Design, synthesis, and biological evaluation of (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as modulators of human P-glycoprotein (ABCB1)

Article abstract of DOI:10.1002/cbic.201300565

Multidrug resistance caused by ATP binding cassette transporter P-glycoprotein (P-gp) through extrusion of anticancer drugs from the cells is a major cause of failure in cancer chemotherapy. Previously, selenazole-containing cyclic peptides were reported

Full text of DOI:10.1002/cbic.201300565

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DOI: 10.1002/cbic.201300565
Design, Synthesis, and Biological Evaluation of (S)-Valine
Thiazole-Derived Cyclic and Noncyclic Peptidomimetic
Oligomers as Modulators of Human P-Glycoprotein
(ABCB1)
Satyakam Singh,[a] Nagarajan Rajendra Prasad,[b, c] Khyati Kapoor,[b] Eduardo E. Chufan,[b]
Bhargav A. Patel,[a] Suresh V. Ambudkar,*[b] and Tanaji T. Talele*[a]
Multidrug resistance caused by ATP binding cassette transport-
er P-glycoprotein (P-gp) through extrusion of anticancer drugs
from the cells is a major cause of failure in cancer chemothera-
py. Previously, selenazole-containing cyclic peptides were re-
ported as P-gp inhibitors and were also used for co-crystalliza-
tion with mouse P-gp, which has 87% homology to human P-
gp. It has been reported that human P-gp can simultaneously
accommodate two to three moderately sized molecules at the
drug binding pocket. Our in silico analysis, based on the ho-
mology model of human P-gp, spurred our efforts to investi-
gate the optimal size of (S)-valine-derived thiazole units that
can be accommodated at the drug binding pocket. Towards
this goal, we synthesized varying lengths of linear and cyclic
derivatives of (S)-valine-derived thiazole units to investigate
the optimal size, lipophilicity, and structural form (linear or
cyclic) of valine-derived thiazole peptides that can be accom-
modated in the P-gp binding pocket and affects its activity,
previously an unexplored concept. Among these oligomers,
lipophilic linear (13) and cyclic trimer (17) derivatives of
QZ59S-SSS were found to be the most and equally potent in-
hibitors of human P-gp (IC50 =1.5 mm). As the cyclic trimer and
linear trimer compounds are equipotent, future studies should
focus on noncyclic counterparts of cyclic peptides maintaining
linear trimer length. A binding model of the linear trimer 13
within the drug binding site on the homology model of
human P-gp represents an opportunity for future optimization,
specifically replacing valine and thiazole groups in the non-
cyclic form.
Introduction
P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1),
encoded by ABCB1, is a plasma membrane-bound ATP-binding
cassette (ABC) transporter. P-gp catalyzes the ATP-dependent
efflux of a highly diverse set of compounds, including amphi-
pathic, neutral, or weakly basic compounds with molecular
weights ranging from less than 200 to 2000 Da.[1–3] The 1280-
residue human P-gp consists of two transmembrane domains
(TMDs), each with six a-helices and two nucleotide-binding do-
mains (NBDs). The drug/substrate binding sites are predicted
to be located in the TMDs.[4,5] It has been well established that
the drug binding pocket is even capable of binding to two to
three molecules simultaneously.[4,6–9] Drug transport by P-gp is
driven by hydrolysis of ATP at NBDs. The close conformation of
NBDs generated by ATP binding/hydrolysis mediates substrate
translocation from the drug binding sites in TMDs, thus trig-
gering release of the substrate to the extracellular face of the
membrane.[10,11]
In cancer patients, resistance to chemotherapy is a critical
issue and can be due to various mechanisms. Overexpression
of multidrug resistance (MDR) proteins such as P-gp is one of
the well-studied mechanisms of drug resistance.[12] Upregula-
tion of ABC drug transporters has been demonstrated in a vari-
ety of cancer types and has been shown to result in reduced
intracellular concentration of chemotherapeutic drugs. P-gp is
largely recognized for its role in enabling cancer cells to evade
response to treatment through the efflux of chemotherapeutic
agents. This multidrug resistance impedes the clinical efficiency
of chemotherapy. Therefore, many researchers have been en-
gaged in developing human P-gp inhibitors to reverse the che-
motherapeutic drug resistance. As there is no high-resolution
crystal structure of human P-gp yet available, a homology
model of human P-gp based on the crystal structure of P-gp
from mouse[13] serves as a tool for structure-based drug design
of P-gp inhibitors. Although various strategies, such as random
and focused screening, systematic chemical modifications, and
[a] Dr. S. Singh, B. A. Patel, Dr. T. T. Talele
Department of Pharmaceutical Sciences
College of Pharmacy and Health Sciences, St. John’s University
8000 Utopia Parkway, Queens, NY 11439 (USA)
E-mail: talelet@stjohns.edu
[b] Dr. N. R. Prasad, Dr. K. Kapoor, Dr. E. E. Chufan, Dr. S. V. Ambudkar
Laboratory of Cell Biology, Center for Cancer Research
National Cancer Institute, National Institutes of Health
37 Convent Drive, Bethesda, Maryland 20892-4256 (USA)
E-mail: ambudkar@mail.nih.gov
[c] Dr. N. R. Prasad
Current address:
Department of Biochemistry and Biotechnology, Annamalai University
Annamalai Nagar, Annamalai 608002, Tamilnadu (India)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300565.
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combinatorial chemistry have been pursued thus far to devel-
op the first three generations of P-gp inhibitors, their toxicity
and drug interaction profiles are still undesirable. Therefore,
new strategies leading to the development of fourth genera-
tion P-gp inhibitors (isolation of natural products or chemically
modified natural product analogues) with high P-gp selectivity
and potency seems to be a novel approach.[14]
residues of the drug binding site and is four times more
potent than the corresponding QZ59Se-RRR isomer. Therefore,
in our design strategy described below, we chose to maintain
S stereochemistry at all chiral centers. 3) Comparison of inhibi-
tory activity of QZ59S-SSS for Chinese hamster or mouse P-gp
(IC50 =2.7 mm)[15] with that of potent inhibitor valspodar[16] sug-
gests a role of macrocyclic peptidic nature of valspodar for its
high potency as compared to QZ59Se-SSS (Figure 1). During
ongoing phase III clinical trials, valspodar has shown very re-
stricted oral bioavailability which, along with its efficacy and
safety concerns, further suggested the need for optimiza-
tion.[17]
Analysis of QZ59-selenazole cyclic peptides (QZ59Se-SSS and
QZ59Se-RRR)[5] within the drug binding cavity of a human P-gp
homology model (built previously by using mouse P-gp)[13] led
to the following observations: 1) two copies of QZ59Se-SSS
were found at the QZ59Se-SSS binding site[5] (Figure 1); more-
over, it has been reported that human P-gp can simultaneously
accommodate two to three molecules at the drug binding
transmembrane domain.[4,6–9] 2) P-gp can distinguish between
the stereoisomers of cyclic peptides such that QZ59Se-SSS
forms extensive hydrophobic contacts with the hydrophobic
Unlike valspodar and cyclosporine A, both of which are mac-
rocyclic undecamer peptides, our peptidomimetic oligomers
incorporate insertion of thiazole ring between the Ca-carbon
and carbonyl carbon, thus making them non-natural peptides
that could be potentially devoid of immunosuppressant activi-
Figure 1. Design strategy: A) schematic structure of mouse P-gp crystal structure bound to QZ59Se-SSS[5] and chemical structures of QZ59Se-SSS and valspo-
dar; B) human P-gp homology model generated from mouse P-gp (PDB ID: 3G60), cyclic trimer QZ59S-SSS along with its structural (shape) variant linear
trimer and the cyclic hexamer.
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ty as well as resistant to hydrolytic cleavage by proteases. The
constrained five-membered thiazole ring imposes conforma-
tional restrictions that lead to entropically favorable binding at
the drug binding site of P-gp. With this backdrop, herein we
report the effect of structural form, length, and flexibility of
the designed (S)-valine thiazole-derived peptidomimetic oligo-
mers on human P-gp function.
Compound 5 was synthesized by a modified Hantzsch
method.[24,25] This procedure involves a reaction between com-
pound 3 and ethyl bromopyruvate to give the cyclocondensed
intermediate 4,5-dihydrothiazole 4, which, upon treatment
with trifluoroacetic anhydride (TFAA), furnished aromatized
thiazole product 5 in low yields.[21] Here, highly acidic reaction
conditions in the later step present a possibility for N-trifluor-
oacetylation of amide NH. For this reason, the crude product
obtained was further treated with freshly prepared sodium
ethoxide in ethanol to produce the desired product 5 in appre-
ciable yields without affecting the stereocenter adjacent to the
2-position of the thiazole ring. Next, saponification of com-
pound 5 with sodium hydroxide produced the monomeric
acid (6), and deprotection of the Boc-NH in compound 5 by
using trifluoroacetic acid (TFA) produced the monomeric
amine TFA salt (7). Further, Boc-deprotection of carboxylic acid
derivative 6 yielded zwitterion product 8 (Scheme 1).
