Synthesis and biological evaluation of a series of novel inhibitor of Nek2/Hec1 analogues

Article abstract of DOI:10.1021/jm8015969

High expression in cancer 1 (Hec1) is an oncogene overly expressed in many human cancers. Small molecule inhibitor of Nek2/Hec1 (INH) targeting the Hec1 and its regulator, Nek2, in the mitotic pathway, was identified to inactivate Hec1/Nek2 function media

Full text of DOI:10.1021/jm8015969

J. Med. Chem. 2009, 52, 1757–1767
1757
Synthesis and Biological Evaluation of a Series of Novel Inhibitor of Nek2/Hec1 Analogues
Xiao-Long Qiu,†,‡ Guideng Li,†,‡ Guikai Wu,†,‡ Jiewen Zhu,‡ Longen Zhou,‡ Phang-Lang Chen,‡ A. Richard Chamberlin,§ and
Wen-Hwa Lee*,‡
Department of Biological Chemistry, School of Medicine, UniVersity of California, IrVine, IrVine, California 92697, Department of Chemistry,
UniVersity of California, IrVine, IrVine, California 92697
ReceiVed December 17, 2008
High expression in cancer 1 (Hec1) is an oncogene overly expressed in many human cancers. Small molecule
inhibitor of Nek2/Hec1 (INH) targeting the Hec1 and its regulator, Nek2, in the mitotic pathway, was
identified to inactivate Hec1/Nek2 function mediated by protein degradation that subsequently leads to
chromosome mis-segregation and cell death. To further improve the efficacy of INH, a series of INH
analogues were designed, synthesized, and evaluated. Among these 33 newly synthesized analogues, three
of them, 6, 13, and 21, have 6-8 fold more potent cell killing activity than the previous lead compound
INH1. Compounds 6 and 21 were chosen for analyzing the underlying action mechanism. They target directly
the Hec1/Nek2 pathway and cause chromosome mis-alignment as well as cell death, a mechanism similar
to that of INH1. This initial exploration of structural/functional relationship of INH may advance the progress
for developing clinically applicable INH analogue.
Introduction
Breast cancer has been one of the leading causes of death in
American females for many years. Although multiple combined
modality treatment strategies are available and continue to be
improved, seeking for alternative therapeutics with a better
therapeutic index remains a top priority of the drug development
endeavors. Many of the established cancer therapeutics, repre-
sented by the microtubule poison taxane and Vinca alkaloids,
inhibit cancer cell growth via direct inhibition of mitosis.1,2
Mitosis is a highly dynamic and delicate process that requires
multiple layers of regulation by a variety of molecules. Several
innovative agents targeting the key mitotic regulators are
currently in preclinical or clinical development, including those
inhibiting the spindle-associated kinesin KSP/Eg5, kinetochore-
associated kinesin CENP-E, and the mitotic kinase Aurora.3,4
However, whether the downstream substrate of mitotic kinases
may serve as a potential target remains to be explored.
Figure 1. Structures of INH1 and INH2 and the scaffold of INH
analogues.
mitotic pathway.10 Our leads, INH1 and INH2, sharing an aryl
thiazolyl benzamide scaffold, came from screening a library of
∼24000 synthetic compounds through an inducible reverse yeast
two-hybrid system. Although both of them inhibit the prolifera-
tion of multiple human breast cancer cell lines in culture, the
potency in killing cancer cells and the water solubility remain
to be improved for better efficacy. As part of a broad chemical
strategy aimed toward the discovery of novel INH analogues,
we designed, synthesized, and evaluated a series of novel INH
analogues. The structure-activity relationship (SAR) of INH
scaffold was also illustrated. INH1 and INH2 both have a core
phenyl-thiazolyl-benzamide structure with additional groups on
both sides of the thiazolyl rings (Figure 1). The main scaffold
of INH was composed of three parts: left part (hydrophobic
aryl ring), middle part (thiazolyl ring), and right part (arylamide
polar part). The modification of INH analogues was mainly
focused on these three parts.
One of the critical mitotic regulators is Hec1,a an oncogene
that modulates proper spindle formation between kinetochores
and centrosomes. Importantly, overexpression of Hec1 has been
documented to associate with poor clinical outcomes of primary
breast cancers as well as cases with multiple cancers.5,6 Inducible
overexpression of Hec1 in a transgenic mouse model led to
evident tumor formation, primarily lung adenoma and hepato-
cellular adenoma.7 On the other hand, phosphorylation of Hec1
by Nek2 is known to be critical for its mitotic function and cell
survival.8,9 We have recently identified small molecules INH1
and INH2 that can effectively suppress tumor cell growth in
culture and in animals via directly targeting the Hec1/Nek2
* To whom correspondence should be addressed. Phone: (949)-824-4492.
Fax: (949)-824-9767. E-mail: whlee@uci.edu. Address: Department of
Biological Chemistry, School of Medicine, University of California, Irvine,
124 Sprague Hall, Irvine, CA 92697.
Results and Discussion
Chemistry. Our synthesis embarked on the preparation of
two intermediates 3 and 4 followed by the general synthetic
procedure shown in Scheme 1. That is, treatment of bromides
1 and 2 with thiourea in refluxing EtOH afforded 4-(2,4-
dimethylphenyl)thiazol-2-ylamine (3) and 4-(2,4,6-trimethylphe-
nyl)thiazol-2-ylamine (4) in 89% and 99% yields, respectively.
Acylation of amines 3 and 4 with a series of aryl acyl chloride
provided the corresponding INH analogues 5-19 in medium
†
These authors contributed equally to this work.
Department of Biological Chemistry, School of Medicine, University
‡
of California, Irvine.
§
Department of Chemistry, University of California, Irvine.
a Abbreviations: Hec1, high expression in cancer 1; Nek2, NIMA (never
in mitosis gene a)-related kinase 2; INH, Inhibitor of Nek2/Hec1; RNAi,
RNA interference; SAR, structure-activity relationship; DAPI, 4′,6-
diamidino-2-phenylindole; DMAP, 4,4-dimethylaminopyridine.
10.1021/jm8015969 CCC: $40.75
2009 American Chemical Society
Published on Web 02/25/2009

1758 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Qiu et al.
Scheme 1. General Synthetic Procedure for the Preparation of Compounds 5-23a
a Reagents and conditions: (a) Thiourea, EtOH, reflux (89% for 3, 99% for 4). (b) For 5: 2,4-dimethoxy benzoyl chloride, DMAP, CH2Cl2, rt
(67%); for 6: BzCl, Et3N, dioxane, reflux (48%); for 7: 2-methoxyl benzoyl chloride, DMAP, CH2Cl2, 0 °C to rt (77%); for 8: 2-trifluoromethylbenzoyl
chloride, DMAP, CH2Cl2, 0 °C to rt (52%). (c) For 9: picolinoyl chloride hydrochloride, DMAP, CH2Cl2, 0 °C to rt (87%); for 10: picolinoyl chloride
hydrochloride, DMAP, CH2Cl2, 0 °C to rt (93%); for 11: nicotinoyl chloride hydrochloride, DMAP, CH2Cl2, 0 °C to rt (64%); for 12: nicotinoyl
chloride hydrochloride, DMAP, CH2Cl2, 0 °C to rt (73%); for 13: isonicotinoyl chloride hydrochloride, DMAP, CH2Cl2, 0 °C to rt (88%); for 14:
pyrazine-2-carbonyl chloride, DMAP, CH2Cl2, 0 °C to rt (77%); for 15: pyrazine-2-carbonyl chloride, DMAP, CH2Cl2, 0 °C to rt (93%). (d) 1-Methyl-
1H-imidazole-5-carbonyl chloride hydrochloride, DMAP, CH2Cl2, 0 °C to rt (37% for 16, 28% for 17). (e) 1,3-Thiazole-2-carbonyl chloride, DMAP,
CH2Cl2, 0 °C to rt (76% for 18, 89% for 19). (f) 4-Morpholinecarbonyl chloride, DMAP, CH2Cl2, 0 °C to rt (63% for 20, 39% for 21). (g) For 22:
PhSO2Cl, DMAP, pyridine, 0 °C to rt (68%); for 23: BnSO2Cl, Et3N, DMAP, THF, 0 °C to rt (63%).
