Isoindoline scaffold-based dual inhibitors of HDAC6 and HSP90 suppressing the growth of lung cancer in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2020.112086

This study reports the synthesis of a series of 2-aroylisoindoline hydroxamic acids employing N-benzyl, long alkyl chain and acrylamide units as diverse linkers. In-vitro studies led to the identification of N-benzyl linker-bearing compound (10) and long

Full text of DOI:10.1016/j.ejmech.2020.112086

Journal Pre-proof
Isoindoline scaffold-based dual inhibitors of HDAC6 and HSP90 suppressing the
growth of lung cancer in vitro and in vivo
Ritu Ojha, Kunal Nepali, Chun-Han Chen, Kuo-Hsiang Chuang, Tung-Yun Wu, Tony
Eight Lin, Kai-Cheng Hsu, Min-Wu Chao, Mei-Jung Lai, Mei-Hsiang Lin, Han-Li
Huang, Chao-Di Chang, Shiow-Lin Pan, Mei-Chuan Chen, Jing-Ping Liou
PII:
S0223-5234(20)30053-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112086
EJMECH 112086
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 November 2019
Revised Date: 18 January 2020
Accepted Date: 20 January 2020
Please cite this article as: R. Ojha, K. Nepali, C.-H. Chen, K.-H. Chuang, T.-Y. Wu, T.E. Lin, K.-C.
Hsu, M.-W. Chao, M.-J. Lai, M.-H. Lin, H.-L. Huang, C.-D. Chang, S.-L. Pan, M.-C. Chen, J.-P. Liou,
Isoindoline scaffold-based dual inhibitors of HDAC6 and HSP90 suppressing the growth of lung cancer
in vitro and in vivo, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112086.
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Graphical abstract
Compound 17 downregulated PD-L1 expression in INF-γ treated H1975 lung cancer cells.
Compound 17 in combination with afatinib enhanced antitumor efficacy.

Revised manuscript for Article
Isoindoline scaffold-based dual inhibitors of HDAC6 and HSP90 suppressing
the growth of lung cancer in vitro and in vivo
a,1
a,1
f
c,b
b
Ritu Ojha, Kunal Nepali, Chun-Han Chen, Kuo-Hsiang Chuang, Tung-Yun Wu, Tony
d
d
d
e
a
e
Eight Lin, Kai-Cheng Hsu, Min-Wu Chao, Mei-Jung Lai, Mei-Hsiang Lin, Han-Li Huang,
g
d,g
b,h,*
*,a,e,g
Chao-Di Chang, Shiow-Lin Pan, Mei-Chuan Chen,
Jing-Ping Liou
1
These authors contributed equally to this work
*
To whom correspondence should be addressed. M. C. Chen: (Phone) 886-2-7361661 ext 6184;
(e-mail) mcchen1250@tmu.edu.tw; J. P. Liou: (Phone) 886-2-2736-1661 ext 6130; (e-mail)
jpl@tmu.edu.tw
a
School of Pharmacy, College of Pharmacy, Taipei Medical University.
b
Ph.D. Program in Clinical Drug Development of Herbal Medicine, College of Pharmacy, Taipei
Medical University.
c
Graduate Institute of Pharmacognosy, Taipei Medical University
Graduate Institute of Cancer Molecular Biology and Drug Discovery, College of Medical
.
d
Science and Technology, Taipei Medical University.
e
TMU Biomedical Commercialization Center, Taipei Medical University.
Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical
f
University.
g
Ph.D. Program in Biotechnology Research and Development, College of Pharmacy, Taipei
Medical University.
h
Traditional Herbal Medicine Research Center of Taipei Medical University Hospital, Taipei
1
1031, Taiwan.

Abstract: This study reports the synthesis of a series of 1-aroylisoindoline hydroxamic acids
employing N-benzyl, long alkyl chain and acrylamide units as diverse linkers. In-vitro studies led
to the identification of N-benzyl linker-bearing compound (10) and long chain linker-containing
compound (17) as dual selective HDAC6/HSP90 inhibitors. Compound 17 displays potent
inhibition of HDAC6 isoform (IC = 4.3 nM) and HSP90a inhibition (IC = 46.8 nM) along with
5
0
50
substantial cell growth inhibitory effects with GI = 0.76 µM (lung A549) and GI = 0.52 µM
5
0
50
(lung EGFR resistant H1975). Compound 10 displays potent antiproliferative activity against lung
A549 (GI50 = 0.37 µM) and lung H1975 cell lines (GI50 = 0.13 µM) mediated through selective
HDAC6 inhibition (IC50 = 33.3 nM) and HSP90 inhibition (IC50 = 66 nM). In addition,
compound 17 also modulated the expression of signatory biomarkers associated with HDAC6 and
HSP90 inhibition. In the in vivo efficacy evaluation in human H1975 xenografts, 17 induced
slightly remarkable suppression of tumor growth both in monotherapy as well as the combination
therapy with afatinib (20 mg/kg). Moreover, compound 17 could effectively reduce programmed
death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose dependent manner
suggesting that dual inhibition of HDAC6 and HSP90 can modulate immunosuppressive ability of
tumor area.
Keywords: Isoindoline, programmed death-ligand 1 (PD-L1), Histone deacetylase, Heat shock
protein, lung cancer

1
. Introduction
Non-Small Cell Lung Cancer (NSCLC) accounts for more than 85-90 % of all lung cancers, a
1
-3
disease which is the leading cause of cancer-related mortality. Arising from a complex series of
genetic and epigenetic changes, NSCLC leads to uncontrolled cell growth and metastases and has
4
-5
a poor prognosis. Several chemotherapy regimens have demonstrated efficacy, but the existing
therapies have not been able to obtain satisfactory results in improving survival of NSCLC
3
patients . Often subclassified as molecularly defined oncogene addicted tumors, NSCLC suffers
6
,7
from the development of therapeutic resistance to targeted agents. These factors clearly confirm
the need for exploration of diverse mechanisms to accomplish therapeutic benefits in lung cancer
along with a comprehensive understanding of the molecular heterogeneity in NSCLC that is
necessary to design new anticancer agents.
The last decade has evidenced an exponential increase in the number of investigations on
NSCLC by targeting of histone deacetylase (HDAC) enzymes and heat shock protein 90 (HSP90)
chaperone proteins. HDAC inhibitors induce relief of transcriptional repression in various
8
,9
leukemias and are considered to be a key to epigenetic cancer therapy. Several FDA approved
1
0
11
12
13,14
HDAC inhibitors such as SAHA (1) , PXD101 (2) , LBH589 (3) and Valproic acid (4)
have been extensively investigated in NSCLC at the clinical and the preclinical level. In addition
1
5
to induction of apoptosis in NSCLC or demonstration of synergistic antitumor efficacy in
1
2, 16-19
combination studies with established therapeutics
, HDAC inhibitors have also exhibited
20
potential for sensitizing NSCLC to ionizing radiations. These facts place this therapeutic class in
the forefront of the investigations presently being conducted for NSCLC. Concomitantly HSP90
inhibition has also emerged as a logical strategy in drug design for NSCLC. HSP90 is a chaperone
protein that protects other cellular proteins from degradation by the ubiquitin-proteasome system

