Benzothiazolyl and Benzoxazolyl Hydrazones Function as Zinc Metallochaperones to Reactivate Mutant p53

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Benzothiazolyl and Benzoxazolyl Hydrazones Function as Zinc

Metallochaperones to Reactivate Mutant p53

¶

John A. Gilleran, Xin Yu, Alan J. Blayney, Anthony F. Bencivenga, Bing Na, David J. Augeri,

&

,&

Adam R. Blanden, S. David Kimball, Stewart N. Loh, Jacques Y. Roberge, and Darren R. Carpizo*

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ABSTRACT: We identiﬁed a set of thiosemicarbazone (TSC) metal ion chelators that reactivate speciﬁc zinc-deﬁcient p53 mutants

using a mechanism called zinc metallochaperones (ZMCs) that restore zinc binding by shuttling zinc into cells. We deﬁned

biophysical and cellular assays necessary for structure−activity relationship studies using this mechanism. We investigated an

alternative class of zinc scaﬀolds that diﬀer from TSCs by substitution of the thiocarbamoyl moiety with benzothiazolyl,

benzoxazolyl, and benzimidazolyl hydrazones. Members of this series bound zinc with similar aﬃnity and functioned to reactivate

mutant p53 comparable to the TSCs. Acute toxicity and eﬃcacy assays in rodents demonstrated C1 to be signiﬁcantly less toxic than

the TSCs while demonstrating equivalent growth inhibition. We identiﬁed C85 as a ZMC with diminished copper binding that

functions as a chemotherapy and radiation sensitizer. We conclude that the benzothiazolyl, benzoxazolyl, and benzimidazolyl

hydrazones can function as ZMCs to reactivate mutant p53 in vitro and in vivo.

INTRODUCTION

then allows the protein to adopt a wild-type conformation.

■

These zinc-deﬁcient mutant p53’s are best exempliﬁed by the

Mutant p53 remains one of the most signiﬁcant targets in the

ﬁeld of cancer drug development for several reasons: (1) p53 is

the most commonly mutated gene in human cancer; (2) the

majority of mutations (>70%) are missense, leading to a single

amino acid change that generates a defective protein found at

higher levels in cancer cells (therefore being druggable); and

R175H

most common p53 missense mutant, p53

. We initially

discovered a set of thiosemicarbazone (TSC) metal ion

chelators that could reactivate zinc-deﬁcient mutant p53’s

and elucidated their novel mechanism of action, which we

6

−8

termed “ZMCs”.

As ZMCs, these compounds bind

extracellular zinc and function as zinc ionophores to raise

intracellular zinc levels suﬃciently to overcome the zinc-

binding impairment induced by mutation in the p53 protein.

This allows zinc to bind in its native p53 ligation site and

induce a wild-type conformational change. We have validated

this mechanism in vivo using genetically engineered murine

models of cancer by showing that the lead TSC (ZMC1) has

antitumor activity in mice harboring zinc-deﬁcient p53

mutations while having no eﬃcacy in mice with nonzinc-

(

3) restoring wild-type p53 function in mouse cancer models is

highly therapeutic. Small-molecule compounds that can restore

the wild-type structure and function to these mutant p53s are

often called mutant p53 reactivators. One recent and successful

strategy to reactivate mutant p53 is using zinc metal-

lochaperones (ZMCs). The p53 protein requires the binding

of a single zinc ion in order to facilitate proper folding.

Experimentally, it has been shown that removing zinc will

cause it to misfold and that by adding zinc back it will refold

1

,2

8

−10

3

−5

deﬁcient p53 mutations.

We have also shown that we can

properly.

This inherent “malleability” of the protein is what

allows the ZMC mechanism to function. A large number of

mutations in p53 are located near the zinc coordination site

and cause the protein to misfold by impairing zinc binding

Received: September 8, 2020

Published: February 4, 2021

(

reducing its K for zinc). The strong dependence of p53

d

protein folding on zinc binding serves as the basis for ZMC

therapy. These compounds increase intracellular zinc, facilitat-

ing binding in the native zinc ligation site on the protein, which

©

2021 American Chemical Society

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Figure 1. Development of a panel of assays for SAR.

improve eﬃcacy in these models using a novel formulation of

zinc-loaded ZMC1 in which we synthesized a 2:1 complex of

ZMC1 and zinc.

chemotherapy and radiation sensitizers to cancer cells

harboring zinc-deﬁcient p53 missense mutations.

1

0

RESULTS AND DISCUSSION

Our research thus far has demonstrated that the ZMC

pathway represents a viable strategy for mutant p53-targeted

anticancer drug development. One of the main objectives to

move this ﬁeld forward is to develop molecules with properties

optimized for clinical development (solubility, potency,

eﬃcacy, and toxicity). The clinical utility of the class of

thiosemicarbazones (TSCs) as anticancer drugs has been

■

Unique Mechanism of Action Requires Development

of Assays to Conduct SAR. The delivery of a metal ion (like

zinc) to restore the normal structure of a mutant protein is an

entirely novel mechanism of action for a small-molecule drug.

We further elucidated this novel mechanism to show that not

only chaperoning of the metal ion through the plasma

membrane was important (ionophore activity) but also

11,12

limited by toxicity and poor solubility.

The toxicity is

6

,14

increasing ROS levels was integral to its function

(Figure

thought to be attributed mainly to the chelation of redox-active

13

1A). The ionophore activity (Ji drug/Ji DMSO) as −log(mol/

metals such as iron. Iron chelation has been shown to cause

alternate mechanisms of toxicity including inhibition of

ribonucleotide reductase (RR), the key iron-dependent

enzyme that mediates the synthesis of deoxyribonucleotides,

2

m s) of the compounds is presented in Table 1 where a larger

number indicates weaker ionophore activity. The major

mechanism of regulation of p53 function is through post-

translational modiﬁcations (PTMs) that not only guide which

p53 targets are transcribed but also enhance or inhibit the

transcription factor’s ability to recruit the transcriptional

12

and methemoglobinemia. Based on these limitations of

TSCs, we sought to investigate an alternative zinc scaﬀold as

potential clinical lead candidates that could perform this

unique mechanism. Here, we designed and evaluated a new

class of metal ion chelators (benzothiazolyl and benzoxazolyl

hydrazones) with zinc-binding properties similar to TSCs that

function as mutant p53 reactivators with better pharmaco-

logical properties. Given the novelty of the ZMC mechanism,

we deﬁned the assays required to conduct structure−activity

relationship (SAR) studies and identiﬁed several candidate

compounds, which were evaluated in vitro and in vivo

preclinical studies. Lastly, we identiﬁed a new class of ZMCs

with diminished copper-binding aﬃnity that can function as

15

machinery. We demonstrated that the ZMC-induced ROS

signal (likely through redox-active copper chelation) serves to

induce the PTMs on p53 that typically results in an apoptotic

program. In other words, without the ROS eﬀect, a ZMC

would induce a wild-type conformational change but would

not induce an apoptotic program. To conduct SAR around this

unique mechanism, we needed to ﬁrst deﬁne the assays that

would yield the necessary functional information to discrim-

inate structural chemotype changes and second deﬁne the

appropriate range in zinc binding for the small-molecule metal

chelators that would induce conformational changes in p53.

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,

a b

Table 1. A- and C-Series Structure−Activity Exploration

a

b

ND, not detected. NT, not tested.

These compounds are intermediate strength chelators such

serum albumin (K_Z_n≈ 100 nM), which is the major source of

that their aﬃnity for zinc is tight enough to steal zinc from

available zinc in the blood, but weak enough to donate it to

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1

6−19

mutant p53 (K_Z_n≈ 0.01−100 pM).

We previously

Scheme 1. (i) KOH, N H ·H O, i-PrOH, and H O; (ii) MeI;

2 4 2 2

(iii) EtOH; (iv) Azetidine or Dimethylamine and EtOH;

and (v) ZMC1, MeI, EtOH, and DCM

determined the ZMC1 K_Z_nto be 30 nM, but the range of zinc

aﬃnity suﬃcient for zinc metallochaperone function was

unclear. To investigate this, we performed a rigorous study

of a small set of ZMC1 derivatives, using the following assays

R175H

to guide synthesis: (1) K_Z_nin vitro, (2) EC₅₀in p53

expressing cancer cells, (3) cellular immunoﬂuorescence using

p53 conformation-speciﬁc antibodies to monitor conforma-

tional change (PAB1620 for wild type and PAB240 for mutant

conformation) (Figure 1B), (4) ROS change by ﬂow

cytometry analysis using a ﬂuorescent probe, and (5)

membrane permeability using Caco-2 assays. We ﬁrst validated

the SAR assays of ZMC1 and its analogues (A1 and A6, Table

1

4

1

)

and found that for SAR, the most useful assays were (1)

R175H

zinc aﬃnity (K ), (2) EC for a p53

cell line, and (3)

Zn

50

with N,N-diimidazothiocarbonyl and hydrazine followed by

the displacement of the remaining imidazole with the selected

amine (Scheme 2). The resulting hydrazinocarbothioamides

are coupled with ketones to give the thiosemicarbazones.

The carbonyl analogue of A6 was prepared via addition of

azetidine to the nitrophenoxyacylated 2-acetylpyridine hydra-

zone (Scheme 3).

evidence of conformational change (refolding). We reasoned

^t^h^a^t^t^h^e^E^C⁵0 was a reasonable indicator of the ionophore

activity as any change that impaired the compound’s ability to

traverse the plasma membrane would negatively aﬀect its EC ;

5

0

moreover, any change that limited the ROS level would also

limit its transcriptional activity and hence its eﬃcacy in

inducing an apoptotic program. We next evaluated a number of

structural modiﬁcations that we thought would signiﬁcantly

impact zinc binding in a focused SAR study (Table 1;

molecular changes are highlighted). All the compounds in

Benzothiazolyl and Benzoxazolyl Hydrazones: An

Alternative ZMC. To identify a nonthiosemicarbazone ZMC,

we turned to another class of zinc chelators (benzothiazolyl C1

and benzoxazolyl hydrazones C2 (Figure 2B)), which were

designed with the goals of decreasing oﬀ-target toxicity and

6

14

Table 1 are new except for ZMC1, Zn-1, and A6. We found,

22

^f^o^r^t^h^e^m^o^s^t^p^a^r^t^,^t^h^a^t^a^l^l^s^t^r^u^c^t^u^r^e^s^t^h^a^t^b^o^uⁿ^d^zⁱⁿ^c^wⁱ^t^h^KZn

2

<

increasing potency over the TSCs. α-(N)-Heteroaromatic

TSCs (i.e., ZMC1) have a characteristic N−NH−C−S

backbone, while the benzothiazolyl, benzoxazolyl, and

benzimidazolyl (C9) hydrazones would be expected to have

a N−NH−C−N backbone with the last nitrogen mimicking

the thiocarbonyl. This is demonstrated through the crystal

00 nM (A1, ZMC1, A10, and A11) induced a wild-type

conformational change and prompted cell killing at a level

similar to or greater than ZMC1. The exception was A14,

which did not induce a conformational change yet maintained

signiﬁcant cytotoxity against the mutant p53 cell line. Later

results suggested that A14 induced cell death through an

alternate mechanism likely through the chelation of redox-

23

structure determination shown in Figure 2. pK calculations

a

of the NH for TSCs (∼11.8) showed that they are in a range

similar to the benzimidazole (∼13.7) but diﬀerent from the

more acidic benzothiazoles (7−9) and benzoxazoles (∼7).

Aside from toxicity due to copper or iron chelation, it was

20

active metals such as copper. Likewise, compounds with a

K_Z_n> 200 nM (A4, 5, 6, 9, 12, and 13) could no longer induce

a conformational change and were consequently not cytotoxic

with the exception of A12. The eﬀective upper limit of K_Z_nis

thought that part of the toxicity of the TSCs is related to the

2

+

24

set by the dissociation constant of serum albumin for Zn

release of H S during metabolism. In fact, structure−activity

2

(

∼100 nM) and is thus likely to be similar for all zinc

relationship (SAR) studies in which the thiocarbamoyl

structure was replaced by a 2-pyridinyl moiety showed a

drastic reduction in toxicity and retention of biological

metallochaperones. The observed cutoﬀ of K ≈ 200 nM is

Zn

expected as compounds with larger K_Z_nvalues bind zinc too

weakly to compete with BSA in our assays. These results,

illustrated in Figure 1C, indicate that we have deﬁned an upper

limit for the optimal K_Z_nrange for a zinc metallochaperone at

approximately 200 nM. Although there were exceptions, we

could use this number as a cutoﬀ point to save resources. In

terms of SAR, we found that replacing the thiourea moiety

with a urea (A6), blocking the thiol via alkylation with a

methyl (A5), or replacing the pyridine with a phenyl (A9)

increased the K_Z_nto >400 nM. This resulted in a subsequent

loss of its ability to induce a conformational change in p53 and

hence its activity as a cytotoxic agent (Table 1 and Figure 1C).

Replacement of the pyridine with the much less basic

25

activity. The general design principle behind this class of

chelators is similar in that the thiocarbamoyl moiety is replaced

2

by a benzothiazole or benzoxazolyl. The two scaﬀolds have

very similar interatomic spacing and binding elements that

allow for nearly equivalent zinc-binding aﬃnities. Studies on

these compounds in cancer cells revealed increased potency

over TSCs, but interestingly, they did not inhibit RR, and thus,

26

their exact mode of action is currently still unknown.

Synthesis of C-Series Compounds. Selective alkylation

of 2-aminobenzo[d]thiazol-6-ol followed by substitution of the

amino group with hydrazine provided the substituted

hydrazine intermediate 9 (Scheme 4). The benzimidazole

intermediates 11 and 15 are prepared by NH alkylation using

sodium hydride on 2-chlorobenzimidazole and then displace-

ment of the chloride with hydrazine.

2

1

piperazine (A10) and methylation at C-6 of the pyridine

A11) were both tolerated. Replacement of the azetidine or

(

dimethylamine with substituted aminopiperazine (A4) and

addition of bulky groups that would likely reduce zinc binding

The 4-O-alkylated-2-acetylpyridine was prepared in a

(

A12 and A13) prevented the refolding of p53.

Synthesis of A-Series Compounds. ZMC1 and A1 were

straightforward fashion according to Scheme 5. S Ar on 4-

N

chloro-2-cyanopyridine gave dimethylamine 12 and THP-

protected ether 16. Both were subjected to the methylmagne-

sium iodide addition and hydrolysis. Compound 12 gave the

desired ketone 13, while the ketone derived from 16 was

prepared by displacing the methylthiol of intermediate 2, and

A5 was obtained via S-methylation of ZMC1 (Scheme 1). The

thiocarbonyl in compounds A4 and A9−A14 is introduced

2

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Scheme 2. (i) TEA and THF; (ii) N H ·H O and EtOH; and (iii) DCM and AcOH

(cat.)

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2

4

2

Scheme 3. (i) N H ·H O and EtOH; (ii) 4-Nitrophenyl

structure of C1 in complex with zinc and found it to bind as a

2:1 octahedral complex with zinc (Zn-2, Figure 2B), just as

had been previously published for ZMC1 (Zn-1, Figure 2B).

2

4

2

Carbonochloridate and DCM; and (iii) Azetidine, TEA, and

DCM

6

Furthermore, the internal carbonyl CN coordinates zinc in

this 2:1 complex analogously to the C−S of ZMC1 with S−Zn

being slightly longer than N−Zn of the benzothiazole in the

crystal structures (2.45 and 2.10 Å, respectively, see Scheme

9

). The two hydrazinyl moeities of the ligands are each

deprotonated and each bear one negative charge that balances

2

+

the Zn ion charge. The remaining pyridine and benzothiazole

rings contain CN: nitrogen atoms with a nitrogen lone pair

available for d-orbital insertion. This neutral complex is then

capable of ionophore activity to traverse the plasma membrane

of the cell to deliver zinc intracellularly. Donation of zinc

within the cell is followed by tetrahedral coordination with key

Cys176−His179 of L2 and Cys238−Cys242 of L3 residues on

further hydrolyzed to give alcohol 17. 17 was mesylated, and

the mesylate 18 was displaced with 4-methylpiperazine to give

1

9.

The synthesis of C65 is shown in Scheme 6, where the

bromoacetylpyridine is simultaneously methoxylated while the

hydrazone is formed.

The majority of the hydrazone preparations were done in

methanol with acetic acid or with additional hydrochloric acid

to give the HCl salt, as shown in Scheme 7.

The syntheses of the substituted azole analogues (C69, C78,

C85, and C89) are shown in Scheme 8. Alkylation of

benzimidazoles (21 and 25) is followed by lithiation and

addition to di-tert-butyl-azodicarboxylate, providing the di-

Boc-substituted hydrazines (22, 26, and 27). Simultaneous

hydrazine deprotection and condensation with the appropriate

ketone provided the desired products.