to good yields. In addition, treatment of amines 3 and 4 with
4-morpholinecarbonyl chloride led to the compounds 20 and
21 in 63% and 39% yields, respectively. Sulphonylation of 3
with benzenesulfonyl chloride and benzylsulfonyl chloride
furnished the compounds 22 and 23 in 68% and 63% yields,
respectively.
in CH2Cl2 for 2 h smoothly gave the phenol 32 in 72% yield
along with diphenol 33 in 3% yield. Further, subjecting INH2
to BBr3 (6.5 equiv) in CH2Cl2 for 3 h delivered 32 in 20% yield
and the diphenol 33 in 64% yield. Synthesis of compound 36
started from 2′-hydroxy-4′,6′-dimethylacetophenone (34), which
was converted to the 2-aminothiazoline 35 in three steps
including methylation with Me2SO4/K2CO3, bromination with
HBr/BuOOH, and final condensation of the resultant R-bro-
moacetone with thiourea. Benzoylation of amine 35 smoothly
gave the compound 36 in 93% yield. Exposure of compound
36 to BBr3/CH2Cl2 at room temperature provided the phenol
analogue 37 in 90% yield.
Substitution of an oxazolyl ring for the thiazolinyl ring in
INH1 was also accomplished (Scheme 4). Reaction of bromide
1 with urea in refluxing EtOH gave the corresponding 2-ami-
nooxazole 38, which was benzoylated to afford the 2-aminoox-
azolyl derivative 39. In addition, replacement of amide group
in INH1 with thioamide group was performed through treatment
with INH1 with Lawesson’s reagent in refluxing toluene and
the desired thioamide derivative 40 was obtained in 79% yield.
SAR Analysis of the INH Analogues. Synthesized analogues
3-27, 30-33, 36-37, and 39-40 were tested for their killing
activities on human breast cancer cell lines MDA-MB231 and
MDA-MB468, human cervical cancer line HeLa, and human
erythromyeloblastoid leukemia cell line K562. The results are
outlined in Table 1, which are the basis of the current SAR
analysis. Upon removal of the benzoyl groups from INH1 and
INH2, the resultant thiazolyl amine 3 and 4 did not exhibit any
antiproliferative activity even at >40 µM, indicating that benzoyl
moiety is indispensable. Remarkably, the hybrid compound 5
containing the left moiety of INH1 and the right moiety of INH2
To explore the influence of the hydrogen atom of amide
groups on bioactivities, methylated INH analogues 24-26 were
prepared (Scheme 2). Treatment of INH1 and compound 6 with
NaH/MeI/THF gave the methylated compounds 24 and 25 as
the only product in 74% and 89% yields, respectively. Interest-
ingly, subjecting INH2 to the same condition (NaH/MeI/THF)
gave two separable methylated compounds 26 and 27 in 26%
and 63% yields, respectively. In our opinion, the generation of
compound 27 was ascribed to the electron-donating properties
of two MeO groups. That is, once INH2 was treated with NaH
in THF, the two MeO groups would result in the formation of
two intermediates 28 and 29 in tautomeric equilibrium. Inter-
mediate 29 was thought to be preferable to the intermediate 28
because the electronegative center in 29 was more remote from
the two electron donating MeO groups than that in the
intermediate 28, which was consistent with the higher yield of
product 27. Furthermore, a steric contribution to the observed
selectivity (higher yield of 27) should also be taken into account
as the ortho methoxy group would be occupying the same place
as the methyl added to the amide nitrogen. Additionally, amide-
protected compound 30 was prepared in 72% yield via treatment
of compound 6 with NaH/BrCH2CO2Et/THF. Saponification of
the ester 30 with LiOH afforded the acid 31 in 77% yield.
Some phenolic group-containing derivatives were also pre-
pared (Scheme 3). Treatment of INH2 with BBr3 (1.5 equiv)

Synthesis and Biological EValuation of INH Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1759
Scheme 2a
a Reagents and conditions: (a) (i) NaH, THF, 0 °C; (ii) MeI, 0 °C to rt (74% for 24, 89% for 25, 26% for 26, 63% for 27). (b) (i) NaH, DMF, 0 to 90
°C; (ii) BrCH2CO2Et, 90 °C (72%). (c) LiOH, H2O, MeOH, rt (77%).
Scheme 3a
a
t
Reagents and conditions: (a) BBr3, CH2Cl2, 0 °C to rt (20% for 32, 64% for 33). (b) (i) Me2SO4, K2CO3, acetone, reflux; (ii) HBr, BuOOH, dioxane,
reflux; (iii) thiourea, EtOH, reflux (20% over 3 steps). (c) BzCl, DMAP, CH2Cl2, 0 °C to rt (93%); (d) BBr3, CH2Cl2, 0 °C to rt (90%).
Scheme 4a
loss of bioactivity (7) and replacement of the methoxyl group
in 7 with trifluoromethyl group would efficiently improve the
bioactivity by approximately 1.5-2 fold (8). On the basis of
the above results and in light of the well-known strong electron-
withdrawing properties of trifluoromethyl group that renders
neighboring group polar, we proposed that 2,4,6-trimethyl
phenyl is preferred to 2,4-dimethyl phenyl, probably due to less
rotational flexibility, and that introduction of a more polar group
on the right moiety of scaffold seems to improve the bioactivity.
A series of analogues 9-21 containing different polar groups
on the right part of scaffold was prepared and the biological
a Reagents and conditions: (a) urea, EtOH, reflux; (b) BzCl, DMAP,
activities were evaluated. As expected, most analogues exhibited
CH2Cl2, 0 °C to rt (7% in 2 steps); (c) Lawesson reagent, toluene, reflux
better cell killing activity than the original leads. For most cases,
2,4,6-trimethyl phenyl group was more active than the 2,4-
dimethyl phenyl group. Apparently, the isonicotinoyl group (13)
was preferred to pyridine-2-carboxyl group (9, 10) and nicoti-
noyl group (11, 12). Substitution of pyrazinyl group for
isonicotinoyl group led to a considerable reduction in bioactivity
(13 vs 14), although compound 15 still exhibited good bioac-
tivity. The bioactivity variation between 16 and 17, which
(79%).
exhibited very low antiproliferation activities against all the
tested cell lines. To our delight, the other hybrid compound 6,
containing the right moiety of INH2 and the left moiety of
INH1, exhibited significantly improved antiproliferation activi-
ties compared with our leads INH1 and 2. Additionally, deletion
of the para-methoxyl group in INH2 resulted in the dramatic

1760 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 1. Activity of INH1, INH2, and Their Analogues
Qiu et al.

Synthesis and Biological EValuation of INH Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1761
contain 1-methyl-1H-imidazolyl groups, further consolidated our
conclusion that 2,4,6-trimethyl phenyl group was preferred to
2,4-dimethyl phenyl group. Introduction of 1,3-thiazolyl groups
did not change the bioactivities significantly (18, 19). Notably,
the replacement of phenyl group in lead INH1 with a mor-
pholinyl group efficiently improved the activity. Among the
analogues made, 6, 13, and 21 displayed the strongest activity,
the IC50 values of which were improved by 6-8 fold in
comparison to INH1. The para position of the heteroatom
(N, O) on the six-membered ring of compounds 13 and 21
clearly provides advantages over the lead INH1 and the
pyridine-2-carboxyl derivatives 9-10 (with heteroatom N at
ortho position), suggesting possible existence of an extended
hydrophilic groove or pocket in the target binding site.
treatment at 25 µM with a similar period of time (Figure 2D).