21
in conditions of stress . Preclinical HSP90 inhibitors are endowed with significant potential in
2
2
NSCLC, either alone or in combination. In particular, second generation HSP90 inhibitors such
2
3
24
25
26
27
as IPI-504 (5) , BIIB021 (6) , AUY922 (7) , Ganetespib (8) and AT13387 (9) have shown
promise in distinct molecular subtypes, such as ALK, KRAS, EGFR, and HER2 in early clinical
trials. Despite the documented promise in preclinical studies and early phase clinical trials, HSP-
2
2, 28-
9
0 inhibitors have not been able to replicate their effectiveness at higher stage clinical stages.
3
0
Thus, fabrication of rationally designed strategies to extract the potential of HSP90 inhibition is
required to ascertain conclusive benefits in NSCLC.
It has been well reported that HDAC inhibitors induce acetylation and inhibit the ATP binding
and chaperone functions of HSP90 leading to the degradation of pro-survival client proteins in
3
1-33
human leukemia cells.
Synergistic reports of pan HDAC and selective HDAC6 inhibitors with
3
4
HSP90 inhibitors further rationalizes this biochemical relation . Moreover, a recent investigation
by Deskin et al. reported that targeting the HDAC6/HSP90 Axis to destabilize the notch1 receptor
in NSCLC may offer therapeutic benefits in NSCLC as both HDAC6 and HSP90 regulate Notch1
3
5
receptor levels through the ubiquitin proteasome system. In light of these optimistic revelations,
development of dual HDAC-HSP90 inhibitors is anticipated to yield favorable outcomes in
NSCLC therapy.
Our previous investigations on small molecule HDAC inhibitors revealed key structural
insights critical for modulating the enzymatic activity and influencing the cellular activity. These
identified structural features include a flexibility in the chemical nature of the CAP constructs that
tolerates variability from bicyclic planar to non-planar/partially hydrogenated (6,6/6,5-fused)
heteroaryl rings as well as the linkers and the critical point of attachment of the zinc binding motif
3
6, 37
to the heteroaryl rings.
Capitalizing on this structural information, the present study broadens

the investigation parameters of our ongoing drug discovery program towards the design of HDAC
inhibitors capable of modulating the HSP90 chaperone. The design strategy involved the
utilization of amide tethered resorcinol-isoindoline functionality as an extended CAP component
of the HDAC inhibitory scaffold. The resorcinol fragment has been recognized as a crucial
2
3, 25, 26, 27
structural unit present in several second generation HSP90 inhibitors
and serves as a key
binder associated with the ATP binding site of HSP90 proteins by means of an appropriate fit in
3
8
the hydrophilic and hydrophobic regions of the protein . Specifically, the selection of isoindoline
-
resorcinol adduct as the surface recognition part was influenced due to the following reasons : a)
3
9
recent optimistic reports of N-heterocyclic ring linked resorcinol adducts as HSP90 inhibitors b)
existence of the structural unit composed of isoindoline core condensed with resorcinol ring in the
chemical architecture of AT-13387, a second generation HSP90 inhibitor undergoing clinical
2
7
studies in NSCLC c) recent study by our research group demonstrating the potential of indoline
3
6
based hydroxamic acids as dual HDAC/HSP90 inhibitors . Moreover, several alterations were
attempted in the chemical architecture of designed hybrids in the context of the linker type (long
chain alkyl, benzyl and acrylamide functionality), however the positioning of zinc binding motif
on the non-planar isoindoline ring using diverse linkers was kept constant. With this background,
we report herein the synthesis and biological evaluation of a series of 1-aroylisoindoline-
hydroxamic acids as dual inhibitors of HDAC and HSP90. In addition to enzymatic and cellular
activity (in-vitro and in-vivo), the potential of the synthetic compounds to
upregulate/downregulate the expressions of signatory biomarkers associated with HDAC and
HSP 90 inhibition was investigated. A docking study was also performed to examine the key
interactions of the designed synthetic compounds, the selected compounds, with the amino acid
residues and support the basis of inhibition of HDAC6 isoform along with the HSP90 chaperone.

The effect of one of the potent hydroxamic acid was also studied on the IFN-γ induced PDL-1
expression in H1975 lung cancer cells to evaluate the potential of dual HDAC/HSP90 inhibition
towards the modulation of immunosuppressive ability of tumor area.
2
2
. Results
.1 Chemistry
Synthetic routes for the designed compounds are depicted in Schemes 1, 2 and 3. Scheme 1
illustrates the synthesis of the N-benzyl linker-based target compound and the methodology starts
with the nitration of isoindoline (19) which yields nitroisoindoline (20, yield 50 %) with a
moderate yield. This nitrated isoindoline was subjected to EDC/HOBT assisted amidation with
2
,4-bis(benzyloxy)-5-isopropylbenzoic acid to generate the amide (21, yield 70 %) which
underwent Fe/NH Cl mediated nitro reduction to produce the primary amine (22, yield 80 %)
4
which on reductive amination yielded the secondary amine (23, yield 75 %). Lithium hydroxide
assisted ester hydrolysis of 23 produced the carboxylic acid (24, yield 96 %) which upon
2 3
amidation with NH OBn followed by BCl -catalyzed debenzylation furnished the target
hydroxamic acid (10, yield 97 %).
To establish the influence of various linkers on the bioactivity, long chain linker based
hydroxamic acids (11-17) were synthesized using the strategy shown in Scheme 2. The
implementation of the similar methodology i.e EDC/HOBT mediated amide bond formation with
alkoxyalkanoic acids (25-31, yields 73-86 %), ester hydrolysis (32-38, yields 91-97 %) and
amidation with NH OBn afforded the intermediates 39-45 (yields 71-82 %) which were
2
2
debenzylated using 10% Pd/C in MeOH in a hydrogenation vessel with H at 40 - 42 psi to afford
the desired hydroxamic acids (11-17, yields 70-78 %). Attempts to further ascertain the linker-

activity correlation led us synthesize a short linker, an acrylamide bearing a hydroxamic acid (18).
The synthetic route to the target compound (18) begins with the NaBH reduction of 5-
4
bromoisoindoline-1,3-dione (46) to 5-bromoisoindoline (47, yield 59 %)
. Amidation of 47 with
2
,4-bis(benzyloxy)-5-isopropylbenzoic acid (48, yield 75 %) followed by Pd(OAc) -catalyzed
2
Heck olefination with methyl acrylate yielded the intermediate 49 (yield 53 %). The Heck
coupled product (49) underwent lithium hydroxide assisted ester hydrolysis, amidation with
2 3
NH OBn and BCl catalyzed debenzylation to produce the target hydroxamic acid (18, yield 66
%).
2
2
.2 Biological evaluation
.2.1 In vitro cytotoxicity studies
The synthesized compounds were assayed for growth inhibition of non-small cell lung cancer
A549 and H1975 cells. The GI values, defined as concentrations of the compounds that induced
5
0
5
0% cell growth inhibition were determined and the summarized results are shown in Table 1.
SAHA (1), an FDA approved HDAC inhibitor and BIIB021 (6), an orally available small-
molecule HSP90 inhibitor were employed as standards in the study. The synthetic compounds
were designed to ascertain the impact of diverse linkers, i.e the N-benzyl, long alkyl chain and
acrylamide linker on the cellular activity. Among the tested compounds, compound 10 bearing the
N-benzyl linker exhibited the most substantial cytotoxic effects against A549 cell lines and
H1975 cell line with GI values of 0.37 µM and 0.13 µM, respectively. It was interesting to
5
0
observe that the cell growth inhibitory effects of 10 against A549 and H1975 cell lines were more
pronounced than those of 1, which was the standard employed. Specifically, the effects of 10
against non-small cell lung cancer cell lines H1975 cell lines were higher than those of both the