1

mutant p53. The tetracoordinated residues then trigger a

wild-type conformational change that leads to a p53-mediated

apoptotic cellular death. We tested C1 and C2 for their aﬃnity

for zinc as well as in cell growth inhibition assays using ZMC1

as a control. We found the K_Z_nfor C1 and C2 to be 141 and 90

ⁿ^M^,^r^e^s^p^e^c^tⁱ^v^e^l^y^,^w^hⁱ^c^hⁱ^s^wⁱ^t^hⁱⁿ^t^h^e^r^aⁿ^g^e^o^f^t^h^e^KZn we

determined in Table 1. Both C1 and C2 showed toxicity for

R175H

the p53

mutants with similar potency to ZMC1 (Table 1)

and exhibited allele selectivity as they demonstrated almost no

cell kill in the p53 wild type and p53 null cell lines (Suppl.

Heterocycles such as benzothiazole C1, benzoxazole C2, and

N-methylbenzimidazole C9 contain a CN double bond and

an acidic NH that once deprotonated functions to coordinate

Figure S1). Furthermore, we found that they both induced a

R175H

wild-type conformational change in the p53

(Table 1).

2

+

Zn ions analogously to that of the C−S thiolate of a

Taking all three factors into account, we concluded that both

C1 and C2 function as zinc metallochaperones in vitro.

thiosemicarbazone (Figure 2 A). We resolved the X-ray crystal

Figure 2. (A) Simplest C-series analogues of ZMC1. (B) X-ray structures of Zn-1 and Zn-2.

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Scheme 4. (i) 1-Bromo-2-methoxyethane, Cs CO , and DMF; (ii) N H ·H O, HCl (Concd), and Ethylene Glycol; (iii) NaH

2

3

2

4

2

and DMF; and (iv) N H ·H O, i-PrOH, or Ethylene Glycol

2

4

2

Scheme 5. (i) 2-(Dimethylamino)ethan-1-ol, NaH, and DMF; (ii) MeMgI (3 M/Et O) and THF; (iii) 2-((Tetrahydro-2H-

2

pyran-2-yl)oxy)ethan-1-ol, NaH, and DMF; (iv) HCl; (v) MsCl, TEA, and DCM; and (vi) 1-methylpiperazine, KI, K CO , and

2

3

MeCN

Scheme 6. (i) HBr, Br , and AcOH and (ii) AcOH and DCM/MeOH

2

Scheme 7. (i) MeOH and AcOH₍_c_a_t_.₎and (ii) MeOH and AcOH₍_c_a_t_.₎followed by 4 M HCl/dioxane

Synthesis of Zn-1 and Zn-2. The zinc complexes Zn-1

and Zn-2 are prepared by heating in the presence of

triethylamine and zinc chloride in ethanol followed by

recrystallization (Scheme 9).

C-Series Evolution. The evolution of the C-series is shown

in Figure 3. Replacement of the hydrazinothiocarbonyl of

ZMC1 by hydrazinobenzoic heterocycles led to C1 (benzo-

thiazole), C2 (benzoxazole), and C9 (benzimidazole), which

maintained activity but suﬀered from low aqueous solubility.

Addition of solubilizing polyethers (C23) or alkylamines (C27

and C28) resulted in more soluble compounds with better

physical properties. Further optimization of the solubilizing

27

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Scheme 8. (i) K CO and DMF; (ii) BH ·THF and THF; (iii) Di-tert-butyl-azodicarboxylate, n-BuLi (2.5 M in Hexanes), and

2

3

THF; and (iv) MeOH, 4 M HCl/Dioxane (Excess)

Scheme 9. (i) ZnCl , TEA, and EtOH

amines led to compounds like C35, C42, and C69, which had

a mixture of cellular activity and physical properties that made

them attractive to pursue further. Evaluation in the CEREP44

safety screen showed ion channel liabilities with the most

serious one being the potassium channel hERG (from human

2

28

ether-a-go-go-related gene) due to its known association with

̀

serious cardiovascular risks (Table 2). Conversion of the

benzimidazole substituent of C9 to an imidazole (C78)

resulted in a compound with a decreased 1-octanol/water

29

partition coeﬃcient (ClogP). In addition, the removal of one

of the lipophilic bases of the triangular hERG pharmacophore

resulted in a compound devoid of liabilities observed in the

CEREP screen results of earlier analogues.

The 2-quinolinyl heterocycle C22 possesses a polarized

internal carbonyl mimic in the form of the quinoline nitrogen

that binds the zinc ion tightly, probably the consequence of six-

membered ring geometry, with a K_Z_nof 44 nM. However, it

was not as eﬀective as C9 against TOV-112D cells (Table 1).

The dimethylaminoethyloxy substituent appended to the

pyridine ring of C35 proved to be a solubilizing group with

Figure 3. Evolution of the C-series.

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Table 2. CEREP Safety Screening Selected Results

3

0

a favorable topological polar surface area (tPSA) and CLogP,

subsequently leading to mutant p53 protein refolding (the

tPSA and cLogP were calculated using ChemDraw or MOE).

tPSA is also an accurate predictor of permeability and drug

absorption as well as a predictor of absorption across the

not lead to mutant p53 refolding. However, C21 was able to

refold p53 even though the zinc-binding aﬃnity was the

borderline. Morpholine, another widely used solubilizing

group, can be tethered to the benzimidazole nitrogen as

seen in N-ethylmorpholino analogue C42. Functionalized

pyrrolidines (C69 and C85) can also be used as solubilizing

side chains and do not appear to interfere with complex

formation or dissociation. The benzothiazolyl C61 (Table 1)

utilizes a hydroxyethyl appendage to increase solubility, which

could possibly prevent hERG channel inhibition. The

32

31

blood−brain barrier (BBB). In addition, the alkoxy group at

the 4-position of C35 increased the electron density on the

pyridine nitrogen helping to tune the Zn/Cu binding ratio.

This eﬀect was also observed with the dimethylamino

substituent on A14, but unlike in the case for C35, it did

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Figure 4. C-Series compound structures compared with that of serotonin.

Figure 5. Eﬃcacy of C1.

structural elements most commonly associated with this hERG

in protein refolding and were excluded from further

consideration.

liability are the presence of a basic amine (pK > 7.3),

a

lipophilicity (ClogP > 3.7), a lack of negatively ionizable

groups, and a lack of oxygen H-bond acceptors. Potassium

hERG channel inhibitors often have a triangular or pyramidal

pharmacophore with a basic group at the apex and two (or

three) lipophilic groups at the base. These inhibitors are

thought to act as a plug for the channel. A range of

solubilizing functional groups were appended to either the 4-

position of the pyridine ring or to the free NH of the

benzimidazole ring, in search of improved solubility. C61

A standard CEREP safety panel was used to determine the

level of inhibition of most of the ion channels, CNS receptors,

and hERG (Table 2). The results of the screen indicated that

this particular class of chelating ligands inhibits 5-hydroxytrypt-

amine (5HT, serotonin) receptors, opiate μ receptors, hERG,

+

33

Na channel, and even the norepinephrine transporter. It is

evident that two of the C-series benzimidazole analogues, C42

and C69, overlay quite well with 5HT, hence the 100 and 95%

inhibition of the serotonin 5HT receptor, respectively (Figure

4

). Many of the compounds in this series are active against

showed good characteristics and an acceptable zinc K but did

d

targets one wishes to avoid. Besides the 5HT receptor activity

that were observed for C42 and C69, structures with a 2-

carbon tether to a basic amine, C9, C28, C42, C69, and C89

all showed strong hERG inhibition at 10 μM. Several

compounds showed activity against the sodium channel (C1

and C69) and the norepinephrine transporter (C1, C28, C69,

and C77). An ethyl-3,3-diﬂuoropyrrolidine ring was added to

the benzimidazole core to generate C85, reducing the

not initiate mutant protein refolding (Table 1). The N-methyl-

piperizinylethyl analogue C64 showed good zinc binding but

was also unable to demonstrate protein refolding. C69 was able

to demonstrate protein refolding. In addition, it had a favorable

tPSA and ClogP as well as a good balance between aliphatic

and aromatic character and thus was advanced for further

studies. Many of the Table 1 analogues possess favorable

absorption and permeability characteristics but were not active

calculated pK of the side chain’s basic nitrogen by more

b

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Figure 6. Eﬃcacy of the other C compounds.

than 3.2 units to 5.9 as compared to C69 possibly eliminating

the hERG liability and the hydroxytryptamine mimicry.

addition to their induction of DNA damage in cancer cells,

there is a boost of cell killing due to activation of wild-type

p53. This is one of theoretical beneﬁts of combining a putative

mutant p53 reactivator with cytotoxic chemotherapy or

radiation, which is that there will be a boost of wild-type

p53 activity to the eﬀect of chemotherapy or radiation. We

recently investigated this with ZMC1 and found that,

surprisingly, cytotoxic chemotherapy and radiation are not

23

Recent CEREP44 screening results however did not support

the increased acidity hypothesis, with the compound showing

potent hERG inhibition as well as other liabilities (Table 2).

Benzothiazolyl Hydrazones Function as Zinc Metal-

lochaperones In Vivo. To further evaluate the potential of

these compounds for clinical development as zinc metal-

lochaperones, we studied C1 for toxicity and eﬃcacy in mice.

We ﬁrst determined the maximum tolerated dose (MTD)

using acute single-dose exposure toxicity assays for each

selected compound that previously showed low EC₅₀values

36

more eﬀective in combination. We elucidated the mechanism

for this and found that the ROS signal of ZMC1 induced the

same PTMs on p53 that are typically performed by

3

6

chemotherapy or radiation. Since the ROS signal by

2

+

and reactivation of p53 functions in the cellular assays. Then,

ZMC1 is from the chelation of redox-active metals like Cu ,

R175H

we treated the animals bearing the p53

mutant tumors

we measured the aﬃnity of ZMC1 for copper (K ) and

Cu

−

17

8

with these compounds to compare the eﬃcacy. We found that

C1 was well tolerated up to 30 mg/kg without signiﬁcant

weight loss, much better than ZMC1 where the MTD was 5

determined it to be 10 M. This is 10 -fold higher than its

2

+

36

2

aﬃnity for Zn

and 10 -fold higher than high-aﬃnity

37

intracellular copper-binding proteins. We hypothesized that

if we could identify a zinc-binding scaﬀold that bound copper

more weakly (and therefore generated less ROS) but at the

same time kept its aﬃnity for zinc in the same range, the

molecule could induce a wild-type p53 conformation without

generating an ROS signal that would induce PTMs on p53. We

reasoned that such a zinc scaﬀold when combined with

chemotherapy or radiation would be synergistic. As a proof of

1

0

mg/kg (Figure 5A,B). This indicated that the benzothiazolyl

hydrazones are indeed less toxic than the TSCs. To compare

the eﬃcacy of C1 and ZMC1 at 5 mg/kg (a dose which we

know is eﬀective in vivo for ZMC1), we dosed the molar

equivalent of C1 (5.73 mg/kg). We found that, like ZMC1

(

which reduced tumor growth by 63%), C1 treatment also

signiﬁcantly inhibited the tumor growth (reduced tumor

growth by 72%), implying that C1 could be used to reactivate

concept, we tested nitrilotriacetic acid (NTA) whose K (17

Zn

R175H

36

p53

mutation in vivo (Figure 5C,D). We also observed

nM) was in the same range as that of ZMC1. NTA has poor

pharmacological properties including poor cell membrane

permeability due to its anionic character. To overcome this, we

synthesized analogues that replaced the ionizable acids in NTA

with nonionizable esters (NTA-MEE and NTA-DEE) that

were more permeable (Figure 7). While the NTA compounds

that like ZMC1, the tumor growth inhibition by C1 was allele

speciﬁc in that C1 did not inhibit the tumor growth derived

R273H

from p53

, which is not a Zn-deﬁcient mutation (Suppl.

Figure S2).

We then carried out the in vivo eﬃcacy studies for several

candidate compounds selected from the cellular assays. We

conﬁrmed that the doses were not toxic to the normal mice by

2+

bound Zn with aﬃnities similar to that of ZMC1, they also

the MTD assay (Suppl. Figure S3). Then, we treated the

R175H

p53

tumors with compounds C42 and C69 (Figure 6A)

and C35 and C78 (Figure 6B), in comparison to C1. The

studies showed similar eﬃcacy between these compounds over

a 15 day trial with signiﬁcant tumor growth inhibition

(

reduced tumor growth: C42, 59%; C69 , 47%; C35, 56%;

and C78, 57%) (Figure 6 and Suppl. Figure S3).

C-Series Compounds that Function as a New Class of

Zinc Metallochaperones: Chemotherapy/Radiation

Sensitizers. Systemic chemotherapy and radiation have

previously been shown to be more eﬀective in tumors with

wild-type p53 as these therapies activate wild-type p53 through

post-translational modiﬁcations (PTMs) that guide p53 down

34,35

pathways of cell cycle arrest and apoptosis.

Thus, in

Figure 7. Structures of nitriloacetate derivatives.

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6

7

bound copper much more weakly (10 −10 -fold less than

growth inhibition (Figure 8C). Next, we combined the

treatment of C85 and etoposide. The EC₅₀decreased from

0.57 μM (C85 only) to 0.12 μM (C85 + etoposide), 4-fold

more eﬀective in cell growth inhibition (Figure 8D). These

data indicate that C85, with its diminished Cu /Zn binding

ratio, can function as a new type of ZMC that functions as a

chemotherapy or radiation sensitizer in cancer cells with zinc-

deﬁcient mutant p53.

2+

ZMC1). We found that the Cu /Zn selectivity ratio is much

higher for ZMC1 (10 ) than for NTA and its esters (10−

0 ). At this ratio, the approximately 10 -fold greater

8

3

36

5

1

2

+

2+

availability of Zn in serum would be expected to largely

preclude the formation of Cu²complexes in the blood. We

found that, indeed, these NTA esters could function as zinc

metallochaperones to refold mutant p53, and they did produce

less of an ROS signal. This decreased ROS signal lead to less

cell killing due to a lack of induction of p53 PTMs. When

combined with cytotoxic chemotherapy and radiation,

however, we observed synergy in cell killing. Thus, we

concluded that these zinc scaﬀolds, with a lower aﬃnity for

+

17

ZMC1 and C85 Hydrolytic Stability Studies. Many

38

hydrazines are known to be toxic. They are found in toxic

39

mushrooms and even in some common button mushrooms.

Not all hydrazines are toxic, and many are approved drugs like

carbidopa for Parkinson’s disease, isoniazid against malaria,

and phenelzine sulfate for depression. Nevertheless, the

hydrolytic stability of our hydrazones is still a safety concern.

We conducted stability studies with ZMC1 and C85 as

representatives of the A-series and C-series in simulated gastric

ﬂuids (SGF, pH 1−2) and in phosphate-buﬀered saline (PBS,

pH 7.4) at room temperature and at 37 °C when the

compounds were found to be stable at room temperature (∼23

2+

3

copper and a Cu /Zn selectivity in the range of 10−10 ,

would represent a new class of ZMCs that would function as

cytotoxic chemotherapy and radiation sensitizers in cancer cells

with zinc-deﬁcient mutant p53.

Synthesis of the Nitriloacetates. The nitrolotriacetate

derivatives, NTA-MEE and NTA-DEE, were prepared via

selective esteriﬁcation or via alkylation, as shown in Scheme

°

C (Supporting Information)). We used HPLC analysis at 220

1

0.

and 254 nm and conﬁrmed the peak identity by LC−MS. We

found that ZMC1 was not stable in SGF at room temperature,

reaching an equilibrium with ∼22% of ZMC1 remaining after 7

h. C85 was much more stable, with >92 and >82% remaining

after 6 h in SGF at room temperature and 37 °C, respectively.

To avoid the acidic environment of the gut, iv administration

for therapeutic dosing of our compounds is under consid-

eration. Stability in PBS is used as a rough model for plasma

stability. The greater hydrolytic stability of C85 in SGF

demonstrates an additional advantage of the C-series over the

A-series for potential development. Metabolic stability of our

compounds has not yet been fully established, but Pelivan et al.

evaluated the metabolic stability of A1 (their compound 9) by

incubation for 2 h with cell-free human liver microsomes

followed by analysis with HPLC−HRMS and did not report

Scheme 10. (i) Ac O, Pyridine, and DMF; (ii) EtOH; and

2

(

iii) Dioxane

24

observing hydrazine or ketone released from the reactions.

Many metabolites were observed, but the release of hydrazine

was not among them.

C85 Combination Therapy. In an eﬀort to identify a zinc

scaﬀold with better drug-like properties that could function as

this new type of ZMC, we turned to the C-series. We selected

a compound C85 that had similar properties to C69 but with

the addition of the diﬂuoromethylene (Figure 8A). We found

from our SAR analysis that C85 could induce a wild-type

conformational change in the mutant p53 protein (Figure 8B);

however, TOV-112D cells were less sensitive to C85 than

ZMC1 (EC₅₀of C85 as 151 nM vs ZMC1 as 5 nM, 31-fold

less than ZMC1). Likewise using the CellROX Green

ﬂuorescent agent in p53 null cells, we found that C85 did in

fact induce a smaller ROS signal in comparison to ZMC1 (77%

relative to ZMC1) or C1 (170% relative to ZMC1). We

CONCLUSIONS

■

Due to the high frequency of missense p53 mutations in many

types of cancers, the discovery and development of mutant p53

reactivators remains a large unmet need. Zinc metallochaper-

ones represent a novel and viable strategy to meet this need;

while thiosemicarbazones may still be a viable path forward,

there remains a need to continue to identify alternative zinc

scaﬀolds that can function as ZMCs. As the mechanism of

action of ZMCs is novel, here, we provide evidence that the

benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones

do in fact function as zinc metallochaperones both in vitro and

in vivo.

