Synthesis, activity, and pharmacophore development for isatin-β- thiosemicarbazones with selective activity toward multidrug-resistant cells

Article abstract of DOI:10.1021/jm800861c

We have recently identified a new class of compounds that selectively kill cells that express P-glycoprotein (P-gp, MDR1), the ATPase efflux pump that confers multidrug resistance on cancer cells. Several isatin-β- thiosemicarbazones from our initial stud

Full text of DOI:10.1021/jm800861c

J. Med. Chem. 2009, 52, 3191–3204
3191
Synthesis, Activity, and Pharmacophore Development for Isatin-ꢀ-thiosemicarbazones with
Selective Activity toward Multidrug-Resistant Cells
Matthew D. Hall,†,# Noeris K. Salam,‡,# Jennifer L. Hellawell,† Henry M. Fales,§ Caroline B. Kensler,§ Joseph A. Ludwig,|
Gergely Szaka´cs, David E. Hibbs,‡ and Michael M. Gottesman*,†
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Group in Biomolecular
Structure and Informatics, Pharmaceutical Chemistry DiVision, Faculty of Pharmacy, The UniVersity of Sydney, NSW 2006, Australia,
Laboratory of Applied Mass Spectrometry, National Heart, Lung & Blood Institute, National Institutes of Health, Bethesda, Maryland 20892,
Department of Sarcoma Medical Oncology, UniVersity of TexassMD Anderson Cancer Center, Houston, Texas 70030, and Institute of
Enzymology, Hungarian Academy of Sciences, 1113 Karolina ut 29, Budapest, Hungary
ReceiVed January 22, 2009
We have recently identified a new class of compounds that selectively kill cells that express P-glycoprotein
(P-gp, MDR1), the ATPase efflux pump that confers multidrug resistance on cancer cells. Several isatin-
ꢀ-thiosemicarbazones from our initial study have been validated and a range of analogues synthesized and
tested. A number demonstrated improved MDR1-selective activity over the lead, NSC73306 (1).
Pharmacophores for cytotoxicity and MDR1 selectivity were generated to delineate the structural features
required for activity. The MDR1-selective pharmacophore highlights the importance of aromatic/hydrophobic
features at the N4 position of the thiosemicarbazone and the reliance on the isatin moiety as key bioisosteric
contributors. Additionally, a quantitative structure-activity relationship (QSAR) model that yielded a cross-
validated correlation coefficient of 0.85 effectively predicts the cytotoxicity of untested thiosemicarbazones.
Together, the models serve as effective approaches for predicting structures with MDR1-selective activity
and aid in directing the search for the mechanism of action of 1.
Introduction
unique third-generation inhibitors were designed specifically for
their capacity to inhibit P-gp; yet despite their enhanced efficacy
(and in part because of it), the latest generation inhibitors
frequently altered the pharmacokinetic profile of the coadmin-
istered chemotherapy leading to reduced efficacy and increased
side effects.1,3 Given the problems identified above, in addition
to problems relating to poor trial design, the clinical benefit of
direct P-gp inhibitors remains to be proven.
Multidrug resistance (MDRa) conferred by the ABC trans-
porter family that includes MDR1 (ABCB1, P-glycoprotein,
P-gp) presents a significant clinical challenge for drug design
and development.1 P-gp expression is well-characterized in
hematological malignancies, sarcomas, and other solid cancers
and in those tumor types is frequently correlated with poor
clinical response to chemotherapy.2 Strategies employed to
circumvent the reduced drug accumulation conferred by these
polyspecific efflux transporters have relied heavily on the
development of clinical inhibitors of P-gp for concurrent
administration with chemotherapeutics. Although a number of
these have shown promise in vitro, translation to the clinic has
taken longer than may have been expected,1,3 in part because
of pharmacokinetic effects caused by inhibition of endogenous
function,1 and alternative strategies are required.
One strategy to circumvent problems associated with P-gp
inhibition and resolve the emergence of clinical MDR is to
develop drugs that exploit the expression of P-gp, thus turning
a mechanism of drug resistance into a weakness.5,6 To this end,
we have previously profiled mRNA expression of all 48 known
and predicted human ABC transporters in the National Cancer
Institute 60 cell line panel (NCI-60), used by the NCI to screen
over 100 000 compounds for anticancer activity.6 These data
were used to identify the individual ABC transporters that
conferred multidrug resistance on cells.6 Furthermore, bioin-
formatic correlation of gene expression in the NCI-60 cell lines
with cytotoxicity of drugs against the NCI-60 cell lines identified
compounds whose activity was potentiated rather than dimin-
ished by the expression of P-gp and that were recently
validated.7
Early inhibitors were “off-the-shelf” drugs used for other
medical conditions that had limited activity in vivo (e.g,
verapamil), leading to second-generation inhibitors that were
structurally related to first-generation compounds but altered
chemically to improve their affinity for P-gp.4 Later, structurally
* To whom correspondence should be addressed. Phone: 1-301-496-1530.
Fax: 1-301-402-0450. E-mail: gottesmm@mail.nih.gov.
Of the 60 compounds whose activity was inversely related
to P-gp expression (MDR1-selective agents), 10 possessed a
thiosemicarbazone functional group and 7 contained a 1-isatin-
3-thiosemicarbazone (isatin-ꢀ-thiosemicarbazone) moiety; 1
†
National Cancer Institute.
These authors contributed equally to this work.
The University of Sydney.
National Heart, Lung & Blood Institute.
#
‡
§
(NSC73306),8
2
(NSC658339),8
3
(NSC716765),8
4
|
University of TexassMD Anderson Cancer Center.
(NSC716766),8 5 (NSC716768),8 6 (NSC716771),8 and 7
(NSC716772)8 shown in Figure 1.6 The remarkable result that
7 isostructural compounds would be present in the 50 most
statistically significant compounds led us to select 1 as a lead
compound to validate its MDR1-selective properties and
understand its mechanism of action.5 While biochemical assays
have shown that 1 does not interact with P-gp as either a
Institute of Enzymology.
a Abbreviations: APCI-MS, atmospheric pressure chemical ionization
mass spectrometry; 3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicar-
bazone; A, hydrogen bond acceptor; ABC, ATP-binding cassette; DMSO,
dimethyl sulfoxide; ESI-MS, electrospray ionization mass spectrometry; H,
hydrophobic domain; MAIQ, 4-methyl-5-amino-1-formylisoquinoline thi-
osemicarbazone; MDR, multidrug resistance; NCI, National Cancer Institute;
NSC, National Service Center; R, phenyl ring; RR, ribonucleotide reductase;
TKI, tyrosine kinase inhibitor; X, thiosemicarbazone.
10.1021/jm800861c CCC: $40.75
2009 American Chemical Society
Published on Web 04/27/2009

3192 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Hall et al.
Figure 1. Structures of methisazone, 9, and the seven isatin-ꢀ-thiosemicarbazones (1-7) identified in a bioinformatics screen as having activity
that is potentiated rather than inhibited by expression of the multidrug transporter P-gp. 1 is being treated as a lead compound to understand the
mechanism of action of the compounds. An overlay of the seven NSC compounds identified in the bioinformatics screen demonstrates the common
structural features associated with them.
substrate or inhibitor, the effectiveness of 1 against MDR cell
lines correlates with their expression of P-gp.5 Importantly, from
a clinical perspective, cell lines selected for resistance to 1 show
loss of P-gp. As such, 1 represents an exciting prospect for
resolving multidrug resistance in the clinic by selectively killing
cells that express high levels of P-gp and resensitizing residual
cells to conventional chemotherapeutics. This strategy is cur-
rently being assessed using P-gp-mediated drug resistant human
cancer xenografts in the mouse.
The biological activity of thiosemicarbazones has been known
for a considerable period of time, both as anticancer (1956)9
and antiviral (1973)10,9 drugs.11,12 Methisazone (N-methylisatin-
ꢀ-thiosemicarbazone), for example, was effective as prophylaxis
against smallpox and vaccinia viruses,11 and 3-aminopyridine-
2-carboxaldehyde thiosemicarbazone (9, 3-AP) is currently being
evaluated in clinical trials against several malignancies including
leukemia (Figure 1).13,14 At least some of the biological activity
of thiosemicarbazones has been shown to involve interaction
with metal ions,15,16 and a number of mechanisms of action
have been identified including ribonucleotide reductase inhibi-
tion, metal dependent radical damage, DNA binding, and
inhibition of protein synthesis.17-19 The metal chelates of
thiosemicarbazones administered to cells are regularly more
active than the drug alone.16
1 is currently undergoing preclinical evaluation. However,
its nonoptimal aqueous solubility (a feature for which thiosemi-
carbazones are notorious20) has led to a search for more soluble
derivatives, and while 1 is several-fold more active against P-gp-
expressing cell lines than their parental lines, we desire to
develop a yet more selective derivative based on this compound.
This requires an understanding of the relative importance of
the key structural features of 1 and its congeners that must be
retained to maintain MDR1-selective activity. To this end, we
have designed and synthesized a series of compounds and tested
them against a parental HeLa-derived cervical cancer cell line
(KB-3-1) and its vinblastine-selected derivative that highly

