Design, synthesis and structure-activity relationship study of novel urea compounds as FGFR1 inhibitors to treat metastatic triple-negative breast cancer

Article abstract of DOI:10.1016/j.ejmech.2020.112866

Triple-negative breast cancer (TNBC) is an aggressive type of cancer characterized by higher metastatic and reoccurrence rates, where approximately one-third of TNBC patients suffer from the metastasis in the brain. At the same time, TNBC shows good responses to chemotherapy, a feature that fuels the search for novel compounds with therapeutic potential in this area. Recently, we have identified novel urea-based compounds with cytotoxicity against selected cell lines and with the ability to cross the blood-brain barrier in vivo. We have synthesized and analyzed a library of more than 40 compounds to elucidate the key features responsible for the observed activity. We have also identified FGFR1 as a molecular target that is affected by the presence of these compounds, confirming our data using in silico model. Overall, we envision that these compounds can be further developed for the potential treatment of metastatic breast cancer.

Full text of DOI:10.1016/j.ejmech.2020.112866

Journal Pre-proof
Design, synthesis and structure-activity relationship study of novel urea compounds
as FGFR1 inhibitors to treat metastatic triple-negative breast cancer
Md Ashraf-Uz-Zaman, Sadisna Shahi, Racheal Akwii, Md Sanaullah Sajib,
Mohammad Jodeiri Farshbaf, Raja Reddy Kallem, William Putnam, Wei Wang,
Ruiwen Zhang, Karina Alvina, Paul C. Trippier, Constantinos M. Mikelis, Nadezhda A.
German
PII:
S0223-5234(20)30838-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112866
EJMECH 112866
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 July 2020
Revised Date: 13 September 2020
Accepted Date: 16 September 2020
Please cite this article as: M. Ashraf-Uz-Zaman, S. Shahi, R. Akwii, M.S. Sajib, M.J. Farshbaf, R.R.
Kallem, W. Putnam, W. Wang, R. Zhang, K. Alvina, P.C. Trippier, C.M. Mikelis, N.A. German, Design,
synthesis and structure-activity relationship study of novel urea compounds as FGFR1 inhibitors
to treat metastatic triple-negative breast cancer, European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.112866.
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2020 Published by Elsevier Masson SAS.

Design, synthesis and structure-activity relationship study of novel urea compounds as FGFR1
inhibitors to treat metastatic triple-negative breast cancer
1
1
1
1
5
Md Ashraf-Uz-Zaman , Sadisna Shahi , Racheal Akwii , Md Sanaullah Sajib , Mohammad Jodeiri Farshbaf ,
3
3
2
2
4,5,6
7,8,9
Raja Reddy Kallem , William Putnam , Wei Wang , Ruiwen Zhang , Karina Alvina , Paul C. Trippier
,
1
1,4.*
Constantinos M. Mikelis , Nadezhda A. German
.
1
Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences
Center, Amarillo, TX, USA.
2
College of Pharmacy, University of Houston, Houston, TX, USA.
3
Clinical Pharmacology & Experimental Therapeutics Center, Texas Tech University Health Sciences Center, Dallas, TX,
USA.
4
Center of Excellence for Translational Neuroscience and Therapeutics, Texas Tech University Health Sciences Center,
Lubbock, TX, USA
5
Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA
6
Department of Neuroscience, University of Florida, Gainesville, FL, USA
7
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE,
USA
8
Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA.
9
UNMC Center for Drug Discovery, University of Nebraska Medical Center, Omaha, NE, USA.
*
Corresponding author, Tel: 806-414-9190, email: nadezhda.german@ttuhsc.edu
1

ABSTRACT
Triple-negative breast cancer (TNBC) is an aggressive type of cancer characterized by higher metastatic and
reoccurrence rates, where approximately one-third of TNBC patients suffer from the metastasis in the brain. At
the same time, TNBC shows good responses to chemotherapy, a feature that fuels the search for novel
compounds with therapeutic potential in this area. Recently, we have identified novel urea-based compounds
with cytotoxicity against selected cell lines and with the ability to cross the blood-brain barrier in vivo. We have
synthesized and analyzed a library of more than 40 compounds to elucidate the key features responsible for
the observed activity. We have also identified FGFR1 as a molecular target that is affected by the presence of
these compounds, confirming our data using in silico model. Overall, we envision that these compounds can
be further developed for the potential treatment of metastatic breast cancer.
INTRODUCTION
Breast cancer is the second leading cause of cancer-related death in women. Despite the tremendous
advances in cancer treatment in recent decades, over 42,000 women were expected to die of breast cancer in
1
2
020. Triple-negative breast cancer (TNBC) is the subset of breast cancer that is characterized by the
absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2
2
3
(
HER2). It accounts for approximately 15–20% of all breast cancer diagnoses, and has been categorized as
4
the most aggressive subtype, correlated with poor prognosis. Triple-negative tumors possess a higher risk of
developing distant metastases, recurrence, and lower five-year survival compared with other subtypes of
5
, 6
breast cancer. TNBC has a unique propensity for metastasis to the lung and brain that contribute to its lower
7
survival rates.
In the absence of viable targets for the endocrine and HER-2 targeted therapies, chemotherapy is the
2
mainstream treatment for TNBC. Several retrospective studies have concluded that chemotherapy, in
combination with whole-brain radiation therapy, gives the most prolonged median survival. At the same time,
8
9
CALGB trial and WSG AM-01 study emphasized the benefits of dose-dense and dose-intensive
chemotherapeutic regimens in the treatment of TNBC. A retrospective analysis of trastuzumab treatment has
identified that this otherwise potent anticancer agent has increased brain metastasis incidents at a rate of
10
1
2.6% to 34%. The reason for such an increase is seen as “driving” cancer cells to the brain as a sanctuary
2