standards employed (1 and 6). Use of the alkyl chain in place of the N-benzyl linker failed to
yield favorable results in the context of potentiating the antiproliferative effects in a relative
comparison. Among the alkyl chain linker bearing compounds, compound 11 (n = 2) exhibited
significant cell growth inhibitory effects against both NSCLC cell lines with higher sensitivity
towards H1975 cell lines (IC value = 0.39 µM). In general, a variable cytotoxic profile of alkyl
5
0
chain linker based compounds (11-17) was observed towards the cell lines tested. It was observed
that extending the linker length to n = 3 led to dramatic loss of activity as evidenced by compound
1
1
2. Potency was found to be regenerated by extending the methylene chain length as in compound
3 (n = 4) but an exact dependence of linker length and a compound’s ability to exhibit cellular
activity was found to be variable for compounds 13-17, (n = 4-8). Compound 17 (n = 8) displayed
a balanced cytotoxic profile against both the NSCLC cell lines with GI = 0.76 µM in A549 cells
5
0
and GI = 0.52 µM in H1975 cells. Further introduction of an acrylamide linker at position 5 of
5
0
the isoindoline ring (18) led to selective antiproliferative effects about 9-fold better against H1975
cell line (GI = 0.32 µM) than against the A549 cell line (GI = 2.74 µM), however it turned out
5
0
50
to be less effective than its counterpart (compound 10) bearing the N-benzyl linker. In general, all
the compounds except compounds 13 and 15 displayed a similar trend of higher effectiveness
towards H1975 cell lines. Overall, compounds 10, 11 and 17 have significant inhibitory potential
against both the NSCLC cell lines employed.
2
.2.2 HeLa nuclear HDAC enzyme inhibition and Heat Shock protein 90 inhibition
Evaluation of HDAC inhibitory activity using HeLa nuclear extract as the HDAC source was
conducted (Table 2). The correlation of the HDAC inhibitory activity of the synthetic compounds
with the linker length led to interesting results and demonstrated that the length of the linker for

compound 11 - 17 was instrumental in modulating the HDAC inhibition. It was observed that
compounds 11 - 13 (n = 2-4) were devoid of HDAC inhibition and extension of linker length
beyond n = 4 induced the inhibitory potential towards the HeLa HDAC. The long chain linker 15,
bearing a hydroxamic acid (n = 6) and possessing the same combination of the linker and the zinc
binding motif as that of compound 1 displayed the most potent inhibitory activity with IC =
5
0
1
68.3 nM. Among the three most cytotoxic compounds 10, 11, 17, only 17 (n = 8, IC = 388.8
50
nM) exhibited HeLa nuclear HDAC inhibitory potential. The hydroxamic acids 10 (with benzyl
linker) and 11 (n = 2) displayed diminished activity against the HeLa HDAC. Contrary to the
results of HeLa HDAC inhibition, an HSP90 inhibitory assay, employing compound 6 as a
standard indicated that all the compounds are potent HSP90 inhibitors with IC values ranging
5
0
from 42.2 - 135.6 nM (Table 2). In particular, the pronounced effects of compounds 14-17 (n = 5-
, IC = 42.2 – 46.8 nM) against the chaperone protein are higher than those of the standard (6).
8
5
0
Compounds 15 [(IC = 43.9 nM (HSP90), 168.3 nM (HDAC)] and 16 [(IC = 42.2 nM (HSP90),
5
0
50
1
98.9 nM (HDAC)] were found to be the most potent dual inhibitors of HeLa HDAC and HSP90.
Overall, the ability of the compounds to inhibit HSP90 protein was influenced only marginally by
the linker length and could be attributed to the presence of isopropyl resorcinol fragment in the
chemical architecture of the synthesized compounds. All the three potent antiproliferatives 10, 11
and 17 modulated the activity of HSP90 with IC values of 66, 123.1 and 46.8 nM, respectively.
5
0
Compound 18 failed to alter the activity of the HeLa HDAC enzyme and only modulated the
HSP90 activity.
2
.2.3 HDAC isoform inhibition

Isoform selective inhibitory effects were examined for the synthesized adducts towards the
HDAC 1, 3, 6 and 8 isoform employing trichostatin A as the reference compound. The results
summarized in Table 3 indicate that HDAC6 isoform was the most sensitive to the exposure of all
the compounds. The impact of the linker variation on HDAC6 inhibition was clearly observed
with the N-benzyl group and a long chain linker (n = 5-8) demonstrating favorable trends towards
the inhibition of HDAC6 isoform. Compound 10 bearing the N-benzyl linker was effective in
inhibiting the HDAC6 isoform (IC50 = 33.3 nM) with a selectivity of 300 and 296 fold over
HDAC1 and 3. In addition, adduct 10 also moderately inhibited the HDAC8 isoform.
Concomitant evaluation of the HDAC isoform inhibitory ability of the long chain linker bearing
hydroxamic acids (11-17) revealed an interesting linker-HDAC6 inhibition relationship. The
correlation of results presented in Table 1, 2 and 3 showed that compound 11 (n = 2) induces cell
growth inhibitory effects against the NSCLC solely through HSP90 inhibition and is devoid of
inhibitory effects against the HDAC isoforms. A dramatic amplification in the HDAC6 inhibitory
activity was observed on extension of the methylene chain from 11 (n = 2, IC 50 >10,000 nM), to
1
2 (n = 3, IC50 = 68.6 nM), but a further increase in the chain length (n = 4, IC50 = 330 nM) did
not replicate the similar positive trends towards HDAC 6 inhibition. A continuing variable pattern
was seen in the influence of chain length on enzymatic activity and lengthening the methylene
chain from n = 4 to n = 5-8 led to marked increase in the activity profile of hydroxamic acids (14-
1
7) against the HDAC6 isoform. The hydroxamic acids (14-17) were endowed with IC values in
50
single digit nanomolar range (HDAC6 inhibition). Overall, a linker length of n = 6 - 7 was the
most suitable for inducing HDAC 6 inhibition and compounds 15 (n = 6, IC = 2.21 nM) and 16
5
0
(n = 7, IC50 = 2.12 nM) were identified as the most potent HDAC6 inhibitors exhibiting higher
HDAC6 inhibitory effects than even the standard trichostatin A (1, IC50 = 2.60 nM). Compound

1
5 displayed 250, 175 and 683 fold higher selectivity for HDAC 6 than the HDAC1, 3 and 8
isoforms. Similarly, inhibitor 16 also exerted preferential effects against the HDAC6 isoform with
a selectivity ratio of 507 for HDAC1/HDAC6, 233 for HDAC3/HDAC6 and >4716 for
HDAC8/HDAC6. Among the three most potent cytotoxic compounds 10, 11 and 17, the HDAC6
isoform was most significantly inhibited by 17 with an IC value of 4.32 nM. Compound 17 was
5
0
also highly selective against HDAC6 in comparison to HDAC1, HDAC3 and HDAC8 by factors
of 434, 303 and 861 respectively. The insertion of the -C=C- linkage between N-
hydroxyaminocarbonyl and isoindoline ring (18) led to significant loss of the enzymatic potency
(HDAC6) and the acrylamide linker containing hydroxamic acid (18) displayed only moderate
inhibition of the HDAC6. In summary, this study confirms the influence of diverse linkers on the
selective HDAC 6 inhibition.
2
.2.4 Western Blot Analysis
In view of the balanced modulation of HeLa HDAC, HDAC6 isoform and HSP90 along with
the potent antiproliferative effects against NSCLC, the ability of compound 17 to
downregulate/upregulate the protein levels of important biomarkers associated with intracellular
HDAC inhibition was determined. For the western blot analysis, H1975 cells were treated with 17
or SAHA at the indicated concentrations for 24 h (Figure 2). The results demonstrate that
compound 17 induced upregulation of acetylated α-tubulin in a dose-dependent manner and this
feature is consistent with the signatory feature of HDAC6 inhibitors. Western blot analysis was
also conducted to evaluate the degradation of multiple HSP90 client proteins triggered by
compound 17 in concentration and time-dependent manners. Figure 3A presents the results
obtained when H1975 cells were treated with DMSO or 0.03-3 µM of test compounds (17,