Isatin-ꢀ-thiosemicarbazones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3193
expresses P-gp (KB-V1). Both cytotoxicity and MDR1 selectiv-
ity were correlated with structural variations in an effort to
construct an active pharmacophore to be used as the basis for
further drug design.
Compounds 12 and 13 in Table 1 were prepared by
condensation of 2-indanone and 1-indanone (in lieu of isatin)
with 4-methoxyphenyl-3-thiosemicarbazide. Compound 14 was
prepared by reacting thiosemicarbazide with isatin. The isatin-
ꢀ-semicarbazones 15 and 25 were prepared by the reaction of
the corresponding isatin with semicarbazide in lieu of their
respective thiosemicarbazides. Compounds 18, 19, 20, 22, and
23 were prepared by condensation of the starting material (5-
fluoroisatin, N-methylisatin, sodium isatin-5-sulfonate, ben-
z(g)indole-2,3-dione, and 5-nitroisatin, respectively) with 4-meth-
oxyphenyl-3-thiosemicarbazide.
Results and Discussion
Characterization of Compounds from the DTP Library.
Given that seven isatin-ꢀ-thiosemicarbazones were predicted in
the initial bioinformatics screen to identify MDR1-selective
compounds, they were of primary interest for validation. 1 was
selected as a lead compound primarily because of its availability,
and the structure was initially believed to incorporate a 5,7-
dichloroisatin moiety (MW ) 395.3 g mol-1) when submitted
to the NCI for testing. However, APCI-MS analysis of the
sample from the DTP library revealed it to be the nonsubstituted
isatin derivative, whose structure is correctly shown in Figure
1, and additional quantities were synthesized for subsequent
testing.5 Of the seven isatin-ꢀ-thiosemicarbazones predicted in
the DTP compound library to demonstrate MDR1-selective
activity, only 1 and 2 were available for testing while the
remaining five compounds were not publicly available from
the DTP for biological testing. However, it was possible to
Cytotoxicity. To ascertain which structural features are
responsible for the selective cytotoxicity of 1, the structurally
diverse compounds described above were designed and syn-
thesized for testing (Table 1). Some of the synthesized
compounds were analogues of 1 that incorporated simple
substitutions on the isatin framework, whereas others broadly
modify the structure to test which structural elements were
required for activity. We focused our interest, principally, on
understanding two biological properties of 1. (1) The first is
the absolute cytotoxicity (measured using the MTT assay) of
compounds against parental P-gp-negative KB-3-1 adenocar-
cinoma cells. This was tested to determine whether compounds
were active and, if so, how this activity compared with that of
the lead compound 1. (2) The second is MDR1 selectivity, as
indicated by greater sensitivity in KB-V1 cells compared to KB-
3-1 cells, and we tested whether greater selectivity for MDR1-
expressing cells than that of 1 could be achieved. To this end,
compounds were also tested against the KB-V1 cervical
adenocarcinoma cell line that expresses high levels of P-gp (the
protein product of MDR1). The KB-V1 cell line was originally
developed by stepwise selection of KB-3-1 cells in the drug
vinblastine.28 MDR1 selectivity is determined by the ratio of
IC50 against KB-3-1 cells divided by its IC50 against KB-V1
cells. A value of >1 indicates that the compound kills P-gp-
expressing cells more effectively than parental cells, resulting
in so-called MDR1-selective activity.5 Alternatively, a value of
<1 indicates that the P-gp-expressing cells are resistant to the
compound, relative to parental cells, as is normally observed
for drugs effluxed by P-gp.29
1
arrange for H NMR spectra of these five samples to establish
the structure and purity of the compounds as submitted. ESI-
MS of the sample of 2 (expected MW ) 390.1 g mol-1) revealed
a (M + H)+ ion at 221.0, indicating the absence of the ethyl
malonate moiety. MS2 confirmed the loss of a -HNCdS
fragment revealing the likely structure to be the isatin-ꢀ-
thiosemicarbazone 3 (Table 1), more of which was subsequently
synthesized for this study. Analysis of compounds 3, 4, 5, 6,
and 7 by 1H NMR spectra in DMSO-d6 revealed that 3, 4, and
6 were relatively pure (∼95%); however, solutions of 5 and 7
returned spectra showing many impurities.
We synthesized (described below) the correct structures of 1
(Figure 1) and 2 (shown correctly as 3 in Table 1) and 3, 4, 6,
and 7 as reported. 5 was not synthesized. Since neither the NCI
chemical library nor most other publicly available chemical
repositories are validated for structure or purity following
submission, our experience strongly suggests that chemicals
obtained from these sources must be validated for purity.
Synthesis. The straightforward synthesis of isatin-ꢀ-thiosemi-
carbazones by the condensation of isatin and a thiosemicarbazide
has been known for more than 50 years (Figure 2c).21,22
Compounds 3, 4, 5, 6, and 7 have previously been reported by
Karali and co-workers in the context of antiviral and anticancer
screening results and were synthesized by such a method,23-25
though the preparation of the precursor thiosemicarbazides was
not described.
Of the five DTP compounds identified in the bioinformatics
screen and synthesized as described above, four compounds (1,
4, 6, and 7, Table 1) demonstrated varying levels of MDR1
selectivity, with only 7 showing a greater MDR1 selectivity
value than 1 (7.1 and 4.3, respectively). 3, the only DTP
compound possessing an alkenyl rather than aryl moiety at the
N4 position, was not selective for KB-V1 cells, and 14, which
was identified as the true structure of 2 from the DTP collection,
was found to be inactive against both cell lines (IC50 > 50 µM).
Two thiosemicarbazones that have been previously tested
extensively in vitro and in the clinic are the anticancer
compounds 3-AP (9, 3-aminopyridine-2-carboxaldehyde thi-
osemicarbazone) and MAIQ (10, 4-methyl-5-amino-1-formyl-
isoquinoline thiosemicarbazone), and these were chosen to
determine whether MDR1 selectivity is a common feature of
biologically active thiosemicarbazones (Table 1). Neither com-
pound is substituted at the N4 position, and they possess a
pyridine and isoquinoline group, respectively, in lieu of isatin,
resulting in an NNS tridentate moiety capable of tightly chelating
iron, thereby inhibiting ribonucleotide reductase (RR).30 Neither
was found to be MDR1-selective, though their cytotoxicities
against the KB-3-1 line were approximately an order of
magnitude greater than that of 1 (1.4 and 1.9 versus 14.2 µM).
The resistance of KB-V1 cells to these compounds was not
A number of preparative approaches are available for thi-
osemicarbazides, one of the more straightforward being the
addition of hydrazine to an isothiocyanate (Figure 2a), which
we generally employed in our preparations.26 In synthesizing
6, the preparation of 4-fluorophenylthiosemicarbazide from
4-fluorophenyl isothiocyanate and hydrazine yielded an insoluble
1
product that was identified by H NMR to involve reaction of
2 mol of thiocyanate with 1 mol of hydrazine (Figure 2b).
Dropwise addition of hydrazine to an excess of thiocyanate
could not ameliorate the formation of this product. In order to
prevent this, t-Boc protected hydrazine, tert-butyl carbazate,27
was reacted with 4-fluorophenyl isothiocyanate resulting in t-Boc
protected 4-fluorophenylthiosemicarbazide that could be readily
deprotected with acid (Figure 2c). This synthetic route was used
subsequently to ensure the ready preparation of pure thiosemi-
carbazides.

