Optimization of Drug Candidates That Inhibit the D-Loop Activity of RAD51

Article abstract of DOI:10.1002/cmdc.201900075

RAD51 is the central protein in homologous recombination (HR) repair, where it first binds ssDNA and then catalyzes strand invasion via a D-loop intermediate. Additionally, RAD51 plays a role in faithful DNA replication by protecting stalled replication forks; this requires RAD51 to bind DNA but may not require the strand invasion activity of RAD51. We previously described a small-molecule inhibitor of RAD51 named RI(dl)-2 (RAD51 inhibitor of D-loop formation #2, hereafter called 2 h), which inhibits D-loop activity while sparing ssDNA binding. However, 2 h is limited in its ability to inhibit HR in vivo, preventing only about 50 % of total HR events in cells. We sought to improve upon this by performing a structure–activity relationship (SAR) campaign for more potent analogues of 2 h. Most compounds were prepared from 1-(2-aminophenyl)pyrroles by forming the quinoxaline moiety either by condensation with aldehydes, then dehydrogenation of the resulting 4,5-dihydro intermediates, or by condensation with N,N′-carbonyldiimidazole, chlorination, and installation of the 4-substituent through Suzuki–Miyaura coupling. Many analogues exhibited enhanced activity against human RAD51, but in several of these compounds the increased inhibition was due to the introduction of dsDNA intercalation activity. We developed a sensitive assay to measure dsDNA intercalation, and identified two analogues of 2 h that promote complete HR inhibition in cells while exerting minimal intercalation activity.

Full text of DOI:10.1002/cmdc.201900075

Accepted Article
Title: Optimization of Drug Candidates that Inhibit the D-loop Activity of
RAD51
Authors: Brian Budke, Werner Tueckmantel, Kelsey Miles, Alan P.
Kozikowski, and Philip P. Connell
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201900075
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Optimization of Drug Candidates that Inhibit the D-loop Activity of
RAD51
Brian Budke[a], Werner Tueckmantel[b], Kelsey Miles[b], Alan P. Kozikowski[b], Philip P. Connell[a]*
[a]
Dr. Brian Budke (ORCID 0000-0001-6051-8416), Prof. Philip P. Connell
Department of Radiation and Cellular Oncology
University of Chicago
Chicago, IL 60637, United States
E-mail: pconnell@radonc.uchicago.edu
Dr. Werner Tueckmantel, Dr. Kelsey Miles, Dr. Alan P. Kozikowski
StarWise Therapeutics LLC
[b]
Madison, WI 53719, United States
Supporting information for this article is given via a link at the end of the document.
Abstract: RAD51 is the central protein in homologous
recombination repair (HR), where it first binds ssDNA and then
catalyzes strand invasion via a D-loop intermediate. Additionally,
RAD51 plays a role in faithful DNA replication by protecting stalled
replication forks; this requires RAD51 to bind DNA but may not
require the strand invasion activity of RAD51. We previously
described a small-molecule inhibitor of RAD51 named RI(dl)-2
(RAD51 inhibitor of D-loop formation #2 hereafter called 2h), which
inhibits D-loop activity while sparing ssDNA binding. However, 2h is
limited in its ability to inhibit HR in vivo, preventing only about 50% of
total HR events in cells. We sought to improve upon this by
performing a structure-activity-relationship (SAR) campaign for more
potent analogs of 2h. Most compounds were prepared from 1-(2-
aminophenyl)pyrroles by forming the quinoxaline moiety either by
condensation with aldehydes, then dehydrogenation of the resulting
4,5-dihydro intermediates, or by condensation with N,N'-
carbonyldiimidazole, chlorination, and installation of the 4-substituent
through Suzuki-Miyaura coupling. Many analogs exhibited enhanced
activity against human RAD51, but in several of these compounds
the increased inhibition was due to the introduction of dsDNA
intercalation activity. We developed a sensitive assay to measure
dsDNA intercalation, and identified two analogs of 2h that promote
complete HR inhibition in cells while exerting minimal intercalation
activity.
In addition to its role in DSB repair, RAD51
nucleofilaments also serve a critical function in protecting stalled
DNA replication forks from MRE11-mediated degradation.[3-4]
RAD51 helps accomplish this by promoting the reversal of
replication forks into four-way junctions (or so called ‘chicken
feet’), by re-annealing the parental DNA strands and promoting
annealing of nascent DNA strands.[5] Although some uncertainty
remains regarding the influence this process has in cellular
resistance to DNA damaging treatments, recent work from Maria
Jasin suggests that fork protection plays only a minor role in the
repair of cisplatin-induced DNA damage.[6-7]
RAD51 is commonly over-expressed in various types of
human cancer cells[8-9], leading to elevated HR efficiency and
associated resistance to oncologic therapies.[10-13] Inhibition of
RAD51 with siRNA or chemical antagonists can overcome this
resistance to therapy, thereby rendering tumor cells more
susceptible to chemotherapy and radiotherapy.[14-16] Several
small molecule RAD51 inhibitors have been described, most of
which act by preventing the formation of RAD51 filaments on
17-20]
ssDNA.[14,
It is important to note, however, that complete
loss of RAD51 function eliminates both of its known functions:
DSB repair and replication fork stabilization. Therefore, we
generated a novel class of RAD51 inhibitors, capable of
preventing D-loop formation (and hence HR-mediated DSB
repair) while permitting assembly of RAD51 at stalled replication
forks.[21]
The preferred compound from our initial campaign (RI(dl)-
2, hereafter termed compound 2h) was proven capable of
inhibiting RAD51’s D-loop activity while having minimal effects
on RAD51’s ssDNA binding activity.[21] While 2h is capable of
inhibiting most HR events within the first 24 hours following the
induction of a DSB, we noted that it is less effective at blocking
HR events that occur at later time points. Therefore, we sought
to develop improved compounds that inhibit total HR events to a
greater degree. Our SAR campaign (summarized in Figure 1)
sought to access a large diversity of analogs while maintaining
ease of synthesis, and towards this goal explored substitution in
the carbocyclic portion of the quinoxaline ring system (ring A)
and modifications to the pyrrole ring (ring B) and the 4-aryl group
(ring C). Modifications of the latter type eventually became the
focus of the present work. Several compounds were identified
that were more potent inhibitors of RAD51 protein. However,
many of these more potent inhibitors acquired significant DNA
intercalation activity and associated cellular toxicity; therefore,
Introduction
Homologous recombination (HR) is an essential DNA repair
process, which ensures genomic stability by repairing DNA
double strand breaks (DSBs). Unlike other pathways of DSB
repair like the error-prone non-homologous end-joining (NHEJ)
pathway, HR repairs DNA damage by utilizing an undamaged
homologous DNA template to guide the repair process.[1-2]
Initiation of HR requires the processing of DSB ends to generate
a 3' single-stranded DNA (ssDNA) tail at the site of DNA
damage, which is subsequently coated with RAD51 to form a
helical nucleoprotein filament. This resulting RAD51
nucleofilament is capable of searching for a homologous DNA
sequence and invading it to form a joint molecule intermediate,
termed a D-loop. Additional related proteins subsequently
promote accurate DNA synthesis using the undamaged
homologous sequence as a template.
