N-alkyl-hydroxybenzoyl anilide hydroxamates as dual inhibitors of HDAC and HSP90, downregulating IFN-γ induced PD-L1 expression

Article abstract of DOI:10.1016/j.ejmech.2019.111725

Novel dual inhibitors of histone deacetylase (HDAC) and heat-shock protein 90 (HSP90) are synthesized and evaluated. These compounds are endowed with potent HDAC and HSP90 inhibitory activities with IC₅₀ values in nanomolar range with Compound 20 (HDAC IC₅₀ = 194 nM; HSP90α IC₅₀ = 153 nM) and compound 26 (HDAC IC₅₀ = 360 nM; HSP90α IC₅₀ = 77 nM) displaying most potent HDAC and HSP90α inhibitory activities. Both of these compounds induce HSP70 expression and down regulate HSP90 client proteins which play important roles in the regulation of survival and invasiveness in cancer cells. In addition, compounds 20 and 26 induce acetylation of α-tubulin and histone H3. Significantly, compounds 20 and 26 could effectively reduce programmed death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose dependent manner. These findings suggest that dual inhibition of HDAC and HSP90 that can modulate immunosuppressive ability of tumor area may provide a better therapeutic strategy for cancer treatment in the future.

Full text of DOI:10.1016/j.ejmech.2019.111725

Journal Pre-proof
N-alkyl-hydroxybenzoyl anilide hydroxamates as dual inhibitors of HDAC and HSP90,
downregulating IFN-γ induced PDL-1 expression
Samir Mehndiratta, Mei-Hsiang Lin, Yi-Wen Wu, Chun-Han Chen, Tung-Yun Wu,
Kuo-Hsiang Chuang, Min-Wu Chao, Yi-Ying Chen, Shiow-Lin Pan, Mei-Chuan Chen,
Jing-Ping Liou
PII:
S0223-5234(19)30877-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111725
EJMECH 111725
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 July 2019
Revised Date: 5 September 2019
Accepted Date: 20 September 2019
Please cite this article as: S. Mehndiratta, M.-H. Lin, Y.-W. Wu, C.-H. Chen, T.-Y. Wu, K.-H. Chuang, M.-
W. Chao, Y.-Y. Chen, S.-L. Pan, M.-C. Chen, J.-P. Liou, N-alkyl-hydroxybenzoyl anilide hydroxamates
as dual inhibitors of HDAC and HSP90, downregulating IFN-γ induced PDL-1 expression, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111725.
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Graphical abstract
Downregulation of PD-L1 expression in IFN-γ treated H1975 lung cancer cells.

European Journal of Medicinal Chemistry
N-alkyl-hydroxybenzoyl anilide hydroxamates as dual inhibitors of HDAC and
HSP90, downregulating IFN-γ induced PDL-1 expression
Samir Mehndiratta,a Mei-Hsiang Lin,a Yi-Wen Wu,b Chun-Han Chen,c Tung-Yun Wu,h Kuo-Hsiang
Chuang,g,h Min-Wu Chao,d Yi-Ying Chen,d Shiow-Lin Pan,d Mei-Chuan Chen,*,h Jing-Ping Liou*,a,e,f
* To whom correspondence should be addressed. For M. C. Chen: (Phone) 886-2-27361661 ext
6184, (e-mail) mcchen1250@tmu.edu.tw; For 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, Taiwan.
bPhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology,
Taipei Medical University, Taiwan.
cDepartment of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University,
Taipei, Taiwan.
d Graduate Institute of Cancer Molecular Biology and Drug Discovery, College of Medical Science
and Technology, Taipei Medical University, Taipei, Taiwan.
e School of Pharmacy, National Defense Medical Center, Taipei, Taiwan.
f TMU Biomedical Commercialization Center, Taipei Medical University, Taiwan.
g
Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei,
Taiwan
h Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei
Medical University, Taipei, Taiwan

Abstract: Novel dual inhibitors of histone deacetylase (HDAC) and heat-shock protein 90 (HSP90)
are synthesized and evaluated. These compounds are endowed with potent HDAC and HSP90
inhibitory activities with IC50 values in nanomolar range with Compound 20 (HDAC IC50 = 194 nM;
HSP90α IC50 = 153 nM) and compound 26 (HDAC IC50 = 360 nM; HSP90α IC50 = 77 nM)
displaying most potent HDAC and HSP90α inhibitory activities. Both of these compounds induce
HSP70 expression and down regulate HSP90 client proteins which play important roles in the
regulation of survival and invasiveness in cancer cells. In addition, compounds 20 and 26 induce
acetylation of α-tubulin and histone H3. Significantly, compounds 20 and 26 could effectively reduce
programmed death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose
dependent manner. These findings suggest that dual inhibition of HDAC and HSP90 that can
modulate immunosuppressive ability of tumor area may provide a better therapeutic strategy for
cancer treatment in the future.
Key words: Histone deacetylase inhibitors, Dual inhibitors, Design Multiple Ligand, Lung Cancer,
Heat shock protein, programmed death-ligand 1 (PD-L1)

1. Introduction
With the advancement in the knowledge of epigenetics it has become more evident that histone
deacetylases (HDACs) are potential therapeutic targets with the ability to reverse aberrant epigenetic
states associated with cancer. Various studies in cancer cell lines and tumor tissues revealed changes
in the acetylation levels and the expression of the HDAC enzymes and it can be anticipated that
HDAC inhibitors (HDIs) could be therapeutically useful as a single agent or in combination with
other therapies, such as chemotherapy, immunotherapy or radiotherapy.1-4 The approval of
suberoylanilide hydroxamic acid (SAHA) by the FDA as first HDI in 2006 has accelerated the search
for more potent and more selective HDIs. Many pharmaceutical companies have developed potent
HDIs and till date four of these have been granted FDA approval (Figure 1). SAHA with simple
chemical architect and easy synthesis has received much attention for further modifications. Many
compounds based on SAHA with various heteroatoms as the surface recognition cap, exhibiting
potent HDAC inhibitory activity have been reported including ACY-241 (6) (Figure 2).5-9 Recently,
much attention has been given to the designed multiple ligand (DML) approach for the development
of compounds comprising two or more pharmacophores, multi-component ligands and multiple-
ligands within the same molecule to achieve multi-target modulations. The deliberate use of dual
inhibition for enzyme inhibition in cancer treatment is relatively new. Design of such multiple
ligands is based on various interdependent intrinsic cellular pathways where one target could be a
downstream target or client protein or co-chaperone of another or where inhibitors of two different
targets have some synergistic effects. Since HDACs play crucial role in various cellular pathways, it
is useful to evaluate HDIs not only in various combination therapies but also as compounds with dual
or multitarget inhibition, achievable by modified chemical templates of HDIs. There have been some
recent reports of dual inhibitors of inosine monophosphate dehydrogenase and histone deacetylase10,
potent multi-acting HDAC, EGFR, and HER2 inhibitors11, dual inhibitors of histone deacetylase and
Topoisomerase II12 and Toposiomerase I and II13., histone deacetylase inhibitors equipped with

