Development of HDAC Inhibitors Exhibiting Therapeutic Potential in T-Cell Prolymphocytic Leukemia

pubs.acs.org/jmc

Article

Development of HDAC Inhibitors Exhibiting Therapeutic Potential in

T‑Cell Prolymphocytic Leukemia

Published as part of the Journal of Medicinal Chemistry special issue “Epigenetics 2022”.

Krimo Toutah,^▲Nabanita Nawar,^▲Sanna Timonen, Helena Sorger, Yasir S. Raouf, Shazreh Bukhari,

Jana von Jan, Aleksandr Ianevski, Justyna M. Gawel, Olasunkanmi O. Olaoye, Mulu Geletu,

Ayah Abdeldayem, Johan Israelian, Tudor B. Radu, Abootaleb Sedighi, Muza ﬀ ar N. Bhatti,

Muhammad Murtaza Hassan, Pimyupa Manaswiyoungkul, Andrew E. Shouksmith, Heidi A. Neubauer,

Elvin D. de Araujo, Tero Aittokallio, Oliver H. Kr ä mer, Richard Moriggl,* Satu Mustjoki,*

Marco Herling,* and Patrick T. Gunning *

Cite This: J. Med. Chem. 2021, 64, 8486 −8509

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Epigenetic targeting has emerged as an eﬃcacious therapy for

hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL)

is known for its aggressive clinical course. Current epigenetic agents such as histone

deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a

structure−activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531,

which exhibited higher potency in T-PLL compared to other hematological cancers. KT-

531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological

activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL

patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong

biological responses, and safety in healthy donor samples. Notably, combination studies in

T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs,

bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL

could aﬀord suﬃcient therapeutic windows to achieve durable remission either as stand-

alone or in combination with targeted drugs.

The human HDAC superfamily consists of 18 nuclear and

cytoplasmic proteins, distributed into 4 distinct classes

according to their sequence similarity; Class I (1, 2, 3, 8),

Class IIa (4, 5, 7, 9), Class IIb (6, 10), Class III (NAD⁺-

dependent Sir2), and Class IV (11). Deﬁned nonhistone

substrates have been identiﬁed for certain HDAC iso-

forms.^3,4,7,9,10In particular, HDAC6 is responsible for the

deacetylation of α-tubulin, HSP90, Cortactin, and Peroxir-

edoxin. HDAC6 is also recognized as a KDAC (lysine

deacetylase). As such, HDAC6 plays a key role in microtubule

dynamics and chaperone activities, DNA damage repair

pathways, and oncogenic stress responses through the

regulation of cell migration, immune-cell synapse formation,

protein traﬃcking, and degradation.¹¹⁻¹³The upregulation of

INTRODUCTION

■

Epigenetic regulation of gene expression in the onset and

progression of cancer has fueled therapeutic strategies against a

number of molecular targets. Histone deacetylases (HDACs)

have been identiﬁed as targets for reversing cancer-associated

epigenetic states.¹⁻⁴HDACs remove acetyl groups from the

amino-terminal lysine residues of histones and nonhistone

proteins. This can lead to the formation of a condensed

chromatin structure that consequently limits the binding of

transcription factors to promoter sequences. Conversely,

histone acetyltransferases (HATs) reverse this activity by

catalyzing the transfer of an acetyl group from acetyl-CoA to

lysine residues.^2,5These post-translational modiﬁcations have

been identiﬁed as key regulatory strategies for a multitude of

processes such as signaling networks, gene expression,

transcription, cell cycle, and metabolism pathways. The activity

of key regulatory proteins such as the central tumor suppressor

p53, STATs, or HIF family members as well as key epigenetic

regulatory proteins such as p300/CBP HATs themselves is

also modulated by acetylation reactions. Hence, they also

represent important targets of HDAC inhibitors (HDACi).⁶⁻⁸

Received: March 8, 2021

Published: June 8, 2021

American Chemical Society

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

8486

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. HDACi 1−14 and their in vitro activity and selectivity proﬁles. (a) Chemical structures of HDACi 1−14. (b) Biochemical HDAC

inhibition against recombinant enzymes (Nanosyn) and cellular cytotoxicity results in MV4-11 (AML) and MRC-9 (healthy ﬁbroblasts). Note:

**not HDAC6 selective, *selectivity ≥ indicated value (beyond limit of experiment). A color gradient is applied for visualization of diﬀerential

HDAC6 activity.

HDAC6 activity has been directly correlated with cellular

proliferation, metastasis, and mitosis, highlighting the impact of

this epigenetic regulator in cancer pathogenesis.^11,12,14

In the past two decades, successful eﬀorts have led to four

HDACi receiving U.S. Food and Drug Administration (FDA)

approval for hematological cancers: vorinostat (SAHA, a),

romidepsin (FK-228, b), belinostat (PXD-101, c), and

panobinostat (LBH-589, d), with several in clinical trials

(quisinostat, e) (Figure S1a). In general, these HDACi exhibit

broad spectrum HDAC activity, which limits their therapeutic

tolerability owing to side eﬀects such as fatigue, diarrhea,

vomiting, anorexia, asthenia, weight loss, and thrombocytope-

nia.^19,20Selective HDACi are hypothesized to minimize side

eﬀects associated with pan-inhibitors.^2,15,16Since the identi-

8487

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 1. Synthetic Conditions for Preparation of 2−14

a

(a) Reagents and conditions to synthesize 2−9 and 11−14. (i) BnBr, Cs₂CO₃, DMF, RT, 24 h; (a) (i) R−NH₂, AcOH, DCE, RT, 2 h; (ii)

NaBH(OAc)₃, RT, 16 h; (b) R′SO₂Cl, Et₃N, CH₂Cl₂, 3−16 h, RT; (c) H₂, 10% Pd/C, THF/MeOH (2:1), RT, 16 h; (g) HSO₃Cl, 3 h, 150°C; (b)

RNH₂, Et₃N, CH₂Cl₂, RT, 3−16 h d) C₁₂H₁₅BrO₂, Cs₂CO₃, DMF, RT, 24 h; e) 4M HCl/dioxane, 0°C-RT, 3 h; (f) (i) (COCl)₂, THF, DMF,

i

0°C, 1 h; (ii) H₂N-OTHP, Pr₂NEt, THF, RT, 16 h; (f′) H₂N-OBn, EDCI, HOBt, Et₃N, DMF, RT, 16−24 h; (c) H₂, 10% Pd/C, THF/MeOH

(2:1), RT, 16−24 h; (e) 4M HCl/dioxane, 0°C-RT, 3 h. b Reagents and conditions to synthesize 10. (i) BnBr, Cs₂CO₃, DMF, RT, 24 h; a) (i) ^cPr-

NH₂, AcOH, DCE, RT, 2 h; (ii) NaBH(OAc)₃, RT, 16 h; (b) PFBSCl, Et₃N, CH₂Cl₂, 3−16 h, RT; (e) H₂, 10% Pd/C, THF/MeOH (2:1), RT, 18

i

h; (f) (i) (COCl)₂, THF, DMF, 0°C, 1 h; (ii) H₂N-OTHP, Pr₂NEt, THF, RT, 16 h; (e) H₂, 10% Pd/C, THF/MeOH (2:1), RT, 6 h.

ﬁcation of the ﬁrst selective HDAC6 inhibitor tubacin (f) in

2003, a number of HDAC6-selective inhibitors have been

developed, notably tubastatin A (g), ricolinostat (ACY-1215,

h), citarinostat (ACY-241, i) (Figure S1a), KA2507, and

marbostat-100. Ricolinostat, citarinostat, and KA2507 are

currently under evaluation in advanced clinical trials for

hematological and solid tumors.¹⁷⁻¹⁹Although modest

selectivity toward HDAC6 (5−6-fold) has been achieved

over the nearest HDAC family member in vitro, the selectivity

proﬁle of these clinical candidates in vivo remains largely

indiscriminatory toward other HDACs.²⁰Newer inhibitors

such as marbostat-100 have attained higher selectivity than

current clinical candidates and are well-tolerated in vitro and in

vivo.^21,22The benign phenotype of HDAC6 knockout mice

suggests that HDAC6 inhibition is a safe therapeutic strategy.²³

Overexpression of HDAC6 and conﬁrmed dependency proﬁles

in multiple hematological malignancies have accelerated

preclinical and clinical studies of HDAC6-selective inhibitors

8488

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

as single agents, and in combination with lenalidomide,

pomalidomide, paclitaxel, bortezomib, or dexametha-

sone.^{18,20,24−26}

promising drug sensitivity scores (DSS), with a therapeutic

window for T-PLL over healthy donor-derived peripheral

blood mononuclear cells (PBMCs). Finally, KT-531 showed

high synergy with chemotherapeutic agents idasanutlin,

bendamustine, and venetoclax in T-PLL patient samples,

representing a novel, eﬃcacious, and potentially safer

combination for T-PLL treatment. To the best of our

knowledge, KT-531 is the ﬁrst HDAC6 inhibitor to show

eﬃcacy in T-PLL patient samples.

T-cell prolymphocytic leukemia (T-PLL) is one of the most

aggressive forms of hematological neoplasms.²⁷Systematic

analysis, largely by sequencing eﬀorts of T-PLL, has revealed

insights into the molecular landscape of this condition, notably

through recognition of recurrent T-cell leukemia 1A (TCL1A)

oncogene rearrangements, damaging lesions of the ataxia

telangiectasia mutated (ATM) tumor suppressor gene, and gain-

of-function mutations of Janus-activated kinase−signal trans-

ducer and activator of transcription factor (JAK-STAT)

molecules.²⁸⁻³³These functional cancer genomic insights in

T-PLL were also validated biochemically, suggesting that

aberrant DNA damage and hyper-cytokine and -growth factor

responses trigger neoplastic T-cell outgrowth. These responses

are associated with fulminant migratory properties of T-PLL

cells, and this migratory T-cell phenotype could be under

HDAC6 control.^27,32,34Improved biological understanding has

yet to translate into a therapeutic application, as patient

outcomes remain poor (median overall survival <20 months),

owing to the aggressive tumor growth and insuﬃcient

responses to conventional chemotherapy. The current ﬁrst-

line treatment for T-PLL, the monoclonal anti-CD52 antibody

alemtuzumab, induces high response rates, although eventual

relapses are dominant.³⁵Pan-HDACi have received FDA

approval for the treatment of other mature T-cell neoplasms

such as cutaneous T-cell lymphomas (CTCL) and peripheral

T-cell lymphomas (PTCL) but not for T-PLL.^2,36−39

In the absence of drugs that confer sustained tumor control

in T-PLL, we focused on studying novel HDACi scaﬀolds for

potential therapeutic use in these patients. Recent in vitro

studies in T-PLL have also shown impressive eﬃcacy of pan-

HDACi and marked synergisms with the MDM2 inhibitor

idasanutlin.^29,40The intertwined mechanisms of HDAC

inhibition with the activity of tumor suppressor protein p53,

whose repression is a hallmark in T-PLL, may be contributing

to the therapeutic beneﬁt of epigenetic modulation in this

aggressive diseased state.⁴⁰⁻⁴²Moreover, the BH3-mimetic

venetoclax (ABT-199) has emerged as a breakthrough in the

treatment of hematologic neoplasms. Venetoclax can induce

apoptosis by targeting the B-cell lymphoma 2 (BCL-2) family

of proteins. Ex vivo and human studies of T-PLL patients have

highlighted an enhanced sensitivity to venetoclax.^41,43−45

Here, we report the identiﬁcation of a novel perﬂuorinated

benzenesulfonamide HDAC inhibitor, KT-531 (14), which

showed high in vitro HDAC6 selectivity (39-fold HDAC6

selectivity; compared to ∼6-fold selectivity of clinical

candidates citarinostat and ricolinostat) with low nanomolar

potency (IC₅₀= 8.5 nM) against HDAC6 in a functional in

vitro activity assay. Furthermore, KT-531 demonstrated

biological potency in multiple hematological cancer cell models

(acute myeloid leukemia (AML), PTCL, and T-cell acute

lymphoblastic leukemia (T-ALL)) and limited cytotoxicity in

nonmalignant cell types as well as no observable toxicity in vivo

(CD-1 mice). Notably, KT-531 exhibited strong potency (IC₅₀

= 0.42 μM) in the T-ALL/T-PLL-like cell line SUP-T11. Data

mining using Oncomine revealed HDAC6 was the only HDAC

member to be selectively overexpressed in T-PLL patient

samples but not in other mature or immature T-cell

malignancies, supporting the rationale for HDAC6-targeting

in treatment of T-PLL.⁴⁶KT-531 was subsequently tested in

ten primary T-PLL patient samples, where it exhibited

RESULTS

■

Chemistry. Herein, we describe a new class of perﬂuori-

nated benzenesulfonamide HDAC6-selective inhibitors, which

originated from our recently reported HDAC6-selective

inhibitor, JG-265 (1).⁴⁷A comprehensive structure−activity

relationship (SAR) study of 1 was designed to identify

inhibitors that retained/ampliﬁed HDAC6 potency and

selectivity, while improving the limited pharmacokinetic

(PK) proﬁle which precluded its advancement to preclinical

studies (Figure 1a,b).⁴⁷

Synthetic pathways used to prepare the ﬁnal compounds

(Figure 1a) are outlined in Scheme 1. In order to synthesize a

subset of the desired inhibitor library using Scheme 1a (upper

branch), 4-formyl benzoic acid was protected using benzyl

bromide to form benzyl 4-formylbenzoate (S1) (70%), which

was reductively aminated with the appropriate amines to form

the desired secondary amine (S2, S2c) (83%). The

corresponding amine was coupled to polyhalogenated

benzenesulfonyl chloride (S3) via an amine sulfonylation

reaction to yield the sulfonamide precursors (S4a, S4c−S4g,

S4i) (61−91%). If the precursor polyhalogenated benzene-

sulfonyl chloride (S3) was commercially unavailable, it was

synthesized via the sulfonylation of the corresponding

polyhalogenated benzene with chlorosulfonic acid at 150 °C

for 2 h. After removal of the benzylic ester protecting group

(S5a, S5c−S5g, S 5f’, S5i, S5l) (71−98%) from the

sulfonamide precursors, it was coupled to tetrahydropyranyl

(THP) or benzyl-protected hydroxylamines (S6a, S6c−S6g,

S6f’, S6i, S6l) (61−76%). A ﬁnal deprotection using H₂(g),

Pd/C, or 4 M HCl (35−81%) yielded the ﬁnal molecules (2, 3,

5, 7, 9, 11−13).

The alternate strategy (lower branch of Scheme 1a) was

used to design compounds 4, 6, and 14. Polyﬂuorobenzene-

sulfonyl chloride (prepared as previously described if not

commercially available) was coupled to the respective amines

to generate the desired sulfonamides (S9b−d) (68−83%),

which were subsequently coupled to 4-bromomethylbenzoic

tert-butyl ester to form the corresponding tert-butyl ester-

protected benzoic acids (S4b, S11a,b) (27−82%). Following

acid-mediated deprotection (S5b,j,k) (99%), coupling to O-

(Tetrahydro-2H-pyran-2-yl)hydroxylamine yielded the pro-

tected hydroxamic acid (S6b,j,k) (88−98%) which was readily

deprotected to reveal the ﬁnal compounds 4 (50%), 6 (49%),

and 14 (60%). Compound 1 was generated as previously

reported.⁴⁷

A similar synthetic route (Scheme 1b) was utilized to furnish

3-(((N-cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)sulfonamido)-

methyl)-N-hydroxybenzamide, by beginning with the benzyl

ester protection of 3-carboxybenzaldehyde (S1b) (98%) and

reductively aminating with cyclopropylamine to yield benzyl 3-

((cyclopropylamino)methyl)benzoate (S2b) (93%). Coupling

to pentaﬂuorobenzenesulfonyl chloride (S4h) (78%) and

following the aforementioned deprotection yielded 3-(((N-

8489

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. In vitro and in silico conﬁrmation of superior HDAC6-selectivity and potency of compound 14 (KT-531). (a) X-ray crystal structure of

14. (b) Dose−response curve representing the percent inhibition and HDAC6 selectivity of citarinostat against HDAC2−4,6,8,11 via EMSA

activity assay (Nanosyn). (c) Dose−response curve representing the percent inhibition and HDAC6-selectivity of 14 against HDAC1−11. (d) In

vitro HDAC inhibition IC₅₀values of citarinostat (i) and 14 against HDAC1−11. A color gradient is applied for visualization of diﬀerential activity.

(e) In silico docking binding pose of 5 in human HDAC6 (PDB 5EDU), with Zn²⁺(yellow sphere), hydrogen bonds (yellow dashed line), π−π

stacking (green dashed line), and salt bridges (purple dashed line) (N, blue; O, red; C, green; F, blue-green). Catalytic triad residues and portions

of the protein have been omitted for clarity. (f) In silico docking binding pose of 14 in human HDAC6 (PDB 5EDU), with Zn²⁺(yellow sphere),

hydrogen bonds (yellow dashed line), π−π stacking (green dashed line), and salt bridges (purple dashed line) (N, blue; O, red; C, yellow; F,

green). Catalytic triad residues and portions of the protein have been omitted for clarity. (g) In silico docking binding pose of 14 in human HDAC8

(PDB 1T64).

cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)sulfonamido)methyl)-

benzoic acid (S5h) (98%). The synthesis was completed

through conversion to the hydroxamate ester (S6h) (70%) and

deprotecting with acid to furnish compound 10 (66.7%).

Structure−Activity Relationships, In Vitro HDAC

Inhibition, and In Cellulo Cytotoxicity. The prepared

library (Figure 1a) was screened against HDAC3, 6, 8, 11

(representative of groups I, II, and IV) to determine in vitro

activity inhibition proﬁles (Figure 1b).^48,49Concurrently,

cellular activity was analyzed in a model cancer cell line

(MV4-11) and healthy ﬁbroblasts (MRC-9) to correlate

biochemical inhibition with cellular potency and therapeutic

window, and other HDACi (SAHA/vorinostat, ricolinostat,

and citarinostat), as well as parent compound 1, were included

for parallel comparison.

First, the role of the pentaﬂuorobenzamide moiety was

investigated by substituting it with a pentaﬂuorobenzenesulfo-

namide (PFBS), 2. This resulted in a substantial loss of activity

8490

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

against HDAC6 (IC₅₀[HDAC6] = 389 nM) vs 1

(IC₅₀[HDAC6] = 5.9 nM) and a subsequent loss in cellular

potency (IC₅₀[MV4-11] = 11.07 μM) vs 1 (IC₅₀[MV4-11] =

1.85 μM). The introduction of an acidic sulfonamide group

likely incurs unfavorable electrostatic interactions with tunnel

residues. This hypothesis is supported by the N-methylated

analogue, 3, being ∼200 fold more potent. Moreover,

substitutions on the N-sulfonamide with alkyl groups of

diﬀerent sizes (−CH₃, 3; −ⁱPr, 4; −^cPr, 5) showed improved

HDAC6 potency (∼200-fold for 3, ∼100-fold for 4, and ∼430-

fold for 5) and cellular IC₅₀’s (0.31−1.04 μM). One of the

most notable improvements included the selectivity jump from

36-fold for the parent compound 1 to 163-fold for 4 (Figure

1b). Furthermore, 5, with an N-^cPr substituent, exhibited

picomolar inhibitory activity (IC₅₀= 0.9 nM) toward HDAC6.

The N-^cPr group of 5 was retained, and the importance of

the halogenation and electrophilicity was explored next.

Diminishing cellular cytotoxicity in MV4-11 cells was observed

by successively removing of ﬂuorine atoms from 5. Although

mono-, tri-, and tetraﬂuorobenzenesulfonamides (6−9)

retained biochemical HDAC6 inhibition (IC₅₀= 8−14 nM),

cellular eﬃcacy was reduced by ∼5−10-fold (IC₅₀= 5−9 μM),

suggesting that substitution of the phenyl sulfonamide ring is

important for biological activity. HDAC selectivity proﬁles

ranged from 20-fold for 8 to 163-fold for 4. Selectivity toward

HDAC isoforms was modulated by varying the position of the

hydroxamic acid, speciﬁcally, para- to meta-, which switched

selectivity toward HDAC8 (4-fold) (10).

Although covalent modiﬁcation of HDAC6 was not

observed (Figure S2), the PFBS group is susceptible to

nucleophilic aromatic substitution by biological nucleophiles.

A previously described high-throughput in vitro glutathione-

reactivity assay was conducted, where compounds were

incubated in the presence of glutathione (GSH) and the

formation of a GSH-compound adduct was monitored over

time via LC/MS/MS.⁵⁰Fluorinated analogues 3 and 4 were

found to react readily with glutathione, which precluded

further advancement (Table S1a). The exceptional picomolar

HDAC6 potency of 5 and moderate GSH stability (half-life of

20 min) warranted further ADME analysis. In hepatocytes, 5

was cleared rapidly, with a half-life of 3 min (Table S1b). In

contrast, 6, lacking the para F, was unreactive toward GSH

(Table S1a). However, in mouse plasma, 6 had a low half-life

of 25 min (Table S2a).

We next investigated strategies to improve stability by

reducing the electrophilicity of the perﬂuorinated cap group.