The co-crystal structure of QZ59Se-SSS and mouse P-gp has
been previously reported.[5] The sulfur analogue was also
shown to have an IC50 value of 2.7 mm against mouse P-gp;[15]
however, the study reported here illustrates the inhibitory ac-
tivity of QZ59S-SSS against human P-gp. Replacement of sele-
nium with sulfur could be an effective strategy for designing
natural product analogues, which are better tolerated by
human cells. Thiazole structures are abundant in natural prod-
ucts and have direct applications in drug discovery.[18] Further-
more, we performed docking experiments on these analogues
at all of the possible binding sites of homology modeled
human P-gp.
Next, through quick initial experiments, we optimized the
coupling of valine-derived monomeric thiazole acid (6)
through activation with BOP (benzotriazol-1-yl-oxy)-tris(dimeth-
ylamino)phosphonium hexafluorophosphate reagent), HOBt,
and DIEA in CH2Cl2/DMF (4:1) with an excess of monomer thi-
azole amine (7) to produce the required dimer (9) in moderate
yield, as shown in Scheme 2. This may be attributed to the
sterically demanding isopropyl group adjacent to the amine
functionality.[26,27] Compounds 10 and 11 were synthesized by
basic and acidic hydrolysis of dimer 9, respectively. Further, de-
protected compound 12 was made by treating dimer acid 10
with TFA in DCM (Scheme 2). The synthetic route to trimer de-
rivatives is portrayed in Scheme 3. Monomeric amine 7 and di-
meric acid 10 were coupled by using the BOP-HOBt method to
obtain the desired linear trimer (13). Compound 13 was hydro-
lyzed by sodium hydroxide and TFA, respectively, to give tri-
meric acid 14 and trimeric amine 15. Acid 14 was again treat-
ed with TFA in DCM to obtain intermediate trimeric zwitterion
16, which was carried forward without further purification. In-
termediate 16 was cyclized in
Results and Discussion
Chemistry
The thiazole derivatives were synthesized with diminutive var-
iations in the procedures reported by Bertram et al. and as
shown in Schemes 1 to 6.[19,20] Compounds with one thiazole
unit, both linear and cyclic, are referred to as monomeric deriv-
atives, those with two thiazole units are called dimeric, and so
forth (trimeric, tetrameric, and hexameric). Scheme 1 shows
the synthesis of monomeric derivatives, starting with commer-
cially available N-Boc-(S)-valine (1), which was converted to
amide 2 by a general mixed anhydride/aqueous ammonia
method in quantitative yield (Scheme 1). Amide 2 was then
transformed to thioamide 3 by a Holpfazel–Hantzsch proce-
dure[21] using Lawesson’s reagent in tetrahydrofuran (THF).[22,23]
the presence of pentafluoro-
phenyl
diphenylphosphinate
(FDPP), anhydrous ZnCl2, and
DIEA in CH2Cl2/DMF (4:1) to pro-
vide the target cyclic trimer (17;
QZ59S-SSS).[28]
Alternatively,
cyclic trimer 17 was prepared by
coupling and subsequent cycli-
zation of three molecules of mo-
nomer zwitterion compound 8
according to the above men-
tioned procedure. The above
compounds were found to be
optically pure (eeꢀ95%), based
on analysis by chiral HPLC
(single peak when using acetoni-
trile/water/TFA 85:15:0.1). After
exploring a number of different
conditions to prepare linear pep-
tides, we found that coupling re-
Scheme 1. Reagents and conditions: a) i: isobutyl chloroformate, N-methyl morpholine, THF, ꢁ208C, 4 h; ii: 30%
NH4OH in excess, ꢁ208C to RT, 4 h; b) Lawesson’s reagent, THF, 508C, 16 h; c) ethyl bromopyruvate, KHCO3, DME,
ꢁ158C to RT, 12 h; d) i: TFAA, 2,6-lutidine, DME, ꢁ158C to RT, 15 h; ii: NaOEt, EtOH, ꢁ158C to RT, 6 h; e) NaOH,
THF/MeOH/H2O (10:2:3), RT, 4 h; f) TFA, CH2Cl2, RT, 12 h.
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Scheme 2. Reagents and conditions: a) BOP, HOBt, DIEA, CH2Cl2/DMF (4:1), RT, 18 h; b) NaOH, THF/MeOH/H2O (10:2:3), RT, 4 h; c) TFA, CH2Cl2, RT, 12 h.
Scheme 3. Reagents and conditions: a) BOP, HOBt, DIEA, CH2Cl2/DMF (4:1), RT, 18 h; b) NaOH, THF/MeOH/H2O (10:2:3), RT, 4 h; c) TFA, CH2Cl2, RT, 12 h;
d) FDPP, DIEA, anhydrous ZnCl2, CH2Cl2/DMF (4:1), RT, 5 d.
actions of thiazole acid in the presence of 1.5 equiv of BOP, investigation, the carboxy terminus of valine-derived bis-thia-
1.5 equiv of HOBt, and 1.5 equiv of N,N-diisopropylethylamine zole acid (10) was coupled with commercially available arylalk-
(DIEA), with valine-derived thiazole amine TFA salt in excess yl amines 22a and 23a by using coupling reagents HCTU,
amounts (1.3–1.5 equiv) in CH2Cl2/DMF (4:1) worked satisfacto- HOBt, and DIEA in DMA to obtain compounds 22 and 23 in
rily. Linear derivatives of tetramer 18 and hexamer 19 were moderate yield, as shown in Scheme 6. The activity of the syn-
prepared from corresponding thiazole starting materials thesized analogues was assessed for P-gp modulation by using
(Scheme 4). Compound 18 was prepared by coupling 10 and calcein-AM and BODIPY-FL-Prazosin efflux assays, inhibition
11; linear hexamer 19 was synthesized beginning from 14 and of photolabeling of P-gp with
[
125I]iodoarylazidoprazosin
15. The cyclic tetramer (20) and cyclic hexamer (21) were syn- ([125I]IAAP), and an ATPase activity assay.
thesized starting from dimer zwitterion (12), giving both prod-
ucts in one step (Scheme 5). The tetramer and hexamer deriva-
Structure–activity relationships
tives were produced in very low yields (Schemes 4 and 5);
therefore, the desired products were purified and characterized The thiazole-containing peptides were studied for their modu-
by means of preparative HPLC with subsequent electron spray latory effects on the transport function of human P-gp. Herein,
ionization-mass spectrometry (ESI-MS) analysis. In our further we wanted to investigate the optimal size, lipophilicity, and
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photolabeling of P-gp [125I]IAAP, with 5 mm tariquidar
giving complete (100%) inhibition (see Table 2).
The monomeric compounds comprising a single
thiazole unit (5–8) were devoid of P-gp inhibitory ac-
tivity, as indicated by data obtained from the efflux
assays (<5–10% inhibition). These results were also
corroborated by [125I]IAAP % inhibition. Ineffective in-
hibition of P-gp efflux function by monomers 5–8
might possibly be due to their smaller size and limit-
ed hydrophobic surface area. In this regard, increas-
ing the number of thiazole units to two in com-
pound 5, thus resulting in compound 9, was found
to result in substantial improvement in P-gp inhibi-
tion. Dimeric compound 9 inhibited calcein-AM efflux
by 55% and [125I]IAAP labeling by 87%, as compared
to <5% and 20%, respectively, for monomeric com-
pound 5. In contrast, BODIPY-FL-Prazosin efflux analy-
sis (20% inhibition) showed modest improvement for
compound 9. However, compounds 10 and 12, the
corresponding acid and zwitterion derivatives, re-
spectively, of 9, exhibited complete loss in inhibitory
activity (<5% calcein-AM efflux inhibition; 12% and
9% BODIPY-Prazosin efflux inhibition, respectively).
On the other hand, amine 11 maintained similar in-
hibition potency to that of compound 9 (55% cal-
cein-AM efflux inhibition and 13% BODIPY-FL-Prazo-
sin efflux inhibition). Among the dimers, a free car-
boxy group proved unfavorable, which might be due
to poor cell penetration. However, dually protected
dimer 9, as well as its Boc-hydrolyzed analogue 11,
both showed identical inhibition of P-gp efflux func-
tion as compared to acid 10 and zwitterion 12. The
Scheme 4. Reagents and conditions: a) BOP, HOBt, DIEA, CH2Cl2/DMF (4:1), RT, 18 h.