1
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OHC
we employed a sensitive and quantitative assay to exclude
compounds exhibiting this off-target activity. The resulting
optimized compounds offer an improved ability to block HR in
vivo while retaining specific inhibition of RAD51’s D-loop activity,
sparing RAD51-ssDNA binding activity, and showing minimal
DNA intercalation activity.
R2
N
N
reduction
(a)
R1
R1 = OH
R1 = OCH2CH2F
NO2
R1
montmorillonite KSF
NH2
PhMe, reflux, -H2O
N,N'-azodicarbonyl-
dipiperidine/P(n-Bu)3
N
N
MnO2
R1
N
H
R1
N
Results and Discussion
R2
R2
(HO)2B
Pd cat., base
R2
C
2
N
O
1)
2) POCl3
N
N
N
(b)
R1
N
Cl
R1
NH2
Scheme 1. Generic approaches to pyrrolo[1,2-a]quinoxalines.
Modification of Ring C. Anilines 1a and 1b were derivatized
into a variety of carboxamides, sulfonamides, and N-alkylation
products (Scheme 2). The amides 2a and 2c-f were obtained by
reacting anilines 1a and 1b with the appropriate carboxylic acid
in the presence of HATU[23] and N,N-diisopropylethylamine.
Hydrolysis of 2a under basic conditions afforded the free alcohol
2b. Reaction of the aniline 1a with methanesulfonyl chloride
provided the sulfonamide 2g. The N-ethyl derivates 2h and 2i,
the N-benzyl derivative 2k, and the N,N-dimethyl derivative 2l
were obtained by reductive alkylation of anilines 1a and 1b with
Figure 1. Proposed modifications of 2h.
General Synthetic Approach. Scheme 1 illustrates the generic
methods employed in synthesizing the compounds of this study.
In most cases,
a modification of the reported synthetic
approach[21] was employed (route a). The introduction of the 2-
fluoroethoxy group through a Mitsunobu reaction gave improved
sodium
triacetoxyborohydride[24]
and
acetaldehyde,
yields
azodicarbonyldipiperidine/P(n-Bu)3,[22] was substituted for the
traditional reagent, diethylazodicarboxylate/PPh3. The
when
the
modified
reagent,
N,N'-
benzaldehyde, and formaldehyde, respectively. The N-tert-butyl
substituted amine 2j was accessed by reaction of aniline 1b with
tert-butyl 2,2,2-trichloroacetimidate and copper triflate in
nitromethane.[25]
generation of the pyrrolo[1,2-a]quinoxaline ring system by
reaction of 1-(2-aminophenyl)pyrroles with aldehydes requires
acid catalysis, and in our earlier work, anhydrous AlCl3 was
employed. The inconvenience of handling this highly moisture-
sensitive material, especially on a small scale, induced us to
search for alternatives. After some experimentation, the
commercial, acid-treated clay mineral, montmorillonite KSF (and
to a lesser extent, montmorillonite K10), was found suitable for
this purpose. The reaction is best performed in boiling toluene
with continuous removal of the generated water, and in
favorable cases affords yields up to 90% after oxidation of the
resulting 4,5-dihydro intermediate with activated MnO2. The
heterogeneous reagents are simply filtered off after completion
of the reaction. For a number of target compounds, it was
advantageous to preform the tricyclic parent structure without
the attached aryl group and to arylate subsequently via a
Suzuki-Miyaura coupling reaction (route b). Syntheses of
compounds with modified B rings follow this coupling approach
as well. The syntheses were in many cases completed by a final
modification of the substituent R2. A nitro group was chosen as
the precursor to not only primary but also several substituted
amines. N-monoalkyl derivatives were formed in only moderate
yields by reductive alkylation of the primary amines, but this
approach nevertheless appears preferable to the previously
employed dealkylation of N,N-dialkyl derivatives in the course of
the MnO2 oxidation.
N
N
N
N
a
R1
R1
2a-l
R3
1a R1 = H
N
NH2
1b R1 = OCH2CH2F
R2
2a R1 = H, R2 = H, R3 = C(O)CH2OAc
2b R1 = H, R2 = H, R3 = C(O)CH2OH
2c R1 = H, R2 = H, R3 = C(O)CH2NH2
2d R1 = H, R2 = H, R3 = C(O)CH2NHAc
2e R1 = H, R2 = H, R3 = C(O)Ph
2g R1 = H, R2 = H, R3 = S(O)2CH3
2h R1 = H, R2 = H, R3 = Et
2i R1 = OCH2CH2F, R2 = H, R3 = Et
2j R1 = OCH2CH2F, R2 = H, R3 = t-Bu
2k R1 = OCH2CH2F, R2 = H, R3 = CH2Ph
2l R1 = OCH2CH2F, R2 = Me, R3 = Me
b
2f R1 = H, R2 = H, R3 = C(O)(3-pyridyl)
Scheme 2. Synthesis of compounds 2a-i. Reagents and conditions: (a) For
2a,c-f: carboxylic acid, HATU, N,N-diisopropylethylamine, DMF, 50 °C, 11-
78%; for 2g: methanesulfonyl chloride, pyridine, CH2Cl2, rt, 9%; for 2h,i,k,l:
aldehyde, NaBH(OAc)3, Na2SO4, CH2Cl2, rt, 44-81%; for 2j: tert-butyl 2,2,2-
trichloroacetimidate, cat. Cu(OTf)2, nitromethane, rt, 16%; (b) 2M aq.
NaOH/MeOH/THF, rt, 83%.