The phenyl group was substituted with a variety of halogen

combinations (11−14). A homologue of 5, missing the 4-

ﬂuoro substituent, 13, showed signiﬁcantly reduced potency

toward HDAC6 (∼700-fold). The loss of HDAC6 inhibition

was concomitant with reduced biological activity in the

cytotoxicity experiments. Analogues where ﬂuorine substitu-

ents were replaced with less electronegative chlorine atoms

exhibited comparable HDAC6 inhibition and cellular activity,

but reduced selectivity. Inhibitor 14 (KT-531) (Figure 2a),

with a tetraﬂuorobenzene substituent led to a substantial

increase in stability. While no discernible reactivity with GSH

was observed after 24 h (Table S1a), KT-531 showed cellular

activity (IC₅₀[MV4-11] = 0.42 μM), single digit nanomolar

potency for HDAC6 (IC₅₀= 8.5 nM), and a promising

selectivity proﬁle (39-fold over the next closest target,

HDAC8) (Figure 1b). KT-531 exhibited a superior in vitro

proﬁle when compared to the current literature HDAC6

inhibitor, Nexturastat, which had comparable HDAC6 potency

(IC₅₀= 12.4 nM) but a lower selectivity margin (19-fold; next

nearest target HDAC3 with IC₅₀= 238 nM) (Table S2b).^51,52

KT-531 also demonstrated higher cytotoxicity than Nextura-

stat in MV4-11 cancer cells (IC_{50‑Nexturastat}= 1.68 μM,

IC_{50‑KT‑531}= 0.42 μM) (Table S2b, Figure 1b).

The binding kinetics proﬁle of KT-531 was also investigated

by determining the association (k_on)/dissociation (k_off) rates as

well as residence time parameters. The in vitro residence time

(1/k_obs) for the KT-531−HDAC6 complex was determined to

be 23.18 min, as compared to 10.72 min for 1. This is a

signiﬁcant improvement over 5, which exhibited a residence

time of 1.09 min (Table S3). Reversible ligand binding was

conﬁrmed for 5 and 14 with unchanged IC₅₀values in the 6 h

time-dependent assay (Figure S2). The K_iof KT-531 for

HDAC6 was determined to be 17.3 nM in a time-dependent

inhibition K_onexperiment (Figure S3).

The selectivity proﬁle of KT-531 for all HDAC families

(Class I, II, and IV) was determined using an activity-based

EMSA assay (Nanosyn, USA). KT-531 showed a clean

selectivity proﬁle, with an IC₅₀of >1000 nM for HDAC1−5,

7, and 9−11 and modest potency against HDAC8 (IC₅₀= 334

nM). This ∼40-fold selectivity was higher than that of

HDAC6-selective clinical candidates, ricolinostat and citarino-

stat (both 5−6-fold selective, Figures 1b and 2b−d). Unlike

KT-531, both clinical candidates inhibit Class I HDACs,

speciﬁcally HDAC3 (IC₅₀of citarinostat = 12.5 nM).

In Silico Modeling and Docking Studies. Docking

simulations between hsHDAC6 (PDB 5EDU, catalytic domain

̈

2) and 5 using Schrodinger’s Maestro v11.9 (Schrodinger,

LLC, New York, 2019) were performed to understand the role

of the PFBS ring in deriving potency toward HDAC6 (Figures

2e and S4).⁵³⁻⁵⁵The core interactions in the catalytic tunnel

included sandwich-type π−π interactions of 5 with Phe620 and

Phe680, coupled with an additional T-shaped π−π stacking of

the PFBS ring with Phe679 (Figure 2e). Hydrogen bonding

interactions between the ﬂuorine substituents and the nearby

backbone of Phe679 likely contributes to the increase in overall

aﬃnity. The ﬂexible sulfonyl center and the benzylic −CH₂−

kink atom serve to accommodate these interactions (ΔG_B=

−8.84 kcal mol⁻¹). The ﬂuorine atoms were deemed crucial for

eﬃcacy and HDAC6 pocket-occupancy, as replacement of the

para ﬂuorine to a chlorine atom led to a signiﬁcant decline in

potency (∼77-fold loss in potency from 0.9 to 70 nM).

Through docking simulations of 13, loss of activity was

predicted to be due to the bulkier chlorine substituent not

being well accommodated in the HDAC6 catalytic tunnel

outlet (ΔG_B= −6.67 kcal mol⁻¹) (Figure S5).

Similar analyses of 14 (KT-531), showed the same key

interactions with Phe620 and Phe680 in the HDAC6 pocket

(ΔG_B= −8.30 kcal mol⁻¹), and hydrogen bonding with the

nearby Phe679 amide, despite the loss of F (Figure 2f). When

docked in the HDAC8 catalytic pocket, KT-531 lost the tunnel

π−π stacking interactions, and only retained a T-shaped

stacking interaction with the proximal Tyr100 (ΔG_B= −5.70

kcal mol⁻¹) (Figure 2g). The ﬂuorinated ring was unable to

eﬀectively bind to the opening of a catalytic tunnel

characterized by an intractable, sterically hindered topology.

Although π−F interactions are also likely, Maestro does not

have the functionality to distinguish such interactions. Overall,

these ﬁndings provide a structural rationale for the high

potency of KT-531 toward HDAC6 (IC₅₀= 8.5 nM), and the

corresponding lower aﬃnity toward HDAC8 (IC₅₀= 334 nM).

8491

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 3. In cellulo pharmacology of compound 14 (KT-531) in comparison to citarinostat. (a) Immunoﬂuorescence analysis of α-tubulin

acetylation and histone H3 acetylation following 6 h treatment of KT-531 in cells. (acetylated α-tubulin in red, acetylated histone H3 in green, and

nuclear stain DAPI in blue). This data is representative of three independent experiments. (b) Quantiﬁcation of ﬂuorescent signals that correspond

to levels of acetylated α-tubulin and acetylated histone H3 in cells dosed with KT-531 at indicated concentrations. (c) Immunoﬂuorescence

analysis of α-tubulin acetylation and histone H3 acetylation following 6 h treatment of citarinostat in cells (acetylated α-tubulin in red, acetylated

histone H3 in green, and nuclear stain DAPI in blue). This data is representative of three independent experiments. (d) Quantiﬁcation of

ﬂuorescent signals that correspond to levels of acetylated α-tubulin and acetylated histone H3 in cells dosed with citarinostat at indicated

concentrations. *p ≤ 0.05; ****p ≤ 0.001; ns, nonsigniﬁcant; two-way ANOVA with Tukey’s multiple comparisons test. (e) Western blot

illustrating α-tubulin acetylation and histone H3 acetylation levels in MV4-11 AML cells following 6 h treatment with varying concentrations of

KT-531 and clinical candidate citarinostat (i). Protein extracts were prepared, resolved by SDS-PAGE and immunoblotted with acetylated α-

tubulin, acetylated histone H3, and HSC70 antibodies. A representative Western blot of three independent experiments is shown.

In Cellulo Pharmacology. KT-531 was examined for its

ability to induce acetylation of α-tubulin, a prominent substrate

of HDAC6, and histone H3 (HDAC Class I substrate).⁵⁶

Initially, model cancer cells (HeLa) were subjected to 6 h

incubation with increasing doses of KT-531 or citarinostat and

target engagement was analyzed via immunoﬂuorescence

assays. In KT-531-treated cells, signiﬁcant acetylation of α-

tubulin was observed, with no observable change in acetylation

of histone H3 (Figure 3a,b). Citarinostat induced weaker

acetylation as compared to KT-531 (Figure 3c,d).

8492

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. Compound 14 (KT-531) induces apoptosis in a dose-dependent manner. (a) Flow cytometric data of MV4-11 cells treated with

increasing concentrations of KT-531 (0−4 μM) for 18 h and analyzed for apoptosis via Annexin V/PI staining. Representative dot blots from three

independent experiments are presented. (b) Percentage of cell populations in healthy, early apoptosis, and late apoptosis phases following

treatment with KT-531 for 18 h at indicated concentrations. (c) Percentage of cell populations in healthy, early apoptosis, and late apoptosis phases

following treatment with citarinostat for 18 h at indicated concentrations. Flow cytometric data for citarinostat is available in Figure S6. (d)

Cleavage of caspase-3 and PARP-1 upon dose-escalation of KT-531 for 6 h in MV4-11 cells. Protein extracts were prepared and subjected to SDS-

PAGE and immunoblotting with procaspase 3, cleaved caspase-3, PARP-1, cleaved PARP-1, and HSC70 antibodies. A representative Western blot

of three independent experiments is shown. Data for citarinostat is available in Figure S7.

8493

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 5. KT-531 shows selective cellular cytotoxicity in PTCL malignancies. Heat map of IC₅₀values calculated from drug response analysis of

KT-531 (14) and citarinostat from one representative out of three independent experiments. Cell lines are classiﬁed according to the respective

disease subtype. Abbreviations: AML, acute myeloid leukemia; ANKL, aggressive NK-leukemia; LGL, large granular lymphocyte leukemia; T-NHL,

T-cell non-Hodgkin’s lymphoma; ALK⁺ALCL, anaplastic large cell lymphoma (anaplastic lymphoma kinase positive); CTCL, cutaneous T-cell

lymphoma; T-ALL, T-cell acute lymphoblastic leukemia.

Figure 6. Preclinical ﬁndings show promising therapeutic window and pharmacokinetic properties of 14 (KT-531). (a) Cytotoxicity of KT-531

(14) and citarinostat in healthy noncancerous cell lines (MRC-9, normal human ﬁbroblasts (NHF), human umbilical vein endothelial cells

(HUVEC), primary pooled ﬁbroblasts from 250 donors (pPF)). (b) Average mouse weight of CD-1 mice (20 mg/kg, P.O.) dosed with KT-531

(14) and vehicle daily for 5 days. (c) Pharmacokinetic assessment of KT-531 (14) in mouse hepatocytes, PAMPA assay, mouse plasma and in vivo

in CD-1 mice (IP, 20 mg/kg).

Next, KT-531-treatment of a hematopoietic cancer cell line,

MV4-11, revealed that, at higher inhibitor concentrations,

acetylated α-tubulin levels were comparable between citarino-

stat and KT-531 (Figures 2d and 3e). Oﬀ-target acetylated

histone H3 levels were higher with citarinostat in comparison

to KT-531, aligning with in vitro ﬁndings (Figures 2d and 3e).

To understand the origin of the cytotoxic activity,

ﬂuorescence-activated cell sorting (FACS) was employed to

evaluate KT-531 (Figure 4a,b). Annexin V/PI staining revealed

15% of cells underwent early apoptosis (Annexin V+/PI) upon

18 h treatment with 0.5 μM KT-531, in contrast to 9% for

citarinostat at the same dose (Figures 4c and S6). At 2 μM KT-

531, only 8% of cells remained healthy and nonapoptotic,

whereas 47% of cells remained as nonapoptotic following 2 μM

treatment with citarinostat. Similar trends were observed for

late apoptotic populations (Annexin V+/PI+) (22% for KT-

8494

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

531 versus 10% for citarinostat at a 1 μM dose). Cleavage of

PARP-1 and Caspase-3 at 0.5 μM treatment of KT-531

conﬁrmed the apoptotic response signature (Figure 4d). The

absence of cleaved Caspase-3 and PARP-1 at higher

concentrations of citarinostat (5 μM) recapitulated FACS

ﬁndings that KT-531 (14) induces apoptosis more strongly

than the clinical molecule (Figure S7).

Therapeutic Potential of KT-531 in T-Cell Neoplasms.

Similarities in the mutational landscape of myeloproliferative

and lymphoid neoplasms suggest that similar treatments might

be eﬀective.^57,58Since biological activity was demonstrated in

AML, KT-531 was further assessed in a collection of PTCL (n

= 8) and T-ALL (n = 3) model cell lines covering various

subtypes to understand if eﬃcacy was transferable (Figure 5).

KT-531 displayed selective cytotoxicity in diﬀerent PTCL

cell lines. A striking observation of this screen in PTCL and T-

ALL models was the indiscriminate behavior of citarinostat

toward all branches of these malignancies, in contrast to the

more selective cellular behavior of KT-531 in certain subtypes.

Unlike citarinostat and FDA-approved pan-HDACi romidepsin

and vorinostat, KT-531 was not very active in CTCL models,

such as Myla and Hut78 (Figure 5).^40,43,59Another distinction

of KT-531 from citarinostat was its strong activity in the T-

ALL/T-PLL-like cell line SUP-T11 (IC₅₀of 0.42 μM; 9-fold

more potent than citarinostat). SUP-T11 (derived from a

patient with mature T-ALL) harbors a TRA/TRD-TCL1A

translocation and consequent aberrant expression of the

TCL1A protein, which is considered the genetic hallmark of

T-PLL. SUP-T11 is, hence, the closest T-PLL-model derived

from a patient so far.^57,59,60These ﬁndings also correlate with

the HDAC6 mRNA expression levels available in Broad

Institute Cancer Cell Line Encyclopedia (CCLE; Figure S8).

The poor activity of KT-531 in Hut78 cells is consistent with

the relatively low HDAC6 expression. The highest sensitivity

to KT-531 was observed for T-ALL cell models SUP-T11,

DND-41 and MOLT4, which had much higher HDAC6

expression levels. Indeed, the sensitivity of HDAC6 inhibitor

KT-531 exhibits signiﬁcant correlation with the available

HDAC6 mRNA expression levels in T-cell lines (R²= 0.9119,

Figure S8b).

The inhibitors were also assessed in nontransformed cellular

systems to determine their therapeutic window. KT-531

displayed an IC₅₀of 22 μM in MRC-9 (lung) cells, revealing

a clear therapeutic window. This supports the improved safety

proﬁle of HDAC6-targeting inhibitors compared to pan-

HDACi. NHF (normal human ﬁbroblasts), HUVEC (primary

human umbilical vein endothelial cells), and pPF (primary

pooled ﬁbroblasts from 250 donors) were also assessed against

citarinostat and KT-531, and while still possessing a

therapeutic window both molecules were signiﬁcantly more

potent in these normal cell types (Figure 6a). The cellular

safety margins for KT-531 are comparable to those of

citarinostat and the FDA-approved HDACi SAHA (vorinostat)

and belinostat (Figure 6a, Table S4). In addition, the safety of

KT-531 was tested in vivo with a 5 day treatment of CD-1 mice

(20 mg/kg via P.O. (oral administration)), which revealed no

visible signs of toxicity or weight loss (Figure 6b).

Figure 7. HDAC6 is signiﬁcantly overexpressed in T-PLL patient

samples, but not in other T-cell cancers. (a) mRNA expression levels

of various HDAC genes (1−6, 9, and 11) in T-cell prolymphocytic

leukemia (T-PLL) patient samples, compared to healthy T-cell

(CD3⁺) control samples. Data were extracted from the Oncomine

Platform, from the Durig Leukemia data set. HDAC7, 8, and 10

expression levels were not reported in this data set. ns; not signiﬁcant

(p > 0.05). (b) mRNA expression levels of HDAC6 in various T-cell

neoplasia patient samples, compared to healthy control cells (from left

to right, Eckerle Lymphoma: NK cell, T-cell, NK/T-cell; Piccaluga

Lymphoma: CD4⁺T-cell, CD8⁺T-cell, T-cell; Zhang Leukemia:

hematogone, bone marrow; Haferlach Leukemia: peripheral blood

mononuclear cells). Data were extracted from the Oncomine

Platform, from the Eckerle Lymphoma, Piccaluga Lymphoma,

Zhang Leukemia, and Haferlach Leukemia data sets. All comparisons

have a p-value > 0.05 and/or a fold-change of ≤1.2 and are not

considered signiﬁcant. Representation: boxes as interquartile range,

horizontal line as the mean, and whiskers as lower and upper limits.

Abbreviations: ALCL, anaplastic large cell lymphoma; ALK, anaplastic

lymphoma kinase; PTCL-NOS, peripheral T-cell lymphoma, not

otherwise speciﬁed; AITL, angioimmunoblastic T-cell lymphoma;

NK, natural killer.

data sets of patients with other mature or immature T-cell

malignancies, including anaplastic large cell lymphoma

(ALCL), angioimmunoblastic T-cell lymphoma (AITL),

PTCL not otherwise speciﬁed (PTCL-NOS), and T-ALL

(Figure 7b), suggesting that HDAC6 overexpression may be

speciﬁc to T-PLL cancer. These data support the hypothesis

that HDAC6 could be a beneﬁcial target in T-PLL, and adverse

eﬀects of pan-HDAC inhibition could be minimized through

the speciﬁc abrogation of HDAC6.

HDAC Expression in T-PLL Tumor Cells. Oncomine

database mining of published T-PLL patient gene expression

data in relation to healthy T-cells revealed speciﬁc over-

expression of HDAC6 (1.7-fold) compared to other HDAC

family members in T-PLL (Figure 7a).^46,59Interestingly, no

increase in HDAC6 expression levels was observed in available

KT-531 Shows Anticancer Activity and Therapeutic

Margin in T-PLL Patient Samples. A high-throughput ex

vivo drug sensitivity and resistance testing (DSRT) platform

8495

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

covering 306 approved and investigational oncology drugs has

identiﬁed a potential therapeutic role for HDACi in T-PLL.⁶¹

Drug sensitivity scores (DSS) that capture and integrate

multiparametric dose−response relationships into a single

metric have previously identiﬁed T-PLL selective drug

responses of FDA-approved HDACi panobinostat and

belinostat.^61,62Therefore, we performed similar studies with

KT-531 on primary T-PLL patient samples.

In comparison to the pan-HDACi, vorinostat (SAHA), KT-

531 resulted in a more potent drug response (p = 0.0007;

Wilcoxon test), which was also comparable to other pan-

HDACi, belinostat and tinostamustine (EDOS101) (Figure

8a). Anticancer eﬀects of HDACi have been synergistically

poor stability proﬁle (t_1/2= ∼20 min in incubation with

GSH, t_1/2< 2.26 min in mouse hepatocytes, t_1/2= 3.84 min in

human hepatocytes, Table S1b). However, the removal of

more than one ﬂuorine from 5 was shown to be detrimental for

antileukemic activity (6−9), leaving KT-531 as the most

prominent lead for initial PK evaluation. Notably, no

discernible S_NAr reaction with GSH was observed with KT-

531 within 24 h of incubation (Table S1a).

The parallel artiﬁcial membrane permeability assay

(PAMPA) and human plasma were used to evaluate the

permeability and stability of compound KT-531, respectively.

A −log P_evalue of 5.44 was reported for KT-531 in the

PAMPA assay, and t_1/2of 37 min in mouse hepatocytes and 41

min in human plasma showed promise for in vivo

bioavailability (Figure 6c). In vivo pharmacokinetic studies

on KT-531 were performed via intraperitoneal (IP) admin-

istration of compound in male CD-1 mice, at a dose of 20 mg/

kg. Compound KT-531 reached a maximum concentration

(C_max) of 493 ng/mL, with t_1/2of 63 min and an overall

exposure of 1576 h·ng/mL (Figure 6c). In comparison, and

under the same conditions, citarinostat was reported to have a

shorter t_1/2of 15 min but higher C_maxand overall exposure

(C_max= 5640 ng/mL, AUC = 4163 h·ng/mL; Figure S9),

supporting the hypothesis that citarinostat is a more readily

absorbed compound.

DISCUSSION AND CONCLUSION

■

T-PLL is a very aggressive T-cell malignancy that does not

beneﬁt from “classical treatments” and urgently demands

rationally designed targeted therapies. Available treatment

options do not prevent inevitable patient relapses, and a novel

treatment approach speciﬁcally tailored to treat T-PLL may

resolve the hurdles of short survival (<20 months) and absence

of curative therapy. The promising eﬃcacy but limited

tolerability of pan-HDAC inhibitors in this group of patients

highlights the need to selectively and safely target the T-PLL-

relevant HDAC isoforms. Critically, only the HDAC6 isozyme

of the HDAC family of enzymes was found to be overex-

pressed in primary T-PLL patient samples.

Figure 8. KT-531 (14) shows promising eﬃcacy and safety in T-PLL

primary patient samples. (a) Drug sensitivity scores (DSS)⁶²of

selected HDACi in T-PLL primary patient samples. (b) Box plot of T-

PLL synergy scores of KT-531 (14) with bendamustine, idasanutlin,

and venetoclax in T-PLL patient samples. (c) Box plot of combination

potency cropped to TC50 (toxic concentration in 50% of PBMC

population) of KT-531 (14) and venetoclax in T-PLL patient samples

and healthy PBMC controls.

A comprehensive SAR study led to the identiﬁcation of a

preclinical candidate, KT-531 which demonstrated exceptional

anticancer eﬀects in T-PLL. This lead candidate exhibited (i)

nanomolar inhibition of HDAC6 in a functional EMSA assay

(IC₅₀= 8.5 nM), (ii) 39-fold in vitro selectivity for HDAC6

over other HDAC isozymes, and (iii) inhibition of cellular

proliferation in a variety of hematologic model cell lines (AML,

PTCL, T-ALL) while possessing a therapeutic window in

heathy cells (MRC-9, NHF, pPF, HUVEC) and safety in 5 day

toxicity trials in CD-1 mice. Since HDAC8 was the next

nearest target of KT-531, HDAC8 selective inhibitors

published in the literature were tested and used as control

compounds to ensure that biological activity is not reliant on

HDAC8 inhibition. Both PCI-34051 (120-fold selective for

HDAC8) and MMH-410 (15-fold selective for HDAC8)

exhibited nanomolar HDAC8 potency but no observable

activity in MV4-11 and MRC9 cells (Table S5).^68,69Given the

established pathogenetic role of HDAC modulation in AML

and PTCL, cell lines representing these disease types were

used to proﬁle the library and investigate the cellular

pharmacology of KT-531.

potentiated by other chemotherapeutic agents in hemato-

poietic cancers.⁶³⁻⁶⁶Combinations of KT-531 with promising

investigational drugs for T-PLL, notably idasanutlin, bend-

amustine, and venetoclax, were performed to identify potential

synergisms. All combinations revealed synergistic relationships

in patient samples, highlighting that HDAC inhibition may

oﬀer potential advantages in T-PLL (Figure 8b).⁶⁷Compar-

ison of the potency in T-PLL patient samples to potency in

PBMCs from healthy donors revealed a therapeutic margin for

this novel combination of HDACi and venetoclax (Figure 8c).