[
125I]IAAP labeling inhibition results for these dimeric
derivatives (10, <10%; 11, 87%; 12, 23%) corre-
sponded to data from the efflux assays. These results
suggest that the free acid group is not accepted;
however, the free amino group is rather well tolerat-
ed in the P-gp binding pocket, as this moiety is fre-
quently seen in P-gp modulators.[32] Next, further in-
Scheme 5. Reagents and conditions: a) FDPP, DIEA, CH2Cl2/DMF (4:1), RT, 5 d.
creasing the length to a tripeptide, compound 13
showed impressive activity with 98% inhibition of
structural form (linear and cyclic) of these peptides that can be
accommodated well in the P-gp drug binding pocket and
affect P-gp activity.
calcein-AM efflux by P-gp, along with 75% inhibition of
BODIPY-FL-Prazosin transport. The dose–response curve for
this analogue against calcein-AM efflux function revealed an
IC50 of 1.5 mm. In affirmation of this activity, [125I]IAAP labeling
was inhibited by a significant extent (91%). In addition to this
substantial enhancement, deprotection of compound 13 was
effected at both ends. As observed for compound 10, acid 14
was devoid of calcein-AM or BODIPY-FL-Prazosin efflux inhibi-
tion potential (<10%) but showed moderate (60%) inhibition
of [125I]IAAP labeling of P-gp. From these results, it is evident
that larger molecular size, coupled with extended hydrophobic
surface area and lipophilic characteristics are the key features
contributing towards increased inhibition of P-gp efflux func-
tion. The increase in the [125I]IAAP labeling inhibition by com-
pound 14 might be due to an increase in the hydrophobic
segment as compared to dimer acid 10, and lack of transport
The synthesized (S)-valine-derived thiazole products (5–15,
17–23) were evaluated for their effect on human P-gp activity
utilizing multiple assays. Table 1 represents the percent inhibi-
tion values for all thiazole derivatives obtained from calcein-
AM and BODIPY-FL-Prazosin efflux assays in HeLa cells express-
ing human P-gp. In Table 1, inhibition of P-gp transport activity
by the synthesized compounds was expressed as relative to
the percent inhibition caused by 1 mm tariquidar, a known
third generation P-gp inhibitor.[29–31] All the synthesized deriva-
tives were tested for their effects on photoaffinity labeling of
P-gp with [125I]IAAP, which is a transport substrate for P-gp; re-
sults are given in Table 2. For comparison of inhibitory potency
of our derivatives, we used tariquidar as a standard inhibitor of
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function inhibitory activity could
be attributed to the fact that
these cell-based assays require
compounds to permeate the lip-
ophilic cell bilayer. Amine deriva-
tive 15 showed moderate inhibi-
tory activity of 55 and 67% for
transport, yet still maintained
91% inhibition of [125I]IAAP label-
ing. Subsequently, trimer 13 was
cyclized to obtain compound 17
(i.e., QZ59S-SSS) and evaluated
against human P-gp efflux func-
tion. The observed results sug-
gest that the linear and cyclic
forms of the tripeptide structure
are equipotent, based on the
inhibition of calcein-AM efflux
(inhibition=97%, IC50 =1.5 mm),
Scheme 6. Reagents and conditions: a) HCTU, HOBt, DIEA, DMA, RT, 18 h.
BODIPY-FL-Prazosin assay (89%),
and the [125I]IAAP labeling assay
(87%). The results obtained from trimeric analogues further re-
inforce the notion that increased molecular size and hydropho-
bicity contribute towards inhibition of P-gp efflux function. As
increasing molecular size and hydrophobicity proved beneficial
to inhibit P-gp efflux activity, we decided to hereafter synthe-
Table 1. Effect of (S)-valine-based thiazole derivatives on the transport
function of P-gp in HeLa cells.
Tested Inhibition of efflux[a] [%]
Compound Common name
conc. Calcein-
BODIPY-
Prazosin
[mm]
AM
Table 2. Effect of (S)-valine-based thiazole derivatives on photolabeling
5
6
7
8
9
10
11
12
13
14
15
17
18
19
20
21
22
monomer
20
20
20
20
20
20
20
20
10
10
10
10
10
10
10
10
10
<5
<5
<5
<5
55
<5
55
<5
98[b]
<5
55
97[b]
59
<5
62
<5
97[b]
10
10
<5
n.d.[c]
20
12
13
9
75
10
67
89
81
12
79
15
88
of P-glycoprotein with [125I]IAAP.
monomer acid
monomer amine
monomer zwitterion
dimer
dimer acid
dimer amine
dimer zwitterions
linear trimer
trimer acid
Compound
Common name
Tested conc.
[mm]
[
125I]IAAP
inhibition[a] [%]
5
6
7
8
9
10
11
12
13
14
15
17
18
19
20
21
22
monomer
20
20
20
20
20
20
20
20
10
10
10
10
10
10
10
10
10
20
<10
<10
<10
87
<10
87
23
91
60
91
87
monomer acid
monomer amine
monomer zwitterion
dimer
dimer acid
dimer amine
dimer zwitterion
linear trimer
trimer acid
trimer amine
cyclic trimer (QZ59S-SSS)
linear tetramer
linear hexamer
cyclic tetramer
cyclic hexamer
R-1-(4-methoxy-phenyl)-
ethyl dimer acid
2,4-dimethoxy benzyl
amine dimer acid
trimer amine
cyclic trimer (QZ59S-SSS)
linear tetramer
linear hexamer
cyclic tetramer
cyclic hexamer
R-1-(4-methoxy-phenyl)-
ethyl dimer acid
2,4-dimethoxybenzyl-
amine dimer acid
99
94
95
91
23
10
91[b]
87
[a] BacMam-P-gp virus-transduced HeLa cells were incubated with 0.5 mm
calcein-AM for 10 min or BODIPY-FL-Prazosin for 45 min at 378C in the
dark in the presence and absence of 10 mm (S)-valine-based thiazole de-
rivatives. Cells were washed once with IMDM medium and data acquired
in FL-1 channel in a flow cytometer. The percentage of transport inhibi-
tion was derived by taking the level of inhibition obtained with a known
P-gp inhibitor, tariquidar, at 1 mm equal to 100%, and the values shown
are an average of two independent experiments done in triplicate.
[b] The IC50 values of inhibition of calcein-AM efflux for selected com-
pounds: 13 (linear trimer)=1.5 mm; 17 (cyclic trimer)=1.5 mm; 22 (R-1-(4-
methoxyphenyl)ethyl dimer acid)=2.0 mm; and 23 (2,4-dimethoxybenzyl
amine dimer acid)=2.0 mm. These values were determined by taking the
average of at least two independent experiments done in triplicate.
[c] Not determined.
100
23
10
95
[a] Crude membranes (50–75 mg protein) from P-gp-expressing High-Five
insect cells were incubated in the presence and absence of 10 mm thi-
azole derivatives in 50 mm Tris·HCl (pH 7.5) and 4–6 nm
[
125I]IAAP
(2200 Cimmolꢁ1). The samples were then photo-crosslinked by exposure
to 366 nm UV light for 10 min, and incorporation of [125I]IAAP was deter-
mined on a phosphorimager as described previously.[45] The positive con-
trol (5 mm tariquidar) inhibited IAAP incorporation into P-gp by 100%.
The results are given as percent inhibition of [125I]IAAP incorporation into
P-gp by taking the average of two independent experiments.
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size only the lipophilic linear structure protected at both ends
and the cyclic form for higher analogues such as tetramers
and hexamers. In this direction, four units of thiazole-contain-
ing linear tetramer 18 were tested to find that the cell-based
inhibitory activity (59 and 81%), decreased by 1.6-fold as com-
pared to that of homologue 13 despite an increase in
with results from the labeling assay; this suggests that these
compounds interact with P-gp. For comparison, we used the
well-established substrate verapamil,[35] which is also a good
stimulator of ATPase activity,[33,34] and found that 50 mm verapa-
mil gave 2.5-fold stimulation of P-gp ATPase activity (see
Table 3). Nonetheless, the results presented here suggest that
the ability to cross the cell membrane decreases with an in-
crease in the size of the molecule above that of the trimer-
length structures. Furthermore, to understand the binding
mechanism of oligomers to the drug binding pocket of the
homology model of human P-gp[13,36] at molecular level, we
performed glide docking using ABCB1-QZ59Se-RRR (site 1),
ABCB1-QZ59Se-SSS (site 2), ABCB1-verapamil (site 3), a site
common to the above three sites (site 4)[5] and the ATP binding
site, following the protocol mentioned in our previous stud-
ies.[13,36] Binding energy data for the docked poses of oligomers
were compared at all sites, and the QZ59Se-RRR binding site of
P-gp (site 1) was found to be the most favorable site for bind-
ing of linear trimer analogue 13, which was also supported by
[
125I]IAAP labeling inhibition (99%). Surprisingly, the linear
hexamer (19) lacked any significant P-gp efflux inhibitory activ-
ity (5 and 12%) in transport assays. Additionally, cyclic tetramer
20, which showed moderate inhibition of efflux transport (62
and 79%) demonstrated appreciable inhibition of [125I]IAAP la-
beling (95%) of P-gp. Similar to its linear analogue, cyclic hexa-
mer (21) also proved to be ineffective in transport assays (<5
and 15%). It was interesting to note that linear and cyclic hex-
amers (19 and 21) did not inhibit calcein-AM or BODIPY-FL-Pra-
zosin transport significantly, although these derivatives signifi-
cantly inhibited photoaffinity labeling of [125I]IAAP (94% for 19
and 91% for 21). This suggests that the hexameric compounds
interact with P-gp, contrary to the transport assay results.
These discrepancies may be ascribed to decreased ability of
the higher analogues of the trimeric compounds to cross the
cell membrane in short-term transport assays.
the photoaffinity labeling assay with substrate
(Table 2).
[
125I]IAAP
The flow cytometer-based transport assay was used with
fluorescent substrates to test the potency of synthesized deriv-
atives. Figure 2A shows a typical histogram of the effect of
selected compounds on calcein-AM efflux by P-gp. It is clear
from the Figure that linear trimer 13 is as effective in its cyclic
form (17) at 5 mm, whereas the dimer (9) is moderately and the
monomer (5) is least effective. For comparison, inhibition of
calcein-AM efflux with 1 mm tariquidar is also shown in Fig-
ure 2A (tainted filled trace). Figure 2B depicts the concentra-
tion-dependent effect of these three compounds and, from
the inhibition curves, IC50 values (concentrations required for
50% inhibition) were determined. The IC50 values for the linear
(13) and cyclic (17) trimers were similar (1.5 mm; see Table 1),
whereas for the dimer (9), it was much higher (20 mm; Fig-
ure 2B).