The N-acetyl derivatives 5a,b were synthesized from the
corresponding N-(2-aminophenyl)pyrroles 3a,b by reaction with
4-acetamidobenzaldehyde (4) in the presence of montmorillonite
K10 (Scheme 3). The initial mixtures of the desired products
5a,b with their 4,5-dihydro derivatives (a result of partial air
oxidation of the latter) were treated with MnO2 to provide the
fully oxidized amides.
2
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pyrrolidone, pyrazole, imidazole,[30] and ethanolamine[31]
provided 10a, 10b, 10c, and 10e, respectively. The morpholine
derivative 10d was accessed via a Pd-catalyzed coupling of 9
with morpholine[32]. The ethylene glycol derivative 10f was
synthesized via a Cu-catalyzed C-O coupling reaction of 9 with
ethylene glycol. Cu-catalyzed cyanation[33] of 9 provided the
nitrile 10g. The homologous nitrile 10i was accessed via
coupling of 9 with trimethylsilylacetonitrile.[34] Subsequent borane
reduction of either nitrile provided the amines 10h and 10j,
respectively, in only moderate yields, the main complication
again being reduction of the C(4)=N double bond.
N
N
a
R1
NH2
3a,b
R1
N
O
OHC
O
5a,b
N
H
CH3
N
H
CH3
4
a
R1 = H
R1 = OCH2CH2F
b
Scheme 3. Synthesis of compounds 5a-b. Reagents and conditions: (a)
montmorillonite K10, toluene, reflux, then MnO2, CHCl3, reflux, 6-19%.
Functionalization of ring C was further explored by Suzuki-
Miyaura coupling of aryl chlorides 6a and 6b with boronic acids
(Scheme 4). Initial attempts with Pd(PPh3)4 as catalyst[26]
provided mostly low yields of the desired products. Changing the
N
N
N
N
b, c, d, e,
f, or g
F
F
O
O
R2
9
10a-j
Br
catalyst
system
to
a
trialkylphosphine
ligand,
R2 = OCH2CH2OH
tricyclohexylphosphine (PCy3), with a Pd(0) source[27-28] provided
higher yields of the desired biaryls. The carboxamides 8a,b and
acetamidomethyl derivatives 8c,d were synthesized in good
yields under these conditions. Coupling of indole-5-boronic acid
with 6a using Pd(PPh3)4 as catalyst provided a moderate yield of
8e, and subsequent reduction of the indole moiety with
NaBH3CN[29] provided indoline 8f. As a side reaction, reduction
of the C(4)=N double bond in the pyrroloquinoxaline moiety was
observed, and we found it more efficient to prepare 8f via its N-
Boc derivative 8l, which resulted from coupling of 6a with the
appropriate pinacolboronate. Pyrimidine 8g was accessed in low
yield with the Pd(0)/PCy3 catalyst system using 2-
aminopyrimidine-5-boronic acid as the coupling partner. The
Pd(0)/PCy3 catalyst system provided also good yields of the N-
Boc-piperidine derivatives 8h and 8j. Removal of the Boc groups
with trifluoroacetic acid provided the desired free amines 8f, 8i,
and 8k.
a
b
c
d
e
R2 = 2-oxo-1-pyrrolidinyl
R2 = 1-pyrazolyl
R2 = 1-imidazolyl
R2 = 4-morpholinyl
R2 = NHCH2CH2OH
f
R2 = CN
a
g
h
i
h
R2 = CH2NH2
R2 = CH2CN
R2 = CH2CH2NH2
3b
h
j
Scheme 5. Synthesis of compounds 10a-j. Reagents and conditions: (a) p-
bromobenzaldehyde dimethyl acetal, montmorillonite KSF, 1,2,4-
trichlorobenzene, 100-120 °C; MnO2, CHCl3, reflux, 79%; (b) for 10a-c:
nitrogen-containing building block, K2CO3, cat. CuI/hexamethylenetetramine,
DMF, 140 °C, 14-69%; (c) for 10d: morpholine, cat. Pd(OAc)2/Xantphos,
toluene, 100 °C, 61%; (d) for 10e: 2-aminoethanol, K3PO4, cat. CuI/L-proline,
DMSO, 90 °C, 48%; (e) for 10f: ethylene glycol, K3PO4, cat. CuI/8-
hydroxyquinoline, 110 °C, 28%; (f) for 10g: K4[Fe(CN)6], cat. CuI, 1-
methylimidazole, 160 °C, 47%; (g) for 10i: Me3SiCH2CN, ZnF2, cat.
Pd2dba3/Xantphos, DMF, 100 °C, 51%; (h) BH3·THF, rt, 36-43%.
We used the gel-based D-loop assay to measure the
ability of compounds to inhibit the homologous strand exchange
activity of purified human RAD51 protein.[21] Compounds that
showed significant inhibition of D-loop activity in this biochemical
assay were prioritized for testing in the DR-GFP assay, which
measures HR activity in cells.[35] In this assay, 293-HEK cells
containing an integrated DR-GFP reporter are transfected with a
plasmid that expresses the I-SceI endonuclease, which
generates a DSB in the DR-GFP reporter cassette. Cells are
subsequently grown for 48 hours in the presence of candidate
small molecules. Results of these assays are displayed in Table
1, Supporting Information Figure 1, and Supporting Information
Figure 2, demonstrating that in most cases compounds with R1 =
fluoroethoxy showed little difference in their inhibitory activity
against purified RAD51 compared to otherwise identical
compounds without this substituent. However, for 1a and 2h,
introduction of a 7-fluoroethoxy substituent yielded compounds
that showed greater inhibition of cellular HR (compare 1a to 1b
and 2h to 2i). Additionally, the fluoroethoxy-substituted
compound 10e showed strong inhibition of cellular HR similar to
2i. Finally, all aromatic R2 substituents except in the case of 8f
and 8g resulted in a loss of RAD51 D-loop inhibitory activity.
Other bulky modifications to R2 such as those on 8h and 10d
resulted in a loss of activity, suggesting that steric hinderance at
this site may contribute to the lack of RAD51 inhibition by these
compounds.