As with any drug development program, establishing assays

for SAR is paramount for successful identiﬁcation of a clinical

candidate. However, due to the uniqueness and complexity of

the ZMC mechanism, the strategy of which assays would be

best suited for this was not immediately obvious. We think that

measured the K of C85 to be 10⁻M and determined the

15

Cu

2

+

2+

7

Cu /Zn selectivity ratio to be 10 , one order of magnitude

less than ZMC1 (Figure 9). Although this reduction in

selectivity does not meet the threshold for complete

5

2+

elimination of copper binding (10 ), the Cu aﬃnity of this

improved drug for copper no longer exceeds that of

2

+

intracellular Cu -binding proteins. Next, we performed

combinatorial experiments with C85 and γ-irradiation or

etoposide in TOV-112D cells. We ﬁrst subjected TOV-112D

cells to C85 and 5 Gy of radiation for 72 h and observed

synergy. The EC decreased from 0.71 μM (C85 only) to 0.18

at minimum, the measurements of the K and the EC₅₀in a

Zn

R175H

p53

tumor cell line (and perhaps a p53 null cell line to

demonstrate speciﬁcity), along with the conformational

antibody assay, are most important initially for SAR develop-

ment. We determined that the upper limit of range in K_Z_nfor a

scaﬀold to function as a ZMC is approximately 200 nM, which

5

0

μM (C85 + 5 Gy of irradiation), 4-fold more eﬀective in cell

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Figure 8. Combinatorial therapy of C85 and irradiation and chemotherapy drug.

Figure 9. Copper aﬃnity of C85.

is very helpful. We cannot, at this time, determine the lower

limit of this range with high accuracy as none of the

modiﬁcations we designed in the SAR around ZMC1 increased

its aﬃnity for zinc signiﬁcantly. In addition, we have not

identify other metal ion chelator scaﬀolds that bound zinc

more tightly than ZMC1, with the exception of EDTA. EDTA

is one in which we have found that zinc binds so tightly that it

imidazole compounds as possible zinc-binding and p53

refolding agents. Although we have not yet exhausted all the

possible optimized analogues around these fragments, we did

not identify analogues that were better than ZMC1. Initially,

the benzothiazole C-series appeared to be the most promising,

but its low solubility led us to add solubilizing groups that

introduced liabilities in the hERG channel and other receptors.

ZMC1 and other highly active analogues are more hydrophilic

than the benzothiazoles, benzimidazoles, and to a lower extent

to the benzoxazoles. In future work, more eﬀorts would be put

into development of the benzoxazole series in addition to

exploring more carefully the type and spacing of solubilizing

−

16

will not donate zinc to p53; however, its K is 10 M (as

Zn

−

8

opposed to 10 M for ZMC1).

Thiosemicarbazones are relatively easy to prepare, but the

molecules can exist in many interchangeable stereoisomers,

conformers, and metal complexes, which sometimes makes

their chemical analysis and biological eﬀects diﬃcult to

deconvolute. Very few drugs (4 hits in DrugBank) incorporat-

ing a thiosemicarbazone functional group have made it to the

41

groups. Bioisosteres of the thiosemicarbazones will also be

explored.

In theory, improving the ionophore activity to increase

intracellular zinc will drive potency. We previously demon-

strated this by synthesizing a neutral 2:1 complex of ZMC1 to

40

market and stayed on the market. Their interactions with

metals ions are numerous, and diﬀerences between complexes

can be subtle. We explored using (pyridinyl)ethylidene)-

hydrazineyl)benzothiazole, benzoxazole, benzimidazole, and

2

+

Zn (Zn(ZMC1) ) and found enhancement in potency as

2

10

compared to ZMC1 alone, both in vitro and in vivo. However

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7.26; ¹³C: δ 77.00), DMSO-d

(¹H: δ 2.50; ¹³C: δ 39.52), or

it is important to understand that with this mechanism, there

are other cellular factors that can contribute to potency, such

as expression of zinc homeostatic genes that regulate

intracellular zinc levels. Zinc homeostatic mechanisms are

dysregulated in certain cancers and particularly in breast cancer

where zinc levels are higher in cancerous cells compared to

normal mammary epithelial cells. In addition, the expression of

zinc homeostatic genes is diﬀerent across breast cancer

6

1

13

methanol-d ( H: δ 3.31; C: δ 49.00). The HRMS analyses were

4

performed using a Bruker Apex 7T FTMS, using an ESI ion source in

a positive mode (capillary exit voltage: 330 V).

Unless otherwise stated, the purities of the ﬁnal compounds were

equal to or greater than 95% by LC−MS analysis. The purities were

1

conﬁrmed with H NMR to look for residual solvents or non-UV-

active impurities. In addition, HRMS of most of the key compounds

was obtained.

42

subtypes (luminal, basal, and triple-negative breast cancer).

Compounds Were Synthesized by the Rutgers Molecular Design

and Synthesis Group. Methyl Hydrazinecarbodithioate (1). To a

solution of KOH (5.6 g, 100 mmol, 1 equiv) in water (10 mL) was

added i-PrOH (10 mL). The solution was cooled to 0 °C followed by

addition of hydrazine hydrate (79% solution in water) (6.15 mL, 100

mmol, 1 equiv). Carbon disulﬁde (6.0 mL, 100 mmol, 1 equiv) was

added dropwise over 30 min, maintaining temperature below 10 °C,

and the reaction was stirred for an additional hour at 0 °C. While

remaining in an ice bath, methyl iodide (6.2 mL, 100 mmol, 1 equiv)

was added over 30 min and the reaction was allowed to stir for an

additional hour at 0 °C. The white solid precipitated from solution

was ﬁltered, washed with cold water, and dried under vacuum. The

solid was washed through the ﬁlter and recrystallized in DCM to

aﬀord the title compound (1) (5.25 g, 43 mmol, 43% yield) as a white

solid after ﬁltration and drying under vacuum.

Thus, it is possible that variations in expression of these genes

among tumors may cause variation in response to ZMCs.

The ﬁnding that C1 is less toxic than ZMC1 in acute in vivo

toxicity assays is a validation of the general design principle

that replacing the thiocarbamoyl moiety of a TSC with a

benzothiazolyl, benzoxazolyl, or benzimidazolyl moiety reduces

toxicity (as well as producing an increase in hydrolytic

stability). The high degree of binding of many of the C-series

analogues to 5HT receptors, opiate μ receptors, hERG, and ion

channels is a complication that may be addressed by careful

modiﬁcations to the scaﬀold.

2

+

The ﬁnding that C85 exhibits the type of reduced Cu

2

+

2+

aﬃnity and Cu /Zn selectivity ratio that would allow it to

function as a chemotherapy and radiation sensitizer is a

signiﬁcant ﬁnding in this study. Up to now, the compounds we

have identiﬁed that could provide this proof of concept were

not suitable pharmacological agents. Using the 3,3′-diﬂuor-

omethylpiperazine group of C85 was an attempt to reduce the

basicity of nitrogen, thereby reducing the risks of interacting

with hERG and other receptors. Unfortunately, the CEREP

screen revealed that this change was not suﬃcient to avoid

these issues. While we still need to conduct screens for

alternative zinc-binding scaﬀolds with diminished copper

selectivity, we can conclude that simply preventing the

compound from competing for intracellular copper produces

powerful synergy. This new class of ZMC that functions as

chemotherapy and radiation sensitizers would presumably lead

to compounds that would be better tolerated. Moreover,

because cytotoxic chemotherapy and radiation still remain the

most common therapeutic modalities in oncology today, such

a ZMC would potentially have a signiﬁcant impact in

oncology.

Methyl (E)-2-(1-(Pyridin-2-yl)ethylidene)hydrazine-1-carbodi-

thioate (2). To a solution of methyl hydrazinecarbodithioate (1)

(

19.4 g, 160 mmol) in i-PrOH (50 mL) was added 1-(pyridin-2-

yl)ethan-1-one (19.62 g, 162 mmol). After stirring for 2 h at 60 °C,

the reaction was cooled to room temperature. The precipitated

product was ﬁltered and washed with i-PrOH. The solid was dried

under vacuum to aﬀord methyl (E)-2-(1-(pyridin-2-yl)ethylidene)-

hydrazine-1-carbodithioate (2) (37.2 g) as a light yellow solid. MS:

+

2

26.2 [M + H] .

(

E)-N′-(1-(Pyridin-2-yl)ethylidene)azetidine-1-carbothiohydra-

zide (ZMC1). Methyl (E)-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-

carbodithioate (2) (19.7 g, 87.6 mmol) was dissolved in MeOH (400

mL) with heating. Azetidine (6 g, 105 mmol) was added dropwise

over 15 min, and the reaction was stirred for 16 h at 60 °C. Overnight

crystallization at 0 °C aﬀorded (E)-N′-(1-(pyridin-2-yl)ethylidene)-

azetidine-1-carbothiohydrazide (ZMC1) (16.5 g, 70.5 mmol, 97.4%

purity by HPLC) as a crystalline white solid after being ﬁltered,

1

washed with i-PrOH, and dried under vacuum. H NMR (400 MHz,

DMSO-d ): δ 2.26 (m, 2H), 2.36 (s, 3H), 4.12 (m, 2H), 4.59 (m,

6

2

H), 7.39 (m, 1H), 7.83 (dt, J = 7.96, 1.76 Hz, 1H), 7.94 (d, J = 8.04

Hz, 1H), 8.60 (d, J = 4.00 Hz, 1H), 10.24 (s, 1H, NH). MS: 235.1 [M

+

H] . HRMS (MALDI) (m/z): [2M + Na ] calcd for (C H N S)

11 14 4 2

+

Na , 491.17710; found, 491.17710.

(E)-N,N-Dimethyl-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-car-

EXPERIMENTAL SECTION

■

Cell Lines and Chemicals. TOV-112D (p53R175H₎_a_n_d_M_D_A_-

bothioamide (A1). To a solution of methyl (E)-2-(1-(pyridin-2-

yl)ethylidene)hydrazine-1-carbodithioate (2) (378 mg, 1.68 mmol, 1

equiv) in EtOH (5 mL) was added dimethylamine (40% in water) (1

mL). The reaction was heated overnight at 40 °C. After cooling to

room temperature, the precipitated solid was ﬁltered, washed with

EtOH, and dried under vacuum to aﬀord (E)-N,N-dimethyl-2-(1-

(pyridin-2-yl)ethylidene)hydrazine-1-carbothioamide (A1) (235 mg,

R273H

MB-468 (p53

) were purchased from ATCC and cultured in the

DMEM with 10% FBS. Cell lines were authenticated by examination

of morphology and growth characteristics. All chemicals were

purchased from Sigma-Aldrich or otherwise indicated.

Synthesis of the Compounds. General Information. The

reagents were purchased and used without additional puriﬁcations.

LC−MS was performed on a Shimadzu system: LC−MS 2010A using

a 10 μL injection on a SunFire C18 column (3.5 μm, 4.6 × 50 mm) at

a temperature of 40 °C with a 4 min gradient from 5% A to 100% B

1

1.06 mmol, 99.6% purity by HPLC) as a yellow solid. H NMR (500

MHz, DMSO-d ): δ 9.61 (s, 1H), 8.80−8.59 (m, 1H), 8.16−7.95 (m,

6

1H), 7.92−7.77 (m, 1H), 7.61−7.34 (m, 1H), 3.36−3.27 (m, 6H),

+

(solvent A: 0.1% TFA in water and solvent B: 0.1% TFA in

2.63−2.39 (m, 3H). MS: 223.1 [M + H] . Note: peaks have complex

acetonitrile) at a ﬂow rate of 2 mL/min. The detection used dual

channel UV at 220 and 254 nm (mass detection CDL voltage: −10

V). Alternatively, LC−MS was performed on an Agilent 1100 system

with a Waters Micromass ZQ spectrometer using a 5 μL injection on

an XBridge C18 (3.5 μm, 4.6 × 50 mm) column at a temperature of

splitting from multiple conformational isomers. HRMS (MALDI)

+

(m/z): [2M + Na ] calcd for (C10

H

14

N

4

S)

+ Na , 467.17720; found,

2

467.17710.

N-(1-(Pyridin-2-yl)piperidin-4-yl)hydrazinecarbothioamide (3).

To a suspension of di(1H-imidazol-1-yl)methanethione (199 mg,

1.17 mmol, 1 equiv) and 1-(pyridin-2-yl)piperidin-4-amine (250 mg,

1.17 mmol, 1 equiv) in THF (3 mL) was added triethylamine (195

μL, 1.4 mmol, 1.2 equiv). After stirring for 3 h at room temperature, a

solution of hydrazine hydrate (79% solution in water) (284 μL, 5.85

mmol, 5 equiv) in methanol (10 mL) was added and the reaction was

allowed to stir overnight at room temperature. The reaction was

4

0 °C with a 3 min gradient from 5% A to 95% B (solvent A: 10 mM

ammonium formate in water and solvent B: acetonitrile) at a ﬂow rate

of 2 mL/min. The detection used a diode array scanning from 190 to

6

00 nm or with dual wavelength detectors at 220 and 254 nm (mass

detection cone voltage: 30 V). NMR was obtained on a Varian

VNMRS 500 MHz or Bruker NMR 400 MHz in chloroform-d ( H: δ

1

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1

determined complete by TLC (50% EtOAc/Hex) and concentrated

mg, 0.47 mmol, 99.0% purity by HPLC) as a white solid. H NMR

under reduced pressure. The residue was partitioned in EtOAc/H O

(400 MHz, CDCl ): δ 2.28 (s, 3H), 2.32 (t, J = 7.68 Hz, 1H), 2.36 (t,

2

3

and extracted 4× with EtOAc. The combined organics were dried

J = 7.80 Hz, 1H), 4.35 (t, J = 7.36 Hz, 4H), 7.24 (ddd, J = 3.52, 2.48,

1.08Hz, 1H), 7.66 (dt, J = 7.84, 1.76 Hz, 1H), 7.81 (s, 1H, NH), 7.91

over Na SO , ﬁltered, and concentrated. The product was recrystal-

2

4

+

lized from EtOAc, ﬁltered, and dried under vacuum to give N-(1-

pyridin-2-yl)piperidin-4-yl)hydrazinecarbothioamide (3) (189 mg,

.75 mmol) as a light yellow solid.

E)-2-(1-(Pyridin-2-yl)ethylidene)-N-(1-(pyridin-2-yl)piperidin-4-

(d, J = 8.08 Hz, 1H), 8.57 (d, J = 4.20 Hz, 1H). MS: 219 [M + H] .

(

Azetidine-1-carbothiohydrazide (6). To a mixture of di(1H-

imidazol-1-yl)methanethione (4.38 g, 24.6 mmol, 1.15 equiv) and

azetidine HCl (2.0 g, 21.4 mmol, 1 equiv) in THF (20 mL) was

added triethylamine (4.47 mL, 32.1 mmol, 1.5 equiv). The reaction

was stirred for 5 days at room temperature followed by removal of

THF under reduced pressure to give a crude mixture of azetidin-1-

yl(1H-imidazol-1-yl)methanethione. This mixture was taken up in

EtOH (20 mL) followed by addition of hydrazine hydrate (2 mL).

The reaction was heated at reﬂux for 3 h, cooled to room temperature,

and concentrated under reduced pressure. The residue was

0

(

yl)hydrazine-1-carbothioamide (A4). To a solution of 1-(pyridin-2-

yl)ethan-1-one (87 mg, 0.72 mmol, 1 equiv) and N-(1-(pyridin-2-

yl)piperidin-4-yl)hydrazinecarbothioamide (3) in DCM (5 mL) was

added AcOH (4 drops). The reaction was stirred overnight at room

temperature and determined complete by TLC (10% MeOH/DCM).

The crude reaction was partitioned in DCM and NaHCO (sat, aq)

3

and extracted 3× with DCM. The combined organics were dried over

Na SO , ﬁltered, and concentrated. The residue was puriﬁed by silica

partitioned in DCM/H O, and the DCM layer was washed 2× with

2

4

2

gel chromatography (EtOAc → 5% MeOH/DCM). Product

containing fractions were concentrated and recrystallized from

EtOAc to give a white solid, (E)-2-(1-(pyridin-2-yl)ethylidene)-N-

water, dried over Na SO , ﬁltered, and evaporated to give azetidine-1-

2

4

carbothiohydrazide (6) (917 mg, 7 mmol, 32% yield) as a white solid

that was used without further puriﬁcation.

