Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase

Article abstract of DOI:10.1016/j.bmcl.2013.08.025

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of >355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome.

Full text of DOI:10.1016/j.bmcl.2013.08.025

Accepted Manuscript
Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine deriv‐
atives as potent inhibitors of Bloom Helicase
Andrew S. Rosenthal, Thomas S. Dexheimer, Opher Gileadi, Giang H. Nguyen,
Wai Kit Chu, Ian Hickson, Ajit Jadhav, Anton Simeonov, David J. Maloney
PII:
S0960-894X(13)00954-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.025
BMCL 20763
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
26 June 2013
26 July 2013
5 August 2013
Please cite this article as: Rosenthal, A.S., Dexheimer, T.S., Gileadi, O., Nguyen, G.H., Chu, W.K., Hickson, I.,
Jadhav, A., Simeonov, A., Maloney, D.J., Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine
derivatives as potent inhibitors of Bloom Helicase, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://
dx.doi.org/10.1016/j.bmcl.2013.08.025
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Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-
amine derivatives as potent inhibitors of Bloom Helicase
Andrew S. Rosenthal a‡, Thomas S. Dexheimera‡, Opher Gileadib, Giang H. Nguyenc,d, Wai Kit
Chue, Ian Hicksonc,e, Ajit Jadhava, Anton Simeonova, David J. Maloneya*
a National Center for Advancing Translational Sciences, National Institutes of Health, 9800
Medical Center Drive, Rockville, MD, 20850. bStructural Genomics Consortium, University of
Oxford, Oxford OX3 7DQ, UK. c Department of Medical Oncology, Weatherall Institute of
Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK. d
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD. e Department of Cellular and Molecular Medicine,
Nordea Center for Healthy Aging, University of Copenhagen, Denmark.
Send proofs to:
Dr. David J. Maloney
National Center for Advancing Translational Sciences
National Institutes of Health
9800 Medical Center Dr.
Rockville, MD, 20850
301-217-4381; Fax : 301-217-5736
Email : maloneyd@mail.nih.gov
*To whom correspondence should be addressed.
‡These authors contributed equally to this work.
KEYWORDS. Bloom Helicase, Small molecule, inhibitor

ABBREVIATIONS: BLM, Bloom helicase; SAR, Structure Activity Relationship; HTS, High
Throughput Screen; MIC, minimum inhibitory concentration; ADME, absorption, distribution,
metabolism and excretion; MLM, mouse liver microsomes; NADPH, nicotinamide adenine
dinucleotide phosphate; PBS, phosphate buffered saline; BS, Bloom syndrome; SCE, sister
chromatid exchanges; HR, homologous recombination; DMF, dimethylformamide

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant
mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ
family of DNA helicases, which includes the Bloom helicase (BLM), plays an important
function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical
DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom
syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-
deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective
BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this
work, we describe the medicinal chemistry optimization of the “hit” molecule following a
quantitative high-throughput screen of >355,000 compounds. These efforts lead to the
identification of ML216 and related analogs, which possess sub-micromolar BLM inhibition and
exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular
activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome.

The human genome is constantly being threatened by potentially harmful DNA-damaging
events. In order to maintain genome integrity, cells have developed various mechanisms for
detecting DNA damage and coordinating its repair. A fundamental feature of DNA repair, as
well as other cellular processes involving nucleic acids, is the transient separation or unwinding
of the DNA double helix into complementary strands permitting access to genetic information.
DNA helicases are a continually expanding group of enzymes that catalyze this strand separation
reaction.1 Indeed, helicases are predicted to constitute approximately 1% of the open reading
frames in the human genome. One highly conserved family of DNA helicases, termed the RecQ
helicases, plays an important function in genome preservation through diverse roles in DNA
repair, recombination, and replication (for review, see 2). The human genome encodes five RecQ
helicases and genetic defects in at least three of these genes, BLM, WRN, and RECQ4, are
responsible for distinct human disorders that cause premature aging and/or cancer predisposition,
among other phenotypes (for review, see 3). For example, Bloom syndrome (BS) is a rare
autosomal recessive disorder originating from loss-of-function mutations in the BLM gene.4 BS
clinically exhibits a pleiotropic phenotype characterized by proportional dwarfism, sun-sensitive
telangiectatic erythema, fertility defects, immunodeficiency, and shortened lifespan, which is
typically cancer-related.5 Cells from BS patients are characterized by an elevated level of
genomic instability and a genome-wide increase in sister chromatid exchanges (SCE), which is a
key feature used in the clinical diagnosis of the disorder.6
The BLM gene product is an ATP-dependent DNA helicase that translocates in the 3’–5’
direction.7 BLM helicase has been shown to resolve a wide variety of DNA structures, including
3′-tailed duplexes, bubble and splayed arm DNA structures, DNA displacement loops (D-loops),
four-way Holliday junctions, and G-quadruplex structures.8 In addition, BLM forms a
multiprotein complex with RMI1, RMI2, and topoisomerase III that functions in the dissolution