N
N
N
N
N
N
a
b
c, d,
or e
R1
N
H
O
R1
R1
R2
R1
NH2
Cl
6a,b
8a-l
3a,b
7a,b
3,6,7a R1 = H; 3,6,7b R1 = OCH2CH2F
O
N
8a R1 = H, R2
8b R1 = OCH2CH2F, R2
8c R1 = H, R2
8d R1 = OCH2CH2F, R2
=
8g R1 = H, R2
8h R1 = H, R2
=
=
NH
2
NH
N
2
O
=
N
N
N
N
Boc
H
NH
2
g
O
=
8i R1 = H, R2
=
N
H
CH
3
8j R1 = OCH2CH2F, R2
=
N
N
Boc
H
O
=
N
g
H
CH
3
H
8e R1 = H, R2
8f R1 = H, R2
=
N
8k R1 = OCH2CH2F, R2
=
N
N
H
N
Boc
f
g
N
=
8l R1 = H, R2
=
Scheme 4. Synthesis of compounds 8a-i. Reagents and conditions: (a) CDI,
o-dichlorobenzene, reflux; (b) POCl3, reflux, 70-95% over 2 steps; (c) for 8a-
d,g,h,j: R2B(OH)2, aq. K3PO4, cat. Pd2(dba)3/PCy3, dioxane, 99 °C, 16-94%;
(d) for 8e: Na2CO3, cat. Pd(PPh3)4, toluene/EtOH/H2O (15:1:4), reflux, 44%; (e)
for 8l: pinacol N-Boc-indoline-5-boronate, aq. K3PO4, cat. Pd2(dba)3/PCy3,
dioxane, 99 °C, 91%; (f) NaBH3CN, AcOH, rt, 21%; (g) TFA, CH2Cl2, rt, 64-
81%.
An additional variety of substitutions on ring C were
examined for the fluoroethoxy-substituted ring A as shown in
Scheme 5. The aryl bromide 9 was obtained through a variant of
our clay-catalyzed cyclization reaction employing the dimethyl
acetal rather than the free aldehyde and distilling off the MeOH
formed. Cu-catalyzed C-N coupling reactions of 9 with 2-
3
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8j
> 100
46 ± 3 %
Table 1. Summary of Ring C Modifications. Measurements are reported as the
indicated values ± standard error, with each data set testing at least six
compound concentrations. NT = Not tested. NA = Not enough cells recovered
for testing due to high compound toxicity.
8k
4.40 ± 0.15
> 100
NA
NT
10a
N
N
N
N
R1
R1
R2
10b
10c
10d
> 100
NT
NT
NT
R2
1a-b, 2a-l, 5a-b, 8a-d, 8h-k, 10a-j
80 ± 185
> 100
8e-g
D-loop
IC50 (μM)
6.01 ± 2.27
5.54 ± 0.71
Max HR
Compound
R1
R2
Inhibition[a]
43 ± 2 %
10e
6.98 ± 1.93
92 ± 3 %
1a
1b
H
NH2
NH2
63 ± 3 %
10f
10h
10i
> 100
NT
2a
H
13.0 ± 5.5
38 ± 5 %
1.50 ± 0.15
> 100
38 ± 3 %
NT
2b
2c
2d
H
H
H
> 100
16 ± 0 %
46 ± 2 %
43 ± 2 %
10j
2.34 ± 0.28
31 ± 0 %
[a] HR inhibition observed at the lesser of either the maximum tested
concentration of 40 μM or the maximum tolerated dose (20 μM for 10h, 10 μM
for 8i and 10j, and 5 μM for 8k).
2.33 ± 0.37
11.5 ± 2.3
2e
2f
H
H
> 100
> 100
78 ± 2 %
83 ± 2 %
Modification of Ring B. This ring was replaced with various
aromatics as shown in Schemes 6-8. Reacting commercially
available 6-chlorophenanthridine with 4-nitrobenzeneboronic
acid under standard Suzuki-Miyaura coupling conditions yielded
13a (Scheme 6). Reduction of the nitro group via catalytic
hydrogenation provided 13b, and subsequent reductive
alkylation of the aniline with acetaldehyde provided the N-ethyl
derivative 13c.
2g
H
H
19.8 ± 13.7
43 ± 3 %
2h
2i
6.64 ± 1.19
6.46 ± 1.22
> 100
56 ± 4 %
93 ± 4 %
62 ± 5 %
2j
(HO)2B
a
2k
> 100
NT
+
N
Cl
N
NO2
R1
2l
45 ± 294
17 ± 0 %
27 ± 4 %
13a-c
12
11
5a
H
25.4 ± 2.3
a
b
c
R1 = NO2
R1 = NH2
R1 = NHEt
b
c
5b
> 100
NT
8a
8b
8c
H
H
16.0 ± 11.3
54.9 ± 90.6
17.9 ± 10.3
8 ± 0 %
NT
Scheme 6. Synthesis of compounds 13a-c. Reagents and conditions: (a)
Na2CO3, cat. Pd(PPh3)4, toluene/EtOH/H2O (15:1:4), reflux, 43%; (b) H2 (1
bar), 20% Pd(OH)2/C, MeOH, 81%; (c) CH3CHO, NaBH(OAc)3, Na2SO4,
CH2Cl2, rt, 51%.
18 ± 2 %
8d
8e
> 100
> 100
NT
NT
Replacement of ring B with an imidazole ring was
accomplished as shown in Scheme 7. Copper-catalyzed
coupling of imidazole with 2-chloronitrobenzene (14) followed by
hydrogenation of the nitro group yielded 2-(1H-imidazol-1-
yl)aniline (15). Reaction of this intermediate with N,N'-
carbonyldiimidazole (CDI) followed by chlorination with POCl3
yielded 4-chloroimidazo[1,2-a]quinoxaline (16). Suzuki-Miyaura
coupling of 16 with 4-nitrobenzeneboronic acid (12) provided
compound 17a. Hydrogenation of the nitro group afforded the
aniline 17b, and subsequent reductive alkylation with
acetaldehyde and sodium cyanoborohydride provided the N-
ethyl derivative 17c in moderate yield.
H
H
8f
8.88 ± 2.16
21 ± 0 %
8g
8h
H
H
12.8 ± 2.7
> 114
49 ± 2 %
99 ± 1 %
8i
H
1.73 ± 0.19
52 ± 1 %
4
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assays are displayed in Table 2, Supporting Information Figure
1, and Supporting Information Figure 2, demonstrating that
chlorination of the B-ring leads to a loss of D-loop inhibition
(compare 1a and 19b). Also, while replacement of the
pyrroloquinoxaline core of 2h with phenanthridine has no
discernible effect on RAD51 D-loop inhibition, cellular HR
inhibition is greatly weakened (compare 2h and 13c). Finally,
replacement of the B-ring with imidazole leads to a loss of
RAD51 D-loop inhibitory activity.