site, suggesting the treatment of TNBC would be more successful with the use of chemotherapeutic agents
with the ability to cross the blood-brain-barrier (BBB). Most available chemotherapies are not able to cross the
11, 12
BBB, not even if the barrier is disrupted by tumor invasion.
As a result, there is an ongoing drive to develop
novel chemotherapeutic agents that are BBB-permeable for the treatment of TNBC patients.
We have targeted the optimization of penfluridol (PFL), an antipsychotic drug, for potential anticancer use. This
compound demonstrates antitumor properties in various cancer cell lines such as breast, pancreatic,
13-16
glioblastoma, and lung cancer cells.
The anticancer effects of PFL were further confirmed in vivo in a
17
variety of models, including the metastatic heart to brain TNBC model. Therefore, penfluridol has the
18, 19
potential to be repurposed as a novel antitumor agent.
Moreover, the ability of PFL to cross the BBB,
makes it an attractive candidate to develop anticancer therapeutics to treat brain metastasized cancers.
However, this compound has extensive interactions with the majority of G-protein coupled receptors (GPCRs)
20
(
Fig. 2A) at levels that correspond to the proposed anticancer dosing (estimated as 50 mg of daily dosing in
14, 16, 17, 21
human
). Thus, an off-target CNS activity of penfluridol as an anticancer agent will lead to intensified
22-25
neurological side effects associated with this agent.
Figure 1. Search for a new hit molecule using scaffold hopping approach. IC50 indicates compound
concentration required to inhibit viability of the MDA-MB-231 triple-negative breast cancer cells by 50%. Data
are expressed as the mean of three independent experiments, each performed in sextet.
Our group has previously reported the employment of PFL as the hit molecule that can be further optimized for
20
its anticancer properties and the removal of GPCR-related activity. We observed that the 4-hydroxypiperidine
3

26, 27
moiety that was previously shown to be important for its cytotoxicity
is also essential for the interaction with
GPCRs. Here, we report our next step in the optimization of the PFL structure, where a topology-based
28-31
scaffold hopping approach
was used to identify the second generation of analogs with the retained
anticancer activity and alleviated affinity to the GPCRs. To achieve this goal, a set of six diverse chemical
fragments was designed to replace the substituted 4-hydroxy-piperidine moiety of PFL (hereafter referred to as
the “head” moiety). Selected structural features of the original “head” moiety, such as the presence of a phenyl
ring and abundance of heteroatoms, were incorporated in the design of new fragments. The diphenyl-butyl
2
0, 26
chain (hereafter referred to as the “tail” moiety) was kept unchanged due to its auxophoric character.
molecules were evaluated for their anticancer properties using cytotoxicity assay (see supplemental materials)
Fig. 1), where compound 4a (IC50 = 7.7 M) was identified as our new “hit” molecule for further evaluation.
In the next step, compound 4a was assessed for the ability to interact with the GPCRs expressed in the brain
Fig. 2B). We found a significant reduction in the interaction of 4a with the GPCRs, where only six receptors
All six
(
μ
(
were inhibited at the level above 50%. Under the same test conditions, PFL was shown to inhibit 28 GPCRs at
the level above 50%. Previously, it has been demonstrated that neurological side effects of PFL are associated
24, 32, 33
with the inhibition of serotonin and dopamine receptors.
Our hit compound (4a) had no significant
interaction with the dopamine receptors (subtypes D1-D5) at 10 µM concentration. Similarly, it has no activity
at serotonin receptors, except for 5HT-2C, which is a subtype with low expression levels, and its potential role
in CNS-induced
Figure 2. Inhibitory activity of penfluridol (A) and compound 4a (B) at selected CNS receptors. Primary binding
assay was performed at 10
μM concentration and data are expressed as the mean ± SEM of four independent
4

experiments. The red dotted line identifies groups of the receptors being inhibited by >90% and by >50% in the
presence of these compounds.
34
toxicity can be considered insignificant. Thus, for compound 4a, only inhibition of the dopamine transporter
DAT) and histamine 1 receptor (H1R) was noted as sources of possible neurotoxicity.
Further, we evaluated compound 4a for its ability to cross the BBB in vivo, where mice were injected with a
(
35
dose of 10 mg/kg through intraperitoneal (i.p.) administration and sacrificed at selected time points. We
observed that 4a reaches the peak values of approximately 1000 nM at 1-hour post-injection (Fig. 3), similarly
36
to the data found for PFL, confirming its retained activity to cross the BBB. In summary, our scaffold-hopping
design identified compound 4a with cytotoxicity against MDA-MB-231 cells retained the ability to cross the BBB
in vivo and limited inhibitory activity on GPCRs. Hence, we envisioned that this compound can be further
evaluated as an anticancer agent with the potential therapeutic application in the treatment of the metastatic
TNBC. Our current report presents an analysis of the performed structure-activity relationship studies, where a
library of 45 analogs was prepared and tested for the anticancer activity. In addition, we investigated potential
unwanted overlap in structural requirements for the cytotoxic activity of 4a and its ability to interact with the
DAT.
Figure 3. Distribution of 4a in plasma and brain. Concentration (nM) of compound 4a in plasma and brain at 0,
0.5, 1, 6, 24, and 48 hours after single i.p. administration (10 mg/kg) was measured by liquid chromatography-
tandem mass spectrometry. Data are expressed as the mean ± SEM of 2-4 independent experiments.
CHEMISTRY
5