estrogen receptor modulation activity14., Janus Kinase 2 (JAK2) and histone deacetylase bispecific
inhibitors15 and, c-Met/ HDIs based on pyridazinone derivatives.16 Figure 3 illustrates the various
examples of compounds that target dual or multiple signaling pathways.
HDIs increase acetylation of both histone and non-histone proteins, such as HSP 90. HSP90 is a
reported downstream target of HDAC6 and inhibitors of HDAC6 lead to destabilization of the
HSP90 chaperone function resulting in protein degradation.17 Various HDIs have been used
synergistically with HSP90 inhibitors (Figure 4) in studies like; HSP90 inhibitor NVP-AUY922 (I) +
PXD101 (3) for anaplastic thyroid carcinoma18, HSP90 inhibitor SNX5422 (II) + PXD101
(3)/SAHA (1)/Trichostatin A for treatment of anaplastic thyroid carcinoma19, 17AAG (III) a HSP90
inhibitor + SAHA (I)/sodium butyrate to induce apoptosis in human leukemia cells20 and, histone
deacetylase inhibitor LBH589 (4) + HSP90 inhibitor 17-AAG for human CML-BC cells and AML
cells with activating mutation of FLT-3.21
1.1 Design Rationale.
In the field of drug discovery, a single compound that simultaneously competes with multiple
targets is an emerging paradigm. Multiple ligands designed in this way offer a cost-effective strategy
over multi-drug combinations and are less likely to have drug-drug interactions. Reports have
suggested that treatment with HDIs re-sensitizes resistant cells that have acquired resistance towards
not only compound III but also towards other HSP90 inhibitors after treatment with III. This further
supports the idea of using HSP90 and HDIs as a combination therapy.22 Consequently, we used
DML strategy to discover dual inhibitors of HSP90 and HDAC using SAHA (1) as a template with
modified surface recognition cap using resorcinol (Figure 5). Due to better stability, resorcinol
moiety has been preferably used for development of various HSP90 inhibitors (figure 4). Thus
synthesized multiple ligands have potencies in the nanomolar range against various cancer cell lines,

downregulate various client proteins of HSP90 and HDAC, and are lead compounds for further
modifications in the development of more potent dual inhibitors of HDAC and HSP90.
Results and discussion
2.1 Chemistry.
Syntheses of various t-butoxycarbamate anilines (34-41) are shown in Scheme 1. TEA
(Triethylamine) and DMAP (4-Dimethylaminopyridine) were added to a solution of the appropriate
nitroaniline (28-30) in Dichloromethane (DCM) to which was added Boc anhydride (Di-tert-butyl
dicarbonate) dissolved in DCM. Thus obtained various t-Boc-nitroanilines were alkylated using
o
sodium hydride and various alkyl halides at 0 C in DMF. The resulting alkyl halides were reduced
using Fe/NH4Cl to give the corresponding anilines (34-41) in up to 80% yield.
Syntheses of various hydroxamates (17–27) are illustrated in Scheme 2. Esters were synthesized
using HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), DIPEA
(N,N-Diisopropylethylamine) and appropriate aliphatic acid (C-5 to C-8) added to a solution of the
appropriate amine (34-41) in DMF. Amidation using 1,4-dibenzyloxy-5-isopropyl benzoic acid using
HBTU and DIPEA provided corresponding amides (42-52) with yields of 49-65%. 1,4-dibenzyloxy-
5-isopropyl benzoic acid was prepared following the published procedure23.
°
A mixture of 1N LiOH (Lithium hydroxide) and 42-52 was stirred at 40 C for 2 h. Precipitates
obtained after work up were filtered, dried, dissolved in DMF and amidation was accomplished with
NH2OBn using EDC•HCl (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide HCl), HOBt (1-
hydroxybenzotriazole) and NMM (N-methylmorpholine). De-protection of NH2OBn was achieved
using 10% Pd/C in methanol under H2 at room temperature. The deprotected compound was purified
to yield compounds 17-27 in 49-63% yield.
2.2 Biological evaluation.
2.2.1 Inhibition of HeLa nuclear HDAC enzyme and HSP90α.

Compounds 17–27 were evaluated for their HDAC inhibitory activity using HeLa nuclear extract
as HDAC source. They were also examined for activity against HSP90α (Table 1). Compound 17, in
which R1 = H and there is an alkyl chain of six carbons (C-6) attached to the p-position of the
benzamide, displays HDAC inhibitory activity (IC50 = 415 nM) but does not exhibit activity against
HSP90α (IC50 = >1000 nM). SAHA, a HDI, has IC50 = 98 nM against HDAC HeLa extract but no
HSP90α inhibitory activity >1000 nM. On the other hand, 17-AAG, a HSP 90 inhibitor, exhibits
potent HSP90α inhibition (IC50 = 98 nM) but no HDAC inhibitory activity (IC50 >1000 nM). To
investigate the regiomeric effect of the alkyl chain with respect to the benzamide, compounds 18
(meta) and 19 (ortho) were synthesized and evaluated. Compound 18 has increased HDAC inhibitor
activity (IC50 = 126 nM) but there was no change with respect to the activity against HSP90α (IC50
>1000 nM) as compared with 17. Compound 19 fails to show any apparent potency against HDAC
and HSP90α. To evaluate the effect of N-alkyl substitution of benzamide (R1 ≠ H), compounds 20 -
24 were synthesized. Compounds 20 (para, N-Me, whose IC50 with HSP90α is IC50 = 153 nM), 21
(meta, N-Me, IC50 = 302 nM), 22 (para, N-Et, IC50 = 186 nM) and 23 (meta, N-Et, IC50 = 145 nM)
show significant improvement in their HSP90a inhibitory compared to that of their precursors. For
the activity against HDAC of para-substituted compounds, the inhibitory activity of N-Me
substitution (20, IC50 = 194 nM) was better than for the N-Et compound (22, IC50 = 461 nM). The
reverse order was favored for meta-substituted compounds, and thus 23 (N-Et, IC50 = 126 nM) is less
active than 21 (N-Me, IC50 = 211 nM). These results indicate the favorability of compounds
containing an N-substituted benzamide group for dual inhibition of HDAC and HSP90α. The N-
isopropylamide (24) displays diminished activity (HDAC and HSP90α IC50 >1000 nM) suggesting
that a bulky group is not favored. To evaluate the effect of alkyl chain length on the activity of this
series, compounds 25 (C-5), 26 (C-7) and 27 (C-8) were synthesized and evaluated. These
compounds maintain HDAC inhibitory activity with compound 26 exhibiting the best HSP90α
inhibition (IC50 = 77 nM) amongst all the compounds. The lower HDAC inhibitory activity of 27 (C-

8; IC50 = 671 nM) compared to its precursor 26 (C-7; IC50 = 360 nM) indicates that a chain length ≤
C-7 is optimum for maintaining activity.
2.2.2 In Vitro Cell Growth Inhibitory Activity.
The anti-proliferative activity of the synthesized hydroxamates (17–27) against lung carcinoma
A549 cells, human colon cancer HCT116 cells and human non-small cell lung EGFR-resistant
cancer cells H1975 was evaluated (Table 1). Cancer cell growth inhibitory results are consistent with
the HDAC and HSP90α inhibitory activities. Compounds 17-19 (R1 = H); fail to show potent activity
in cancer cell lines with exception of the human colon cancer HCT116 cells {17 (GI50 = 5.43 µM),
18 (GI50 = 6.23 µM), 19 (GI50 = 4.92 µM)}. N-alkylated compounds (20 – 23) showed marked
improvement in cancer cell growth inhibitory activities and their results are consistent with the
HDAC and HSP90α inhibition. Compound 20 (para, N-Me) displayed potent activity in the sub-
micromolar range (A549 GI50 = 0.77 µM; HCT116 GI50 = 0.83 µM and H1975 GI50 = 0.69 µM).
Cell growth inhibitory activities followed the same trend as that of HDAC and HSP90α inhibition
with a para N-Me compound 20 (A549 GI50 = 0.77 µM; HCT116 GI50= 0.83 µM and H1975 GI50 =
0.69 µM) > 22 (A549 GI50 = 5.63 µM; HCT116 GI50= 3.56 µM and H1975 GI50 = 1.38 µM) and the
N-Et substituted meta derivative 23 (A549 GI50 = 1.59 µM; HCT116 GI50 = 1.12 µM and H1975
GI50 = 0.94 µM) > 21 (A549 GI50 = 2.30 µM; HCT116 GI50 = 1.17 µM and H1975 GI50 = 1.66 µM).
Compound 24 was active against only HCT116 cells with IC50 = 6.23 µM. To explore the effect of
alkyl side chain on the SAR, compounds 25 - 27 were synthesized and evaluated. Each of these
compounds showed potent cell growth inhibition with 26 (A549 GI50 = 0.44 µM; HCT116 GI50 =
1.06 µM and H1975 GI50 = 0.40 µM) maintaining an appropriate balance between cell growth
inhibitory activity and HDAC and HSP90α inhibitory activity.
2.2.3 HDAC Isoform Inhibition.