of double Holliday junctions,9 which are prominent intermediates in the homologous
recombination (HR) repair pathway.10 The involvement of BLM in double-strand break repair is
corroborated by its interaction with RAD51 recombinase, which is the essential enzyme in HR
that catalyzes homology-dependent strand invasion.11 Current research is also establishing the
role of BLM in telomere maintenance12 as well as the processing and re-initiation of stalled
replication forks.13
Figure 1. Structures of previously identified RecQ helicase inhibitors. Recently published WRN inhibitor, left, and
BLM inhibitors (middle, HTS hit and right, optimized probe).
Prior reports have revealed only non-specific, weakly active RecQ helicase inhibitors. For
example, several clinically used DNA-binding compounds have been described as non-specific
inhibitors of both BLM and WRN-catalyzed DNA unwinding activity.14 A recent screen of the
NCI diversity set identified NSC19630 (Figure 1) as a small molecule inhibitor of WRN
helicase.15 Although this maleimide-containing compound potentially suffers from promiscuity
given the known reactivity of such moities with cysteine residues, it does highlight the growing
interest in the helicase field.16 More recently, we described the discovery and biological activity
of ML216 (Figure 1), a novel small molecule inhibitor of BLM helicase. ML216 was found to
possess potent (1-3 µM) inhibition of the DNA unwinding activity of BLM, induce sister
chromatid exchanges, and demonstrate selective antiproliferative activity in BLM-positive
cells.17 Herein, we detail the medicinal chemistry efforts that led to the nomination of ML216 as
a chemical probe and provide selectivity information and in vitro ADME data for additional
analogs.

Before initiating an extensive medicinal chemistry effort, we first sought to resynthesize and
confirm the activity of the HTS hit molecule MLS000559245 (1) (Figure 1). Pure solid
compound was prepared in one-step by heating (90 ºC) commercially available starting
materials, 2-Amino-5-(4-pyridinyl)-1,3,4-thiadiazole and 3,4-dichlorophenyl isocyanate, in
DMF.17 Subsequent testing of this resynthesized compound (now denoted as 1) in a
conventional gel-based DNA unwinding assay for BLM inhibitory activity confirmed the HTS
activity with an IC50 value of 1.4 µM (Table 1). The general synthetic procedure for the
preparation of analogs 4a-g is outlined in Scheme 1. A variety of thiadiazole-2-amines (2a-h)
are commercially available and thus rapid SAR could be obtained for this region of the molecule
using the previously described method.
Scheme 1. Reagent and conditions: Method A: DMF, 90 oC, 1h (See supplemental information for details)
Our first area of SAR exploration involved modification of the 4-pyridine moiety on the western
region of the molecule (Table 1). These compounds were synthesized via reaction of the
requisite thiadiazole-2-amine derivative with 3,4-dichlorophenylisocyante as shown in Scheme
1. Changing the pyridine group to a phenyl (4a) and changing the position of the pyridine
nitrogen from the 4-position to the 2-position (4b) led to inactivity. Interestingly, the 3-pyridine
(4c) had comparable activity to the lead compound with an IC50 value of 3.5 µM. As

anticipated given the inactivity of phenyl analogue 4a, the cyclohexane derivative (4d) was also
inactive. These data suggest a preference for a basic nitrogen group in this region of the
molecule; however, all attempts to mimic this interaction with other nitrogen containing
heterocycles were largely unsuccessful (4e-h). Only the 4-aminophenyl derivative (4e) exhibited
very modest inhibitory activity (IC50 = 49 µM).
Table 1. SAR of the 4-pyridine moiety (analogs 1, 4a-h).
a Determined by gel-based BLM DNA unwinding assay.
Having established the importance of the 4-pyridine moiety on the western region of the
molecule, we then turned our attention towards modification of the 1,3,4-thiadiazole core.
Surprisingly, this region was also very intolerant to structural modification. The synthesis of
these compounds was achieved through the reaction of the requisite amino derivative (5a-j) and