In contrast, 22 showed no effect on Nek2 level at any time point
tested. In addition, 6 and 21 seemed to trigger a slight Hec1
decrease over time, unlike the treatment with INH1, although
the significance of partial Hec1 reduction is not clear at present.
These results indicate that 6 and 21 are more efficient in
targeting the Hec1/Nek2 complex for degradation.
Compounds 6 and 21 Triggered Mitotic Abnormalities.
The Hec1/Nek2 complex functions primarily during G2 and M
phases. Perturbation of the Hec1 or Nek2 function by antagonists
(RNAi or antibody) leads to mitotic abnormalities represented
by spindle configuration changes and chromosome misalign-
ment, which contribute to mitotic catastrophe.11-18 Treating cells
with INH1 similarly led to mitotic abnormalities, albeit to a
lesser extent.10 To test whether compound 6 and 21 can elicit
similar mitotic phenotypes via inhibiting Hec1/Nek2, cells were
treated with 6, 21, or the negative compound 22. In comparison
to the mock or 22-treated cells, 6 or 21 treated cells exhibited
increased mitotic population with multipolar spindle configura-
tions, presumably because of defective Hec1/Nek2 functionality
(Figure 3A). Consistently, an increased rate of chromosome
misalignment was detected upon treatment with 6 or 21 of HeLa
cells expressing the chromosome marker protein H2B-GFP
(Figure 3B). At the dosage of 6.25 µM, 6 and 21 elicited mitotic
abnormalities to a similar extent as INH1 treatment at 25 µM,
further confirming the improved efficacy of compound 6 and
21.
Treatment of Cells with Compounds 6 and 21 Induced
Apoptosis. Mitotic abnormalities often lead to mitotic arrest,
often followed by mitotic slippage and consequent mitotic
catastrophe (i.e., cell death due to abnormal mitosis). Lead
compound INH1 is known to trigger mitotic catastrophe. To
examine whether compounds 6 and 21 may trigger mitotic
catastrophe, a time lapse experiment was performed to monitor
the cell morphological changes and potential apoptosis. As
expected, 6 or 21 treated cells showed progressive morphological
changes characteristic of dying cells (e.g., membrane bubbling),
which is further confirmed by cell cycle profiling with FACS
analysis (Figure 4A,B). For instance, approximately 20% of
compound 6 or 22 treated cells were apoptotic 72 h after
treatment. The above results are consistent with the mitosis-
targeting activity of compounds 6 and 21 with improved
efficacy.
Replacement of amide moiety in INH1 with sulfonyl amide
reduced the activity significantly (22-23, IC50 > 40 µM). Upon
methylation of amide groups of INH1 or 2 and compound 6,
the resultant derivatives 24-26 showed weakened bioactivities
against all the tested cell lines, indicating that methylation of
the amide group negatively affects compound activity. Although
compound 30 derived from ethoxycarbonylmethylating com-
pound 6 still displayed weak activities, its saponified product
31 is almost inactive (IC50 > 40 µM). Demethylated derivative
33 of INH2 showed very weak activity, but monophenol
compound 32 still exhibited slightly weak bioactivity compared
with INH2, which indicated that phenol group on the para
position was not preferred. Interestingly, compounds 36-37,
derived from substitution of methoxyl group and hydroxyl group
for C6 methyl group in 6, were also found to be active although
bioactivity decreased significantly. Additionally, we also found
that conversion of the thiazolyl ring in INH1 to an oxazolyl
ring (39) or transformation of amide group in INH1 to thioamide
group (40) also made the bioactivity decrease, which demon-
strated that thiazolyl ring and amide group were important for
the cytotoxicity.
Target Validation of INH Analogues. Next, target validation
was performed by using an affinity pulldown assay described
previously.10 Because compounds 6, 13, and 21 showed most
improved cell killing activities with similar IC50 values in the
SAR analysis, they were chosen for target validation by using
compound 22 as a negative control. More specifically, 6 was
used as a positive compound and 22 as a negative control in
the affinity pulldown assay. In principle, compounds 6 and 22
were individually conjugated to an affi-gel matrix through a
multistep synthetic route (Supporting Information) (Figure
2A).10 HeLa cell extract was prepared and subjected to affinity
pulldown experiments with control or compound-conjugated
matrices. Shown in Figure 2B, the 6-conjugated matrix, but not
the 22-conjugated or unconjugated matrix, selectively copre-
cipitated with cellular Hec1. In contrast, none of them copre-
cipitated with cellular Nek2. The results suggest that compound
6 binds to cellular Hec1, similar to the lead, INH1.
A hallmark mechanism of action by INH1 is to trigger
dramatic reduction of Nek2 protein level.10 To determine
whether INH analogues retain such bioactivity in cells, both
time and dosage-dependent kinetic studies were carried out in
HeLa cells. The protein levels of Hec1 and Nek2 were examined
by Western blot. In a dose-dependent study, Nek2 was found
to be undetectable on treating cells with INH1 (25 µM), or lower
dosages of 6 or 21 (both at 6.25 µM), but not by the negative
compound 22 (up to 25 µM) (Figure 2C). For the time-
dependent study, cells were treated with 6.25 µM of compound
6 or 21. Notably, the Nek2 level was reduced by ∼50% at 8-11
h after treatment with 6 or 21, which was comparable to INH1
Conclusion
This study has identified several INH analogues that show a
6-8-fold increase in cellular killing activity by targeting the
Hec1/Nek2 pathway. The SAR study identified a few key
structural features that may serve as a guideline for further
improvement of the current compounds using medicinal chemi-
cal approaches. In essence, the presence of a hydrophobic ring
with less rotational freedom, such as 2,4,6-trimethyl phenyl, is
preferred at the left part of the scaffold (Figure 1). Introducing
hetero atoms (N, O) or a polar group to the para positions of
the six-membered ring on the right side of the scaffold seems
to dramatically improve both solubility and bioactivity. Finally,
the integrity of the thiazolyl ring and the amide bond in the
middle of the scaffold is important for maintaining compound
activity.
Two of the improved versions, compounds 6 and 21, are
capable of targeting the cellular Hec1/Nek2 complex, triggering
mitotic abnormalities and cell death at a much lower dosage
than the lead INH1. The structural features identified in this
study may serve as a foundation for designing, synthesis, and

1762 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Qiu et al.
Figure 2. Target validation. (A) Illustrations of compound conjugated affi-gel matrices. (B) HeLa cell extract was used for affinity binding by
control matrix, INH22, or INH6 conjugated matrix. Resultant precipitates after sufficient washing were subjected to SDS-PAGE analysis and
Western blot to detect Hec1 and Nek2. Whole cell extract was used as input in duplicates. (C) Dosage-dependent kinetic study of compound
treatment. HeLa cells were treated with 6.25 µM INH6 or INH21 and harvested at various time points for detecting Hec1 and Nek2 by Western
blot. P84 is a nuclear matrix protein used as a loading control. (D) Time-dependent kinetic study with varying dosages of the indicated compounds.
Western blot was similarly performed as in (C).
The absorption at 595 nm was measured with a plate reader and
converted to cell survival percentages in comparison to mock treated
groups.
evaluation of the next generation of INH derivatives with further
refinement for potential clinical applications.
Experimental Section
Microscopy and FACS Analysis. Immunostaining, image
processing, and FACS assays were done as detailed previously.19
Chemistry. All reagents were used as received from commercial
sources, unless specified otherwise, or prepared as described in the
literature. Reactions requiring anhydrous conditions were performed
in vacuum heat-dried glassware under nitrogen atmosphere. Reac-
tion mixtures were stirred magnetically. DMF, dichloromethane,
and pyridine were distilled from CaH2. 1H NMR spectra were
recorded at either 400 or 500 MHz. 13C NMR spectra were recorded
at either 125 or 100 MHz. 19F NMR spectra were recorded at 376
MHz with FCCl3 as external standard and low field is positive.