Collectively, the data supports the hypothesis that HDAC6

inhibition, alone or in drug combination, could be a promising

therapy for T-PLL.

Pharmacokinetic Studies. A preliminary in vitro ADME

assessment of selected inhibitors was conducted. The

pentaﬂuorobenzene-sulfonamide analogue (5) exhibited a

The promising cellular potency of KT-531 in the T-PLL

quasi-model SUP-T11 prompted further investigation in this

novel indication. The absence of activity in some branches of

8496

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

atom % D), or MeOH-d₄(99.8 atom % D), as indicated (¹H at 400

MHz, ¹³C at 100 MHz, and ¹⁹F at 54 MHz, unless otherwise stated).

Samples were analyzed with 16 scans for ¹H, 256 scans for ¹³C, and 8

scans for ¹⁹F, in 8” 3 mm A-grade glass tubes. Chemical shifts (δ) are

reported in parts per million (ppm), after calibration to residual

isotopic solvent, and coupling constants (J) are reported in hertz

(Hz). Multiplicities are described as singlet (s), doublet (d), triplet

(t), quartet (q), pentet (p), sextet (sex), septet (sep), multiplet (m),

broad (br), or a combination of these. Low-resolution mass

spectrometry (LRMS) was carried out using a Waters LC-MS system

in ESI mode, ﬁtted with a Micromass ZQ MS and an Alliance 2690

LC. Analysis was performed on a Waters Xterra C18 3.0 × 150 mm

column containing 3.5 μm beads by direct injection into the mass

spectrometer. High-resolution mass spectrometry (HRMS) was

carried out using an Agilent 6538 UHD Q-TOF MS system in ESI

mode with a mass accuracy 1 mDa.

Synthetic Procedures. Reductive Amination (a). The appro-

priate alkyl amine (1.1 equiv) was added in one go to a solution of the

appropriate aldehyde (1.0 equiv) in 1,2-DCE (0.1 M) and stirred at

RT for 10 min before addition of glacial acetic acid (2.0 equiv) and

further stirring for 30 min. Sodium triacetoxyborohydride (2.0 equiv)

was added in one go, and the reaction was stirred for 24 h before

pouring over saturated aqueous sodium bicarbonate. The layers were

separated, and the aqueous layer was extracted with CH₂Cl₂. The

combined organic layer was washed with saturated aqueous sodium

bicarbonate, followed by brine, dried (MgSO₄), ﬁltered, and

concentrated in vacuo. Column chromatography isolated the target

compound.

Amine Sulfonylation (b). The sulfonyl chloride (1.0−1.1 equiv)

was added in one go to an ice-cooled solution of the appropriate

amine (1.1 equiv) and triethylamine or DIPEA (2.0 equiv) in THF,

CH₂Cl₂, or CH₃Cl (0.1−0.2 M) and stirred at room temperature, and

the disappearance of the limiting agent was monitored by TLC

(hexanes/EtOAc 8:2). After 3−16 h, the reaction was quenched with

1 M HCl. The layers were separated, and the aqueous layer was

extracted with CH₂Cl₂. The organic layer was dried over MgSO₄,

ﬁltered, concentrated in vacuo, and puriﬁed by column chromatog-

raphy to isolate the target compound.

Benzyl Deprotection by Hydrogenation (c). To a nitrogen-purged

solution of the benzyl or hydroxamate ester (1.0 equiv) in 2:1 mixture

of THF and methanol or 100% methanol (0.05−0.1 M) was charged

10% Pd/C (0.1 equiv). The ﬂask was purged with hydrogen, and then

the reaction was stirred for 2 h under hydrogen atmosphere (balloon)

before ﬁltration through Celite, washed with THF, and concentrated

in vacuo. Carboxylic acids were isolated without further puriﬁcation.

Preparative HPLC was used to isolate hydroxamic acids.

SN₂Substitution with Benzyl Bromides/Iodides (d). Benzyl

bromide/iodide (1.0 equiv) was added to a solution of the amine

(1 equiv) and Cs₂CO₃(1.5 equiv) in DMF (0.1 M). After 3−16 h

(reaction monitored by TLC, hexanes/EtOAc 6:4). The reaction

mixture was then diluted in EtOAc and saturated aqueous sodium

bicarbonate. The organic layer was washed with saturated aqueous

sodium bicarbonate (1×), water (3×), and brine (1×), and the

aqueous layer was extracted once with EtOAc. The combined organic

layer was dried over MgSO₄, ﬁltered, and concentrated in vacuo to

isolate the target compound without further puriﬁcation.

Acid-Mediated Hydrolysis of Carboxylate or Hydroxamate Esters

(e). The carboxylate or hydroxamate ester was charged in a round-

bottom ﬂask with 4 M HCl in dioxane (0.3 M ﬁnal concentration) at

RT in air. After 3−16 h, the solvent was removed in vacuo. Carboxylic

acid intermediates were used in the next step without further

puriﬁcation, whereas ﬁnal hydroxamic acids were puriﬁed using

preparative HPLC.

Formation of Hydroxamate Esters (f). Oxalyl chloride (4 equiv)

was added dropwise to a solution of the appropriate carboxylic acid

(1.0 equiv) in THF (0.05−0.2 M) and DMF (1 to 2 drops) at 0 °C

and stirred for 50 min −3 h. The reaction was concentrated to dryness

in vacuo before redissolving in dry THF (0.2 M) and mixing with

diisopropylethylamine or triethylamine (2.0 equiv) followed by the O-

protected hydroxylamine or its hydrochloride salt (1.0−2.0 equiv).

hematological neoplasms, such as CTCL, indicated that the

higher selectivity of KT-531 in contrast to FDA-approved pan-

HDAC drugs may be driving its unique and selective

cytotoxicity proﬁle. KT-531 exhibited comparable potency to

clinically used HDACi, belinostat and vorinostat, in T-PLL

patient cells, with a signiﬁcant therapeutic window when

compared to cytotoxicity in treated PBMCs from healthy

donors. Given the well-validated synergism displayed by

epigenetic agents with other compounds, KT-531 was tested

in combination with promising investigational drugs for T-

PLL, namely, idasanutlin, bendamustine, and venetoclax. The

marked synergy of all pairings supports the clinical

combination of more selective HDAC6i with these chemo-

therapeutic agents.

When coupled with the reported increased frequency of

upregulated HDAC6, as compared to other HDAC isoforms in

primary T-PLL patient cells, HDAC6i is a potential therapeutic

avenue in this incurable condition and warrants more in-depth

exploration. Further studies are necessary to explain the

superior eﬀects of KT-531 over citarinostat. Although the in

cellulo and in vitro studies support KT-531 as a more HDAC6-

selective inhibitor than citarinostat, the ∼39-fold HDAC6

enzymatic selectivity of KT-531 is not fully translated with

same selectivity margins in cells. This is evident from the

Western blots, where acetylation levels of histone H3 were

concomitant with acetylation levels of α-tubulin, suggesting

that the mechanism of action may not be solely dependent on

HDAC6 inhibition. Such weak cellular selectivity proﬁles are

common, as observed in multiple clinical trials of the modestly

HDAC6-selective drug candidate ricolinostat where acetylation

of histones was similar to acetylation of α-tubulin.^20,26Based

on the data generated, an HDACi combination approach will

be investigated in preclinical models of T-PLL, speciﬁcally

through development of HDAC6 inhibitors with greater

cellular selectivity and enhanced pharmacokinetic properties.

Subsequent studies will be focused on deciphering the

functional signiﬁcance and mechanism of action of selective

HDAC6 inhibition in T-PLL.

EXPERIMENTAL SECTION

■

General Procedures. All solvents and chemicals were used as

purchased without further puriﬁcation. Reactions were carried out in

oven-dried glassware and monitored by thin-layer chromatography

(TLC) using Merck silica gel 60 F₂₅₄on aluminum sheets (visualized

by 254/365 nm UV light and/or staining with KMnO₄). Column

chromatography was carried out using Biotage Isolera One and Isolera

Prime puriﬁcation systems, with industry-standard SNAP 10, 25, 50,

and 100 g cartridges loaded with 40−60 μm silica gel from VWR

International (average pore size 60 Å), eluting at 12−40 mL/min and

detecting compounds by UV measuring at 254 and 298 nm.

Semipreparative HPLC was conducted using a Waters 2487 Dual λ

Absorbance Detector, equipped with a Symmetry C18 4.6 mm × 150

mm cartridge. Compounds were detected by UV at 254 nm with

sensitivity set to between 0.0001 and 4.0000 AUFS (default settings),

eluting at 20 mL/min and using gradient mixtures of (A) Milli-Q

water with 0.1% (v/v) TFA and (B) HPLC-grade acetonitrile.

Inhibitor purity was evaluated by using a Hewlett-Packard Series

1100 analytical HPLC system ﬁtted with a Phenomenex Luna 5.0 μm

C18 4.6 mm × 150 mm cartridge, with eluent ﬂow set at 1.200 mL/

min and using gradient mixtures of (A) Milli-Q water with 0.1% (v/v)

TFA and (B) HPLC-grade acetonitrile. Retention times for the target

compound are given, followed by purities in their respective order.

Biologically evaluated compounds are ≥95% chemically pure.

1

A 400 MHz Bruker NMR instrument was utilized to obtain H,

¹³C, and ¹⁹F NMR spectra in CDCl₃(99.8 atom % D), CD₃CN (99.8

8497

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

After 16 h, the reaction was quenched with 1 M HCl and the layers

were separated. The organic layer was washed with 1 M HCl and the

combined aqueous layer was extracted with EtOAc or CH₂Cl₂. The

organic layer was dried (MgSO₄), ﬁltered, and concentrated in vacuo,

and puriﬁed by column chromatography to isolate the target

compound.

2H), hydroxamic acid OH, NH, and sulfonamide NH protons were not

observed. ¹³C NMR (126 MHz, cd₃cn) δ 162.6, 140.0, 131.7, 130.1,

129.0, 126.5, 126.3, 125.0, 46.2. ¹⁹F NMR (376 MHz, acetone-d₆) δ

−136.68 to −139.04 (m, 2F), −151.43 to −153.09 (m, 1F), −161.00

to −162.87 (m, 2F). HRMS (ESI⁺) m/z calcd for [C₁₄H₁₀F₅N₂O₄S]⁺:

397.03, found: 397.0269. HPLC (I) t_R= 16.99 min (97%).

Alternate Procedure for Formation of Hydroxamate Esters (f′).

Triethylamine (3.0 equiv) was charged in one go to a solution of the

appropriate carboxylic acid (1.0 equiv), 1-(3-(dimethylamino)-

propyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl, 1.5 equiv),

and 1-hydroxybenzotriazole monohydrate (HOBt, 1.1 equiv) in DMF

(0.1−0.2 M) at RT in air. After 10 min, the O-protected

hydroxylamine (2.0 equiv) was added in one go, and the reaction

was stirred for 16−24 h before quenching with 0.1 M HCl. The layers

were separated, the organic layer was washed with 0.1 M HCl, and the

aqueous layer was extracted with EtOAc or CH₂Cl₂. The organic layer

was dried (MgSO₄), ﬁltered, and concentrated in vacuo, and puriﬁed

by column chromatography to isolate the target compound.

Preparation of Sulfonyl Chlorides S3 from Polyhalogenated

Benzene Rings (g). The polyhalogenated benzene ring was added to

neat chlorosulfonic acid (5 equiv) in a round-bottom ﬂask under

argon atmosphere, and the resulting solution stirred at RT for 5 min.

The reaction was then heated to reﬂux (150 °C) for 2 h. The reaction

turned dark over this period of time. The reaction was cooled to RT,

and the crude material was added slowly to a mixture of ice and water.

The aqueous phase was extracted three times with EtOAc, washed

with brine, and concentrated to aﬀord a dark brown oil that was

ﬂushed through a bed of silica and used without puriﬁcation in

subsequent step.

tert-Butyloxycarbonyl (Boc) Protection (h). Di-tert-butyl dicar-

bonate (2.0 equiv) in THF (0.63 M) was added in one go to a

solution of the appropriate amine hydrochloride (1.0 equiv) in THF

and distilled water (1:1). Sodium bicarbonate (3.0 equiv) in distilled

water (0.95 M) was added to the reaction mixture in one go and

stirred at RT in air. After 19 h, the reaction was quenched by addition

of 1 M HCl until pH 3 and the layers were separated. The aqueous

layer was extracted with EtOAc and the combined organic layer was

dried (MgSO₄), ﬁltered, and concentrated in vacuo to isolate the

target compound without further puriﬁcation.

Carboxyl Benzylation (i). The appropriate benzoic acid (1.0 equiv)

and cesium carbonate (1.1−1.2 equiv) were suspended in DMF

(0.33−0.5 M) and stirred at RT for 20 min in air, before addition of

benzyl bromide (1.0 equiv) in one go. After 24 h, the reaction was

concentrated in vacuo and partitioned between EtOAc and distilled

water. The layers were separated, and the aqueous layer was extracted

with EtOAc. The combined organic layer was dried (MgSO₄), ﬁltered,

and concentrated in vacuo. Column chromatography isolated the

target compound.

Boc Deprotection (j). The appropriate carbamate (1.0 equiv) was

charged with ice-cooled 4 M HCl in dioxane (0.1 M) and stirred at

RT in air. After 3 h, the solvent was removed in vacuo, azeotroped

with CH₂Cl₂, to isolate the target compound without further

puriﬁcation.

N-Hydroxy-4-(((2,3,4,5,6-pentaﬂuoro-N-methylphenyl)-

sulfonamido)methyl)benzamide (3). The product was obtained

using synthetic procedure (c) after puriﬁcation using preparative

1

HPLC and lyophilization as a white powder (35.2%). H NMR (400

MHz, CD₃CN) δ 7.83 (d, J = 8.4 Hz, 2H rotamer #1), 7.78 (d, J = 8.3

Hz, 2H rotamer#2), 7.47 (d, J = 8.3 Hz, 2H rotamer#2), 7.37 (d, J =

8.2 Hz, 2H rotamer#1), 4.48 (s, 2H, rotamer#2), 4.44 (s, 2H

rotamer#1), 2.85 (s, 3H), hydroxamic acid NH and OH protons were

not observed. ¹³C NMR (126 MHz, acetonitrile-d₃) δ 160.6, 139.5,

136.6, 133.6, 131.5, 129.0, 128.2, 53.2, 34.1, 23.9. ¹⁹F NMR (376

MHz, chloroform-d) δ −134.19 to −136.14 (m, 2F), −146.11 (tt, J =

21.1, 6.7 Hz, 1F), −157.50 to −159.50 (m, 2F). HRMS (ESI⁺) m/z

calcd for [C₁₅H₁₂F₅N₂O₄S]⁺: 411.04; found 411.0425. HPLC (I) t_R=

14.54 min (97%).

N-Hydroxy-4-(((2,3,4,5,6-pentaﬂuoro-N-isopropylphenyl)-

sulfonamido)methyl)benzamide (4). The product was obtained

using synthetic procedure (e) after puriﬁcation using preparative

1

HPLC and lyophilization as a white powder (50%). H NMR (400

MHz, acetonitrile-d₃) δ 9.76 (s, 1H), 7.72 (d, 2H), 7.51 (d, 2H), 4.60

(s, 2H), 4.31 (p, J = 6.8 Hz, 1H), 1.11 (d, J = 6.8 Hz, 6H), hydroxamic

acid OH proton was not observed. ¹³C NMR (126 MHz, acetonitrile-

d₃) δ 165.1, 145.7, 143.7, 142.9, 130.9, 127.9, 127.0, 125.6, 117.4,

51.4, 46.3, 20.4. ¹⁹F NMR (376 MHz, acetonitrile-d₃) δ −137.37 to

−137.57 (m, 2F), −149.44 (tt, J = 20.2, 6.6 Hz, 1F), −161.26 to

−161.46 (m, 2F). HRMS (ESI⁺) m/z calcd for [C₁₇H₁₆F₅N₂O₄S]⁺:

439.07; found 439.0740. HPLC (I) t_R= 14.24 min (100%).

4-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)-

sulfonamido)methyl)-N-hydroxybenzamide (5). The product

was obtained using synthetic procedure (c) after puriﬁcation using

1

preparative HPLC and lyophilization as a white powder (53%). H

NMR (400 MHz, acetone-d₆) δ: 10.83 (s, 1H), 7.87 (d, J = 8.3 Hz,

2H), [7.52 (d, J = 8.3 Hz, 2H rotamer #1), 7.46 (d, J = 8.0 Hz, 2H

rotamer #2)], [4.68 (s, 2H rotamer #1), 4.65 (s, 2H rotamer #2)],

2.59−2.49 (m, 1H), 0.86−0.51 (m, 4H), hydroxamic acid OH proton

was not observed. ¹³C NMR (101 MHz, acetone) δ 160.1, 144.4, 141.5,

133.9, 129.8, 128.0, 124.6, 54.2, 31.1, 13.1, 8.2. ¹⁹F NMR (376 MHz,

CDCl₃) δ: −134.84 to −135.26 (m, 2F), −145.36 (t, J = 20.5 Hz,

1F), −157.84 to −158.96 (m, 2F). HRMS (ESI⁺) m/z calcd for

[C₁₇H₁₄F₅N₂O₄S]⁺: 437.0589, found: 437.0588. HPLC (I) t_R= 20.23

min (97.5%).

4-(((N-Cyclopropyl-2,3,5,6-tetraﬂuorophenyl)sulfonamido)-

methyl)-N-hydroxybenzamide (6). The product was obtained

using synthetic procedure (e) after puriﬁcation using preparative

1

HPLC and lyophilization as a white powder (49%). H NMR (400

MHz, acetone-d₆) δ 10.81 (s, 1H), 8.02−7.79 (m, 3H), 7.53 (d, J =

7.9 Hz, 2H rotamer#1), 7.46 (d, J = 7.7 Hz, 2H rotamer#2), 4.70 (s,

1H) (rotamer #1), 4.67 (s, 1H) (rotamer #2), 2.56 (p, J = 6.4, 3.5, 2.8

Hz, 1H), 0.80−0.61 (m, 4H), hydroxamic acid OH proton was not

observed. ¹³C NMR (126 MHz, acetonitrile-d₃) δ 161.8, 147.6, 140.9,

138.4, 131.2, 128.3, 127.2, 125.7, 120.5, 111.1, 53.7, 30.4, 6.4. ¹⁹F

NMR (376 MHz, acetone-d₆) δ −137.15 to −137.73 (m, 2F),

−137.79 to −138.20 (m, 2F). LRMS (ESI⁻) m/z calcd for

[C₁₇H₁₃F₄N₂O₄S]⁻: 417.05, found: 417.48. HRMS (ESI⁺) m/z

calcd for [C₁₇H₁₄F₄N₂O₄S]⁺: 419.0683, found: 419.0677. HPLC (I)

t_R= 20.29 min (100%).

4-(((N-Cyclopropyl-4-ﬂuorophenyl)sulfonamido)methyl)-N-

hydroxybenzamide (7). The product was obtained using synthetic

procedure (c) after puriﬁcation using preparative HPLC and

lyophilization as a white powder (59%). ¹H NMR (400 MHz,

acetone-d₆) δ: 10.28 (s, 1H), 8.03 (d, J = 8.9, 5.2 Hz, 2H), 7.85 (d, J =

8.3 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 4.52

(s, 2H), 2.26−2.19 (m, 1H), 0.79−0.68 (m, 4H), hydroxamic acid OH

proton was not observed. ¹³C NMR (101 MHz, acetone-d₆) δ: 130.7,

130.6, 128.5, 127.0, 116.4, 116.2, 54.1, 31.1, 6.8. ¹⁹F NMR (376 MHz,

2,3,4,5,6-Pentaﬂuoro-N-(4-(hydroxycarbamoyl)benzyl)-

benzamide (1). The product was obtained using synthetic procedure

1

(c) as a white-solid (39%). H NMR δ/ppm (400 MHz, DMSO-d₆)

4.54 (d, J = 5.9 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.3 Hz,

2H), 9.02 (s, 1H), 9.51 (t, J = 5.9 Hz, 1H), 11.19 (s, 1H). ¹³C NMR

δ/ppm (100 MHz, DMSO-d₆) 42.5, 127.1, 127.1, 131.7, 135.8, 138.1,

141.4, 141.9, 144.2, 156.8, 164.0. ¹⁹F NMR δ/ppm (54 MHz, DMSO-

d₆) −161.1 to −161.3 (m, 2F), −152.9 (t, J = 22.1 Hz, 1F), −142.0 to

−142.2 (m, 2F). LRMS (ESI⁺) m/z calcd for [C₁₅H₉F₅N₂O₃Na]⁺:

383.04, found: 383.16. HRMS (ESI⁺) m/z calcd for

[C₁₅H₁₀F₅N₂O₃]⁺: 361.0606, found: 361.0607. HPLC (I) t_R

=

10.48 min (98.7%).

N-Hydroxy-4-(((perﬂuorophenyl)sulfonamido)methyl)-

benzamide (2). The product was obtained using synthetic procedure

(e) after puriﬁcation using preparative HPLC and lyophilization as a

1

white powder (66.7%). H NMR (500 MHz, acetonitrile-d₃) δ 10.73

(s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 4.47 (s,

8498

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

acetone-d₆) δ: −107.29 to −107.46 (m, 1F). HRMS (ESI⁺) m/z calcd

was obtained using synthetic procedure (e) after puriﬁcation using

preparative HPLC and lyophilization as a white powder (71%). H

1

for [C₁₇H₁₈FN₂O₄S]⁺: 365.0966, found: 365.0969. HPLC (I) t_R

=

23.04 min (99%).