Compounds 20 and 26 were tested for enzymatic activities of HDAC isoforms, including HDAC
isoform 1-11 and, SIRT1 (Table 2). The results indicated that both 20 and 26 decrease enzymatic
activities of various HDAC isoforms with 20 exhibiting at least 22 times more selectivity for HDAC
6 isoform with an IC50 value of 0.04 µM.
2.2.4 Effects of Test Compounds on HSP90-Regulated Client Proteins
HSP 90 chaperone inhibitors reportedly cause pronounced induction of HSP70, and quantification
of HSP70 levels is related to the potency of HSP90 inhibition. Thus, HSP70 induction can be a
useful marker to predict the effects of HSP90 inhibitors.24,25 We explored the protein levels of HSP70
and well-known HSP90 clients after drug treatment. Exposure to test compounds induces HSP70
expression and downregulates the protein levels of client proteins, such as EGFR, Src, FAK, and Rb.
Figure 6 shows the HSP90 inhibitory activity of compounds 20 and 26.
2.2.5 Effect of α-Tubulin and Histone H3 Acetylation on Human Non-Small Cell Lung Cancer
Cells.
Compounds 20 and 26 were evaluated by western blot analysis for their ability to acetylate α-
Tubulin and Histone H3. The expression of these HDAC-inhibition biomarkers was observed in a
concentration-dependent fashion upon treatment with 20 or 26 (Figure 7). The results indicate that
these compounds are potent inhibitors of HDACs in human non-small cell H1975 lung cancer cells.
2.2.6 Effect of cell cycle distribution and cell death on Human Non-Small Cell Lung Cancer Cells.
We evaluated cell cycle distribution alteration in response to 20 and 26 by Flow Cytometry. As
shown in Figure 8A, treatment with higher concentrations of 20 and 26 induce severe subG1 phase
cells accumulation, suggesting these two compounds generate dramatic cell death in H1975 cells.
Further, 20 and 26 induce apoptosis by activating caspase 3,8,9, PARP, and γH2AX (Figure 8B).
These results indicate 20 and 26 are potent inducers of apoptotic cell death in human non-small cell

H1975 lung cancer cells.
2.2.7 Effect of IFN-γ induced PDL-1 expression in Human Non-small Cell 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)26,27
.
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 cellular
cytotoxicity28-30. To investigate whether the Compounds 20 and 26 prevent the IFN-γ induced PD-L1
up-regulation in tumor cells, PD-L1 expression on human non-small cell lung cancer cells (H1975)
was stimulated by 20 ng/ml of IFN-γ and these cells were co-treated with compounds 20 or 26. The
western blot analysis (Figure 9A) shows that compound 20 and 26 could effectively reduce PD-L1
expression in IFN-γ treated H1975 cells in a dose dependent manner. Moreover, the expression level
of surface PD-L1 on IFN-γ treated H1975 cells could also be inhibited by compounds 20 and 26 with
the concentrations above 0.25µM (Figure 9B, 9C).
3. Conclusion
We have designed a series of novel hydroxamic acids (17-27) as potent dual inhibitors of histone
deacetylase and the HSP90 chaperone. Alkylated compounds (20-23) were more potent than the non-
alkylated compounds (17-19). Compounds 20 (para, N-Me, C-6, HDAC IC50 = 194 nM; HSP90α
IC50 = 153 nM) and 26 (para, N-Me C-7, HDAC IC50 = 360 nM; HSP90α IC50 = 77 nM) exhibited
most potent activities while maintaining inhibition of HDAC and HSP90α. An alkyl side chain of 7
carbons was found to be the maximum tolerable length. Compounds 20 showed potent HDAC 6
inhibition with IC50 value of 0.04 µM and is 26 to 222 times more selective for HDAC 6 isoform
compared to other synthetics. Upregulation of HSP70 and downregulation of well-known HSP90
client proteins such as EGFR, Src, FAK, and Rb further indicates the HSP90 inhibitory potential of

20 and 26. They induce apoptosis by activating caspase 3,8,9, PARP, and γH2AX. Substantially,
compounds 20 and 26 downregulate PD-L1 expression in IFN-γ treated lung H1975 cells in a dose
dependent manner thus could be lead molecules for further development owing to their tumor growth
and immunosuppression inhibitory activities.
4. Experimental section
4.1 Chemistry.
Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were obtained with a Bruker DRX-500
spectrometer (operating at 300 MHz and 500 MHz), with chemical shifts in parts per million (ppm,
δ) downfield from TMS as an internal standard. High-resolution mass spectra (HRMS) were
measured with a JEOL (JMS-700) electron impact (EI) mass spectrometer. Purity of the final
compounds was determined using an Hitachi 2000 series HPLC system using C-18 column (Agilent
ZORBAX Eclipse XDB-C18 5 µM. 4.6 mm × 150 mm) and were found to be ≥ 95%. 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 N2.
N-4-Nitrophenyl-t-butylcarbamate (31). A solution of di-tert-butyl dicarbonate (0.91 g, 4.20 mmol)
in DCM was added dropwise to a solution of 4-nitroaniline (28, 0.5 g, 4.20 mmol), TEA
(trimethylamine, 0.59 mL, 4.20 mmol) and DMAP (4-Dimethylaminopyridine, 0.25 g, 2.10 mmol)
and the mixture was stirred for 3 h. The reaction was then quenched using H2O, extracted with DCM
1
(25 mL × 5) and purified by column chromatography to give 31 in 78% yield, H NMR (300 MHz,
CDCl3): δ 1.55 (s, 9H), 6.82 (bs, 1H), 7.53 (d, J = 9.3 Hz, 2H), 8.20 (d, J = 9.3 Hz, 2H).
N-4-Aminophenyl-t-butylcarbamate (34). 31 (1.1 g, 4.45 mmol) was dissolved in a mixture of IPA:
H2O (isopropyl alcohol: water. 4:1). Iron powder (Fe, 0.74 g, 13.35 mmol), NH4Cl (ammonium
chloride, 0.47 g, 8.89 mmol) were added and the mixture was refluxed for two hours. After cooling
to room temperature, the reaction mixture was filtered through celite and extracted with EtOAc

(ethyl acetate, 25 mL × 3). The combined organic layer was dried over anhydrous MgSO4,
concentrated under reduced pressure and purified by column chromatography to give 34 as white
solid in 91% yield, 1H NMR (300 MHz, CD3OD): δ 1.48 (s, 9H), 7.12. (d, J = 9.0 Hz, 2H), 7.15 (d, J
= 9.3 Hz, 2H).
7-[4-(2,4-Bis-benzyloxy-5-isopropyl-benzoylamino)-phenylcarbamoyl]-heptanoic acid methyl
ester (42). HBTU (1.82 g, 3.59 mmol), DIPEA (0.84 mL, 4.80 mmol) and monomethyl suberate
(0.93 mL, 5.28 mmol) were added to a solution of 34 (1.0 g, 4.80 mmol) in DMF (10 mL) and the
mixture was stirred for 12 h at rt. Then the reaction was quenched with H2O and extracted using
EtOAc, dried over MgSO4 and passed through a filter column to give corresponding ester which was
dissolved in the minimum amount of dioxane. H2O with pH adjusted to 3 using 3N HCl was added
and the reaction mixture was stirred at reflux overnight to yield the corresponding free amine. The
reaction was basified and extracted using EtOAc, then dried, concentrated and passed through a filter
column to give the free amine. To a solution of this free amine (1.0 g, 3.59 mmol) in DMF (10 mL)
was added HBTU (1.36 g, 3.59 mmol), DIPEA (0.66 mL, 3.59 mmol) and 1,4-dibenzyloxy-5-
o
isopropyl benzoic acid (0.76 g, 4.31 mmol) and the solution was stirred for 12 h at 80 C . The
reaction mixture was quenched with H2O and extracted with EtOAc (25 mL × 3). The combined
organic layer was collected, dried over anhydrous MgSO4 and concentrated under reduced pressure
to give a light yellow residue, which was purified by silica gel chromatography (EtOAc:n-hexane = 1:
1
1) to give 42 as a colorless liquid in 65% yield (overall from 34), H NMR (300 MHz, CD3OD): δ
1.27 (t, J = 6.9 Hz, 6H), 1.38- 1.41 (m, 4H), 1.62 – 1.69 (m, 4H), 2.32 - 2.38 (m, 4H), 3.65. (s,
3H),5.25 (s, 2H), 5.29 (s, 2H), 6.92 (s, 1H), 7.18(d, J = 9.0 Hz, 2H), 7.38 – 7.51 (m, 10 H), 7.56 –
7.58 (m, 2H), 7.98 (s, 1H).
Octanedioic acid [4-(2,4-dihydroxy-5-isopropyl-benzoylamino)phenyl]amide hydroxyamide
o
(17). A mixture of 1N LiOH (7 mL) and 42 (1 g, 1.57 mmol) was stirred at 40 C for 2 h. The
reaction was concentrated under reduced pressure and then H2O was added. The mixture was