BIIB021, and Tubastatin A) for 24 h and Figure 3B presents the results for the treatment of
H1975 cells were treated with 3 µM of 17 for indicated times. Acetylated-tubulin was induced
much greater by 17 compared to Tubastatin A at the same concentrations (Figure 3A). A notable
induction of chaperone protein HSP70 and downregulation of the well-known HSP90 client
protein Akt were observed in cells (Figure 3A). These results represents the characteristic
molecular signature of HSP90 inhibition. In addition, activated Akt (p-Akt Ser473) was reduced
at early 6 h treatment time point followed by downregulation of activated Stat3 (p-Stat3 Tyr705)
at 12 h time point. We further compared 17 with another reference compound AT-13387 which is
also consisting of resorcinol tethered isoindoline ring to inhibit HSP90 activity. As shown in
Figure 3C, both of 17 and AT-13387 exhibit potent inhibitory activity on survival signals
(activated Akt and Stat3). Although it has been reported AT-13387 downregulates HDAC6
4
0
expression to stabilize the acetylated α-tubulin , our result shows 17 is a more potent inducer of
acetylated tubulin than AT-13387 at the same concentrations, suggesting 17 may have more
impacts of microtubule-associated proteins to regulate cell migration compared to AT-13387.
Taken together, the results confirm that the cell killing effects of 17 stems from modulation of
HSP90 protein in addition to HDAC inhibition.
Moreover, compound 17 activates cell accumulation at the subG1 phase as shown by
propidium iodide staining and flow cytometry analysis (Fig. 4A), suggesting compound 17
displays potent cytotoxicity against H1975 cancer cells. We further observed that compound 17
induces concentration-dependent activation of caspase 3/8/9, PARP, and γH2AX (Fig. 4B),
indicating that compound 17 is a potent inducer of apoptosis in H1975 lung cancer cells.
2
.2.5 Molecular modeling study

A molecular docking study was performed for compounds 10 and 17 to identify the important
interactions with the amino acid residues of HSP90 and HDAC6. The docking protocol used in
this study was first validated by redocking the co-crystallized ligands of HSP90, HDAC1 and
HDAC6 (Fig. 5). The co-crystalized ligand of the HDAC3 structure was not included for
validation because it is an acetate molecule that was used for the refinement process of
4
1
crystallization . The redocked ligands produced poses similar to those of the co-crystallized
ligands for HSP90, HDAC1 and HDAC6, indicating that a rational docking protocol was used.
It was observed that when compound 17 binds to HSP90, it adopts a U-shape (Fig. 6). The
compound structure can be divided into four groups. Group 1 is composed of 2,4-dihydroxy-5-
isopropylbenzoyl functionality. This group forms hydrogen bonds with residues N51, D93 and
T184 in HSP90 and is also involved in hydrophobic interactions with residues F138 and T184.
Group 2 consists of an N-(2,3-dihydro-1H-isoindol-5-yl)formamide moiety and forms hydrogen
bonds with residues K58 and G108. Both Group 2 and Group 3, which consists of an eight
carbon-chain occupy a hydrophobic pocket and interact with residues A55 and M98. These
interactions sandwich Group 2 and Group 3 along the outer rim of the HSP90 binding site (Fig. 6).
Group 4 consists of a hydroxamic acid moiety and is involved in hydrogen bonding interactions
with residues E47, N51 and G137 of the protein. This moiety binds to the entrance of the binding
site and forms additional hydrogen bonds (Figs. 6A and 6B).
Compound 10 displays a similar docking pose and can also be separated into four groups.
Group 1 consists of a 2,4-dihydroxy-5-isopropylbenzoyl moiety and creates hydrogen bonds with
residues N51, D93 and T184. Group 2 and Group 3 contain a 2,3-dihydro-1H-isoindol-5-amine
and a benzyl moiety, respectively (Fig. 6D). No hydrogen bonds were observed with these groups.
This may account for the weaker activity of compound 10 towards HSP90 as compared to

compound 17. Hydrophobic interactions with residues A55, M98 and T109 sandwich Group 2
and Group 3 along the outer rim of the binding site (Fig. 6C). Finally, Group 4 bearing a
hydroxamic acid moiety forms hydrogen bonds with N51, G135 and F138. These hydrogen bonds
differ from those observed with compound 17. Compound 17 contains an eight-carbon chain at
Group 3 which increases its overall length. As a result, compound 10 is unable to create hydrogen
bonds with the same residues as compound 17. Together, these interactions suggest that
compound 17 and compound 10 anchor to the binding site and thus inhibit HSP90 function.
Compounds 17 and 10 were docked to HDAC6 and the interactions between the compounds
and the catalytic site were analyzed. HDAC inhibitors typically contain three conserved structures:
a cap that obstructs the binding site entrance, a linker that connects the two structures and a zinc
4
2
binding group (ZBG). Both compound 17 and compound 10 contain these structural hallmarks
where Group 1 and Group 2 act as the cap construct, Group 3 forms the linker and Group 4 acts as
a zinc binding group (Fig. 7A-B). The cap of compound 17 creates hydrogen bonds with residues
S564, F566 and I569. The linker of compound 17 spans the hydrophobic channel of HDAC 6 and
hydrophobic interactions are observed with residue F620. The ZBG of compound 17 extends into
the HDAC6 catalytic site. Compound 17 contains a hydroxamate acid as the ZBG and coordinates
to the zinc ion in a bidentate mode (Fig. 7C). Additional hydrogen bonds are formed with nearby
residues. For example, residue H610 forms a hydrogen bond to the hydroxamate N-O group
(distance 3.1Å) and residue G619 forms a hydrogen bond to the hydroxamate NH group (distance
3
.0Å). These interactions suggest that compound 17 creates sufficient interactions within the
HDAC6 catalytic site.
An analysis of compound 10 was performed and compared to compound 17. Compound 10
contains the same cap structure (Fig. 7B). This facilitates similar hydrogen-bonding interactions

with residues S564, F566 and I569 at the periphery of the HDAC6 binding site. The linker of
compound 10 spans the hydrophobic channel of the binding site (Fig. 7B). Compound 10 contains
an aromatic ring-based linker, making it more rigid than compound 17. Hydrophobic interactions
occur between the linker and residues H651, F680 and L749. The ZBG of Compound 10 contains
a hydroxamic moiety attached to a sterically bulky aromatic ring. It has been reported in the
literature that compounds with sterically bulky substituents, such as ACY-1083, coordinates to
2
+
43,44
the HDAC 6 Zn in a monodentate fashion.
A water molecule was added to the binding site
2
+
during the docking analysis. The docking result showed compound 10 coordinates to the Zn in
2
+
the monodentate mode, with one hydroxamate oxygen atom interacting with the Zn atom and
another oxygen atom hydrogen-bonding with a water molecule (distance 2.7Å) (Fig. 7D). An
additional hydrogen bond is formed between the hydroxamate NH group and residue G619
(distance 2.7Å). Together, our analysis shows that compound 17 and compound 10 can bind to
2
+
the HDAC 6 catalytic site and coordinate to the Zn ion, which suggests that it makes favorable
interactions for HDAC6 inhibition.
For HDAC6 inhibition, we found that compound 10 has lower activity when compared to
compound 17. A relative comparison of the docking results of compound 10 and 17 revealed
different interactions along the periphery of the hydrophobic tunnel (Fig. 7E-F). Residue L749 is
found along the hydrophobic channel periphery. Compound 17 contains an eight-carbon chain
linker that forms a hydrophobic interaction with residue L749 (Fig. 7E). In contrast, the linker of
compound 10 contains an aromatic ring and is a relatively more rigid structure. Our docking
result showed that the compound 10 linker does not form interactions with residue L749 (Fig. 7F).
Together, the different interactions between the linker and the hydrophobic tunnel may account
for reduced activity of compound 10.