N
c,d
N
N
N
N
Cl
N
e
a,b
(HO)2B
N
N
Cl
NO2
NH2
R1
17a-c
14
15
16
NO2
12
a
b
c
R1 = NO2
R1 = NH2
R1 = NHEt
f
g
Scheme 7. Synthesis of compounds 17a-c. Reagents and conditions: (a)
imidazole, potassium tert-butoxide, cat. CuI/benzotriazole, DMSO, 120 °C,
44%; (b) H2 (1 bar), 20% Pd(OH)2/C, MeOH, 100%; (c) CDI, o-
dichlorobenzene, reflux, 2 h; (d) POCl3, reflux, 15 h, 55% over 2 steps; (e)
Na2CO3, cat. Pd(PPh3)4, toluene/EtOH/H2O (15:1:4), 100 °C; (f) H2 (1 bar),
20% Pd(OH)2/C, MeOH, rt, 45% over 2 steps; (g) CH3CHO, NaBH(OAc)3,
Na2SO4, CH2Cl2, rt, 51%.
Modification of Ring A. To explore the effects of different
alkoxy groups in ring A, phenol 20[21] was alkylated with benzyl
bromide, ethyl iodide, and isopropyl iodide, respectively
(Scheme 9). Hydrogenation of the nitro group on ring C afforded
the desired anilines 22a-c in good yield.
Ring B was further modified by introducing a chloro
substituent into the 1-position of the parent pyrrolo(1,2-
a)quinoxaline as shown in Scheme 8. Reacting intermediate 6a
with PCl5[36] provided a dichlorinated intermediate 18 which upon
coupling with boronic acid 12 under Suzuki-Miyaura conditions
yielded the nitroarene 19a. Reduction of the nitro group afforded
aniline 19b.
N
N
N
N
a, b,
or c
R1
R1
d or e
HO
O
N
21a-c
O
N
22a-c
20
NO2
NO2
R1 = CH2CH3;
NH2
a
R1 = CH2Ph;
b
c
R1 = CH(CH3)2
Scheme 9. Synthesis of compounds 22a-c. Reagents and conditions: (a) for
21a: BnBr, NaH, DMF, rt, 89%; (b) for 21b: EtI, NaH, DMF, rt, 92%; (c) for
22c: i-PrI, NaH, DMF, rt, 84%; (d) for 22a: (d) H2 (1 bar), 5% Pt/C, MeOH, rt,
82%; (e) for compounds 23b,c: H2 (1 bar), 5% Pd/C, MeOH, rt, 64-76%.
Cl
Cl
N
N
N
N
a
b
(HO)2B
N
N
H
O
Cl
R1
19a,b
R1 = NO2
R1 = NH2
18
12
6a
NO2
The synthesis of the trifluoroethoxy derivative 27 on the
same route failed due to the combination of low solubility of 20
and low reactivity of the alkylating agent. The successful
pathway is shown in Scheme 10. Alkylation of the readily soluble
phenol 23 followed by hydrogenation of the nitroarene provided
the substituted aniline 24. Its reaction with 4-nitrobenzaldehyde
(25) in the presence of montmorillonite KSF followed by
oxidation with MnO2 yielded the nitroarene 26. Pd-catalyzed
hydrogenation of the nitro group afforded the desired aniline 27.
a
b
d
Scheme 8. Synthesis of compounds 19a,b. Reagents and conditions: (a) PCl5,
POCl3, reflux, 41%; (b) Na2CO3, cat. Pd(PPh3)4, toluene/EtOH/H2O (15:1:4),
100 °C; (d) SnCl2·H2O, 1-propanol/chlorobenzene (1:1), 90 °C, 28% over 3
steps.
Table 2. Summary of Ring B Modifications. Measurements are reported as the
indicated values ± standard error, with each data set testing at least six
compound concentrations. NT = Not tested.
N
N
N
c
a, b
F3C
OHC
F3C
O
N
Cl
HO
NO2
O
NH2
R1
24
25
26 R1 = NO2
27 R1 = NH2
23
NO2
d
N
N
N
N
N
N
Scheme 10. Synthesis of compound 27. Reagents and conditions: (a)
F3CCH2I, K2CO3, DMF, 100 °C, 21%; (b) H2 (1 bar), 5% Pd/C, MeOH, rt, then
H2 (1 bar), 10% Pd/C, MeOH, rt, then H2 (1 bar), 20% Pd(OH)2/C, MeOH, rt,
56%; (c) montmorillonite KSF, toluene, reflux, then MnO2, CHCl3, reflux (53%);
(d) H2 (1 bar), 10% Pd/C, MeOH, rt, 88%.
R
R
R
13a-c
17a-c
19a-b
Max HR
Compound
13b
R
D-loop IC50 (μM)
5.10 ± 1.33
7.2 ± 13.6
> 100
Inhibition[a]
NH2
38 ± 5 %
Ring A substitution was further explored by synthesizing
the 6-bromo and 6-n-propyl derivatives as shown in Scheme 11.
The aniline 28 reacted with 4-nitrobenzaldehyde (25) in the
presence of montmorillonite KSF followed by further oxidation
with MnO2 to yield the nitroarene 29a. Selective reduction of the
nitro group with tin(II) chloride[37] produced the desired aniline
29b. Aryl bromide 29a was also converted to the allyl-substituted
intermediate 29c via Stille coupling with allyltributyltin. Pd-
catalyzed hydrogenation of both the nitro and allyl groups
afforded 29d.
13c
0 ± 0 %
21 ± 1 %
NT
17b
NH2
17c
62.2 ± 5.1
> 100
19b
NH2
NT
[a] HR inhibition observed at the maximum tested concentration of 40 μM.