acidified with 3N HCl to give an off-white liquid. The off-white liquid (0.07 g, 0.11 mmol) was
dissolved in DMF (1 mL) and EDC•HCl (0.03 g, 0.16 mmol), HOBt (0.02 g, 0.16 mmol), and NMM
(0.04 mL, 0.38 mmol) were added. After being stirred at room temperature for 30 min, NH2OBn
(0.02 g, 0.12 mmol) was added and the mixture was stirred for an additional 5 h. The reaction
mixture was quenched with H2O and was extracted with EtOAc (25 mL × 3). The combined organic
layer was collected, dried over anhydrous MgSO4 and concentrated under reduced pressure to give a
light yellow residue, which was purified by silica gel chromatography (EtOAc:n-hexane = 1: 1) to
give a colorless liquid. To a solution of the resulting product in methanol (10 mL) was added 10%
Pd/C and the mixture was stirred at room temperature for 3 h under H2. The reaction mixture was
filtered through celite, concentrated under reduced pressure and purified by column to afford the
desired compound (17), in 63% yield (from 42); 1H NMR (300 MHz, CD3OD): δ 1.25 (d, J = 6.9 Hz,
6H), 1.39-1.42 (m, 4H), 1.62-1.72 (m, 4H), 2.12 (t, J = 7.5 Hz, 2H), 2.38 (t, J = 7.5 Hz, 2H), 3.17-
13
3.26 (m, 1H), 6.35 (s, 1H), 7.53-7.60 (m, 4H), 7.75 (s, 1H). C NMR (300 MHz, CD3OD): 25.21,
25.37, 26.59, 28.44, 28.52, 36.44, 102.26, 107.54, 120.22, 121.84, 126.08, 127.30, 134.15, 135.00,
160.07, 173.13. LRMS m/z: calculated 480.49 found 480.2 (M+Na). HRMS (ESI) C24H32N3O6
(M+H+) calcd, 458.2291; found, 458.2287.
N-3-Nitrophenyl-t-butylcarbamate (32). Compound 32 was synthesized using 3-nitroaniline (29,
0.5 g, 4.20 mmol), TEA (0.59 mL, 4.20 mmol), DMAP (0.25 g, 2.10 mmol) and di-tert-butyl
dicarbonate (0.91 g, 4.20 mmol) following the method used for the synthesis of compound 31, to
give 32 in 81% yield, 1H NMR (300 MHz, CDCl3): δ 1.58 (s, 9H), 6.69 (bs, 1H), 7.72 (d, J = 7.8 Hz,
1H), 7.85 – 8.02 (m, 3H).
N-3-Aminophenyl-t-butylcarbamate (38). Compound 38 was synthesized using 32 (1.1 g, 4.45
mmol) dissolved in a mixture of IPA:H2O (4:1), iron powder (0.74 g, 13.35 mmol) and NH4Cl (0.47
g, 8.89 mmol) following the method used for the synthesis of compound 34, to give 38 as white solid
in 79% yield, 1H NMR (300 MHz, CD3OD): δ 1.46 (s, 9H), 6.85 (d, J = 7.8 Hz, 1H), 7.07 – 7.13 (m,

2H), 7.20 (d, J = 8.1 Hz, 1H), 7.26 (s, 1H).
7-[3-(2,4-Bis-benzyloxy-5-isopropyl-benzoylamino)-phenylcarbamoyl]-heptanoic acid methyl
ester (43). Compound 43 was synthesized using 38 (1.0 g, 4.80 mmol) in DMF (10 mL), HBTU
(1.82 g, 3.59 mmol), DIPEA (0.84 mL, 4.80 mmol) and monomethyl suberate (0.93 mL, 5.28 mmol)
following the method used for the synthesis of compound 42, to give 43 as a colorless liquid in 62%
yield (overall from 38); 1H NMR (300 MHz, CDCl3): δ 1.01-1.11 (m, 6H), 1.37-1.40 (m, 4H), 1.77-
1.88 (m, 4H), 2.33 (t, J = 7.2 Hz , 2H), 2.41 – 2.42 (m, 2H), 3.27-3.36 (m, 1H), 3.64. (s, 3H), 5.05(s,
2H), 5.26 (s, 2H), 6.54 (s, 1H), 7.39 – 7.40 (m, 15 H), 7.82 (s, 1H).
Octanedioic acid [3-(2,4-dihydroxy-5-isopropyl-benzoylamino)-phenyl]-amide hydroxyamide
(18). Compound 18 was synthesized using a mixture of 1N LiOH (1 mL) and 43 (0.1 g, 0.15 mmol)
following the method used for the synthesis of compound 17, to afford the desired compound 18 as
an off white liquid, in 59% yield (from 43); 1H NMR (300 MHz, DMSO-d6): δ 1.18 (d, J = 6.9 Hz,
6H), 1.23-1.28 (m, 4H), 1.44-1.59 (m, 4H), 1.93 (t, J = 7.5 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 3.06-
3.24 (m, 1H), 6.37 (s, 1H), 7.22-7.35 (m, 3H), 7.76 (s, 1H), 7.96 (s, 1H), 8.65 (s, 1H), 9.90 (s, 1H),
10.14 (d, J = 8.4 Hz, 2H), 10.33 (s, 1H). 13C NMR (300 MHz, CD3OD): 13.66, 21.98, 24.87, 25.18,
25.43, 26.48, 28.46, 28.52, 28.64, 28.66, 30.48, 32.37, 34.11, 34.51, 35.78, 36.01, 102.52, 107.50,
125.19, 125.38, 125.68, 126.18, 127.62, 130.01, 132.09, 132.14, 155.14, 159.23, 160.33, 163.54,
167.77, 167.90, 171.71, 174.39, 174.45. LRMS m/z: calculated 480.49 found 480.1 (M+Na+). HRMS
(ESI) C24H32N3O6 (M+H+), calcd, 458.2288; found, 458.2286.
N-2-Nitrophenyl-t-butylcarbamate (33). Compound 33 was synthesized using 2-nitroaniline (30,
0.5 g, 4.20 mmol), TEA (0.59 mL, 4.20 mmol) and DMAP (0.25 g, 2.10 mmol) following the method
used for the synthesis of compound 31, to give 33 in 72% yield, 1H NMR (300 MHz, CDCl3): δ 1.56
(s, 9H), 7.07-7.13 (m, 1H), 7.59 -7.76 (m, 1H), 8.20 (dd, J = 8.4, Hz J = 9.9 Hz, 1H), 8.57 (dd, J =
8.4, Hz J = 8.7 Hz, 1H), 9.68 (s, 1H).
N-2-Aminophenyl-t-butylcarbamate (41). Compound 41 was synthesized using 33 (1.0 g, 4.40