(

1-(pyridin-2-yl)piperidin-4-yl)hydrazine-1-carbothioamide (A4)

(E)-N′-(1-Phenylethylidene)azetidine-1-carbothiohydrazide (A9).

To a solution of azetidine-1-carbothiohydrazide (6) (163 mg, 1.24

mmol, 1 equiv) and acetophenone (223 mg, 1.86 mmol, 1.5 equiv) in

DCM (4 mL) was added AcOH (4 drops). After stirring overnight at

room temperature, the reaction was determined complete by TLC

(50% EtOAc/Hex) and concentrated under reduced pressure.

Recrystallization from MeOH yielded the title compound (E)-N′-

107 mg, 0.30 mmol, 99.3% purity by HPLC), which was ﬁltered

1

and dried under vacuum. H NMR (400 MHz, CDCl ): δ 1.76 (ddd,

3

J = 15.33, 11.72, 3.92 Hz, 2H), 2.30 (m, 2H), 2.42 (s, 3H), 3.04 (dt, J

=

13.73, 2.40 Hz, 2H), 3.72 (m, 2H), 4.57 (m, 1H), 7.16 and 7.19 (E/

Z d, 1.56 Hz, 1H), 7.21 and 7.24 (E/Z m, 1H), 7.30 (ddd, J = 5.87,

4

1

.92, 1.04 Hz), 7.52 (br. d, J = 8.16 Hz, 1H, NH), 7.72 (dt, J = 7.76,

.72 Hz, 1H), 7.90 (d, J = 8.04 Hz, 1H), 8.11 (dd, J = 4.44, 1.32 Hz,

H), 8.35 (d, J = 2.68 Hz, 1H), 8.61 (d, J = 4.12 Hz, 1H), 8.68 (br. s,

H, NH). MS: 287.0 [M + H] .

Methyl (Z)-N-((E)-1-(Pyridin-2-yl)ethylidene)azetidine-1-carbo-

(1-phenylethylidene)azetidine-1-carbothiohydrazide (A9) (176 mg,

1

0.76 mmol) as a white solid. H NMR (400 MHz, CDCl ): δ 2.33 (t,

3

+

J = 7.80 Hz, 1H), 2.37 (t, J = 7.88 Hz, 1H), 4.33 (m, 2H), 4.69 (m,

2H), 7.37 (m, 3H), 7.65 (m, 2H), 8.63 (br. s, 1H, NH). MS: 234.1

+

hydrazonothioate (A5). To a solution of (E)-N′-(1-(pyridin-2-

yl)ethylidene)azetidine-1-carbothiohydrazide (ZMC1) (248 mg, 1

mmol, 1 equiv) in EtOH (4 mL) and DCM (4 mL) was added MeI

[

M + H] .

(

E)-N′-(1-(Pyrazin-2-yl)ethylidene)azetidine-1-carbothiohydra-

zide (A10). To a solution of azetidine-1-carbothiohydrazide (6) (156

mg, 1.19 mmol, 1.0 equiv) and 1-(pyrazin-2-yl)ethan-1-one (152 mg,

1.25 mmol, 1.05 equiv) in DCM (6 mL) was added AcOH (4 drops).

After stirring overnight at room temperature, the reaction was

concentrated under reduced pressure and recrystallized from MeOH

to aﬀord (E)-N′-(1-(pyrazin-2-yl)ethylidene)azetidine-1-carbothiohy-

(68.5 μL, 1.1 mmol, 1.1 equiv). After stirring for 2 h at room

temperature, the reaction was determined complete by TLC (5%

MeOH/DCM) and concentrated under reduced pressure. The

product was recrystallized from EtOH, sonicated, and ﬁltered. The

solid was dried under high vacuum to aﬀord the title compound

methyl (Z)-N-((E)-1-(pyridin-2-yl)ethylidene)azetidine-1-carbohy-

drazide (A10) (132 mg, 0.56 mmol, 99.2% purity by HPLC) as a

crystalline white solid. H NMR (400 MHz, CDCl ): δ 2.38 (t, J =

1

drazonothioate (A5) (237.2 mg, 0.96 mmol, 100% purity by

3

1

HPLC) as a light orange solid. H NMR (400 MHz, DMSO-d ): δ

7.72 Hz, 1H), 2.42 (t, J = 7.88 Hz, 1H), 4.36 (br. t, J = 7.52 Hz, 1H),

4.73 (br. t, J = 7.40 Hz, 1H), 8.50 (d, J = 2.56 Hz, 1H), 8.53 (m, 1H),

6

8

1

7

2

.55 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.99 (m, 1H), 7.76 (q, J = 8.1 Hz,

H), 7.32 (q, J = 7.0, 6.6 Hz, 1H), 4.26 (t, J = 7.6 Hz, 2H), 4.12 (t, J =

.7 Hz, 2H), 2.39 (d, J = 2.4 Hz, 3H), 2.33 (s, 3H), 2.32−2.20 (m,

+

8.78 (s, 1H, NH), 9.13 (m, 1H). MS: 236.1 [M + H] .

(E)-N′-(1-(6-Methylpyridin-2-yl)ethylidene)azetidine-1-carbo-

thiohydrazide (A11). To a solution of azetidine-1-carbothiohydrazide

(6) (102 mg, 0.78 mmol, 1 equiv) and 1-(6-methylpyridin-2-yl)ethan-

1-one (126 mg, 0.93 mmol, 1.2 equiv) in DCM (5 mL) was added

AcOH (4 drops). After stirring overnight at room temperature, the

reaction was determined complete by TLC (50% EtOAc/Hex) and

concentrated under reduced pressure. The resultant solid was

recrystallized from MeOH, ﬁltered, and dried under vacuum to give

the title compound (E)-N′-(1-(6-methylpyridin-2-yl)ethylidene)-

+

H). MS: 248.95 [M + H] .

(E)-2-(1-Hydrazineylideneethyl)pyridine (4). To a solution of 1-

(pyridin-2-yl)ethan-1-one (2.0 g, 16.5 mmol, 1 equiv) in EtOH (5

mL) was added hydrazine hydrate (79% solution in water) (5 mL).

The reaction was heated 4 h at 90 °C, determined complete by TLC,

concentrated under reduced pressure, and dried overnight under high

vacuum to aﬀord (E)-2-(1-hydrazineylideneethyl)pyridine (4) (2.1 g,

1

5.5 mmol) as a white solid that was used without further puriﬁcation.

E)-N′-(1-(Pyridin-2-yl)ethylidene)azetidine-1-carbohydrazide

A6). To a solution of (E)-2-(1-hydrazineylideneethyl)pyridine (4)

300 mg, 2.2 mmol, 1 equiv) in DCM (3 mL) at 0 °C was added a

(

azetidine-1-carbothiohydrazide (A11) (30 mg, 0.12 mmol, 100%

1

(

purity) as a white solid. H NMR (400 MHz, CDCl ): δ 2.35 (m,

3

5H), 2.56 and 2.67 (E/Z s, 3H), 4.34 (m, 2H), 4.70 (m, 2H), 7.11

and 7.18 (E/Z d, J = 7.52 Hz, 1H), 7.30 and 7.65 (E/Z d, J = 8.00 Hz,

1H), 7.56 and 7.73 (E/Z t, J = 7.84 Hz, 1H), 8.71 (s, 1H, NH). MS:

solution of 4-nitrophenyl carbonochloridate (447 mg, 2.2 mmol, 1

equiv) in DCM (3 mL) dropwise over 10 min. The reaction was

warmed to room temperature and stirred for 90 min to give 4-

nitrophenyl (E)-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-carboxy-

+

248.9 [M + H] .

(E)-N′-(1-(3-Methylpyridin-2-yl)ethylidene)azetidine-1-carbo-

thiohydrazide (A12). To a solution of azetidine-1-carbothiohydrazide

(6) (100 mg, 0.77 mmol, 1 equiv) and 1-(6-methylpyridin-2-yl)ethan-

1-one (123 mg, 0.91 mmol, 1.2 equiv) in DCM (5 mL) was added

AcOH (4 drops). After stirring overnight at room temperature, the

reaction was determined complete by TLC (50% EtOAc/Hex) and

concentrated under reduced pressure. The resultant solid was

recrystallized from MeOH, ﬁltered, and dried under vacuum to give

the title compound (E)-N′-(1-(3-methylpyridin-2-yl)ethylidene)-

azetidine-1-carbothiohydrazide (A12) (23 mg, 0.093 mmol, 99.0%

purity by HPLC) as a white solid after recrystallization from MeOH.

+

late (5). MS: 300.90 [M + H] . The reaction was cooled to 0 °C

followed by addition of triethylamine (930 μL, 6.7 mmol, 3 equiv)

and azetidine (300 μL, 4.4 mmol, 1 equiv). The reaction was stirred

overnight, slowly warming to room temperature. The reaction was

determined complete by LC−MS and partitioned in DCM/H O. The

2

aqueous layer was extracted 2× with DCM, dried over Na SO ,

2

4

ﬁltered, and concentrated. The residue was puriﬁed by silica gel

chromatography (1% → 2% → 5% MeOH/DCM). Product fractions

were concentrated followed by recrystallization in EtOAc. The solid

was ﬁltered, washed with EtOAc, and dried under vacuum to give (E)-

N′-(1-(pyridin-2-yl)ethylidene)azetidine-1-carbohydrazide (A6) (103

1

Note: a signiﬁcant amount of product was washed into the ﬁltrate. H

2

037

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Article

NMR (400 MHz, CDCl ): δ 2.27 (m, 6H), 2.43 (m, 2H), 4.37 (m,

(E)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)benzo[d]oxazole

(C2) (1.81 g, 7.17 mmol, 100% purity by HPLC) as a white solid

after drying the solid under high vacuum. H NMR (400 MHz,

3

4

H), 7.19 and 7.26 (E/Z dd, J = 7.72, 4.72 Hz, 1H), 7.56 and 7.61

1

(E/Z d, J = 7.68 Hz, 1H), 8.40 and 8.66 (E/Z br. s, 1H, NH), 8.46

+

and 8.55 (E/Z d, J = 3.76 Hz, 1H). MS: 248.9 [M + H] .

CDCl ): δ 2.48 (s, 3H), 7.16 (br. t, J = 7.28 Hz, 1H), 7.28 (m, 2H),

3

(

E)-N′-(2,2-Dimethyl-1-(pyridin-2-yl)propylidene)azetidine-1-car-

7.44 (br. d, J = 7.28 Hz, 1H), 7.51 (br. d, J = 7.04 Hz), 7.73 (t, J =

7.40 Hz, 1H), 8.27 (br. d, J = 7.28 Hz, 1H), 8.60, (d, J = 4.72 Hz,

bothiohydrazide (A13). To a solution of azetidine-1-carbothiohy-

drazide (6) (116 mg, 0.88 mmol, 1 equiv) and 2,2-dimethyl-1-

(

+

1H), 8.85 (br. s, 1H, NH). MS: 253.1 [M + H] . HRMS (MALDI)

+

pyridin-2-yl)propan-1-one (123 mg, 0.91 mmol, 1.2 equiv) in DCM

5 mL) was added AcOH (4 drops). After stirring overnight at room

(m/z): [2M + H ] calcd for (C H N O) + H , 505.20930; found,

14 12 4 2

505.20950.

temperature, the reaction was determined complete by TLC (50%

EtOAc/Hex) and concentrated under reduced pressure. The crude

residue was puriﬁed by silica gel chromatography (20% → 30%

EtOAc/Hex), and the concentrated product fractions were recrystal-

lized from EtOAc/Hex. The crystallized solid was ﬁltered and dried

under high vacuum to aﬀord (E)-N′-(2,2-dimethyl-1-(pyridin-2-

(E)-1-Methyl-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)-1H-

benzo[d]imidazole (C9). 2-Hydrazinyl-1-methyl-1H-benzo[d]-

imidazole (109 mg, 0.68 mmol, 1 equiv) and 1-(pyridin-2-yl)ethan-

1-one (76 μL, 0.68 mmol, 1 equiv) were dissolved in EtOH (5 mL)

followed by addition of AcOH (4 drops). After stirring overnight at

room temperature, the reaction was determined complete by LC−

MS. Puriﬁcation by silica gel chromatography (50% EtOAc/Hex →

EtOAc) followed by recrystallization from 5:1 Hex/EtOAc yielded

the title compound (E)-1-methyl-2-(2-(1-(pyridin-2-yl)ethylidene)-

hydrazinyl)-1H-benzo[d]imidazole (C9) (110 mg, 0.41 mmol, 99%

yl)propylidene)azetidine-1-carbothiohydrazide (A13) (9.3 mg, 0.034

1

mmol, 99.0% purity by HPLC). H NMR (400 MHz, CDCl ): δ 1.18

3

(

=

s, 9H), 2.29 (t, J = 7.76 Hz, 1H), 2.33 (t, J = 7.84 Hz, 1H), 4.26 (t, J

7.36 Hz, 1H), 4.66 (t, J = 7.72 Hz, 1H), 7.18 (d, J = 7.76 Hz, 1H),

7

1

H] .

.35 (ddd, J = 6.00, 4.92 , 1.12 Hz, 1H), 7.81 (dt, J = 7.76, 1.76 Hz,

purity by HPLC) as a yellow solid after drying the solid under high

1

H), 8.26 (br. s, 1H, NH), 8.76 (d, J = 4.80 Hz, 1H). MS: 277.1 [M +

vacuum. H NMR (400 MHz, CDCl

3

): δ 2.59 (s, 3H), 3.54 (s, 3H),

+

7.02 (m, 4H), 7.21 (br. t, J = 5.50 Hz, 1H), 7.67 (t, J = 7.88 Hz, 1H),

8.08 (d, J = 8.00 Hz, 1H), 8.60 (d, J = 4.72 Hz, 1H), 9.10 (br. s, 1H,

1

-(4-(Dimethylamino)pyridin-2-yl)ethan-1-one (7). 1-(4-Chloro-

+

pyridin-2-yl)ethan-1-one (500 mg, 3.2 mmol, 1 equiv) was taken up

in dimethylamine (40% in water) (3.5 mL) and heated overnight at

NH). MS: 266.3 [M + H] . HRMS (MALDI) (m/z): [2M + H ]

+

calcd for (C15H N ) + H , 531.27240; found, 531.27280.

15 5 2

1

00 °C in a sealed reaction vial. The reaction was determined

complete by TLC (50% EtOAc/Hex) and concentrated under

reduced pressure. Silica gel chromatography (50% EtOAc/Hex →

EtOAc) aﬀorded 1-(4-(dimethylamino)pyridin-2-yl)ethan-1-one (7)

(357 mg, 2.2 mmol) as a light yellow oil after concentration of

product fractions.

(E)-N′-(1-(4-(Dimethylamino)pyridin-2-yl)ethylidene)azetidine-1-

carbothiohydrazide (A14). To a solution of azetidine-1-carbothiohy-

drazide (6) (139 mg, 1.06 mmol, 1.1 equiv) and 1-(4-

(dimethylamino)pyridin-2-yl)ethan-1-one (7) (150 mg, 0.96 mmol,

1

equiv) in DCM (4 mL) was added AcOH (4 drops). After stirring

puriﬁed by silica gel chromatography (2% → 5% → 10% MeOH/

DCM). The combined product containing fractions were concen-

trated under reduced pressure and recrystallized from EtOH. The

crystalline solid was ﬁltered and washed with EtOH, and the solid was

dried under high vacuum to aﬀord (E)-2-(1-(2-(benzo[d]thiazol-2-

yl)hydrazineylidene)ethyl)-N,N-dimethylpyridin-4-amine (C21)

overnight, the reaction was determined complete by TLC (10%

MeOH/DCM) and concentrated under reduced pressure. Puriﬁca-

tion by silica gel chromatography (5% → 10% MeOH/DCM)

followed by recrystallization from MeOH yielded the title compound

(

E)-N′-(1-(4-(dimethylamino)pyridin-2-yl)ethylidene)azetidine-1-

(

52.3 mg, 0.17 mmol, 96.9% purity by HPLC) as a pale red solid.

carbothiohydrazide (A14) (27.4 mg, 0.1 mmol, 100% purity by

1

H NMR (400 MHz, chloroform-d): δ 8.91 (s, 1H), 8.26 (dd, J =

19.8, 6.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H),

7.42 (s, 1H), 7.33 (dt, J = 17.4, 7.9 Hz, 1H), 7.15 (dt, J = 24.7, 7.4 Hz,

1H), 6.53 (t, J = 5.5 Hz, 1H), 3.09 (d, J = 11.3 Hz, 6H), 2.41 (s, 3H).

HPLC) as a white solid. H NMR (400 MHz, CDCl ): δ 2.34 (m,

3

5

H), 3.02 (s, 6H), 4.34 (m, 2H), 4.70 (m, 2H), 6.49 and 6.54 (E/Z

dd, J = 6.04, 2.64 Hz, 1H), 6.62 and 7.08 (E/Z d, J = 2.44 Hz, 1H),

+

8

.26 (m, 1H), 8.71 (br. s, 1H, NH). MS: 278.0 [M + H] .