Functionally, P-gp possesses ATPase activity that is stimulat-
ed by many substrates.[33,34] ATPase stimulation activity for se-
lected derivatives (13–15, 17–23) was determined by using
membranes of P-gp-expressing High-Five insect cells (Table 3).
Here as well, the linear (13, fold stimulation=2.80) and the
cyclic (17, fold stimulation=2.81) trimer analogues were found
to stimulate the ATPase activity equivalently. Corresponding to
the above transport assays, the trimeric acid (14, fold stimula-
tion=1.47) was less effective, whereas an increase in ATPase
activity by the amine derivative (15, fold stimulation=2.16)
was comparable to that of compound 13. For the higher ana-
logues, the ATPase activity fold stimulation data was consistent
Table 3. Effect of selected thiazole derivatives on vanadate-sensitive
The data obtained thus far suggests that trimeric length,
both for linear and cyclic compounds, was optimal for blocking
P-gp efflux activity. To this end, we synthesized two derivatives
(22 and 23) of dimer acid 10 by coupling with the correspond-
ing methoxy-substituted arylalkyl amines to mimic the trimer
length. A critical role of methoxy-substituted arylalkyl amines
in tariquidar and elacridar towards P-gp inhibition prompted
us to choose these pharmacophores. Further, we chose a di-
meric acid, in spite of its inactivity, because protected dimer 9
was found to be active, thus indicating a possible role for the
carboxy terminal extension in enhanced binding. Also, we
wanted to limit the size of the resulting chain to a trimer
because analogues higher than a trimer proved ineffective.
Indeed, these compounds showed significant efflux inhibition
of both calcein-AM and BODIPY-FL-Prazosin (22, inhibition=97
and 88%; 23, inhibition=91 and 87%) as well as [125I]IAAP
labeling of P-gp (22, inhibition=100%; 23, inhibition=95%).
The IC50 values for inhibition of calcein-AM efflux for both 22
and 23 were found to be 2.0 mm (see Table 1). Both 22 and 23
also stimulated ATPase activity of P-gp by 3.31- and 3.42-fold,
respectively.
ATPase activity of P-glycoprotein.
Compound
ATPase activity
[nmol Pi minꢁ1 per mg protein][a]
Fold stimulation
13
14
15
17
18
19
20
21
22
23
78.00
41.40
60.70
78.75
83.70
54.30
42.15
77.20
89.40
92.30
2.80
1.47
2.16
2.81
2.98
1.93
1.50
2.75
3.31
3.42
[a] Crude membranes (100 mg proteinmLꢁ1) from P-gp expressing High-
Five cells were incubated at 378C with 2.5 mm of selected derivatives in
the presence and absence of 0.3 mm sodium orthovanadate in ATPase
assay buffer for 5 min, then the vanadate-sensitive ATPase activity of P-
gp was determined as described.[44] Vanadate-sensitive ATPase activity
was calculated as nmol Pi per min per mg protein and expressed as fold
stimulation with respect to basal ATPase activity (in the presence of
DMSO solvent) taken as 1.0. For the same membranes, 50 mm verapamil
stimulated the ATPase activity of P-gp by 2.5-fold (70 nmol Pi minꢁ1 mgꢁ1
protein). The values are an average of two independent experiments.
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inhibition of [125I]IAAP incorporation and stimulation of ATPase
activity, which are not transport assays, the same does not
hold true.
Molecular docking
The binding model of linear trimer 13 (labeled as X1) is shown
in Figure 3A and B. The Boc tert-butyl group is involved in hy-
drophobic contacts with the side chains of residues L304 and
M986, whereas the carbamate group of Boc enters into elec-
trostatic interactions with Q725 (ꢁNH···O(H2N)C-Q725, 3.2 ꢁ)
and Q990 (ꢁC(H3C)3ꢁO···H2N(O)C-Q990, 2.8 ꢁ). The thiazole ring
nitrogen atom next to the carbamate group is involved in elec-
trostatic interactions with Q725 (N···H2N(O)C-Q725, 2.2 ꢁ). Fur-
ther, the carbonyl oxygen atom attached to the same thiazole
ring was shown to have a critical hydrogen bonding interac-
tion with Y307 (ꢁCO···HO-Y307, 2.0 ꢁ). The thiazole rings and
isopropyl groups are responsible for the significant interactions
with M69, F72, F336, F343, F728, A729, F732, F957, F978 and
V982. The ethyl group of the ester functionality is located in
the vicinity of hydrophobic residues L65, M68 and M949. The
thiazole ring nitrogen atom near the ester group is shown to
have another hydrogen bonding interaction with Y953 (ꢁCO···
HO-Y953, 2.4 ꢁ).
Among these residues, Y307, Q725, F728, and V982 might
have a significant role in interactions, as these residues were
found to be involved with most of the ligands during docking
to different drug binding sites of human P-gp. These residues
were also shown to be critical based on labeling with metha-
nethiosulfonate (MTS)-verapamil.[40]
Figure 2. Inhibition of P-gp-mediated calcein-AM transport by various deriv-
atives. HeLa cells transduced with wild-type P-gp BacMam virus were evalu-
ated for transport function by using calcein-AM (0.5 mm) as described in the
Experimental Section. A) Reversal of calcein-AM transport was carried out in
the presence of solvent DMSO (control, solid line), or in the presence of vari-
ous synthesized derivatives at 5 mm. The reversal by monomer 5 (dashed
line), dimer 9 (complex line), linear trimer 13 (long dashed line), and cyclic
trimer 17 (QZ59S-SSS; dotted line) were compared with tariquidar (1 mm),
which is a known inhibitor of P-gp (tinted filled trace). B) BacMam P-gp-
transduced HeLa cells were assayed for calcein-AM transport in the presence
Conclusions
We synthesized a series of (S)-valine-derived thiazole-contain-
ing cyclic and noncyclic peptidomimetic oligomers to identify
the optimal structural requirements for potent inhibition of
human P-gp. Based on a set of 17 compounds from monomer
to hexamer, it was revealed that linear (13) and cyclic (17)
trimer oligomers of (S)-valine-derived thiazole units were found
to be the most potent inhibitors of human P-gp (IC50 =1.5 mm),
with potency comparable to third generation inhibitors (e.g.,
tariquidar) of this transporter. In addition, our analysis also indi-
cated the importance of the molecular size, lipophilicity, and
hydrophobic contact surface area of the synthesized com-
pounds. As the cyclic trimer and linear trimer were equipotent,
future studies should focus on noncyclic versions of various
known cyclic peptides. The binding model of compound 13
within the drug binding site on the homology model of
human P-gp represents an opportunity for future optimization
with linear trimers based on the present interactions. Two rep-
resentative dimer analogues (22 and 23) were also synthesized
to verify the hypothesis that maintaining a length up to trimer
improves inhibition of P-gp efflux function. Indeed, 22 and 23
were shown to have significant inhibitory capacity on human
P-gp efflux transport. Therefore, future SAR will be mainly de-
voted to dimer derivatives modified to mimic a trimer length
to obtain highly active inhibitors specific for human P-gp.
~
&
of increasing concentrations of monomer 5 ( ), dimer 9 ( ), linear trimer 13
!
*
), and cyclic trimer 17 (QZ59S-SSS; ). Transport in the absence of these
(
compounds was taken as 100%. The results are represented as an average
of two independent experiments. The IC50 value for the dimer (9) was
20 mm, and the value for both the linear trimer (13) and the cyclic trimer
(17) was 1.5 mm.
In general, logP values of resulting compounds could have
a major role in the activity of compounds, as observed in vari-
ous QSAR analyses of P-gp inhibitors.[37–39] It is evident that lip-
ophilicity contributes significantly to high P-gp inhibitory activ-
ity. These studies have also described that molecules with logP
values between 3 and 6 have been shown to exhibit higher
mean efflux ratios.[37–39] Interestingly, the QikProp-derived
clogP values of potent trimer analogues (13, 17, 22, and 23)
were found to be within the range of 3 and 6. However, the
higher oligomers, namely tetramer and hexamer derivatives
18–21 were in a higher range of clogP values (6–9), which
could have been a possible barrier for the inhibitory activity of
P-gp, whereas inactive zwitterion compounds were found to
have negative clogP values. This is also evident from the
graphs illustrated in Figure S1A–D (see the Supporting Infor-
mation). There is a clear correlation between size and potency
of derivatives until ~850 Da for inhibition of transport, but for
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were determined on
a Thomas
Hoover capillary melting point
apparatus and are uncorrected.
NMR spectra were recorded on
a Bruker 400 Avance DPX spec-
trometer (1H at 400 MHz) outfitted
with a z-axis gradient probe. The
chemical shifts (d) for 1H NMR are
reported in parts per million (ppm)
downfield from tetramethylsilane
(TMS) as an internal standard.