3194 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Hall et al.
Table 1. Structures of compounds considered in this study, along with IC50 values determined against the parental KB-3-1 cell line, and the
P-glycoprotein-expressing cell line KB-V1a

Isatin-ꢀ-thiosemicarbazones
Table 1. Continued
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3195
a The MDR1 selectivity is calculated as the ratio of a compound’s IC50 against KB-3-1 cells divided by its IC50 against KB-V1 cells. A value of >1
indicates that the compound kills P-gp-expressing cells more effectively than parental cells, so-called MDR1-selective activity. A value of <1 indicates that
the P-gp-expressing cells are resistant to the compound, relative to parental cells, as is normally observed for P-gp substrates. “n/a” denotes compounds, as
described in the discussion section, that were not tested for their biological activity because they were shown to have been incorrectly identified or impure.
surprising given that L1210 murine leukemia cells selected for
resistance to 10 (L1210 MQ-580) have been shown to express
P-gp31 and demonstrated cross-resistance to 932 (1 is not a P-gp
substrate5). The antitubercular thiosemicarbazone, thiacetazone
(11, 1-(4′-formylacylamine)-3-thiosemicarbazone, Table 1),
which is also unsubstituted at the thiosemicarbazone N4 position
and contains a phenyl group rather than isatin, was inactive
against both cell lines. Isatin itself was also shown to be inactive.
When the p-methoxyphenyl group of 1 was replaced with an
alkenyl group in 24 (Table 1), significant activity was lost (14.2
vs 26.1 µM), along with MDR1 selectivity (4.2 versus 1.4)
which, along with the observation that 3 was not selective,

3196 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Hall et al.
Figure 2. Scheme showing general synthetic steps in the preparation of isatin-ꢀ-thiosemicarbazones. (a) General synthetic method used for the
synthesis of isatin-ꢀ-thiosemicarbazones, the example here being 1. Equimolar quantities of isatin (or a substituted isatin) and a thiosemicarbazide
(here, 4-(4-methoxyphenyl)-3-thiosemicarbazide) are stirred in hot ethanol, a few drops of glacial acetic acid are added, and the solution is refluxed
for a short period that on cooling resulted in the precipitation of the final product. (b) The phenylthiosemicarbazide is generally reported to be
synthesized by the reaction of hydrazine with the substituted phenyl isothiocyanate of choice, such as p-methoxypheny isothiocyanate shown here,
yielding 4-(4-methoxyphenyl)-3-thiosemicarbazide. (c) In our hands the reaction between hydrazine and p-halogen phenyl isothiocyanates such as
p-fluorophenyl isothiocyanate, did not proceed as expected but resulted (d) in an insoluble precipitate that was unable to undergo further reaction
with isatin. It was identified by mass spectrometry to be a 2:1 combination of thiocyanate and hydrazine (MW ) 338 g mol-1) that formed even
when hydrazine was added dropwise to the excess thiocyanate. Using a t-Boc protected form of hydrazine, tert-butyl carbazate, which is effectively
monofunctional hydrazine, the t-Boc protected thiosemicarbazide could be produced, from which the thiosemicarbazone was recovered under acidic
conditions.
suggests that an aromatic group at the N4 position improves
selectivity. Given that KB-V1 cells were resistant to the RR
inhibitors 9 and 10 and that these N4 unsubstituted compounds
are known to be P-gp substrates, we incorporated a p-
methoxyphenyl group at the N4 position of PT (pyridine-2-
carboxaldehyde thiosemicarbazone), an analogue of 9, forming
21. Not only did compound 21 show superior cytotoxicity to 1
(3.4 versus 14.2 µM), KB-V1 cells were not resistant to it but
were modestly (1.4-fold) sensitive.
Of the compounds synthesized for testing, 12 and 13 contain
indanone moieties conjugated to the thiosemicarbazone at
positions 1 and 2, lacking the hydrogen bonding potential of
the original isatin. Compound 13 removes the lactam moiety
of the isatin in 1. Despite minimal structural disruption, both
compounds were inactive against both cell lines. The semicar-
bazone derivatives 15 (structurally analogous to 14) and 25
(based on 1) were designed to test whether the thiosemicarba-
zone (a strong metal coordination functional group) was essential
to activity. Like its thiosemicarbazone counterpart, 24 was
inactive against both cell lines, but 25 was weakly active (IC50
) 27.7 µM) only against the KB-3-1 parental line.
A number of substitutions on the isatin group of 1 were
explored to examine the effect on activity (the 5,7-dichloroisatin
derivative of 1, 17, was not tested, as it was found to be insoluble
in the solvents used including DMSO, ethanol, and water). The
5-fluoroisatin (18) analogue demonstrated lower activity against
the parental KB-3-1 line (39.3 µM) coupled with greater
selectivity than 1 for P-gp-expressing cells (7.5 vs 4.3).
Likewise, the 5-nitroisatin analogue 23 showed slightly lower
activity, but similar selectivity, compared to 1. The 5-isatin-
sulfonic acid derivative of 1 (20) was prepared to markedly
increase aqueous solubility (which it did); however, the
compound was inactive, possibly because of the overall negative
charge on the molecule in solution resulting in poor cellular
uptake. The N-methyl derivative of 1 (analogous to methisa-
zone), 19, showed lower activity and selectivity relative to the
parent compound (IC50 ) 4.4 µM, selectivity of 2.1). Compound
22 was prepared using benz(g)indole-2,3-dione, and while active
(IC50 ) 4.4 µM), it was not selective against the V1 line (IC50
) 30.3 µM), suggesting it is a P-gp substrate.
Disregarding the anionic sulfonate, it appears that small
substitutions at position 5 of the isatin nucleus on 1 do not

Isatin-ꢀ-thiosemicarbazones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3197
abolish its MDR1 selectivity, and indeed in the case of 18 the
electron-withdrawing fluorine appears to improve its selectivity.
N-Methylation reduced selectivity but did not abolish it. The
isatin feature is required for selectivity; simply retaining the
six- and five-membered ring system as in 12 and 13 is not
sufficient for activity. Incorporating steric bulk onto the isatin
moiety in 22 appears to improve cytotoxicity. However, its
MDR1-selective activity is lost. Collectively, these compounds
suggest that an isatin is required for MDR1-selective activity
and that greater selectivity may be possible by substituting
electron-withdrawing substituents on the isatin (though the
5-nitroisatin substitution in 23 does not enhance activity over
1). A diverse range of substitutions at both the isatin and phenyl
ring will be examined in a subsequent study in a quest to
uncover optimal selectivity.
Importantly, it seems that the range of activity of current RR
inhibitor thiosemicarbazones may be enhanced by substitution
at the thiosemicarbazone N4 position with a hydrophobic or
aromatic group. Subsequent to our report of the MDR1
selectivity of the thiosemicarbazone 1, Whitnall et al. demon-
strated that a dipyridyl thiosemicarbazone with a dimethyl
substitution at the N4 position also demonstrated MDR1-
selective activity, though the selectivity of other compounds
reported was not demonstrated.33
Figure 3. Pharmacophores that contribute cytotoxicity and selectivity.
(a) Pharmacophore features required for cytotoxicity, indicated by the
activity of compounds tested against the parental KB-3-1 cell line. Pink
sphere ) A, H-bond acceptor, with the sphere located at the projected
point of a hydrogen bond donor. Green sphere ) H, hydrophobic group.
Ring ) aromatic ring. Blue sphere ) X, thiosemicarbazone functional
group. The pharmacophore sites are underlaid with the structure of 10
to aid in relating the pharmacophore features to structural features of
the active compounds. (b) Pharmacophore features required for selectiv-
ity against the P-gp-expressing KB-V1 cell line: AH1H2H3R1R2X. Pink
sphere ) A, H-bond acceptor, with the sphere located at the projected
point of a hydrogen bond donor. Green sphere ) H, hydrophobic group.
Ring ) aromatic ring. Blue sphere ) X, thiosemicarbazone functional
group. Yellow sphere ) exclusion sphere. The pharmacophore sites
are underlaid with the structure of 1 to aid in relating the pharmacophore
features to structural features of the active compounds.
Structure-Activity Studies. The poor water solubility of
otherwise promising thiosemicarbazones is a well-known chal-
lenge to their clinical use.20 By identifying the key structural
features required for MDR1 selectivity, we sought to design
and synthesize variants that display a greater selectivity for
resistant cells and that have improved aqueous solubility.
Commensurate with observations for 10 and 1, pharmacophore
modeling and quantitative structure-activity relationships (QSAR)
were employed as two quantitative measures used to gauge the
structural relationships of the compounds described above with
respect to cytoxicity and MDR1 selectivity.
In this study, the generation of a pharmacophore for the
cytotoxicity of compounds against the parental line KB-3-1
(AHRX, Figure 3a) allowed us to determine what molecular
components were required for cytotoxicity, compared with those
additionally required for MDR1 selectivity. The need for a
thiosemicarbazone site (X) is apparent, as compounds that lack
this feature (15 and 25) proved inactive. The thiosemicarbazone
functional group was assigned a single site, X, for two reasons;
first, assigning individual bonding features of the thiosemicar-
bazone would potentially result in a large number of extra sites
common to most molecules, and second, it is possible the
thiosemicarbazone group is coordinating to metal ions to effect
its cytotoxicity and/or MDR1 selectivity as a bidentate ligand,
as is the case for other classes of thiosemicarbazones.30
The hydrogen bond acceptor site (A), corresponding to the
lactam oxygen of isatin-ꢀ-thiosemicarbazones or the aromatic
nitrogen of 9, 10, and 21, was deemed essential for cytotoxicity,
along with their associated aromatic ring/hydrophobic sites (R/
H). Molecules lacking any one of the features (A, H, R, or X)
are inactive, and the failure of thiacetazone, 12, 13, 16, and 26
to match the KB-3-1 cytotoxicity pharmacophore can be
understood in terms of missing the hydrogen bonding site, A.
The use of a projected point for the hydrogen bond acceptor
(simulating the corresponding hydrogen bond donor in the
receptor) was introduced to allow structurally dissimilar active
compounds to form hydrogen bonds to the same location,
regardless of their point of origin and directionality. Such is
the case with the isatin-ꢀ-thiosemicarbazones, 9, 10, and 21, in
which the hydrogen bond acceptors originate in different
locations of the ring/hydrophobic region but are still capable
of hydrogen-bonding to a common site.
Apart from correctly identifying all of the active thiosemi-
carbazones, the KB-3-1 pharmacophore was used to prealign
the molecules for QSAR analysis. The performance of the
single-factor atom-based QSAR model on the training and test
set molecules is illustrated in Figure 4a. The scatter plot indicates
exceptionally high correlation (r2 ) 0.96) and cross-validated
correlation (q2 ) 0.85) coefficients, indicative of a model with
strong predictive power and significance. Figure 4a also
compares experimental and predicted pIC50 values for both the
training and test set molecules, showing that activity was
effectively predicted. This observation further supports the
validity of the pharmacophore model, suggesting the spatial
arrangement of chemical features, when aligned by the phar-
macophore, is indicative of the probable active conformation
of the molecule. The KB-3-1 cytoxicity QSAR model is intended
as a preliminary effort to quantitatively infer how cytotoxic new
compounds might be, and although strong predictive power is
evident, the low number of training/test set compounds warrants
caution when using this model for this purpose. Of course, the
predictive accuracy will improve once more compounds are
added to training and test sets.
In the definition of the pharmacophore for the MDR1
selectivity demonstrated by 1, 4, 6, 7, 18, 19, 21, 23, and 24
(Table 1), three additional features (R2, H2, and H3) were
incorporated into the pharmacophore (described above); all
represent electron-rich substitution at the N4 position of
thiosemicarbazones common to MDR1-selective compounds.
These features, which match well with 1 (Figure 3b), resulted