The synthesis of compounds designed for the scaffold-hopping study is depicted in scheme 1. We have
coupled commercially available 4-fluorobenzaldehyde (30), and 4-chlorophenol (31) afforded aryl ether 32
(
Scheme 1A) which was subsequently reduced in the presence of lithium aluminum hydride to yield
intermediate alcohol 33 and was further coupled with diphenyl alkyl intermediate 27a (scheme 2) to access
3
7, 38
compound 2.
Commercially available phenol 34 was protected by benzylation (35), followed by base-
3
9
mediated hydrolysis of the ester functionality to obtain carboxylic acid intermediate 36 (Scheme 1B). A
40
Steglich esterification successfully afforded compound 3. Chloro-phenyl isocyanate 48a was coupled with
diphenyl alkyl intermediate 27a to afford urea compound 4a (Scheme 1C). A condensation reaction between
glycerol (37) and benzaldehyde (38) under acidic conditions afforded 2-phenyl,1,3-dioxane-2-ol 39 (Scheme
4
1
42
1
D), that was coupled with 27a to form compound 5. Synthesis of analog 6 (Scheme 1E) began with the
43
Buchwald-Hartwig coupling of aryl iodide 41 using 1,1′-bis(diphenylphosphino)ferrocene (DPPF) as a catalyst
44
to afford diphenylamine intermediate 42. Next, coupling with 27a afforded compound 6. Finally, key
6

4
5
intermediate 45 was obtained by sulfonylation of piperazine (44) with substituted arylsulfonyl chloride 43, and
26
was further coupled with the tail moiety 27a to form compound 7 (Scheme 1F).
Scheme 1. Synthesis of compounds selected for a scaffold-hopping study. Reagents and conditions: (a) Cs CO , DMF,
2
3
8
5 ºC; (b) LiAlH , THF, rt; (c) 27a, NaH, DMF, 80 °C; (d) Benzyl bromide, K CO , DMF, rt; (e) KOH, ethanol, reflux; (f)
4 2 3
2
5b, DCC, DMAP, DCM, rt to 32 ºC; (g) 29a, DCM, 0 °C to 32 °C; (h) conc. H
(dppf)• CH Cl , KOtBu, anhydrous THF, 100 ºC; (k) 27a, NaH, DMF, rt; (l) DIPEA, DCM, rt; (m) 27a, Na
KI (cat.), acetonitrile, reflux.
2
SO
4
, 85 ºC; (i) 27a, NaH, DMF, rt; (j) dppf
(
cat.), PdCl
2
2
2
2
CO
3
,
7

The preparation of diphenyl alkyl intermediates (“tail” moieties) that were used to synthesize the final
compounds depicted in schemes 1 and 3-6 is outlined in scheme 2. Suitably substituted aryl bromides (21a-e)
were converted to the corresponding Grignard reagents, which underwent a double addition reaction with the
respective lactones 22a-c to afford dihydroxyl intermediates 23a-g. Acid-catalyzed dehydration of the tertiary
alcohol afforded unsaturated aliphatic alcohols 24a-g. Intermediates 26a-g were obtained by subjecting
46
alcohols 24a-g to Appel conditions. A palladium-carbon mediated hydrogenation reaction was utilized to
26
obtain saturated intermediates 25a-c, 27a-e, and 27g-h. Compound 27f was prepared from 27e using
47, 48
modified nitration conditions to avoid unwanted ortho-substitutions.
Aliphatic bromides 27a-h were
converted to primary amines 29a-h by treatment with ammonia or by Staudinger reduction via the azide
49, 50
intermediates 28a, d-f.
Scheme 2. Synthesis of diphenyl alkyl chain intermediates. Reagents and conditions: (a) Mg, I , THF, reflux; (b) EtOH,
2
HClconc, reflux; (c) CBr , Ph P, DCM, 0 °C to rt; (d) Pd/C (cat.), H (80 psi), EtOH, rt; (e) NaN acetone: water (2.2:1), rt; (f)
4
3
2
3,
Ph P, anhydrous ether, water; (g) NH OH, dioxane, 100 °C; (h) conc. H SO , conc. HNO , -15 °C.
3
4
2
4
3
8

Synthesis of a subgroup of analogs with substitutions at the “head” moiety is depicted in Scheme 3. Diphenyl-
51
alkyl amine 29a (Scheme 2) was coupled with freshly prepared isocyanates 48a-q, to obtain desired urea
compounds 4a-d, 4g-m, and 4p-u. Amino substituted derivatives 4e, and 4n were prepared using standard
Raney-Ni-catalyzed reduction conditions from the respective nitro-precursors 4d and 4m. They were further
acetylated to afford analogs 4f and 4o. Prior to being converted to isocyanates 51a,b via exposure to
52
53
54
triphosgene, benzoic acid 49 was protected as the methyl, or benzyl, ester (Scheme 3B). Coupling of
55
5
1a,b with 29a afforded analogs 4v-w. The final compound in this scheme, acid 4x, was obtained by
reductive deprotection of the benzyl ester of 4w.
Scheme 3. Synthesis of analogues with substitutions at monophenyl “head” moiety. Reagents and conditions: (a)
triphosgene, DCM, rt; (b) 29a, DCM, 0 °C to 32 °C; (c) H N-NH , Raney-nickel, ethanol, 50 °C; (d) acetic anhydride, DCM.
2
2
(
(
e) SOCl , MeOH, reflux; (f) benzyl bromide, K CO , DMF, 100 ºC; (g) triphosgene, DCM, r.t; (h) 29a, DCM, 0 °C to 32 °C;
2
2
3
i) Pd/C, NaBH , MeOH.
4
9

Analogs with varied substitution at the diphenyl (“tail”) moiety were synthesized according to Scheme 4. The
diphenyl-alkyl amine intermediates 29b-f were coupled with the freshly prepared selected isocyanates 48a and
4
8j-k to produce desired urea compounds 8a-g. Raney-Ni catalyzed reduction was utilized to make compound
h, which was converted to an acetylated analog 8i. To prepare N-linked diphenyl analogs, 9a-e diphenyl
8
amine 42 was coupled with butyl bromide to obtain 52, which was further converted to the primary alkylamine
53. Final compounds 9a-e were synthesized using 53 and freshly prepared isocyanates 48a, 48b, 48j, 48n,
and 48q (Scheme 4B). Synthesis of the last analog (Scheme 4C), compound 10, began with the reduction of 4-
56
fluorophenyl valeric acid (54) with LiAlH using a modified literature procedure. The resultant primary alcohol
4
55 was converted to the desired analog 10 in three subsequent steps involving Appel reaction, amination, and
urea formation.
10

