Discovery of 7-(4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N- hydroxyheptanamide (CUDC-101) as a potent multi-acting HDAC, EGFR, and HER2 inhibitor for the treatment of cancer

Article abstract of DOI:10.1021/jm901453q

By incorporating histone deacetylase (HDAC) inhibitory functionality into the pharmacophore of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) inhibitors, we synthesized a novel series of compounds with potent, multiacting HDAC, EGFR, and HER2 inhibition and identified 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N- hydroxyheptanamide 8 (CUDC-101) as a drug candidate, which is now in clinical development. 8 displays potent in vitro inhibitory activity against HDAC, EGFR, and HER2 with an IC₅₀ of 4.4, 2.4, and 15.7 nM, respectively. In most tumor cell lines tested, 8 exhibits efficient antiproliferative activity with greater potency than vorinostat (SAHA), erlotinib, lapatinib, and combinations of vorinostat/erlotinib and vorinostat/lapatinib. In vivo, 8 promotes tumor regression or inhibition in various cancer xenograft models including nonsmall cell lung cancer (NSCLC), liver, breast, head and neck, colon, and pancreatic cancers. These results suggest that a single compound that simultaneously inhibits HDAC, EGFR, and HER2 may offer greater therapeutic benefits in cancer over single-acting agents through the interference with multiple pathways and potential synergy among HDAC and EGFR/HER2 inhibitors.

Full text of DOI:10.1021/jm901453q

2000 J. Med. Chem. 2010, 53, 2000–2009
DOI: 10.1021/jm901453q
Discovery of 7-(4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide
(CUDC-101) as a Potent Multi-Acting HDAC, EGFR, and HER2 Inhibitor for the Treatment of
Cancer
Xiong Cai,* Hai-Xiao Zhai, Jing Wang, Jeffrey Forrester, Hui Qu, Ling Yin, Cheng-Jung Lai, Rudi Bao, and Changgeng Qian
Curis Inc., 45 Moulton Street, Cambridge, Massachusetts 02138
Received October 1, 2009
By incorporating histone deacetylase (HDAC) inhibitory functionality into the pharmacophore of the
epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)
inhibitors, we synthesized a novel series of compounds with potent, multiacting HDAC, EGFR, and
HER2 inhibition and identified 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydro-
xyheptanamide 8 (CUDC-101) as a drug candidate, which is now in clinical development. 8 displays
potent in vitro inhibitory activity against HDAC, EGFR, and HER2 with an IC50 of 4.4, 2.4, and 15.7
nM, respectively. In most tumor cell lines tested, 8 exhibits efficient antiproliferative activity with
greater potency than vorinostat (SAHA), erlotinib, lapatinib, and combinations of vorinostat/erlotinib
and vorinostat/lapatinib. In vivo, 8 promotes tumor regression or inhibition in various cancer xenograft
models including nonsmall cell lung cancer (NSCLC), liver, breast, head and neck, colon, and
pancreatic cancers. These results suggest that a single compound that simultaneously inhibits HDAC,
EGFR, and HER2 may offer greater therapeutic benefits in cancer over single-acting agents through the
interference with multiple pathways and potential synergy among HDAC and EGFR/HER2 inhibitors.
Introduction
HDAC2, HDAC3, and HDAC8) and II (HDAC4, HDAC5,
HDAC6, HDAC7, HDAC9, and HDAC10) zinc-containing
hydrolases. HDAC inhibitors can impact a variety of cell
functions by blocking the deacetylation of histone or non-
histone proteins, such as HSP90 and tubulin, causing cell cycle
arrest, differentiation, and/or apoptosis.13 The HDAC inhi-
bitor vorinostat (SAHA) (Chart 1) is an FDA-approved drug
for the treatment of cutaneous T-cell lymphoma (CTCL).14
HDAC inhibitors have also been shown to synergize with
other agents, including RTK inhibitors, to suppress prolifera-
tion and induce apoptosis in tumor cells.15-21 In our own
study, two reference compounds, vorinostat and erlotinib, were
used to achieve HDAC and EGFR inhibition, respectively, and
a well-established mathematical model for studying multidrug
interactions22 was applied to assess whether there is a synergis-
tic effect between HDAC and RTK inhibition.20 Using this
mathematical model, IC50s of growth inhibition of several
cancer cells treated with vorinostat, erlotinib, or their combina-
tion were compared, and combination indices were calculated
to interpret the effect of concurrent inhibition of the HDAC
and RTK targets. Our results show that cotreatment with
vorinostat and erlotinib (1:1 ratio) results in greater growth
inhibition and with combination indices well below 1, indicat-
ing considerable synergy between the inhibition of these two
targets (Supporting Information Table S1). We also carried out
similar studies by applying varying ratios of vorinostat and
erlotinib. In all cases, the combination indices were calculated
to be well below 1,20 indicating that the inhibition between these
two targets is synergistic and that this synergy is not affected by
the specific dosing ratio. This result suggests that a single
molecule that simultaneously inhibits HDAC and RTK acti-
vities could act synergistically in treated cancer cells. With
Thehumanepidermal growthfactorreceptor(HER) family
of receptor tyrosine kinases (RTKsa) is recognized as a key
mediator of cancer progression.1-4 The HER tyrosine kinase
family consists of four structurally related cellular receptors:
the epidermal growth factor receptor (EGFR; HER1), HER2
(ErbB2), HER3 (ErbB3), and HER4.2,5 The EGFR inhibitors
erlotinib and gefitinib as well as the dual EGFR/HER2
inhibitor lapatinib (Chart 1) are FDA-approved cancer drugs
that are effective against multiple solid tumor cancers.6 How-
ever, their efficacy is restricted to a small subset of patients due
tomolecularheterogeneity amongand withintumors.7,8 Their
effectiveness is also limited by the drug resistance that fre-
quently emerges following treatment.9,10
To overcome the low response rate and acquired resistance
to RTK inhibitors, a number of strategies have been tested,
including combination therapies and multitargeted inhibitors
to inhibit multiple pathogenic pathways.11 One particularly
promising approach is the modulation of RTK pathways by
the inhibition of histone deacetylases (HDACs). HDACs
comprise a family of 18 genes in humans and are divided into
four classes.12 Among them are the Class I (HDAC1,
*To whom correspondence should be addressed. Phone: 617-503-
6656. Fax: 617-503-6501. E-mail: xcai@curis.com.
a Abbreviations: HDAC(s), histone deacetylase(s); RTKs, receptor
tyrosine kinases; EGFR, epidermal growth factor receptor; HER, hu-
man epidermal growth factor receptor; HER2, human epidermal growth
factor receptor 2; NSCLC, nonsmall cell lung cancer; HNSCC, head and
neck squamous cell carcinoma; CTCL, cutaneous T-cell lymphoma;
SAR, structure-activity relationship; HDLP, HDAC like protein; PK,
pharmacokinetics; PD, pharmacodynamics; MTD, maximum tolerated
dose; PDB, protein data bank.
r
pubs.acs.org/jmc
Published on Web 02/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2001
Figure 1. Design of multitargeted EGFR/HER2/HDAC inhibitors.
Chart 1. Structure of Known Inhibitors
adenine region of the ATP binding site and forming two hydro-
gen bonds at N1 and N3, the phenyl-amino group occupying the
hydrophobic pocket, and the two methoxy-ethoxy groups at
C-6 and C-7 of the quinazoline ring sticking out of the recep-
tor.24,27 The cocrystal structure of trichostatin A (TSA) or
vorinostat with HDLP (an HDAC-like protein) reveals that
both compounds bind to HDLP with their large hydrophobic
groups (phenyl) binding to the hydrophobic part of the enzyme,
˚
the aliphatic chain sitting in the 11 A tube-like channel through
multiple contacts with hydrophobic residues, and the terminal
hydroxamic acid interacting with zinc at the active site to disrupt
enzyme activity.28,29 Since the methoxy-ethoxy groups extend
outside of the EGFR binding pocket and are flexible, we
reasoned that the modification of these side chains should not
dramatically affect binding to EGFR. Through careful analysis
of the ligand-protein interactions, we deduced that a group
containing a side-chain of a certain length that terminates with a
hydroxamic acid is essential for HDAC binding and that such a
group is well-suited to be incorporated into the quinazoline-
based EGFR/HER2 inhibitors. We predicted that the best place
to introduce hydroxamic acid functionality without sacrificing
EGFR/HER2 binding affinity is the site in which the methoxy-
ethoxy groups of erlotinib are located (C-6 or C-7 of the
quinazoline ring). We also predicted that the hydrophobic
pocket within the HDAC dimeric interface should be large
enough to accommodate the phenylaminoquinazoline back-
bone of EGFR/HER2 inhibitors, and that the incorporated side
chain with the terminal hydroxamic acid should be able to reach
the active site and interact with zinc as required to disrupt the
HDAC enzyme activity. Therefore, to obtain and optimize
HDAC, EGFR, and HER2 inhibitory activities in a single
molecule, we incorporated hydroxamic acid functionality into
the quinazoline pharmacophore of EGFR/HER2 inhibitors
with a proper spacer, which served as a bridge to properly
display HDAC and EGFR/HER2 inhibitory pharmacophores
(Figure 1). This novel design strategy proved to be highly
effective in producing single molecules with potent multiacting
HDAC/EGFR/HER2 inhibition and low molecular weights.
combined inhibitory activities and synergistic effects, such a
multiacting single molecule may not only enhance drug efficacy
but also overcome the current clinical limitations of EGFR/
HER2 inhibitors, such as poor overall response rates and the
rapid emergence of drug resistance. Unlike combinations of
two or more EGFR/HER2 and HDAC inhibitors, a single
agent with EGFR/HER2 and HDAC inhibitory activities
offers key advantages, including concurrent pharmacokinetics,
minimized off-target adverse effects, and drug-drug interac-
tions caused by multiple agents, as well as increased patient
compliance and reduced drug cost.