Chemical shifts (δ) are reported in ppm, and coupling constants
(J) are in Hz. The following abbreviations were used to explain
the multiplicities: s ) singlet, d ) doublet, t ) triplet, q ) quartet,
m ) multiplet.
Cell Lines and Antibodies. Human breast cancer cell lines
MDA-MB231 and MDA-MB468, and the cervical adenocarcinoma
line HeLa, were maintained in DMEM medium (Invitrogen)
supplemented with 10% FBS. Antibody sources were: mouse anti-
Hec1 clone 9G3 and mouse anti-p84 (Genetex), rabbit anti-Nek2
polyclonal antibodies (Santa Cruz Biotechnology), mouse anti-R-
tubulin and rabbit anti-γ-tubulin (Sigma Aldrich), and secondary
antibodies conjugated with Alexa dyes (Invitrogen).
Cell Killing Assay. Standard XTT assays with a four-day drug
treatment procedure were performed to measure the dose-dependent
cytotoxicity of INH analogues in cultured cells. Triplicate sets were
measured and compiled for final data presentation. The assay was
performed by using a commercial kit (Roche Scientific) by
following the instructions. In principle, cells were plated on 96-
well dishes one day before the drug treatment, followed by drug
treatment on day 2 and XTT assay on day 5 after drug addition.
4-(2,4-Dimethyl-phenyl)thiazol-2-ylamine (3). The mixture of
compound 1 (1.28 g, 5.63 mmol) and thiourea (450 mg, 5.91 mmol)

Synthesis and Biological EValuation of INH Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1763
Figure 3. Mitotic abnormality induced by compound 6 and 21 but not 22 and the mock (buffer control). (A) Immunofluorescent staining of HeLa
cells treated with mock or compounds 22, 6, or 21 (each at 3.75 µM) for 24 h. Right column showed merged images of R-tubulin, γ-tubulin, and
the DAPI dye (DNA). Arrows indicated spindle poles. Note the multipolar spindle configurations in 6 and 21 treated cells. Scale bar, 10 µM. (B)
incidence of metaphase chromosome alignment in cells treated individual compounds (2.5 µM each). Triplicate sets of compound-treated HeLa
cells expressing the chromosome marker H2B-GFP were measured to obtain the averages and deviations.
Figure 4. Apoptosis of cells treated with compounds 6 and 21 but not 22 and the mock (buffer control). (A) phase contrast images of cells at
various time points after treatment with mock or individual compounds (each at 2.5 µM). (B) FACS analysis results of cells treated as in (A). Sub
G1 populations in circles represented apoptotic cells at 72 hr time point.
in anhydrous EtOH (20 mL) was heated to reflux for 30 min. After
that, the solvent was removed in vacuo and the saturated aqueous
NaHCO3 was added to make the mixture basic (pH ) 8-9). Then
the mixture was extracted with CH2Cl2 (3 × 30 mL). The combined
organic phases were dried with anhydrous Na2SO4. After removal
of all the solvent, the residue was purified by silica gel chroma-
tography (hexane/EtOAc ) 2:1) to afford product 3 (1.03 g, 89%)
as a solid; mp 84.5-85.5 °C. 1H NMR (400 MHz, CDCl3) δ 7.43
(d, J ) 8.0 Hz, 1H), 7.06-7.03 (m, 2H), 6.42 (s, 1H), 5.29 (br,
2H), 2.42 (s, 3H), 2.35 (s, 3H). MS (ESI) m/z 205 (M + H+).
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]-2,4-dimethoxybenz-
amide (5). The solution of compound 3 (82 mg, 0.40 mmol),
DMAP (55 mg, 0.44 mmol), and 2, 4-dimethoxy benzoyl chloride
(83 mg, 0.42 mmol) in CH2Cl2 (3 mL) was stirred for 1 h. After
that, the mixture was directly subjected to purification by silica
gel chromatography (hexane/EtOH ) 3:1) to give compound 5 (98
mg, 67%) as a solid; mp 159-160 °C. 1H NMR (400 MHz, CDCl3)
δ 10.99 (s, 1H), 8.30 (d, J ) 8.8 Hz, 1H), 7.49 (d, J ) 7.6 Hz,
1H), 7.09-7.06 (m, 2H), 6.88 (s, 1H), 6.69 (dd, J ) 8.8, 2.4 Hz,
1H), 6.56 (s, J ) 2.4 Hz, 1H), 4.07 (s, 3H), 3.90 (s, 3H), 2.43 (s,
3H), 2.36 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 164.9, 162.7,
159.4, 157.3, 150.4, 137.9, 136.1, 134.7, 132.3, 131.7, 129.8, 126.8,
112.4, 110.6, 106.2, 98.9, 56.5, 55.9, 21.3, 21.2. MS (ESI) m/z 369
(M + H+), 391 (M + Na+). HRMS calcd for C20H20N2O3SNa
(M + Na+), 391.1092; found, 391.1095.
N-[4-(2,4,6-Trimethylphenyl)thiazol-2-yl]benzamide (6). The
solution of compound 4 (105 mg, 0.48 mmol), Et3N (0.30 mL, 2.15
mmol) and BzCl (75 mg, 0.53 mmol) in dioxane (5 mL) was heated
to reflux for 2.5 h. After that, the mixture was cooled to room
temperature and all the solvent was removed in vacuo to afford a
residue, which was extracted with EtOAc (3 × 30 mL). The
combined organic phases were dried with anhydrous Na2SO4.
Removal of all the solvent in vacuo resulted in a residue, which
was purified with silica gel chromatography (hexane/EtOAc ) 15:
1) to give compound 6 (48 mg, 31%) as a white solid; mp 192-193
1
°C. H NMR (400 MHz, CDCl3) δ 11.63 (s, 1H), 7.52-7.73 (m,
2H), 7.51 (tt, J ) 7.6, 1.2 Hz, 1H), 7.41-7.37 (m, 2H), 6.74 (s,
1H), 6.62 (s, 2H), 2.18 (s, 3H), 1.93 (s, 6H). 13C NMR (100.5 MHz,
CDCl3) δ 165.3, 159.1, 148.8, 137.7, 137.0, 132.6, 132.0, 131.5,
128.6, 128.4, 127.7, 111.6, 21.2, 20.5. MS (ESI) m/z 323 (M +
H+), 345 (M + Na+); 645 (2 M + H+), 667 (M + 2Na+). HRMS
calcd for C19H19N2OS (M + H+), 323.1218; found, 323.1212.

1764 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Qiu et al.
2-Methoxy-N-[4-(2,4,6-trimethylphenyl)thiazol-2-yl]benz-
amide (7). The solution of amine 4 (120 mg, 0.55 mmol) and
DMAP (110 mg, 0.90 mmol) in CH2Cl2 (2 mL) was added to a
solution of 2-methoxyl benzoyl chloride (120 mg, 0.70 mmol) at 0
°C. The resultant mixture was warmed to room temperature and
stirred for 20 min. After that, H2O (20 mL) was added to quench
the reaction and the mixture was extracted with CH2Cl2 (3 × 30
mL). The combined organic phases were dried with anhydrous
Na2SO4. Removal of all the solvent in vacuo resulted in a residue,
which was purified with silica gel chromatography (hexane/EtOAc
) 4:1) to give compound 7 (150 mg, 77%) as a white solid; mp
1H), 9.51 (s, 1H), 8.85 (d, J ) 2.0 Hz, 1H), 8.61 (t, J ) 1.5 Hz,
1H), 7.48 (d, J ) 8.0 Hz, 1H), 7.07-7.05 (m, 2H), 6.99 (s, 1H),
2.44 (s, 3H), 2.35 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 160.9,
155.9, 151.0, 148.5, 145.0, 143.2, 142.9, 138.1, 136.0, 131.8, 131.7,
129.7, 126.8, 111.2, 21.3, 21.2. MS (ESI) m/z 311 (M + H+), 333
(M + Na+). HRMS calcd for C16H14N4OSNa (M + Na+), 333.0786;
found, 333.0781.