3198 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Hall et al.
Figure 4. Pharmacophores predict cytotoxicity and selectivity. (a) Scatter plot and comparison of the KB-3-1 cytoxicity QSAR model applied to
13 active thiosemicarbazones. The training set correlation is characterized by one partial least-squares (PLS) factor (SD ) 0.12, R2 ) 0.96). The
test set correlation is characterized by one PLS factor (root-mean-square error (rmse) ) 0.16, q2 ) 0.85, Pearson R ) 0.95). Experimental and
calculated pIC50 values are shown for the QSAR training and test set. (b) Scatter plot and comparison of the KB-V1 cytoxicity QSAR model
applied to 12 active thiosemicarbazones. The training set correlation is characterized by one PLS factor (SD ) 0.11, R2 ) 0.91). The test set
correlation is characterized by one PLS factor (rmse ) 0.29, q2 ) 0.42, Pearson R ) 0.70). Experimental and calculated pIC50 values are shown
for the QSAR training and test sets.
in the MDR1-selectivity pharmacophore AH1H2H3R1R2X. Set-
ting a minimum of three sites for matching, corresponding to
AH1X, the MDR1-selectivity pharmacophore can identify
compounds that are cytotoxic but not necessarily selective
(overlaid in Figure 5a). By incorporation of further constraints
by increasing the minimum number of required sites to the full
seven descriptors (AH1H2H3R1R2X), the pharmacophore high-
lights compounds that show selectivity for KB-V1 cells.
Employing all seven sites predicts only compound 21 and the
isatin-ꢀ-thiosemicarbazones possessing either a p-methoxyphe-
nyl or p-fluorophenyl group at the N4 position (Figure 5b). The
performance of the single-factor atom-based QSAR model on
the training and test set molecules for prediction of cytotoxicity
against the MDR1-expressing KB-V1 line is illustrated in Figure
4b. The scatter plot indicates a high correlation coefficient (r2
) 0.91), although the cross-validated correlation coefficient
is lower (q2 ) 0.42). Experimental and predicted pIC50 values
against KB-V1 cells for both the training and test set molecules
are also shown in Figure 4b, showing that activity was
effectively predicted.
Figure 5. Refined pharmacophore predicting sensitivity of KB-V1
cells. (a) Pharmacophore for activity against KB-V1 cells with overlay
of compounds that match three of seven pharmacophore features
(AH1X). (b) Pharmacophore for activity against KB-V1 cells with
overlay of compounds that satisfy all pharmacophore features
(AH1H2H3R1R2X). The compounds that meet these requirements are
the isatin-ꢀ-thiosemicarbazones and compound 21.
Table 2 demonstrates the pharmacophore site match combina-
tions for MDR1 selectivity and the compounds that satisfy each
set of criteria. A minimal requirement of three sites (AH1X)
satisfies all compounds listed. However, three sites alone cannot
discriminate between compounds that do and do not demonstrate
selectivity for KB-V1 cells, while including all seven sites
misses some compounds with KB-V1-selective activity (>1-
fold experimental selectivity). Incorporating all seven sites
(AH1H2H3R1R2X) reveals only MDR1-selective compounds, but
three (4, 7, and 24) are excluded. A combination of five or six
features (AH1H2R2X and AH1H2R1R2X), both incorporating R2
and H2, correctly selects all MDR1-selective compounds but
24, the false negative being due to the lack of a phenyl ring at
N4. By use of the predicted activity of compounds against KB-