mmol) which was dissolved in a mixture of IPA:H2O (4:1) and iron powder (0.69 g, 12.88 mmol),
NH4Cl (0.39 g, 8.72 mmol) following the method used for the synthesis of compound 34, to give 41
as white solid in 77% yield, 1H NMR (300 MHz, CD3OD): δ 1.48 (s, 9H),7.14 - 7.24 (m, 2H) 7.35. (d,
J = 8.1 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H).
7-[2-(2,4-Bis-benzyloxy-5-isopropyl-benzoylamino)-phenylcarbamoyl]-heptanoic acid methyl
ester (44). Compound 44 was synthesized using 41 (1.0 g, 4.80 mmol) in DMF (10 mL), HBTU
(1.82 g, 3.59 mmol), DIPEA (0.84 mL, 4.80 mmol) and monomethyl suberate (0.93 mL, 5.28 mmol)
following the method used for the synthesis of compound 42, to give 44 as a colorless liquid in 57%
1
yield (overall from 41), H NMR (300 MHz, CD3OD): δ 1.11- 1.26 (m, 6H), 1.31-1.36 (m, 4H),
1.52-1.74 (m, 4H), 2.29-2.25 (m, 2H), 2.37-2.45 (m, 2H), 3.10-3.21 (m, 1H), 3.66. (s, 3H), 5.15(s,
2H), 5.31 (s, 2H), 6.78 (s, 1H), 7.14-7.22 (m, 2 H), 7.35-7.54 (s, 14H).
Octanedioic acid [2-(2,4-dihydroxy-5-isopropyl-benzoylamino)-phenyl]-amide hydroxyamide
(19). Compound 19 was synthesized using 1N LiOH (1 mL) and 44 (0.1 g, 0.15 mmol) following the
method used for the synthesis of compound 17, to give 19 as a colorless liquid in 49% yield (from
44); 1H NMR (300 MHz, CD3OD): δ 1.25 (d, J = 6.9 Hz, 7H), 1.29-1.34 (m, 4H), 1.49-1.58 (m, 2H),
1.65-1.72 (m, 2H) 2.02-2.04 (m, 2H), 2.44 (t, J = 7.5 Hz, 2H), 3.17-3.26 (m, 1H), 6.37 (s, 1H), 7.20-
7.25 (m, 1H), 7.28-7.33 (m, 2H), 7.68 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H). 13C NMR (300 MHz,
DMSO-d6): 23.13, 25.47, 25.55, 26.39, 28.87, 31.26, 32.71, 36.17, 36.26, 103.05, 108.48, 124.71,
124.91, 126.32, 126.86, 127.01, 130.30, 159.06, 160.08, 162.81, 166.60, 169.61, 172.87. LRMS m/z:
calculated 480.49 found 480.2 (M+Na). HRMS (ESI) C24H32N3O6 (M+H+) calcd, 458.2287; found,
458.2286
N-4-Aminophenyl-N-methyl-t-butylcarbamate (35). 60% NaH (0.36 g, 9 mmol) was added at 0 oC
to a solution of 31 (1.4 g, 6 mmol) in DMF and the mixture was stirred for 5 min before adding CH3I
(0.75 mL, 9 mmol). The reaction mixture was stirred for another 30 min at rt, then quenched by slow
addition of H2O and extracted with EtOAc (25 mL × 3). The combined organic layer was dried over

anhydrous MgSO4 and concentrated under reduced pressure. This dried product (1.6 g, 6.46 mmol)
was dissolved in a mixture of IPA:H2O (4:1) then iron powder (1.1 g, 19.38 mmol) and NH4Cl (0.7 g,
12.92 mmol) were added following the procedure used for the synthesis of compound 34, to give 35
as white solid in 79% yield (from 31), 1H NMR (300 MHz, CD3OD): δ 1.48 (s, 9H), 2.59 (s, 3H),
6.82. (d, J = 9.0 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H).
7-{4-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-methyl-amino]-phenylcarbamoyl}-heptanoic
acid methyl ester (45). Compound 45 was synthesized using 35 (1.0 g, 4.50 mmol) in DMF (10 mL),
HBTU (1.71 g, 4.50 mmol), DIPEA (0.78 mL, 4.50 mmol) and monomethyl suberate (0.87 mL, 4.95
mmol), following the method used for synthesis of compound 42, to 45 give as a colorless liquid in
o
1
61% yield (overall from 35), mp: 138-140 C. H NMR (300 MHz, CD3OD): δ 1.07 (bs, 6H), 1.31-
1.38 (m, 4H), 1.63 – 1.68 (m, 4H), 2.32 - 2.34 (m, 4H), 3.12-3.20 (m, 1H), 3.59 (s, 3H), 3.64. (s, 3H),
4.95(bs, 4H), 6.46 (s, 1H), 6.93 – 6.97 (m, 3H), 7.33-7.47 (m, 12 H).
Octanedioic
acid
{4-[(2,4-dihydroxy-5-isopropyl-benzoyl)-methyl-amino]-phenyl}-amide
hydroxyamide (20). Compound 20 was synthesized using 1N LiOH (7 mL) and 45 (1 g, 1.53 mmol)
following the method used for synthesis of compound 17, to give 20 as white solid, in 64% yield
(from 45); mp: 164-165 oC. 1H NMR (300 MHz, CD3OD): δ 0.82 (d, J = 6.6 Hz, 6H), 1.31-1.38 (m,
4H), 1.64-1.69 (m, 4H), 2.10 (t, J = 7.2 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.88-2.97 (m, 1H), 3.41 (s,
13
3H), 6.19 (s, 1H), 6.61 (s, 1H), 7.12 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H). C NMR (300
MHz, CD3OD): 21.53, 25.21, 25.31, 25.48, 28.46, 32.32, 36.45, 37.71, 101.93, 109.02, 120.41,
125.61, 126.87, 128.03, 137.44, 141.09, 158.07, 158.33, 171.92, 173.19. LRMS m/z: calculated
494.52 found 494.2 (M+Na). HRMS (ESI) C25H34N3O6 (M+H+) calcd, 472.2448; found, 472.2444.
N-3-Aminophenyl- N-methyl-t-butylcarbamate (39). Compound 39 was synthesized using 32 (1.4
g, 6 mmol) in DMF, 60% NaH (0.36 g, 9 mmol) and CH3I (0.75 mL, 9 mmol) following the method
used for the synthesis of compound 35, to give 39 as white solid in 72% yield (from 32), 1H NMR
(300 MHz, CDCl3): δ 1.42 (s, 9H), 3.23 (s, 3H), 6.96-6.70. (m, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.35-