3-Methyl-3H-imidazole-4-carboxylic Acid [4-(2,4-Dimeth-
ylphenyl)thiazol-2-yl]amide (16). To a 0 °C solution of amine 3
(85 mg, 0.42 mmol) in CH2Cl2 (2 mL) was added DMAP (118
mg, 0.97 mmol) followed by 1-methyl-1H-imidazole-5-carbonyl
chloride hydrochloride (180 mg, 0.99 mmol). Then the mixture was
stirred at room temperature for 2 h. After that, the whole mixture
was subjected to purification by silica gel chromatography to give
compound 16 (49 mg, 37%) as a solid; mp 250 °C (decomposed).
1H NMR (500 MHz, DMSO-d6) δ 12.55 (br, 1H), 8.11 (s, 1H),
7.92 (s, 1H), 7.53 (d, J ) 8.0 Hz, 1H), 7.22 (s, 1H), 7.10 (s, 1H),
7.06 (d, J ) 7.5 Hz, 1H), 3.91 (s, 3H), 2.41 (s, 3H), 2.30 (s, 3H).
13C NMR (125 MHz, DMSO-d6) δ 157.9, 156.9, 149.1, 143.6,
136.9, 135.1, 131.7, 131.4, 129.4, 126.4, 124.0, 110.5, 34.0, 21.0,
20.7. MS (ESI) m/z 313 (M + H+), 335 (M + Na+). HRMS calcd
for C16H17N4OS (M + H+), 313.1123; found, 313.1131.
1
218.5-219.5 °C. H NMR (400 MHz, CDCl3) δ 11.11 (s, 1H),
8.34 (dd, J ) 7.6, 1.2 Hz, 1H), 7.55 (td, J ) 7.6, 1.6 Hz, 1H), 7.16
(t, J ) 7.6 Hz, 1H), 7.05 (d, J ) 8.4 Hz, 1H), 6.92 (s, 2H), 6.75
(s, 1H), 4.05 (s, 3H), 2.31 (s, 3H), 2.12 (s, 6H). 13C NMR (100.5
MHz, CDCl3) δ 162.5, 157.7, 157.3, 149.1, 137.7, 137.3, 134.3,
132.7, 132.3, 128.2, 121.7, 119.2, 111.5, 111.1, 56.2, 21.1, 20.3.
MS (ESI) m/z 353 (M + H+), 375 (M + Na+); 705 (2 M + H+),
727 (M + 2Na+). HRMS calcd for C20H20N2O2SNa (M + Na+),
375.1143; found, 375.1139.
Pyridine-2-carboxylic Acid [4-(2,4-Dimethylphenyl)thiazol-
2-yl]amide (9). To a 0 °C solution of amine 3 (90 mg, 0.44 mmol)
in CH2Cl2 (4 mL) was added DMAP (120 mg, 0.98 mmol) followed
by picolinoyl chloride hydrochloride (80 mg, 0.45 mmol). Then
the mixture was stirred at room temperature for 4 h. After that, the
whole mixture was subjected to purification by silica gel chroma-
tography to give compound 9 (118 mg, 87%) as a solid; mp
Thiazole-2-carboxylic Acid [4-(2,4-Dimethylphenyl)thiazol-
2-yl]amide (18). To a 0 °C solution of amine 3 (86 mg, 0.42 mmol)
in CH2Cl2 (2 mL) was added DMAP (62 mg, 0.51 mmol) followed
by 1, 3-thiazole-2-carbonyl chloride (75 mg, 0.51 mmol). Then the
mixture was stirred at room temperature for 15 min. After that, the
whole mixture was subjected to purification by silica gel chroma-
tography to give compound 18 (100 mg, 76%) as a solid; mp
1
212-213 °C. H NMR (400 MHz, CDCl3) δ 11.22 (br, 1H), 8.65
(d, J ) 4.0 Hz, 1H), 8.31 (d, J ) 8.0 Hz, 1H), 7.93 (t, J ) 7.6 Hz,
1H), 7.53 (d, J ) 7.2 Hz, 2H), 7.10-7.07 (m, 2H), 6.97 (s, 1H),
2.46 (s, 3H), 2.37 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 162.1,
156.3, 150.8, 148.7, 148.0, 137.9, 136.0, 131.9, 131.8, 129.7, 127.4,
126.8, 122.9, 110.8, 21.3, 21.3. MS (ESI) m/z 310 (M + H+), 332
(M + Na+). HRMS calcd for C17H15N3OSNa (M + Na+), 332.0833;
found, 332.0825.
1
146.5-147.5 °C. H NMR (500 MHz, CDCl3) δ 10.60 (br, 1H),
7.98 (d, J ) 2.5 Hz, 1H), 7.71 (d, J ) 3.0 Hz, 1H), 7.48 (d, J )
7.5 Hz, 1H), 7.08-7.06 (m, 2H), 6.98 (s, 1H), 2.44 (s, 3H), 2.36
(s, 3H). 13C NMR (125 MHz, CDCl3) δ 161.1, 157.1, 155.6, 151.0,
144.4, 138.0, 136.0, 131.8, 131.7, 129.7, 126.8, 126.3, 111.2, 21.3,
21.2. MS (ESI) m/z 316 (M + H+), 338 (M + Na+). HRMS calcd
for C15H13N3OS2Na (M + Na+), 338.0398; found, 338.0391.
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]nicotinamide (11). To
a 0 °C solution of amine 3 (97 mg, 0.47 mmol) in CH2Cl2 (2 mL)
was added DMAP (120 mg, 0.98 mmol) followed by nicotinoyl
chloride hydrochloride (108 mg, 0.60 mmol). Then the mixture was
stirred at room temperature for 20 min. After that, the whole mixture
was subjected to purification by silica gel chromatography to give
compound 11 (93 mg, 64%) as a solid; mp 210-211 °C. 1H NMR
(500 MHz, CDCl3) δ 12.55 (br, 1H), 8.64 (s, 1H), 8.61 (s, 1H),
7.93 (d, J ) 8.0 Hz, 1H), 7.20-7.15 (m, 2H), 6.92 (s, 1H),
6.82-6.79 (m, 2H), 2.23 (s, 6H). 13C NMR (125 MHz, CDCl3) δ
164.3, 159.3, 152.9, 148.9, 138.2, 135.7, 135.2, 131.6, 130.9, 129.6,
128.2, 126.7, 123.3, 111.0, 21.2, 20.7. MS (ESI) m/z 310 (M +
H+), 332 (M + Na+). HRMS calcd for C17H15N3OSNa (M + Na+),
332.0833; found, 332.0835.
Morpholine-4-carboxylic Acid [4-(2,4-Dimethylphenyl)thi-
azol-2-yl]amide (20). To a 0 °C solution of amine 3 (94 mg, 0.46
mmol) in CH2Cl2 (2 mL) was added DMAP (281 mg, 2.30 mmol)
followed by 4-morpholinecarbonyl chloride (117 mg, 0.78 mmol).
Then the mixture was stirred at room temperature for 3 days. After
that, the whole mixture was subjected to purification by silica gel
chromatography to give compound 20 (92 mg, 63%) as a solid;
1
mp 168-169 °C. H NMR (500 MHz, CDCl3) δ 10.41 (br, 1H),
7.34 (d, J ) 8.0 Hz, 1H), 7.07 (s, 1H), 7.04 (d, J ) 7.5 Hz, 1H),
6.80 (s, 1H), 3.43 (t, J ) 4.5 Hz, 4H), 3.21 (t, J ) 4.0 Hz, 4H),
2.34 (s, 3H), 2.33 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 161.4,
154.2, 149.1, 138.1, 136.0, 132.0, 131.7, 129.6, 126.9, 110.1, 66.4,
44.1, 21.3, 20.9. MS (ESI) m/z 318 (M + H+), 340 (M + Na+).