NMR (400 MHz, acetone) δ 7.87 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.6

Hz, 2H), 4.69 (s, 2H), 2.89−2.45 (m, 1H), 1.05−0.52 (m, 4H). ¹⁹F

NMR (376 MHz, acetone) δ −136.45 to −136.81 (m, 2F), −140.16

to −140.46 (m, 2F). ¹³C NMR (101 MHz, acetone) δ 146.0, 145.8,

143.0, 140.7, 131.7, 128.3, 127.2, 118.6, 53.7, 30.3, 6.4. HRMS (ESI⁺)

m/z calcd for [C₁₇H₁₄F₄ClN₂O₄S]⁺: 453.0293, found: 453.0294.

HPLC (I) t_R= 20.45 min (100%).

4-(((N-Cyclopropyl-3,4,5-triﬂuorophenyl)sulfonamido)-

methyl)-N-hydroxybenzamide (8). The product was obtained

using synthetic procedure (c) after puriﬁcation using preparative

1

HPLC and lyophilization as a white powder (60%). H NMR (400

MHz, acetone-d₆) δ: 10.83 (s, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.79−

7.69 (m, 2H), [7.48 (d, J = 8.3 Hz, 2H rotamer #1), 7.42 (d, J = 8.0

Hz, 2H rotamer #2)], [4.53 (s, 2H rotamer #1), 4.50 (s, 2H rotamer

#2)], 2.35−2.25 (m, 1H), 0.77−0.64 (m, 4H), hydroxamic acid OH

proton was not observed. ¹³C NMR (101 MHz, acetone) δ 202.5,

170.7, 152.6, 142.0, 136.9, 129.3, 127.9, 114.0, 113.7, 55.0, 31.9, 7.6.

¹⁹F NMR (376 MHz, acetone-d₆) δ: −132.40 to −132.79 (m, 1F),

−155.19 to −155.55 (m, 2F). HRMS (ESI⁺) m/z calcd for

4-(((N-Cyclopropyl-2,3,4,5-tetraﬂuorophenyl)sulfonamido)-

methyl)-N-hydroxybenzamide (14). The product was obtained

using synthetic procedure (e) after puriﬁcation using preparative

1

HPLC and lyophilization as a white powder (60%). H NMR (400

MHz, DMSO) δ 11.28 (s, 1H), 8.04−7.84 (m, 1H), 7.81−7.69 (m,

2H), 7.58−7.20 (m, 2H), 4.59 (s, 2H), 2.48 (p, J = 7.1, 3.9 Hz, 1H),

0.76−0.61 (m, 4H), hydroxamic acid OH proton was not observed. ¹³C

NMR (126 MHz, acetone) δ 141.0, 132.5, 130.8, 129.2, 129.1, 128.0,

126.2, 113.7, 113.5, 53.5, 31.2, 31.0, 28.1, 7.4. ¹⁹F NMR (376 MHz,

acetone-d₆) δ: −134.84 (ddt, J = 21.3, 14.0, 7.4 Hz, 1F), −138.26 to

−138.43 (m, 1F), −149.58 (tt, J = 20.0, 8.2 Hz, 1F), −153.95 (ddt, J

= 21.9, 19.2, 3.4 Hz, 1F). HRMS (ESI⁺) m/z calcd for

[C₁₇H₁₆F₃N₂O₄S]⁺: 401.0777, found: 401.0772. HPLC (I) t_R

=

26.05 min (98.8%).

4-(((N-Cyclopropyl-2,4,6-triﬂuorophenyl)sulfonamido)-

methyl)-N-hydroxybenzamide (9). The product was obtained

using synthetic procedure (c) after puriﬁcation using preparative

1

HPLC and lyophilization as a white powder (63%). H NMR (400

[C₁₇H₁₅F₄N₂O₄S]⁺: 419.0681, found: 419.0683. HPLC (I) t_R

=

MHz, acetone-d₆) δ: 10.86 (s, 1H), 7.89−7.82 (m, 2H), 7.52 (d, J =

8.0 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.28−7.18 (m, 2H), [4.66 (s,

2H rotamer #1), 4.63 (s, 2H rotamer #2)], 2.62−2.37 (m, 1H), 0.70−

0.62 (m, 4H), hydroxamic acid OH proton was not observed. ¹³C NMR

(101 MHz, acetone-d₆) δ: 160.5, 141.2, 138.8, 131.6, 128.3, 128.2,

127.1, 125.7, 102.8, 53.6, 30.1, 6.2. ¹⁹F NMR (376 MHz, acetone-d₆)

δ: −100.83 to −101.14 (m, 1F), −103.06 (dt, J = 39.8, 10.5 Hz, 2F).

HRMS (ESI⁺) m/z calcd for [C₁₇H₁₆F₃N₂O₄S]⁺: 401.0777, found:

401.0772. HPLC (I) t_R= 15.16 min (99%).

20.43 min (100%).

Intermediate Compound Characterization for All New

Molecules. Benzyl 4-Formylbenzoate (S1). The product was

obtained using synthetic procedure (i) as a light yellow oil (70%).

¹H (400 MHz, CDCl₃) δ: 10.10 (s, 1H); 8.23 (d, J = 8.3 Hz, 2H),

7.95 (d, J = 8.5 Hz, 2H), 7.35−7.47 (m, 5H), 5.40 (s, 2H). ¹³C (101

MHz, CDCl₃) δ: 191.6, 165.4, 139.2, 135.6, 135.1, 130.3, 128.7,

128.5, 128.4, 67.3. LRMS (ESI) mass not observed.

Benzyl 4-((Cyclopropylamino)methyl)benzoate (S2). The product

was obtained using synthetic procedure (a) as a pale yellow oil (83%).

¹H (400 MHz, CDCl₃) δ: 8.07 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 7.3

Hz, 2H), 7.45−7.36 (m, 5H), 5.39 (s, 2H), 3.92 (s, 2H), 2.16 (dd, J =

6.6, 3.7 Hz, 1H), 0.46 (s, 2H), 0.41 (s, 2H). ¹³C (101 MHz, CDCl₃)

δ: 166.4, 146.2, 136.2, 129.8, 128.7, 128.6, 128.2, 128.1, 66.6, 53.4,

53.1, 51.6. LRMS (ESI+) m/z calcd for [C₁₈H₂₀NO₂]⁺: 282.14,

found: 282.30.

3-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)-

sulfonamido)methyl)-N-hydroxybenzamide (10). The product

was obtained using synthetic procedure (e) after puriﬁcation using

1

preparative HPLC and lyophilization as a white powder (66.7%). H

NMR (400 MHz, MeOD) δ 7.78 (s, 1H), 7.70 (d, J = 7.7, 1.5 Hz,

1H), 7.61 (d, J = 7.7, 1.6 Hz, 1H), 7.48 (dd, J = 7.7 Hz, 1H), 4.64 (s,

2H), 2.48 (p, J = 6.4, 3.2 Hz, 1H), 0.80−0.61 (m, 4H). hydroxamic

acid OH and NH protons were not observed. ¹³C NMR (101 MHz,

chloroform-d) δ 139.4, 135.6, 130.8, 87.8, 31.6, 30.9, 30.4, 22.7, 14.1,

7.0. ¹⁹F NMR (376 MHz, chloroform-d) δ −134.85 (2F), −145.19

(1F), −158.25 (d, J = 18.1 Hz, 2F). HRMS (ESI⁺) m/z calcd for

[C₁₇H₁₃F₅N₂O₄S]⁺: 436.05, found: 437.05, HPLC (I) t_R= 19.59 min

(98.1%).

N-Cyclopropyl-2,3,4,5,6-pentaﬂuorobenzenesulfonamide (S9a).

The product was obtained using synthetic procedure (b) as a clear oil

1

without further puriﬁcation (80%). H NMR (400 MHz, CDCl₃) δ:

5.47 (s, 1H), 2.47−2.39 (m, 1H), 0.82−0.70 (m, 4H). ¹³C NMR (101

MHz, CDCl₃) δ: 156.8, 145.5, 144.8, 116.5, 39.9, 27.8. ¹⁹F NMR (376

MHz, CDCl₃) δ: −137.91 to −138.14 (m, 2F), −148.02 (s, 1F),

−160.05 to −161.11 (m, 2F). LRMS (ESI+) m/z calcd for

[C₉H₆F₅NO₂SNa]⁺: 309.99, found: 310.24.

4-(((3,5-Dichloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)-N-hydroxybenzamide (11). The product

was obtained using synthetic procedure (e) after puriﬁcation using

1

preparative HPLC and lyophilization as a white powder (81%). H

N-Cyclopropyl-2,3,4,5-tetraﬂuorobenzenesulfonamide (S9b).

The product was obtained using synthetic procedure (b) as a yellow

oil without further puriﬁcation (83%). ¹H NMR (400 MHz, CD₃CN)

δ: 7.92−7.30 (m, 1H), 6.40 (s, 1H), 2.34 (p, J = 6.9, 3.6 Hz, 1H),

0.65−0.53 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 156.8, 145.5,

144.8, 140.4, 117.3, 23.9, 4.9. ¹⁹F NMR (376 MHz, CDCl₃) δ:

−135.25 (s, 1F), −135.39 to −135.78 (m, 1F), −146.02 to −147.09

(m, 1F), −151.30 (s, 1F). LRMS (ESI-) m/z calcd for

[C₉H₆F₄NO₂S]⁻: 268.01, found: 268.02.

NMR (400 MHz, CD₃CN) δ 9.91 (s, 1H), 7.75 (d, J = 7.8 Hz, 2H),

7.48 (d, J = 7.7 Hz, 2H), 4.63 (s, 2H), 2.49 (p, J = 6.6, 3.5 Hz, 1H),

0.74−0.65 (m, 4H), hydroxamic acid OH proton was not observed. ¹³C

NMR (101 MHz, CD₃CN) δ 177.2, 157.5, 157.1, 153.2, 140.3, 129.2,

128.1, 54.7, 31.4, 7.4. ¹⁹F NMR (376 MHz, CD₃CN) δ −105.08 (t, J

= 6.9 Hz, 1F), −107.87 (d, J = 6.9 Hz, 2F). HRMS (ESI⁺) m/z calcd

for [C₁₇H₁₄F₃Cl₂N₂O₄S]⁺: 468.9998, found: 468.9997. HPLC (I) t_R=

20.78 min (100%).

2,3,4,5-Tetraﬂuorobenzenesulfonyl Chloride Used for S9b. The

product was obtained using synthetic procedure (g) as a clear, yellow

oil without further puriﬁcation (80%). ¹H NMR (400 MHz,

acetonitrile-d₃) δ 7.92−7.66 (m, 1H). ¹³C NMR (101 MHz,

acetonitrile-d₃) δ 147.8, 147.3, 146.7, 145.3, 144.6, 140.5, 116.6. ¹⁹F

NMR (376 MHz, acetonitrile-d₃) δ −133.82 to −134.01 (m, 1F),

−135.65 to −136.10 (m, 1F), −142.87 (s, 1F), −151.06 to −151.57

(m, 1F). LRMS (ESI+) found 229.04 (corresponding to M-1 for the

acid showing rapid compound hydrolysis under MS conditions).

Benzyl 4-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)-

sulfonamido)methyl)benzoate (S4a). The product was obtained

4-(((3-Chloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)-N-hydroxybenzamide (12). The product

was obtained using synthetic procedure (e) after puriﬁcation using

1

preparative HPLC and lyophilization as a white powder (77%). H

NMR (400 MHz, CD₃CN) δ: 7.75 (d, J = 7.8 Hz, 2H), 7.48 (d, J =

7.6 Hz, 2H), 7.29−7.16 (m, 1H), 4.61 (s, 2H), 2.59−2.40 (m, 1H),

0.77−0.56 (m, 4H), hydroxamic acid OH proton was not observed. ¹³C

NMR (101 MHz, CD₃CN) δ 148.0, 145.6, 144.6, 141.5, 132.1, 129.8,

128.1, 104.5, 104.0, 103.6, 54.6, 30.8, 6.7. ¹⁹F NMR (376 MHz,

CD₃CN) δ: −104.04 (q, J = 7.5 Hz, 1F), −105.46 (t, J = 10.4 Hz,

1F), −105.71 (d, J = 7.7 Hz, 1F). HRMS (ESI⁺) m/z calcd for

1

[C₁₇H₁₅F₃ClN₂O₄S]⁺: 435.0388, found: 435.0392. HPLC (I) t_R

=

using synthetic procedure (b) as a viscous pale yellow oil (75%). H

NMR (400 MHz, CDCl₃) δ: 8.09 (d, J = 8.3 Hz, 2H), 7.53−7.46 (m,

4H), 7.45−7.35 (m, 3H), 5.40 (s, 2H), 4.62 (s, 2H), 2.39 (p, J = 5.3

Hz, 1H), 0.72 (s, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 166.0, 141.4,

19.19 min (99.2%).

4-(((4-Chloro-N-cyclopropyl-2,3,5,6-tetraﬂuorophenyl)-

sulfonamido)methyl)-N-hydroxybenzamide (13). The product

8499

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

135.9, 130.1, 129.9, 128.6, 128.3, 128.3, 128.2, 66.8, 54.2, 30.4, 6.9.

130.0, 128.4, 53.6, 30.7, 6.8. ¹⁹F NMR (376 MHz, CDCl₃) δ: −134.65

to −134.83 (m, 2F), −145.18 (s, 1F), −158.16 to −159.22 (m, 2F).

LRMS (ESI-) m/z calcd for [C₁₇H₁₁F₅NO₄S]⁻: 420.03, found:

420.38.

¹⁹F NMR (376 MHz, CDCl₃) δ: −134.77 (s, 2F), −145.18 to

−145.41 (m, 1F), −158.10 to −158.36 (m, 2F). LRMS (ESI+) m/z

calcd for [C₂₄H₁₈F₅NO₄SNa]⁺: 534.08, found: 534.32.

4-(((N-Cyclopropyl-3,4,5-triﬂuorophenyl)sulfonamido)methyl)-

benzoic Acid (S5c). The product was obtained using synthetic

procedure (c) as an amorphous solid in a quantitative yield. ¹H NMR

(400 MHz, DMSO-d₆) δ: 7.92 (d, J = 8.0 Hz, 2H), 7.84 (m, 2H),

7.40 (d, J = 8.0 Hz, 2H), 4.46 (s, 2H), 2.25 (p, J = 8.8, 6.8, 3.7 Hz,

1H), 0.74−0.53 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 129.7,

128.3, 113.5, 97.6, 66.1, 54.1, 33.6, 31.4, 23.7, 7.2. ¹⁹F NMR (376

MHz, DMSO-d₆) δ: −130.95 to −131.32 (m, 2F), −153.44 (s, 1F).

LRMS (ESI-) m/z calcd for [C₁₇H₁₃F₃NO₄S]⁻: 384.05, found:

383.98.

4-(((N-Cyclopropyl-2,4,6-triﬂuorophenyl)sulfonamido)methyl)-

benzoic Acid (S5d). The product was obtained using synthetic

procedure (c) as an amorphous solid in a quantitative yield. ¹H NMR

(400 MHz, THF-d₈) δ: 8.03 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 7.9 Hz,

2H), 7.17 (t, J = 9.4 Hz, 2H), 4.63 (s, 2H), 2.20 (p, J = 7.6 Hz, 1H),

0.94−0.47 (m, 2H). ¹³C NMR (101 MHz, CD₃CN) δ: 169.2, 142.4,

140.7, 140.1, 139.8, 130.1, 110.0, 60.6, 30.5, 6.2. ¹⁹F NMR (376 MHz,

THF-d₈) δ: −100.67 (s, 1F), −102.55 (s, 2F). LRMS (ESI-) m/z

calcd for [C₁₇H₁₃F₃NO₄S]⁻: 384.05, found: 384.21.

tert-Butyl 4-(((N-Cyclopropyl-2,3,4,5-tetraﬂuorophenyl)-

sulfonamido)methyl)benzoate (S4b). The product was obtained

using synthetic procedure (d) as a viscous pale yellow viscous oil

1

(82%). H NMR (400 MHz, CDCl₃) δ: 8.09 (d, J = 8.3 Hz, 2H),

7.68−7.55 (m, 1H), 7.51−7.45 (m, 4H), 7.45−7.33 (m, 3H), 5.40 (s,

2H), 4.61 (s, 2H), 2.32 (p, J = 6.8, 5.6, 4.5, 1.0 Hz, 1H), 0.65 (d, J =

4.4 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 166.0, 141.7, 136.0,

130.3, 130.0, 129.8, 128.6, 128.3, 128.2, 128.1, 112.7, 66.7, 54.1, 30.3,

7.1. ¹⁹F NMR (376 MHz, CDCl₃) δ: −133.29 (s, 1F), −134.73 to

−136.53 (m, 1F), −145.42 to −146.92 (m, 1F), −150.93 to −151.01

(m, 1F). LRMS (ESI+) m/z calcd for [C₂₄H₁₉F₄NO₄SNa]⁺: 516.09,

found: 516.36.

Benzyl 4-(((N-Cyclopropyl-3,4,5-triﬂuorophenyl)sulfonamido)-

methyl)benzoate (S4c). The product was obtained using synthetic

procedure (b) as a yellow oil (91%). ¹H NMR (400 MHz, THF-d₈) δ:

8.07 (d, J = 8.3 Hz, 2H), 7.76 (t, J = 6.6 Hz, 2H), 7.55−7.49 (m, 4H),

7.45−7.09 (m, 3H), 5.40 (s, 2H), 4.51 (s, 2H), 2.20 (tt, J = 7.0, 3.4

Hz, 1H), 0.85−0.48 (m, 2H). ¹³C NMR (101 MHz, THF-d₈) δ:

165.2, 150.1, 143.7, 142.6, 141.2, 136.5, 135.1, 129.7, 129.5, 128.4,

128.3, 127.9, 112.9, 54.2, 27.7, 6.7. ¹⁹F NMR (376 MHz, THF-d₈) δ:

−132.18 (s, 1F), −154.98 (s, 2F). LRMS (ESI+) m/z calcd for

[C₂₄H₂₀F₃NO₄SNa]⁺: 498.10, found: 497.60.

Benzyl 4-(((N-Cyclopropyl-2,4,6-triﬂuorophenyl)sulfonamido)-

methyl)benzoate (S4d). The product was obtained using synthetic

procedure (b) as a yellow oil (81%). ¹H NMR (400 MHz, CDCl₃) δ:

8.08 (d, J = 8.3 Hz, 2H), 7.51−7.45 (m, 2H), 7.44−7.33 (m, 2H),

6.81 (t, J = 8.6 Hz, 1H), 5.39 (s, 2H), 4.61 (s, 2H), 2.36 (p, J = 7.0,

3.9 Hz, 1H), 0.97−0.56 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ:

166.0, 142.3, 136.0, 129.9, 129.6, 128.6, 128.2, 128.1, 126.5, 120.8,

102.1, 66.7, 54.0, 30.3, 6.8. ¹⁹F NMR (376 MHz, CDCl₃) δ: −98.44

(s, 1F), −101.59 (s, 2F). LRMS (ESI+) m/z calcd for

[C₂₄H₂₀F₃NO₄SNa]⁺: 498.10, found: 498.30.

4-(((3,5-Dichloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)benzoic Acid (S5f). The product was obtained

using synthetic procedure (c) as an amorphous solid. S5f and

byproduct S5f’ were isolated by reverse phase chromatography water/

ACN (100:0 → 0:100) as amorphous solids in a quantitative yield, in

1

a 75:25 proportion, respectively. H NMR (400 MHz, methanol-d₄)

δ: 8.05 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 4.69 (s, 2H),

2.55 (p, J = 6.7, 4.9, 3.6 Hz, 1H), 0.80−0.66 (m, 4H). ¹³C NMR (101

MHz, CD₃CN) δ: 170.0, 141.1, 140.5, 140.1, 133.2, 132.1, 130.0,

130.1, 111.5, 70.9, 31.3, 7.0. ¹⁹F NMR (376 MHz, methanol-d₄) δ:

−105.06 (s, 1F), −107.80 (s, 2F). LRMS (ESI+) m/z calcd for

[C₁₆H₁₁BrF₅NO₂SNa]⁺: 454.96, found: 454.31.

4-(((4-Chloro-N-cyclopropyl-2,3,5,6-tetraﬂuorophenyl)-

sulfonamido)methyl)benzoic Acid (S5g). The product was obtained

using synthetic procedure (c) as an amorphous solid in a quantitative

yield. ¹H NMR (400 MHz, CDCl₃) δ: 8.10 (d, J = 7.9 Hz, 2H), 7.50

(d, J = 7.9 Hz, 2H), 4.64 (s, 2H), 2.48−2.32 (m, 1H) 0.79−0.63 (m,

4H). ¹³C NMR (101 MHz, CDCl₃) δ: 161.2, 150.9, 150.8, 145.6,

145.2, 107.7, 107.4, 106.1, 67.5, 29.0, 23.7. ¹⁹F NMR (376 MHz,

CDCl₃) δ: −135.11 to −135.34 (m, 2F), −137.65 to −137.84 (m,

2F). LRMS (ESI-) m/z calcd for [C₁₇H₁₁ClF₄NO₄S]⁻: 436.00, found:

436.31.

N-(Benzyloxy)-4-(((N-cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)-

sulfonamido)methyl)benzamide (S6a). The product was obtained

using synthetic procedure (f) as an amorphous solid (62%). ¹H NMR

(400 MHz, CDCl₃) δ: 9.11 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.52−

7.31 (m, 7H), 5.03 (s, 2H), 4.57 (s, 2H), 2.37 (p, J = 5.5, 4.8 Hz,

1H), 0.73−0.64 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 163.4,

155.9, 154.3, 145.6, 140.4, 135.2, 131.6, 129.3, 128.8, 128.6, 128.4,

127.5, 78.4, 54.1, 30.4, 6.9. ¹⁹F NMR (376 MHz, CDCl₃) δ: −134.90

(s, 2F), −145.01 to −145.29 (m, 1F), −158.00 to −158.30 (m, 2F).