To further identify the reasons HDAC6 selectivity towards, we docked compound 17 into
HDAC1 and HDAC3. Superimposing the HDAC1, 3 and 6 binding site revealed a specific pocket
observed in HDAC6 (Fig. 8A). The compound 17 cap forms hydrogen bonds with residues S564,
F566 and I569 (Fig. 8B). In addition, the hydrophobic channel consists of residues that create a
larger cavity at the outer rim for the compound 17 cap. HDAC1 and HDAC3, in contrast, do not
have a corresponding pocket on the surface and contain a loop which interferes with the cap
interactions (Fig. 8B). As a result, compound 17 show greater specificity towards HDAC6.
To summarize, the docking study indicates that compound 17 and compound 10 target both the
HSP90 chaperone and the HDAC6 isoform. The two active compounds share structures and
interactions that can be separated into four groups. Group 1 enters the HSP90 binding pocket.
The co-crystallized HSP90 ligand, 2D9, contains a similar sub-structure to Group 1 of compound
4
5
1
7 and compound 10 that is buried deep into the HSP90 binding site. However, in HDAC6,
Group 1 is located in the periphery and forms part of the cap seen in traditional HDAC inhibitors.
In contrast, Group 4 bearing the vital zinc binding group for HDAC6 inhibition is located near the
outer margin of the HSP90 binding site. Group 2 and Group 3 link the terminal ends of both
compounds in HSP90. Hydrophobic interactions were observed sandwiching these structures in
compound 17 and 10. In addition, the Group 2 of compound 17 forms hydrogen bonds with
residues K58 and G108. Group 2 of both active compounds form part of the cap in HDAC6 and
group 3 forms the linker, which spans the HDAC6 hydrophobic tunnel.
The variation in the activity profile of compound 10 and 17 can be attributed to the nature of
the linker. Residue L749 along the outer rim of the HDAC6 hydrophobic channel (Fig. 8). This
residue has the potential to obstruct the entrance of the zinc binding site. Compound 17 is more
flexible and does not clash with residue L749. However, compound 10 contains an aromatic ring

that occupies a space close to residue L749. The aromatic ring also forms hydrophobic
interactions with the periphery residues. Thus, the variation in the linker may account for the
reduced activity seen with compound 10. The results of the docking study revealed key
interactions that could be responsible for dual inhibition of HSP90 and HDAC6.
Our assays found compound 17 and compound 10 to have greater selectivity towards HDAC6.
A molecular docking analysis was performed to further study HDAC selectivity. While the ZBG
plays a crucial role in inhibiting HDACs, it is thought that the HDAC inhibitor cap determines the
4
2, 46
selectivity.
We found that the cap region of compound 17 targets residues S564, F566 and
I569 (Fig. 8). This area constitutes a binding pocket absent from HDAC1 or HDAC3. The binding
pockets for the cap in HDAC1 and HDAC3 do not present optimal binding of the two compounds
created for this study.
2
.2.6 Growth Inhibition of human EGFR-resistance NSCLC H1975 xenograft model in vivo.
For the in vivo studies, Fig. 9 shows the in vivo activity of compound 17 in human NSCLC
H1975 xenograft model employing Afatinib as a reference compound. The results have indicated
that administration of 17 (50 mg/kg, ip, qd) reduced the tumor volume (TGI = 44.8%, *p < 0.05).
However, the inhibitory effect of 17 at 50 mg/kg were less significant than afatinib (20 mg/kg,
*
1
**p<0.001). The in-vivo potential of compound 17 was also investigated at the concentration of
00 mg/kg which led to slightly higher suppression of tumor growth (TGI = 69.7%) than afatinib
(TGI = 63.8%, 20 mg/Kg). The combination treatment of Afatinib with compound 17 (50 mg/kg,
ip, qd, Afatinib – 20 mg/kg, ***p<0.001) was also evaluated which led to marginally higher
tumor growth inhibition than produced by Afatinib alone. The most substantial in-vivo efficacy
(combination studies) was exhibited by the combination of compound 17 and Afatinib (compound

1
7 - 50 mg/kg, ip, qd, Afatinib - 20 mg/kg, ***p<0.001) and showed enhancement of tumor
suppression by a 72.3% tumor growth inhibition (TGI). In addition, no significant differences in
weight loss were observed during all the treatments.
2
.2.7 Downregulation of IFN-γ induced PD-L1 expression in H1975 lung cancer cells
T cells, especially cytotoxic T cells, can infiltrate into the tumor area to eliminate tumor cells
by releasing cytotoxins (perforins and granzymes) and inflammatory cytokines (IFN-γ and TNF-
α). However, IFN-γ also triggers the expression of programmed death-ligand 1 (PD-L1) on the
surface of many tumor cells, leading T-cell suppression and immune evasion of tumor cells from
4
7,48
cellular cytotoxicity
. To investigate whether compound 17 affects the INFγ-induced PD-L1
expression, H1975 human non-small cell lung cancer cells were cotreated with variant
concentrations of compound 17 and 20 ng/ml of IFN-γ. The Western blot analysis (Figure 10 A)
shows that compound 17 effectively reduced PD-L1 expression in IFN-γ treated H1975 cells in a
dose-dependent manner. To further elucidate the inhibited level of surface PD-L1 expression,
treated H1975 cells were analyzed with flow cytometry (Fig. 10 B and C). The PD-L1 expression
on cell surface induced by IFN-γ was significantly decreased after the treatment with the
compound 17 at concentration above 0.3 µM. These results indicate that the compound 17
reversed the IFN-γ-mediated upregulation of PD-L1, suggesting a possible function of the
compound 17 in destroying the immunosuppressive environment of tumor area. Since the hostile
tumor microenvironment of solid tumors remains a challenge to T cell therapy, a combination of
compound 17 and T cell immunotherapy might be worth investigating to develop an approach
generating synergistic antitumor effects of chemotherapy and immunotherapy.