Investigating the structural requirements of EGFR/HER2
and HDAC inhibitors led us to design and synthesize a series of
novel, potent multitargeted inhibitors. Here, we describe the
design, synthesis, and structure-activity relationship (SAR) of
these novel compounds and the identification of 8(CUDC-101)
as a clinical candidate currently in phase I clinical trials.
Compound Design
Several EGFR, HER2, and HDAC inhibitors have been
reported.23,24 These studies reveal hydroxamic acid as a broad
class of HDAC inhibitors with high affinity for HDACs that
depend on zinc chelation for their activity.25 Additionally,
quinazolines are a well-characterized scaffold of kinase inhibi-
tors, including EGFR and HER2 inhibitors.26 The X-ray
cocrystal structures of EGFR with erlotinib and HDAC with
vorinostat have been reported.24,27,28 Our compound design
strategy was to fully analyze the structure requirements of these
ligand-protein interactions and design our own novel multi-
targeted inhibitors through a structure-based rational drug
design approach. It has been reported that erlotinib binds to
the EGFR binding domain with quinazoline occupying the
Chemistry
The preparation of analogues with various chain lengths and
aniline substitution (1-11) is shown in Scheme 1. Compounds
31-35 were prepared through the coupling of anilines 27-30
with either quinazolines 2530 or 26.31 Hydrolysis of the acetyl
group on C-6 of compounds 31-35 using lithium hydroxide
gave corresponding C6-OH quinazolines 36-40. Alkylation of
the C6-OH of 36-40 with various chain lengths of ethyl or methyl
bromoalkanoate yielded ester products 41-51. Conversion

2002 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Cai et al.
Scheme 1a
a (a) Isopropanol, reflux; (b) LiOH, CH3OH, H2O; (c) ethyl or methyl bromoalkanoate, K2CO3, 40 °C; (d) NH2OH, CH3OH, 0 °C to rt.
Scheme 2a
a (a) CH3I or 2-methyoxyethyl-4-methylbenzenesulfonate, K2CO3, DMF; (b) ethyl 7-bromoheptanoate, K2CO3, DMF, 60 °C; (c) HNO3, HOAc, 20 °C;
(d) Fe, HCl, EtOH, H2O, reflux; (e) HCONH2, HCOONH4, 180 °C; (f) PCl3, reflux; (g) 27 or 28, isopropanol, reflux; (h) NH2OH, CH3OH, 0 °C to rt.
of esters 41-51 to hydroxamic acids using freshly prepared
hydroxylamine yielded final compounds 1-11. Alternatively,
these compounds may be prepared by installing the ester side
chain prior to constructing the quinazoline core as shown in
Scheme 2. This process is more suited to the synthesis of certain
analogues and is easy to scale up. Compound 52 was treated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2003
Scheme 3a
a (a) Na, CH3OH, sealed tube; (b) POCl3, reflux; (c) 27, isopropanol, reflux; (d) Fe, HCl, EtOH, H2O, reflux; (e) methyl 5-chloro-5-oxopentanoate,
Et3N, CH2Cl2; (f) NH2OH, CH3OH, 0 °C to rt.
Scheme 4a
a (a) NaNO2, H2SO4, AcOH, KI; (b) thiobenzoic acid, 1,10-phenathroline, CuI, DIPEA, toluene, 110 °C; (c) ethyl 7-bromoheptanoate, K2CO3,
DMF, 50 °C; (d) KMnO4, CH3CN, H2O, 65 °C; (e) NH2OH, CH3OH, 0 °C to rt.
with iodomethane or 2-methoxyethyl-4-methylbenzenesulfo-
nate to give corresponding intermediates 53 and 54. Alkylation
of 53 and 54 with ethyl 7-bromoheptanoate gave intermediates
55 and 56, which were then treated with fuming nitric acid to
introduce a nitro group onto the phenyl ring to yield 57 and 58.
Reduction of nitro compounds 57 and 58 with iron/hydrochlo-
ric acid gave corresponding amines 59 and 60. Cyclization of 59
and 60 using formamide and ammonium formate gave quinazo-
line intermediates 61 and 62. Chlorination of 61 and 62 using
phosphoryl chloride yielded the desired intermediates 63 and 64.
The coupling of 63 and 64 with 27 and 28 gave corresponding
esters 48 and 65, which were finally reacted with freshly prepared
hydroxylamine to form final product 8 and 12, respectively.
The preparation of amide linker compound 13 is shown in
Scheme 3. Conversion of the chloro group of 66 to the
methoxy group using in situ formed sodium methoxide gave
the corresponding methoxy compound 67. Treatment of 67
with phosphoryl chloride gave the chloride 68. Coupling of 68
with 27 gave nitro compound 69. Reduction of 69 with iron/
hydrochloric acid gave the corresponding amine 70. Coupling
of70withmethyl5-chloro-5-oxopentanoateyieldedamide71.
Final compound 13 was obtained by the treatment of 71 with
freshly prepared hydroxylamine.
The sulfur and sulfone-linked side chain compounds 14 and
15 were synthesized according to Scheme 4. Conversion of
aminogroup of70toiodogroup was achievedby first forming

2004 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Cai et al.