E)-2-(2-(1-(Pyridin-2-yl)ethylidene)hydrazinyl)benzo[d]thiazole

C1). 1-(Pyridin-2-yl)ethan-1-one (204 μL, 1.82 mmol, 1 equiv) and

-hydrazinylbenzo[d]thiazole (300 mg, 1.82 mmol, 1 equiv) were

+

(

MS: 312.1 [M + H] .

(

2

(E)-2-(2-(1-(Pyridin-2-yl)ethylidene)hydrazineyl)quinoline (C22).

2-Hydrazineylquinoline (100 mg, 0.63 mmol, 1 equiv) and 1-

(pyridin-2-yl)ethan-1-one (71 μL, 0.63 mmol, 1 equiv) were dissolved

in EtOH (5 mL) followed by addition of AcOH (4 drops). The

reaction was determined complete by LC−MS, and the precipitated

solid was ﬁltered. Recrystallization of the resultant solid from MeOH

gave the title compound (E)-2-(2-(1-(pyridin-2-yl)ethylidene)-

hydrazineyl)quinoline (C22) (65 mg, 0.25 mmol, 99.4% purity by

HPLC) as a yellow/orange solid after drying the solid under high

dissolved in EtOH (10 mL) followed by addition of AcOH (10

drops). The reaction was stirred overnight at room temperature and

determined complete by TLC (50% EtOAc/Hex). The crude reaction

was concentrated under reduced pressure, recrystallized from MeOH,

sonicated, and ﬁltered to give the title compound (E)-2-(2-(1-

(pyridin-2-yl)ethylidene)hydrazinyl)benzo[d]thiazole (C1) (324 mg,

1

.21 mmol, 100% purity by HPLC) as a white solid after drying under

1

high vacuum. H NMR (400 MHz, CDCl ): δ 2.44 (s, 3H), 7.19 (dt,

vacuum. H NMR (400 MHz, chloroform-d): δ 8.64−8.58 (m, 1H),

3

J = 7.2, 1.01 Hz, 1H), 7.26 (m, 1H), 7.36 (dt, J = 7.2, 1.01 Hz, 1H),

8.55 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.9 Hz, 1H), 7.85−

7.68 (m, 4H), 7.62 (ddt, J = 8.5, 7.1, 1.6 Hz, 1H), 7.41−7.30 (m,

7

1

.62 (d, J = 7.96 Hz, 1H), 7.71 (d, J = 7.08 Hz, 1H), 7.74 (dt, J =

.76, 1.76 Hz, 1H), 8.18 (d, J = 8.12 Hz, 1H), 8.60 (br. d, J = 4.32 Hz,

H), 9.14 (br. s, 1H, NH). MS: 269.0 [M + H] . HRMS (MALDI)

+

1H), 7.25−7.19 (m, 1H), 2.48 (s, 3H). MS: 262.9 [M + H] .

+

6-(2-Methoxyethoxy)benzo[d]thiazol-2-amine (8). To a solution

of 2-aminobenzo[d]thiazol-6-ol (500 mg, 3.0 mmol, 1 equiv) in DMF

+

(m/z): [M + H ] calcd for C H N S + H , 269.08620; found,

14 12 4

2

69.08550.

E)-2-(2-(1-(Pyridin-2-yl)ethylidene)hydrazinyl)benzo[d]oxazole

C2). 2-Hydrazinylbenzo[d]oxazole (1.33 g, 8.9 mmol, 1 equiv) and 1-

pyridin-2-yl)ethan-1-one (1.0 mL, 8.92 mmol, 1 equiv) were

(30 mL) was added Cs CO (4.9 g, 15 mmol, 5 equiv) followed by 1-

2 3

(

bromo-2-methoxyethane (310 μL, 3.3 mmol, 1.1 equiv). The reaction

was stirred overnight at room temperature and determined complete

by TLC (10% MeOH/DCM). The crude reaction was partitioned in

(

dissolved in EtOH (20 mL) followed by addition of AcOH (10

drops). After stirring overnight at room temperature, the reaction was

determined complete by LC−MS and the precipitated solid was

ﬁltered. Recrystallization from MeOH yielded the title compound

EtOAc/H O. After removal of the layer, the organic was further

2

washed 2× with H O and 1× with brine. The organic was dried over

2

Na SO , ﬁltered, and concentrated. The residue was puriﬁed by silica

2

4

gel chromatography (2% → 5% MeOH/DCM) followed by (25% →

2

038

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5

0% EtOAc/Hex → EtOAc). Product containing fractions were

dimethylethan-1-amine (11) (crude from the previous reaction) in

EtOH (10 mL) was added 1-(pyridin-2-yl)ethan-1-one (300 μL,

excess) followed by AcOH (10 drops). The reaction was stirred

overnight at room temperature and was determined complete by LC−

concentrated under reduced pressure and recrystallized from EtOAc.

The solid was ﬁltered and dried under high vacuum to aﬀord 6-(2-

methoxyethoxy)benzo[d]thiazol-2-amine (8) (251 mg, 1.1 mmol) as

a white solid.

MS. The crude reaction was partitioned in DCM/NaHCO (sat, aq),

3

2

-Hydrazineyl-6-(2-methoxyethoxy)benzo[d]thiazole (9). To a

and the aqueous was extracted 3× with DCM. The combined organic

solution of 6-(2-methoxyethoxy)benzo[d]thiazol-2-amine (8) (150

mg, 0.67 mmol, 1 equiv) in ethylene glycol (3 mL) was added

hydrazine hydrate (195 μL, 4.0 mmol, 6 equiv) followed by concd

HCl (37% aq) (195 μL, 2.3 mmol, 3.5 equiv). The reaction was

heated overnight at 110 °C. There was very little conversion to the

desired product, so a mixture of hydrazine hydrate (400 μL, 8.2

mmol, 12 equiv) and concd HCl (37% aq) (400 μL, 4.6 mmol, 7

equiv) was added and the reaction was heated overnight at 140 °C in

a sealed reaction vial. The reaction was determined complete by TLC

was dried over Na SO , ﬁltered, and concentrated. The residue was

2

4

puriﬁed by basic alumina chromatography (50% EtOAc/Hex →

EtOAc), and product fractions were concentrated and repuriﬁed by

silica gel chromatography (2% → 5% → 10% MeOH/DCM) to give

the title compound (E)-N,N-dimethyl-2-(2-(2-(1-(pyridin-2-yl)-

ethylidene)hydrazineyl)-1H-benzo[d]imidazol-1-yl)ethan-1-amine

(C27) (56.6 mg, free base) as a red/orange oil. This oil was dissolved

in MeOH (3 mL), acidiﬁed with HCl (2 M in Et

concentrated under reduced pressure. The concentrate was recrystal-

lized from MeOH/Et O, ﬁltered, and dried under vacuum to give (E)-

O) (1 mL), and

2

(

5% MeOH/DCM), and the reaction was partitioned in DCM/H O

2

and extracted 3× with DCM. The combined organic was dried over

N,N-dimethyl-2-(2-(2-(1-(pyridin-2-yl)ethylidene)hydrazineyl)-1H-

benzo[d]imidazol-1-yl)ethan-1-amine HCl (C27) (52 mg, 0.13

mmol, 100% purity by HPLC) as an orange solid. H NMR (HCl

Na SO , ﬁltered, and concentrated. The residue was recrystallized

2

4

1

from EtOAc to aﬀord the title compound 2-hydrazineyl-6-(2-

methoxyethoxy)benzo[d]thiazole (9) (77.7 mg, 0.32 mmol) as a

light brown solid.

salt) (500 MHz, DMSO-d ): δ 13.24 (s, 1H), 10.28 (s, 1H), 8.78 (dd,

6

J = 6.0, 1.6 Hz, 1H), 8.52 (t, J = 7.9 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H),

7.82 (t, J = 6.6 Hz, 1H), 7.56−7.49 (m, 1H), 7.30 (dt, J = 7.6, 3.1 Hz,

1H), 7.21−7.13 (m, 2H), 4.63−4.51 (m, 2H), 3.54 (q, J = 5.7 Hz,

(

E)-6-(2-Methoxyethoxy)-2-(2-(1-(pyridin-2-yl)ethylidene)-

hydrazineyl)benzo[d]thiazole AcOH (C23). 2-Hydrazineyl-6-(2-

methoxyethoxy)benzo[d]thiazole (9) (60 mg, 0.25 mmol, 1 equiv)

and 1-(pyridin-2-yl)ethan-1-one (28 μL, 0.25 mmol, 1 equiv) were

dissolved in EtOH (5 mL) followed by addition of AcOH (4 drops).

The reaction was determined complete by LC−MS and concentrated

under reduced pressure. The resultant solid was puriﬁed by silica gel

chromatography (25% → 50% EtOAc/Hex → EtOAc) followed by

recrystallization of product fractions from EtOH to give the title

compound (E)-6-(2-methoxyethoxy)-2-(2-(1-(pyridin-2-yl)-

ethylidene)hydrazineyl)benzo[d]thiazole AcOH (C23) (44.7 mg,

+

2H), 2.91 (d, J = 3.8 Hz, 6H), 2.42 (s, 3H). MS: 323.1 [M + H] .

+

HRMS (MALDI) (m/z): [M + H ] calcd for C H N + H ,

18

22

6

323.19850; found, 323.19750.

4-(2-(Dimethylamino)ethoxy)picolinonitrile (12). 2-

(Dimethylamino)ethan-1-ol (289 μL, 2.89 mmol, 1 equiv) was

added dropwise to a mixture of NaH (60% dispersion) (138 mg, 3.46

mmol, 1.2 equiv) in DMF (12 mL) at 0 °C. The solution was allowed

to warm to room temperature, and 4-chloropicolinonitrile (400 mg,

2.89 mmol, 1 equiv) was added. The reaction was stirred overnight at

room temperature and poured over water. The resultant mixture was

diluted in brine and extracted 3× with EtOAc. The combined

0

.11 mmol, 99.3% purity by HPLC) as a white solid after drying the

1

solid under high vacuum. H NMR (acetate salt) (400 MHz,

chloroform-d): δ 8.68−8.56 (m, 1H), 8.15 (d, J = 8.3 Hz, 1H), 7.72

organics were washed with brine, dried over Na SO , ﬁltered, and

2 4

(

td, J = 7.7, 1.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.27−7.22 (m,

concentrated. The crude product was puriﬁed by silica gel

chromatography (50% EtOAc/Hex → EtOAc → (5% MeOH, 2%

TEA)/DCM) to give 4-(2-(dimethylamino)ethoxy)picolinonitrile

(12) (295 mg, 1.54 mmol, 53% yield) as a light yellow oil.

2

H), 6.99 (dd, J = 8.8, 2.5 Hz, 1H), 4.26−4.11 (m, 2H), 3.88−3.71

(m, 2H), 3.47 (s, 3H), 2.48 (s, 3H), 2.14(s, 3H). MS: 342.95 [M +

+

H] .

2

-(2-Chloro-1H-benzo[d]imidazol-1-yl)-N,N-dimethylethan-1-

1-(4-(2-(Dimethylamino)ethoxy)pyridin-2-yl)ethan-1-one (13).

4-(2-(Dimethylamino)ethoxy)picolinonitrile (12) (295 mg, 1.54

mmol, 1 equiv) was dissolved in THF (3 mL) and cooled to 0 °C

under N . MeMgI (3 M in Et O) (770 μL, 2.32 mmol, 1.5 equiv) was

amine (10). To a solution of 2-chloro-1H-benzo[d]imidazole (500

mg, 3.3 mmol, 1 equiv) in DMF (7 mL) at 0 °C was added NaH

(

60% dispersion) (394 mg, 9.8 mmol, 3 equiv). After stirring for 10

min, 2-chloro-N,N-dimethylethan-1-amine HCl (709 mg, 4.92 mmol,

equiv) was added and the reaction was allowed to stir overnight as it

2

added dropwise, and the reaction was continuously stirred at 0 °C

until complete by LC−MS. Water (5 mL) was added to quench the

reaction followed by acidiﬁcation to pH = 1 with 1 M HCl(aq). The

mixture was stirred for 20 min, and the aqueous was subsequently

washed with EtOAc to remove nonbasic impurities. The aqueous was

basiﬁed to pH = 10 with 1 M NaOH and extracted 3× with EtOAc.

The combined organics were dried over Na SO , ﬁltered, and

1

slowly warmed to room temperature. The reaction proceeded to

roughly 60% completion by LC−MS but was quenched with water

and partitioned in EtOAc/H O. The organic was washed 2× with

2

water and 1× with brine, dried over Na SO , ﬁltered, and

2

4

concentrated. The residue was puriﬁed by silica gel chromatography

2

4

(

EtOAc → 5% MeOH/DCM), and product containing fractions were

concentrated to give 1-(4-(2-(dimethylamino)ethoxy)pyridin-2-yl)-

ethan-1-one (13) (288 mg) as a yellow oil that was carried on crude

without further puriﬁcation.

concentrated to give 2-(2-chloro-1H-benzo[d]imidazol-1-yl)-N,N-

dimethylethan-1-amine (10) (300 mg, 70% purity by LC−MS),

which was used without further puriﬁcation in the subsequent

(E)-2-((2-(1-(2-(Benzo[d]thiazol-2-yl)hydrazineylidene)ethyl)-

pyridin-4-yl)oxy)-N,N-dimethylethan-1-amine HCl (C28). To a

solution of 1-(4-(2-(dimethylamino)ethoxy)pyridin-2-yl)ethan-1-one

(13) (120 mg, 0.576 mmol, 1 equiv) and 2-hydrazinylbenzo[d]-

thiazole (105 mg, 0.634 mmol, 1.1 equiv) in EtOH (5 mL) was added

AcOH (3 drops). The reaction was stirred overnight at room

temperature, determined complete by TLC, and concentrated under

reduced pressure. Puriﬁcation by silica gel chromatography (EtOAc

→ (5% MeOH, 2% TEA)/DCM) gave the free base of C28 as a

yellow oil. The yellow oil was dissolved in MeOH and acidiﬁed with

+

reaction. MS: 223.85 [M + H] .

2

-(2-Hydrazineyl-1H-benzo[d]imidazol-1-yl)-N,N-dimethyle-

than-1-amine (11). A mixture of 2-(2-chloro-1H-benzo[d]imidazol-

-yl)-N,N-dimethylethan-1-amine (10) (300 mg, 70% purity by LC−

1

MS) in i-PrOH (2 mL) and hydrazine hydrate (2 mL) was heated

overnight at 140 °C in a sealed reaction vial. The reaction was

determined complete by LC−MS and concentrated under reduced

pressure. The crude product was redissolved in i-PrOH and

reconcentrated repeatedly until there was conﬁdence that a majority

of the excess hydrazine hydrate was removed. The title compound 2-

HCl (2 M in Et

recrystallized in MeOH/Et

2

O) (excess). The solution was concentrated and

2

(

2-hydrazineyl-1H-benzo[d]imidazol-1-yl)-N,N-dimethylethan-1-

O, and subsequently, the solid was ﬁltered

amine (11) was used as is in the next reaction, assuming quantitative

yield. MS: 219.90 [M + H] .

and dried under vacuum to give the title compound (E)-2-((2-(1-(2-

(benzo[d]thiazol-2-yl)hydrazineylidene)ethyl)pyridin-4-yl)oxy)-N,N-

dimethylethan-1-amine HCl (C28) (58.2 mg, 0.136 mmol, 98%

+

(

E)-N,N-Dimethyl-2-(2-(2-(1-(pyridin-2-yl)ethylidene)-

1

hydrazineyl)-1H-benzo[d]imidazol-1-yl)ethan-1-amine HCl (C27).