LRMS (ESI+) m/z calcd for [C₂₄H₁₉F₅N₂O₄SNa]⁺: 549.09, found:

549.43.

Benzyl 4-(((N-Cyclopropyl-4-ﬂuorophenyl)sulfonamido)methyl)-

benzoate (S4e). The product was obtained using synthetic procedure

(b) as a yellow oil (92%). ¹H NMR (400 MHz, THF) δ 8.07 (d, J =

8.3 Hz, 2H), 8.03−7.97 (m, 2H), 7.57−7.48 (m, 4H), 7.46−7.32 (m,

5H), 5.40 (s, 2H), 4.46 (s, 2H), 2.15−2.04 (m, 1H), 0.71−0.53 (m,

4H). ¹³C NMR (101 MHz, THF-d₈) δ: 166.4, 165.3, 163.9, 136.6,

135.0, 130.5, 129.5, 129.5, 128.4, 128.0, 127.9, 116.1, 115.8, 54.1,

31.0, 6.6. ¹⁹F NMR (376 MHz, THF-d₈) δ: −107.05 (s, 1F). LRMS

(ESI+) m/z calcd for [C₂₄H₂₂FNO₄SNa]⁺: 462.11, found: 462.46.

Benzyl 4-(((3,5-Dichloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)benzoate (S4f). The product was obtained

1

using synthetic procedure (b) as a yellow oil (61%). H NMR (400

MHz, CDCl₃) δ: 8.09 (s, 2H), 7.57−7.44 (m, 4H), 7.45−7.34 (m,

3H), 5.40 (s, 2H), 4.63 (s, 2H), 2.41 (p, J = 5.8, 5.3 Hz, 1H), 0.99−

0.20 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 130.0, 128.6, 128.2,

128.2, 126.7, 125.6, 123.4, 107.8, 67.9, 67.7, 29.1, 23.9, 7.0. ¹⁹F NMR

(376 MHz, CDCl₃) δ: −101.81 (s, 1F), −105.89 (s, 2F). LRMS

(ESI) mass not observed.

Benzyl 4-(((4-Chloro-N-cyclopropyl-2,3,5,6-tetraﬂuorophenyl)-

sulfonamido)methyl)benzoate (S4g). The product was obtained

4-(((3,5-Dichloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-

benzamide (S6b). The product was obtained using synthetic

1

using synthetic procedure (b) as a yellow oil (61%). H NMR (400

MHz, CDCl₃) δ: 8.09 (d, J = 8.3 Hz, 2H), 7.53−7.46 (m, 4H), 7.46−

7.34 (m, 3H), 5.40 (s, 2H), 4.63 (s, 2H), 2.46−2.37 (m, 1H), 0.77−

0.67 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 165.9, 141.4, 135.9,

134.5, 130.0, 129.9, 129.5, 128.6, 128.3, 128.2, 66.8, 54.2, 30.4, 7.0.

¹⁹F NMR (376 MHz, CDCl₃) δ: −134.91 to −135.16 (m, 2F),

1

procedure (f) as an amorphous solid (88%). H NMR (400 MHz,

CDCl₃) δ: 9.24 (s, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.61 (m, 1H), 7.43

(d, J = 8.0 Hz, 2H), 5.08 (s, 1H), 4.56 (s, 2H), 4.03 (dd, J = 10.4, 9.1,

2.9 Hz, 1H), 3.70−3.61 (m, 1H), 1.99−1.75 (m, J = 11.0, 9.8, 5.3 Hz,

3H), 1.75−1.47 (m, J = 14.5, 10.6, 5.1 Hz, 4H), 1.33−1.17 (m, 1H),

0.69−0.49 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 140.7, 131.6,

128.5, 127.6, 112.7, 102.7, 68.5, 62.6, 54.0, 30.3, 28.0, 27.7, 25.0, 22.1,

18.6, 7.0. ¹⁹F NMR (376 MHz, CDCl₃) δ: −133.36 to −133.98 (m,

1F), −135.31 (s, 1F), −145.79 to −146.76 (m, 1F), −150.49 to

−151.20 (m, 1F). LRMS (ESI) mass not observed.

−137.36 to −137.61 (m, 2F). LRMS (ESI+) m/z calcd for

[C₂₄H₁₈ClF₄NO₄SNa]⁺: 550.05, found: 550.30.

4-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)sulfonamido)-

methyl)benzoic Acid (S5a). The product was obtained using

synthetic procedure (c) as a white solid (98%). ¹H NMR (400

MHz, CDCl₃) δ: 8.12 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H),

4.65 (s, 2H), 2.42 (p, J = 5.2 Hz, 1H), 0.83−0.63 (m, 4H). ¹³C NMR

(101 MHz, DMSO-d₆) δ: 167.5, 165.6, 165.4, 150.9, 142.1, 130.7,

N-(Benzyloxy)-4-(((N-cyclopropyl-3,4,5-triﬂuorophenyl)-

sulfonamido)methyl)benzamide (S6c). The product was obtained

8500

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

using synthetic procedure (f) as an amorphous solid (71%). ¹H NMR

(400 MHz, CD₃CN) δ: 9.99 (s, 1H), 7.76−7.67 (m, 2H), 7.67−7.57

(m, 2H), 7.53−7.46 (m, 2H), 7.47−7.36 (m, 5H), 4.99 (s, 2H), 4.43

(s, 2H), 2.26−2.18 (m, 1H), 0.72−0.64 (m, 4H). ¹³C NMR (101

MHz, CD₃CN) δ: 164.9, 152.2, 149.7, 144.0, 141.1, 136.0, 134.4,

131.7, 129.2, 128.5, 128.4, 127.2, 114.6, 77.7, 54.1, 31.1, 6.8. ¹⁹F

NMR (376 MHz, CD₃CN) δ: −132.13 to −132.99 (m, 2F), −154.83

(s, 1F). LRMS (ESI-) m/z calcd for [C₂₄H₂₁F₃N₂O₄SCl]⁻: 525.09,

found: 525.19.

Benzyl 4-(((Perﬂuorophenyl)sulfonamido)methyl)benzoate (S4i).

The product was obtained using synthetic procedure (b) as a light

1

yellow solid (69%). H δ/ppm (400 MHz, CDCl₃) 4.67 (d, J = 5.9

Hz, 2H), 5.34 (s, 2H), 6.51 (s, 1H), 7.33−7.45 (m, 7H), 8.03 (d, J =

8.3 Hz, 2H); ¹³C δ/ppm (100 MHz, CDCl₃) 44.0, 67.0, 117.4, 127.6,

128.3, 128.5, 128.8, 129.9, 130.4, 136.0, 138.9, 142.2, 143.2, 145.6,

157.6, 166.2; ¹⁹F δ/ppm (376 MHz, CDCl₃) −159.6 to −160.0 (m,

2F), - 150.1 (s, 1F), −140.2 to −140.4 (m, 2F); LRMS (ESI+) m/z

calcd for [C₂₂H₁₄F₅NO₃Na]⁺: 458.08, found: 458.41.

tert-Butyl 4-(((2,3,4,5,6-pentaﬂuoro-N-isopropylphenyl)-

N-(Benzyloxy)-4-(((N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)benzoate (S11a). The product was obtained

sulfonamido)methyl)benzamide (S6d). The product was obtained

1

using synthetic procedure (d) as a viscous pale yellow oil (27%). H

using synthetic procedure (f) as an oﬀ-white solid (61%). H NMR

NMR (400 MHz, acetonitrile-d₃) δ 7.92 (d, J = 8.4 Hz, 2H), 7.48 (d, J

= 8.4 Hz, 2H), 4.60 (s, 2H), 4.33 (p, J = 6.8 Hz, 1H), 1.60 (s, 9H),

1.11 (d, J = 6.8 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ: 165.50,

141.43, 135.97, 130.10, 129.93, 128.64, 128.31, 128.29, 127.60, 65.01,

54.25, 32.24, 7.07. ¹³C NMR (101 MHz, CDCl₃) δ 165.5, 147.4,

147.3, 145.8, 145.0, 138.8, 133.6, 133.5, 112.0, 112., 39.71, 39.6, 23.6,

17.6, 12.3. ¹⁹F NMR (376 MHz, acetonitrile-d₃) δ −137.37 to

−137.57 (m, 2F), −149.44 (s, 1F), −161.26 to −161.46 (m, 2F).

LRMS (ESI-) m/z calcd for [C₂₁H₂₂ClF₅NO₄S]⁺: 514.09, found:

514.40.

(400 MHz, CD₃CN) δ: 10.01 (s, 1H), 7.80−7.65 (m, 2H), 7.57−7.34

(m,7H), 7.12−6.97 (m, 2H), 4.99 (s, 2H), 4.59 (s, 2H), 2.27−2.16

(m, 1H), 0.72−0.58 (m, 4H). ¹³C NMR (101 MHz, CD₃CN) δ:

178.0, 154.3, 151.0, 150.4, 141.4, 135.9, 131.7, 129.2, 128.5, 128.4,

127.3, 117.3, 77.8, 53.5, 30.2, 6.3. ¹⁹F NMR (376 MHz, CD₃CN) δ:

−100.83 to −101.19 (m, 1F), −103.33 to −103.70 (m, 2F). LRMS

(ESI-) m/z calcd for [C₂₄H₂₁F₃N₂O₄SCl]⁻: 525.09, found: 525.33.

N-(Benzyloxy)-4-(((N-cyclopropyl-4-ﬂuorophenyl)sulfonamido)-

methyl)benzamide (S6e). The product was obtained using synthetic

procedure (f) as a yellow solid (67%). ¹H NMR (400 MHz, CD₃CN)

δ: 7.96−7.83 (m, 2H), 7.71−7.63 (m, 2H), 7.52−7.46 (m, 2H),

7.45−7.29 (m, 7H), 4.40 (s, 2H), 2.17−2.05 (m, 1H), 0.67−0.57 (m,

4H). ¹³C NMR (101 MHz, CD₃CN) δ: 169.9, 152.3, 151.9, 151.8,

151.5, 150.9, 142.2, 136.4, 131.0, 129.0, 128.3, 112.5, 112.3, 77.8,

53.0, 23.3, 21.5, 13.9, 6.0. ¹⁹F NMR (376 MHz, acetone-d₆) δ:

−107.10 to −107.39 (m, 1F). LRMS (ESI-) m/z calcd for

[C₂₄H₂₃FN₂O₄SCl]⁻: 489.11, found: 489.35.

4-(((2,3,4,5,6-Pentaﬂuoro-N-isopropylphenyl)sulfonamido)-

methyl)benzoic Acid (S5j). The product was obtained using synthetic

1

procedure (e) as an oﬀ-white solid in a quantitative yield. H NMR

(400 MHz, acetonitrile-d₃) δ 7.98 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3

Hz, 2H), 4.62 (s, 2H), 4.32 (p, J = 6.8 Hz, 1H), 1.10 (d, J = 6.8 Hz,

6H). ¹³C NMR (101 MHz, CDCl₃) δ: 165.5, 141.4, 135.9, 135.7,

129.0, 128.9, 128.3, 127.9, 124.1, 72.0, 50.5, 20.3. ¹⁹F NMR (376

MHz, acetonitrile-d₃) δ −137.37 to −137.57 (m, 2F), −149.44 (s,

1F), −161.26 to −161.46 (m, 2F). LRMS (ESI) mass not observed.

N-Cyclopropyl-2,3,5,6-tetraﬂuorobenzenesulfonamide (S9d).

The product was obtained using synthetic procedure (b) as a yellow

oil without further puriﬁcation (68%). ¹H NMR (400 MHz,

chloroform-d) 7.39−7.28 (m, 1H), 2.47−2.35 (m, 1H), 0.74−0.63

(m, 4H). ¹³C NMR (101 MHz, CD₃CN) δ: 140.3, 133.9, 120.3, 15.7,

4.9. ¹⁹F NMR (376 MHz, acetonitrile-d₃) δ −137.67 to −138.01 (m,

2F), −137.79 to −138.07 (m, 2F). LRMS (ESI) mass not observed.

tert-Butyl 4-(((N-Cyclopropyl-2,3,5,6-tetraﬂuorophenyl)-

sulfonamido)methyl)benzoate (S11b). The product was obtained

using synthetic procedure (d) as a pale yellow viscous oil (77.4%). ¹H

NMR (400 MHz, chloroform-d) δ 7.97 (d, J = 8.2 Hz, 2H), 7.44 (d, J

= 8.2 Hz, 2H), 7.39−7.28 (m, 1H), 4.61 (s, 2H), 2.44−2.33 (m, 1H),

1.60 (s, 9H), 0.74−0.63 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ:

166.0, 141.7, 136.0, 130.3, 130.0, 129.8, 128.6, 128.3, 128.2, 128.1,

67.7, 54.1, 30.2, 7.0. ¹⁹F NMR (376 MHz, chloroform-d) δ −135.41

(s, 2F), −135.86 to −136.09 (m, 2F). LRMS (ESI) mass not observed.

Benzyl 3-Formylbenzoate (S1b). The product was obtained using

4-(((3,5-Dichloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)-

sulfonamido)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-

benzamide (S6f). The product was obtained using synthetic

1

procedure (f) as an amorphous solid (82%). H NMR (400 MHz,

CDCl₃) δ: 9.14 (s, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 8.0 Hz,

2H), 5.10 (s, 1H), 4.60 (s, 2H), 4.12−3.91 (m, 1H), 3.71−3.62 (m,

1H), 1.97−1.82 (m, 3H), 1.74−1.56 (m, 3H), 1.33−1.23 (m, 1H),

0.69 (d, J = 5.3 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 156.4,

145.4, 143.2, 140.5, 131.7, 128.5, 127.6, 102.7, 62.6, 54.2, 30.4, 28.0,

25.0, 18.6, 7.0. ¹⁹F NMR (376 MHz, CDCl₃) δ: −101.73 (s, 1F),

−105.97 to −106.12 (m, 2F). LRMS (ESI) mass not observed.

4-(((3-Chloro-N-cyclopropyl-2,4,6-triﬂuorophenyl)sulfonamido)-

methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (S6f’). The

product was obtained using synthetic procedure (f) as an amorphous

solid (73%). ¹H NMR (400 MHz, CDCl₃) δ: 9.12 (s, 1H), 7.76 (d, J

= 8.2 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 6.96 (m, 1H), 5.09 (s, 1H),

4.59 (s, 2H), 4.12−3.95 (m, 1H), 3.73−3.57 (m, 1H), 2.36 (p, J = 5.3

Hz, 1H), 1.99−1.74 (m, 4H), 1.74−1.47 (m, 4H), 0.75−0.58 (m, J =

5.3 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃) δ: 165.7, 145.1, 144.2,

144.0, 140.8, 131.6, 127.6, 102.7, 62.6, 54.1, 30.3, 28.0, 25.0, 18.6, 6.9.

¹⁹F NMR (376 MHz, CDCl₃) δ: −101.01 (s, 1F), −103.18 to

1

synthetic procedure (i) as a light yellow oil (98%). H NMR (400

MHz, chloroform-d) δ 10.07 (s, 1H), 8.56 (m, 1H), 8.34 (dd, J = 7.7,

1.5 Hz, 1H), 8.09 (dd, J = 7.7, 1.5 Hz, 1H), 7.63 (m, 1H), 7.51−7.46

(m, 2H), 7.45−7.36 (m, 3H), 5.42 (s, 2H). ¹³C NMR (101 MHz,

chloroform-d) δ 191.3, 165.3, 136.6, 135.6, 135.2, 133.1, 131.3, 131.2,

129.3, 128.7, 128.5, 128.4, 67.2. LRMS (ESI+) m/z calcd for

[C₁₅H₁₂O₃]⁺: 240.08, found: 241.11.

−104.54 (m, 1F), −104.00 (t, J = 9.9 Hz, 1F). LRMS (ESI) mass not

observed.

4-(((Perﬂuorophenyl)sulfonamido)methyl)-N-((tetrahydro-2H-

pyran-2-yl)oxy)benzamide (S6l). The product was obtained using

1

synthetic procedure (f) as an oﬀ-white solid (70%). H NMR (400

Benzyl 3-((Cyclopropylamino)methyl)benzoate (S2b). The prod-

uct was obtained using synthetic procedure (a) as a pale yellow oil

(93%). ¹H NMR (400 MHz, chloroform-d) δ 8.06 (s, 1H), 8.00 (dt, J

= 7.8, 1.5 Hz, 1H), 7.55 (dt, J = 7.7, 1.5 Hz, 1H), 7.49 (d, 2H), 7.42

(dt, J = 7.7, 1.9 Hz, 3H), 7.38 (d, 1H), 5.40 (s, 2H), 3.90 (s, 2H),

2.16 (p, 1H), 0.48−0.45 (m, 2H), 0.45−0.40 (m, 2H). ¹³C NMR

(101 MHz, chloroform-d) δ 166.5, 141.0, 136.1, 133.0, 130.2, 129.4,

128.6, 128.4, 128.3, 128.2, 128.2, 66.7, 53.3, 43.5, 30.1, 6.5. LRMS

(ESI+) m/z calcd for [C₁₈H₁₉NO₂]⁺: 281.36, found: 282.11.

Benzyl 3-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)-

sulfonamido)methyl)benzoate (S4h). The product was obtained

using synthetic procedure (b) 78%. ¹H NMR (400 MHz, chloroform-

d) δ 8.07−8.02 (m, 2H), 7.67 (m, 1H), 7.50 (s, 1H), 7.48 (s, 2H),

7.45−7.40 (m, 3H), 5.40 (s, 2H), 4.62 (s, 2H), 2.40 (p, 1H), 0.74 (s,

2H), 0.73 (s, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 166.0,

MHz, acetone-d₆) δ 7.77 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H),

5.08 (s, 1H), 4.46 (s, 2H), 2.13−1.28 (m, 8H). ¹³C NMR (101 MHz,

acetone) δ 205.5, 154.2, 145.7, 140.3, 139.1, 132.0, 127.9, 102.0, 62.3,

47.9, 28.0, 25.4, 25.3, 25.0, 19.9, 18.4. ¹⁹F NMR (376 MHz, acetone-

d₆) δ −138.08 to −139.04 (m, 2F), −150.03 (s, 1F), −161.43 to

−162.65 (m, 2F). LRMS (ESI-) m/z calcd for [C₁₉H₁₆F₅N₂O5_S]⁻:

479.07, found: 479.38.

Benzyl 4-(Aminomethyl)benzoate Hydrochloride (S2c). The

product was obtained using synthetic procedure (i) as a white solid

(83%). ¹H δ/ppm (400 MHz, DMSO-d6) 4.10 (s, 2H), 5.36 (s, 2H),

7.34−7.48 (m, 5H), 7.66 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz,

2H); ¹³C δ/ppm (100 MHz, DMSO-d6) 41.7, 66.3, 127.9, 128.1,

128.5, 129.2, 129.4, 129.5, 136.1, 139.6, 165.2; LRMS (ESI+) m/z

calcd for [C₁₅H₁₅NO₂Na]⁺: 264.10, found: 264.08.

8501

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

136.4, 135.9, 133.0, 130.5, 129.5, 129.3, 128.9, 128.6, 128.3, 128.2,

66.8, 54.1, 30.3, 7.1. ¹⁹F NMR (376 MHz, chloroform-d) δ −134.65

(dq, J = 21.0, 6.9, 6.0 Hz, 2F), −143.09 to −149.37 (m, 1F), −156.12

to −161.92 (m, 2F). LRMS (ESI+) m/z calcd for [C₂₄H₁₈F₅NO₄S]⁺:

511.09, found: 512.11.

3-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)sulfonamido)-

methyl)benzoic Acid (S5h). The product was obtained using

synthetic procedure (c) as an amorphous white solid in a quantitative

yield (98%). ¹H NMR (400 MHz, acetone-d₆) δ 8.06 (s, 1H), 8.01 (d,

J = 7.8 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H),

4.70 (s, 2H), 2.54 (p, J = 6.9, 3.8 Hz, 1H), 0.78−0.61 (m, 4H). ¹³C

NMR (101 MHz, acetone-d₆) δ 167.4, 137.2, 132.4, 132.0, 129.4,

128.9, 128.6, 53.7, 30.1, 6.4. ¹⁹F NMR (376 MHz, acetone-d₆) δ

−136.80 (s, 2F), −145.37 to −152.60 (m, 1F), −161.51 to −162.30

(m, 2F). LRMS (ESI+) m/z calcd for [C₁₇H₁₂F₅NO₄S]⁺: 421.04,

found: 422.21.

3-(((N-Cyclopropyl-2,3,4,5,6-pentaﬂuorophenyl)sulfonamido)-

methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (S6h). The

product was obtained using synthetic procedure (f) as a clear oil

(84.5%). ¹H NMR (400 MHz, chloroform-d) δ 9.32 (s, 1H), 7.73 (s,

1H), 7.68 (d, J = 7.7 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.7

Hz, 1H), 5.08 (s, 1H), 4.56 (s, 2H), 1.93−1.75 (m, 4H), 1.73−1.53

(m, 4H), 0.68 (d, J = 5.7 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 146.0, 143.4, 139.5, 138.1, 133.1, 131.6, 129.0, 127.4, 126.8, 115.1,

101.5, 61.9, 53.7, 30.6, 28.4, 25.2, 18., 14.25, 6.9. ¹⁹F NMR (376

MHz, chloroform-d) δ −134.85 to −135.69 (m, 2F), −145.52 to

−146.03 (m, 1F), −154.98 to −162.40 (m, 2F). LRMS (ESI+) m/z

calcd for [C₂₂H₂₁F₅N₂O₅S]⁺: 520.11, found: 520.54.