Flash chromatography was per-
formed by using silica gel (0.060–
0.200 mm) obtained from Dynamic
adsorbents. HPLC was performed
by using an Agilent 1260 Infinity
system employing
silica gel cartridge
a
Dynamax
column
(30 cmꢂ10 mm internal diameter).
Elutions were monitored at UV=
254 nm. Optical rotations of the
chiral compounds were measured
on a PerkinElmer 241 Polarimeter
with chloroform as solvent; con-
centration (c) is expressed as g/
100 mL. Chiral HPLC analysis was
performed on
a
Dionex Ulti-
mate 3000 Series instrument. The
compounds were dissolved in
MeOH and injected (20 mL each)
into a Chiralpak 1A column (Daicel
Corp., Fort Lee, NJ) with stationary
phase as amylose tris (3,5-dime-
thylphenylcarbamate) immobilized
on 5 mm silica gel. Chiral homoge-
neity was ensured by using an iso-
cratic mobile phase (n-hexane/
EtOAc 1:1), eluting at a flow rate of
1 mLminꢁ1 and monitored at UV=
370 nm.
Synthesis
Method A: General procedures for
ester hydrolysis of thiazole amino
acid derivatives: Thiazole ethyl
ester derivatives (0.01m) were
added in a mixture of solvents
[THF/MeOH/water (10:2:3)], and
cooled to 08C. NaOH (10 equiv)
was added, and the mixture was
stirred at RT for 12 h. The reaction
mixture was then concentrated in
vacuo and partitioned between
EtOAc (30 mL) and water (20 mL).
The aqueous phase containing
compound was collected and
Figure 3. XP-Glide-predicted binding mode of compound 13 with homology modeled P-gp. A) 3D model of the
binding mode of linear trimer 13 in the homology modeled human P-gp drug binding cavity. Important amino
acids present within 4 ꢁ from the ligand are depicted as sticks with the atoms colored as: carbon: green, hydro-
gen: white, nitrogen: blue, oxygen: red, sulfur: yellow), whereas the inhibitor is shown as a ball-and-stick model
with the same color scheme as above except that carbon atoms are represented in orange. Electrostatic and hy-
drogen bonding interactions are shown in ꢁ with the distances in dotted lines. B) Schematic representation dia-
gram of the 3D model with important interactions observed in the complex of liner trimer 13 with the drug bind-
ing site residues of human P-gp. Electrostatic (blue dotted lines) and hydrogen bonding (red dotted lines) interac-
tions are shown with distances in ꢁ. (For the references to color in this figure legend, the reader is referred to the
Supporting Information.)
acidified to pH 4 with 10% KHSO4, then extracted with EtOAc (3ꢂ
20 mL). The organic fractions were dried over sodium sulfate
(Na2SO4) and concentrated under reduced pressure to yield the
carboxylic acid derivatives.
Experimental Section
Chemistry–general: Chemicals were purchased from Aldrich, AK
Scientific (Union City, CA), Oakwood Products (West Columbia, SC),
Alfa Aesar (Ward Hill, MA), and TCI America (Portland, OR) and
were used as received. All compounds were checked for homoge-
neity by TLC with silica gel as the stationary phase. Melting points
Method B: General procedures for Boc-N-deprotection of thiazole
amino acid derivatives: Trifluoroacetic acid (TFA; 42 equiv) was
added dropwise to a solution of N-Boc protected thiazoles in
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CH2Cl2 (1 mLmmolꢁ1) at 08C and the solution was stirred at RT
under nitrogen atmosphere for 12 h. After completion of the reac-
tion, the solvent was removed in vacuo, followed by coevaporation
of the residual solvent with toluene (3ꢂ30 mL). The remaining
crude mass was added to water (20 mL), acidified with 2m HCl,
and extracted with EtOAc (30 mL). Saturated aqueous NaHCO3 was
added to the aqueous phase containing amine salt and extracted
with EtOAc (3ꢂ30 mL). The organic layers were washed with brine,
dried over Na2SO4, and evaporated. The resulting concentrate was
triturated with diethyl ether and dried in vacuo to afford free
amine derivatives.
lized by trituration with EtOAc/n-hexane (3:8) to provide the amide
(2) as a white solid (2.87 g, 96%): m.p. 156–1598C (lit. 160–
1618C);[25] 1H NMR (400 MHz; [D6]DMSO; TMS): d=7.29 (s, 1H), 7.03
(s, 1H), 6.54 (d, J=8.9 Hz, 1H), 3.73 (t, J=8.9 Hz, 1H), 1.91–1.89 (m,
1H), 1.38 (s, 9H), 0.85 (d, J=7.1 Hz, 3H), 0.81 ppm (d, J=7.1 Hz,
3H).
(S)-(2-Methyl-1-thiocarbamoylpropyl)carbamic acid tert-butyl
ester (3): Compound 2 (2.50 g, 11.56 mmol) was dissolved in dry
THF (30 mL) under nitrogen atmosphere. Lawesson’s reagent
(9.35 g, 23.12 mmol) was added under a well-ventilated fume
hood, and the resulting suspension was stirred at 508C for 16 h.
After cooling to RT, the reaction was quenched with saturated
aqueous NaHCO3 (20 mL), then diluted with EtOAc (40 mL) and
stirred at RT for additional 2 h. The layers were separated, and the
organic layer was washed with brine (20 mL), dried over Na2SO4,
and concentrated in vacuo. Purification by flash column chroma-
tography (5–25% EtOAc/n-hexane) yielded title compound (3) as
a yellow solid (2.15 g, 80%): m.p. 72–768C (lit. 84–858C);[21] Rf =0.6
Method C: General procedures for peptide coupling to obtain linear
thiazole derivatives: Di-isopropylethylamine (DIEA; 1.5 equiv) was
added to the stirred suspension of the carboxylic acid derivatives
(1.0 equiv) in 4:1 CH2Cl2/DMF (0.25m) or dimethyl acetamide
(DMA; 0.10m). The mixture was cooled to 08C, then BOP reagent
(1.5 equiv) in DMF or HCTU (1.5 equiv) in DMA, followed by HOBt
(1.5 equiv), were added. The solution was stirred at 08C for 10 min,
then a pre-cooled solution of the thiazole amine TFA salt (1.3–
1.5 equiv) in DMF (or DMA) and DIEA (1.5 equiv) was added and
stirred at RT for 12–15 h. The reaction mixture was then concen-
trated in a vacuum rotary evaporator, followed by partitioning of
the residual mass between EtOAc and aqueous citric acid (10%,
w/v). The aqueous layer was repeatedly extracted with EtOAc (4ꢂ
20 mL). The combined organic fractions were washed sequentially
with saturated aqueous NaHCO3 and brine. This mixture was then
dried over Na2SO4 with subsequent evaporation under reduced
pressure. The residue was purified by flash chromatography on
silica gel with n-hexane/EtOAc (1:1) as eluent to give the required
compounds.
1
(EtOAc/n-hexane 2:3); H NMR (400 MHz; [D6]DMSO; TMS): d=9.63
(s, 1H), 9.18 (s, 1H), 6.52 (d, J=9.1 Hz, 1H), 3.55–3.53 (m, 1H),
1.90–1.88 (m, 1H), 1.43 (s, 9H), 0.87 (d, J=7.1 Hz, 3H), 0.84 ppm (d,
J=7.1 Hz, 3H).
(S)-2-(1-tert-Butoxycarbonylamino-2-methylpropyl)thiazole-4-car-
boxylic acid ethyl ester (5):[23] The solution of
3 (2.00 g,
8.61 mmol) in dry DME (30 mL) was cooled to ꢁ158C with subse-
quent addition of KHCO3 (7.76 g, 77.49 mmol) under nitrogen at-
mosphere. The resulting suspension was vigorously stirred for
15 min, followed by dropwise addition of ethyl bromopyruvate
(2.01 g, 10.33 mmol) under inert atmosphere. The reaction was
then continued at ꢁ158C for 1 h after which it was allowed to
warm gradually to RT and stirred for 12 h. The mixture was then fil-
tered through a celite pad. The filtrate was concentrated in vacuo
at 408C to yield a dark colored crude mass of hydroxythiazoline
intermediate 4. The crude mass was further dissolved per se in dry
DME (20 mL) under nitrogen, and the resulting solution was
cooled to ꢁ158C. To this, a solution of 2,6-lutidine (7.38 g,
68.88 mmol) and TFAA (9.04 g, 43.05 mmol) in dry DME (10 mL)
was added dropwise over a period of 40 min (large amounts of
CO2 were released). The solution was stirred at ꢁ158C for 3 h and
then allowed to stir at RT for an additional 12 h; afterwards, the re-
action mixture was concentrated and then stirred with freshly pre-
pared sodium ethoxide (3 equiv) in EtOH (20 mL). After 6 h, EtOH
was evaporated under reduced pressure. The residual product was
added to a saturated aqueous NaHCO3 (10 mL) solution. The result-
ing mixture was extracted with EtOAc (3ꢂ30 mL). The combined
organic layer was washed with citric acid (10%) and brine, dried
over Na2SO4, and concentrated in vacuo. Purification of the residue
by column chromatography (5–35% EtOAc/n-hexane) afforded the
title compound (5) as a white solid (1.04 g, 37%): m.p. 112–1148C
(lit. 114–1168C);[21] Rf =0.5 (EtOAc/n-hexane 2:3); purity (HPLC,
Method D: General procedures for cyclization through peptide cou-
pling of thiazole amino acids: The zwitterion derivatives were sus-
pended in a mixture of anhydrous CH2Cl2/DMF (4:1; 20 mLmmolꢁ1
)
under inert atmosphere with subsequent addition of DIEA
(3 equiv), pentafluorophenyl diphenylphosphinate (FDPP; 3 equiv)
and anhydrous ZnCl2 (1 equiv; to induce cyclization of the
trimer).[28] The reaction mixture was stirred at RT and monitored by
TLC. Upon disappearance of the starting compound (five days), the
reaction was quenched with saturated aqueous NaHCO3, and the
solvent was concentrated in vacuo. The residual mixture was parti-
tioned between CH2Cl2 (50 mL) and water (25 mL). The aqueous
fraction was back-extracted with CH2Cl2 (2ꢂ50 mL), and combined
organic layers were washed successively with 10% aqueous citric
acid (2ꢂ25 mL), water (25 mL) and brine (50 mL), dried over
Na2SO4, and evaporated under reduced pressure to leave a mixture
of cyclic peptide products, which were separated by silica gel
column chromatography (EtOAc/n-hexane 7:3) or by preparative
HPLC with a gradient solvent system (60–90% CH3CN in mixture of
0.1% TFA in water) with a flow rate of 1 mLminꢁ1 to obtain the
pure cyclic peptides.