3
. Conclusion
In this study, a series of 1-aroylisoindoline-hydroxamic acids has been synthesized. The impact
of diverse linkers connecting the 1-aroylisoindoline moiety to the zinc binding motif on the
bioactivity was studied. In-vitro cytotoxicity studies revealed that the N-benzyl linker-based
inhibitor (10) was the most effective in suppressing cell growth in both the NSCLC cell lines
employed. Incorporation of the alkyl chain and acrylamide linker in the chemical architecture of
the designed synthetics failed to enhance the cytotoxic potential as seen in the relative comparison
with compound 10. Compounds 11 and 17 displayed the most substantial cell growth inhibitory
effects among the hydroxamic acids bearing an alkyl chain linker. It was interesting to observe
that compound 11 exercised its cytotoxicity solely through HSP90 inhibition as evidenced by
enzymatic assays, whereas the inhibitor 17 was found to have a balanced antiproliferative and
enzyme inhibitory profile towards both the targets. In general, all the compounds exhibit selective
inhibitory activity against HDAC 6 isoform with few exceptions. an important finding of this
study is that the most pronounced dual HDAC6/HSP90 inhibition induced by a compound bearing
a long chain linker was by 15 (n = 6) which possesses the same combination of linker and zinc
binding group as that of SAHA, however the cell growth inhibitory effects of 15 were not
significant. The main finding of this study is the identification of compound 10 and 17 as dual
selective inhibitors of the HDAC6 isoform as well as the HSP90 protein, inducing significant
antiproliferative effects against the NSCLC cell lines employed with higher efficacy against
H1975 cell lines. Compound 17 also induced upregulation of important biomarkers associated
with intracellular HDAC inhibition along with the concomitant induction of chaperone protein
HSP70 and degradation of HSP90 client proteins which are considered to be a molecular
signature of HSP90 inhibitors. Molecular modeling studies were employed to rationalize and

support the experimental results and the key interactions of compound 10 and 17 with HSP90
chaperone along with the HDAC6 isoform were rationalized. The docking study suggested
several notions for the differences in the activity profile of compounds 10 and 17 along with the
selectivity of these compounds towards the HDAC6 isoform. The in vivo efficacy of compound
1
7 was also evaluated in human EGFR-resistance NSCLC H1975 xenograft model and it was
found that this inhibitor, both alone and in combination with Afatinib induced significant tumor
growth inhibition. Moreover, compound 17 could effectively reduce (PD-L1) expression in IFN-γ
treated lung H1975 cells in a dose dependent manner suggesting that dual inhibition of HDAC6
and HSP90 can modulate immunosuppressive ability of tumor area. This also indicates that
compound 17 in combination with T cell immunotherapy might be worth investigating to develop
an approach generating synergistic antitumor effects of chemotherapy and immunotherapy.
Overall, the cumulative results indicates that hydroxamic acids such as 17 could emerge as
potential leads for further investigation to develop selective HDAC6/HSP90 inhibitors as potent
anticancer agents for lung cancer and immunosuppression inhibitory activity. Thus this study
concludes and ascertains that the selective HDAC6/HSP90 inhibition appears to be a potentially
effective strategy to target NSCLC.
4
4
. Experimental
.1 Chemistry
Nuclear magnetic resonance spectra were obtained with a Bruker DRX-500 spectrometer
operating at 300 MHz, with chemical shifts reported in parts per million (ppm, d) downfield from
TMS as an internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL
(JMS-700) electron impact (EI) mass spectrometer. The purity of the final compounds was

determined to be ≥ 95%, using an Agilent 1100 series HPLC system using C-18 column (Agilent
ZORBAX Eclipse XDB-C18 5 µm, 4.6 mm × 150 mm). Flash column chromatography was done
using silica gel (Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM). All reactions were carried
out under an atmosphere of dry nitrogen
5
-Nitroisoindoline (20)
Conc. H SO (8 mL) was added dropwise at 0° C to a solution of isoindoline (19) (2 g) in
DCM (10 mL), and the reaction mixture was stirred at rt for 10 min. DCM was evaporated under
vacuum from the reaction mixture and conc. HNO (2 mL) was added dropwise at 0 °C. The
2
4
3
reaction mixture was stirred for 2 h at rt. Ice water was added to the reaction mixture then the
reaction mixture was basified with sodium bicarbonate. Extraction was done with EtOAc (100
mL x 3) and the combined organic layer was dried over anhydrous MgSO and concentrated
4
1
under reduced pressure to give 20 in 50% yield; H NMR (300 MHz, CDCl ): δ 8.07 - 8.12 (m, 2
3
H), 7.34 (d, J = 8.5 Hz, 1H), 4.32 (s, 4H).
(
2,4-Bis(benzyloxy)-5-isopropylphenyl) (5-nitroisoindolin-2-yl) methanone (21)
A mixture of 3,5-bis(benzyloxy)-2-isopropylbenzoic acid (2.29 g, 6.09 mmol), EDC.HCl (1.88g,
2.18 mmol), HOBt (1.39 g, 9.13 mmol) and DIPEA (1.96 g, 15.22 mmol) in DCM (10 mL) was
1
stirred at rt for 30 min before adding 5-nitroisoindoline (20) (1 g, 6.09 mmol). After being stirred
for a further 5 h, the reaction mixture was quenched with H O and extracted with EtOAc (50 mL
2
x 3). The combined organic layer was dried over anhydrous MgSO and concentrated under
4
reduced pressure. The residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:4)
1
to give 21 in 70% yield. H NMR (300 MHz, CDCl
3
): δ 8.19-8.20 (m, 2H), 7.98 (s, 1H), 7.10 -

7
.45 (m, 11 H), 6.59 (s, 1H), 5.05 (s, 4H), 4.87 (s, 2H), 4.42 (s, 2H), 3.34 (m, 1H), 1.22 (d, J = 6.9
Hz, 6H).
2,4-Bis(benzyloxy)-5-isopropylphenyl) (5-aminoisoindolin-2-yl) methanone (22)
(
A mixture of compound 21 (1.5 g, 2.8 mmol), Fe powder (801 mg, 14.3 mmol) and NH Cl (304
4
mg, 5.7 mmol) in EtOH (40 mL) and H O (10 mL) was refluxed for 2 h. The progress of the
2
reaction was monitored by TLC. After completion of the reaction, the reaction mixture was
filtered over celite. To the filtrate, H
2
O (50 mL) was added and extraction was done with EtOAc
and concentrated
(50 mL x 3). The combined organic layer was dried over anhydrous MgSO
4
under reduced pressure. The residue was purified by silica gel chromatography (EtOAc:n-hexane
1
=
2:3) to give 22 in 80% yield. H NMR (300 MHz, CD OD): δ 7.10 -7.45 (m, 14 H), 6.56 (s, 1H),
3
5
.16 (s, 2H), 5.12 (s, 2H), 4.75 (s, 2H), 4.39 (s, 2H), 3.32 (1H, m), 1.23 (d, J = 6.9 Hz, 6H).
4
-{[2-(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-2,3-dihydro-1H-isoindol-5-ylamino]-methyl}-
benzoic acid methyl ester (23)
Methyl 4-formylbenzoate (366 mg, 2.23 mmol) and a few drops of glacial AcOH were added to
the solution of compound 22 (1 g, 2.03 mmol) in EtOH. The reaction mixture was stirred at rt for
3
3
0 min then NaBH CN (191 mg, 3.04 mmol) was added to the reaction mixture and stirring was
continued for 4 h at rt. The reaction mixture was quenched with H O and extracted with EtOAc
2
(
100 mL x 3). The combined organic layer was dried over anhydrous MgSO and concentrated
4
under reduced pressure. The residue was purified by silica gel chromatography (EtOAc:n-hexane
1
=
3:2) to give 23 in 75% yield. H NMR (300 MHz, CD OD): δ 7.99 (d, J =8.1 Hz, 2H), 7.13 -
3
7
3
.47 (m, 16 H), 6.80 (s, 1H), 5.05 (s, 2H), 5.03 (s, 2H), 4.87 (s, 2H), 4.42 (s, 2H), 4.40 (s, 2H),
.91 (s, 3H), 3.35 (1H, m), 1.20 (d, J = 6.9 Hz, 6H).