Isatin-ꢀ-thiosemicarbazones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3199
Table 2. KB-V1 Pharmacophore Site Matches for Compounds Examined and the Site Feature Combinations for Which They Satisfy Selectivity for
KB-V1 Cellsa
pharmacophore site matches
fold selectivity
exptl calcd
7.6 7.4
>1-fold selectivity
compd
AH1X
AH1H2X
AH1H2R2X
AH1H2R1R2X
AH1H2H3R1R2X
exptl
calcd
1
6
18
19
21
23
4
7
3
24
9
10
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
no
4.3
3.6
2.1
2.3
1.4
7.1
1.4
1.3
0.5
0.9
0.2
2.2
2.6
2.0
3.1
1.8
5.4
2.6
0.9
1.0
0.4
0.2
no
no
no
no
no
a Setting a minimal requirement for matching, such as AH1X, satisfies all compounds listed but cannot discriminate between compounds that do and do
not demonstrate selectivity for KB-V1 cells, while setting all seven sites misses some compounds with KB-V1 selective activity. The AH1H2X and AH1H2R2X
site combinations both predict for all KB-V1-selective compounds, though each with one false positive. The QSAR model correctly predicts the selectivity
of 11 of 12 compounds listed, though it performs less well at predicting by how much a compound will select for KB-V1.
3-1 (parental) and KB-V1 (P-gp-expressing) cells, the predicted
MDR1 selectivity can be determined (Table 2). The QSAR
model correctly predicts the selectivity of 11 of 12 compounds
listed and makes no false positive predictions, though the
magnitude of selectivity is not well predicted. The cytotoxicity
and MDR1-selective pharmacophores and QSAR models will
be employed to aid in presynthetic screening of drug design
candidates. On the basis of the AH1H2H3R1R2X pharmacophore,
isatin-ꢀ-thiosemicarbazones and other highly active thiosemi-
carbazones with aromatic substitution at the N4 position will
be developed and validated and the pharmacophore further
optimized.
Given that the pharmacophore was developed on the basis
of an initial observation of structural commonality from a cohort
of 60 compounds identified by a bioinformatics screen, the
pharmacophore site features were queried against the remaining
53 compounds in the library (other than the isatin-ꢀ-thiosemi-
carbazones) to ascertain whether there was an underlying
structural commonality to them. Querying the geometry-
optimized structures of the bioinformatics hits for those that
satisfy at least AH1X identified the seven isatin-ꢀ-thiosemicar-
bazones (Figure 1) from which the pharmacophore was partially
derived, as well as the three remaining thiosemicarbazones in
the set: NSC669341 (27, six site matches, AH1H2H3R2X),
NSC695331 (28, four sites, AH1R2X), and NSC695333 (29, six
site matches, AH1H2H3R2X). Each geometry optimized structure
is overlaid with the MDR1-selective pharmacophore in Figure
6, and the structures show phenyl substitution at the N4 position
(with an electron withdrawing substituent in each case), as well
as hydrogen bond acceptors (A1) with a hydrophobic region (H1)
flanking the thiosemicarbazone (X) functional group. One of
the three thiosemicarbazones was available, and while it was
not highly active (∼20 µM), its cytotoxicity was effectively
equipotent against both the multidrug-resistant and parental lines
(Figure 6a) as expected from the pharmacophore.
As with the thiosemicarbazones described above, aromatic
regions connected by a linker are common to many of the
structures, often with further hydrogen bonding potential in the
linker. Validation of the available non-thiosemicarbazone 30
revealed an MDR1 selectivity ratio of 3.3, reinforcing the
pharmacophore’s potential applicability outside thiosemicarba-
zones.
The additional features on the MDR1-selectivity pharma-
cophore and the exciting fact that non-thiosemicarbazones from
our data set of MDR1-selective compounds satisfy the phar-
macophore developed here suggest that the selectivity is not
mediated by the thiosemicarbazone functional group. While
thiosemicarbazones are known for the metal-chelation-mediated
mechanisms of action, the isatins also have a diverse range of
biological properties such as tyrosine kinase inhibition (TKI),
particularly when conjugated as indolin-2-ones (as is the case
here).34
It has not escaped our attention that 5-halogen substituted
indolin-2-one moieties are reported to be efficacious TKIs and
demonstrate greater cytotoxicity than their unhalogenated ana-
logues35 (as is the case for the isatins36). For example, sunitinib
(5-[5-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-di-
methyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)a-
mide) is a rationally designed multitargeted TKI approved for
the treatment of gastrointestinal stromal cancers and advanced
renal-cell carcinoma37,38 that possesses a 5-fluoroindolin-2-one
moiety (like that of 18). Sunitinib’s TKI activity against VEGF-
R2 and PDGF is an order of magnitude greater than that of the
nonfluorinated analogue (as are the 5-chloro and 5- bromo
examples35), and its activity is mediated by the adenine mimic
properties of the substituted isatin that binds to the kinase active
site.39 Meijer and co-workers have also examined a series of
indirubin natural products (indirubins contain an indone-2-one
moiety) and shown that derivatives with 5- and 6-halogenated
indolin-2-one moieties demonstrate better kinase inhibitory
activity (CDK1, CDK5, GSK-3) than their nonsubstituted
counterparts,40,41 and cocrystallization studies showed that the
indone-2-one group is binding in the active site.41
Querying the structural database for matches that are not
thiosemicarbazones, by excluding X from the search criteria
but requiring at least three other site matches, gave a further 13
non-thiosemicarbazone NSC compounds from our data set that
appear to satisfy the structural criteria for MDR1 selectivity.
Two examples are shown in Figure 6b; NSC168468 (30,
2-hydroxy-3-(hydroxy(oxido)amino)benzaldehyde-2-quinolinyl-
hydrazone, five site matches, AH1H2R1R2) and NSC621481 (31,
3-(2-chlorophenyl)-1-(1,4-dihydroxy-3-methyl-15,45-quinoxalin-
2-yl)-2-propen-1-one, five site matches, AH1H2R1R2), and the
remaining structures are given in the Supporting Information.
Given these precedents, the kinase inhibitory activity of 1
was examined against a representative panel of 50 kinases.
While the positive control staurosporine demonstrated strong
inhibition of most kinases in the panel, 1 showed no
inhibitory activity at the highest dose concentration of 20
µM against the majority of kinases (Supporting Information
Table S1), and an IC50 could only be calculated for RSK1
(ribosomal S6 kinase 1) with a relatively high value of 34.9

3200 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Hall et al.
Figure 6. The pharmacophore for activity against KB-V1 cells was tested against the 60 compounds previously identified in a bioinformatic
analysis for compounds that were predicted to be selectively active against P-gp-expressing cells. The activities of two available compounds, the
thiosemicarbazone 27 and 30, are given. (a) Querying the structural database for compounds that satisfy at least AH1 X (X ) thiosemicarbazone)
identified the seven NSC isatin-ꢀ-thiosemicarbazones shown in Figure 1, from which the pharmacophore was partially derived, and the three
remaining thiosemicarbazones in the set, 27, 28, and 29, shown over the site features demonstrating the good fit of structural features of these
molecules with the pharmacophore. (b) Querying the structural database for compounds that satisfy at least three out of seven sites, including
AH1R2 but not the thiosemicarbazone feature (X), gave a further 13 non-thiosemicarbazone NSC compounds from our data set that appear to satisfy
the structural criteria for selectively killing KB-V1 cells. Two examples are shown: 30 and 31.
µM. The cytotoxicity of sunitinib itself was examined against
the cell line pair employed here and found not to display
MDR1-selective activity (KB-3-1 IC50 ) 2.3 µM, KB-V1
IC50 ) 4.1 µM, RR IC50 ) 0.6), reinforcing that kinase
inhibitory activity alone is not adequate for conferring
MDR1-selective cytotoxicity.
Unlike high-throughput screens that identify molecules with
activity against a specific biological assay, bioinformatics