1
UV=254 nm): 95%; H NMR (400 MHz; [D6]DMSO; TMS): d=8.40 (s,
(S)-(1-Carbamoyl-2-methylpropyl)carbamic acid tert-butyl ester
(2): Isobutyl chloroformate (2.26 g, 16.56 mmol) and N-methylmor-
pholine (NMM; 1.68 g, 16.56 mmol) were added to a stirred solu-
tion of N-Boc-(S)-valine (1; 3.00 g, 13.80 mmol) in anhydrous THF
(20 mL) under nitrogen atmosphere and was allowed to stir for 4 h
at ꢁ208C. Afterwards, excess (20 mL) of aqueous ammonia (30%)
was added, and the resulting biphasic mixture was stirred at room
temperature for 4 h. After completion of the reaction, THF was re-
moved under reduced pressure. The residual mixture was extracted
with EtOAc (3ꢂ30 mL). The combined organic fractions were
washed with 1n KHSO4 and brine, then dried over anhydrous
Na2SO4. After evaporation of the solvent, the product was recrystal-
1H), 7.72 (d, J=8.4 Hz, 1H), 4.61 (t, J=7.5 Hz, 1H), 4.31–4.27 (m,
2H), 2.21–2.19 (m, 1H) 1.39 (s, 9H), 1.29 (t, J=7.1 Hz, 3H), 0.88 (d,
J=6.7 Hz, 3H), 0.84 ppm (d, J=6.7 Hz, 3H).
(S)-2-(1-tert-Butoxycarbonylamino-2-methylpropyl)thiazole-4-car-
boxylic acid (6): Compound 5 (0.90 g, 2.74 mmol) was hydrolyzed
to obtain compound 6, according to method A, as a white solid
(0.75 g, 91%): m.p. 131–1338C; Rf =0.20 (MeOH/CH2Cl2 5:95);
purity (HPLC, UV=254 nm): 97%; 1H NMR (400 MHz; [D6]DMSO;
TMS): d=12.34 (s, 1H), 8.32 (s, 1H), 7.70 (d, J=8.4 Hz, 1H), 4.60 (t,
J=7.5 Hz, 1H), 2.21–2.19 (m, 1H) 1.39 (s, 9H), 0.88 (d, J=6.7 Hz,
3H), 0.84 ppm (d, J=6.7 Hz, 3H).
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(S)-2-(1-Amino-2-methylpropyl)thiazole-4-carboxylic acid ethyl
ester (7): Compound 5 (0.80 g, 3.50 mmol) was used to obtain
compound 7 as a yellow solid (0.47 g, 85%) by using Method B:
m.p. 38–398C; Rf =0.30 (MeOH/CH2Cl2 5:95); purity (HPLC, UV
8.40 (s, 1H), 5.18–5.12 (m, 1H), 4.70 (t, J=7.6 Hz, 1H), 2.47–2.45 (m,
1H), 2.25–2.3 (m, 1H), 1.01–0.90 ppm (m, 12H) (amine and acid
peaks were missing); ESI-MS(+): m/z found for C16H22N4O3S2Na:
382.11 [M+Na]+.
1
254 nm): 95%; H NMR (400 MHz; [D6]DMSO; TMS): d=8.50 (s, 1H),
(S)-2-(1-{[2-(1-{[2-(1-tert-Butoxycarbonylamino-2-methylpro-
pyl)thiazole-4-carbonyl]amino}-(S)-2-methylpropyl)thiazole-4-car-
bonyl]amino}-(S)-2-methylpropyl)thiazole-4-carboxylic acid ethyl
ester (13): Linear trimer 13 was obtained from dimer acid 10
(0.30 g, 0.62 mmol) and monomer amine 7 (0.17 g, 0.74 mmol), by
following Method C, as a white solid (0.16 g, 36%): Rf =0.40
(EtOAc/n-hexane 1:1); purity (HPLC, UV 254 nm): 98%; 1H NMR
(400 MHz; CDCl3; TMS): d=8.07 (s, 1H), 8.06 (s, 1H), 8.04 (s, 1H),
7.95 (d, J=9.2 Hz, 1H), 7.82 (d, J=9.2 Hz, 1H), 5.39–5.32 (m, 2H),
5.11 (d, J=8.7 Hz, 1H), 4.89 (brs, 1H), 4.43 (q, J=7.1 Hz, 2H), 2.71–
2.60 (m, 2H), 2.41–2.30 (m, 1H), 1.45 (s, 9H), 1.40 (t, J=7.1 Hz, 3H),
1.08–1.00 (m, 15H), 0.94–0.87 ppm (d, J=6.8 Hz, 3H); ESI-MS(+):
m/z found for C31H45N6O6S3: 693.26 [M+H]+.
5.10–4.80 (m, 1H), 4.39 (q, J=7.0 Hz, 2H), 2.44–2.42 (m, 1H), 2.1
(brs, 2H), 1.38 (t, J=7.0 Hz, 3H), 0.89 (d, J=6.7 Hz, 3H), 0.86 ppm
(d, J=6.8 Hz, 3H).
(S)-2-(1-Amino-2-methylpropyl)thiazole-4-carboxylic acid (8):
Compound 6 (0.50 g, 1.66 mmol) was first dissolved in CH2Cl2
(15 mL) under nitrogen atmosphere and cooled to 08C. TFA
(42 equiv) was added dropwise, and the solution was stirred at RT
under nitrogen atmosphere for 12 h. After removal of CH2Cl2, the
residual mixture was coevaporated with toluene (3ꢂ10 mL) to
obtain the salt form of compound 8, which was used without fur-
ther purification.
(S)-2-(1-{[2-(1-tert-Butoxycarbonylamino-2-methylpropyl)thia-
zole-4-carbonyl]amino}-(S)-2-methylpropyl)thiazole-4-carboxylic
acid ethyl ester (9): Dimer 9 was obtained from compounds 6
(0.45 g, 1.99 mmol) and 7 (0.50 g, 1.66 mmol), by following Meth-
od C, as a white solid (0.45 g, 53%): m.p. 90–928C (lit. 91–938C);[20]
Rf =0.40 (EtOAc/n-hexane 2:3); purity (HPLC, UV=254 nm): 98%;
1H NMR (400 MHz; [D6]DMSO; TMS): d=8.73 (d, J=8.4 Hz, 1H),
8.46 (s, 1H), 8.23 (s, 1H), 7.74 (t, J=6.9 Hz, 1H), 5.15–5.06 (m, 1H),
4.70 (brs, 1H), 4.32 (q, J=7.0 Hz, 2H), 2.47–2.45 (m, 1H), 2.24–2.22
(m, 1H), 1.40 (s, 9H), 1.30 (t, J=7.1 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H),
0.90–0.88 ppm (m, 9H); ESI-MS(+): m/z found for C23H34N4O5S2Na:
533.18 [M+Na]+.
(S)-2-(1-{[2-(1-{[2-(1-tert-Butoxycarbonylamino-2-methylpro-
pyl)thiazole-4-carbonyl]amino}-(S)-2-methylpropyl)thiazole-4-car-
bonyl]amino}-(S)-2-methylpropyl)thiazole-4-carboxylic acid (14):
Trimer acid 14 was obtained from linear trimer 13 (0.30 g,
0.43 mmol), by following Method A, as a white solid (0.24 g, 83%):
Rf =0.20 (MeOH/CH2Cl2 5:95); purity (HPLC, UV 254 nm): 98%;
1H NMR (400 MHz; [D6]DMSO; TMS): d=12.95 (s, 1H), 8.73 (s, 2H),
8.38 (s, 1H), 8.28 (d, J=5.6 Hz, 1H), 8.23 (brs, 1H), 7.74 (d, J=
9.2 Hz, 1H), 5.18 (d, J=8.7 Hz, 2H), 4.68 (brs, 1H), 2.47–2.31 (m,
2H), 2.24–2.21 (m, 1H), 1.41 (s, 9H), 1.04–0.86 ppm (m, 18H); ESI-
MS(+): m/z found for C29H40N6O6S3Na: 687.21 [M+Na]+.