HRMS calcd for C16H19N3O2SNa (M + Na+), 340.1096; found,
340.1092.
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]isonicotinamide (13).
To a 0 °C solution of amine 3 (75 mg, 0.37 mmol) in CH2Cl2
(2 mL) was added DMAP (90 mg, 0.74 mmol) followed by
isonicotinoyl chloride hydrochloride (85 mg, 0.48 mmol). Then the
mixture was stirred at room temperature for 20 min. After that, the
whole mixture was subjected to purification by silica gel chroma-
tography to give compound 13 (101 mg, 88%) as a solid; mp
Morpholine-4-carboxylic Acid [4-(2,4,6-Trimethylphenyl)thi-
azol-2-yl]amide (21). To a solution of amine 4 (112 mg, 0.51
mmol) in anhydrous CH2Cl2 (4 mL) was added DMAP (69 mg,
0.56 mmol). The resultant solution was cooled to 0 °C and then a
solution of 4-morpholinecarbonyl chloride (80 mg, 0.54 mmol) in
anhydrous CH2Cl2 (1 mL) was added dropwise. The mixture was
warmed up to room temperature and stirred for 1 h. After that,
DMAP (150 mg, 1.23 mmol) and 4-morpholinecarbonyl chloride
(0.67 mmol) were added. The whole mixture was further stirred
for 7 h. H2O (20 mL) was added to quench the reaction, and the
mixture was extracted with CH2Cl2 (3 × 20 mL). The combined
organic phases were washed with brine and dried with anhydrous
Na2SO4. After removal of all the solvent, the residue was purified
by silica gel chromatography (hexane/EtOAc ) 3:1) to give
compound 21 (66 mg, 39%) as a white solid; mp 183-184 °C. 1H
NMR (400 MHz, CDCl3) δ 10.62 (s, 1H), 6.91 (s, 2H), 6.60 (s,
1H), 3.53 (s, 4H), 3.21 (s, 4H), 2.32 (s, 3H), 2.01 (s, 6H). 13C NMR
(125 MHz, CDCl3) δ 162.41, 154.59, 146.50, 138.08, 137.39,
131.47, 128.60, 110.77, 66.54, 44.00, 21.24, 20.37. MS (ESI) m/z
1
148-149 °C. H NMR (500 MHz, CDCl3) δ 12.96 (br, 1H), 8.53
(d, J ) 4.5 Hz, 2H), 7.38 (d, J ) 4.5 Hz, 2H), 7.08 (d, J ) 8.0 Hz,
1H), 6.94 (s, 1H), 6.79-6.76 (m, 2H), 2.21 (s, 6H). 13C NMR (100.5
MHz, CDCl3) δ 164.4, 159.2, 150.3, 150.1, 139.0, 138.2, 135.5,
131.7, 130.8, 129.5, 126.8, 121.1, 111.2, 21.1, 20.6. MS (ESI) m/z
310 (M + H+), 332 (M + Na+). HRMS calcd for C17H15N3OSNa
(M + Na+), 332.0833; found, 332.0826.
Pyrazine-2-carboxylic Acid [4-(2,4-Dimethylphenyl)thiazol-
2-yl]amide (14). To a 0 °C solution of amine 3 (103 mg, 0.51
mmol) in CH2Cl2 (2 mL) was added DMAP (75 mg, 0.61 mmol)
followed by pyrazine-2-carbonyl chloride (87 mg, 0.61 mmol).
Then, the mixture was stirred at room temperature for 30 min. After
that, the whole mixture was subjected to purification by silica gel
chromatography to give compound 14 (122 mg, 77%) as a solid;
1
mp 168.5-169.5 °C. H NMR (500 MHz, CDCl3) δ 11.05 (br,

Synthesis and Biological EValuation of INH Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1765
332 (M + H+), 354 (M + Na+), 662 (2 M + H+), 684 (M + 2Na+).
HRMS calcd for C17H22N3O2S (M + H+), 332.1433; found,
332.1429.
of all the solvent, the residue was purified by silica gel chroma-
tography (hexane/EtOAc ) 4:1 to 2:1) to afford compound 26 (less
polar, 38 mg, 26%) as a solid and compound 27 (more polar, 93
1
mg, 63%) as a solid. Compound 26: mp 156-157 °C; H NMR
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]benzenesulfonamide (22).
To a 0 °C solution of amine 3 (99 mg, 0.485 mmol) in pyridine (3
mL) was added DMAP (20 mg, 0.16 mmol) followed by PhSO2Cl
(95 mg, 0.53 mmol). Then the mixture was stirred at room
temperature overnight. After that, the mixture was poured into a
solution of aqueous 1 N HCl (50 mL). The mixture was extracted
with CH2Cl2 (3 × 30 mL). The combined organic phases were dried
with anhydrous Na2SO4. Removal of all the solvent in vacuo
resulted in a residue, which was purified with silica gel chroma-
tography (hexane/EtOAc ) 2:1) to give compound 22 (113 mg,
(400 MHz, CDCl3) δ 7.35 (d. J ) 8.4 Hz, 1H), 6.94 (s, 2H), 6.79
(s, 1H), 6.59 (dd, J ) 10.4, 2.0 Hz, 1H), 6.52 (d, J ) 2.0 Hz, 1H),
3.87 (s, 3H), 3.86 (s, 3H), 3.55 (s, 3H), 2.32 (s, 3H), 2.15 (s, 6H);
1C NMR (125 MHz, CDCl3) δ 168.9, 162.9, 157.4, 148.5, 137.8,
137.6, 133.0, 131.1, 130.1, 128.5, 117.6, 112.6, 105.2, 98.8, 55.8,
55.7, 37.1, 21.3, 20.7; MS (ESI) m/z 397 (M + H+), 419 (M +
Na+); HRMS calcd for C22H24N2O3SNa (M + Na+), 419.1405,
found, 419.1390. Compound 27: mp 161-162 °C; 8.28 (d, J )
9.6 Hz, 1H), 6.99 (s, 2H), 6.54 (s, 1H), 6.53 (d, J ) 6.4 Hz, 1H),
6.39 (s, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 3.44 (s, 3H), 2.35 (s, 3H),
1
68%) as a solid; mp 188-189 °C. H NMR (400 MHz, CDCl3) δ
1
2.07 (s, 6H); C NMR (125 MHz, CDCl3) δ 173.9, 168.1, 139.9,
9.93 (br, 1H), 7.93 (d, J ) 7.6 Hz, 2H), 7.55-7.43 (m, 3H), 7.17
(d, J ) 7.6 Hz, 1H), 7.09-7.04 (m, 2H), 6.26 (s, 1H), 2.34, 2.33
(2s, 6H). 13C NMR (100.5 MHz, CDCl3) δ 168.9, 142.1, 140.3,
136.7, 136.3, 132.4, 132.2, 129.2, 128.9, 127.4, 126.7, 125.9, 103.9,
21.4, 20.6. MS (ESI) m/z 345 (M + H+), 367 (M + Na+). HRMS
calcd for C17H16N2O2S2Na (M + Na+), 367.0551; found, 367.0551.
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]-C-phenyl-methane-
sulfonamide (23). To a 0 °C solution of compound 3 (116 mg,
0.57 mmol) in THF (5 mL) was added Et3N (0.23 mL, 1.64 mmol)
and DMAP (10 mg, 0.08 mmol). Then BnSO2Cl (130 mg, 0.68
mmol) was added in one portion. The resultant mixture was warmed
up to room temperature and stirred for 1.5 h. The reaction was
quenched with H2O (5 mL), and H2O (30 mL) was added. The
mixture was extracted with CH2Cl2 (3 × 20 mL). The combined
organic phases were dried with anhydrous Na2SO4. After removal
of all the solvent, the residue was purified by silica gel chroma-
tography (hexane/EtOAc ) 5:1 to 2:1) to afford product 23 (129
mg, 63%) as a solid; mp 162-163 °C. 1H NMR (400 MHz, CDCl3)
δ 10.20 (br, 1H), 7.42-7.40 (m, 2H), 7.30-7.24 (m, 3H),
7.05-7.01 (m, 3H), 6.08 (s, 1H), 4.29 (s, 2H), 2.34 (s, 3H), 2.22
(s, 3H). 13C NMR (100.5 MHz, CDCl3) δ 170.6, 140.1, 136.6, 136.3,
132.0, 131.4, 129.9, 129.1, 128.6, 128.5, 127.3, 126.1, 103.3, 60.2,
21.4, 20.5. MS (ESI) m/z 359 (M + H+), 381 (M + Na+). HRMS
calcd for C18H19N2O2S (M + H+), 359.0888; found, 359.0880.