Isatin-ꢀ-thiosemicarbazones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3201
crystallized; if it did not, water was added to encourage it. The
best solvent for recrystallization of thiosemicarbazones was invari-
ably DMSO with small amounts of water. With one exception
(below), the (M + H)+ and (M - H)- ions lost the elements of the
corresponding RNCS on MS2, although (M)- ions were also
detected. In the case of isatin allylthiosemicarbazone (compound
24), the (M + H)+ ion lost the elements of allyl thiourea. The (M
- H)- ion lost allylisothiocyanate as expected. Low resolution mass
spectra (LRMS) were collected on a Thermo LCQ Classic
spectrometer (Madison, WI) and high resolution mass spectra
(HRMS) with a Waters LCT Premier spectrometer (Milford, MA).
NMR spectra were taken at 300 MHz in deuterated dimethyl
sulfoxide using a Varian 300 Gemini spectrometer (Palo Alto, CA).
In each case, the purity of recrystallized compounds described below
was confirmed to be g95% by the GC/MS gas chromatograph trace
displaying only a single peak during analysis. Furthermore, in each
screens based on correlation with gene expression identify
candidates whose activity can be validated but is not indicative
of the mechanisms of action of hits, which can be diverse. While
QSAR studies cannot reveal mechanism of action, they aid in
understanding the structural features required for biological
activity. In future studies, the pharmacophore identified in this
report, along with the underlying understanding that isatin,
conjugated as indolin-2-one to thiosemicarbazones, can result
in compounds that selectively kill multidrug-resistant (P-gp-
expressing) cells, will be used to methodically produce improved
analogues of 1. Substitution at the 5 and 6 positions of isatin
and around the phenyl ring should allow us to obtain maximal
selectivity for P-gp-expressing cells.
Additionally, incorporating an amine into the system would
theoretically help to solubilize the drug as a hydrochloride salt;
while no reliable pharmacophore for substrates of P-gp exists,
it generally transports neutral and cationic organic molecules.42
Thiosemicarbazones such as 9 and 10 that are P-gp substrates
(discussed above) are not MDR1-selective, and 1 cannot be
effluxed by P-gp and shows MDR1-selective activity. While
the site of action of 1 has not yet been identified, this suggests
that MDR1-selective compounds cannot be P-gp substrates and
that thiosemicarbazones such as 14 and 22 are inactive or
nonselective because chemical substitution has sensitized them
to detection and efflux by P-gp. This may mean that small
substitutions on a MDR1-selective pharmacophore will reverse
the selectivity, and a comprehensive library of compounds will
allow us to probe this feature. Taken together, the information
presented within this report should facilitate the design of 1
analogues that are both more selective and soluble, thereby
accelerating their transition from bench to bed.
1
case H NMR spectra revealed no impurities.
Compound 1. 1-Isatin-4-(4′methoxyphenyl)thiosemicarba-
zone. 1 was prepared by reacting isatin with 4-(4′methoxyphenyl)-
3-thiosemicarbazide. Yield 87%, light-yellow needles, mp 220-248
°C dec. 1H NMR (DMSO-d6) δ ) 3.79 (3H, s), 6.98 (2H, dt), 6.98
(2H,dt), 7.45 (2H,dt), 6.95 (1H, d), 7.37 (1H,dt), 7.11 (1H, dt),
7.76 (1H, d), 12.7 (1H,s), 10.7 (1H,s), 11.25 (1H,s). LRMS m/z
327 (M + H)+, m/z 162 (MS2 of 327), m/z 326 (M)-, m/z 325 (M
- H)-, m/z 160 (MS2 of 325). HRMS m/z 327.0917 (M + H)+,
calcd C16H15N4O2S 327.0916.
Comopund 3. 1-(5′-Nitroisatin)-4-allyl-3-thiosemicarbazone.
3 was prepared by reacting 5-nitroisatin with 4-allyl-3-thiosemi-
carbazide. Yield 49%, light-brown plates, mp 226-230 °C dec.
1H NMR (DMSO-d6) δ ) 4.28 (2H, t), 5.18 (1H, d), 5.19 (1H, d),
5.92 (1H, m), 7.12 (1H, d), 8.27 (1H, dd), 8.57 (1H, d), 9.78 (1H,
t), 11.83 (1H, bs), 12.38 (1H, s). LRMS m/z 306 (M + H)+, m/z
270 (MS2 of 306), m/z 304 (M - H)-, m/z 205 (MS2 of 304).
HRMS m/z 306.0649 (M + H)+, calcd C12H12N5O3S 306.0661.
Compound 4. 1-(5′-Nitroisatin)-4-phenyl-3-thiosemicarba-
zone. 4 was prepared by reacting 5-nitroisatin with 4-phenyl-3-
thiosemicarbazide. Yield 70%, light-brown plates, mp 250-252
Conclusions
The models developed here, the first of their kind for
predicting cytotoxicity and MDR1 selectivity, constitute valuable
tools that will aid in the design of new MDR1-selective
cytotoxins based on isatin-ꢀ-thiosemicarbazones. The necessity
for isatin, and enhancement with halogenation to achieve greater
P-gp-targeted selectivity, will serve as the foundation in the
search for potential drug targets. Of great interest to those
devoted to overcoming P-gp-mediated drug resistance, the
pharmacophore we describe suggests that R-N-heterocyclic
carboxaldehyde thiosemicarbazones such as 3-AP could be
substantially improved. Here, we have laid a foundation upon
which to build more soluble and P-gp-selective analogues of 1.
Evolution of this chemical family offers the promise of curing
those cancers most resistant to chemotherapy.
1
°C dec. H NMR (DMSO-d6) δ ) 7.31 (1H, t), 7.45 (2H, t), 7.59
(2H, d), 7.17 (1H, d), 8.28 (1H, d), 8.70, 1H, d), 11.1 (1H, s), 12.55
(1H, s), 11.86 (1H, br s). LRMS m/z 342 (M + H)+, m/z 207 (MS2
of 342, m/z 340 (M - H)-, m/z 205 (MS2 of 340). HRMS m/z
342.0663 (M + H)+, calcd C15H12N5O3S 342.0661.
1-(tert-Butoxycarbonyl)-4-(4′-fluorophenyl)-3-thiosemicarba-
zide. The compound (Figure 2c) was prepared by reaction of tert-
butylcarbazate with 4-fluorophenyl isothiocyanate. Yield 90%,
colorless prisms, mp 158-160 °C. HRMS m/z 286.1019 (M + H)+,
calcd C12H17N3O2FS 286.1026. The t-Boc group was removed by
acid hydrolysis, and the resulting 4-(4′-fluorophenyl)-3-thiosemi-
carbazide was used for the synthesis of 6. Reaction of the simple
hydrazine with 4-fluorophenylisothiocyanate yielded 1,2-bis(4′-
fluorophenylthiocyanato)hydrazine (Figure 2b). Yield 89%, color-
less prisms, mp 204-205 °C. HRMS m/z 339.0557 (M + H)+,
calcd C14H13N4F2S2 339.0550; m/z 337.0388 (M - H)-, calcd C14
H11N4F2S2 337.0393.
Experimental Section
Compound 6. 1-(5′-Bromoisatin)-4-(4′-fluorophenyl)-3-thi-
osemicarbazone. 6 was prepared by reacting 5-bromoisatin with
4-(4′-fluorophenyl)-3-thiosemicarbazide. Yield 97%, orange needles,
mp 238-240 °C dec. 1H NMR (DMSO-d6) δ ) 6.91 (1H, d), 7.53
(1H, dd), 7.97 (1H, d), 7.59 (2H, m), 7.28 (2H, t), 10.89 (1H, s),
11.36 (1H, s), 12.61 1H, s). HRMS m/z 392.9821 (M + H)+, calcd
Materials and Methods. Synthetic materials were sourced from
Aldrich unless otherwise noted. Triapine (9, 3-AP, 3-aminopyridine-
2-carboxaldehyde thiosemicarbazone) was generously provided by
Vion Pharmaceuticals, CT. MAIQ (10, NSC246112, 2-[(5-amino-
4-methyl-1-isoquinolinyl)methylene]) was provided by the Devel-
opmental Therapeutics Program (DTP), National Institutes of
Health. Thiacetazone (11) was purchased from Sigma. Sunitinib
was purchased from Toronto Chemicals, Toronto, Canada. Stock
solutions of compounds for biological assays were prepared in
DMSO and stored in frozen aliquots until use. The kinase inhibitory
activity of 1 against a panel of 50 kinases was measured by Reaction
Biology Corp, Malvern, PA.
Synthesis of Thiosemicarbazones. The thiosemicarbazones were
prepared by combining equimolar quantities of the isatins and
thiosemicarbazides dissolved in large amounts of ethanol with
addition of a few drops of acetic acid to initiate the reaction. When
the mixture was heated to boiling, the thiosemicarbazone often
C
15H11N4OFSBr 392.9821.
Compound 7. 1-(5′-Bromoisatin)-4-(4′-nitrophenyl)-3-thi-
osemicarbazone. 7 was prepared by reacting 5-bromoisatin with
4-(4′-nitrophenyl)-3-thiosemicarbazide. Yield 83%, orange needles,
mp 270-275 °C dec. 1H NMR (DMSO-d6) δ ) 6.92 (1H, d), 7.55
(1H, dd), 8.00 (1H, d), 8.07 (2H, d), 8.31 (2H, d), 11.14 (1H, s),
11.41 (1H, s), 12.82 (1H, s). HRMS m/z 417.9608 (M + H)+, calcd
C15H9N5O3SBr 417.9609.
Compound 12. 1-(2′-Indanone)-4-(4′-methoxyphenyl)-3-thi-
osemicarbazone. 12 was prepared by reacting 2-indanone with
4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield 76%, white needles,