(S)-2-(1-{[2-(1-{[2-(1-Amino-2-methylpropyl)thiazole-4-carbonyl]a-
mino}-(S)-2-methylpropyl)thiazole-4-carbonyl]amino}-(S)-2-meth-
ylpropyl)thiazole-4-carboxylic acid ethyl ester (15): Trimer amine
15 was obtained from linear trimer 13 (0.40 g, 0.58 mmol), by fol-
lowing Method B, as a white solid (0.11 g, 31%): Rf =0.20 (MeOH/
CH2Cl2 5:95); purity (HPLC, UV 254 nm): 95%; 1H NMR (400 MHz;
[D6]DMSO; TMS): d=8.74–8.68 (m, 2H), 8.62 (brs, 2H), 8.47 (s, 2H),
8.30 (s, 1H), 5.21–5.10 (m, 2H), 4.74 (brs, 1H), 4.29 (q, J=7.1 Hz,
2H), 2.47–2.31 (m, 2H), 2.29–2.27 (m, 1H), 1.28 (t, J=7.1 Hz, 3H),
1.32–0.87 ppm (m, 18H); ESI-MS(+): m/z 593.00 (C26H37N6O4S3,
593.20, [M+H]+).
(S)-2-(1-{[2-(1-tert-Butoxycarbonylamino-2-methylpropyl)thia-
zole-4-carbonyl]amino}-(S)-2-methylpropyl)thiazole-4-carboxylic
acid (10): Dimer acid 10 was obtained, by hydrolyzing dimer 9
(0.40 g, 0.78 mmol) according to Method A, as a white solid
(0.33 g, 79%): m.p. 101–1038C (lit. 101–1068C);[20] Rf =0.20 (MeOH/
1
CH2Cl2 5:95); purity (HPLC, UV=254 nm)>99%; H NMR (400 MHz;
[D6]DMSO; TMS): d=8.69 (d, J=8.4 Hz, 1H), 8.38 (s, 1H), 8.23 (s,
1H), 7.74 (d, J=8.1 Hz, 1H), 5.11 (t, J=8.4 Hz, 1H), 4.70 (t, J=
7.6 Hz, 1H), 2.47–2.45 (m, 1H), 2.25–2.23 (m, 1H), 1.40 (s, 9H), 0.97
(d, J=6.8 Hz, 3H), 0.92–0.85 ppm (m, 9H) (acid peak was missing);
ESI-MS(+): m/z found for C21H30N4O5S2Na: 505.15 [M+Na]+.
(S)-2-(1-{[2-(1-{[2-(1-Amino-2-methylpropyl)thiazole-4-carbonyl]ami-
no}-(S)-2-methylpropyl)thiazole-4-carbonyl]amino}-(S)-2-methylpro-
pyl)thiazole-4-carboxylic acid (16): TFA (42 equiv) was added drop-
wise to a solution of trimer acid 14 in CH2Cl2 (15 mL) at 08C, and the
solution was stirred at RT under nitrogen atmosphere for 15 h. After
completion of the reaction, the solvent was coevaporated with tolu-
ene (3ꢂ). The resulting product was used without further purification.
ESI-MS(+): m/z found for C24H33N6O4S3: 565.18 [M+H]+.
(S)-2-(1-{[2-(1-Amino-2-methylpropyl)thiazole-4-carbonyl]amino}-
(S)-2-methylpropyl)thiazole-4-carboxylic acid ethyl ester (11):
Dimer amine 11 was obtained from dimer 9 (0.40 g, 0.78 mmol), by
following Method B, as a yellowish-white solid (0.12 g, 36%): m.p.
119–1228C (lit. 120–1238C);[20] Rf =0.20 (MeOH/CH2Cl2 5:95); purity
(HPLC, UV=254 nm): 95%; 1H NMR (400 MHz; CD3OD; TMS): d=
8.62 (d, J=8.7 Hz, 1H), 8.42 (s, 1H), 8.21 (s, 1H), 5.15–5.11 (m, 2H),
4.32 (q, J=7.1 Hz, 2H), 2.47–2.40 (m, 2H), 1.30 (t, J=7.1 Hz, 3H),
1.16 ppm (m, 12H) (amine peak was missing); ESI-MS(+): m/z
found for C18H26N4O3S2Na: 433.18 [M+Na]+.
Cyclic tris-thiazole amino acid (QZ59S-SSS; 17): Compound 17
was obtained from compound 16 (0.80 g, 1.42 mmol), by following
Method D, as a white solid (0.25 g, 32%): m.p. 243–2478C; Rf =0.45
(S)-2-(1-{[2-(1-Amino-2-methylpropyl)thiazole-4-carbonyl]amino}-
(S)-2-methylpropyl)thiazole-4-carboxylic acid (12): TFA (42 equiv)
was added dropwise to the suspension of dimer acid 10 (0.30 g,
0.62 mmol) in CH2Cl2 (15 mL) at 08C. After completion of the reac-
tion, the solvent was coevaporated with toluene (45 mL). The
crude mass was dissolved in EtOAc and washed with 10% aqueous
NaHCO3 (2ꢂ10 mL) and brine (2ꢂ10 mL). The organic extract was
dried over Na2SO4 and concentrated in vacuo. The residual mass
was triturated with ether and hexane to afford the title com-
pound 12 as a white solid (0.21 g, 86%): Rf =0.20 (MeOH/CH2Cl2
5:95); purity (HPLC, UV=254 nm): 98%; 1H NMR (400 MHz;
[D6]DMSO; TMS): d=8.69 (t, J=8.9 Hz, 1H), 8.45 (d, J=3.3 Hz, 1H),
25
(EtOAc/n-hexane 1:1); purity (HPLC, UV=254 nm)ꢀ98%; ½aꢂD
=
1
ꢁ102.648 (c=5.3, CHCl3); H NMR (400 MHz; CDCl3; TMS): d=8.47
(d, J=9.1 Hz, 3H), 8.09 (s, 3H), 5.43 (dd, J=9.3 Hz, 5.8 Hz, 3H),
2.34–2.27 (m, 3H), 1.10 (d, J=6.8 Hz, 9H), 1.05 ppm (d, J=6.8 Hz,
9H); ESI-MS(+): m/z found for C24H31N6O3S3: 547.15 [M+H]+.
Preparation of 17 through coupling and cyclization of 8: Alterna-
tively, compound 17 was obtained from compound 8 (0.75 g,
2.45 mmol) as a white solid (0.11 g, 24%), by following method D.
(S)-2-(1-{[2-(1-{[2-(1-{[2-(1-tert-Butoxycarbonylamino-2-methyl-
propyl)thiazole-4-carbonyl]amino}-(S)-2-methylpropyl)thiazole-4-
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1
carbonyl]amino}-(S)-2-methylpropyl)thiazole-4-carbonyl]amino}-
(S)-2-methylpropyl)thiazole-4-carboxylic acid ethyl ester (18):
Dimer acid 10 (0.40 g, 0.46 mmol) and dimer amine 11 (0.23 g,
0.55 mmol) were coupled according to Method C to give linear
tetramer 18 as a white solid (0.23 g, 32%): purity (HPLC, UV=
254 nm): 98%; 1H NMR (400 MHz; [D6]DMSO; TMS): d=8.77–8.70
(m, 3H), 8.45 (s, 1H), 8.27 (brs, 2H), 8.23 (s, 1H), 7.76 (d, J=7.9 Hz,
1H), 5.19–5.07 (m, 3H), 4.70 (brs, 1H), 4.28 (q, J=7.2 Hz, 2H), 2.47–
2.42 (m, 3H), 2.27–2.23 (m, 1H), 1.39 (s, 9H), 1.31 (t, J=7.1 Hz, 3H),
1.10–0.95 (m, 9H), 0.94–0.82 ppm (m, 15H); ESI-MS(+): m/z found
for C39H54N8O7S4Na: 897.2871 [M+Na]+.
(HPLC, UV 254 nm): 98%; H NMR (400 MHz; [D6]DMSO; TMS): d=
8.68 (brs, 1H), 8.39 (d, J=5.8 Hz, 1H), 8.23 (s, 1H), 8.19 (d, J=
3.6 Hz, 1H), 7.75 (brs, 1H), 7.08 (d, J=8.2 Hz, 1H), 6.56 (s, 1H), 6.47
(d, J=8.4 Hz, 1H), 5.17–5.07 (m, 1H), 4.69–4.67 (m, 1H), 4.37 (d, J=
5.8 Hz, 2H), 3.80 (s, 3H), 3.73 (s, 3H), 2.48–2.46 (m, 1H), 2.25–2.22
(m, 1H), 1.40 (s, 9H), 1.12–1.05 ppm (m, 12H); ESI-MS(+): m/z
found for C30H41N5O6S2Na: 654.2399 [M+Na]+.