Western Blotting. Brieﬂy, MV4-11 cells were incubated with

inhibitors prior to cell lysis with radioimmunoprecipitation assay

(RIPA) buﬀer (20 mM Tris pH 7.4, 150 mM NaCl, 0.5%

deoxycholate, 1% Triton X-100, and 0.1% sodium dodecyl sulfate

(SDS)). Total protein content was determined through a BCA assay

(ThermoFisher), resolved via a 4−20% polyacrylamide SDS gel, and

transferred to a nitrocellulose membrane (Bio-Rad). The membranes

were blocked with a 5% solution (skimmed milk powder in PBS-T).

This was followed by incubation at 4 °C (overnight) with the

following antibodies: acetylated α-tubulin mouse monoclonal

(MABT868, EMD Millipore), acetylated histone H3 (Ac-Lys18,

07−354, Sigma), PARP-1 (ab227244, Abcam), apoptosis Western

blot cocktail (136812, Abcam), cleaved PARP-1 (ab32561, Abcam),

and HSC70 (sc-7298, Santa Cruz). Following overnight incubation,

horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG

secondary antibody (7076, Cell Signaling) or HRP-linked anti-rabbit

IgG secondary antibody (7074, Cell Signaling) was applied to the

membrane in a 1:5000 dilution. The bands were visualized using

clarity Western ECL substrate luminal/enhancer solution and

peroxide solution. Western blotting analysis was carried out using

Image lab software (Bio-Rad).^47,50,70

T11, and YT cell lines were obtained from the Deutsche Sammlung

von Mikroorganismen and Zellkulturen GmbH (DSMZ, Germany).

SNK6 and NK-YS were a generous gift from Dr. Wing C. Chan (City

of Hope Medical Center, Duarte, CA, USA), and the DND-41 cell

line was a generous gift from A. Thomas Look (Dana-Farber Cancer

Institute, Boston, MA, USA). Myla cells were a generous gift from Dr.

K. Kaltoft, Institute of Human Genetics, University of Aarhus

(Aarhus, Denmark) via European Collection of Authenticated Cell

Cultures (ECACC). MOLT4 cells were obtained from ATCC, USA.

NHF and pPF were grown with Cell System growth medium,

supplemented with Culture Boost, whereas the U87-MG cells were

grown in DMEM (and 10% FBS). HUVECs were grown in Vascular

Cell Basal medium supplemented with Endothelial Cell Grow Kit-

VEGF. NHF and pPF were obtained from Cell Systems, USA. U87-

MG and HUVEC cell lines were obtained from ATCC, USA.

Cell lines were regularly tested for mycoplasma using the

MycoAlert mycoplasma detection kit (Lonza Group AG, Switzer-

land). All cell lines were cultured at 37 °C in a humidiﬁed atmosphere

containing 5% CO₂. Experiments were performed within 20 passages

after cell resuscitation. None of the above-mentioned cell lines are

listed in the register of cell lines that are known to be misidentiﬁed

through cross-contamination.

Cells were plated in 96-well ﬂat-bottom sterile culture plates with

low-evaporation lids (Costar #3997). The inhibitors and a vehicle

control (0.5% DMSO) were added to the cells following 24 h. After

72 h, Cell Titer-Blue (Promega #G808A) was added to each well (20

μL), and the ﬂuorescence was measured at 560/590 nm using a

Cytation S63 spectrophotometer (BioTek) or a GloMax Discover

Microplate Reader (Promega, Madison, WI, USA). GraphPad Prism

6.0 (GraphPad Software Inc.) was used to determine the IC₅₀values.

Immunoﬂuorescence Assay. HeLa cells were plated to

subconﬂuency on a 96-well plate (clear bottom black from Fisher

Scientiﬁc). As above, inhibitors were introduced following 24 h. The

cells were washed with 1× PBS, ﬁxed with 4% formaldehyde

(Millipore Sigma), permeabilized with 1% Triton X-100 (Millipore

Sigma), and blocked in 5% bovine serum albumin (BSA) (BioShop)

for 1 h at room temperature. Following this treatment, the cells were

incubated in a cocktail composed of acetylated α-tubulin mouse

monoclonal (1:100 dilution, MD Millipore) and acetylated histone

H3 (1:50 dilution, Ac-Lys18, Sigma) antibodies. Secondary antibodies

anti-mouse Alexa Fluor 647 and anti-rabbit Alexa Fluor 488 were used

for immunoﬂuorescent detection. Cells were counterstained for

nucleic acids using 4′,6-diamidino-2-phenylindole (DAPI) (Thermo-

Fisher Scientiﬁc). The cells were imaged with a Cytation S63

spectrophotometer and the ﬂuorescence intensity was determined

using ImageJ (n = 3). Two-way ANOVA with Tukey’s multiple

comparisons test were performed using GraphPad Prism 6.0

(GraphPad Software Inc.). A p-value ≤ 0.05 was considered

signiﬁcant.

FACS Apoptosis Detection Assay. MV4-11 cells were prepared,

dosed, and washed with cold 1× PBS. The resulting cell pellets were

resuspended in 1× Binding Buﬀer (1 × 10⁶cell/mL) from the FITC

Annexin V Apoptosis Detection Kit I (BD Pharmingen). Sub-

sequently, the dyes annexin V (5 μL) and propidium iodide (PI, 5

μL) were added to 2.5 × 10⁵cells (250 μL). The suspension was

thoroughly mixed and incubated in the dark for 15 min. Following the

addition of 250 μL of 1× binding buﬀer, the cells were analyzed by

ﬂow cytometry within 1 h using the Cytoﬂex S system (Beckman

Coulter).

Maximum Tolerated Dose Studies. Acute toxicity of the lead

inhibitors was studied using 4−12 week old male CD-1 mice (Charles

River). A concentration of 20 mg/kg of inhibitor via oral gavage was

administered to mice (n = 5) over 5 days. The vehicle consisted of

10% DMSO, 5% Cremophor EL, and 85% saline. Mice were

monitored daily for toxicity eﬀects including excessive changes in

weight and hair as well as changes to posture and inactivity. All mouse

studies were approved by the Local Animal Care Committee at the

University of Toronto and Oﬃce of Laboratory Animal Welfare

(OLAW). The mice were housed in Tecniplast Blueline ventilated

Cytotoxicity Assays. HeLa cells were grown in Dulbecco’s

modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal

bovine serum (FBS) (Sigma-Aldrich). MV4-11 were grown in Iscove’s

modiﬁed Dulbecco’s medium (IMDM) supplemented with 10% FBS.

MOLM-13 and MRC-9 cells were maintained in RPMI-1640

supplemented with 10% FBS. HeLa, MOLM-13, MRC-9, and MV4-

11 were obtained from the American Type Culture Collection

(ATCC, USA).

The PTCL cell lines, KHYG-1, NK-92, SNK6, DERL-2, Mac1,

1305 Myla, Hut78, SU-DHL-1 or T-ALL cell lines SUP-T11, DND-

41, and MOLT4 were maintained in RPMI-1640 supplemented with

10% FBS, 0.06 g/L penicillin/0.1 g/L streptomycin (Pen/Strep,

Gibco), and 2 mM ^L-glutamine (Gibco). Culture media of KHYG-1,

NK-92, SNK6, and DERL-2 cells was additionally supplemented with

2.5 ng/mL recombinant human IL-2 (ImmunoTools GmbH,

Germany). The authenticity of PTCL cell lines was conﬁrmed by

analysis of highly polymorphic short tandem repeat loci (STR) using

the PowerPlex 16 HS System (Promega; performed by Microsynth

AG, Switzerland).

Hut78 cells were obtained from CLS Cell Lines Service GmbH,

Germany. Mac1, SU-DHL-1, HH, DERL-2, KHYG-1, NK-92, SUP-

8502

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

cages with environmental enrichment and fed a Teklad 2019 diet

supplemented with municipal water.

Computational Modeling and Docking. Computational

alprazolam, 200 nM caﬀeine, 200 nM Labetalol, and 100 nM

tolbutamide). Samples were vortexed and centrifuged for 45 min at

3220g. Aliquots were diluted by ultrapure water, and used for LC/

MS/MS analysis. Peak area ratios were determined and percent

compounds remaining was calculated by the following equation:

̈

modeling and docking was performed with Schrodinger Maestro

11.9.011 software using Glide, Epik, Optimized Potentials for Liquid

Simulations 3e (OPLS3e) force-ﬁeld, Glide, LigPrep and the Protein

Preparation Wizard. All ligand poses as well as protein structure

images were generated with Maestro version11.9.011. Relevant

compounds were prepared via a ligand preparation workﬂow (Epik

to generate possible protonation states at target pH 7.0 2.0) and

generating tautomers. Protein structures were retrieved from the

protein data bank (https://www.rcsb.org): HDAC6, 5EDU; HDAC8,

1T64. For protein model preparation, appropriate bond orders were

assigned as well as including hydrogen atoms and creating zero-order

bonds to any metal centers. Any of the loops and side chains that were

not present in the protein structures were added using Prime.

Furthermore, any water molecules that were located >5.0 Å from

heteroatoms were removed. All residues with steric clashes were

individually minimized (OPLS3e). Ramachandran plot analysis was

used to examine the structure output. Receptor grids (10 × 10 × 10

Å³) were deﬁned surrounding the cocrystallized ligands (Trichostatin

A, TSA) within the HDAC active site during docking. The relevant

HDACi were screened against HDAC6 and HDAC8 using standard

precision (SP) docking, and the top 25 binding poses were generated.

In all simulations, the ligands were ﬂexible and the protein remained

static (excluding rotatable groups). After postdocking minimizations,

the top binding pose for each ligand was produced and analyzed for

protein−ligand interactions using Maestro 11.9.011 and Ligand

Interaction Diagrams.

Permeability Determination by Lipid-PAMPA. A 1.8%

solution (w/v) of lecithin in dodecane was added to each acceptor

plate well (top), followed by application of the artiﬁcial membrane

and addition of 300 μL of PBS (pH 7.4) solution to each well of the

acceptor plate. Compounds were added to the donor plate and

incubated at 25 °C, 60 rpm for 16 h. After incubation, aliquots of 50

μL from each acceptor well and donor plate were transferred into a

96-well plate, vortexed at 750 rpm for 100 s and centrifuged at 3220g

for 20 min. Compound concentrations and eﬀective permeability (P_e)

were determined by LC/MS/MS.

Stability in Mouse Plasma. The plasma stability of the

compounds was evaluated in mouse plasma provided from Sigma-

Aldrich, Oakville, Canada. The main stock solutions of the

compounds were prepared at 10 mM in DMSO, which was diluted

to 100 μM in 50% acetonitrile (ACN)/50% water. Aliquots of 356.5

μL of plasma proteins were placed in a heating block and allowed to

equilibrate at 37 °C for 5 min. Then, 3.5 μL of 100 μM compound

solutions were added to each vial (ﬁnal concentration of 1 μM).

Three quality control (QC) samples at 100, 500, and 1000 nM were

prepared in 0.5% ACN. The vial contents were transferred in 50 μL

aliquots at time points of 0.5, 5, 10, 20, 30, and 60 mi to a 96-well

autosampler plate containing 150 μL of protein precipitation solution.

Ice-cold ACN containing internals standards (100 nM glyburide)

were used as the protein precipitation solution. After centrifugation at

4 °C, 5500 rpm for 15 min, the supernatant was diluted in MQ water,

and injected into the LC/MS/MS for quantitative analysis.

The mobile phase consisted of (A) 0.1% (v/v) formic acid in Milli-

Q water; (B) 0.1% (v/v) formic acid in acetonitrile. Gradients were

run over 15 min and proceeded as follows: A:B, 85:15, 0.0−1 min,

85:15 → 10:90, 1−7 min, 10:90, 7−9 min, 10:90 → 85:15, 9−9.5

min, 85:15, 9.5−15 min. The analytical column was a Waters T3 HSS

iKey 1.7 μm (50 × 0.15 mm) column. The MS data was collected via

multiple reaction monitoring in positive ion mode. All calculations

were carried out using Microsoft Excel. Peak areas were determined

from extracted ion chromatograms. The in vitro half-life (t_1/2) of

parent compounds were determined by regression analysis of the

percent parent disappearance vs time curve.

peak area ratio (t min)

peak area ratio (0 min)

remaining percentage_tmin(%) =

× 100

where peak area ratio (t min) is the peak area ratio of control and test

compounds at t min; peak area ratio_0minis the peak area ratio of

control and test compounds at the zero time point. In vitro half-life

(t_1/2) = −(0.693/k).

Reversibility of Inhibition (K_offKinetics). The enzyme was

preincubated with compound or DMSO, followed by dilution of the

[compound/enzyme] complex into buﬀer with a substrate peptide to

bring the compound concentration ∼10× below its respective IC₅₀

value. Progress curves were obtained after dilution, and enzyme

recovery was ﬁtted with the following equation: % conv = V_s× t + (V_i

− V_s)/K_obsapp × (1 − exp(−K_obsapp × t), in which V_iis the initial

velocity, V_sis steady state velocity, and K_obsis the observed rate

constant. The obtained K_obsrate constant was used to determine the

reversibility of inhibition and calculate residence time as 1/2T_res= ln2

× 1/K_obs

.

Determination of K_i(K_onKinetics). Serial dilutions of

compound were mixed with 3 nM HDAC6 enzyme and substrate

peptide in assay buﬀer. Progress curves were acquired without

preincubation for ∼5 h on a Labchip3000 instrument. Progress curves

in the presence of compound were ﬁt with the following equation:

((A + (V_s× x)) + (((V_o− V_s) × (1 − exp((−1 × K_obs) × t)))/K_obs)).

To determine observed rate of inhibition, V_owas locked to that in

control (V_i, DMSO only). Fractional steady state velocity (V_s/V_i) and

K_obsvalues were plotted against [compound] to determine K_iand

residence time.

In Vitro Stability in Mouse Hepatocytes. The inhibitors (and

controls) were formulated to concentrations of 100 μM in 50%

acetonitrile/50% water. The medium (William’s E Medium

supplemented with GlutaMAX) and hepatocyte thawing medium

were allowed to reach room temperature and the hepatocytes and

thawing medium and centrifuged at 100 g for 10 min. The

supernatant was removed, and hepatocytes resuspended in the

incubation medium (∼1.5 × 10⁶cells/mL). The cells were diluted

to 0.5 × 10⁶viable cells/mL. A volume of 198 μL of hepatocytes was

transferred to a 96-well noncoated plate and incubated at 37 °C for 10

min. Following this, 2 μL of the 100 μM test compounds or positive

control was transferred into respective wells to start the reaction. At

diﬀerent time points (0, 15, 30, 60, 90, and 120 min), 25 μL aliquots

were mixed with 6 volumes (150 μL) of acetonitrile containing

internal standard, IS (100 nM alprazolam, 200 nM labetalol, 200 nM

caﬀeine, and 200 nM diclofenac) to terminate the reaction. Samples

were vortexed for 5 min and centrifuged for 45 min at 3220g, diluted

by ultrapure water, followed by LC/MS/MS analysis. The in vitro

half-life (T_1/2) was determined by regression analysis of the percent

parent disappearance vs time curve (T_1/2= 0.693/k) using Microsoft

Excel. Conversion of the in vitro T_1/2into the in vitro intrinsic

clearance (in vitro CL_int.in μL/min/10⁶cells) was performed using

the following equation (mean of duplicate determinations): CL_int

=

kV/N, where V = incubation volume (0.2 mL) and N = number of

hepatocytes per well (0.1 × 10⁶cells)

In Vivo PK Study in CD-1 Male Mice (Pharmaron, MA, USA).

The test compounds were formulated within a 4 mg/mL solution

(10% DMA, 65% PEG400, 25% saline). CD-1 mice were

administered the test compound (20 mg/kg, I.P) once, and blood

samples were obtained from each mouse at 0.25, 0.5, 1, 2, 4, 8, and 24

h postdose. The working solutions of 5 μL at diﬀerent concentrations

(2, 4, 10, 20, 100, 200, 1000, 2000 ng/mL) were added to CD-1

mouse plasma (10 μL) to generate calibration standards of 1, 2, 5, 10,

50, 100, 500, and 1000 ng/mL. Four quality control (QC) samples at

2, 5, 50, and 800 ng/mL for plasma were prepared independently of

calibration curves. Standards, QC samples, and unknown samples

Glutathione Stability Assay. Compound was added to PBS and

5 mM GSH to reach a ﬁnal concentration of 5 μM. Samples were

incubated at 25 °C at 60 rpm, and aliquots were taken at 0, 30, 60,

and 120 min and quenched with internal standard, IS (100 nM

8503

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(total volume 15 μL) were added to acetonitrile (200 μL) containing

IS (2 ng/mL verapamil, and 50 ng/mL dexamethasone) for

precipitation of protein. Samples were vortexed and centrifuged (4

°C, 3900 rpm, 15 min), and the supernatant was diluted 3-fold with

ultrapure water. Diluted supernatant was injected into the LC/MS/

MS system for quantitative analysis.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00420.

■

sı

Raw data, structures of inhibitors, additional biochemical

assay results, docking results, ﬂow cytometry, pharma-

cokinetic evaluations, cytotoxicity assays, and compound

characterizations (PDF)

Molecular formula strings (CSV)

In silico docking binding pose of 5 in human HDAC6

(PDB)

In silico docking binding pose of 14 in human HDAC6

(PDB)

In silico docking binding pose of 14 in human HDAC8

(PDB)

In vitro IC₅₀values for 1−14, SAHA, ricolinostat, and

citarinostat (XLSX)

HDAC Target Engagement (Nanosyn, CA, USA). The full-

length human HDACs were recombinantly expressed and puriﬁed

from SF9 insect cells. All compounds were serially prediluted in

DMSO from a top concentration of 10 or 1 μM and introduced into

the 384-well plates through an acoustic dispenser (Labcyte550) into

the reaction buﬀer (100 mM HEPES, pH 7.5, 25 mM KCl, 0.1% BSA,

0.01% Triton X-100, and enzyme). The total concentration of DMSO

was 1% in all wells. The deacetylation reaction was initiated by

introduction of a FAM-labeled acetylated peptide substrate. The

reaction was monitored through the change in the relative

ﬂuorescence intensity of the substrate and product peaks. The

activity was determined as the product sum ratio (PSR): P/(S + P),

where P = product peak height and S = substrate peak height. The

negative control (0% inhibition in absence of inhibitor, DMSO only)

and positive control (100% inhibition in absence of enzyme) were

determined from n = 4 samples. Percent inhibition (P_inh) was

In vitro IC₅₀values for 14 and citarinostat (XLSX)

Pharmacology of 14 and citarinostat (XLSX)

Cellular IC₅₀values for 14 and citarinostat (XLSX)

Pharmacokinetic parameters of 14 (XLSX)

determined through the equation: P_inh= (PSR_0%− PSR_inh)/(PSR_0%

−

PSR_100%) × 100, where PSR_inhis the product sum ratio in the

presence of inhibitor, PSR_0%is the product sum ratio in absence of

inhibitor, and PSR_100%is the product sum ratio in 100% inhibition

samples. Inhibition curves (P_inhvs inhibitor concentration) were ﬁtted

by a four-parameter sigmoid dose−response model using XLﬁt

software (IDBS) to determine the IC₅₀values.

Primary T-PLL Patient Sample Studies. Primary T-PLL cells

were isolated from peripheral blood (PB) of 10 T-PLL patients. All

patients were diagnosed according to WHO criteria based on clinical

features, immunophenotyping (ﬂow cytometry and histochemisty;

including TCL1 expression), and FISH/karyotyping.^71,72Samples

were obtained from patients under IRB-approved protocols following

written informed consent according to the Declaration of Helsinki.

Collection and use of patient material have been approved for

research purpose by the ethics committee of the University Hospital

of Cologne (EudraCT-Nr.: #2008-001421-34 and AZ11-319) and

Helsinki (303/13/03/01/2011).

All patients except for one (patient 9: alemtuzumab and

bendamustine) were untreated before the time of sample collection,

and all the patients, except for two (Patient 8 (50%) and Patient 10

(25%)) had a > 80% T-cell fraction of their PB leukocytes. PB

mononuclear cells (PBMCs) of T-PLL samples were obtained by

density gradient centrifugation (Histopaque; Sigma-Aldrich). All drug

screening in primary T-PLL patient samples were conducted as

previously described.^34,73

Oncomine Gene Expression Analysis. HDAC gene expression

data and associated statistical analyses were extracted from the

Oncomine Research Premium Edition database (Thermo Fisher, Ann

Arbor, MI),^46,59from the data sets described in Figure 7 using the

following reporters: HDAC1 (201209_at), HDAC2 (201833_at),

HDAC3 (216326_s_at), HDAC4 (204225_at), HDAC5

(202455_at), HDAC6 (206846_s_at), HDAC9 (205659_at), and

HDAC11 (219847_at).