Scheme 4. Synthesis of analogs with substitution at diphenyl (“tail”) moiety. Reagents and conditions: (a) 48a, 48j-k,
DCM, 0 °C to 32 °C; (b) H N-NH , Raney nickel, ethanol, 50 °C; (c) acetic anhydride, DCM; (d) NaH, 1, 4-dibromobutane,
2
2
THF, rt; (e) NH OH, dioxane, 100 °C; (f) 48a, 48b, 48j, 48n, 48q, DCM, 0 °C to 32 °C; (g) LiAlH , 1N HCl, THF; (h) CBr ,
4
4
4
Ph P, DCM, 0 °C to rt; (i) 48j, DCM, 0 °C to 32 °C.
3
Scheme 5. Synthesis of analogs with structural modifications at the linker motif. Reagents and conditions: (a) 48j, DCM, 0
C to 32 °C; (b) DCM, 0 °C to 32 °C; (c) TEA, CCl , rt.
°
4
Several analogs bearing modifications of the linker chain were prepared using conditions outlined in Scheme 5.
Alterations in the length of the linker were achieved by reacting intermediates 29g or 29h with isocyanide 48j.
The similar synthetic procedure was employed for the preparation of the thiourea analog 12 (Scheme 5B),
where thioisocyanate 57 was used as starting material and for the introduction of the carbamate bioisostere 13
57
(
Scheme 5C), where primary alcohol 25a reacted with isocyanate 48j under basic condition.
11

Scheme 6. Synthesis of analogs containing methylated urea functionality and analogs with the amide linker. Reagents
and conditions: (a) Boc anhydride, TEA, DCM; (b) NaH, CH I, DMF; (c) TFA, DCM; (d) 48j, DCM. 0 °C to 32 °C; (e) NaH,
3
CH I, DMF; (f) NaHCO , isocyanuric chloride, TEMPO (cat.), NaBr (cat.), water: acetone (1:3.5), 0 °C to 25 °C; (g) i) oxalyl
3
3
chloride, DMF, ii) 47j, TEA, DCM; (h) oxalyl chloride, DMF (cat.), DCM, rt, 1 hr; (i) 29a, TEA, DCM, rt, 12 hrs.
Scheme 6 depicts reaction conditions for the preparation of mono- and dimethyl substituted urea analogs 14
58,57
(
Scheme 6A) and 15 (Scheme 6B).
In addition, it describes final modifications of the linker performed in this
study, such as the synthesis of N’-phenyl and C’-phenyl amides 16 (Scheme 6C) and 17 (Scheme 6D). To
59
prepare 16, primary alcohol 25c was oxidized under metal-free conditions, to afford acid 59, which was
60
subsequently activated as the acid chloride and coupled with substituted aniline 47j.
Analog 17, was
60
accessed by the acid chloride of free acid 60 reacting with diphenyl alkyl amine 29a.
To evaluate the effect of a constrained linker on the anticancer activity of compounds, we prepared analogs 18
52, 61, 62
and 19a-b following reported literature procedures.
As depicted in scheme 7, Grignard reaction of
substituted Boc-protected piperidine 61a-b with 4-fluorophenylmagnesium bromide afforded tertiary alcohol 62.
This compound underwent simultaneous dehydration and Boc-deprotection in the presence of HClconc to obtain
unsaturated intermediates 63a-b. Palladium assisted hydrogenation conditions were used to convert 63b to 4-
substituted piperidine intermediate 64b. Final compounds 18 and 19b were synthesized by reacting an
isocyanate with the respective piperidine intermediates 64a and 64b, whereas the preparation of 19a required
treatment of 62a with trifluoroacetic acid and NaBH to prepare piperidine 64a prior to the coupling reaction.
4
Scheme 7. Synthesis analogues with cyclic alkyl chain. Reagents and conditions: (a) THF, reflux; (b) HClconc, EtOH,
reflux; (c) NaBH , TFA, DCM, 0 °C to rt; (d) H (80 psi), Pd/C (cat.), EtOH; (e) 48j, DCM, rt
4
2
12

RESULTS AND DISCUSSION
In vitro cytotoxic activity of synthesized analogs
In our SAR studies, we have used a fragment-based approach to design and synthesize a series of 49 analogs
of the hit compound 4a that were further evaluated for cytotoxic potential using the MDA-MB-231 triple-
negative breast cancer line (see supplemental materials). The structural modifications were performed in three
areas of the hit compound: monophenyl (“head”) moiety, urea linker, and diphenyl (“tail”) moiety (Fig. 4). Thus,
we have grouped our in vitro data according to the site of the performed modification.
Figure 4. Areas of the proposed structural modifications.
Modifications in the mono-phenyl “head” moiety and their effect on the cytotoxicity of analogs. As shown in
table 1, a mono-substitution pattern favors the presence of an electron-withdrawing group located meta- to the
urea moiety (compound 4g, 4i-4k) or strong electron-withdrawing functionality at the para-position (4h).
Electron-donating groups in both para- (4c, 4e) and meta- (4k) positions cause abrogation of cytotoxic activity.
Interestingly, the presence of a hydrogen-bond acceptor is well tolerated in the meta -position (4g vs. 4j), but
causes increase in IC50 values in compounds bearing mono para-substitution (4d vs. 4h). We observed that
increase in the size of the para-substituents leads to decreased (4b) or complete loss (4f) of cytotoxicity.
Another factor that might lead to the observed changes is the loss in the electron-withdrawing properties of
substituents when moving from 4d to 4a to 4b, and, finally, to 4f.
Table 1. In vitro cytotoxic activity of analogs with the mono-substitution at the “head” moiety.
13