3,4-dichlorophenylisocyanate (3) as shown in Scheme 2. Amino derivatives (5a-h) were all
commercially available; however, pyridine- (5i) and pyridimide- (5j) amines were prepared via a
Suzuki coupling of 4-pyridine boronic acid with 6-chloropyridine-2-amine and 2-
chloropyrimidine-4-amine, respectively. Changing the thiadiazole to the thiophene derivative
(6a) resulted in a loss of activity. Interestingly, the 4-substituted thiazole (6b, IC50 = 28 µM)
maintained some BLM inhibitory activity, yet the 5-substituted thiazole (6c) was completely
inactive despite being more structurally similar to the original HTS hit. Replacement of the
1,3,4-thiadiazole for an 1,3,4-oxadiazole (6d) or other nitrogen containing heterocycles (6e-h)
also resulted in loss of activity. Pyridine analog (6i) was the only core modification that was
tolerated with an IC50 comparable to 1 of 4.0 µM, as even the related pyrimidine analog (6j) was
inactive.
Scheme 2: Reagents and conditions (See supplemental data): Method A: DMF, 90 oC, 1h; Method B: analog 6i: 6-
chloropyridine-2-amine, 6j: 2-chloropyridimide-4-amine; Na2CO3 (3.0 eq.), Pd(PPh3)4 (10 mol%), DME:H2O
(10:1), 150 oC, 1 h.

Table 2. SAR of the thiadiazole moiety (analogs 1, 6a-j).
a Determined by gel-based BLM DNA unwinding assay.
Next we explored the role of the urea moiety by replacement with comparable functional
groups and known urea bioisosteres as shown in Table 3. The compounds were synthesized via a
variety of methods with additional details provided in the Supporting Information. Converting
the urea to a thiourea (7a) was tolerated (IC50 = 4.0 µM), however, given the comparable activity
and that thioureas represent a potential toxicophore, additional analogs with this moiety were not
pursued.18 Replacement of the urea with a guanidine (7b), amide (7c), carbomate (7d)
sulfonamide (7e) and cyanoguanidine (7f) all yielded weakly active or inactive molecules.
Interestingly, squarate (7g) a known urea bioisostere, demonstrated comparable activity with an
IC50 value of 4.3 M, however the solubility of this compound was not improved over (1). Even
mono-methylation of one of the urea nitrogens (7h) resulted in a complete loss of activity. Due
to the lack of noticeable improvements in activity via modification at this particular part of the
lead molecule, we continued our chemical efforts with the urea linker in place.

Table 3. SAR of the urea moiety (analogs 1, 7a-h).
a Determined by gel-based BLM DNA unwinding assay.
Thus far, investigations of the initial compound (1), yielded tractable SAR with definite trends,
but no analogs with improved potency were found and a general lack of tolerance for structural
modifications was observed (see Tables 1-3). Our first experience with potency improvement
came in the SAR studies of the eastern di-chlorophenyl moiety as shown in Table 4. These
compounds were synthesized using the same method described in Scheme 1. Note that for

compounds in which the desired phenylisocyanate derivative was not commercially available, we
synthesized it in situ via reaction of the requisite aniline with triphosgene.19
Table 4. SAR of the di-chlorophenyl moiety (analogs 1, 8-36)
a Determined by gel-based BLM DNA unwinding assay.
We found that removal of one (8) or both (9) of the chloro atoms led to a drastic loss in activity.
However, a noticeable improvement in potency (4-fold) was observed when the 3-Cl was
replaced with a cyano group (10, IC50 = 0.1 µM). A similar trend was observed with the 3-CN,
4-Br-phenyl derivative (23, IC50 = 0.11 µM) and the 3-Br,4-CN analog (24) was also well
tolerated with an IC50 value of 0.91 µM. Changing the initial bis-chlorophenyl ring to the
isosteric 2-naphthalene moiety resulted in very little change in potency (data not shown) and