3202 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Hall et al.
1
browning in air, mp 152-4 °C. H NMR (DMSO-d6) δ ) 3.75
(DMSO-d6) δ ) 3.79, (3H, s), 6.99 (2H, d), 7.48 (2H, m), 7.60
(2H, d), 7.66 (1H, d). 7.88 (1H, d), 7.96 (1H, dd), 8.12 (1H, dd)
10.78 (1H, s), 11.97 (1H, s), 12.78 (1H, s). LRMS m/z 377 (M +
H)+, m/z 212 (MS2 of 377), m/z 375 (M - H)-, m/z 210 (MS2 of
375). HRMS m/z 377.1073 (M + H)+, calcd C20H17N4O2S
377.1072.
Compound 23. 1-(5′-Nitroisatin)-4-(4′-methoxyphenyl)-3-thi-
osemicarbazone. 23 was prepared by reacting 5-nitroisatin with
4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield 63%, light-yellow
needles, mp 220-248 °C dec. 1H NMR (DMSO-d6) δ ) 3.79 (3H,
s), 7.00 (2H, d), 7.14 (1H, d), 7.44 (2H, d), 8.28 (1H, dd), 8.69
(1H, d), 11.01 (1H, s), 11.86 (1H, s), 12.51 (1H, s). LRMS m/z
372 (M + H)+, m/z 207 (MS2 of 372), m/ z 370 (M - H)-, m/z
205 (MS2 of 370). HRMS m/z 372.0764 (M + H)+, calcd C
16H14N5O4S 372.0767.
(3H, s), 3.87 (4H, d), 6.82 (2H, d), 7.29 (2H, m), 7.32 (2H, m),
7.41 (2H, d), 9.81 (1H, s), 10.38 (1H, s). HRMS m/z 312.1179 (M
+ H)+, calcd C 17H17N3OS 312.1171.
Compound 13. 1-(1′-Indanone)-4-(4′-methoxyphenyl)-3-thi-
osemicarbazone. 13 was prepared by reacting 1-indanone with
4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield 96%, white needles,
mp 185-6 °C. 1H NMR (DMSO-d6) δ ) 2.95 (2H, m), 3.08 (2H,
m), 3.79 (3H, s), 6.92 (2H, d), 7.30 (1H, m), 7.38 (1H, d), 7.40
(1H, m), 7.41 (2H, d), 8.04 (1H, d), 9.99 (1H, s), 10.53 (1H, s).
HRMS m/z 312.1179 (M + H)+, calcd C17H17N3OS 312.1171.
Compound 14. 1-Isatin-3-thiosemicarbazone. 14 was prepared
by reacting isatin with 3-thiosemicarbazide. Yield 92%, fine yellow
1
needles, mp 210-240 °C dec. H NMR (DMSO-d6) δ ) 6.93
(1H,d), 7.09 (1H, t), 7.36 (1H, t), 7.65 (1H, d), 8.68 (1H, s), 9.03
(1H, s), 11.21 (1H, s), 12.47 (1H, s). LRMS m/z 221 (M + H)+,
m/z 160 (MS2 of 221), m/z 219, (M - H)-, m/z 160 (MS2 of 219).
HRMS m/z 219.0331 (M + H)+, calcd C9H7N4SO 219.0340.
Compound 15. 1-Isatin-3-semicarbazone. 15 was prepared by
reacting isatin with 3-semicarbazide. Yield 85%, light-yellow
needles, dec. 1H NMR (DMSO-d6) δ ) 6.89 (1H, d), 6.91 (1H, s),
7.03 (1H, t), 7.34 (1H, t), 8.06 (1H, d), 10.18 (1H, s), 10.73 (1H,
Compound 24. 1-Isatin-4-allyl-3-thiosemicarbazone. 24 was
prepared by reacting isatin with 4-allyl-3-thiosemicarbazide. Yield
1
90%, light-yellow needles, mp 210 °C dec. H NMR (DMSO-d6)
δ ) 4.25 (2H, t), 5.92 (1H, t), 5.18 (1H, m), 5.19 (1H, d), 6.93
(1H,dd), 7.35 (1H, dt), 7.09 (1H, dt), 7.67 (1H, dd), 9.45 (1H, t),
11.21 (1H, s), 12.60 (1H, s). LRMS no (M + H)+ ion, m/z 145
(loss of NHCSNHCH2CH(CH3)), m/z 259 (M - H)-, m/z 160 (MS2
of 259). HRMS m/z 261.0808 (M + H)+, calcd C12H13N4OS
261.0810.
s). HRMS m/z 205.0707 (M + H)+, calcd C H9N4O2 205.0726.
Compound 16. 4-(4′-Methoxyphenyl)-3-thiosemicarbazide. 16
9
was obtained from Trans World Chemical (Rockville, MD). Mp
154-156 °C. H NMR (DMSO-d6) δ 3.73 (3H, s), 4.76, (2H, br
Compound 25. 1-Isatin-4-(4′-methoxyphenyl)-3-semicarba-
zone. 25 was prepared by reacting isatin with 4-(4′-methoxyphenyl)-
3-semicarbazide. Yield 35%, yellow needles, mp 182-6 °C. H
1
1
s), 6.86 (2H, d), 7.44 (2H, d), 8.72 (1H, br s), 9.50 (1H, br s).
Compound 17. 1-(4′,7′-Dichloroisatin)-4-(4′-methoxyphenyl)-
3-thiosemicarbazone. 17 was prepared by reacting 4,7-dichloroi-
satin with 4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield 89%,
NMR (DMSO-d6) δ ) 3.74 (3H, s), 6.92 (2 h, d), 7.49 (2H, d),
6.92 (1H, d), 7.39 (1H, m), 7.06 (1H, m), 8.09 (1H, d), 10.8 (1H,
s). LRMS m/z 311 (M + H)+, m/z 162 (MS2 of 311), m/z 309 (M
- H)-, m/z 160 (MS2 of 309). HRMS m/z 311.1138 (M + H)+,
calcd C16H15N4O3 311.1144.
Compound 26. 1-(4′-Acetylaminobenzaldehyde)-4-(4′-meth-
oxyphenyl)-3-thiosemicarbazone. 26 was prepared by reacting
4-acetylaminobenzaldehyde with 4-(4′-methoxyphenyl)-3-thiosemi-
carbazide. Yield 98%, light-yellow needles, mp 208-9 °C. 1H NMR
(DMSO-d6) δ ) 2.05 (3H, s), 3.77 (3H, s), 6.92 (2H, d), 7.39 (2H,
d), 7.63 (2H, d), 7.82 (2H, d), 9.93 (1H, s), 10.11 (1H, s), 11.68
(1H, s). HRMS m/z 343.1230 (M + H)+, calcd C17H19N4O2S
343.1229.
Cell Lines and MTT Cytotoxicity Assay. KB-3-1 cells (a HeLa
deriviative) and its MDR derivative (KB-V1) were grown as
previously described.5 Cell survival was measured by the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
as previously described.5 Briefly, cells were seeded in 100 µL of
growth medium at a density of 5000 cells/well in 96-well plates
and allowed to establish for 24 h, at which time serially diluted
drugs were added in an additional 100 µL of growth medium. Cells
were then incubated for 72 h at 37 °C in humidified 5% CO2, at
which time the growth medium was drawn and replaced with MTT
in IMDM growth medium and incubated for 4 h. The MTT solution
was then drawn from the wells, and 100 µL of acidified ethanol
solution was added to each well and after 15 min absorption at
560 nm was measured. IC50 cytotoxicity values were determined
as the drug concentration that reduced the absorbance to 50% of
that in untreated control wells and derived from at least three
separate experiments.
Pharmacophore Perception and 3D-QSAR. Table 1 shows the
compounds tested for the generation of two three-dimensional (3D)
pharmacophore models in order to rationalize chemical structure
with the observed cytotoxicity and selectivity of the isatin-ꢀ-
thiosemicarbazones. In the development of a pharmacophore for
cytotoxicity against the parental KB-3-1 cell line, compounds were
divided into an active class (13 compounds) and an inactive class
(8 compounds) based on an arbitrary activity threshold of 50 µM.
The pharmacophore relating structure to MDR1-selective cytotox-
icity against the P-gp-expressing KB-V1 cell line was similarly
created with the above thresholds, though 22 was removed from
the active class and placed into the inactive class given that it was
inactive (IC50 > 50 mM) against KB-V1 cells. It is worth stating
that the inactivity of a molecule may be due to a range of reasons
1
bright-orange needles, mp 280 °C dec. H NMR (DMSO-d6) δ )
7.03 (2H, d), 7.55 (2H, d), 7.50 (1H, s), 7.19 (1H, s), 10.1 (2H, br
s), 12.0 (1H, s). HRMS m/z 395.0142 (M + H)+, calcd C
16H13N4O2SCl2 395.0136.
Compound 18. 1-(5′-Fluoroisatin)-4-(4′-methoxyphenyl)-3-
thiosemicarbazone. 18 was prepared by reacting 5-fluoroisatin with
4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield 61%, orange
needles, mp 238-240 °C dec. 1H NMR (DMSO-d6) δ ) 3.79 (3H,
s), 6.94 (1H, dd), 6.99 (2H, d), 7.21 (H, dt), 7.46 (2H, d), 7.62
(1H, dd), 10.78 (1H, s), 11.25 (1H, s), 12.63 (1H, s). LRMS m/z
345 (M + H)+, m/z 180 (MS2 of 345), m/z 343, (M - H)-, m/z
178, MS2 of 343. HRMS m/z 345.0830 (M + H)+, calcd
C16H14N4O2FS 345.0822.
Compound 19. 1-(N-Methylisatin)-4-(4′-methoxyphenyl)-3-
thiosemicarbazone. 19 was prepared by reacting N-methylisatin
with 4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield 87%, short
1
yellow prisms, mp 216-17 °C. H NMR (DMSO-d6) δ ) 3.78
(3H, s), 3.24, (3H, s), 6.98 (2H, d), 7.46 (2H, d), 7.16 (1H, d),
7.46 (1H, t), 7.17 (1H, t), 7.80 (1H, t), 10.75 (1 h, s), 12.68 (1H,
s). LRMS m/z 341 (M + H)+, m/z 176 (MS2 of 341), m/z 339 (M
- H)-, m/z 174 (MS2 of 339). HRMS m/z 341.1078 (M + H)+,
calcd C17H17N4O2S 341.1072.
Compound 20. 1-(Isatin 5′-sulfonic acid)-4-(4′-methoxyphe-
nyl)-3-thiosemicarbazone. 20 was prepared by reacting isatin-5-
sulfonic acid with 4-(4′-methoxyphenyl)-3-thiosemicarbazide. Yield
1
98%, dark-yellow needles, mp >300 °C. H NMR (DMSO-d6) δ
) 3.79 (3H, s), 6.97 (2H, dd), 7.44 (2H, dd), 6.86 (1H, d), 7.62
(1H, dd), 8.13 (1H, br s), 10.95 (1H, s), 11.28 (1H, s), 12.70 (1H,
s). LRMS no positive or negative ions. HRMS m/z 407.0469 (M
+ H)+, calcd C16H15N4O5S2 407.0469.
Compound 21. 1-(2′-Pyridinecarboxaldehyde)-4-(4′-methox-
yphenyl)-3-thiosemicarbazone. 21 was prepared by reacting
2-pyridinecarboxaldehyde with 4-(4′-methoxyphenyl)-3-thiosemi-
1
carbazide. Yield 66%, mp 173-4 °C. H NMR (DMSO-d6) δ )
3.76, (3H, s), 6.95 (2H, d), 7.39 (3H, d,m), 7.84 (1H, t), 8.18 (1H,
s), 8.43 (1H,d), 8.58 (1H, d), 10.17 (1H, S), 11.95 (1H, s). HRMS
m/z 287.0968 (M + H)+, calcd C14H15N4OS 287.0967.
Compound 22. 1-(1H′-Benz[g]indole-2′,3′-dione)-4(4′-meth-
oxyphenyl)-3-semicarbazone. 22 was prepared by reacting 1H-
benz[g]indole-2,3-dione with 4-(4′-methoxyphenyl)-3-thiosemicar-
bazide. Yield 66%, dark-red prisms, mp 240-268 °C dec. 1H NMR