MDA-MB-231
MDA-MB-231
R
R
a
a
IC50 (µM)
IC50 (µM)
4
a
b
7.68 ± 0.21
4g
4h
3.38 ± 0.08
2.78 ± 0.09
4
11.79 ± 0.40
4c
4d
4e
>20.00
6.47 ± 0.84
>20.0
4i
4j
5.07 ± 0.33
3.79 ± 0.22
>20.0
4k
4
f
>20.0
a
Data are expressed as the mean ± SEM of three independent experiments, each performed in a
quartet.
Given that electron-withdrawing groups at para and meta positions were favored, we next prepared a series of
di-substituted analogs that possess similar electronic characteristics. As shown in Table 2, a combination of 4g
and 4a yielded compound 4l with an activity slightly better than 4g but with a two-fold increase in activity when
compared to our hit molecule 4a. Substitution of the trifluoromethyl moiety (4l) with the hydrogen-bond
acceptor nitro functionality (4m) led to a slight decrease in overall activity, which was restored in the positional
isomer 4l. This data suggests that the meta position prefers non-hydrogen bonding functional groups, a
conclusion that was further confirmed by the observed change in the activity of 4o. Although the para position
tolerates the presence of H-bond acceptor groups (4l, 4q, 4v), the introduction of a carboxylic acid, an
ionizable H-bond donor moiety (4x), led to the loss of cytotoxic activity. We also observed a possible inverse
correlation between the size of the meta-substituent and the activity of compounds (4l vs. 4q), suggesting
potential size limitations in this part of the binding pocket. Di-substitution with electron-donating groups (4r)
14

abolished the desired activity, whereas a combination of the meta-amino moiety and para-chloro group
regained some activity. Finally, we have explored the possibility of ortho-, para- di-substitution pattern, but all
of the compounds in this subgroup (4s-4u) were inactive, irrelevant to the electronic properties of substituents.
Overall, we have concluded that di-substitution provides compounds with increased cytotoxicity if the meta-,
para- pattern is preserved.
Table 2. In vitro cytotoxic activity of analogs with the di-substitution at the “head” moiety.
MDA-MB-231
MDA-MB-231
R
R
a
a
IC50 (µM)
IC50 (µM)
4
l
3.15 ± 0.11
4.06 ± 0.74
9.24 ± 0.29
9.12 ± 0.43
3.47 ± 0.14
4.55 ± 0.12
4r
4s
4t
>20.0
>20.0
4
m
4
4
4
4
n
o
p
q
>20.0
4u
4v
4x
>20.0
4.43 ± 0.23
>20.0
a
Data are expressed as the mean ± SEM of three independent experiments, each performed in a
quartet.
Our structure-activity relationship studies on the “tail” di-phenyl moiety allowed us to draw four main
conclusions. First, we observed that the electronic properties of para-substituents do not play a defining role in
the activity of the studied analogs, as seen by comparing compounds 4l, 8a, and 8c. However, strong electron-
15

withdrawing groups are favored (8e and 8g). In the subgroup of analogs bearing electron-donating groups of
similar size, 8a, and 8h, the ability to form H-bonds was disfavored, although not forbidden. Second, we noted
that the size of the para-substitution might be inversely linked to the activity of compounds, as seen in pairs 8a
vs. 8c and 8e vs. 8i. Finally, we observed that compounds containing an N-linker have less activity when
compared to their carbon-containing bioisosteres (see pairs 9c vs. 4l, 9a vs. 4a, 9b vs. 4b). Finally, compound
1
0 designed to determine the necessity of the diphenyl moiety showed just a slight decrease in the overall
activity, indicating that the diphenyl moiety plays an auxiliary role in binding. However, it might affect the ability
of a compound to penetrate the BBB, a hypothesis that is currently under investigation in our laboratory.
Table 3. In vitro cytotoxic activity of analogs with the modifications at the “tail” di-phenyl moiety.
MDA-MB-231
R
1
R
2
a
IC50 (µM)
4
l
4-Cl, 3-CF3
4-Cl, 3-CF3
4-Cl
3.15 ± 0.11
2.51 ± 0.09
17.93 ± 0.37
8
a
b
8
8
c
d
4-Cl, 3-CF3
4-Cl, 3-NO2
3.30 ± 0.32
3.99 ± 0.44
8
8
e
4-Cl, 3-CF3
4-Cl, 3-CF3
4-Cl, 3-CF3
4-Cl, 3-CF3
1.69 ± 0.11
5.72 ± 0.26
1.23 ± 0.20
12.20 ± 0.59
8
f
8
8
g
h
8
i
4-Cl, 3-CF3
4-Cl
>20.0
>20.0
9
a
9
b
c
4-I
> 20.0
9
4-Cl, 3-CF3
5.04 ± 0.14
16

9
d
e
4-CH , 3-CH3
>20.0
3
9
4-Br, 2-I
> 20.0
1
0
4-Cl, 3-CF3
5.44 ± 0.59
a
Data are expressed as the mean ± SEM of three independent experiments, each performed in a
quartet.
The result of structural modifications in the alkyl-urea linker is depicted in Table 4. First, we observed that
increase in the length of the linker results in a slight increase in cytotoxicity (4l vs. 11b). Limiting the flexibility
of the linker chain by locking it into cyclic urea (19b) and by adding an olefin moiety (18) resulted in a minor
decrease of activity, which became more prominent with the simultaneous reduction in the linker length (19a).
It is known that substitution at the nitrogen atom of the urea functionality plays an important role in the
63
conformational preferences of these compounds. Thus, we have designed and evaluated a subgroup of
analogs; the mono-methylated (14, corresponding to the trans,cis configuration) and di-methylated (15,
corresponding to the cis,cis configuration) urea, where our results showed a clear preference for the
trans,trans configuration of the unsubstituted functionality (4l). These results also show a potential involvement
of the phenyl substituted nitrogen in the urea functionality in the hydrogen-bond interaction within the binding
pocket. Finally, a substitution of the urea moiety to a thiourea functionality (12) or a carbamate (13) had no
significant impact on the activity of the compound. In contrast, amide-containing linkers in 16 and 17 caused a
reduction in the cytotoxicity of studied analogs.
Table 4. In vitro inhibitory activity of analogs with modifications in the linker part.
MDA-MB-231
MDA-MB-231
a
a
IC50 (µM)
IC50 (µM)
4
l
3.15 ± 0.11
15
16
> 20.0
1
1a
3.50 ± 0.08
11.44 ± 0.40
17