Scheme 5a
a (a) Isopropanol, 60 °C; (b) ethyl 7-bromoheptanoate, K2CO3, DMF, 60 °C; (c) NH2OH, CH3OH, 0 °C to rt.
a diazonium salt and subsequently reacting it with cuprous
iodide to yield compound 72. Treatment of compound 72 with
thiobenzoic acid gave compound 73. Hydrolysis of the thioe-
ster 73 using potassium carbonate generated free thio com-
pound, which was then reacted with ethyl 7-bromoheptanoate
to give compound 74. Oxidation of 74 with KMNO4 gave the
sulfone intermediate 75. Final hydroxamic acid compounds
14 and 15 were obtained by reacting esters 74 and 75 with
freshly prepared hydroxylamine, respectively.
The synthesis of the benzylamine series of analogues 16-22
is shown in Scheme 5. Compounds 83-89 were synthesized by
the coupling of compound 25 with commercially available
amines 76-82. In the reaction condition, the C-6 acetyl group
was cleaved to form C-6 OH. Alkylation of the C-6 OH of
83-89 with ethyl 7-bromoheptanoate gave compounds
90-96. Final hydroxamic acids 16-22 were prepared through
the conversion of esters 90-96 using freshly prepared hydro-
xylamine.
The preparationofposition analogues23and 24 isshownin
Scheme 6. Alkylation of the C-4 OH of 52 with ethyl 7-
bromoheptanoate gave compound 97. Methylation of the
C-3 OH of 97 using iodomethane gave compound 98. Intro-
duction of a nitro group onto compound 98 to form com-
pound 99 was accomplished by treating98 with fuming HNO3
and AcOH. Reduction of 99 using iron/hydrochloric acid
gave its corresponding amine 100. Cyclization of 100 using
formamide and ammonium formate gave the quinazoline
intermediate 101. Chlorination of 101 using phosphoryl
chloride gave intermediate 102. Coupling of 102 with anilines
27 and 28 yielded compounds 103 and 104, respectively.
Treatment of 103 and 104 with freshly prepared hydroxyla-
mine gave the final position analogues 23 and 24.
ring substitution analogues, benzylamine analogues, and
position analogues. The in vitro enzymatic inhibition assay
results of these four types of compounds are summarized in
Tables 1-4.
Chain Length and Phenyl Ring Substitution Analogues
(1-10, Table 1). Our data suggest that increasing the length
of the hydroxamic acid side chain increases HDAC inhibi-
tory activity. The short chain analogues (compounds 1 and
2) have weak HDAC inhibitory activity. Appreciable HDAC
inhibition was observed only when the carbon chain length
between the quinazoline C-6 oxygen and the hydroxamic
acid carbonyl group reached four carbons (n = 3)
(compounds 3 and 4). The HDAC inhibitory activity further
increased with a carbon chain length of five carbons (n = 4)
(compounds 5 and 6). The optimal carbon chain length is six
(n = 5), which gives an IC50 of HDAC inhibition in the
single-digit nanomolar range (compounds 7, 8, and 9). Small
substituents on the phenyl ring do not affect the HDAC
inhibitory activity, but a large group, such as 3-fluorophe-
nylmethoxy decreases potency of HDAC inhibition (10,
IC50 = 58.1 nM). These results suggest that the length of
the hydroxamic acid side chain is important for HDAC
inhibition. In contrast, EGFR and HER2 inhibition were
largely unaffected by the change in carbon chain length and
phenyl ring substitution (EGFR inhibition IC50=2.4 nM-
15.1 nM; HER2 inhibition IC50=10.1 nM - 38.4 nM) with
the exception of compound 9, which had relatively low
inhibitory activities against EGFR (IC50 = 28.8 nM) and
HER2 (IC50 = 76.5 nM). These data suggest that the side
chain on the quinazoline ring and substitution on the phenyl
ring are not critical for EGFR/HER2 inhibition, and that the
optimal carbon chain length to achieve maximum inhibitory
activities against HDAC and EGFR/HER2 is six carbons
(n = 5). Among four six-carbon chain (n = 5) compounds
(7, 8, 9, 10), 7 and 8 are more promising, with potent
inhibitory activities against all three targets, HDAC, EGFR,
and HER2. Compared with reference compounds, 7 and 8
inhibit HDAC and EGFR activity with higher potency
than vorinostat and erlotinib (or lapatinib), respectively.
Results and Discussion
Tostudy structure-activityrelationships, compoundswere
evaluated in EGFR and HER2 kinase activity assays, as well
as an HDAC enzyme assay. For the purpose of SAR analysis,
all compounds were assigned to four groups including chain
length and phenyl ring substitution analogues, quinazoline

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2005
Scheme 6a
a (a) Ethyl 7-bromoheptanoate, K2CO3, DMF; (b) CH3I, K2CO3, DMF, 80 °C; (c) HNO3, AcOH, 20 °C; (d) Fe, HCl, EtOH, reflux; (e) HCONH2,
HCOONH4, 180 °C; (f) POCl3, reflux; (g) 27 or 28, isopropanol, reflux; (h) NH2OH, CH3OH, 0 °C to rt.