Isatin-ꢀ-thiosemicarbazones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3203
including a large desolvation penalty, excessive entropy loss upon
receptor binding, and poor membrane permeability or active
efflux.43 As a result, some molecules will fail to show any activity
while containing all the necessary features in identical spatial
arrangements (sites) as the active compounds. Consequently, their
inclusion in pharmacophore creation may unfairly jeopardize the
selection of the correct features and sites. Thus, inactive compounds
14 and 20 were excluded in this pharmacophore study. Other
compounds were excluded, as biological data could not be
determined for reasons of purity and solubility as described below:
2, 5, and 17. Other structurally diverse compounds that were inactive
were included in pharmacophore creation as the members of the
inactive class of drugs.
A maximum of 100 conformers were generated for each of the
21 molecules, sampling the conformational space that may represent
possible active structures. Conformers not within 5 kJ/mol of the
global minimum were automatically discarded. Visual inspection
and careful analysis of chemical features were conducted to guide
the pharmacophore development process. Particular attention was
paid to compounds 10 and 18, as they represent the optimal
reference structures for KB-3-1 cytoxicity and selective MDR1-
selective cytoxicity, respectively, based on their activity (Table 1).
Individual features that compose the thiosemicarbazone functional
group were excluded and replaced with a single, custom group-
feature (X) representing this functional group. Pharmacophore
variants considered for cytotoxicity were composed of four sites
with the following chemical features: hydrogen-bond acceptor (A,
pink sphere), aromatic ring (R, ring), hydrophobic (H, green sphere),
and thiosemicarbazone group (X, blue sphere). Model variant
AHRX was preselected, as it was visually apparent that all active
compounds possessed these features.
cessfully matches the MDR1-selective thiosemicarbazones. Further
specifications for the search criteria implemented here include
distance matching tolerances of 2.0 Å and feature matching
tolerances for hydrogen bond acceptors of 2.0 Å, with hydrophobic,
aromatic ring, and thiosemicarbazone tolerances each set to 1.5 Å.
QSAR Modeling. Attempts to statistically analyze three-
dimensional molecular fields by means of a QSAR analysis were
made, correlating chemical structure with either the KB-3-1
cytotoxicity or MDR1 selectivity data. The QSAR models for KB-
3-1 cytotoxicity and MDR1 selectivity were developed using
molecules capable of matching either of the two pharmacophores.
Molecules were placed into training and test sets once aligned onto
the pharmacophore with an effort to minimize structural redundancy
while maximizing coverage of the experimental activities. Figure
5 summarizes the compounds investigated. Atom based QSAR
models were built for the two pharmacophores using a grid spacing
of 0.5 Å and one partial least-squares (PLS) factor.
Computational Details. All molecules investigated in this study
were built with the Maestro 7.5 graphical interface.45 Each structure
was subsequently minimized using the OPLS_200546 with a
constant dielectric of 1.0. Rapid torsional sampling was achieved
using MacroModel 9.145 using the OPLS_2005 force field with
minimum atom deviation of 0.5 Å and a distant-dependent dielectric
solvation treatment. The maximum relative energy difference was
set to 10.0 kcal/mol. Pharmacophore development and subsequent
searches, site point superpositions, vector characteristics alignment,
molecular volume overlap, penalty scoring, and QSAR model
building were carried out using phase 2.0.43,44 All calculations were
performed in Maestro 7.5 on an AMD64+ workstation using the
SUSE10.1 Linux operating system.
Acknowledgment. D.E.H. acknowledges Australian Re-
search Council for funding. N.K.S. acknowledges Faculty of
Pharmacy, University of Sydney, for award of a scholarship.
J.L.H. acknowledges the Howard Hughes Medical Institute for
award of a HHMI Fellowship. G.S. is an EMBO SDIG fellow
and a Special Fellow of the Leukemia and Lymphoma Society.
The authors thank Edward Sokoloski and Noel Whittaker for
assistance in obtaining NMR and mass spectra. This research
was supported by the Intramural Research Program of the
National Institutes of Health, National Cancer Institute.
In the search for a pharmacophore predictive of cytotoxicity
against KB-3-1 cells, common pharmacophores exhibiting AHRX
were searched in at least 3 of the 13 active structures with a final
box size and minimum intersite distance of 1.0 Å. Several
hypotheses were generated of which the best that mapped onto 10,
based on the PHASE scoring functions,43,44 was chosen as the final
KB-3-1 pharmacophore for use in subsequent screening and QSAR
investigations. Similarly, pharmacophore variants considered for
MDR1 selectivity consisted of the same above features; however,
the number of sites increased to seven (covering the additional
phenyl ring and methoxy group as H2/R2 and H3 features,
respectively) to place greater weight on mapping the isatin-ꢀ-
thiosemcarbazones, specifically that of 18 for the reason outlined
previously. Common pharmacophore features from at least 3 of
the 12 active compounds were searched, revealing several hypoth-
eses of variant AH1H2H3R1R2X. The hypothesis that best mapped
onto 18 was chosen. A single exclusion-volume of radius 1.5 Å
was added to prohibit 22 from matching the final MDR1 selectivity
pharmacophore.
Quality of the Pharmacophoric Hypotheses. The quality of
each pharmacophore is first measured in three ways based on the
alignments to the input structures: (1) the alignment score, which
is the root-mean-squared deviation (rmsd) in the site-point positions;
(2) the vector score, which is the average cosine of the angles
formed by corresponding pairs of vector features (acceptors, donors,
and aromatic rings) in the aligned structures; (3) a volume score
based on the overlap of van der Waals models of the non-hydrogen
atoms in each pair of structures. For details, see Dixon et al.43 We
also assigned higher scores to hypotheses that match a greater
number of the chosen actives.
Supporting Information Available: Structures of compounds
that matched pharmacophore screen; results of kinase inhibition
screen of 1. This material is available free of charge via the Internet
at http://pubs.acs.org.
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