To a solution of 2-(2-hydrazineyl-1H-benzo[d]imidazol-1-yl)-N,N-

purity by HPLC) as a light yellow solid. H NMR (HCl salt) (500

MHz, methanol-d ): δ 8.69 (d, J = 6.9 Hz, 1H), 7.82 (d, J = 2.6 Hz,

4

2

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1

8

4

3

H), 7.71−7.65 (m, 1H), 7.55 (dd, J = 6.9, 2.6 Hz, 1H), 7.46 (d, J =

.1 Hz, 1H), 7.43−7.37 (m, 1H), 7.24 (td, J = 7.6, 1.2 Hz, 1H), 4.84−

.83 (m, 2H), 3.82−3.70 (m, 2H), 3.06 (s, 6H), 2.53 (s, 3H). MS:

was recrystallized from MeOH/Et O, ﬁltered, and dried under

vacuum to give (E)-4-(2-(2-(2-(1-(pyridin-2-yl)ethylidene)-

hydrazineyl)-1H-benzo[d]imidazol-1-yl)ethyl)morpholine HCl

2

+

56.1 [M + H] . HRMS (MALDI) (m/z): [M + H ] calcd for

(C42) (1.78 g, 4.07 mmol, 100% purity by HPLC) as a light orange

+

1

C H N OS + H , 356.15450; found, 356.15400.

solid. H NMR (HCl salt) (400 MHz, methanol-d ): δ 8.79 (ddd, J =

18

21

5

4

(

E)-2-((2-(1-(2-(Benzo[d]oxazol-2-yl)hydrazineylidene)ethyl)-

5.9, 1.6, 0.7 Hz, 1H), 8.61 (ddd, J = 8.2, 7.7, 1.6 Hz, 1H), 8.32−8.27

(m, 1H), 7.94 (ddd, J = 7.6, 5.8, 1.1 Hz, 1H), 7.55 (dd, J = 5.9, 3.2

Hz, 1H), 7.48−7.42 (m, 1H), 7.28 (dd, J = 5.9, 3.1 Hz, 2H), 4.81 (t, J

= 6.4 Hz, 2H), 3.97 (s, 4H), 3.76 (t, J = 6.4 Hz, 2H), 3.59 (s, 4H),

pyridin-4-yl)oxy)-N,N-dimethylethan-1-amine HCl (C35). To a

solution of 1-(4-(2-(dimethylamino)ethoxy)pyridin-2-yl)ethan-1-one

13) (60 mg, 0.29 mmol, 1 equiv) and 2-hydrazinylbenzo[d]oxazole

45 mg, 0.32 mmol, 1.1 equiv) in MeOH (3 mL) was added AcOH (3

drops). Puriﬁcation by silica gel chromatography (5% MeOH/DCM

(5% MeOH, 5% TEA)/DCM) gave the free base of C35 as a

yellow oil. The yellow oil was dissolved in MeOH and acidiﬁed with

HCl (2 M in Et O) (excess). The solution was concentrated and

recrystallized in MeOH/Et O, and subsequently, the solid was ﬁltered

and dried under vacuum to give the title compound (E)-2-((2-(1-(2-

benzo[d]oxazol-2-yl)hydrazineylidene)ethyl)pyridin-4-yl)oxy)-N,N-

dimethylethan-1-amine HCl (C35) (31 mg, 0.075 mmol, 100% purity

by HPLC) as a bright yellow solid. H NMR (HCl salt) (500 MHz,

methanol-d ): δ 8.75 (d, J = 6.8 Hz, 1H), 7.84 (d, J = 2.6 Hz, 1H),

(

+

2.57 (s, 3H). MS: 365.15 [M + H] . HRMS (MALDI) (m/z): [M +

+

H ] calcd for C H N O + H , 365.20880; found, 365.20840.

20 24 6

→

4-(2-((Tetrahydro-2H-pyran-2-yl)oxy)ethoxy)picolinonitrile (16).

To a solution of NaH (60% dispersion) (1.04 g, 26 mmol, 1.2 equiv)

in DMF (50 mL) at 0 °C was added 2-((tetrahydro-2H-pyran-2-

yl)oxy)ethan-1-ol (3.16 g, 21.7 mmol, 1 equiv). The solution was

allowed to warm to room temperature and stirred for 1 h. 4-

Chloropicolinonitrile (3.0 g, 21.7 mmol, 1 equiv) was added, and the

reaction was allowed to stir overnight at room temperature. The

resultant mixture was diluted in brine and extracted 3× with EtOAc.

The combined organics were washed 1× with water and 3× with

brine, dried over Na SO , ﬁltered, and concentrated. The crude

2

(

1

4

7

1

3

.56 (dd, J = 6.8, 2.6 Hz, 1H), 7.51−7.44 (m, 2H), 7.34 (td, J = 7.7,

2

4

.1 Hz, 1H), 7.26 (td, J = 7.8, 1.3 Hz, 1H), 4.85−4.82 (m, 2H), 3.82−

product was puriﬁed by silica gel chromatography (35% → 65%

EtOAc/Hex) to give 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-

picolinonitrile (16) (3.33 g, 13.4 mmol, 62% yield) as a yellow oil.

1-(4-(2-Hydroxyethoxy)pyridin-2-yl)ethan-1-one (17). 4-(2-

((Tetrahydro-2H-pyran-2-yl)oxy)ethoxy)picolinonitrile (16) (248

mg, 1.0 mmol, 1 equiv) was dissolved in THF (2 mL) and cooled

to 0 °C under N . MeMgI (3 M in Et O) (500 μL, 1.5 mmol, 1.5

+

.74 (m, 2H), 3.06 (s, 6H), 2.50 (s, 3H). MS: 339.8 [M + H] .

+

HRMS (MALDI) (m/z): [2M + H ] calcd for (C H N O ) + H ,

1

8

21

5

2 2

6

79.34390; found, 679.34630.

-(2-(2-Chloro-1H-benzo[d]imidazol-1-yl)ethyl)morpholine (14).

To a solution of 2-chloro-1H-benzo[d]imidazole (2.47 g, 16.2 mmol,

equiv) in DMF (35 mL) at 0 °C was added NaH (60% dispersion)

1.94 g, 24.3 mmol, 3 equiv) in multiple portions. After stirring for 10

4

1

(

2

equiv) was added dropwise, and the reaction was allowed to warm to

room temperature. After 3 h, the reaction was determined complete

by LC−MS, quenched with water (5 mL), and acidiﬁed to pH = 1

with 1 M HCl(aq). The mixture was stirred for 2 h to give complete

deprotection of the THP protecting group in situ. The mixture was

basiﬁed to pH = 7 with 1 M NaOH and extracted 3× with EtOAc.

The combined organics were dried over Na SO , ﬁltered, and

min at 0 °C, 4-(2-chloroethyl)morpholine (3.5 g, 24.3 mmol, 1.5

equiv) was added and the reaction was allowed to stir overnight as it

slowly warmed to room temperature. The reaction proceeded to

roughly 80% completion by LC−MS but was quenched with water

and partitioned in EtOAc/H O. The organic was washed 2× with

2

water and 1× with brine, dried over Na SO , ﬁltered, and

2

4

2

4

concentrated. The residue was puriﬁed by silica gel chromatography

concentrated to give 1-(4-(2-hydroxyethoxy)pyridin-2-yl)ethan-1-one

(17) (154 mg) as an oil that was carried on crude without further

puriﬁcation.

(

50% EtOAc/Hex → EtOAc), and product containing fractions were

concentrated to give 4-(2-(2-chloro-1H-benzo[d]imidazol-1-yl)-

ethyl)morpholine (14) (3.57 g, 13.4 mmol) as a viscous pink oil

that was dried under high vacuum and used without further

(E)-2-((2-(1-(2-(Benzo[d]thiazol-2-yl)hydrazineylidene)ethyl)-

pyridin-4-yl)oxy)ethan-1-ol (C61). To a solution of 1-(4-(2-

hydroxyethoxy)pyridin-2-yl)ethan-1-one (17) (150 mg, 0.83 mmol,

1 equiv) and 2-hydrazinylbenzo[d]thiazole (137 mg, 0.83 mmol, 1

equiv) in DCM (2 mL) and MeOH (2 mL) was added AcOH (4

drops). The reaction was stirred overnight, determined complete by

LC−MS, and concentrated under reduced pressure. The residual solid

was recrystallized from MeOH to give the title compound (E)-2-((2-

(1-(2-(benzo[d]thiazol-2-yl)hydrazineylidene)ethyl)pyridin-4-yl)-

+

puriﬁcation in the subsequent reaction. MS: 265.9 [M + H] .

4

-(2-(2-Hydrazineyl-1H-benzo[d]imidazol-1-yl)ethyl)morpholine

(

15). To a solution of 4-(2-(2-chloro-1H-benzo[d]imidazol-1-yl)-

ethyl)morpholine (14) (3.5 g, 13.2 mmol) in ethylene glycol (10 mL)

was added hydrazine hydrate (2 mL). The reaction was heated for 2 h

at 140 °C and was determined complete by TLC (5% MeOH/DCM).

The reaction was concentrated under reduced pressure, redissolved in

i-PrOH, and reconcentrated 4× to remove excess hydrazine. The title

compound 4-(2-(2-hydrazineyl-1H-benzo[d]imidazol-1-yl)ethyl)-

morpholine (15) in 10 mL ethylene glycol was used as is in the

oxy)ethan-1-ol (C61) (71 mg, 0.22 mmol, 99.1% purity by HPLC) as

1

a white solid. H NMR (500 MHz, methanol-d ): δ 8.38 (d, J = 5.8

4

Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.72 (s, 1H), 7.54 (s, 1H), 7.39−

7.29 (br. m, 1H), 7.17 (br. m, 1H), 7.01 (dd, J = 5.8, 2.6 Hz, 1H),

4.29−4.22 (m, 2H), 4.01−3.94 (m, 2H), 2.45 (s, 3H). MS: 328.9 [M

+

next reaction, assuming quantitative yield. MS: 261.9 [M + H] .

(

E)-4-(2-(2-(2-(1-(Pyridin-2-yl)ethylidene)hydrazineyl)-1H-benzo-

+

[

(

d]imidazol-1-yl)ethyl)morpholine HCl (C42). To a solution of 4-(2-

+ H] .

2-hydrazineyl-1H-benzo[d]imidazol-1-yl)ethyl)morpholine (15)

crude from the previous reaction) in EtOH (10 mL) and ethylene

glycol (10 mL, remaining from the previous reaction) was added 1-

pyridin-2-yl)ethan-1-one (2 mL, 17.6 mmol, 1.33 equiv) followed by

AcOH (20 drops). The reaction was stirred overnight at room

temperature, determined complete by LC−MS, and concentrated

under reduced pressure. The crude reaction was partitioned in DCM/

NaHCO (sat, aq), and the aqueous was extracted 3× with DCM. The

combined organic was dried over Na SO , ﬁltered, and concentrated.

2-((2-Acetylpyridin-4-yl)oxy)ethyl Methanesulfonate (18). To a

solution of 1-(4-(2-hydroxyethoxy)pyridin-2-yl)ethan-1-one (17)

(933 mg, 5.15 mmol, 1 equiv) in DCM (20 mL) was added TEA

(1.08 mL, 7.7 mmol, 1.5 equiv) followed by methanesulfonyl chloride

(dropwise) (600 μL, 7.7 mmol, 1.5 equiv). After stirring for 2 h at

(

room temperature, the reaction was quenched with NaHCO (sat, aq)

3

and partitioned in EtOAc/H O. The aqueous was extracted 3× with

2

3

EtOAc, and the combined organics were washed 1× with brine, dried

2

4

over Na SO , ﬁltered, and concentrated to give 2-((2-acetylpyridin-4-

2

4

The residue was puriﬁed by basic alumina chromatography (50%

EtOAc/Hex → EtOAc), and product fractions were concentrated and

repuriﬁed by silica gel chromatography (EtOAc → 2% MeOH/

EtOAc) to give the title compound (E)-4-(2-(2-(2-(1-(pyridin-2-

yl)ethylidene)hydrazineyl)-1H-benzo[d]imidazol-1-yl)ethyl)-

morpholine (C42) (1.51 g, free base) as an orange foam. This foam

yl)oxy)ethyl methanesulfonate (18) (1.16 g) as a brown oil that

solidiﬁed on standing. This product was used without further

puriﬁcation in subsequent reactions.

1-(4-(2-(4-Methylpiperazin-1-yl)ethoxy)pyridin-2-yl)ethan-1-one

(19). To a solution of 2-((2-acetylpyridin-4-yl)oxy)ethyl methanesul-

fonate (18) (200 mg, 0.77 mmol, 1 equiv) in MeCN (15 mL) was

added 1-methylpiperazine (171 μL, 1.54 mmol, 2 equiv) followed by

KI (128 mg, 0.77 mmol, 1 equiv) and K CO (213 mg, 1.54 mmol, 2

was dissolved in MeOH (20 mL), acidiﬁed with HCl (2 M in Et O)

2

(7 mL), and concentrated under reduced pressure. The concentrate

2

3

2

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equiv). After heating for 6 h at 80 °C, the reaction was determined

complete by LC−MS, cooled to room temperature, and poured into

water. The resulting mixture was extracted 3× with EtOAc, and the

combined organics were dried over Na SO , ﬁltered, and concen-

trated to give 1-(4-(2-(4-methylpiperazin-1-yl)ethoxy)pyridin-2-yl)-

ethan-1-one (19) (145 mg, 0.55 mmol) as a pale orange oil that was

used crude without further puriﬁcation.

Di-tert-butyl 1-(1-(2-(Pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazol-

2-yl)hydrazine-1,2-dicarboxylate (22). To a solution of 1-(2-

(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole (21) (1.70 g, 7.9

mmol, 1 equiv) in THF (25 mL) at −78 °C was added n-BuLi

(2.5 M in hexanes) (3.5 mL, 8.7 mmol, 1.1 equiv) dropwise over 10

min. After stirring for 10 min at −78 °C, a solution of di-tert-butyl-

azodicarboxylate (2.0 g, 8.7 mmol, 1.1 equiv) in THF (7 mL) was

added dropwise over 10 min. After stirring for an additional 10 min at

−78 °C, the reaction was allowed to warm to room temperature and

was stirred for an additional 30 min. The reaction was observed as

approximately 90% complete by LC−MS and was quenched with

2

4

(

E)-2-(2-(1-(4-(2-(4-Methylpiperazin-1-yl)ethoxy)pyridin-2-yl)-

ethylidene)hydrazineyl)benzo[d]thiazole HCl (C64). To a solution

of 1-(4-(2-(4-methylpiperazin-1-yl)ethoxy)pyridin-2-yl)ethan-1-one

(19) (145 mg, 0.55 mmol, 1 equiv) in DCM (2 mL) and MeOH

(2 mL) was added 2-hydrazinylbenzo[d]thiazole (91 mg, 0.55 mmol,

H

2

O. The mixture was partitioned in EtOAc/H

2

O, and the organic

SO

1

equiv). AcOH (4 drops) was added, and the reaction was stirred

was washed 2× with H O and 1× with brine, dried over Na

2

,

4

overnight at room temperature. The reaction was determined

complete by LC−MS and concentrated under reduced pressure.

The product was redissolved in MeOH and neutralized with TEA (0.1

mL) in order to generate the free base for column chromatography.

After concentration, the product was puriﬁed by silica gel

chromatography (2% MeOH/DCM → (5% MeOH, 2% TEA)/

DCM)). Product containing fractions were concentrated under

reduced pressure. The residue was redissolved in MeOH and

ﬁltered, and concentrated. The residue was puriﬁed by silica gel

chromatography (50% EtOAc/Hex → EtOAc), and product

containing fractions were concentrated to give di-tert-butyl 1-(1-(2-

(

pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazol-2-yl)hydrazine-1,2-di-

carboxylate (22) (2.35 g, 5.3 mmol) as an orange solid. MS: 446.15

+

[

M + H] .

E)-2-(2-(1-(Pyridin-2-yl)ethylidene)hydrazineyl)-1-(2-(pyrrolidin-

-yl)ethyl)-1H-benzo[d]imidazole HCl (C69). To a solution of di-tert-

(

1

butyl 1-(1-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazol-2-yl)-

hydrazine-1,2-dicarboxylate (22) in MeOH (30 mL) was added 1-

converted to the HCl salt with the addition of 2 M HCl/Et O (1

2

mL). The mixture was concentrated and recrystallized from MeOH to

aﬀord the title compound (E)-2-(2-(1-(4-(2-(4-methylpiperazin-1-

yl)ethoxy)pyridin-2-yl)ethylidene)hydrazineyl)benzo[d]thiazole HCl

(pyridin-2-yl)ethan-1-one (591 μL, 5.27 mmol, 1 equiv). HCl (4 M in

dioxane) (8 mL) was added, and the reaction was stirred overnight at

room temperature. The reaction was determined complete by LC−

MS, and the precipitated solid was ﬁltered. The ﬁltrate was

concentrated and recrystallized from i-PrOH to collect a second

crop of solid. The combined solids were recrystallized from MeOH/

EtOAc, sonicated, and ﬁltered. The solid was dried under high

vacuum to aﬀord (E)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazineyl)-

(

C64) (73 mg, 0.15 mmol, 98.9% purity by HPLC) as a yellow solid.

1

H NMR (500 MHz, deuterium oxide): δ 8.41 (d, J = 6.9 Hz, 1H),

7

.60 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 2.6 Hz, 1H), 7.38 (d, J = 7.8 Hz,

H), 7.30 (ddd, J = 8.3, 7.4, 1.2 Hz, 1H), 7.27 (dd, J = 7.0, 2.7 Hz,

H), 7.16−7.12 (m, 1H), 4.51−4.47 (m, 2H), 3.37 (br. s, 4H), 3.27−

1

3

.23 (m, 2H), 3.13 (br. s, 4H), 2.84 (s, 3H), 2.28 (s, 3H). MS: 411.1

1

-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole HCl (C69) (1.35

+

[

M + H] .