Moreover, 7 and 8 exhibit greater or equal potency of HER2
inhibition compared to erlotinib or lapatinib, respectively.
Quinazoline Ring Substitution Analogues (7, 11-15,
Table 2). Keeping the optimal carbon chain length of six
(n = 5), our next step was to examine the influence of C-7
substitution (R3) and the C-6 oxygen atom of the quinazoline
ring on inhibitory activities. When X is an oxygen, varying
R3 does not affect HDAC inhibition (all such compounds
displayed a single-digit nanomolar IC50), while the potency
of EGFR or HER2 inhibition has the following order:
OCH3 (compound 7) > OCH2CH2OCH3 (compound 12)>H
(compound 11). When R3 is fixed to be OCH3, the linker X
does affect HDAC, EGFR, and HER2 inhibitory activities.
For HDAC inhibition, an oxygen linker (compound 7) is more
potent than an amide linker (compound 13), which is more
potent than a sulfur linker (compound 14), while a sulfone
linker 15 has lowest potency. For EGFR and HER2 inhibition,
7, 13, and14 have similar potency, but sulfone linker 15 showed
reduced potency in EGFR inhibition (IC50 = 15.4 nM) and
greatly reduced HER2 inhibition (IC50>2000 nM).
Benzylamine Analogues (16-22, Table 3). Unlike the ani-
line analogues discussed earlier, the benzylamine series dis-
rupted the conjugation of the quinazoline ring with the phenyl
ring. The HDAC inhibition drops to half when changing ani-
line (7, IC50=6.5 nM) to benzylamine (16, IC50=11.3 nM), but
is restored when R1 and R2 are both H (17, IC50=4.2 nM) or R1
is F and R2 is H (18, IC50=5.6 nM). When a methyl group is
added onto the benzylamine methelene of compound 17 to
create a pair of enantiomers, the S isomer shows a 6-fold drop
(19, IC50=25.7 nM) and the R isomer shows a 23-fold drop (20,
IC50=98.4 nM) in HDAC inhibition compared to 17. When
changing H on R1 of compound 20 to F (21) or Cl (22), an
additional 1.5- to 2-fold drop of HDAC inhibition is observed
compared to compound 20. For EGFR and HER2 inhibition,
the replacement of aniline (7) with benzylamine (16) resulted in
a more than 20-fold drop in EGFR and HER2 inhibition.
Unlike HDAC inhibition, the R isomers (20, 21, and 22) all
have much higher EGFR and HER2 inhibitory activities than
the S-isomer (19), which shows very weak inhibitory activity
against EGFR (IC50=5077 nM) and HER2 (IC50 >2000 nM).
Among the three R isomers, the potency of EGFR and HER2
inhibition is related to the phenyl ring substitution and has the
following order: unsubstituted 20 > monofluoro-substituted
21>monochloro-substituted 22. Overall, the changing of an
aniline to a benzylamine resulted in less potent compounds.
Position Analogues (7, 8, 23, 24, Table 4). To investigate
the effect of the hydroxamic acid side chain position on
inhibitory activities, we synthesized 23 (a position analogue
of 7) and 24 (a position analogue of 8). Compound 23 showed
a 10-fold drop in HDAC inhibition, a 2.2-fold drop in EGFR
inhibition, and a 3.5-fold drop in HER2 inhibition compared
to 7. Similarly, compound 24 showed a 8-fold drop in HDAC
inhibition, a 3.4-fold drop in EGFR inhibition, and a 3.3-fold
drop in HER2 inhibition compared to compound 8.
The SAR study led to the identification of compound 7 and 8
as the most potent compounds in inhibiting HDAC, EGFR,
and HER2. In addition, these two compounds display balanced
inhibitory activities against all three targets, a desirable feature

2006 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Cai et al.
Table 1. Structure and In Vitro Activity of Chain Length and Ring Substitution Analogues
a Synthesized in-house. b HDAC inhibitory activity was determined using the Biomol Color de Lys system. c EGFR and HER2 kinase activity was
measured using HTScan EGF receptor and HER2 kinase assay kits (Cell Signaling Technology).
Table 2. Structure and In Vitro Activity of Quinazoline Ring Substitu-
tion Analogues
Table 3. Structure and In Vitro Activity of Right-Side Phenyl Ring
Analogues
IC50 (nM) in Enzyme Assays
compd
Y
R1 R2
HDACa
EGFRb
HER2b
IC50 (nM) in enzyme assays
7
direct bond
CH2
CH2
F
Cl
Cl
H
H
H
H
H
H
6.5
11.3
4.2
3.1
72.0
19.0
478.2
225.2
295.4
>2000
27.8
compd
X
R3
HDACa EGFRb
HER2b
16
17
18
19
20
21
22
F
7
O
O
O
CH3O-
H-
CH3OCH2CH2O-
6.5
4.4
3.1
84.2
10.4
2.1
19.0
43.5
H
F
99.4
11
12
13
14
15
CH2
5.6
268.0
5077.0
12.8
8.4
24.0
20.1
(S)-CHCH3
(R)-CHCH3
(R)-CHCH3
H
H
F
25.7
98.4
165.3
188.0
CONH CH3O-
15.3
39.6
121.7
S
SO2
CH3O-
CH3O-
1.99
15.4
24.2
125.6
614.0
175.0
556.9
>2000
(R)-CHCH3 Cl
a HDAC inhibitory activity was determined using the Biomol Color
de Lys system. b EGFR and HER2 kinase activity was measured using
HTScan EGF receptor and HER2 kinase assay kits (Cell Signaling
Technology).