Biological procedures
Cell lines: HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 1% glutamine, and 1% penicillin as described previously.[3]
Linear hexamer (19): The trimer acid (14; 0.10 g, 0.15 mmol) and
the trimer amine (15; 0.11 g, 0.18 mmol) were coupled according
to Method C, and the crude product was purified by preparative
HPLC with a gradient solvent system of 60–90% CH3CN in water
with 0.1% TFA. The peak separated at 51 min was characterized as
linear hexamer 19 by ESI-MS analysis and obtained as a white solid
(0.002 g, 0.60%): purity (HPLC, UV=254 nm) ꢀ99%; ESI-MS(+):
m/z found for C55H74N12O9S6Na: 1261.48 [M+Na]+.
Cloning and amplification of BacMam-P-gp virus and transduction of
HeLa cells: The expression clones for P-gp were generated in
pDest-625, as described previously.[3] These were then transformed
into E. coli DH10Bac cells (Invitrogen) and plated on gentamycin-,
kanamycin-, tetracycline-, IPTG-, and X-gal-selective media as per
the manufacturers’ protocols. White colonies were then selected
from these plates, and bacmid DNA was prepared by the alkaline
lysis method, which was further verified by PCR amplification
across the bacmid junctions.
Cyclic tetramer 20 and cyclic hexamer 21: Cyclic tetramer 20 and
cyclic hexamer 21 were obtained from dimer zwitterions (0.90 g,
2.74 mmol) by following Method D and were separated by prepa-
rative HPLC with a gradient solvent system of 60–90% CH3CN in
water with 0.1% TFA at a flow rate of 1 mLminꢁ1. Both compounds
were confirmed by single peaks in the ESI-MS spectrum. The ex-
tremely low yield of cyclic hexamer permitted us to evaluate ESI-
MS and biological activity only.
HeLa cells (2.5 million) were transduced with BacMam WT-P-gp
virus at a titer of 50–60 viral particles per cell in DMEM (3 mL).[3]
DMEM (17 mL) was added after 1 h, and the cells were further in-
cubated at 378C in 5% CO2. After 3–4 h, 10 mm butyric acid was
added, and the cells were grown overnight at 378C. After 24 h, the
cells were trypsinized, washed, counted, and analyzed by flow
cytometry to test the potency of various derivatives to inhibit
transport function of P-gp.
Cyclic tetramer 20: The peak separated at 19 min was cyclic tetra-
mer 20 (0.001 g, 0.11%): m.p. 150–1538C; Rf =0.45 (EtOAc/n-
1
Fluorescent substrate transport assay: The ability of various deriva-
tives to inhibit the transport function of human P-gp was evaluat-
ed with fluorescent substrates by flow cytometry, as described pre-
viously.[41,42] Briefly, the baculovirus-transduced cells were trypsi-
nized and resuspended in IMDM medium containing 5% FBS. Cells
were incubated with the indicated concentration of the com-
pounds or 1 mm tariquidar, followed by calcein-AM (0.5 mm) for
10 min or BODIPY-FL-Prazosin (0.5 mm) for 45 min at 378C, as
described previously.[3] The cells were washed with cold PBS, resus-
pended in 300 mL of PBS with 0.1% bovine serum albumin, and
analyzed. Fluorescence of calcein or BODIPY-FL-Prazosin was mea-
sured with a FACSort flow cytometer, equipped with a 488 nm
argon laser and 530 nm bandpass filter. The percentage of trans-
port inhibition was derived when compared with inhibition ob-
tained by using the standard inhibitor, tariquidar, at 1 mm. The re-
sults are plotted as an average of two experiments. The IC50 values
for inhibition of calcein-AM efflux by selected derivatives were cal-
culated by using GraphPad Prism 5.0.
hexane 2:1); purity (HPLC, UV=254 nm): 99%; H NMR (400 MHz;
CDCl3; TMS): d=8.66 (d, J=8.9 Hz, 1H), 8.31 (d, J=8.6 Hz, 1H),
8.18 (s, 1H), 8.15 (brs, 2H), 8.13 (s, 1H), 8.01 (d, J=7.3 Hz, 1H),
7.89 (d, J=10.0 Hz, 1H), 5.52–5.48 (m, 1H), 5.36 (t, J=9.8 Hz, 1H),
5.25 (t, J=7.8 Hz, 1H), 5.03 (t, J=7.8 Hz, 1H), 2.69–2.67 (m, 1H),
2.43–2.41 (m, 2H), 2.29–2.27 (m, 1H), 1.23 (d, J=6.7 Hz, 3H) 1.17
(d, J=6.7 Hz, 3H), 1.12 (d, J=6.52 Hz, 3H), 1.07 (d, J=6.8 Hz, 3H),
1.04–0.90 (m, 9H), 0.87 ppm (d, J=6.7 Hz, 3H); ESI-MS(+): m/z
found for C32H41N8O4S4: 729.00 [M+H]+.
Cyclic hexamer 21: The peak separated at 55 min was cyclic hexa-
mer 21 (0.001 g, 0.11%): purity (HPLC, UV=254 nm)ꢀ98%; ESI-
MS(+): m/z found for C48H60N12O6S6: 1094 [M+H]+.
{1-[4-(1-{4-[(R)-(+)-1-(4-Methoxyphenyl)ethylcarbamoyl]thiazol-2-
yl}-(S)-2-methylpropylcarbamoyl)thiazol-2-yl]-(S)-2-methylpro-
pyl}carbamic acid tert-butyl ester (22): Compounds 10 (0.10 g,
0.21 mmol) and 22a (0.06 g, 0.42 mmol) were reacted together by
following Method C with HCTU to obtain compound 22 as a yel-
lowish-white solid (0.08 g, 63%): m.p. 64–668C; Rf =0.55 (EtOAc/n-
Photoaffinity labeling of P-gp with [125I]IAAP: Crude membranes (1 mg
protein per mL) from P-gp-expressing High-Five insect cells were
photolabeled with [125I]IAAP (2200 Cimmolꢁ1) in the absence or pres-
ence of tested compounds, as described previously.[43] Briefly, crude
membranes from P-gp-expressing High-Five insect cells were incu-
bated in the presence and absence of 10 mm thiazole derivatives or
5 mm tariquidar in 50 mm Tris·HCl (pH 7.5) and 6 nm [125I]IAAP
(2200 Cimmolꢁ1). The samples were then photo-crosslinked, and
images were developed on a phosphorimager. The radioactivity asso-
ciated with the P-gp band was quantified, and percent inhibition of
IAAP incorporation was calculated. The results are given as percent
inhibition as an average of two independent experiments.
1
hexane 1:1); purity (HPLC, UV=254 nm): 96%; H NMR (400 MHz;
CDCl3; TMS): d=8.66–8.61 (m, 1H), 8.43–8.36 (m, 1H), 8.22 (s, 1H),
8.16 (s, 1H), 7.74 (d, J=7.8 Hz, 1H), 7.32 (d, J=8.4 Hz, 2H), 6.88 (d,
J=8.4 Hz, 2H), 5.18–5.08 (m, 2H), 4.70–4.66 (m, 1H), 3.72 (s, 3H),
2.47–2.44 (m, 1H), 2.27–2.21 (m, 1H), 1.48 (d, J=6.9 Hz, 3H), 1.40
(s, 9H), 0.97 (d, J=6.2 Hz, 3H), 0.90–0.86 ppm (m, 9H); ESI-MS(+):
m/z found for C30H41N5O5S2Na: 638.2430 [M+Na]+.
[1-(4-{1-[4-(2,4-Dimethoxybenzylcarbamoyl)thiazol-2-yl]-(S)-2-
methylpropylcarbamoyl}thiazol-2-yl)-(S)-2-methylpropyl]carba-
mic acid tert-butyl ester (23): Compounds 10 (0.10 g, 0.21 mmol)
and 23a (0.07 g, 0.42 mmol) were reacted together according to
Method C by using HCTU to obtain compound 23 as a white solid
(0.09 g, 68%): m.p. 70–748C; Rf =0.55 (EtOAc/n-hexane 1:1); purity
ATPase assay: Crude membrane protein (100 mg proteinmLꢁ1) from
High-Five insect cells expressing P-gp was incubated at 378C with
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 157 – 169 168

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Molecular modeling
Ligand preparation: The structures of (S)-valine-based thiazole de-
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Protein preparation: The X-ray crystal structure of mouse P-gp in
the apo state (PDB ID: 3G5U) and in complex with inhibitors
QZ59Se-RRR (PDB ID: 3G60), QZ59Se-SSS (PDB ID: 3G61)[5] and ATP-
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This research was supported by the Department of Pharmaceuti-
cal Sciences of St. John’s University and St. John’s University Seed
Grant No. 579-1110 (to T.T.T.). N.R.P., K.K., E.E.C., and S.V.A. were
supported by the Intramural Research Program of the NIH, Na-
tional Cancer Institute, Center for Cancer Research. Financial sup-
port from the Indian Council of Medical Research, New Delhi in
the form of an International Fellowship for Young Indian Biomed-
ical Scientists to N.R.P. is gratefully acknowledged (Indo/FRC/
452(Y-19)/2012-13IHD). S.S. and T.T.T. are thankful to Dr. Sanjai
Kumar and Dibyendu Dana for assisting in preparative HPLC, ESI-
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Keywords: ABC transporters · molecular modeling · multidrug
resistance · peptide mimics · P-glycoprotein
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Received: August 30, 2013
Published online on November 29, 2013
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