only added to the lipophilicity of the molecule so additional derivatives around the 2-napthalene
were not explored. Replacing the chloro functionality with Br (20) or F (25) was tolerated, but
did not result in improved potency with IC50 values of 5.0 and 8.9 µM, respectively. A variety of
other aromatic substitutions were tried (entries 11-19) and most were tolerated, but did not result
in an increase in potency. Generally, electron-withdrawing groups at the 3-position [NO2 (13),
CN (10), Br (20)] were preferred over electron-donating groups [CH3 (11), NH2 (14)]. Despite
the fact that introduction of the fluoro group appeared to result in a less desirable gel assay IC50
when compared to Br or Cl containing analogs, the 3-CF3, 4-F-phenyl analog (ML216)
demonstrated good activity that was reproducible in orthogonal bioassays and had other
favorable pharamacokinetic properties. For example, while we were able to identify compounds
more potent than ML216 (e.g. analogs 10, 23, 24), these compounds had noticeably diminished
aqueous/DMSO solubility. Accordingly, we declared ML216 as our chemical probe for the
Molecular Library program given the optimal balance of potency and physicochemical properties
in relation to other analogs. Subsequent to these studies, we remained interested in further
improving the physicochemical properties of ML216, and more specifically the aqueous
solubility. Therefore, we sought to introduce nitrogen containing heterocycles in an effort to
increase the basicity/polarity of the molecules.20 Preliminary investigations towards this end
revealed that the 4-position of the eastern phenyl group was more tolerant to these modifications
(data not shown). The synthesis of these compounds was accomplished through the requisite 4-
Br derivative via Suzuki coupling (for pyrazoles) or Cu-catalyzed coupling (triazoles and
imidazoles) as shown in Scheme 3. Introduction of the triazole moiety (entries 30, 31) was
tolerated yet resulted in a significant loss of potency with IC50 values of 36 and 14.1 µM,
respectively. Imidazole (entry 35) and pyrrole (entry 36) derivatives both had good activity,
however we were particularly intrigued by the activity of the 3-CN, 4-pyrazole phenyl derivative

(33) which possessed comparable activity to the probe compound. Importantly, analog 33 has a
much improved CLog P value compared to ML216 of 2.0 vs. 3.8 as shown below in Table 6.
Scheme 3. Synthesis of heterocyclic derivatives at the 4-position. Reagent and conditions (See Supporting
Information for details): (a) analogs 30 and 31: aniline [X = N and R = CF3 and F, respectively], analog 35: aniline
[X = C, R = F]; triphosgene (1.0 eq.), NEt3 (3.0 eq.), CH2Cl2, rt, 3h, then concentrate and add 5-(pyridin-4-yl)-1,3,4-
thiadiazol-2-amine (1.0 eq.), DMF, heat to 90 oC, 1h; (b) analog 32-34: [R1 = CH3 (32), CN (33), CF3 (34); R2 = 4-
(Boc-1H-pyrazole)]; analog 36: [R1 = CN, R2 = 2-(1H-pyrrole)] K3PO4 (2.5 eq.), Pd(PPh3)4 (10 mol %),
Dioxane/DMSO/water (1/0.3/0.2), 100 oC, 3 hr. For analogs (32-34): (c) TFA, CH2Cl2, rt.
After completing our initial SAR exploration of the HTS “hit” molecule (1), we aimed to
determine selectivity of top compounds against related RecQ helicases. As summarized in Table
5, several compounds demonstrated both improved potency and selectivity for BLM compared to
ML216. For example, the pyrazole-containing compound 33 exhibited an IC50 value of 1.1 M
against BLM and a greater than 6-fold selectivity over WRN (Figure S1). While compound 10
showed particularly good activity (IC50 = 0.10 M) and a nearly 16-fold selectivity for BLM, it
exhibited diminished solubility compared with 33. All compounds showed only weak inhibition
of RECQ1 at 50 M. Based on its potency, selectivity, and CLog P, compound 33 was chosen to

further investigate the underlying mechanism of action and cellular activity for this class of
compounds.
Table 5. RecQ helicase selectivity
a Determined by gel-based DNA unwinding assay.
ND, not determined
Like most helicases, BLM exhibits intrinsic DNA-dependent ATPase activity, which provides
energy for DNA strand separation. It can be envisioned that reduction in the accessibility of the
ATP-binding site may lead to decreased ATP hydrolysis and an equivalent reduction of the DNA
unwinding rate. To investigate this type of inhibition mechanism, we measured BLM-catalyzed
ATP hydrolysis rates using a single-stranded oligonucleotide as the DNA effector in the presence
of 33. As shown in Figure 2A, kinetic analysis reveals that compound 33 does not affect the
apparent Km value of BLM ATPase activity. A Lineweaver-Burk double reciprocal plot of the
curves derived from control and inhibitor-containing reactions gave regression lines that intersect
at a point on the x-axis indicating that 33 is a non-ATP competitive BLM inhibitor (Figure 2B).
These results also suggest that 33 acts by preventing BLM from binding to DNA, as
demonstrated previously for ML216.17 Indeed, dose-dependent inhibition of BLM binding to
single-stranded DNA by 33 was confirmed using fluorescence polarization analysis (Figure S2).