a HDAC inhibitory activity was determined using the Biomol Color de
Lys system. b EGFR and HER2 kinase activity was measured using HTScan
EGF receptor and HER2 kinase assay kits (Cell Signaling Technology).
for a multitargeted inhibitor. We further evaluated these two
compounds, along with other selected compounds from the
above SAR studies, for their antiproliferative effects on tumor
cells. In NSCLC, liver, breast, and pancreatic cancer cell lines,
compound 8 consistently exhibits greater potency than com-
pound 7 (Table 5). Compound 8 was further evaluated in a
series of in vitro cell-based assays, selectivity assays, in vivo effi-
cacy models and PK/PD, metabolism, and toxicology studies.20
Here, we describe the salient properties of this compound.
In vitro, 8 inhibits both class I and class II HDACs (Sup-
porting Information Table S2), but not class III, Sir-type
HDACs (data not shown). 8 displays broad antiproliferative

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2007
Table 4. Structure and In Vitro Activity of Side Chain Position Analogues
IC50 (nM) in enzyme assays
compd
R1
R2
Cl
HDACa
EGFRb
HER2b
7
F
6.5
66.6
4.4
3.1
7.0
2.4
8.2
19.0
71.2
15.7
51.6
23
8
F
Cl
CꢀCH
CꢀCH
H
H
24
34.1
a HDAC inhibitory activity was determined using the Biomol Color de Lys system. b EGFR and HER2 kinase activity was measured using HTScan
EGF receptor and HER2 kinase assay kits (Cell Signaling Technology).
Table 5. Inhibition of Proliferation in Selected Human Cancer Cell Lines by Compound 8, 7, and Reference Compounds
IC50 (uM)a
cancer type
cell line
HCC827
H358
8
7
vorinostat
erlotinib
vorinostat þ erlotinib
2.3
lapatinib
vorinostat þ lapatinib
NSCLC
NSCLC
NSCLC
Liver
0.60
0.40
0.70
0.13
0.23
0.22
0.80
0.27
0.55
0.10
0.04
0.93
0.71
0.90
0.29
0.60
0.33
1.73
0.38
1.10
0.37
0.17
1.80
2.50
1.70
1.66
2.44
3.46
7.30
2.70
2.80
2.11
1.19
7.50
6.00
1.10
1.40
1.95
2.64
3.97
4.40
1.00
2.70
2.00
0.90
H460
8.20
HepG2
Hep3B2
Sk-Hep-1
Capan1
BxPc3
>20
>20
10.37
>20
7.60
6.27
5.49
5.30
1.23
2.36
1.50
Liver
Liver
Pancreatic
Pancreatic
Breast
MCF-7
MDA-MB-231
Sk-Br-3
>20
>20
1.56
6.60
5.40
0.04
2.60
1.80
0.05
Breast
Breast
a Cancer cell lines were plated at 5000 to 10 000 cells per well in 96-well flat-bottomed plates with varying concentrations of compounds. The cells were
incubated with compounds for 72 h in the presence of 0.5% of fetal bovine serum. Growth inhibition was assessed by an adenosine triphosphate (ATP)
content assay using the Perkin-Elmer (Waltham, MA) ATPlite kit.
activity in many human cancer cell types and in most cases has a
higher potency than erlotinib, lapatinib, and combinations of
vorinostat with either erlotinib or lapatinib (Table 5).20 8 is a
potent and selective HDAC, EGFR, and HER2 inhibitor with
only weak inhibition of the following protein kinases (IC50):
KDR (VEGFR2) (849 nM), Src (11000 nM), Lyn (840 nM),
Lck (5910 nM), Abl-1 (2890 nM), FGFR-2 (3430 nM), Flt-3
(1500 nM), and Ret (3200 nM).
In vivo, 8 induces tumor regression in the Hep-G2 liver
cancer model and is more efficacious than erlotinib at its
maximum tolerated dose (MTD)20 and vorinostat at an
equimolar concentration dose (Figure 2, Table 6). 8 is also
highly efficacious in a number of other xenograft models
(Table 6). In the erlotinib-resistant A549 NSCLC xenograft
model, 8 shows potent inhibition of tumor growth. In the
erlotinib-sensitive H358 NSCLC models, 8 inhibits tumor
growth in a dose-dependent manner. 8 causes significant
tumor regression in the lapatinib-resistant, HER2-negative,
EGFR-overexpressing MDA-MB-468 breast cancer model
and the EGFR-overexpressing CAL-27 head and neck squa-
Figure 2. 8, dosed at 120 mg/kg, iv, daily, induced tumor regression
in the Hep-G2 liver cancer model and was more efficacious than
mous cell carcinoma (HNSCC) model. 8 also inhibits tumor
growth in the K-ras mutant HCT116 colorectal and EGFR/
HER2 (neu)-expressing HPAC pancreatic cancer models.