7.39 (m, 1H), 7.59 (t, J = 2.1 Hz, 1H).
7-{3-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-methyl-amino]-phenylcarbamoyl}-heptanoic
acid methyl ester (46). Compound 46 was synthesized using 39 (1.0 g, 4.50 mmol) in DMF (10 mL),
HBTU (1.71 g, 4.50 mmol), DIPEA (0.78 mL, 4.50 mmol) and monomethyl suberate (0.87 mL, 4.95
mmol) following the method used for synthesis of compound 42, to give 46 as a colorless liquid in
56% yield (overall from 39), 1H NMR (300 MHz, CD3OD): δ 1.07-1.09 (m, 6H), 1.31-1.36 (m, 4H),
1.59-1.68 (m, 4H), 2.88-2.34 (m, 4H), 3.12-3.19 (m, 1H), 3.41 (s, 3H), 3.64. (s, 3H), 4.92-4.97 (m,
4H), 6.46 (s, 1H), 6.74 (s, 1H), 6.99-6.71 (m, 2H), 7.24 -7.39 (m, 10 H), 7.56 (s, 1H).
Octanedioic
acid
{3-[(2,4-dihydroxy-5-isopropyl-benzoyl)-methyl-amino]-phenyl}-amide
hydroxyamide (21). Compound 21 was synthesized using 1N LiOH (1 mL) and 46 (0.1 g, 0.15
mmol) following the method used for the synthesis of compound 17, to give 21, in 61% yield (from
o
46); mp: 173-174 C 1H NMR (300 MHz, DMSO-d6): δ 0.77 (d, J = 6.9 Hz, 6H), 1.23 (bs, 8H),
1.42-1.53 (m, 4H), 1.90-1.95 (m, 2H), 2.24 (t, J = 7.5 Hz, 2H), 2.79-2.86 (m, 1H), 3.29 (s, 3H), 6.19
(s, 1H), 6.60 (s, 1H), 6.86 (d, J = 7.8 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.51
(s, 1H), 8.64 (s,1H), 9.72 (s,1H), 9.78 (s,1H), 10.31 (s, 1H), 10.70 (s, 1H). 13C NMR (300 MHz,
CD3OD): 21.60, 25.13, 25.24, 25.32, 25.49, 28.46, 28.55, 28.71, 30.38, 32.37, 34.05, 35.00, 35.68,
36.51, 37.88, 102.03, 108.51, 117.95, 118.08, 121.84, 125.65, 128.17, 129.54, 139.92, 146.03,
158.29, 158.80, 163.52, 171.64, 171.92, 173.33. LRMS m/z: calculated 494.52 found 494.2 (M+Na+).
HRMS (ESI) C25H34N3O6 (M+H+) calcd, 472.2444; found, 472.2442.
N-4-Aminophenyl-N-ethyl-t-butylcarbamate (36). Compound 36 was synthesized using 31 (0.93 g,
3.90 mmol) in DMF, 60% NaH (0.24 g, 5.85 mmol) and ethyl iodide (0.62 mL, 3.90 mmol)
following the method used for the synthesis of compound 35, to give 36 as white solid in 81% yield
(from 31), 1H NMR (300 MHz, CD3OD): δ 1.23 (t, J = 7.2 Hz, 3H), 1.42 (s, 9H) 3.10-3.13 (m, 2H),
6.63. (d, J = 9.0 Hz, 2H), 7.28 (d, J = 9.0 Hz, 2H).
7-{4-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-ethyl-amino]-phenylcarbamoyl}-heptanoic acid

methyl ester (47). Compound 47 was synthesized using 36 (1.0 g, 4.23 mmol) in DMF (10 mL),
HBTU (1.60 g, 4.23 mmol), DIPEA (0.74 mL, 4.23 mmol) and monomethyl suberate (0.82 mL, 4.65
mmol) following the method used for the synthesis of compound 42, to give 47 as a colorless liquid
in 54% yield (overall from 36), 1H NMR (300 MHz, CD3OD): δ 0.78 (d, J = 7.2 Hz, 6H), 1.23 (t, J =
7.2 Hz, 3H), 1.22-1.27 (m, 4H), 1.31-1.41 (m, 4H), 2.30 - 2.34 (m, 4H), 3.06-3.08 (m, 1H), 3.09-3.14
(m, 2H), 3.66 (s, 3H), 5.15(s, 2H), 5.21 (s, 2H), 6.89 (s, 1H), 7.10 (d, J = 8.2 Hz, 2H), 7.33-7.52 (m,
13H).
Octanedioic
acid
{4-[(2,4-dihydroxy-5-isopropyl-benzoyl)-ethyl-amino]-phenyl}-amide
hydroxyamide (22). Compound 22 was synthesized using 1N LiOH (7 mL) and 47 (1 g, 1.50 mmol)
following the method used for the synthesis of compound 17, to give 22, in 59% yield (from 47); 1H
NMR (300 MHz, CD3OD): δ 0.82 (d, J = 6.9 Hz, 6H), 1.21 (t, J = 6.3 Hz, 3H), 1.38-1.40 (m, 4H),
1.62-1.70 (m, 4H), 2.11 (t, J = 6.9 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.90-2.97 (m, 1H), 3.89-3.96 (m,
13
2H), 6.19 (t, J = 0.9 Hz, 1H), 6.61 (s, 1H), 7.11 (d, J = 7.8 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H). C
NMR (300 MHz, CD3OD): 11.78, 21.70, 25.05, 25.27, 25.37, 25.53, 28.48, 28.49, 28.60, 28.69,
32.41, 34.77, 35.79, 36.55, 45.50, 102.09, 109.16, 120.48, 120.58, 125.61, 127.85, 128.05, 137.56,
139.18, 158.01, 158.53, 171.44, 171.69, 173.29. LRMS (M+Na) m/z: calculated 508.55 found 508.2.
HRMS (ESI) C26H36N3O6 (M+H+) calcd, 486.2599; found, 486.2601.
N-3-Aminophenyl- N-ethyl-t-butylcarbamate (40). Compound 40 was synthesized using 32 (1.0 g,
3.98 mmol) in DMF, 60% NaH (0.25 g, 5.92 mmol) and C2H5I (0.66 mL, 4.10 mmol) following the
method used for the synthesis of compound 35, to give 40 as white solid in 74% yield (from 32), 1H
NMR (300 MHz, CDCl3): δ 1.15 (t, J = 7.2 Hz, 3H), 1.42 (s, 9H) 3.42-3.13 (m, 2H), 6.50-6.57. (m,
1H), 7.13-7.14 (m, 1H), 7.32-7.38 (m, 1H), 7.41 (t, J =2.1 Hz, 1H).
7-{3-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-ethyl-amino]-phenylcarbamoyl}-heptanoic acid
methyl ester (48). Compound 48 was synthesized using 40 (1.0 g, 4.23 mmol) in DMF (10 mL),
HBTU (1.60 g, 4.23 mmol), DIPEA (0.74 mL, 4.23 mmol) and monomethyl suberate (0.82 mL, 4.65

mmol) following the method used for synthesis of compound 42, to give 48 as a colorless liquid in
1
49% yield (overall from 40), H NMR (300 MHz, DMSO-d6): δ 0.98-1.06 (m, 9H), 1.23-1.26 (m,
4H), 1.50- 1.52 (m, 4H), 2.21-2.30 (m, 4H), 3.02-3.10 (m, 1H), 3.56 (s, 3H), 3.72-3.80 (m, 2H),
5.01-5.14 (m, 4H), 6.64-6.65 (m, 2H), 6.86-6.88 (m, 1H), 7.07-7.46 (m, 1H), 7.23-7.46 (m, 13H),
7.57 (s, 1H), 9.81 (s, 1H).
Octanedioic
acid
{3-[(2,4-dihydroxy-5-isopropyl-benzoyl)-ethyl-amino]-phenyl}-amide
hydroxyamide (23). Compound 23 was synthesized using 1N LiOH (7 mL) and 48 (1 g, 1.50 mmol)
following the method used for the synthesis of compound 17, to give 23 in 55% yield (from 48); 1H
NMR (300 MHz, DMSO-d6): δ 0.75 (d, J = 6.9 Hz, 6H), 1.09 (t, J = 7.2 Hz, 3H), 1.23 (bs, 9H),
1.42-1.53 (m, 4H), 1.90-1.97 (m, 2H), 2.25 (t, J = 7.2 Hz, 2H), 2.78-2.85 (m, 1H), 3.74-3.81 (m, 2H),
6.18 (s, 1H), 6.57 (s, 1H), 6.83 (d, J = 9.0 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 7.44-7.49 (m, 2H), 8.64
(s, 1H), 9.72 (s, 1H), 9.90 (s, 1H), 10.32 (s, 1H), 10.84 (s, 1H). 13C NMR (300 MHz, CD3OD): 11.74,
21.59, 25.23, 25.31, 25.47, 28.45, 28.54, 32.36, 36.51, 45.58, 101.98, 108.62, 118.02, 118.87, 122.76,
125.55, 128.11, 129.45, 139.94, 144.31, 158.20, 159.02, 171.44, 171.64, 173.34. LRMS m/z:
calculated 508.55 found 508.2 (M+Na). HRMS (ESI) C26H36N3O6 (M+H+) calcd, 486.2599; found,
486.2601.
N-4-Aminophenyl- N-isopropyl-t-butylcarbamate (37). 60% NaH (0.25 g, 6.28 mmol) was added
at 0 oC to a solution of 31 (1 g, 4.19 mmol) in DMF and stirred for 5 mins before adding C2H5I (0.62
o
mL, 3.90 mmol). The reaction mixture was stirred for another 30 min at rt and then heated at 50 C
for 5 h following the method used for synthesis of compound 35, to give 37 as an off white solid in
69% yield (from 31), 1H NMR (300 MHz, CDCl3): δ 1.07 (d, J = 6.6 Hz, 6H), 1.38 (s, 9H), 4.44-4.45
(m, 1H), 6.88-6.95 (m, 4H).
7-{4-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-isopropyl-amino]-phenylcarbamoyl}-heptanoic
acid methyl ester (49). Compound 49 was synthesized using 37 (0.28 g, 1.12 mmol) in DMF (5 mL),
HBTU (0.42 g, 1.12 mmol), DIPEA (0.19 mL, 1.12 mmol) and monomethyl suberate (0.22 mL, 1.23