1

g, 3.2 mmol, 98.4% purity by HPLC) as a light orange solid. H NMR

2

-Bromo-1-(pyridin-2-yl)ethan-1-one HBr (20). 1-(Pyridin-2-yl)-

(

500 MHz, methanol-d ): δ 8.80 (ddd, J = 5.8, 1.6, 0.6 Hz, 1H), 8.62

ddd, J = 8.3, 7.7, 1.6 Hz, 1H), 8.31 (d, J = 8.3 Hz, 1H), 7.95 (ddd, J

4

ethan-1-one (930 μL, 8.25 mmol, 1 equiv) was dissolved in a mixture

of HBr (33%, aq) (1.4 mL) and AcOH (3.7 mL). A pyridinium salt

quickly precipitated from solution. To this mixture under vigorous

=

7.5, 5.8, 1.1 Hz, 1H), 7.58−7.53 (m, 1H), 7.49−7.44 (m, 1H),

7

.34−7.28 (m, 2H), 4.75 (t, J = 6.3 Hz, 2H), 3.89 (s, 2H), 3.81 (t, J =

stirring was added a mixture of Br (0.42 mL, 8.25 mmol, 1 equiv) in

2

6.3 Hz, 2H), 3.27 (s, 2H), 2.60 (s, 3H), 2.21 (s, 2H), 2.04 (s, 2H).

AcOH (0.92 mL). After stirring overnight at room temperature, the

+

MS: 348.95 [M + H] . HRMS (MALDI) (m/z): [M + H ] calcd for

C H N + H , 349.21410; found, 349.21350.

reaction was diluted in Et O and the precipitated solid was ﬁltered

+

2

0

24

6

and washed with additional Et O. Drying the solid under high vacuum

2

(

E)-2-(2-(1-(Pyrimidin-2-yl)ethylidene)hydrazineyl)benzo[d]-

yielded 2-bromo-1-(pyridin-2-yl)ethan-1-one (20) (2.26 g, HBr salt,

thiazole AcOH (C77). To a solution of 1-(pyrimidin-2-yl)ethan-1-one

8

.1 mmol) as a white solid.

(74 mg, 0.61 mmol, 1 equiv) and 2-hydrazinylbenzo[d]thiazole (100

(

Z)-2-(2-(2-Methoxy-1-(pyridin-2-yl)ethylidene)hydrazineyl)-

mg, 0.61 mmol, 1 equiv) in MeOH (5 mL) was added AcOH (5

drops). The reaction was stirred overnight at room temperature, and

the precipitated solid was ﬁltered, washed with MeOH, and dried

under high vacuum to aﬀord (E)-2-(2-(1-(pyrimidin-2-yl)ethylidene)-

benzo[d]thiazole (C65). To a mixture of 2-bromo-1-(pyridin-2-

yl)ethan-1-one HBr (20) (281 mg, 1 mmol, 1 equiv) and 2-

hydrazinylbenzo[d]thiazole (165 mg, 1 mmol, 1 equiv) in DCM (4

mL) and MeOH (2 mL) was added AcOH (4 drops). The reaction

was stirred overnight at room temperature and determined complete

by LC−MS. The orange precipitate was ﬁltered, subsequently

recrystallized from MeOH, ﬁltered, washed with MeOH, and dried

under vacuum to give (Z)-2-(2-(2-methoxy-1-(pyridin-2-yl)-

hydrazineyl)benzo[d]thiazole AcOH (C77) (47.6 mg, 0.15 mmol,

1

9

8.9% purity by HPLC) as a light brown solid. H NMR (acetate salt)

(

500 MHz, DMSO-d ): δ 11.92 (s, 2H), 8.89 (d, J = 4.8 Hz, 2H),

.77 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 4.8 Hz, 1H), 7.44 (d, J = 7.8 Hz,

6

7

1

3

H), 7.36−7.27 (m, 1H), 7.17−7.10 (m, 1H), 2.45 (s, 3H), 1.91 (s,

ethylidene)hydrazineyl)benzo[d]thiazole (C65) (47 mg, 0.16 mmol,

+

H, acetate CH ). MS: 330.0 [M + H] . HRMS (MALDI) (m/z):

3

1

9

7.1% purity by HPLC) as an orange solid. H NMR (400 MHz,

+

[

2M + H ] calcd for (C H N S) + H , 539.15310; found,

13 11 5 2

methanol-d ): δ 8.77 (ddd, J = 5.9, 1.6, 0.7 Hz, 1H), 8.60 (ddd, J =

4

539.15430.

8.3, 7.6, 1.6 Hz, 1H), 8.45 (ddd, J = 8.3, 1.2, 0.8 Hz, 1H), 7.97 (ddd, J

Di-tert-butyl 1-(1-Methyl-1H-imidazol-2-yl)hydrazine-1,2-dicar-

boxylate (23). To a solution of 1-methyl-1H-imidazole (1) (1.0 g,

12.2 mmol, 1 equiv) in THF (10 mL) at −78 °C was added n-BuLi

(2.5 M in hexanes, 5.4 mL, 13.4 mmol, 1.1 equiv). After stirring for 10

min at −78 °C, a solution of di-tert-butyl-azodicarboxylate (3.09 g,

13.4 mmol, 1.1 equiv) in THF (5 mL) was added dropwise over 5

min. The reaction was allowed to stir for 30 min at −78 °C before

being warmed to room temperature and quenched with water. The

reaction was diluted in EtOAc, washed 1× with water and 1× with

brine, dried over Na SO , ﬁltered, and concentrated. The crude

=

7.5, 5.9, 1.3 Hz, 1H), 7.70−7.62 (m, 1H), 7.43−7.35 (m, 2H), 7.24

(ddd, J = 7.9, 6.8, 1.7 Hz, 1H), 4.96 (s, 2H), 3.47 (s, 3H). MS: 298.85

+

[

M + H] .

1

-(2-(Pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole (21). To a

solution of 1H-benzo[d]imidazole (3.0 g, 25.4 mmol, 1 equiv) in

DMF (30 mL) was added 1-(2-chloroethyl)pyrrolidine HCl (6.48 g,

3

8.1 mmol, 1.5 equiv) followed by K CO (10.5 g, 76.2 mmol, 3

2 3

equiv). After stirring for 2 h at 60 °C, the reaction was determined

complete by TLC (10% MeOH/DCM). The reaction was partitioned

in EtOAc/H O, and the organic was washed 3× H O and 1× brine,

2

4

2

mixture was puriﬁed by silica gel chromatography, eluting in 50% →

75% EtOAc/Hex. Product containing fractions were combined and

concentrated to aﬀord the title compound di-tert-butyl 1-(1-methyl-

1H-imidazol-2-yl)hydrazine-1,2-dicarboxylate (23) (1.54 g, 40%

dried over Na SO , ﬁltered, and concentrated. Silica gel chromatog-

2

4

raphy (5% → 10% MeOH/DCM) yielded 1-(2-(pyrrolidin-1-

yl)ethyl)-1H-benzo[d]imidazole (21) (2.94 g, 13.7 mmol) after

+

concentration of product fractions. MS: 215.95 [M + H] .

yield) as a white crystalline solid. MS: 313.1 [M + H] .

2

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2

-(1-(2-(1-Methyl-1H-imidazol-2-yl)hydrazono)ethyl)pyridine

(1 mL). After stirring for 1 h at 60 °C, the reaction was determined

complete by LC−MS and cooled to room temperature. The

precipitated solid was ﬁltered, washed with MeOH, and dried under

vacuum to give (E)-1-(2-(3,3-diﬂuoropyrrolidin-1-yl)ethyl)-2-(2-(1-

(pyridin-2-yl)ethylidene)hydrazineyl)-1H-benzo[d]imidazole HCl

HCl (C78). Di-tert-butyl 1-(1-methyl-1H-imidazol-2-yl)hydrazine-1,2-

dicarboxylate (23) (800 mg, 2.56 mmol, 1 equiv) and 1-(pyridin-2-

yl)ethan-1-one (287 μL, 2.56 mmol, 1 equiv) were dissolved in

MeOH (15 mL). Following addition of 4 N HCl/dioxane (4.0 mL,

1

6.0 mmol, 6.25 equiv), the reaction was allowed to stir for 16 h at

(C85) (137 mg, 0.30 mmol, 99.2% purity by HPLC) as a light

1

room temperature. The crude reaction mixture was concentrated and

recrystallized from a mixture of MeOH and EtOAc to give the title

compound 2-(1-(2-(1-methyl-1H-imidazol-2-yl)hydrazono)ethyl)-

pyridine HCl (C78) (571 mg, 2.27 mmol, 100% purity by HPLC)

as a yellow solid. H NMR (400 MHz, methanol-d ): δ 8.91 (ddd, J =

.8, 1.6, 0.7 Hz, 1H), 8.69 (td, J = 8.0, 1.6 Hz, 1H), 8.47 (dt, J = 8.2,

.9 Hz, 1H), 8.08 (ddd, J = 7.7, 5.7, 1.2 Hz, 1H), 7.28 (d, J = 2.3 Hz,

H), 7.23 (d, J = 2.3 Hz, 1H), 3.82 (s, 3H), 2.62 (s, 3H). MS: 216.15

orange solid. H NMR (400 MHz, methanol-d ): δ 8.76 (ddd, J = 5.8,

4

1.6, 0.7 Hz, 1H), 8.60 (ddd, J = 8.3, 7.7, 1.6 Hz, 1H), 8.28 (dt, J = 8.3,

1.0 Hz, 1H), 7.91 (ddd, J = 7.6, 5.9, 1.2 Hz, 1H), 7.53−7.45 (m, 1H),

7.45−7.37 (m, 1H), 7.28−7.22 (m, 2H), 4.71 (t, J = 6.0 Hz, 2H),

4.27 (t, J = 11.6 Hz, 2H), 3.97−3.87 (m, 4H), 2.79−2.64 (m, 2H),

1

4

+

5

0

1

[

(

2.56 (s, 3H). MS: 384.80 [M + H] . HRMS (MALDI) (m/z): [M +

+

H ] calcd for C H F N + H , 385.19520; found, 385.19470.

2

0

22

2

6

Di-tert-butyl 1-(1-Methyl-1H-benzo[d]imidazol-2-yl)hydrazine-

1,2-dicarboxylate (27). To a solution of 1-methyl-1H-benzo[d]-

imidazole (1.54 g, 11.6 mmol, 1 equiv) in THF (35 mL) at −78 °C

was added n-BuLi (2.5 M in hexanes) (5.1 mL, 12.8 mmol, 1.1 equiv).

After stirring for 15 min at −78 °C, a solution of di-tert-butyl-

azodicarboxylate (2.95 g, 12.8 mmol, 1 equiv) in THF (11 mL) was

added and the reaction was allowed to stir overnight while slowly

warming to room temperature. The reaction was determined

complete by LC−MS and poured into water. The resulting solution

was extracted 3× with EtOAc, and the combined organics were dried

over Na SO , ﬁltered, and concentrated. The brown residual oil was

+

M + H] . HRMS (MALDI) (m/z): [2M + H ] calcd for

+

C H N ) + H , 431.24180; found, 431.24150.

11

13

5 2

2

-(1H-Benzo[d]imidazol-1-yl)-1-(3,3-diﬂuoropyrrolidin-1-yl)-

ethan-1-one (24). A mixture of 1H-benzo[d]imidazole (514 mg, 4.35

mmol, 1 equiv), 2-chloro-1-(3,3-diﬂuoropyrrolidin-1-yl)ethan-1-one

(

800 mg, 4.35 mmol, 1 equiv), and K CO (1.2 g, 8.7 mmol, 2 equiv)

in DMF (15 mL) was stirred overnight at 60 °C. The reaction was

determined complete by LC−MS and partitioned in EtOAc/H O.

The aqueous was extracted 2× with EtOAc, and the combined

organic was dried over Na SO , ﬁltered, and concentrated. The

2 3

2

4

2

4

residue was puriﬁed by silica gel chromatography (EtOAc → 2%

MeOH/EtOAc → 5% MeOH/EtOAc) to aﬀord 2-(1H-benzo[d]-

imidazol-1-yl)-1-(3,3-diﬂuoropyrrolidin-1-yl)ethan-1-one (24) (705

mg, 2.7 mmol) as a white solid after concentration. MS: 265.65 [M +

puriﬁed by silica gel chromatography (50% EtOAc/Hex) to give the

title compound di-tert-butyl 1-(1-methyl-1H-benzo[d]imidazol-2-yl)-

hydrazine-1,2-dicarboxylate (27) (1.78 g, 4.9 mmol) as an orange oil

after concentration of product fractions.

(E)-1-Methyl-2-(2-(1-(pyrimidin-2-yl)ethylidene)hydrazineyl)-1H-

benzo[d]imidazole HCl (C89). To a mixture of di-tert-butyl 1-(1-

methyl-1H-benzo[d]imidazol-2-yl)hydrazine-1,2-dicarboxylate (27)

(100 mg, 0.82 mmol, 1 equiv) and 1-(pyrimidin-2-yl)ethan-1-one

(297 mg, 0.82 mmol, 1 equiv) in MeOH (5 mL) was added HCl (4

M in dioxane) (1.25 mL, 4.9 mmol, 6 equiv). The mixture was stirred

overnight and determined complete by LC−MS. The precipitated

product was ﬁltered, and the collected solid was recrystallized form

+

H] .

1

-(2-(3,3-Diﬂuoropyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole

(

25). To a solution of 2-(1H-benzo[d]imidazol-1-yl)-1-(3,3-diﬂuor-

opyrrolidin-1-yl)ethan-1-one (24) (400 mg, 1.5 mmol, 1 equiv) in

THF (10 mL) was added BH ·THF (2 M in THF) (2.25 mL, 4.5

3

mmol, 3 equiv). After stirring overnight at room temperature, the

reaction was 1/3 complete by LC−MS. Additional BH ·THF (2 M in

3

THF) (5.0 mL, 10 mmol, 6.7 equiv) was added, and the reaction was

determined complete after 4 h at room temperature. MeOH (10 mL)

and HCl (4 M in dioxane) (10 mL) were added, and the reaction was

stirred overnight. The mixture was concentrated and partitioned in

MeOH/Et O. The recrystallized solid was ﬁltered and dried under

2

vacuum to yield the title compound (E)-1-methyl-2-(2-(1-(pyrimidin-

2-yl)ethylidene)hydrazineyl)-1H-benzo[d]imidazole HCl (C89)

1

EtOAc/H O. The product was partitioned into the mildly acidic

(76.8 mg, 0.25 mmol, 99.3% purity) as a yellow solid. H NMR

2

aqueous, and the organic was extracted 2× with water. The combined

(400 MHz, DMSO-d ): δ 9.08 (d, J = 5.1 Hz, 2H), 7.71 (t, J = 5.0 Hz,

6

aqueous was neutralized with NaHCO (sat, aq) and extracted 3×

1H), 7.58 (s, 1H), 7.46 (s, 1H), 7.33−7.29 (m, 2H), 3.82 (s, 3H),

3

+

with DCM. The combined organics were dried over Na SO , ﬁltered,

2.53 (s, 3H). MS: 289.1 [M + Na] . HRMS (MALDI) (m/z): [2M +

2

4

+

and concentrated. The crude product was puriﬁed by silica gel

chromatography (50% EtOAc/Hex → EtOAc), and product fractions

were concentrated to give 1-(2-(3,3-diﬂuoropyrrolidin-1-yl)ethyl)-

H ] calcd for (C H N ) + H , 533.2629; found, 533.26330.

14 14 6 2

Zn((E)-N′-(1-(pyridin-2-yl)ethylidene)azetidine-1-carbothiohy-

drazide) (Zn-1). To a suspension of (E)-N′-(1-(pyridin-2-yl)-

ethylidene)azetidine-1-carbothiohydrazide (ZMC1) (190.8 mg,

0.814 mmol, 1 equiv) in EtOH (20 mL) was added ZnCl (55.5

2

1

H-benzo[d]imidazole (25) (273 mg, 1.09 mmol) as a white

+

crystalline solid. MS: 251.70 [M + H] .

2

Di-tert-butyl 1-(1-(2-(3,3-Diﬂuoropyrrolidin-1-yl)ethyl)-1H-

benzo[d]imidazol-2-yl)hydrazine-1,2-dicarboxylate (26). To a

solution of 1-(2-(3,3-diﬂuoropyrrolidin-1-yl)ethyl)-1H-benzo[d]-

imidazole (25) (270 mg, 1.07 mmol, 1 equiv) in THF (10 mL) at

mg, 0.407 mmol, 0.5 equiv). After 5 min, TEA (0.80 mL, excess) was

added and the mixture was heated for 2 h at reﬂux under nitrogen.

Upon cooling to ambient temperature, a solid precipitate was

collected by ﬁltration and washed with EtOH followed by Et O.

2

−

78 °C was added n-BuLi (2.5 M in hexanes) (472 μL, 1.18 mmol,

.1 equiv). After 5 min at −78 °C, di-tert-butyl-azodicarboxylate (272

The solids were dried under high vacuum to give Zn((E)-N′-(1-

1

(pyridin-2-yl)ethylidene)azetidine-1-carbothiohydrazide) (Zn-1)

2

1

mg, 1.18 mmol, 1.1 equiv) in THF (2 mL) was added and the

reaction was stirred for 30 min at −78 °C before slowly warming to

room temperature and stirring overnight. The reaction was complete

by LC−MS, quenched with water, and partitioned in EtOAc/H O.