1
1b
2.57 ± 0.07
3.66 ± 0.12
17
18
11.71 ± 0.38
5.65 ± 0.43
1
2
3
4
1
4.58 ± 0.25
6.02 ± 0.09
19a
19b
11.41 ± 0.32
4.84 ± 0.17
1
a
Data are expressed as the mean ± SEM of three independent experiments, each performed in
quartet.
Overall, our structure-activity relationship studies highlighted the importance of para-substitution of the “head”
moiety with electron-withdrawing functionalities. Similar electronic properties were also favored for the para-
substitution of the “tail” moiety. We observed that flexibility of the linker and trans,trans conformation of the
urea moiety are preferred, as well as the presence of the three heteroatom-containing moiety. As a result of
performed structural modifications, we were able to optimize the cytotoxicity of our “hit” molecule 4a by 6-fold
(
8g).
Figure 5. Summary of the SAR data for the anticancer activity of studied urea analogs
In vitro activity of selected analogs against DAT. To evaluate the effect of performed structural
modifications on the ability of compounds to inhibit DAT and estimate the possibility of separating this activity
from cytotoxicity, we have selected 13 analogs with different anticancer profile. As shown in Table 5, for
monosubstituted analogs 4a and 4b, the ability to inhibit DAT decreases as the size of para-substituent goes
18

up. In a subgroup of disubstituted analogs 4l, 4o, and 4r, there is no direct correlation between the electronic
properties of substituents and the observed activity of compounds. We observe that either hydrogen bond
acceptor character of meta-group in 4o or slightly larger size may lead to a 100-fold decrease in affinity to DAT
when compared to 4l. Similar to the observed trend in anticancer activity, ortho-substitution is not favored here
as well (4s and 4t). Unlike in cytotoxicity-related SAR, modifications in the tail moiety (8a-8c) have a notable
impact on the interaction with DAT, providing bases for separating pharmacophores responsible for transporter
inhibition and anticancer activity. Unlike in other analogs (4a vs. 4l, 9a vs. 9c), a di-substitution pattern in 8a,
bearing para-methyl groups at the tail moiety, is less active, than its mono-substituted counterpart 8b. Finally,
selected modifications in the linker moiety affect the ability of compounds 9a-19b to interact with the DAT. For
example, introduction of a nitrogen atom causes a slight decrease in the inhibitory properties of compounds
(
4a vs 9a and 4b vs 9c). Similar activity was observed in a series of analogs with the modified linker length (4a
vs 11a and 11b). Conformational restrictions introduced by the piperidine moiety result in decreased activity in
9a and abolished DAT activity in 19b. Similarly, an unsubstituted urea moiety is required for DAT activity (4a),
1
whereas the mono-methylated analog 14 loses some of this inhibitory activity, and di-methylated compound 15
is inactive as a DAT inhibitor. Substitution of the urea moiety with carbamate (13) or amide (16, 17)
functionalities causes significant loss of DAT activity as well. We have summarized the observed trend in the
activity of this set of compounds in Figure 6.
Table 5. Inhibitory activity of selected analogs at dopamine transporter*.
%
inhibition
% inhibition
at 10 µM
Structure
Ki, nM
Structure
Ki, nM
at 10 µM
4
a
b
82
15
9a
68
405
4
24
90
ND
6.5
9c
85
86
43
4
l
11a
141
19

4
o
76
79
20
58
38
609
80
11b
13
77
49
73
26
33
399
ND
998
ND
ND
4
r
4
s
ND
118
ND
14
4
t
15
8
a
b
16
8
59
535
17
51
603
8
c
d
75
64
40
19a
19b
52
24
1589
ND
8
144
In primary screening assays, compounds were tested in triplicate or quadruplicate at a final concentration of 10 μM.
Compounds with a minimum of 50% antagonist activity were subjected to secondary screening (dose-response) assays.
*Receptor binding profiles were generously provided by the National Institute of Mental Health's Psychoactive Drug
Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth
MD, Ph.D. at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD,
USA. ND − not determined (Ki was not evaluated due to the less than 50% antagonist activity of a compound).
20

Figure 6. Heat map of the observed trends in the cytotoxic activity of selected compounds and their ability to
inhibit DAT (% inhibition was measured at 10 µM level). Compounds with IC50 >20 µM (tables 1-5) were
ascribed IC50value of 30 µM
.
Evaluation of microsomal stability in vitro. As part of the pharmacokinetic assessment of urea analogs in vitro,
we have evaluated the metabolic stability of selected analogs (4l and 4q) using standard microsomal stability
assay (see supplemental material). The compound’s selection was based on two parameters: the IC50 values
and percent of the cell survival 48 hours post-treatment (Fig. S1). If both parameters were similar, we chose
compounds with a more diverse substitution pattern. To validate our assay, we have used the clinically
approved compound verapamil and PFL as the reference compounds. Both new analogs showed preferred
metabolic stability over PFL when analyzed for up to 60 minutes (Fig. 7).
21