AUTHOR INFORMATION

Corresponding Authors

■

Patrick T. Gunning − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada;

Centre for Medicinal Chemistry, University of Toronto

Mississauga, Mississauga, Ontario L5L 1C6, Canada;

orcid.org/0000-0003-0654-735X; Phone: 905-828-

5354; Email: patrick.gunning@utoronto.ca

Marco Herling − Department of Internal Medicine, Center for

Integrated Oncology Aachen-Bonn-Cologne-Duesseldorf (CIO

ABCD), University of Cologne (UoC), 50923 Cologne,

Germany; Excellence Cluster for Cellular Stress Response and

Aging-Associated Diseases (CECAD) and Center for

Molecular Medicine Cologne (CMMC), UoC, 50923

Cologne, Germany; Email: marco.herling@uk-koeln.de

Satu Mustjoki − Hematology Research Unit Helsinki, Helsinki

University Hospital Comprehensive Cancer Center, Helsinki

00029, HUS, Finland; Translational Immunology Research

Program and Department of Clinical Chemistry and

Hematology, University of Helsinki, Helsinki 00014, Finland;

iCAN Digital Precision Cancer Medicine Flagship, 00014

Helsinki, Finland; Email: satu.mustjoki@helsinki.ﬁ

Richard Moriggl − Institute of Animal Breeding and Genetics,

University of Veterinary Medicine Vienna, A-1210 Vienna,

Austria; Email: richard.moriggl@vetmeduni.ac.at

Authors

Krimo Toutah − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada

Statistical Testing. Statistical analysis was performed using two-

way ANOVA with Tukey’s multiple comparisons test in GraphPad

Prism 6.0 (GraphPad Software Inc.). To determine diﬀerences

between drug treatments in T-PLL primary samples, Wilcoxon test

was performed in R with the function wilcox.test. A p-value of <0.05

was considered signiﬁcant. HDAC gene expression data and

associated statistical analyses were extracted from the Oncomine

Research Premium Edition database (Thermo Fisher, Ann Arbor, MI)

from the Durig Leukemia data set.^46,59

Nabanita Nawar − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada

Sanna Timonen − Hematology Research Unit Helsinki,

Helsinki University Hospital Comprehensive Cancer Center,

Helsinki 00029, HUS, Finland; Translational Immunology

Research Program and Department of Clinical Chemistry and

8504

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Hematology, University of Helsinki, Helsinki 00014, Finland;

Institute for Molecular Medicine Finland (FIMM), HiLIFE,

University of Helsinki, Helsinki 00014, Finland

Helena Sorger − Institute of Animal Breeding and Genetics,

University of Veterinary Medicine Vienna, A-1210 Vienna,

Austria

Yasir S. Raouf − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada

Shazreh Bukhari − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada

Jana von Jan − Department of Internal Medicine, Center for

Integrated Oncology Aachen-Bonn-Cologne-Duesseldorf (CIO

ABCD), University of Cologne (UoC), 50923 Cologne,

Germany; Excellence Cluster for Cellular Stress Response and

Aging-Associated Diseases (CECAD) and Center for

Molecular Medicine Cologne (CMMC), UoC, 50923

Cologne, Germany

Aleksandr Ianevski − Institute for Molecular Medicine

Finland (FIMM), HiLIFE, University of Helsinki, Helsinki

00014, Finland

Justyna M. Gawel − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada

Olasunkanmi O. Olaoye − Department of Chemical and

Physical Sciences, University of Toronto Mississauga,

Mississauga, Ontario L5L 1C6, Canada; Department of

Chemistry, University of Toronto, Toronto, Ontario M5S

3H6, Canada

Mulu Geletu − Department of Chemical and Physical Sciences,

University of Toronto Mississauga, Mississauga, Ontario L5L

1C6, Canada

Chemistry, University of Toronto, Toronto, Ontario M5S

3H6, Canada

Andrew E. Shouksmith − Department of Chemical and

Physical Sciences, University of Toronto Mississauga,

Mississauga, Ontario L5L 1C6, Canada

Heidi A. Neubauer − Institute of Animal Breeding and

Genetics, University of Veterinary Medicine Vienna, A-1210

Vienna, Austria

Elvin D. de Araujo − Centre for Medicinal Chemistry,

University of Toronto Mississauga, Mississauga, Ontario L5L

1C6, Canada

Tero Aittokallio − Institute for Molecular Medicine Finland

(FIMM), HiLIFE, University of Helsinki, Helsinki 00014,

Finland; Department of Cancer Genetics, Institute for Cancer

Research, Oslo University Hospital, 0424 Oslo, Norway; Oslo

Centre for Biostatistics and Epidemiology, University of Oslo,

0316 Oslo, Norway; orcid.org/0000-0002-0886-9769

Oliver H. Krämer − Department of Toxicology, University

Medical Center, 55131 Mainz, Germany

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00420

Author Contributions

^▲K.T. and N.N. contributed equally. K.T., N.N., R.M., M.H.,

and P.T.G. conceptualized the study and designed experi-

ments. K.T., N.N., Y.S.R., M.N.B., A.A., O.O.O., and A.E.S.

synthesized/characterized the compounds. N.N., H.S., S.B.,

J.M.G., M.G., H.A.N., and R.M. carried out in cellulo biological

evaluation and analysis. J.I., P.M., O.O.O., T.B.R., A.S.,

M.M.H., and E.D.A. performed biophysical evaluation and

analysis. Y.S.R. performed in silico studies and analysis. H.A.N.

performed Oncomine analysis. J.V.J., A.I., S.T., T.A., S.M., and

M.H. performed synergy studies and analysis. N.N., E.D.A.,

M.H., O.H.K., H.A.N., R.M., and P.T.G. wrote the manuscript

with contributions from all authors.

Ayah Abdeldayem − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada

Johan Israelian − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada

Tudor B. Radu − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; Department of Chemistry,

University of Toronto, Toronto, Ontario M5S 3H6, Canada

Abootaleb Sedighi − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada; orcid.org/0000-0001-5974-

1792

Muzaﬀar N. Bhatti − Department of Chemical and Physical

Sciences, University of Toronto Mississauga, Mississauga,

Ontario L5L 1C6, Canada

Muhammad Murtaza Hassan − Department of Chemical and

Physical Sciences, University of Toronto Mississauga,

Mississauga, Ontario L5L 1C6, Canada; Department of

Chemistry, University of Toronto, Toronto, Ontario M5S

3H6, Canada

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors would like to thank Ruth Villalonga for her

contributions with cytotoxicity assays using NHF, pPF and

HUVEC. They would also like to thank Andrew Sedmihradsky

and family, who have raised funds, awareness for DMD and

generated support for Max’s Big Fellowship. Funding: This

work was supported by the EU consortia ERAPerMed

“JAKSTAT-TARGET” (S.M., H.A.N., M.H., P.T.G.) and the

“ERANETPLL” (M.H., R.M.). S.M. was funded by European

Research Council (M-IMM project), Academy of Finland,

Sigrid Juselius Foundation and the Cancer Foundation

Finland. T.A. was funded by the Academy of Finland (grants

310507, 313267, 326238), Cancer Society of Finland, Sigrid

Jusélius Foundation and Helse Sør-Øst. R.M., H.A.N., and H.S.

were supported by the Austrian Science Fund (FWF) (SFB-

F04707, SFB-F06105). R.M. and H.A.N. were also generously

supported by a private cancer metabolism grant donation from

Liechtenstein. P.T.G is supported by research grants from

NSERC (RGPIN-2014-05767), CIHR (MOP-130424, MOP-

137036), Canada Research Chair (950-232042), Canadian

Cancer Society (703963), Canadian Breast Cancer Foundation

(705456), Leukemia and Lymphoma Society of Canada and

infrastructure grants from CFI (33536) and the Ontario

Research Fund (34876). O.O.O. is supported by an OGS

Pimyupa Manaswiyoungkul − Department of Chemical and

Physical Sciences, University of Toronto Mississauga,

Mississauga, Ontario L5L 1C6, Canada; Department of

8505

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(4) Pandolfi, P. P. Histone Deacetylases and Transcriptional

Therapy with Their Inhibitors. Cancer Chemother. Pharmacol. 2001,

48, S17.

Fellowship. Y.S.R and N.N are supported by grants from

Jesse’s Journey.

(5) Thiagalingam, S.; Cheng, K. H.; Lee, H. J.; Mineva, N.;

Thiagalingam, A.; Ponte, J. F. Histone Deacetylases: Unique Players

in Shaping the Epigenetic Histone Code. Ann. N. Y. Acad. Sci. 2003,

983, 84−100.

(6) Haberland, M.; Montgomery, R. L.; Olson, E. N. The Many

Roles of Histone Deacetylases in Development and Physiology:

Implications for Disease and Therapy. Nat. Rev. Genet. 2009, 10, 32−

42.

(7) Allfrey, V. G.; Faulkner, R.; Mirsky, A. E. Acetylation and

Methylation of Histones and Their Possible Role in the Regulation of

RNA. Proc. Natl. Acad. Sci. U. S. A. 1964, 51, 786−794.

(8) Yan, W.; Herman, J. G.; Guo, M. Epigenome-Based Personalized

Medicine in Human Cancer. Epigenomics 2016, 8, 119−133.

(9) Lombardi, P. M.; Cole, K. E.; Dowling, D. P.; Christianson, D.

W. Structure, Mechanism, and Inhibition of Histone Deacetylases and

Related Metalloenzymes. Curr. Opin. Struct. Biol. 2011, 21, 735−743.

(10) Feinberg, A. P.; Tycko, B. The History of Cancer Epigenetics.

Nat. Rev. Cancer 2004, 4, 143−153.

(11) Tran, A. D. A.; Marmo, T. P.; Salam, A. A.; Che, S.; Finkelstein,

E.; Kabarriti, R.; Xenias, H. S.; Mazitschek, R.; Hubbert, C.;

Kawaguchi, Y.; Sheetz, M. P.; Yao, T.; Bulinski, J. C. HDAC6

Deacetylation of Tubulin Modulates Dynamics of Cellular Adhesions.

J. Cell Sci. 2007, 120, 1469−1479.

(12) Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.;

Nixon, A.; Yoshida, M.; Wang, X. F.; Yao, T. P. HDAC6 Is a

Microtubule-Associated Deacetylase. Nature 2002, 417, 455−458.

(13) Krämer, O. H.; Mahboobi, S.; Sellmer, A. Drugging the

HDAC6-HSP90 Interplay in Malignant Cells. Trends Pharmacol. Sci.

2014, 35, 501−509.

(14) Zhang, Y.; Li, N.; Caron, C.; Matthias, G.; Hess, D.; Khochbin,

S.; Matthias, P. HDAC-6 Interacts with and Deacetylates Tubulin and

Microtubules in Vivo. EMBO J. 2003, 22, 1168−1179.

(15) Cosenza, M.; Pozzi, S. The Therapeutic Strategy of HDAC6

Inhibitors in Lymphoproliferative Disease. Int. J. Mol. Sci. 2018, 19,

2337.

(16) Ellis, L.; Pan, Y.; Smyth, G. K.; George, D. J.; McCormack, C.;

Williams-Truax, R.; Mita, M.; Beck, J.; Burris, H.; Ryan, G.; Atadja, P.;

Butterfoss, D.; Dugan, M.; Culver, K.; Johnstone, R. W.; Prince, H. M.

Histone Deacetylase Inhibitor Panobinostat Induces Clinical

Responses with Associated Alterations in Gene Expression Profiles

in Cutaneous T-Cell Lymphoma. Clin. Cancer Res. 2008, 14, 4500−

4510.

(17) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.;

Schreiber, S. L. Domain-Selective Small-Molecule Inhibitor of

Histone Deacetylase 6 (HDAC6)-Mediated Tubulin Deacetylation.

Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 4389−4394.

(18) Huang, P.; Almeciga-Pinto, I.; Jarpe, M.; van Duzer, J. H.;

Mazitschek, R.; Yang, M.; Jones, S. S.; Quayle, S. N. Selective HDAC

Inhibition by ACY-241 Enhances the Activity of Paclitaxel in Solid

Tumor Models. Oncotarget. 2017, 8, 2694−2707.

(19) Santo, L.; Hideshima, T.; Kung, A. L.; Tseng, J. C.; Tamang,

D.; Yang, M.; Jarpe, M.; Van Duzer, J. H.; Mazitschek, R.; Ogier, W.

C.; Cirstea, D.; Rodig, S.; Eda, H.; Scullen, T.; Canavese, M.; Bradner,

J.; Anderson, K. C.; Jones, S. S.; Raje, N. Preclinical Activity,

Pharmacodynamic, and Pharmacokinetic Properties of a Selective

HDAC6 Inhibitor, ACY-1215, in Combination with Bortezomib in

Multiple Myeloma. Blood 2012, 119, 2579−2589.

ABBREVIATIONS USED

■

ABT-199, venetoclax; AcOH, acetic acid; MeCN, acetonitrile;

ADME, absorption, distribution, metabolism, and excretion;

AITL, angioimmunoblastic T-cell lymphoma; ALCL, Ana-

plastic large cell lymphoma; ALK, anaplastic lymphoma kinase;

AML, acute myeloid leukemia; ATM, ataxia telangiectasia

mutated; AUC, area under the curve; BCL, B-cell lymphoma;

Boc, tert-butoxycarbonyl; FAM, carboxyﬂuorescein; CCLE,

Cancer Cell Line Encyclopedia; CoA, coenzyme A; C_max

,

maximum concentration the drug achieves; Cs₂CO₃, cesium

carbonate; CTCL, cutaneous T-cell lymphomas; DCE,

dichloroethane; DCM and CH₂Cl₂, dichloromethane; DMF,

dimethylformamide; DMSO, dimethyl sulfoxide; DNA, deox-

yribonucleic acid; iPr₂NEt, diisopropylethylamine; DSRT, drug

sensitivity and resistance testing; DSS, drug sensitivity score,

EMSA, electrophoretic mobility shift assay; FACS, ﬂuores-

cence-activated cell sorting; FITC, ﬂuorescein isothiocyanate;

FDA, U.S. Food and Drug administration; GSH, reduced

glutathione; t_1/2, half-life; h, hour; HCl, hydrochloric acid;

HPLC, high-performance liquid chromatography; HRMS,

high-resolution mass spectrometry; HPOB, N-hydroxy-4-(2-

[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide;

HSP, heat shock protein; HUVEC, normal human umbilical

vein endothelial cells; HIF, hypoxia-inducible factors; IC₅₀, half

maximal inhibitory concentration; IS, internal standard; IP,

intraperitoneal; JAK, Janus-activated kinase; LC/MS, liquid

chromatography/mass spectrometry; LRMS, low-resolution

mass spectrometry; MaTCL, mature T-cell leukemias and

lymphomas; MeOH, methanol; min, minutes; NAD⁺,

nicotinamide adenine dinucleotide; NHF, normal human

ﬁbroblasts; NK, natural killer; NMR, nuclear magnetic

resonance; (COCl)₂, oxalyl chloride; p300/CBP, EA1 binding

protein 300/CREB-binding protein; Pd(OAc)₂, palladium

acetate; Pd/C, palladium on carbon; PAMPA, parallel artiﬁcial

membrane permeability assay; PBMCs, peripheral blood

mononuclear cells; P_e, eﬀective permeability; PFBSCl,

pentaﬂuorobenzyl sulfonyl chloride; PTCL, peripheral T-cell

lymphomas; PTCL-NOS, peripheral T-cell lymphoma, not

otherwise speciﬁed; PG, protecting group; PK, pharmacoki-

netic; P.O., oral administration; pPF, primary pooled

ﬁbroblasts; rpm, revolutions per minute; RT, room temper-

ature; Na(AcO)₃BH, SCT, stem cell transplantation; sDSS,

selective drug sensitivity score, sodium triacetoxyborohydride;

SI, Supporting Information; T-ALL, T-cell acute lymphocytic

leukemia; TC50, toxic concentration in 50% of PBMC

population; TCL1A, T-cell leukemia 1A; THF, tetrahydrofur-

an; Et₃N, triethylamine; SAR, structure−activity relationship;

SAHA, suberanilohydroxamic acid; Sir2, sirtuin; STAT, signal

transducer and activator of transcription factor; T-PLL, T-cell

prolymphocytic leukemia; ZBG, zinc-binding group

(20) Vogl, D. T.; Raje, N.; Jagannath, S.; Richardson, P.; Hari, P.;

Orlowski, R.; Supko, J. G.; Tamang, D.; Yang, M.; Jones, S. S.;

Wheeler, C.; Markelewicz, R. J.; Lonial, S. Ricolinostat, the First

Selective Histone Deacetylase 6 Inhibitor, in Combination with

Bortezomib and Dexamethasone for Relapsed or Refractory Multiple

Myeloma. Clin. Cancer Res. 2017, 23, 3307−3315.

(21) Leonhardt, M.; Sellmer, A.; Krämer, O. H.; Dove, S.; Elz, S.;

Kraus, B.; Beyer, M.; Mahboobi, S. Design and Biological Evaluation

of Tetrahydro-β -Carboline Derivatives as Highly Potent Histone

REFERENCES

■

(1) Lund, A. H.; Van Lohuizen, M. Epigenetics and Cancer. Genes

Dev. 2004, 18, 2315−2335.

(2) Eckschlager, T.; Plch, J.; Stiborova, M.; Hrabeta, J. Histone

Deacetylase Inhibitors as Anticancer Drugs. Int. J. Mol. Sci. 2017, 18,

1414.

(3) Tang, J.; Yan, H.; Zhuang, S. Histone Deacetylases as Targets for

Treatment of Multiple Diseases. Clin. Sci. 2013, 124, 651−662.

8506

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Deacetylase 6 (HDAC6) Inhibitors. Eur. J. Med. Chem. 2018, 152,

329−357.

R.; Gunning, P. T. Structural and Functional Consequences of the

STAT5BN642H Driver Mutation. Nat. Commun. 2019, 10, 2517.

(34) He, L.; Tang, J.; Andersson, E. I.; Timonen, S.; Koschmieder,

S.; Wennerberg, K.; Mustjoki, S.; Aittokallio, T. Patient-Customized

Drug Combination Prediction and Testing for t-Cell Prolymphocytic

Leukemia Patients. Cancer Res. 2018, 78, 2407−2418.

(22) Sellmer, A.; Stangl, H.; Beyer, M.; Grunstein, E.; Leonhardt,

̈

M.; Pongratz, H.; Eichhorn, E.; Elz, S.; Striegl, B.; Jenei-Lanzl, Z.;

Dove, S.; Straub, R. H.; Krämer, O. H.; Mahboobi, S. Marbostat-100

Defines a New Class of Potent and Selective Antiinflammatory and

Antirheumatic Histone Deacetylase 6 Inhibitors. J. Med. Chem. 2018,

61, 3454−3477.

(35) Dearden, C. How I Treat Prolymphocytic Leukemia. Blood

2012, 120, 538−551.

(23) Zhang, Y.; Kwon, S.; Yamaguchi, T.; Cubizolles, F.; Rousseaux,

S.; Kneissel, M.; Cao, C.; Li, N.; Cheng, H.-L.; Chua, K.; Lombard,

D.; Mizeracki, A.; Matthias, G.; Alt, F. W.; Khochbin, S.; Matthias, P.

Mice Lacking Histone Deacetylase 6 Have Hyperacetylated Tubulin

but Are Viable and Develop Normally. Mol. Cell. Biol. 2008, 28,

1688−1701.

(24) Hideshima, T.; Cottini, F.; Ohguchi, H.; Jakubikova, J.;

Gorgun, G.; Mimura, N.; Tai, Y. T.; Munshi, N. C.; Richardson, P. G.;

Anderson, K. C. Rational Combination Treatment with Histone

Deacetylase Inhibitors and Immunomodulatory Drugs in Multiple

Myeloma. Blood Cancer J. 2015, 5, e312.

(25) Heider, U.; Metzler, I.; Von Kaiser, M.; Rosche, M.; Sterz, J.;

Rötzer, S.; Rademacher, J.; Jakob, C.; Fleissner, C.; Kuckelkorn, U.;

Kloetzel, P.; Sezer, O. Synergistic Interaction of the Histone

Deacetylase Inhibitor SAHA with the Proteasome Inhibitor

Bortezomib in Mantle Cell Lymphoma. Eur. J. Haematol. 2008, 80,

133−142.

(26) Yee, A. J.; Bensinger, W. I.; Supko, J. G.; Voorhees, P. M.;

Berdeja, J. G.; Richardson, P. G.; Libby, E. N.; Wallace, E. E.; Birrer,

N. E.; Burke, J. N.; Tamang, D. L.; Yang, M.; Jones, S. S.; Wheeler, C.

A.; Markelewicz, R. J.; Raje, N. S. Ricolinostat plus Lenalidomide, and

Dexamethasone in Relapsed or Refractory Multiple Myeloma: A

Multicentre Phase 1b Trial. Lancet Oncol. 2016, 17, 1569−1578.

(27) Herling, M.; Khoury, J. D.; Washington, L. B. T.; Duvic, M.;

Keating, M. J.; Jones, D. A Systematic Approach to Diagnosis of

Mature T-Cell Leukemias Reveals Heterogeneity among WHO

Categories. Blood 2004, 104, 328−335.

(36) Hockly, E.; Richon, V. M.; Woodman, B.; Smith, D. L.; Zhou,

X.; Rosa, E.; Sathasivam, K.; Ghazi-Noori, S.; Mahal, A.; Lowden, P.

A. S.; Steffan, J. S.; Marsh, J. L.; Thompson, L. M.; Lewis, C. M.;

Marks, P. A.; Bates, G. P. Suberoylanilide Hydroxamic Acid, a Histone

Deacetylase Inhibitor, Ameliorates Motor Deficits in a Mouse Model

of Huntington ’s Disease. Proc. Natl. Acad. Sci. U. S. A. 2003, 100,

2041−2046.

(37) Kumagai, T.; Wakimoto, N.; Yin, D.; Gery, S.; Kawamata, N.;

Takai, N.; Komatsu, N.; Chumakov, A.; Imai, Y.; Koeffler, H. P.

Histone Deacetylase Inhibitor, Suberoylanilide Hydroxamic Acid

(Vorinostat, SAHA) Profoundly Inhibits the Growth of Human

Pancreatic Cancer Cells. Int. J. Cancer 2007, 121, 656−665.