Moreover, selective displacement of DNA binding to BLM over related DNA helicases was also
observed for compound 33 (Figure S2).
Figure 2. Inhibition mechanism of compound 33. A) Michaelis-Menten kinetic analysis of BLM ATPase activity in
the absence and presence of compound 33. B) Lineweaver-Burke double reciprocal plot derived from the assay
described in (A). Each data point represents the mean ± SD of quadruplicate determinations. The results are a
representative of three independent experiments.
An elevated frequency of SCEs is a characteristic feature of BS cells. Thus, we next asked
whether 33 could induce similar cytological effects in a BLM-specific manner. Using the
isogenic pair of GM08505-derived BS cells,21 we demonstrate a significant and dose-dependent
induction of SCEs by 33 in cells expressing BLM (PSNF5), while no further increase in SCEs
was observed in cells lacking BLM (PSNG13) (Figure 3). Given the intimate role of BLM in
suppressing SCE formation, these results strongly suggest that the induction of SCEs by 33 is
mediated through inhibition of BLM activity.

Figure 3. Effects of 33 on SCE frequency. BLM-complemented (PSNF5) and BLM-defective (PSNG13) cells
were treated with 33, as indicated below the bars, and the frequency of SCEs per chromosome was quantified. An
asterisk indicates a p-value less than 0.01, while ns denotes no statistical significance (p > 0.5).
In order to gain a better understanding of the potential of these compounds to be used in studies
beyond biochemical and cell-based assays we sought to determine representative in vitro ADME
properties of the our top compounds (ML216 and 33). As shown in Table 6, these compounds
exhibit generally favorable properties; namely microsomal stability, CLog P, and plasma
stability. However, both compounds have low aqueous solubility (PBS buffer, pH 7.4) of 1 and
10 µM for ML216 and 33, respectively. Of note, the compounds show improved solubility in
the assay buffer system (data not shown, see Supporting Information for buffer conditions),
which suggests that the biochemical data for these analogs were not compromised by this
liability. Typically, the improved solubility in the assay buffer is a result of having non-ionic
detergent (Tween-20) present which aids in solubilizing the more lipophilic compounds.
Moreover, both compounds have low Caco-2 permeability which may explain the higher
concentrations of drug required to observe an effect in cell-based studies. The low solubility and
permeability suggest that these compounds could possess poor absorption and oral
bioavailability and further optimization of this chemotype may be required for use in vivo.

Table 6.a In vitro ADME properties for ML216 and 33.
aq.
MLM
Plasma
Stability
(5 h)
Caco-2
Permeability ratio
Efflux
Compound CLog P Solubility Stability
(pH 7.4)
(T1/2)
3.8
2
1 µM
>80 min
>80 min
100%
100%
1 X 10-6 cm/s
ND
2
ML216
33
10 µM
ND
aCLogP values were determined using ChemBioDraw™ 12.0; Solubility refers to the kinetic solubility as
determined by Analiza Inc. (www.analiza.com) using chemiluminescent nitrogen detection (CLND) and in-house
via UV-Vis detection methodology; MLM stability refers to mouse liver microsome stability in the presence of
NADPH; Plasma stability was performed using mouse plasma and the % remaining after 5 hours was determined.
The Caco-2 permeability value represents the Papp from A→B; Efflux ratio is B→A/A→B. Microsomal stability,
Plasma stability and Caco-2 peremeability studies were conducted at Pharmaron Inc.
In summary, the medicinal chemistry optimziation efforts surrounding a 5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-amine based BLM helicase inhibitor is described. Top compounds possess low
micromolar to sub-micromolar potency and good selectivity against other related DNA helicases.
Moreover, the mode of inhibition was investigated and the activity in cell-based assays was
demonstrated by an observation of an increase in SCEs, as anticipated. While some
improvement in the aqueous solubility was achieved with compound 33, compared to the
previously described ML216, this particular ADME attribute remains a liability. We hope that
the compounds described herein provide a means to interrogate BLM helicase biology through
pharmacological inhibition and offer them freely to the research community.

Acknowledgements
This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for
Medical Research (grant U54MH084681), and the National Institutes of Health Intramural
Research Programs of the National Center for Advancing Translational Sciences. I.D.H. was
funded by Cancer Research UK and the Nordea Foundation (Denmark). G.H.N. was funded by
the NIH-Oxford Graduate Partnership Program. The authors wish to thank Sam Michael and
Richard Jones for automation support; Paul Shinn and Danielle van Leer for the assistance with
compound management; William Leister, Heather Baker, Christopher Leclair and Elizabeth
Fernandez for analytical chemistry and compound purification support.
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