We again utilized used the biochemical D-loop assay to
measure the ability of compounds to inhibit the homologous
strand exchange activity of purified human RAD51 protein, and
we used these results to determine which compounds to
advance into cell-based assays of HR activity. Results of these
5
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(Figure 2A). Taken together, this prompted us to question
whether some of these new analogs might promote off-target
activity by intercalating between dsDNA base pairs and affecting
DNA topology. This is an important consideration because
RAD51-catalyzed D-loops are much more stable when formed in
negatively supercoiled dsDNA than linear dsDNA.[38] We tested
this possibility by electrophoresing relaxed covalently-closed
circular dsDNA plasmids through agarose gels that were
impregnated with individual compounds. In this assay, DNA
intercalation activity is detected based on the accumulation of
positive supercoils in the plasmid (Figure 2B). The abundance of
these compound-induced topoisomers enables the quantification
of that compound’s intercalation activity. DMSO vehicle control
was defined as having a score of 0, and 10 μM chloroquine was
defined as generating a score of 1 (i.e. saturation of the
endpoint). Although this method has relatively low throughput, it
provides quantitative measurements that are highly sensitive
and reproducible. We also included compounds having D-loop
IC50 concentrations greater than 100 μM to measure possible
intercalation activity in compounds that shared the flat planar
structure of 2h but showed weak or non-detectable D-loop
inhibitory activity.
N
N
a
R1
N
OHC
Br
NH2
29a-d
25
28
R2
NO2
a R1 = Br; R2 = NO2
b R1 = Br; R2 = NH2
b
c
c R1 = CH2CH2=CH2; R2 = NO2
d R1 = CH2CH2CH3; R2 = NH2
d
Scheme 11. Synthesis of compounds 29b,d. Reagents and conditions: (a)
montmorillonite KSF, toluene, reflux, then MnO2, CHCl3, reflux, 85%; (b)
allyltributyltin, cat. Pd(PPh3)4, toluene, 110 °C, 54%; (c) SnCl2·H2O, PhCl/1-
propanol, 100 °C, 71%; (e) H2 (1 bar), 5% Pd/C, MeOH, rt, 83%.
We again utilized used the biochemical D-loop assay to
measure the ability of compounds to inhibit purified human
RAD51 protein and to inhibit HR activity in cells. Results of these
assays are displayed in Table 3, Supporting Information Figure
1, and Supporting Information Figure 2, showing that bulky,
hydrophobic groups on ring A resulted in a loss of D-loop
inhibitory activity (compare 1a to 22a, 22c, 27, and 29d) and that
smaller groups on ring A caused little change in biochemical or
cellular inhibitory activities (compare 1a to 22b, 29b).
A.
Relaxed
Relaxed
Relaxed
−Sc
−Sc
−Sc
B.
Table 3. Summary of Ring A Modifications. Measurements are reported as the
indicated values ± standard error, with each data set testing at least six
compound concentrations. NT = Not tested.
Nicked
Topo-
isomers
Super-
coiled
Super-
coiled
Super-
coiled
Super-
coiled
Nicked
Nicked
Nicked
Max HR
Compound
22a
R
D-loop IC50 (μM)
Inhibition[a]
> 100
35 ± 10 %
22b
22c
5.17 ± 0.71
> 100
33 ± 4 %
NT
27
> 100
16 ± 1 %
Figure 2. A) Two views of the 3D structure of 2h demonstrate its planar
geometry. B) Representative gel images are displayed for circular plasmids
(mixture of negatively supercoiled [−Sc] and relaxed covalently closed), which
were electrophoresed through agarose gels containing 4% DMSO alone, 10
μM 2h, or 10 μM chloroquine. Densitometry traces are displayed below each
gel. The positions of resulting topoisomers are denoted with blue color, and
the positions of nicked plasmids are shown in green. The degree of DNA
intercalation is denoted by the red line, indicating the median distance of
topoisomer migration relative to the DMSO control.
29b
29d
Br
12.1 ± 5.9
30.2 ± 3.9
24 ± 13 %
NT
[a] HR inhibition observed at the maximum tested concentration of 40 μM.
Identification of Off-Target DNA Intercalation Activity. We
initially prioritized compounds that inhibited RAD51’s D-loop
activity at least as well as the starting compound 2h for further
testing. Several of the above described analogs (2c, 8i, 8k, 10h,
and 10j) appear to elicit unusually strong inhibitory activities,
with IC50 values that approached the concentration of the
RAD51 protein (i.e. the intended target) in the biochemical
assay. Importantly, the starting chemical scaffold of this SAR
campaign exhibits a relatively flat planar central ring structure
Several of the compounds (2c, 8i, 8k, 10h, and 10j) were
found to exhibit significant off-target dsDNA intercalation (Figure
3A, red box). As predicted, these compounds also generate high
levels of inhibition of the D-loop assay, with IC50 values that
approached the concentration of the RAD51 target.
Furthermore, these compounds also highly toxic to cells, with
LD50 values in the 5 – 33 μM range. A second subgroup of sub-
6
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optimal compounds (Figure 3A, blue box) exhibited low
intercalation into dsDNA but a poor ability to inhibit RAD51 in the
D-loop assay; these compounds were similarly deemed as not
suitable for further development.
24 hours of the assay. Among the subset of the 19 best
compounds, 2i and 10e demonstrated a superior ability to inhibit
HR events in the second 24 hours of the assay (Figure 4B). Both
compounds exert a nearly linear dose-dependent inhibition of
HR, approaching complete inhibition at with concentrations in
the 30-40 μM range. Finally, both 2i and 10e showed cellular
LD50 values (11.3 ± 0.7 μM for 2i and 24.3 ± 1.3 μM for 10e) that
were proportional to their HR inhibition IC50 values at 48 hours
following DSB induction (10.9 ± 5.6 μM for 2i and 15.8 ± 8.5 μM
for 10e).
Among remaining 19 compounds (Figure 3A, green box),
we found no significant correlation between intercalation score
and their ability to inhibit D-loop formation (p=0.17, Pearson
product-moment correlation). Furthermore, no significant
correlation exists between intercalation score and compound
toxicity (Figure 3B). By contrast, compound-induced cellular
toxicity does significantly correlate with compounds’ ability to
inhibit D-loop formation and to reduce HR proficiency in cells.
Taken together, these results support the conclusion that cell-
based toxicity for this optimized collection of compounds
represents an on-target effect.
A.
A.
B.
B.
Figure 3. A) Sub-optimal analogs exhibit either off-target dsDNA intercalation
(red box) or poor ability to inhibit RAD51 in the D-loop assay (blue box). For
the remaining 19 compounds (green box), no significant relationship is
observed between intercalation score and ability to inhibit D-loop formation
(Pearson product-moment correlation). B) Inhibition of RAD51’s D-loop activity
and cellular HR activity, but not intercalation activity, correlate with toxicity in
293-HEK cells (Significance determined by Kendall’s tau correlation).