Figure 7. Stability of verapamil, PFL, 4l, and 4q in liver microsomes following 60-min incubation was measured
by liquid chromatography-tandem mass spectrometry. Data are expressed as the mean ± SEM of three
independent experiments, each performed in triplicate.
Molecular target analysis. To elucidate the downstream molecular pathway of 4q-induced antiproliferative
effects observed in the cytotoxicity studies, we first explored whether apoptosis is induced. After 48 hours of 4q
treatment apoptosis was induced in MDA-MB-231 cells in a dose-dependent manner. Even at the 1 μM
concentration, significant induction of apoptosis was observed, whereas 5 μM levels of compound 4q
increased the apoptotic index by 10.6-fold (P < 0.01), compared to control cells (Figure 8A). Further, we
observed that 4q is capable of caspase-3 cleavage and activation (Supplemental material). At the same time,
4q did not affect the cell cycle in MDA-MB-231 cell line (Figure 8B).
Figure 8. Effect of 4q on the induction of apoptosis (A) and cell cycle (B) in MDA-MB-231 triple-negative
breast cancer cells. Data are expressed as the mean ± SEM of three independent experiments, each
performed in triplicate. **P<0.01
64
Fibroblast growth factor receptor (FGFR) pathways are significant driver pathways for breast cancer, and
65
FGFR overexpression has been proposed as a biomarker for TNBC. TNBC presents with gene amplification
and protein overexpression of FGFR1, FGFR2 and bFGF, with the latter being shown to have an autocrine
effect on tumor growth. TNBCs, along with basal-like breast cancers, show sensitivity to FGFR inhibitors, such
as PD173074, while upregulated FGFR signaling is a known resistance mechanism to CDK inhibitors,
66-68
highlighting the FGFR signaling as a potential therapeutic target for breast cancer, among others.
We,
therefore, explored the effect of compound 4q on FGFR1 activation in the presence and absence of bFGF. As
shown in Figure 9, compound 4q completely abrogated phosphorylation of FGFR1 in the presence of its
22

natural ligand bFGF at a concentration of 3 µM and 6 µM, indicating that inhibition of the FGFR1 signaling
pathway is at least one of the molecular mechanisms responsible for the cytotoxic effects of compound 4q.
Figure 9. The expression of FGFR1 was significantly reduced by compound 4q. Representative images (left
panel) and quantification (right panel) of pFGFR1 expression in MDA-MB-231 cells after 48 hours of treatment
with compound 4q at the concentrations depicted in the presence and absence of natural ligand bFGF (n = 3).
NS: no significance; *P < 0.05; **P<0.01.
Molecular Modelling. Compound 4q was selected for docking into the FGFR1 protein to gain insights into
69
binding interactions. The crystal structure of FGFR1 in complex with the potent inhibitor AZD4547 (PDB code
4V05) was prepared for docking by removing the ligand and defining the binding site as the 35 amino acid
residues surrounding the ligand and an additional 23 residues that account for unoccupied space around the
ligand-binding site. Molecular docking simulations afforded binding poses that were scored by HYDE, as
70
described in our previous publication. Predicted protein-ligand interactions (Fig. 10) correlate well with
experimental data obtained from SAR studies. The compound is predicted to occupy the ATP-binding cleft of
69
FGFR1 in a similar position to the known inhibitor AZD4547. The p-NO moiety of 4q is predicted to
2
participate in hydrogen bonding interactions with an active site water molecule forming a bridging network that
incorporates the LYS514, GLU531, and ASP641 residues. This is facilitated by the length of the linker allowing
the bulky diphenyl tail moiety to remain outside the binding pocket and solvent-exposed. The urea moiety of 4q
occupies a similar position within the ATP-binding cleft to the pyrazole moiety of AZD4547 (Fig. 11).
Calculation of hydrogen bonding distance results in potentially favorable proximity and orientation of a urea NH
to the GLU562 residue (3.77 Å), whereas the second urea NH is positioned 4.78 Å away from the ALA564
residue precluding potential binding. This is supported by SAR data that shows a critical role for the hydrogen
23

of the urea nitrogen alpha to the head group. The binding affinity of 4q was predicted to be in the high
nanomolar to a low micromolar range, which compares well to its experimentally derived value (IC50 = 4.55
µM).
Figure 10. Compound 4q (Gold) docked into the ATP-binding cleft of FGFR1 (Grey/Green). The green dashed
lines depict putative binding interactions indicating strong hydrogen bonding. Pink lines represent predicted
distance between hydrogen bond acceptors and donors..
Figure 11. Overlay of compound 4q (Gold) and the known inhibitor AZD4547 (Magenta) in the ATP-binding
site of FGFR1
CONCLUSION
We have described a novel series of cytotoxic compounds that possess cytotoxicity towards the MDA-MB-231
cell line. The first compound in this series, our “hit” molecule 4a, was shown to cross the BBB in vivo and to
have minimum interaction with CNS receptors when compared to the original penfluridol molecule. The first
round of structure-activity relationship studies was designed to understand the structural requirements for the
anticancer activity of these compounds. Observed results highlighted the importance of electron-withdrawing
24