Our studies on the mechanism of action suggest that 8 may
overcome drug resistance via the sustained blockade of
survival pathways. We have demonstrated that besides
EGFR and HER2 inhibition, 8 also suppresses HER3
erlotinib at its maximum tolerated dose (MTD, 25 mg/kg, po, daily)
and vorinostat at an equimolar concentration (72 mg/kg, iv, daily).
expression, Met amplification, and AKT reactivation in
tumor cells.20
In conclusion, compound 8 is a potent HDAC, EGFR,
and HER2 inhibitor with favorable drug-like properties. It

2008 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Cai et al.
Table 6. In Vivo Efficacy of Compound 8 in Mice Human Tumor
Xenograft Models
(45 mL) was stirred at reflux for 3 h. The reaction mixture was
cooled to room temperature and the resultant precipitate was
collected by filtration. The solid was further dried in vacuo to
give the compounds 31-35.
T/C % or
regression (-)b
cancer type
NSCLC
cell line
H358
dosing schedulea
General Procedure for the Synthesis of 36-40. A mixture of
4-phenylamino-quinazoline derivatives (31-35, 3.5 mmol) and
8, 15 mg/kg
8, 30 mg/kg
8, 60 mg/kg
8, 120 mg/kg
41.9
10.3
LiOH H2O (0.5 g, 11.7 mmol) in methanol (100 mL) and H2O
3
6.5
35.9
(100 mL) was stirred at room temperature for 0.5 h. The mixture
was neutralized by the addition of diluted acetic acid. The
resultant precipitate was collected by filtration, washed, and
dried to give compounds 36-40.
A549
breast
MDA-MB468 8, 120 mg/kg
Lapatinib,75 mg/kg
8, 60 mg/kg
-13.7
51.0
35.3
colon
HNSCC
liver
HCT116
CAL-27
HepG2
General Procedure for the Synthesis of 41-51. A mixture of
4-phenylaminoquinazolin-6-ol derivatives (36-40, 1.0 mmol),
appropriate ethyl or methyl bromoalkanoate (1.1 mmol), and
potassium carbonate (323 mg, 2.4 mmol) in DMF (6 mL) was
stirred at 40 °C for 30 min. The reaction mixture was filtered and
the filtrate was concentrated in vacuo. The residue was washed
with diethyl ether and dried to give compounds 41-51.
General Procedure for the Synthesis of 1-24 from Their
Corresponding Esters. Preparation of Fresh Hydroxylamine solu-
tion. A solution of potassium hydroxide (11.2 g, 199.6 mmol)
in methanol (28 mL) was added to a stirred solution of hydro-
xylamine hydrochloride (9.34 g, 134.4 mmol) in methanol (48 mL)
at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. The
resultant precipitate was removed by filtration. The filtrate was
collected to provide free hydroxylamine solution which was stored
in a refrigerator before use.
Preparation of Compounds 1-24. The appropriate esters (11.0
mmol) were added to the above freshly prepared hydroxylamine
solution (30 mL) at 0 °C. The reaction mixture was warmed to
room temperature and stirred at 25 °C for 1 to 24 h until the
reaction was complete (reaction progess was monitored by TLC
or LCMS). The reaction mixture was neutralized with acetic
acid. The formed precipitate was collected by filtration, washed
with water, and dried to give the title compounds. If a precipitate
did not form after neutralization, the solvent was removed to
yield a crude mixture, which was purified by reverse-phase
preparative HPLC to give the title compounds.
EGFR and HER2 Inhibition Assay. EGFR and HER2 kinase
activity were measured using HTScan EGF receptor and HER2
kinase assay kits (Cell Signaling Technology). Briefly, the GST-
EGFR fusion protein was incubated with synthetic biotinylated
peptide substrate and varying concentrations of drugs in the
presence of 400 mM ATP. Phosphorylated substrate was cap-
tured with strapavidin-coated 96-well plates. The level of phos-
phorylation was monitored by antiphospho-tyrosine- and
europium-labeled secondary antibodies (DELFIA, Perkin-
Elmer). The enhancement solution was added at the end of the
assay and enzyme activity was measured in the Wallac Victor II
1420 microplate reader at 615 nM.
HDAC Inhibition Assay. The activities of Class I and II
HDACs were assessed using the Biomol Color de Lys system.
Briefly, HeLa cell nuclear extracts were used as a source of
HDACs. Different concentrations of drugs were added to HeLa
cell nuclear extracts in the presence of a colorimetric artificial
substrate. Developer was added at the end of the assay and
enzyme activity was measured in the Wallac Victor II 1420
microplate reader at 405 nM.