4
-{[2-(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-2,3-dihydro-1H-isoindol-5-ylamino]-methyl}-
benzoic acid (24)
A mixture of compound 23 (800 mg, 1.2 mmol), 1 M LiOH (aq) (6 mL) and dioxane (10 mL) was
stirred at 40 °C for 2 h. The reaction was concentrated under reduced pressure and H O was
2
added. The mixture was acidified with 3 N HCl and extracted with EtOAc (50 mL x 3). The
combined organic layer was dried over anhydrous MgSO and concentrated under reduced
4
1
pressure to yield the acid (24) in 96% yield; H NMR (300 MHz, CD
3
OD): δ 8.02 (d, J =8.1 Hz,
2
H), 7.17 -7.49 (m, 16 H), 6.82 (s, 1H), 5.16 (s, 2H), 5.12 (s, 2H), 4.75 (s, 2H), 4.40 (s, 2H), 4.39
s, 2H), 3.39 (m, 1H), 1.22 (d, J = 6.9 Hz, 6H).
-(((2-(2,4-Dihydroxy-5-isopropylbenzoyl) isoindolin-5-yl) amino) methyl)-N-hydroxy-
(
4
benzamide (10)
A mixture of compound 24 (750 mg, 1.19 mmol), EDC.HCl (494 mg, 2.39 mmol), HOBt (242 mg,
1
.79 mmol) and DIPEA (385 mg, 2.99 mmol) in DMF (5 mL) was stirred at rt for 30 min before
adding NH OBn.HCl After being stirred for a further 5 h, the reaction mixture was quenched with
2
H
2
O and extracted with EtOAc (100 mL x 3). The combined organic layer was dried over
anhydrous MgSO and concentrated under reduced pressure. The residue was purified by silica
4
gel chromatography (EtOAc) to give a semisolid product. The resulting product was dissolved in
DCM (100 mL). BCl (1 M in heptane, 6 eq) was added to this solution at 0 °C and the reaction
3
mixture was stirred for 45 min at the same temperature. The progress of the reaction was
monitored by TLC. The reaction mixture was filtered to collect the precipitated solid (10, 97%
1
yield). HPLC purity: 97.19%. mp: 100-101 °C; H NMR (300 MHz, CD OD): δ 7.81 (d, J = 8.1
3
Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.46-7.47 (m, 2H), 7.36 (d, J = 8.1 Hz, 1 H), 7.18 (s, 1H), 6.43
1
3
(
s, 1H), 5.04 (s, 4H), 4.70 (s, 2H), 3.22 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H); C NMR (75 Hz,

3
CD OD): δ 168.40, 163.21, 155.390, 151.36, 131.77, 131.42, 130.47, 127.96, 125.64, 125.53,
1
24.77, 124.27, 122.95, 121.69, 119.95, 115.11, 109.74, 99.72, 52.28, 23.69, 19.09; HRMS (ESI)
-
for C H N O (M-H) : calcd, 460.1872; found, 460.1867.
2
6
26
3
5
Methyl 4-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-4-oxobutanoate (25)
A mixture of 22 (0.3 g, 0.609 mmol), EDC.HCl (0.232 g, 1.21 mmol), HOBt (0.123 g, 737 mg,
0
.914 mmol), 4-methoxy-4-oxobutanoic acid (0.96 g, 0.727 mmol) and DIPEA (0.265 mL, 1.52
mmol) in DMF (5 mL) was stirred at rt for 5 h. After being stirred for a further 5 h, the reaction
mixture was quenched with H O and extracted with EtOAc (50 mL x 3). The combined organic
layer was dried over anhydrous MgSO
2
4
, concentrated under reduced pressure and purified by
1
silica gel chromatography (hexane : EtOAc = 4 : 1) to give 25 in 80% yield; H NMR (300 MHz,
CDCl ): δ 7.13 -7.54 (m, 14H), 6.48 (s, 1H), 5.14 (s, 4H), 4.74 (bs, 4H), 3.64 (s, 3H), 3.14 (m,
3
1
H), 2.21 (t, J = 6.9 Hz, 2H), 2.15 (t, J = 7.5 Hz, 2H), 1.12 (d, J = 6.9 Hz, 6H).
Methyl 5-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-5-oxopent-
anoate (26)
The title compound 26 was obtained in 82% yield from compound 22 using 5-methoxy-5-
1
oxopentanoic acid in a manner similar to that described for the synthesis of compound 25; H
NMR (300 MHz, CDCl ): δ 7.11 -7.51 (m, 14H), 6.42 (s, 1H), 5.11 (s, 4H), 4.78 (bs, 4H), 3.59 (s,
3
3
H), 3.12 (m, 1H), 2.29 (t, J = 6.9 Hz, 2H), 2.07 (bs, 2H), 1.98 (m, 2H), 1.15 (d, J = 6.9 Hz, 6H).
Methyl 6-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-6-oxo hexan-
ate (27)
The title compound 27 was obtained in 86% yield from compound 22 using 6-methoxy-6-
1
oxohexanoic acid in a manner similar to that described for the synthesis of compound 25; H
3
NMR (300 MHz, CDCl ): δ 7.09 -7.54 (m, 14H), 6.47 (s, 1H), 5.11 (s, 4H), 4.85 (bs, 4H), 3.62 (s,

3
6
H), 3.09 (m, 1H), 2.21 (t, J = 6.9 Hz, 2H), 2.11 (bs, 2H), 1.69-1.75 (m, 4H), 1.18 (d, J = 6.9 Hz,
H).
Methyl 7-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-7-oxoheptanoate
28)
(
The title compound 28 was obtained in 79% yield from compound 22 using 7-methoxy-7-
1
oxoheptanoic acid in a manner similar to that described for the synthesis of compound 25; H
NMR (300 MHz, CDCl
H), 3.11 (m, 1H), 2.13 (t, J = 6.9 Hz, 2H), 2.08 (bs, 2H), 1.64-1.78 (m, 4H), 1.45 (bs, 2H), 1.21
d, J = 6.9 Hz, 6H).
3
): δ 7.12 -7.48 (m, 14H), 6.42 (s, 1H), 5.11 (s, 4H), 4.81 (bs, 4H), 3.61 (s,
3
(
Methyl 8-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-8-oxooctanoate (29)
The title compound 29 was obtained in 76% yield from compound 22 using 8-methoxy-8-
1
oxooctanoic acid in a manner similar to that described for the synthesis of compound 25; H
NMR (300 MHz, CDCl ): δ 7.11 -7.55 (m, 14H), 6.49 (s, 1H), 5. 08 (s, 4H), 4.88 (bs, 4H), 3.65 (s,
3
3
H), 3.15 (m, 1H), 2.20 (bs, 2H), 2.11 (t, J = 6.3 Hz, 2H), 1.58-1.61 (m, 4H), 1.28 (bs, 4H), 1.18
(d, J = 6.6 Hz, 6H).
Methyl 9-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-9-oxonon-
anoate (30)
The title compound 30 was obtained in 86% yield from compound 22 using 9-methoxy-9-
1
oxononanoic acid in a manner similar to that described for the synthesis of compound 25; H
NMR (300 MHz, CDCl ): δ 7.13 -7.51 (m, 14H), 6.43 (s, 1H), 5.12 (s, 4H), 4.82 (bs, 4H), 3.63 (s,
3
3
H), 3.11 (m, 1H), 2.22 (bs, 2H), 2.14 (t, J = 6.3 Hz, 2H), 1.58-1.72 (m, 4H), 1.42 (bs, 6H), 1.21
(d, J = 6.9 Hz, 6H).