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]-N-methylbenzamide (24).
To a cooled mixture of NaH (20 mg, 60% in oil, 0.5 mmol) in
THF (5 mL) was added a solution of compound INH1 (120 mg,
0.39 mmol) in THF (3 mL) dropwise. The mixture was warmed
up to room temperature and stirred for 20 min. After that, the
mixture was cooled to 0 °C again and MeI (32 µL, 0.5 mmol) was
added dropwise. Then the mixture was warmed up to room
temperature and stirred for 2 h. Then H2O (2 mL) was added to
quench the reaction and the mixture was diluted with H2O (30 mL).
The mixture was extracted with CH2Cl2 (3 × 15 mL). The combined
organic phases were dried with anhydrous Na2SO4. After removal
of all the solvent, the residue was purified by silica gel chroma-
tography (hexane/EtOAc ) 4:1) to afford compound 24 (93 mg,
138.5, 136.8, 134.0, 128.7, 127.3, 119.8, 106.2, 104.3, 99.1, 56.2,
55.6, 33.4, 21.4, 20.1; MS (ESI) m/z 397 (M + H+), 419 (M +
Na+); HRMS calcd for C22H25N2O3S (M + H+), 397.1586, found,
397.1584.
{Benzoyl-[4-(2,4,6-trimethylphenyl)thiazol-2-yl]-amino}acetic Acid
Ethyl Ester (30). NaH (38 mg, 60% in oil, 0.95 mmol) was cooled
to 0 °C and a solution of compound 6 (167 mg, 0.52 mmol) in
DMF (6 mL) was added. The mixture was carefully heated to 90
°C and stirred for 1 h. Then the mixture was cooled to 0 °C and
BrCH2CO2Et (0.60 mL, 5.4 mmol) was added dropwise. After that,
the mixture was heated to 90 °C and stirred overnight. H2O (10
mL) was added to quench the reaction, and the mixture was
extracted with CH2Cl2 (3 × 20 mL). The combined organic phases
were dried with anhydrous Na2SO4. Removal of all the solvent in
vacuo resulted in a residue, which was purified with silica gel
chromatography (hexane/EtOAc ) 5:1) to give compound 30 as a
white solid (152 mg, 72%); mp 160-161 °C. 1H NMR (500 MHz,
CDCl3) δ 8.32-8.30 (m, 2H), 7.49 (tt, J ) 7.0, 1.5 Hz, 1H),
7.45-7.42 (m, 2H), 6.99 (s, 2H), 6.47 (s, 1H), 4.50 (s, 2H), 4.16
(q, J ) 7.0 Hz, 2H), 2.36 (s, 3H), 2.13 (s, 6H), 1.20 (t, J ) 7.0 Hz,
3H). 13C NMR (125.5 MHz, CDCl3) δ 174.1, 168.6, 167.3, 140.4,
139.0, 136.9, 136.5, 131.8, 129.5, 129.0, 128.2, 126.0, 106.5, 61.9,
47.9, 21.4, 20.1, 14.3. MS (ESI) m/z 409 (M + H+), 431 (M +
Na+). HRMS calcd for C23H25N2O3S (M + H+), 409.1586; found,
409.1594.
{Benzoyl-[4-(2,4,6-trimethylphenyl)thiazol-2-yl]amino}acetic Acid
(31). Compound 30 (75 mg, 0.18 mmol) was dissolved in CH3CN
(6 mL). Then MeOH (3 mL), H2O (1.5 mL) and LiOH ·H2O (30
mg, 0.71 mmol) were added subsequently. The resultant mixture
was stirred at room temperature for 4 h. H2O (20 mL) was added
to quench the reaction, and HCl (1M, 3 mL) was added. The
resultant mixture was extracted with EtOAc (3 × 20 mL). The
combined organic phases were dried with anhydrous Na2SO4.
Removal of all the solvent in vacuo resulted in a residue, which
was purified with silica gel chromatography (hexane/EtOAc ) 2:1
to 1:2) to give compound 31 (53 mg, 77%) as a white solid; mp
1
1
198-199 °C. H NMR (500 MHz, CDCl3) δ 10.40 (br, 1H), 8.30
74%) as a solid; mp 146-147 °C. H NMR (400 MHz, CDCl3) δ
(d, J ) 7.5 Hz, 2H), 7.49-7.39 (m, 3H), 7.00 (s, 2H), 6.48 (s,
1H), 4.59 (2H), 2.38 (s, 3H), 2.10 (S, 6H). 13C NMR (100.5 MHz,
CDCl3) δ 174.1, 171.1, 168.8, 140.4, 138.8, 136.6, 136.2, 131.8,
129.4, 128.9, 128.2, 125.5, 106.9, 47.7, 21.3, 19.9. MS (ESI) m/z
381 (M + H+), 403 (M + Na+); 761 (2 M + H+), 783 (M + 2Na+).
HRMS calcd for C21H20N2O3SNa (M + Na+), 403.1092; found,
403.1093.
8.40 (m, 2H), 7.46 (m, 3H), 7.15 (m, 3H), 6.49 (s, 1H), 3.56 (s,
3H), 2.41 (s, 3H), 2.16 (s, 3H). 13C NMR (100.5 MHz, CDCl3) δ
174.40, 168.66, 140.44, 138.69, 137.94, 137.30, 131.61, 131.52,
130.77, 129.43, 128.25, 127.47, 127.22, 106.75, 34.14, 21.53, 19.77.
MS (ESI) m/z 323 (M + H+). HRMS calcd for C19H18N2OSNa (M
+ H+), 345.1038; found, 345.1042.
2,4-Dimethoxy-N-methyl-N-[4-(2,4,6-trimethylphenyl)thiazol-
2-yl]benzamide (26) and 2,4-Dimethoxy-N-[3-methyl-4-(2,4,6-
trimethylphenyl)-3H-thiazol-(2Z)-ylidene]benzamide (27). To a
cooled mixture of NaH (100 mg, 60% in oil, 2.5 mmol) in THF (5
mL) was added a solution of compound INH2 (140 mg, 0.37 mmol)
in THF (5 mL) dropwise. The mixture was warmed up to room
temperature and stirred for 30 min. After that, the mixture was
cooled to 0 °C again and MeI (1.0 mL, 16.1 mmol) was added
dropwise. Then the mixture was warmed up to room temperature
and stirred for 2 h. Then H2O (2 mL) was added to quench the
reaction and the mixture was diluted with H2O (30 mL). The
mixture was extracted with CH2Cl2 (3 × 30 mL). The combined
organic phases were dried with anhydrous Na2SO4. After removal
2-Hydroxy-4-methoxy-N-[4-(2,4,6-trimethylphenyl)thiazol-2-
yl]benzamide (32) and 2,4-Dihydroxy-N-[4-(2,4,6-trimethylphe-
nyl)thiazol-2-yl]benzamide (33). To a 0 °C solution of INH2 (148
mg, 0.39 mmol) in CH2Cl2 (2.5 mL) was added BBr3 (2.5 mL, 1
M in CH2Cl2, 2.5 mmol) dropwise. The mixture was warmed up
to room temperature and stirred for 3 h. Then MeOH (1 mL) was
added and all the solvent was removed in vacuo. The procedure of
addition of MeOH (1 mL) was repeated four times. The residue
was purified by silica gel chromatography (hexane/EtOAc ) 4:1)
to give compound 32 (less polar, 28 mg, 20%) as a solid and
compound 33 (more polar, 88 mg, 64%) as a solid. Compound 32:
1
mp 206-207 °C; H NMR (500 MHz, CDCl3) δ 11.83 (s, 2H),

1766 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Qiu et al.