The organic was washed 1× with water and 1× with brine, dried over

Na SO , ﬁltered, and concentrated. Puriﬁcation by silica gel

(215 mg, 0.404 mmol, 99%) as a bright yellow solid. H NMR

(400 MHz, DMSO-d ): δ 2.26 (overlapping tt, J = 7.48, 7.40 Hz, 4H),

6

2.58 (s, 6H), 4.05 (m, 8H), 7.29 (dd, J = 7.28, 5.60 Hz, 2H), 7.75 (m,

4H), 7.88 (dt, J = 8.04, 1.52 Hz, 2H). Slow evaporation of

2

[Zn(ZMC1) ] from a 1:1 mixture of DCM/MeOH aﬀorded yellow

2

crystals that were suitable for X-ray crystallography. HRMS (MALDI)

2

4

+

chromatography (25% EtOAc/Hex) aﬀorded di-tert-butyl 1-(1-(2-

3,3-diﬂuoropyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazol-2-yl)-

(m/z): [M + H ] calcd for C H N S Zn + H , 531.10810; found,

22 26 8 2

(

531.10860.

Zn((E)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)benzo[d]-

thiazole) (Zn-2). To a suspension of (E)-2-(2-(1-(pyridin-2-

hydrazine-1,2-dicarboxylate (26) (267 mg, 0.56 mmol) as a white

solid after concentration. MS: 482.1 [M + H] .

+

2

(

E)-1-(2-(3,3-Diﬂuoropyrrolidin-1-yl)ethyl)-2-(2-(1-(pyridin-2-yl)-

yl)ethylidene)hydrazinyl)benzo[d]thiazole (C1) (75.0 mg, 0.279

ethylidene)hydrazineyl)-1H-benzo[d]imidazole HCl (C85). To a

solution of di-tert-butyl 1-(1-(2-(3,3-diﬂuoropyrrolidin-1-yl)ethyl)-

mmol, 1 equiv) in EtOH (7 mL) was added ZnCl (19.1 mg, 0.140

2

mmol, 0.5 equiv). After 5 min, TEA (0.279 mL, excess) was added

and the mixture was heated for 2 h at reﬂux under nitrogen. Upon

cooling to ambient temperature, a solid precipitate was collected by

ﬁltration and washed with a 1:1 EtOH/water mixture followed by

1

0

H-benzo[d]imidazol-2-yl)hydrazine-1,2-dicarboxylate (26) (150 mg,

.31 mmol, 1 equiv) and 1-(pyridin-2-yl)ethan-1-one (35 μL, 0.31

mmol, 1 equiv) in MeOH (2 mL) was added HCl (4 M in dioxane)

2

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J. Med. Chem. 2021, 64, 2024−2045

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2+

Et O. The solids were dried under high vacuum to give Zn((E)-2-(2-

2

Cu -K Measurements by Competition. Compound (6 μM)

d

(

1-(pyridin-2-yl)ethylidene)hydrazinyl)benzo[d]thiazole)₂(Zn-2)

64.6 mg, 0.108 mmol, 77%) as an orange solid. H NMR (500

and CuSO (8 μM) in 150 mM NaCl and 20 mM Tris (pH 7.2) were

4

1

−1

−7

incubated with EGTA (10 −10 M, 1.8-fold dilution series) at

room temperature in a transparent, ﬂat-bottom 96-well plate.

Absorbance spectra from 200 to 550 nm were acquired after using

a SpectraMax i3x plate reader. The spectra were blank-subtracted

using a matched dilution of EGTA in buﬀer, and the absorbance

MHz, DMSO-d ) δ 7.88 (td, J = 5.8, 5.1, 3.5 Hz, 4H), 7.82 (dd, J =

6

8

5

1

.5, 1.1 Hz, 2H), 7.45 (dd, J = 7.8, 1.2 Hz, 2H), 7.26 (ddd, J = 7.4,

.2, 1.1 Hz, 2H), 6.95 (td, J = 7.7, 7.3, 1.2 Hz, 2H), 6.75 (td, J = 7.6,

.2 Hz, 2H), 6.60 (d, J = 8.0 Hz, 2H), 2.63 (s, 6H). Slow evaporation

2

+

of [Zn(C1) ] from THF aﬀorded orange crystals that were suitable

maximum of the compound with Cu was plotted versus EGTA

concentration. These data were ﬁt to a sigmoid, and the IC₅₀was used

to calculate the K_C_uof the compound as described above. The K_C_uof

EGTA under the experimental conditions was deteremined using

WinMAXC (C. Patton, Stanford University).

2

+

for X-ray crystallography. HRMS (MALDI) (m/z): [M + H ] calcd

for C H N S Zn + H , 599.07500; found, 599.0773.

+

28

22

8 2

2

,2′-((2-Ethoxy-2-oxoethyl)azanediyl)diacetic acid (NTA-MEE).

To a suspension of nitrilotriacetic acid (1.91 g, 10 mmol, 1 equiv)

in DMF (4 mL) was added acetic anhydride (1.13 mL, 12 mmol, 1.2

equiv) and pyridine (0.1 mL). The reaction mixture was heated to 70

Oxidative Stress Detection. The ROS signal was measured

using CellROX Green reagent (Thermo Fisher Scientiﬁc, Carlsbad,

CA) following manufacturer’s protocol. Brieﬂy, H1299 cells were

°C and stirred for 18 h. The reaction vessel was equipped with a

short-path distillation apparatus, and the volatiles were removed by

distillation. EtOH (0.6 mL, 0.46 g, 10 mmol, 1 equiv) was added to

the remaining mother liquor. The solution was sonicated for 30 min

and diluted with water. A solid precipitate was removed by ﬁltration,

and the aqueous ﬁltrate was extracted 3× with EtOAc. The organic

layers were discarded, and the aqueous layer was retained. The water

was removed azeotropically with additional EtOH by rotary

evaporation to give 2,2′-((2-ethoxy-2-oxoethyl)azanediyl)diacetic

5

seeded at 2.2 × 10 per well in 12-well plates overnight and treated

with 0.75 μM corresponding compound for 24 h. CellROX Green (5

μM) was added to each well and incubated for 30 min. The cells were

harvested, and the ﬂuorescence signal was measured using a Cytomics

FC500 ﬂow cytometer. The p53 null cells were used to minimize any

p53 eﬀects.

Cell Growth Inhibition Assay. The cell growth inhibition assay

was performed by MTS (Promega) or Calcein AM assay (Trevigen),

acid (NTA-MEE) as a hygroscopic brown foam (0.232 mgs, 1.06

mmol, 11%). H NMR (500 MHz, DMSO-d ): δ 12.36 (bs, 2H), 4.07

(

3

36

as described previously. Brieﬂy, 5000 cells per well were cultured in

1

6

a 96-well plate to reach the 50% conﬂuence on the second day when

treated with serial dilutions of the compounds. Growth was measured

either by the MTS reagent or Calcein AM assay and Tecan plate

reader instrument (Tecan) after incubation for 3 days. Statistical

signiﬁcance of the data, obtained from three independent experi-

ments, each with triplicates, was calculated with Student’s t test.

Immunoﬂuorescence Staining. Immunoﬂuorescence staining

q, J = 7.1 Hz, 2H), 3.56 (s, 2H), 3.50 (s, 4H), 1.18 (t, J = 7.1 Hz,

H).

Bis(2-ethoxy-2-oxoethyl)glycine (NTA-DEE). To a solution of

diethyl 2,2′-azanediyldiacetate (1.9 mL, 10.6 mmol, 2 equiv) in

dioxane (6.5 mL) was added 2-bromoacetic acid (0.38 mL, 5.3 mmol,

1

equiv). After heating overnight at 60 °C, the crude reaction was

concentrated under reduced pressure. The residual oil was dissolved

8

was performed, as described previously. In brief, cells were grown on

in DCM and washed in succession 1× with NaHCO (sat, aq), 1×

3

coverslips followed by various treatments. The coverslips were ﬁxed

with 4% paraformaldehyde for 10 min and then permeabilized with

with water, and 1× with brine. The organic was dried over Na SO ,

2

4

ﬁltered, concentrated, and dried under high vacuum to give the title

0.5% Triton X-100 for 5 min. The conformation of the mutant and

compound bis(2-ethoxy-2-oxoethyl)glycine (NTA-DEE) as a brown

oil. H NMR (500 MHz, DMSO-d ): δ 4.04 (q, J = 7.1 Hz, 4H), 3.55

1

WT p53 proteins was recognized speciﬁcally by the antibodies

PAB1620 (1:50, recognizing wild-type conformation) and PAB240

6

(s, 4H), 3.47 (s, 2H), 1.16 (t, J = 7.1 Hz, 6H).

(1:400, recognizing misfolded/unfolded conformation) stained over-

K_Z_nand K_C_uMeasurements. K_dmeasurements of zinc and

36

night, respectively. The secondary antibody, goat antimouse IgG, was

incubated for 40 min. PAB1620 and PAB240 were purchased from

EMD Chemicals.

copper were performed, as described previously.

2+

Zn -K Measurements by Competition. The compounds in

d

5

2

0 mM Tris (pH 7.2) and 150 mM NaCl (compound concentrations:

-fold serial dilution from 10,000 to 4.9 nM) were incubated with 30

Eﬄux Analysis. Caco-2 assays were performed by Sai Life

Sciences (Hinjewadi, India). Brieﬂy, Caco-2 cells were seeded in 24-

well Millicell plates and incubated for 21 days. For A−B transport, the

candidate compounds and loperamide were added to the apical side.

For B−A transport, the candidate compounds and loperamide were

added to the basal side. At 0, 15, 30, 60, and 90 min, aliquots of 50 μL

were collected from the receiver compartment for determination of

the candidate compound concentrations by LC−MS/MS.

nM ZnCl₂+ 30 nM FluoZin-3 or RhodZin-3 reporter at room

temperature for 60 min in Costar black polystyrene 96-well plates

(

Corning Inc., Corning, NY), and the ﬂuorescence was measured

using a SpectraMax i3x plate reader (λ_e_x= 500 nm; λ_e_m= 540 nm)

Molecular Devices, LLC, Sunnyvale, CA). The data were plotted on

(

a log axis and ﬁt to a sigmoid to measure IC₅₀

A

Mouse Experiments. Mice are housed and treated according to

guidelines, and all the mouse experiments are done with the approval

of Institutional Animal Care and Use Committee (IACUC) of

Rutgers University. The nude mice NCr nu/nu are purchased from

Taconic. The acute toxicity and eﬃcacy assays were performed, as

^y⁼1

+ e⁻

^x⁻^x⁰b

(

)

(1)

where y is the measured ﬂuorescence, A is the curve amplitude, x is

log[compound], x is the log IC , and b is an empirical steepness

parameter. That IC₅₀was then used in the Munson−Robard solution

to the Cheng−Prussof equation to calculate K_d

0

50

36

described previously. Brieﬂy, for the maximum tolerated dose

assays, 8−12 week old mice (5 mice per dose) were administered by

intraperitoneal injection (ip) once for acute test and health, behavior,

and body weight were monitored. The survival times of these groups

were shown in Kaplan−Meier survival curves with the log-rank test.

Xenograft tumor assays were derived from the human tumor cell lines,

IC₅₀

y

0

K =

− K_d

i

L (y + 2)

T

0

^y0 ⁺²

1

+ ₂

+ y₀

K (y + 1)

d

0

(2)

6

TOV-112D (5 × 10 cells/tumor site/mouse) and MDA-MB-468 (6

where K is the dissociation constant of the DBD, L_Tis the total

i

6

concentration of the reporter, K is the dissociation constant of the

× 10 cells/tumor site/mouse). Tumor dimensions were measured

d

reporter for Zn²⁺(15 and 65 nM for FluoZin-3 and RhodZin-3,

every other day, and their volumes were calculated by length (L) and

2

respectively, per manufacturer), and y is the ratio of bound reporter

to free reporter in the absence of the study compound (e.g., 15 nM/

width (W) using the formula: volume = L × W × π/6. Tumors (ﬁve

0

3

per group) were allowed to grow to 50 mm prior to daily

1

5 nM in the case of FluoZin-3). Curve ﬁtting was done in SigmaPlot

3.0.

administration of each compound administered ip at the doses

detailed in the text.

FluoZin-3 and RhodZin-3 were used interchangeably, depending

on commercial availability.

Hydrolytic Stability Studies. A detailed procedure is reported in

the Supporting Information.

2

043

https://dx.doi.org/10.1021/acs.jmedchem.0c01360

J. Med. Chem. 2021, 64, 2024−2045

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Statistical Methods. The data were analyzed by Student’s t test

with an overall signiﬁcance level of p < 0.05. p values were described

in each ﬁgure and in the text.. *p < 0.05, **p < 0.01, and ***p <

Author Contributions

S.N.L, J.Y.R., and D.R.C. are co-senior authors.

&

Funding

0.001.

This work was supported by grants from the NIH (R01

CA200800 and K08 CA172676), the Breast Cancer Research

Foundation (to D.R.C.), and the NIH (F30GM113299) (to

A.R.B.).

ASSOCIATED CONTENT

■

*

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01360.

Notes

The authors declare the following competing ﬁnancial

interest(s): D.R.C. is the founders of Z53 Therapeutics, Inc.,

a company that aims to develop p53 mutant reactivators such

as zinc metallochaperons into therapeutic agents for the

treatment of cancer and other diseases. J.Y.R. is a paid

consultant, and own options, of Pacylex Pharmaceuticals, a

company developing PCLX-001 for oncology targets. No

potential conﬂicts of interest were disclosed by the other

authors.

Suppl. Figure S1, S2, and S3; Hydrolytic Stability

Studies of ZMC1 and C85; HPLC traces (PDF )

Molecular formula strings of all compounds (CSV)

X-ray crystallographic data of Zn-1 (CIF )

X-ray crystallographic data of Zn-2 (CIF )

AUTHOR INFORMATION

Corresponding Author

Darren R. Carpizo − Division of Surgical Oncology,

Department of Surgery, University of Rochester Medical

Center, Rochester, New York 14642, United States; Wilmot

Cancer Center, University of Rochester, Rochester, New York

■

ABBREVIATIONS

■

ATCC, American Type Culture Collection; Caco-2, human

colorectal adenocarcinoma cells; CDL, curved desolvation line;

DBD, DNA binding domain; DEE, diethyl ester; DMEM,

Dulbecco’s modiﬁed Eagle’s medium; EGTA, egtazic acid;

FBS, fetal bovine serum; Gy, gray; Hex, n-hexanes; IACUC,

Institutional Animal Care and Use Committee; MEE,

monoethyl ester; MsCl, methanesulfonyl chloride; MTS, 3-

1

4642, United States; Email: darren.carpizo@

urmc.rochester.edu

Authors

John A. Gilleran − Rutgers Molecular Design and Synthesis,

Oﬃce of Research and Economic Development, Piscataway,

New Jersey 08854, United States

(

4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium); NTA, nitrilotriacetic acid; Pab,

p53 antibody; PBS, phosphate-buﬀered saline; PTM, post-

translational modiﬁcation; RR, ribonucleotide reductase; SGF,

simulated gastric ﬂuid; TEA, triethylamine; TSC, thiosemi-

carbazone; ZMC, zinc metallochaperone

Xin Yu − Program of Surgical Oncology, Rutgers Cancer

Institute of New Jersey and Department of Surgery, Robert

Wood Johnson Medical School, Rutgers University, New

Brunswick, New Jersey 08901, United States

Alan J. Blayney − Department of Biochemistry and Molecular

Biology, SUNY Upstate Medical University, Syracuse, New

York 13210, United States

Anthony F. Bencivenga − Rutgers Molecular Design and

Synthesis, Oﬃce of Research and Economic Development,

Piscataway, New Jersey 08854, United States

Bing Na − Program of Surgical Oncology, Rutgers Cancer

Institute of New Jersey and Department of Surgery, Robert

Wood Johnson Medical School, Rutgers University, New

Brunswick, New Jersey 08901, United States

David J. Augeri − Rutgers Molecular Design and Synthesis,

Oﬃce of Research and Economic Development, Piscataway,

New Jersey 08854, United States

Adam R. Blanden − Department of Biochemistry and

Molecular Biology, SUNY Upstate Medical University,

Syracuse, New York 13210, United States

S. David Kimball − Rutgers Molecular Design and Synthesis,

Oﬃce of Research and Economic Development, Piscataway,

New Jersey 08854, United States

Stewart N. Loh − Department of Biochemistry and Molecular

Biology, SUNY Upstate Medical University, Syracuse, New

York 13210, United States

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Article Doi

Benzothiazolyl and Benzoxazolyl Hydrazones Function as Zinc Metallochaperones to Reactivate Mutant p53

DOI: 10.1021/acs.jmedchem.0c01360

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