mmol) following the method used for the synthesis of compound 42, to give 49 as a colorless liquid
1
in 49% yield (overall from 37), H NMR (300 MHz, CD3OD): δ 1.01-1.09 (m, 12H), 1.10-1.29 (m,
4H), 1.51-1.58 (m, 4H), 2.02-2.17 (m, 2H), 2.30-2.33 (m, 2H), 3.11-3.12 (m, 1H), 3.69 (s, 3H),
4.93(s, 3H), 4.96 (s, 2H), 6.44 (s, 1H), 6.87-6.92 (m, 3H), 7.30-7.45 (m, 12 H).
Octanedioic acid {4-[(2,4-dihydroxy-5-isopropyl-benzoyl)-isopropyl-amino]-phenyl}-amide
hydroxyamide (24). Compound 24 was synthesized using 1N LiOH (1 mL) and 49 (0.1 g, 1.47
mmol) following the method used for the synthesis of compound 17, to give 24 in 51% yield (from
49); 1H NMR (300 MHz, CD3OD): δ 0.85 (d, J = 6.9 Hz, 6H), 1.18 (d, J = 6.9Hz, 6H), 1.36 (bs, 4H),
1.61-1.64 (m, 4H), 2.00-2.10 (m, 2H), 2.34 (t, J = 7.2 Hz, 2H), 2.85-2.98 (m, 1H), 4.93-4.97 (m, 1H),
13
6.13 (s, 1H), 6.58 (s, 1H), 7.07 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H). C NMR (300 MHz,
CD3OD): 19.95, 21.67, 25.21, 25.31, 25.60, 28.46, 28.55, 36.49, 70.06, 101.87, 119.71, 125.46,
127.16, 130.29, 138.15, 157.17, 171.75, 173.24. LRMS (ESI) m/z: calcd, (M+H+) 500.2 found 500.3.
HRMS (ESI) C27H38N3O6 (M+H+) calcd, 500.2757; found, 500.2755.
6-{4-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-methyl-amino]-phenylcarbamoyl}-hexanoic acid
ethyl ester (50). Compound 50 was synthesized using 35 (0.62 g, 2.78 mmol) in DMF (5 mL),
HBTU (1.05 g, 2.78 mmol), DIPEA (0.48 mL, 2.78 mmol) and monoethyl pimelate (0.54 mL, 2.36
mmol) following the method used for the synthesis of compound 42, to give 50 as a colorless liquid
1
in 63% yield (overall from 35), H NMR (300 MHz, CD3OD): δ 1.11-1.26 (m, 9H), 1.62-1.77 (m,
4H), 2.30-2.38 (m, 4H), 2.37-2.45 (m, 2H), 3.01-3.13 (m, 1H), 3.16-3.32 (m, 2H), 3.42. (s, 3H),
4.08-4.15 (m, 2H), 5.15(s, 2H), 5.28 (s, 2H), 6.78 (s, 1H), 7.14 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.7
Hz, 2H), 7.78-7.84 (m, 12H).
Heptanedioic acid [4-(2,4-dihydroxy-5-isopropyl-benzoylamino)-phenyl]-amide hydroxyamide
(25). Compound 25 was synthesized using 1N LiOH (3 mL) and 50 (0.5 g, 0.84 mmol) following the
method used for the synthesis of compound 17, to give 25, in 53% yield (from 50); 1H NMR (300
MHz, CD3OD): δ 0.82 (d, J = 6.9 Hz, 6H), 1.36-1.44 (m, 2H), 1.61-1.75 (m, 4H), 2.11 (t, J = 7.2 Hz,

2H), 2.36 (t, J = 7.5 Hz, 2H), 2.88-2.97 (m, 1H), 3.40 (s, 3H), 6.20 (s, 1H), 6.62 (s, 1H), 7.11 (d, J =
9 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H). 13C NMR (300 MHz, CD3OD): 21.70, 25.08, 25.56, 32.27, 36.38,
37.93, 102.13, 109.16, 120.55, 125.73, 126.90, 128.09, 137.37, 141.04, 158.06, 158.21, 171.60,
171.92, 173.16. LRMS (M+H+) m/z: calculated 458.2 found 458.2. HRMS (ESI) C24H33N3O6 (M+H+)
calcd, 458.2287; found, 458.2286.
8-{4-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-methyl-amino]-phenylcarbamoyl}-octanoic acid
methyl ester (51). Compound 51 was synthesized using 35 (0.57 g, 2.56 mmol) in DMF (5 mL),
HBTU (0.97 g, 2.56 mmol), DIPEA (0.45 mL, 2.56 mmol) and monomethyl azelate (0.55 mL, 2.84
mmol) following the method used for the synthesis of compound 42, to give colorless liquid as 51 in
63% yield (overall from 35), 1H NMR (300 MHz, CD3OD): δ 1.52-1.53 (m, 4H), 1.76-1.81 (m, 6H),
2.03-2.12 (m, 4H), 2.73-2.78(m, 4H), 3.21-3.25 (m, 1H), 3.77 (m, 3H), 4.08 (s, 3H), 5.40 (bs, 4H),
6.91 (s, 1H), 7.39-7.42 (m, 2 H), 7.77-7.84 (m, 14H).
Nonanedioic acid [4-(2,4-dihydroxy-5-isopropyl-benzoylamino)-phenyl]-amide hydroxyamide
(26). Compound 26 was synthesized using 1N LiOH (2 mL) and 51 (0.4 g, 0.58 mmol) following the
method used for the synthesis of compound 17, to give 26, in 51% yield (from 51); 1H NMR (300
MHz, CD3OD): δ 0.76 (d, J = 6.9 Hz, 6H), 1.25-1.32 (m, 6H), 1.55-1.65 (m, 4H), 2.04 (t, J = 7.5 Hz,
2H), 2.30 (t, J = 7.5 Hz, 2H), 2.83-2.92 (m, 1H), 3.36 (s, 3H), 6.15 (s, 1H), 6.56 (s, 1H), 7.07 (d, J =
13
8.7 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H). C NMR (300 MHz, CD3OD): 21.57, 25.32, 25.45, 25.50,
28.69, 28.68, 28.74, 37.76, 101.97, 120.42, 125.61, 126.88, 128.06, 141.08, 158.08. LRMS (M+H+)
m/z: calcd. 486.5 found 486.2. HRMS (ESI) C26H36N3O6 (M+H+) calcd, 486.2599; found, 486.260.
9-{4-[(2,4-Bis-benzyloxy-5-isopropyl-benzoyl)-methyl-amino]-phenylcarbamoyl}-nonanoic acid
methyl ester (52). Compound 52 was synthesized using 35 (0.6 g, 2.69 mmol) in DMF (5 mL),
HBTU (1.02 g, 2.69 mmol), DIPEA (0.47 mL, 2.69 mmol) and monomethyl sebacate (0.64 g, 2.96
mmol) following the method used for the synthesis of compound 42, to give 52 as a colorless liquid
1
in 63% yield (overall from 35), H NMR (300 MHz, CD3OD): δ 1.06- 1.08 (m, 6H), 1.31-1.34 (s,