7.47 (s, 1H), 6.75 (s, 1H), 6.68 (s, 2H), 6.33 (dd, J ) 2.0, 2.0 Hz,
1H), 6.22 (s, 1H), 3.78 (s, 3H), 2.22 (s, 3H), 1.98 (s, 6H); 13C NMR
(125 MHz, CDCl3) δ 167.15, 165.02, 162.51, 159.27, 147.98,
138.19, 137.04, 130.88, 129.57, 128.34, 111.62, 107.47, 106.91,
101.54, 55.61, 21.16, 20.51; MS (ESI) m/z 369 (M + H+); HRMS
calcd for C20H20N2O3SNa (M + Na+), 391.1092, found, 325.1082.
Compound 33: mp 236.5-237.5 °C; 1H NMR (400 MHz, DMSO-
d6) δ 11.83 (s, 2H), 10.31 (s, 1H), 7.93 (d, J ) 6.4 Hz, 1H), 6.98
(s, 1H), 6.91 (s, 2H), 6.42 (d, J ) 6.4 Hz, 2H), 2.25 (s, 3H), 2.05
(s, 6H); 13C NMR (100.5 MHz, DMSO-d6) δ 164.33, 163.08,
159.88, 156.88, 148.24, 136.62, 132.48, 131.85, 127.89, 111.16,
108.28, 107.38, 102.74, 20.69, 20.04; MS (ESI) m/z 355 (M +
H+); HRMS calcd for C19H18N2O3SNa (M + Na+), 377.0936,
found, 377.0932.
128.5, 128.2, 127.1, 21.5, 21.3. MS (ESI) m/z 292 (M + H+).
HRMS calcd for C18H17N2O2 (M + H+), 293.1290; found,
293.1298.
N-[4-(2,4-Dimethylphenyl)thiazol-2-yl]thiobenzamide (40).
The mixture of INH1 (152 mg, 0.49 mmol) and Lawesson reagent
(210 mg, 0.49 mmol) in toluene (10 mL) was heated to reflux for
2 h. After that, the mixture was cooled to room temperature and
all the solvent was removed in vacuo. The resultant residue was
purified by silica gel chromatography (hexane/EtOAc ) 20:1) to
give compound 40 (126 mg, 79%) as a solid; mp 161.5-162.5 °C.
1H NMR (400 MHz, CDCl3) δ 12.37 (br, 1H), 7.68 (d, J ) 7.6
Hz, 2H), 7.44 (t, J ) 7.2 Hz, 1H), 7.30 (t, J ) 7.6 Hz, 2H), 7.20
(d, J ) 8.0 Hz, 1H), 6.97-6.93 (m, 2H), 6.88 (s, 1H), 2.34 (s,
3H), 2.29 (s, 3H). 13C NMR (100.5 MHz, CDCl3) δ 195.0, 160.5,
149.6, 141.0, 138.1, 135.7, 131.8, 131.8, 130.9, 129.6, 128.6, 127.3,
126.8, 122.2, 109.7, 21.3, 21.0. MS (ESI) m/z 325 (M + H+), 347
(M + Na+). HRMS calcd for C18H16N2S2Na (M + Na+), 347.0653;
found, 347.0644.
4-(2-Methoxy-4,6-dimethylphenyl)thiazol-2-ylamine (35). To
a solution of compound 34 (500 mg, 3.05 mmol) in acetone (30
mL) was added K2CO3 (7.6 g, 55 mmol) and Me2SO4 (4.5 mL,
47.5 mmol). The resultant mixture was heated to reflux for 4 h.
Then the mixture was cooled to room temperature and NaOH (2N,
100 mL) was added. The whole mixture was extracted with CH2Cl2
(3 × 60 mL), and the combined organic phases were dried over
anhydrous Na2SO4. Removal of all the solvent in vacuo gave a
residue, which was dissolved in dioxane (8 mL). To the resultant
solution, HBr (0.6 mL, 5.3 mmol) and tBuOOH (0.6 mL, 5.0-6.0
M in hexane, ∼3.4 mmol) were added subsequently. The mixture
was refluxed overnight. After being cooled to room temperature,
all the solvent was removed in vacuo to give a residue. The residue
was dissolved in H2O (20 mL), and the whole mixture was extracted
with CH2Cl2 (3 × 60 mL). The combined organic layers were dried
over anhydrous Na2SO4. All the solvent was removed in vacuo,
and the resultant residue was purified by silica gel chromatography
(hexane/EtOAc ) 100:1) to give a compound (169 mg) as a oil.
This oil (169 mg) was dissolved in EtOH (4 mL), and thiourea (59
mg, 0.78 mmol) was added. The resultant mixture was heated to
reflux for 1 h. After being cooled to room temperature, all the
solvent was removed in vacuo to provide a residue. To this residue,
saturated aqueous NaHCO3 (15 mL) was added and the mixture
was extracted with CH2Cl2 (3 × 15 mL). The combined organic
layers were dried over anhydrous Na2SO4. All the solvent was
removed in vacuo, and the resultant residue was purified by silica
gel chromatography (hexane/EtOAc ) 2:1 to 1:1) to give compound
Acknowledgment. This work is supported by a NIH grant
to W.-H. Lee (CA107568), a postdoctoral-multidisciplinary
fellowship to X.-L. Qiu (W81XWH-06-1-0418) from the U.S.
Department of Defense Congressionally Directed Medical
Research Programs (DOD CDMRP), a postdoctoral fellowship
to G. Wu (PDF0600907) from the Susan Komen Breast Cancer
Foundation, a postdoctoral fellowship to J. Zhu (#11FB-0117)
from the California Breast Cancer Research Program, and a
postdoctoral fellowship to L. Zhou (W81XWH-04-1-0470) from
U.S. DOD CDMRP.
Supporting Information Available: Analytic data of compounds
4, 8, 10, 12, 15, 17, 19, 25, and 36-37. Detailed procedure for
preparation of compound-conjugated matrix. This material is
available free of charge via the Internet at http://pubs.acs.org.
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1
35 (140 mg, 20%) as a solid. H NMR (400 MHz, CDCl3) δ 6.68
(t, J ) 0.8 Hz, 1H), 6.60 (s, 1H), 6.35 (s, 1H), 5.05 (s, 2H), 3.74
(s, 3H), 2.34 (s, 3H), 2.17 (s, 3H). 13C NMR (100.5 MHz, CDCl3)
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saturated NaHCO3 was added to make the mixture base (PH )
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gel chromatography (hexane/EtOAc ) 3:1) to afford the desired
amine (100 mg) as a solid. A cooled solution of the above solid
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added to a solution of BzCl (100 mg, 0.71 mmol) in CH2Cl2 (1
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and stirred for 40 min. After that, the mixture was poured into H2O
(20 mL) and the resultant mixture was extracted with CH2Cl2 (3 ×
20 mL). The combined organic phases were dried with anhydrous
Na2SO4. After removal of all the solvent, the residue was purified
by silica gel chromatography (hexane/EtOAc ) 8:1) to give the
compound 39 (92 mg) as a solid; mp 137-138 °C. 1H NMR (400
MHz, CDCl3) δ 10.77 (br, 1H), 7.88 (s, 2H), 7.55 (s, 1H), 7.46 (s,
2H), 7.28 (s, 2H), 7.06-7.10 (m, 2H), 2.39 (s, 3H), 2.33 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 138.6, 135.7, 132.8, 131.9, 128.8,

Synthesis and Biological EValuation of INH Analogues
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