Methyl
10-((2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)
isoindolin-5-yl)
amino)-10-
oxodecanoate (31)
The title compound 31 was obtained in 73% yield from compound 22 using 10-methoxy-10-
1
oxodecanoic acid in a manner similar to that described for the synthesis of compound 25; H
NMR (300 MHz, CDCl ): δ 7.12 -7.47 (m, 14H), 6.48 (s, 1H), 5.11 (s, 4H), 4.89 (bs, 4H), 3.61 (s,
3
3
H), 3.14 (m, 1H), 2.24 (bs, 2H), 2.11 (t, J = 6.3 Hz, 2H), 1.57-1.68 (m, 4H), 1.48 (bs, 8H), 1.19
d, J = 6.9 Hz, 6H).
-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-4-oxobutanoic acid (32)
A mixture of 25 (0.3 g, 0.49 mmol), aq. LiOH (1M) (3 mL) and dioxane (5 mL) was stirred at 40
C for 2 h. The reaction was concentrated under reduced pressure and H O was added. The
(
4
°
2
mixture was acidified with 3 N HCl and extracted with EtOAc (50 mL x 3). The combined
organic layer was dried over anhydrous MgSO and concentrated under reduced pressure to yield
4
1
the acid 32 in 96% yield; H NMR (300 MHz, CD OD): δ 7.12 - 7.61 (m, 14H), 6.45 (s, 1H), 5.16
3
(
s, 4H), 4.87 (bs, 4H), 3.11 (m, 1H), 2.11 -2.31 (m, 4H), 1.15 (d, J = 6.9 Hz, 6H).
-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-5-oxopentanoic acid
33)
5
(
The title compound 33 was obtained in 92% yield from compound 26 in a manner similar to that
1
described for the synthesis of compound 32; H NMR (300 MHz, CD OD): δ 7.18 -7.59 (m, 14H),
3
6
.48 (s, 1H), 5.13 (s, 4H), 4.89 (bs, 4H), 3.22 (m, 1H), 2.12 – 2.24 (m, 4H), 1.99 (m, 2H), 1.12 (d,
J = 6.9 Hz, 6H).
-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-6-oxohexanoic acid (34)
6
The title compound 34 was obtained in 95% yield from compound 27 in a manner similar to that
1
described for the synthesis of compound 32; H NMR (300 MHz, CD
3
OD): 7.19 -7.64 (m, 14H),

6
2
7
.56 (s, 1H), 5.12 (s, 4H), 4.81 (bs, 4H), 3.09 (m, 1H), 2.21 (t, J = 6.9 Hz, 2H), 2.18 (t, J = 6.9 Hz,
H), 1.78-1.85 (m, 4H), 1.21 (d, J = 6.9 Hz, 6H).
-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-7-oxoheptanoic acid
(35)
The title compound 35 was obtained in 93% yield from compound 28 in a manner similar to that
1
described for the synthesis of compound 32; H NMR (300 MHz, CD OD): 7.22 -7.67 (m, 14H),
3
6
1
8
.49 (s, 1H), 5.12 (s, 4H), 4.76 (bs, 4H), 3.18 (m, 1H), 2.08 – 2.13 (m, 4H), 1.68-1.85 (m, 4H),
.55 (bs, 2H), 1.15 (d, J = 6.9 Hz, 6H).
-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-8-oxooctanoic acid (36)
The title compound 36 was obtained in 97% yield from compound 29 in a manner similar to that
1
described for the synthesis of compound 32; H NMR (300 MHz, CD OD): 7.12 -7.52 (m, 14H),
3
6
4
9
.54 (s, 1H), 5.14 (s, 4H), 4.82 (bs, 4H), 3.11 (m, 1H), 2.20 (bs, 2H), 2.11 (bs, 2H), 1.52-1.67 (m,
H), 1.43 (bs, 4H), 1.11 (d, J = 6.6 Hz, 6H).
-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-9-oxononanoic acid
(37)
The title compound 37 was obtained in 97% yield from compound 30 in a manner similar to that
1
described for the synthesis of compound 32; H NMR (300 MHz, CD OD): 7.13 -7.58 (m, 14H),
3
6
4
1
.47 (s, 1H), 5.09 (s, 4H), 4.89 (bs, 4H), 3.18 (m, 1H), 2.22 (bs, 2H), 2.14 (bs, 2H), 1.59-1.73 (m,
H), 1.48 (bs, 6H), 1.17 (d, J = 6.9 Hz, 6H).
0-((2-(2,4-Bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) amino)-10-oxodecanoic acid
(38)
The title compound 38 was obtained in 91% yield from compound 31 in a manner similar to that
1
1
3
described for the synthesis of compound 32; H NMR (300 MHz, CD OD): H NMR (300 MHz,

3
CD OD): 7.21 -7.52 (m, 14H), 6.49 (s, 1H), 5.05 (s, 4H), 4.82 (bs, 4H), 3.13 (m, 1H), 2.13 - 2.21
(m, 4H), 1.61-1.71 (m, 4H), 1.42 (bs, 8H), 1.19 (d, J = 6.9 Hz, 6H).
1
4
N -(Benzyloxy)-N -(2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) succinamide
(39)
The title compound 39 was obtained in 82% yield from compound 32 using NH OBn.HCl in a
2
1
manner similar to that described for the synthesis of compound 25. H NMR (300 MHz, CD OD):
3
7
.14 - 7.56 (m, 19H), 6.41 (s, 1H), 5.21 (s, 2H), 5.18 (s, 4H), 4.81 (s, 4H), 3.12 (m, 1H), 2.41-2.44
(m, 4H), 1.21 (d, J = 6.9 Hz, 6H).
1
5
N -(Benzyloxy)-N -(2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl) glutaramide
(40)
The title compound 40 was obtained in 73% yield from compound 33 using NH OBn.HCl in a
2
1
manner similar to that described for the synthesis of compound 25. H NMR (300 MHz, CD OD):
3
7
.12 - 7.54 (m, 19H), 6.49 (s, 1H), 5.19 (s, 2H), 5.14 (s, 4H), 4.89 (s, 4H), 3.16 (m, 1H), 2.21-2.24
(m, 4H), 1.91 (m, 2H), 1.16 (d, J = 6.9 Hz, 6H).
1
6
N -(Benzyloxy)-N -(2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl)adipamide (41)
2
The title compound 41 was obtained in 77% yield from compound 34 using NH OBn.HCl in a
1
manner similar to that described for the synthesis of compound 25. H NMR (300 MHz, CD OD):
3
7
.15 - 7.48 (m, 19H), 6.45 (s, 1H), 5.12 (s, 2H), 5.03 (s, 4H), 4.81 (s, 4H), 3.09 (m, 1H), 2.22-2.29
(m, 4H), 1.78-1.85 (m, 4H), 1.15 (d, J = 6.9 Hz, 6H).
1
7
N -(Benzyloxy)-N -(2-(2,4-bis(benzyloxy)-5-isopropylbenzoyl) isoindolin-5-yl)
heptanediamide (42)
The title compound 42 was obtained in 71% yield from compound 35 using NH
2
OBn.HCl in a
OD):
1
manner similar to that described for the synthesis of compound 25. H NMR (300 MHz, CD
3