8H), 1.59- 1.68 (m, 4H), 2.28-2.36 (m, 4H), 3.11-3.22 (m, 1H), 3.64 (s, 3H), 4.94 (bs, 4H), 6.45 (s,
1H), 6.91-6.97 (m, 2H), 7.32-7.49 (m, 14H).
Decanedioic acid [4-(2,4-dihydroxy-5-isopropyl-benzoylamino)-phenyl]-amide hydroxyamide
(27). Compound 27 was synthesized using 1N LiOH (2 mL) and 52 (0.3 g, 0.44 mmol) following the
method used for the synthesis of compound 17, to give 27, in 53% yield (from 52); 1H NMR (300
MHz, CD3OD): δ 0.82 (d, J = 6.6 Hz, 6H), 1.31-1.35 (m, 10H), 1.60-1.68 (m, 4H), 2.09 (t, J = 7.5
Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 2.89-2.95 (m, 1H), 3.42 (s, 3H), 6.20 (s, 1H), 6.61 (s, 1H), 7.13 (d,
J = 8.7 Hz, 2H), 7.56 (d, J = 9.0 Hz, 2H). 13C NMR (300 MHz, CD3OD): 21.64, 25.07, 25.38, 25.52,
28.73, 28.82, 28.85, 28.91, 32.44, 36.63, 37.87, 102.05, 108.93, 120.47, 125.64, 126.88, 128.10,
137.46, 141.05, 158.12, 158.44, 171.71, 171.91, 173.34. LRMS (ESI) m/z: calcd, (M+H+) 500.2
found 500.3. HRMS (ESI) C27H38N3O6 (M+H+) calcd, 500.2759; found, 500.2755
4.2 Biology
4.2.1 Tumor Cell Culture. All human cancer cells were maintained in RPMI 1640 medium
supplemented with 10% FBS and penicillin (100 units/ml)/streptomycin (100 µg/ml)/amphotericin B
(0.25 µg/ml). All cells were maintained in humidified air containing 5% CO2 at 37°C and cultured
every 2-3 days. All cells were cultured in tissue culture flasks in a humidified air containing 5% CO2
and 95% air at 37 ° C and cultured every 2-3 days.
4.2.2 Sulforhodamine B Assays. Cells were seeded at a density of 5000 cells/well into 96-plate
overnight. Basal cells were fixed with 10% trichloroacetic acid (TCA) to represent cell population at
the time of compound addition (T0). After additional incubation of DMSO (C) or different doses of
test compounds (Tx) for 48 h, cells were fixed with 10 % TCA and stained with sulforhodamine B
(SRB) at 0.4 % (w/v) in 1% AcOH (acetic acid). Unbound SRB was washed out using 1% AcOH and
SRB-bound cells were solubilized with 10 mM Trizma base. The absorbance was read at a
wavelength of 515 nm. The 50% growth inhibition (GI50) was calculated as 100 − [(Tx – T0) / (C –

T0)] × 100.
4.2.3 HeLa Nuclear Extract HDAC Activity Assay. HDAC Fluorescent Activity Assay Kit
(BioVision, CA) was used to detect HeLa nuclear extract HDAC activity according to manufacturer’s
instructions. Briefly, the HDAC fluorometric substrate and assay buffer were added to HeLa nuclear
extracts in a 96-well format and incubated at 37°C for 30 min. The reaction was terminated by
addition of lysine developer, and the mixture was incubated for another 30 min at 37°C. Additional
negative controls included incubation without the nuclear extract, without the substrate, or without
both. A fluorescence plate reader with excitation at 355 nm and emission at 460 nm was used to
quantify HDAC activity.
4.2.4 HDAC Biochemical Assays. Enzyme inhibition assays (HDAC1-11 and SIRT1) were
conducted by the Reaction Biology Corporation, Malvern, PA. (www.reactionbiology.com)
Compounds were dissolved in DMSO and tested in 10-dose IC50 mode with 3-fold serial dilution
starting at 10 µM. HDAC Control Compound trichostatin A was tested in a 10-dose IC50 with 3-fold
serial dilution starting at 10 µM as shown in supporting information.
4.2.5 Heat shock protein (HSP) 90α activity assay. An HSP90α activity assay kit (BPS Bioscience,
San Diego, CA, USA) was used to determine the compound inhibitory effect of HSP90α activity
according to manufacturer's instructions. Briefly, the reactions were conducted at room temperature
for 3ꢀh in a 100ꢀµL mixture containing FITC-labeled geldanamycin, Hsp90α enzyme, and test
compounds in assay buffer. A fluorescence polarization plate reader with excitation at 475-495ꢀnm
and emission at 518–538ꢀnm was used to quantify HSP90α activity.
4.2.6 FACScan Flow Cytometric analysis. Cells were seeded in 6-well plates (2.5 × 105/well) and
incubated overnight. Next day cells were treated with DMSO or indicated compounds at various
concentrations for 48 h. Cells were washed with phosphate-buffered saline, fixed in ice-cold 70%
ethanol at −20 °C overnight, and stained with propidium iodide (80 ug/ml) containing Triton X-100
(0.1%, v/v) and RNase A (100 ug/ml) in phosphate-buffered saline. DNA content was counted and

analyzed with the FACScan and CellQuest software (Becton Dickinson, Mountain View, CA, USA).
4.2.7 Western Blot Analysis. Cells were incubated with indicated compounds for 24 h and lysed
with ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA,
1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 2.5 mM β-glycerolphosphate, 1 mM Na4P2O7, 5
mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail from Millipore) on ice for 30 min followed
by centrifugation at 13000 rpm for 30 min. Protein concentrations were determined and equal
amounts of protein were separated by 8-15% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS–PAGE) and transferred to poly(vinylidene difluoride) (PVDF) membranes.
Membranes were immunoblotted with specific antibodies overnight at 4°C and then applied to
appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies
for 1 h at room temperature. Signals were detected using an enhanced chemiluminescence
(Amersham, Buckinghamshire, UK). Primary antibodies against heat shock protein 70 (HSP70) and
other proteins were purchased from Cell Signalling Technology (Danvers, MA, USA).
4.2.8 Western blot analysis of PD-L1 expression. Cell lysates of H1975 cells which were treated
with DMSO, Compound 20, Compound 26, or IFN-γ at various concentrations were electrophoresed
in 10% reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk
(dissolved in phosphate-buffered saline containing 0.05% Tween 20 (PBST)) and incubated with
mouse anti-PDL-1 antibody (Cat#AW5698;ABGENT, San Diego, CA, U.S.A.) or mouse anti-β-
actin (Cat# A5416; Sigma-Aldrich, St. Louis, MO, U.S.A.) for 1h. HRP-conjugated goat anti-mouse
IgG antibodies (1 µg/ml; Cat# 115-035-003; Jackson ImmunoResearch Laboratories, West Grove, PA,
U.S.A.) and enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Waltham, MA,
U.S.A.) were used to detect protein signals.
4.2.9 Analysis of PD-L1 expression on cell surface by flow cytometry. H1975 cells were seeded in
6 well-plates (3.2 x 105 cells/well) and treated with DMSO, Compound 20, 26, or IFN-γ at various

concentrations for 48h. After removing supernatant, cells were suspended at 3 x 105 cells/tube in
PBS buffer containing 0.05% (w/v) BSA (PBS/BSA) and then incubated with 5 µg/ml of mouse
anti-PD-L1 antibody (Cat# 14-5983-82; Thermo Fisher Scientific) for 1h. Then cells were incubated
with FITC-conjugated goat anti-mouse IgG antibody (Cat# 115-096-071; Jackson ImmunoResearch
Laboratories) for 1h. After the removal of unbound antibodies by extensive washing with
PBS/BSA, forward scatter (FSC) signal, side scatter (SSC) signal, and FITC fluorescence of the
cells were measured by FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The data
and mean fluorescence intensity (MFI) of FITC signal were analyzed by CellQuest software (BD
Biosciences, San Jose, CA, USA).
Acknowledgment This research was supported by the Ministry of Science and Technology, Taiwan
(grant no. MOST 106-2113-M-038 -002; 107-2113-M-038 -001).
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