Figure 4. A) Inhibition of HR activity in 293-HEK cells by 2h at 24 hours or 48
hours following DSB induction. B) Inhibition of HR activity by 2i and 10e at 48
hours following DSB induction. Individual data points from at least four
independent experiments are plotted along with fitted four-parameter log-
logistic curves.
Optimized Analogs Offer Improved Ability to Inhibit HR in
Cells. We used the DR-GFP assay to test the ability of select
compounds to inhibit HR in human cells.[35] Briefly, cells
containing the chromosomally-integrated DR-GFP reporter were
transfected with a plasmid that expresses I-SceI, a rare-cutting
A defining feature of this class of compounds is their ability
to inhibit RAD51’s D-loop activity while having minimal effects on
RAD51’s ssDNA binding activity.[21] Therefore, additional
experiments were performed to confirm that the optimized
analogs 2i and 10e had not gained the ability to interfere with
RAD51’s binding to ssDNA. Using gel shift assays, we
confirmed that neither of these compounds affected the
formation or stability of RAD51-ssDNA nucleoprotein filaments
on ssDNA (Figure 5). By comparison, the more generalized
RAD51 inhibitor RI-1 (which prevents binding of RAD51 to
endonuclease that generates
a DSB within the DR-GFP
cassette. Compounds were added at the time of transfection,
and the cells were outgrown for 48 hours thereafter. Cells
capable of successfully performing HR-mediated DSB repair are
quantified based on their expression of functional green
fluorescent protein (GFP).
ssDNA)
eliminates
the
formation
of
RAD51-ssDNA
As previously reported, the lead compound 2h effectively
inhibits HR in the first 24 hours of the DR-GFP assay.[21]
However, 2h-treated cells regain some HR activity in the second
24 hours (Figure 4A), which is highly relevant since the majority
of I-SceI-dependent conversion events occur within the second
nucleoprotein filaments.[17] Taken together, these results confirm
that optimized drug candidates 2i and 10e act to specifically to
prevent the D-loop formation activity of RAD51.
7
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A.
B.
their oncologic effects over prolonged time intervals. An
additional challenge that arose during this SAR campaign was
the introduction of off-target toxicity, in which some analogs had
ability to intercalate into dsDNA. Having now overcome these
limitations with the optimized compounds 2i and 10e, our future
work will be focused on additional synthetic schemes to improve
upon potency.
There is a growing interest in RAD51 as a potential target
in cancer therapy. Figure 6 shows some of the RAD51-inhibitory
compounds in development by our group and others. The first
RAD51 inhibitors developed by our group, RI-1 (41) and RI-2
(42), represent a class of compounds that block RAD51-ssDNA
C.
D.
nucleoprotein filament formation and subsequent strand
17]
exchange.[14,
Other compounds in this class include DIDS
(43), IBR 120 (44), B02 (45), and a recently-developed analog of
45 with greater single-agent potency against tumor cell lines.[18,
20, 42-43]
Halenaquinone (46), 2h, 2i, and 10e belong to a second
class of compounds purported to target strand exchange while
permitting the binding of RAD51 to ssDNA.[21, 44]
These findings represent
a major advance in the
development of RAD51-targeting small molecules. To our
current knowledge, our series of compounds remain the only
known chemicals capable of inhibiting RAD51’s D-loop activity
without interfering with its ability to bind ssDNA, both in
biochemical systems and in cells. Further work is necessary to
determine which clinical scenarios will be best suited for this
therapeutic approach. Specifically, we are working to define the
optimal classes of DNA-damaging therapies that will offer
maximal synergy in combination with these novel compounds.
Furthermore, we are exploring the utility of these agents as
single-agent treatments in a specific susceptible tumor subtype.
Finally, these compounds present a novel set of tools for basic
scientists who wish to study the distinct functions of RAD51.
Figure 5. RAD51 binding to ssDNA binding is inhibited by RI-1, but not by
optimized compounds 2i or 10e. Representative agarose gels are shown with
data plotted from three independent experiments. Error bars denote the
standard error of the mean.
Conclusions
The over-expression of the RAD51 recombinase protein
represents a common hallmark of malignancy, and tumor cells
are especially dependent upon the HR function of RAD51.
Taken together, RAD51 is a highly attractive therapeutic target
in oncology.[15-16, 18, 39] In addition to its classical role in catalyzing
homologous strand exchange, RAD51 additionally plays a
central role in protecting stalled replication forks from nucleolytic
degradation.[3-4] This protective function in replication requires
that RAD51 is able to bind ssDNA, however it may not require
the strand invasion activity of RAD51. Therefore, our drug
development strategy aims to inhibit RAD51’s D-loop activity
while sparing its ssDNA binding activity.
The role of RAD51 and other HR proteins in replication
fork remodeling during replication stress has been an intense
topic of research in recent years. Further work is underway to
mechanistically dissect the effect of our inhibitory compounds on
RAD51 at the level of the replication fork. However, such effects
may be challenging to investigate using typical nascent DNA
resection assays. This is because MRE11-dependent nucleolytic
degradation of the nascent DNA at stalled forks requires
preceding replication fork reversal, and this reversal process
requires RAD51 protein.[40-41] Therefore, compounds that permit
RAD51-ssDNA binding are expected to permit fork reversal and
to stabilize forks from MRE11-dependent nuclease degradation.
Such an effect would be difficult to distinguish from that caused
by total loss of RAD51 activity, in which fork reversal never
occurs.
Figure 6. Chemical structures for several RAD51 inhibitors.
