.; Volodymir, M. K.; Caroline, T. W.;
Julia, A. K.; Dmitry, V. K. J. Comb. Chem. 2005, 7, 227e235; (d) Eastland, G., Jr.
Drugs Future 1988, 13, 13e15; (e) Dejmek, L. Drugs Future 1990, 15, 126e129.
9. Frank, E. B.; Luca, F. R.; Marco, A.; Stefano, C.; Catherine, D.; Carlo, F.; Mario, G.;
Stefania, G.; Mark, A. L.; Marisa, M.; Guy, M. M.; Nadler, G.; Carlo, P.; Paola, P.;
Henry, M. S.; Mark, A. S.; Douglas, W. P. H.; Giuseppe, A. M. G. J. Med. Chem.
2001, 44, 1675e1689.
CDCl3):
d
3.09 (t, J¼4.8 Hz, 2H), 3.62 (t, J¼4.9 Hz, 2H), 3.67e3.73
(m, 8H), 7.61 (d, J¼8.4 Hz, 2H), 7.84 (s, 1H), 7.92 (dd, J¼9.0, 1.9 Hz,
1H), 8.04 (d, J¼9.0 Hz, 1H), 8.09 (d, J¼8.4 Hz, 2H), 8.43 (d, J¼1.9 Hz,
1H); 13C NMR (DMSO-d6):
d 38.3, 38.8, 66.5, 68.6, 69.3, 69.6, 117.4,
122.1, 125.5, 127.5, 130.9(ꢂ2), 131.8, 132.3, 133.8(ꢂ2), 134.4, 134.5,
144.5, 146.7, 149.6, 163.1, 166.4; HRMS (ES): m/z [MþH]þ calcd for
C23H25BrN3O5S: 534.0698, found 534.0689.
4.1.12. Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)-ethyl)-6-bromo-
4-hydroxyquinoline-2-carboxamide (8j). To
methyl 6-bromo-4-hydroxyquinoline-2-carboxylate
a
stirred solution of
(100 mg,
4
0.355 mmol) in dry methanol (5 mL) were added 2,20-ethylene
dioxy-bis-ethylamine (148 mg, 1.0 mmol) and a catalytic amount of
p-toluenesulfonic acid (10 mg). The resulting solution was stirred at
room temperature for 24 h diluted with saturated NaHCO3 (50 mL),
concentrated in vacuo and the residue extracted with ethyl acetate
(3ꢂ50 mL). The combined organic extracts were washed with brine
(2ꢂ50 mL), dried over MgSO4, and concentrated in vacuo. The resi-
due was purified by flash chromatography (MeOH/EtOAc, 1:1 with
1% Et3N) to give N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-6-bromo-4-
hydroxyquin-oline-2-carboxamide (91 mg, 64%) as an off-white
solid. MP: 229.2e230.6oC; IR (KBr) nmax: 3233, 2928, 1676, 1626,
10. (a) Ned, D. H.; Ibrahim, S. B.; Peter, D. K.; James, M.; Cyrus, J. O.; Sally, M. L.;
Thomas, F. L. J. Med. Chem. 1968, 11, 1218e1221.
11. (a) Ned, D. H.; Ibrahim, S. B.; Thomas, F. L.; Velmer, B. F. J. Org. Chem. 1967, 32,
4155e4157; (b) Edward, C. T.; Ned, D. H. J. Org. Chem. 1967, 32, 3339e3343.
12. The amount of TFA present as a counter ion was quantified by 19F (1H decoupled)
NMR experiment using spiked sample of trifluoroethanol as reference and found
to contain 1:1 ratio of TFA as salt. As a consequence, on a weight basis the lowest
effective concentrations that the active compounds 8b, 8c, 8d, and 8h were
assayed as free quinoline compounds were w20% lower than 80 nM, i.e., 67 nM.
1597, 1509, 1203, 1130, 829, 722 cmꢃ1 1H NMR (MeOD):
; d 3.12 (t,
J¼5.1 Hz, 2H), 3.63 (t, J¼5.1 Hz, 2H), 3.68e3.73 (m, 8H), 6.89 (s, 1H),
7.76 (d, J¼8.9 Hz, 1H), 7.81 (dd, J¼8.9, 2.1 Hz, 1H), 8.31 (d, J¼2.0 Hz,
1H); 13C NMR (D2O):
d 38.6, 39.2, 65.8, 68.0, 69.0, 69.1, 106.2, 117.6,