(38) Ruefli, A. A.; Ausserlechner, M. J.; Bernhard, D.; Sutton, V. R.;

Tainton, K. M.; Kofler, R.; Smyth, M. J.; Johnstone, R. W. The

Histone Deacetylase Inhibitor and Chemotherapeutic Agent Sub-

eroylanilide Hydroxamic Acid (SAHA) Induces a Cell-Death Pathway

Characterized by Cleavage of Bid and Production of Reactive Oxygen

Species. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 10833−10838.

(39) Lee, H. Z.; Kwitkowski, V. E.; Del Valle, P. L.; Ricci, M. S.;

Saber, H.; Habtemariam, B. A.; Bullock, J.; Bloomquist, E.; Shen, Y.

L.; Chen, X. H.; Brown, J.; Mehrotra, N.; Dorff, S.; Charlab, R.; Kane,

R. C.; Kaminskas, E.; Justice, R.; Farrell, A. T.; Pazdur, R. FDA

Approval: Belinostat for the Treatment of Patients with Relapsed or

Refractory Peripheral T-Cell Lymphoma. Clin. Cancer Res. 2015, 21,

2666−2670.

(40) Putzer, S.; Varghese, L.; von Jan, J.; Braun, T.; Giri, A. K.;

̈

Mayer, P.; Riet, N.; Timonen, S.; Oberbeck, S.; Kuusanmäki, H.;

Mustjoki, S.; Stern, M.; Aittokallio, T.; Newrzela, S.; Schrader, A.;

Herling, M. Reinstated P53 Response and High Anti-T-Cell Leukemia

Activity by the Novel Alkylating Deacetylase Inhibitor Tinostamus-

tine. Leukemia 2020, 34, 2513−2518.

(28) Stengel, A.; Kern, W.; Zenger, M.; Perglerová, K.; Schnittger,

S.; Haferlach, T.; Haferlach, C. Genetic Characterization of T-PLL

Reveals Two Major Biologic Subgroups and JAK3 Mutations as

Prognostic Marker. Genes, Chromosomes Cancer 2016, 55, 82−94.

(29) Schrader, A.; Crispatzu, G.; Oberbeck, S.; Mayer, P.; Putzer, S.;

̈

(41) Andersson, E. I.; Putzer, S.; Yadav, B.; Dufva, O.; Khan, S.; He,

̈

Von Jan, J.; Vasyutina, E.; Warner, K.; Weit, N.; Pflug, N.; Braun, T.;

Andersson, E. I.; Yadav, B.; Riabinska, A.; Ferreira, M. S. V.; Beier, F.;

Altmuller, J.; Lanasa, M.; Herling, C. D.; Haferlach, T.; Stilgenbauer,

S.; Hopfinger, G.; Peifer, M.; Brummendorf, T. H.; Nurnberg, P.;

Elenitoba-Johnson, K. S.; Zha, S.; Hallek, M.; Moriggl, R.; Reinhardt,

H. C.; Stern, m. H.; Mustjoki, S.; Newrzela, S.; Frommolt, P.; Herling,

M. Actionable Perturbations of Damage Responses by TCL1/ATM

and Epigenetic Lesions Form the Basis of T-PLL. Nat. Commun.

2018, 9, 1−22.

́

L.; Sellner, L.; Schrader, A.; Crispatzu, G.; Oles, M.; Zhang, H.;

Adnan-Awad, S.; Lagstrom, S.; Bellanger, D.; Mpindi, J. P.; Eldfors, S.;

Pemovska, T.; Pietarinen, P.; Lauhio, A.; Tomska, K.; Cuesta-Mateos,

C.; Faber, E.; Koschmieder, S.; Brummendorf, T. H.; Kytola, S.;

Savolainen, E.; Siitonen, T.; Ellonen, P.; Kallioneimi, O.; Wennerberg,

K.; Ding, W.; Stern, M_H.; Huber, W.; Anders, S.; Tang, J.;

Aittokallio, T.; Zenz, T.; Herling, M.; Mustjoki, S. Discovery of Novel

Drug Sensitivities in T-PLL by High-Throughput Ex Vivo Drug

Testing and Mutation Profiling. Leukemia 2018, 32, 774−787.

(42) Mrakovcic, M.; Kleinheinz, J.; Fröhlich, L. F. P53 at the

Crossroads between Different Types of Hdac Inhibitor-Mediated

Cancer Cell Death. Int. J. Mol. Sci. 2019, 20, 2415.

(30) Herling, M.; Patel, K. A.; Teitell, M. A.; Konopleva, M.;

Ravandi, F.; Kobayashi, R.; Jones, D. High TCL1 Expression and

Intact T-Cell Receptor Signaling Define a Hyperproliferative Subset

of T-Cell Prolymphocytic Leukemia. Blood 2008, 111, 328−337.

(31) Kiel, M. J.; Velusamy, T.; Rolland, D.; Sahasrabuddhe, A. A.;

Chung, F.; Bailey, N. G.; Schrader, A.; Li, B.; Li, J. Z.; Ozel, A. B.;

Betz, B. L.; Miranda, R. N.; Medeiros, L. J.; Zhao, L.; Herling, M.;

Lim, M. S.; Elenitoba-Johnson, K. S. J. Integrated Genomic

Sequencing Reveals Mutational Landscape of T-Cell Prolymphocytic

Leukemia. Blood 2014, 124, 1460−1472.

(32) Wahnschaffe, L.; Braun, T.; Timonen, S.; Giri, A. K.; Schrader,

A.; Wagle, P.; Almusa, H.; Johansson, P.; Bellanger, D.; López, C.;

Haferlach, C.; Stern, M.; Durig, J.; Siebrt, R.; Mustjoki, S.; Aittokallio,

T.; Herling, M. Jak/Stat-Activating Genomic Alterations Are a

Hallmark of T-PLL. Cancers 2019, 11, 1833.

(43) Boidol, B.; Kornauth, C.; Van Der Kouwe, E.; Prutsch, N.;

̈

Kazianka, L.; Gultekin, S.; Hoermann, G.; Mayerhoefer, M. E.;

Hopfinger, G.; Hauswirth, A.; Panny, M.; Aretin, M.-B.; Hilgarth, B.;

Sperr, W. R.; Valent, P.; Simonitsch-Klupp, I.; Moriggl, R.; Merkel, P.;

Kenner, L.; Jager, U.; Kubicek, S.; Staber, P. B. First-in-Human

Response of BCL-2 Inhibitor Venetoclax in T-Cell Prolymphocytic

Leukemia. Blood 2017, 130, 2499−2503.

(44) Souers, A. J; Leverson, J. D; Boghaert, E. R; Ackler, S. L;

Catron, N. D; Chen, J.; Dayton, B. D; Ding, H.; Enschede, S. H;

Fairbrother, W. J; Huang, D. C S; Hymowitz, S. G; Jin, S.; Khaw, S.

L.; Kovar, P. J; Lam, L. T; Lee, J.; Maecker, H. L; Marsh, K. C;

Mason, K. D; Mitten, M. J; Nimmer, P. M; Oleksijew, A.; Park, C. H;

Park, C.-M.; Phillips, D. C; Roberts, A. W; Sampath, D.; Seymour, J.

F; Smith, M. L; Sullivan, G. M; Tahir, S. K; Tse, C.; Wendt, M. D;

Xiao, Y.; Xue, J. C; Zhang, H.; Humerickhouse, R. A; Rosenberg, S.

H; Elmore, S. W ABT-199, a Potent and Selective BCL-2 Inhibitor,

(33) de Araujo, E. D.; Erdogan, F.; Neubauer, H. A.; Meneksedag-

Erol, D.; Manaswiyoungkul, P.; Eram, M. S.; Seo, H.-S.; Qadree, A.

K.; Israelian, J.; Orlova, A.; Suske, T.; Pham, H. T. T.; Boersma, A.;

Tangermann, S.; Kenner, L.; Rulicke, T.; Dong, A.; Ravichandran, M.;

Brown, P. J.; Audette, G. F.; Rauscher, S.; Dhe-Paganon, S.; Moriggl,

8507

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Achieves Antitumor Activity While Sparing Platelets. Nat. Med. 2013,

19, 202−208.

(45) Roberts, A. W.; Davids, M. S.; Pagel, J. M.; Kahl, B. S.; Puvvada,

S. D.; Gerecitano, J. F.; Kipps, T. J.; Anderson, M. A.; Brown, J. R.;

Gressick, L.; et al. Targeting BCL2 with Venetoclax in Relapsed

Chronic Lymphocytic Leukemia. N. Engl. J. Med. 2016, 374, 311−

322.

(46) Rhodes, D. R.; Yu, J.; Shanker, K.; Deshpande, N.; Varambally,

R.; Ghosh, D.; Barrette, T.; Pander, A.; Chinnaiyan, A. M.

ONCOMINE: A Cancer Microarray Database and Integrated Data-

Mining Platform. Neoplasia. 2004, 6, 1−6.

(47) Gawel, J. M.; Shouksmith, A. E.; Raouf, Y. S.; Nawar, N.;

Toutah, K.; Bukhari, S.; Manaswiyoungkul, P.; Olaoye, O. O.;

Israelian, J.; Radu, T. B.; Cabral, A. D.; Sina, D.; Sedighi, A.; de

Araujo, E. D.; Gunning, P. T. PTG-0861: A Novel HDAC6-Selective

Inhibitor as a Therapeutic Strategy in Acute Myeloid Leukaemia. Eur.

J. Med. Chem. 2020, 201, 112411.

Isoform-Selective Inhibitors. J. Biomol. Screening 2015, 20, 1277−

1285.

(57) Warner, K.; Crispatzu, G.; Al-Ghaili, N.; Weit, N.; Florou, V.;

You, M. J.; Newrzela, S.; Herling, M. Models for Mature T-Cell

Lymphomas-A Critical Appraisal of Experimental Systems and Their

Contribution to Current T-Cell Tumorigenic Concepts. Critical

Reviews in Oncology/Hematology. 2013, 88, 680−695.

(58) Orlova, A.; Wingelhofer, B.; Neubauer, H. A.; Maurer, B.;

̋

Berger-Becvar, A.; Miklós Keseru, G.; Gunning, P. T.; Valent, P.;

Moriggl, R. Emerging Therapeutic Targets in Myeloproliferative

Neoplasms and Peripheral T-Cell Leukemia and Lymphomas. Expert

Opin. Ther. Targets 2018, 22, 45−57.

(59) Du

̈

rig, J.; Bug, S.; Klein-Hitpass, L.; Boes, T.; Jöns, T.; Martin-

hrsen, U.; Siebert, R.

Subero, J. I.; Harder, L.; Baudis, M.; Du

̈

Combined Single Nucleotide Polymorphism-Based Genomic Map-

ping and Global Gene Expression Profiling Identifies Novel

Chromosomal Imbalances, Mechanisms and Candidate Genes

Important in the Pathogenesis of T-Cell Prolymphocytic Leukemia

with Inv(14)(Q11q32). Leukemia 2007, 21, 2153−2163.

(48) Shouksmith, A. E.; Gawel, J. M.; Nawar, N.; Sina, D.; Raouf, Y.

S.; Bukhari, S.; He, L.; Johns, A. E.; Manaswiyoungkul, P.; Olaoye, O.

O.; Cabral, A. D.; Sedighi, A.; de Araujo, E. D.; Gunning, P. T. Class

I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and

Therapeutic Efficacy in Acute Myeloid Leukemia. ACS Med. Chem.

Lett. 2020, 11, 56−64.

(60) Patil, P.; Cieslak, A.; Bernhart, S. H.; Toprak, U. H.; Wagener,

̈

R.; López, C.; Wiehle, L.; Bens, S.; Altmuller, J.; Franitza, M.; Scholz,

I.; Jayne, S.; Ahearne, M. J.; Scheffold, A.; Jebaraj, B. M. C.;

Schneider, C.; Costa, D.; Braun, T.; Schrader, A.; Campo, E.; Dyer,

M. J. S.; Nurnberg, P.; Durig, J.; Bottcher, S.; Schesner, M.; Herling,

M.; Stilgenbauer, S.; Macintyre, E.; Siebert, R. Reconstruction of

Rearranged T-Cell Receptor Loci by Whole Genome and Tran-

scriptome Sequencing Gives Insights into the Initial Steps of T-Cell

Prolymphocytic Leukemia. Genes, Chromosomes Cancer 2020, 59,

261−267.

(49) Olaoye, O. O.; Watson, P. R.; Nawar, N.; Geletu, M.; Sedighi,

A.; Bukhari, S.; Raouf, Y. S.; Manaswiyoungkul, P.; Erdogan, F.;

Abdeldayem, A.; Cabral, A. D.; Hassan, M. M.; Toutah, K.;

Shouksmith, A. E.; Gawel, J. M.; Israelian, J.; Radu, T. B.;

Kachhiyapatel, N.; de Araujo, E. D.; Christianson, D. W.; Gunning,

P. T. Unique Molecular Interaction with the Histone Deacetylase 6

Catalytic Tunnel: Crystallographic and Biological Characterization of

a Model Chemotype. J. Med. Chem. 2021, 64, 2691−2704.

(50) Shouksmith, A. E.; Shah, F.; Grimard, M. L.; Gawel, J. M.;

Raouf, Y. S.; Geletu, M.; Berger-Becvar, A.; De Araujo, E. D.;

Luchman, H. A.; Heaton, W. L.; Bakhshinyan, D.; Adile, A. A.;

Venugopal, C.; O’Hare, T.; Deininger, M. W.; Singh, S. K.;

Konieczny, S. F.; Weiss, S.; Fishel, M. L.; Gunning, P. T.

Identification and Characterization of AES-135, a Hydroxamic Acid-

Based HDAC Inhibitor That Prolongs Survival in an Orthotopic

Mouse Model of Pancreatic Cancer. J. Med. Chem. 2019, 62, 2651−

2665.

(51) Bergman, J. A.; Woan, K.; Perez-Villarroel, P.; Villagra, A.;

Sotomayor, E. M.; Kozikowski, A. P. Selective Histone Deacetylase 6

Inhibitors Bearing Substituted Urea Linkers Inhibit Melanoma Cell

Growth. J. Med. Chem. 2012, 55, 9891−9899.

(52) Woan, K. V.; Lienlaf, M.; Perez-Villaroel, P.; Lee, C.; Cheng, F.;

Knox, T.; Woods, D. M.; Barrios, K.; Powers, J.; Sahakian, E.; Wang,

H. W.; Canales, J.; Marante, D.; Malley, K. S. M.; Bergman, J.; Seto,

E.; Kozikowski, A.; Pinilla-Ibarz, J.; Sarnaik, A.; Celis, E.; Weber, J.;

Sotomayor, E. M.; Villagra, A. Targeting Histone Deacetylase 6

Mediates a Dual Anti-Melanoma Effect: Enhanced Antitumor

Immunity and Impaired Cell Proliferation. Mol. Oncol. 2015, 9,

1447−1457.

(61) Andersson, E. I.; Putzer, S.; Yadav, B.; Dufva, O.; Khan, S.; He,

̈

L.; Sellner, L.; Schrader, A.; Crispatzu, G.; Oles, M.; Zhang, H.;

Adnan-Awad, S.; Lagstrom, S.; Bellanger, D.; Mpindi, J. P.; Eldfors, S.;

Pemovska, T.; Pietarinen, S.; Lauhio, A.; Tomska, K.; Cuesta-Matoes,

C.; Faber, E.; Koschmieder, S.; Brummendorf, T. H.; Kytola, S.;

Savolainen, E.-R.; Siitonen, T.; Ellonen, P.; Kallioniemi, O.;

Wennerberg, K.; Ding, W.; Stern, M.; Huber, W.; Anders, S.; Tang,

J.; Aittokallio, T.; Zenz, T.; Herling, M.; Mustjoki, S. Discovery of

Novel Drug Sensitivities in T-PLL by High-Throughput Ex Vivo Drug

Testing and Mutation Profiling. Leukemia 2018, 32, 774−787.

(62) Yadav, B.; Pemovska, T.; Szwajda, A.; Kulesskiy, E.; Kontro,

M.; Karjalainen, R.; Majumder, M.; Malani, D.; Murumägi, A.;

Knowles, J.; Porkka, K.; Heckman, C.; Kallioniemi, O.; Wennerberg,

K.; Aittokallio, T. Quantitative Scoring of Differential Drug Sensitivity

for Individually Optimized Anticancer Therapies. Sci. Rep. 2015, 4,

5193.

(63) Cosenza, M.; Civallero, M.; Marcheselli, L.; Sacchi, S.; Pozzi, S.

Ricolinostat, a Selective HDAC6 Inhibitor, Shows Anti-Lymphoma

Cell Activity Alone and in Combination with Bendamustine. Apoptosis

2017, 22, 827−840.

(64) North, B. J.; Almeciga-Pinto, I.; Tamang, D.; Yang, M.; Jones,

S. S.; Quayle, S. N. Enhancement of Pomalidomide Anti-Tumor

Response with ACY-241, a Selective HDAC6 Inhibitor. PLoS One

2017, 12, No. e0173507.

(65) Zhang, G.; Gan, Y. H. Synergistic Antitumor Effects of the

Combined Treatment with an HDAC6 Inhibitor and a COX-2

Inhibitor through Activation of PTEN. Oncol. Rep. 2017, 38, 2657−

2666.

(53) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;

Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T.

Extra Precision Glide: Docking and Scoring Incorporating a Model of

Hydrophobic Enclosure for Protein-Ligand Complexes. J. Med. Chem.

2006, 49, 6177−6196.

(54) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.;

Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: A New Approach for

Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in

Database Screening. J. Med. Chem. 2004, 47, 1750−1759.

(55) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;

Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.;

Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: A New

Approach for Rapid, Accurate Docking and Scoring. 1. Method and

Assessment of Docking Accuracy. J. Med. Chem. 2004, 47, 1739−

1749.

(66) Hideshima, T.; Qi, J.; Paranal, R. M.; Tang, W.; Greenberg, E.;

West, N.; Colling, M. E.; Estiu, G.; Mazitschek, R.; Perry, J. A.;

Ohguchi, H.; Cottini, F.; Mimura, N.; Gorgun, G.; Tai, Y.-T.;

Richardson, P. G.; Carrasco, R. D.; Wiest, O.; Schreiber, S. L.;

Anderson, K. C.; Bradner, J. E. Discovery of Selective Small-Molecule

HDAC6 Inhibitor for Overcoming Proteasome Inhibitor Resistance in

Multiple Myeloma. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 13162−

13167.

(67) Grant, C.; Rahman, F.; Piekarz, R.; Peer, C.; Frye, R.; Robey, R.

W.; Gardner, E. R.; Figg, W. D.; Bates, S. E. Romidepsin: A New

Therapy for Cutaneous T-Cell Lymphoma and a Potential Therapy

for Solid Tumors. Expert Rev. Anticancer Ther. 2010, 10, 997−1008.

(56) Padige, G.; Negmeldin, A. T.; Pflum, M. K. H. Development of

an ELISA-Based HDAC Activity Assay for Characterization of

8508

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(68) Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner,

E.; Buggy, J. J. A Novel Histone Deacetylase 8 (HDAC8)-Specific

Inhibitor PCI-34051 Induces Apoptosis in T-Cell Lymphomas.

Leukemia 2008, 22, 1026−1034.

(69) Hassan, M. M.; Israelian, J.; Nawar, N.; Ganda, G.;

Manaswiyoungkul, P.; Raouf, Y. S.; Armstrong, D.; Sedighi, A.;

Olaoye, O. O.; Erdogan, F.; Cabral, A. D.; Angeles, F.; Altintas, R.; de

Araujo, E. D.; Gunning, P. T. Characterization of Conformationally

Constrained Benzanilide Scaffolds for Potent and Selective HDAC8

Targeting. J. Med. Chem. 2020, 63, 8634−8648.

(70) Shouksmith, A. E.; Gawel, J. M.; Nawar, N.; Sina, D.; Raouf, Y.

S.; Bukhari, S.; He, L.; Johns, A. E.; Manaswiyoungkul, P.; Olaoye, O.

O.; Cabral, A. D.; Sedighi, A.; de Araujo, E. D.; Gunning, P. T. Class

I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and

Therapeutic Efficacy in Acute Myeloid Leukemia. ACS Med. Chem.

Lett. 2020, 11, 56−64.

(71) Staber, P. B.; Herling, M.; Bellido, M.; Jacobsen, E. D.; Davids,

M. S.; Kadia, T. M.; Shustov, A.; Tournilhac, O.; Bachy, E.; Zaja, F.;

Porkka, K.; Hoermann, G.; Simonitsch-Klupp, I.; Haferlach, C.;

Kubicek, S.; Mayerhoefer, M. E.; Hopfinger, G.; Jaeger, U.; Dearden,

C. Consensus Criteria for Diagnosis, Staging, and Treatment

Response Assessment of T-Cell Prolymphocytic Leukemia. Blood

2019, 134, 1132−1143.

(72) Herling, M.; Khoury, J. D.; Washington, L. B. T.; Duvic, M.;

Keating, M. J.; Jones, D. A Systematic Approach to Diagnosis of

Mature T-Cell Leukemias Reveals Heterogeneity among WHO

Categories. Blood 2004, 104, 328−335.

(73) Ianevski, A.; Timonen, S.; Kononov, A.; Aittokallio, T.; Giri, A.

K. SynToxProfiler: An Interactive Analysis of Drug Combination

Synergy, Toxicity and Efficacy. PLoS Comput. Biol. 2020, 16,

No. e1007604.

8509

https://doi.org/10.1021/acs.jmedchem.1c00420

J. Med. Chem. 2021, 64, 8486−8509

Article Doi

Development of HDAC Inhibitors Exhibiting Therapeutic Potential in T-Cell Prolymphocytic Leukemia

DOI: 10.1021/acs.jmedchem.1c00420

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.1c00420

Full text of DOI:10.1021/acs.jmedchem.1c00420

Products guided by the article

R&D Labs maybe for 591249-21-7

Relevant to this article

Hot Product