Experimental Section
Cells, Media, and Proteins. The 293-HEK cell line containing the
chromosomal DR-GFP reporter cassette along with the I-SceI expressing
pCBASce and pCAGGS empty vector control plasmids were kindly
provided by Jeremy Stark and have been described previously.[35] All cell
lines were maintained in complete DMEM (DMEM + 4.5 g/L D-glucose +
L-glutamine + 10% fetal calf serum + penicillin/streptomycin) and verified
to be free of mycoplasma by monthly testing. Human RAD51 protein was
purified as described previously.[45]
Our initial efforts had identified lead compound 2h, which
satisfied our broad goals.[21] However, 2h proved to be relatively
ineffective at blocking cellular HR events at time points later than
24 hours. This feature of 2h limits its potential utility in clinical
settings, given that many common oncologic therapies exert
8
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D-Loop Assay. Human RAD51 protein in reaction buffer was combined
with compound in DMSO in a total volume of 8 µL, incubated at 37 ºC for
10 minutes. Then 1 µL of 5′-32P-labeled or 5’-Cy5-labeled 90-mer ssDNA
(5′-TACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGT-
TTGAAGCAGGCGGCAGAAGAAGTAACAAAGGAACCTAGAGGCCTT-
TT) with homology to the plasmid pRS306 was added, and the reaction
was incubated at 37 ºC for an additional 5 minutes. Then 1 µL of
supercoiled pRS306 dsDNA plasmid was added, and the reaction was
incubated at 37 ºC for 20 minutes. At this point, reactions containing
human RAD51 protein consisted of 25 mM HEPES-NaOH (pH 7.0), 1
mM ATP, 1 mM MgCl2, 1 mM DTT, 100 µg/mL BSA, 4.5% DMSO, 0.5
µM RAD51, 11 mM NaCl, 1% glycerol, 20 nM ssDNA, and 5 nM pRS306
dsDNA. Reactions using the 5′-32P-labeled ssDNA contained 10 nM
ssDNA oligonucleotides and those using the 5’-Cy5-labeled ssDNA
contained 20 nM ssDNA oligonucleotides; there was no difference in the
ability of compounds to inhibit D-loops between these conditions (data
not shown). The reactions were de-proteinized with 0.8% SDS and 0.8
mg/mL proteinase K, mixed with gel loading buffer, and run on a 0.9%
agarose / 1X TAE gel. The gel was dried under vacuum on a positively-
charged nylon membrane for 2 hours at 80 °C, exposed to a phosphor
screen overnight, and imaged on a Storm 860 or Typhoon 9200 scanner
(Molecular Dynamics). Quantitation of free oligo and D-loop bands was
performed using ImageJ software (NIH, Bethesda, MD).
ATP, 5 mM MgCl2, 1 mM tris(2-carboxyethyl)phosphine, 100 μg/mL BSA,
0.5 μM RAD51, 1.5 μM nucleotide concentration ssDNA, and 4.5%
DMSO. RAD51-ssDNA complexes were then fixed by addition of 2 μL
glutaraldehyde in water to 0.25%, incubated for 5 minutes at 37 °C, and
run on a 1% agarose / 1X TAE gel, which was stained in SYBR Gold
(Molecular Probes) and imaged using
(Molecular Dynamics).
a Typhoon 9200 scanner
Statistical Analysis. All statistical analyses and graphing were
performed using R.[46] LD50, IC50, and maximum inhibition values and
their standard errors were determined by fitting four-parameter log-
logistic curves to the data by least-squares nonlinear regression followed
by inverse estimation using the ‘drc’ package.[47] Baseline correction for
densitometry plots was performed using the ‘baseline’ package.[48]
Supporting Information. Synthetic procedures for compounds 1a – 40
can be found in the Supporting Information on the journal’s website.
Acknowledgments
This work was funded by NIH grant 2R01CA142642 to P.P.C.
and A.P.K. The Bruker Avance III 400 NMR spectrometer was
supported by NSF grant CHE-1048642, and the Bruker Avance
III 500 NMR spectrometer by a generous gift from Paul J. and
Margaret M. Bender (both to the University of Wisconsin at
Madison Department of Chemistry). The purchase of the
Thermo Scientific Q Exactive Plus Orbitrap mass spectrometer
was funded by NIH Award 1S10 OD020022-1 to the University
of Wisconsin at Madison Department of Chemistry.
Quantitation of DNA Repair Efficiency in Cells. 293-DR-GFP cells
were electroporated in Opti-MEM with 37.5 µg/mL of the I-SceI
endonuclease bearing pCBASce plasmid or the pCAGGS empty vector
control in 0.4 cm cuvettes at 325 V, 975 µF and seeded into 6-well plates
with 2.5 mL complete DMEM + 0.5% DMSO with compound and allowed
to outgrow for the indicated time. Following outgrowth, cells were
harvested, suspended in PBS with 1 µg/mL 7-aminoactinomycin D (7-
AAD), and analyzed on a BD LSRII flow cytometer. Dead and apoptotic
cells were gated out based on size, shape, and 7-AAD staining. The
fraction of GFP-positive cells was determined within the population of live
cells.
Keywords: Biological activity • Cancer • DNA repair • Inhibitors •
Structure-activity relationships
Intercalation Assay. Negatively supercoiled pRS306 plasmid DNA was
prepared by standard CsCl gradient purification. Relaxed covalently-
closed circular (rCCC) plasmid DNA was prepared from negatively
supercoiled plasmid DNA by treating with 1.33 units E. coli
topoisomerase I (New England Biolabs) per microgram plasmid DNA at
37 °C for 30 minutes followed by phenol-chloroform extraction and
ethanol precipitation. One-hundred and fifty nanograms of negatively
supercoiled or rCCC plasmid were loaded along with linear DNA size
markers in immediately adjacent lanes into a 0.9% agarose 1X TAE gel,
with 4% DMSO ± 10 μM compound present throughout the gel and the
running buffer. The gel was run at 5 V/cm until the xylene cyanol FF dye
had migrated 5 cm from the well, stained with ethidium bromide, and
photographed under UV transillumination. Densitometry traces were
obtained for each lane using ImageJ, and intercalation scores were
calculated from the lane loaded with rCCC plasmid as the median
distance migrated by topoisomers for each compound relative to the
DMSO-only control from the same experiment.
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RAD51-DNA binding assay. Human RAD51 protein in reaction buffer
was combined with compound at the indicated concentration in DMSO in
a total volume of 9 μL, incubated at 37 °C for 40 minutes, then 1 μL of
4,373-nt closed circular pRS306 virion ssDNA substrate was added, and
the reaction was incubated at 37 °C for an additional 5 minutes. At this
point, the reaction consisted of 25 mM HEPES-NaOH (pH 7.2), 3 mM
9
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Entry for the Table of Contents
We previously described a small-molecule inhibitor of RAD51, which inhibits D-loop activity while sparing the ssDNA binding function.
The resulting lead compound, however, was limited in its ability to completely inhibit HR in vivo. Here we employed a structure-
activity-relationship (SAR) campaign that led to more potent analogs.
11
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