8, 120 mg/kg
8, 120 mg/kg
-36.5
-21.3
50.8
Erlotinib, 25 mg/kg
Vorinostat, 72 mg/kg
8, 120 mg/kg
26.0
35.1
pancreatic HPAC
a Compound 8 was dosed iv, daily, except in the H358 NSCLC model,
where 8 was dosed iv on 7-6-5 (on-off-on) schedule, and in HCT116
colorectal cancer model, 8 was dosed iv on 5-2-5 (on-off-on)
schedule. Lapatinib was dosed po, bid. Erlotinib was dosed po, daily,
and vorinostat was dosed iv, daily. b During each animal study, tumors
were measured with calipers, tumor size determined using the formula:
tumor volume = (length ꢁ width2)/2, and tumor size changes in percent
calculated. In most cases, % T/C values were calculated using the
following formula: % T/C = 100 ꢁ ΔT/ΔC if ΔT>0, where T/C is
the tumor size change ratio in treated animals (T) and control animals
(C), ΔT is the tumor size change in treated animal between last tumor
size measurement and predosing (T0) tumor size measurement, ΔC is the
tumor size change in control animal between last tumor size measure-
ment and predosing tumor size measurement. In cases where tumor
regression occurred (ΔT < 0), however, the following formula was used:
% T/T0 = 100 ꢁ ΔT/T0, where T/T0 is the tumor size change ratio in
treated animal. Values of <42% are considered significant.32
also displays a favorable preclinical safety profile (data not
shown). Its simultaneous blockade of HDAC and RTK
pathways represents a novel approach to cancer therapy
and may provide high efficacy and overcome limitations in
the treatment of certain cancers with RTK inhibitors. There-
fore, compound 8 has advanced to clinical development as a
novel anticancer agent.
Experimental Section
Chemistry. Anhydrous solvents and other solvents were
purchased from commercial suppliers. All other chemicals were
from commercial suppliers and used without further purifica-
tion. 1H NMR spectra were recorded on a Bruker AVANCE III
400 MHz or Varian Mercury-VX-300 300 MHz NMR spectro-
meter at ambient temperature and proton chemical shifts are
relative to TMS as an internal standard. Melting points were
determined on a X-4 micromelting point apparatus in micro-
scope slides and cover glass, or a WRS-1B digital melting point
apparatus in an open capillary tube and are uncorrected (both
melting point apparatuses were manufactured by Shanghai
Precision & Scientific Instrument Co., Ltd.).
LCMS. The purity of the compounds was analyzed by
reversed-phase liquid chromatography and mass spectrometry
(Agilent 6110 quadruple ion trap mass spectrometer or Shimadzu
LCMS-2010EV chromatograph mass spectrometer) with a UV
detector at 214 and 254 nm and electrospray ionization source
(ESI) as described in further detail in the Supporting Informa-
tion. All final compounds (1-24) were assayed by LCMS
methods and showed purity of at least 95%.
Cell Growth, Viability, and Apoptosis Assay. Cancer cell lines
were plated at 5000 to 10 000 cells per well in 96-well flat-
bottomed plates with varying concentrations of compounds.
The cells were incubated with compounds for 72 h in the
presence of 0.5% of fetal bovine serum. Growth inhibition
was assessed by an adenosine triphosphate (ATP) content assay
using the Perkin-Elmer (Waltham, MA) ATPlite kit. Apoptosis
was routinely assessed by measuring the activities of Caspase-3
and -7 using Promega (Madison, WI) Apo-ONE Homogeneous
Assay Kit.
Preparative HPLC. The separation of certain compounds
was accomplished by using reverse-phase preparative HPLC
(Gilson GX-281) with a UV detector at 214 nm as described in
further detail in the Supporting Information.
General Procedure for the Synthesis of 31-35. A mixture of
4-chloroquinazolines derivatives (25-26, 5 mmol) and appro-
priate substituted anilines (27-30, 10 mmol) in isopropanol
Efficacy Studies in Human Cancer Xenograft Model. Four- to
six-week-old female athymic mice (nude nu/nu CD-1, Charles

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2009
River, Wilmington, MA) were inoculated subcutaneously into
the right hind flank region with 1 to 5 ꢁ 106 cells in a medium
suspension of 100-200 μL. For orthotopic implantation of
breast cancer cells, a cell suspension in 100 μL of medium was
injected directly into the mammary fat pads through a 27G
needle. Different doses of 8, standard anticancer agents and
vehicle were administered orally, intraperitoneally, or via tail
vein injection as indicated.
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Acknowledgment. The authors thank Zihong Guo,
Tianhao Wang, Keyou Xue, Hui Liu, Xiaoyue Su, Xiongwen
Yang and Liangbin Jia of Curis chemistry FTE team at
Shanghai ChemPartner Co., Ltd. for conducting chemical
synthesis of compounds, Carmen Pepicelli and Mark Noel of
Curis for helpful discussions and their review of this manu-
script, and Nicole Davis for her assistance in the preparation
of this manuscript.
Supporting Information Available: Details of synthesis and
analytical data, the LCMS method used for the determination of
purity and the HPLC method used for compound separation,
HDAC and RTK inhibition synergy data, HDAC class I and
class II inhibitory activity of 8. This material is available free of
charge via the Internet at http://pubs.acs.org.
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FDA Approval Summary: Vorinostat for Treatment of Advanced