substitution with the hydrogen-bonding properties on both tail and head moieties of studied analogs, as well as
the preferred length of a linker and its flexibility. Selected derivatives were subjected to a primary binding assay
to investigate their affinity to the DAT. Based on the current data, we have concluded that the toluene-like
structure of the tail-moiety will allow preserving the anticancer activity of urea analogs while eliminating
unwanted interactions with the DAT. These results are in line with the reported SAR studies for DAT
71
inhibitors, and we will continue exploring the effect of electron-withdrawing groups in the tail moiety to clarify
DAT-related pharmacophore of the urea compounds. Similarly, certain modification of the linker motif will allow
preservation of the anticancer activity of compounds, while eliminating unwanted interactions with the DAT.
Using a representative molecule, we have shown that this class of agents can induce apoptosis in MDA-MB-
2
31 cell lines in a concentration-dependent manner while having no effect on cell cycle. Further, we observe
that the tested analog was able to cleave caspase-3. Our initial mechanistic studies have identified FGFR1 as
one of the potential targets modulated by this class of agents. Our molecular modeling studies confirmed the
emphasized findings from the SAR studies. We envision that these compounds can be further developed for
the potential treatment of metastatic breast cancer. In vivo validation of the observed in vitro data, as well as
further validation of FGFR1 as a potential target will be reported shortly.
EXPERIMENTAL SECTION
General chemistry procedures. All reactions were carried out in oven- or flame-dried glassware under
positive nitrogen/ argon pressure unless otherwise noted. All solvents and chemicals were reagent grade.
Unless otherwise noted, all reagents and solvents were purchased from commercial vendors and used as
received. The purity and characterization of compounds were established by a combination of methods,
1
13
including TLC, HPLC, mass spectrometry, NMR analysis. H and C NMR spectra were recorded on a Bruker
4
00 MHz Advance III HD spectrometer using chloroform-d, methanol-d, or DMSO-d with tetramethyl (TMS)
6
(
0.00 ppm) or solvent peaks as the internal standard. Chemical shits (
reference signal, and coupling constant (J) values are recorded in hertz (Hz). Multiplicates are indicated by s
singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), br (broad). Thin-layer
ꢀ) are recorded in ppm relative to the
(
chromatography (TLC) was performed on EMD precoated silica gel 6-F254 plates, and spots were visualized
with UV light or iodine staining. Flash column chromatography was performed with silica gel (40-63 µm, 60 Å)
using the mobile phase indicated or on a Teledyne Isco (CombiFlash R UV/Vis). High-resolution mass spectra
f
25

were obtained using TripleTOF 5600 mass spectrometer. The purity of all final compounds was greater than
5%. The purity was determined by Waters Acquity UPLC using C18 column (Cortecs, 1.6µm, 2.1x50 mm):
eluent A, 0.1 % aqueous CF COOH and eluent B. CH CN containing 0.1 % CF COOH, gradient elution (0 min:
5% A, 5% B; 2 min: 50% A, 50% B; 4 min: 50% A, 50% B; 6 min: 10% A, 90% B; 9 min: 10% A, 90% B; 10
min: 95% A, 5%), with a flow rate of 0.2 ml/min.
9
3
3
3
9
General procedure A. A substituted phenyl isocyanate (0.50 mmol) dissolved in dichloromethane (2 mL) was
added to a solution of selected alkyl amine (0.50 mmol) in dichloromethane (3 mL) at 0 °C under inert
environment. After stirring for 6 hours at room temperature, the solvent was evaporated under reduced
pressure, and the formed residue was purified by flash chromatography with ethyl acetate /hexane (1:9 to 1:1)
to obtain the desired compound.
General procedure B. Hydrazine hydrate (0.50 mL) was added to a solution of a nitroaromatic compound
(
1.00 eq, 0.40 mmol) in methanol (10 mL). After stirring for 15 min at 50 °C, an excess of Raney®-Nickel was
®
added (approx. 2.00 eq.). A reaction mixture was kept stirring at 50 °C for an hour then filtered using Celite
bed. The organic solvent was removed under reduced pressure, and formed residue was purified by flash
chromatography with methanol/ dichloromethane (1:99 to 1:19) to obtain the reduced product.
General procedure C. An excess of acetic acid (0.03 mL) was added to a solution of an aniline derivative
(
1.00 equiv., 0.21 mmol) in dichloromethane (10 mL). After overnight stirring at room temperature, the reaction
solvent was removed under reduced pressure and formed residue was purified by flash chromatography using
methanol/ dichloromethane (1:99 to 1:19) to obtain desired N-phenylacetamide derivative.
1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(4-chlorophenyl)urea (4a). Compound 4a was prepared in 56% yield
1
from compound 29a and 48a using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.12-1.27 (m, 2H),
3
H
1
.46 (quin, 2H, J = 7.46 Hz), 1.87-2.01 (m, 2H), 3.11 (q, 2H, J = 6.85 Hz), 3.79 (t, 1H, J = 7.83 Hz), 5.00 (t, 1H,
13
J = 5.62 Hz), 6.83 (s, 1H), 6.89-7.04 (m, 4H) 7.05-7.14 (m, 4H), 7.14-7.23 (m, 4H). C NMR (100 MHz, CDCl )
3
δC 25.18, 30.00, 35.47, 40.11, 49.67, 115.19, 115.40, 121.62, 128.99, 129.07, 129.14, 137.28, 140.36, 140.40,
+
1
9
60.12, 162.55. HRMS-ESI: (m/z) calculated for C24
H
23ClF
2
N
2
O, 429.1545 [M+H] ; found 429.1590. Purity:
8.67%
26

1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(4-iodophenyl)urea (4b). Compound 4b was prepared in 31% yield from
1
compounds 29a and 48b using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.17-1.32 (m, 2H),
3
H
1
6
.47-1.56 (m, 2H), 1.90-2.05 (m, 2H), 3.11-3.23 (m, 2H), 3.83 (t, 1H, J = 7.95 Hz), 4.64 (t, 1H, J = 5.62 Hz),
13
.31 (s, 1H), 6.89-7.00 (m, 4H), 7.00-7.09 (m, 2H), 7.09-7.21 (m, 4H), 7.51-7.63 (m, 2H). C NMR (100 MHz,
CDCl ) δ 25.19, 29.99, 35.48, 40.18, 49.68, 86.48, 115.21, 115.42, 122.27, 129.03, 129.11, 138.08, 138.47,
3
C
+
1
40.42, 160.13, 162.57. HRMS-ESI: (m/z) calculated for C24
23 2 2
H F IN O, 521.0901 [M+H] ; found 521.0897.
Purity: 97.41%.
1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(p-tolyl)urea (4c). Compound 4c was prepared in 84% yield from
1
compounds 29a and 48c using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.13-1.24 (m, 2H), 1.48
3
H
(
quin, 2H, J = 7.40 Hz), 1.88-2.01 (m, 2H), 2.29 (s, 3 H), 3.06-3.20 (m, 2H), 3.80 (t, 1H, J = 7.83 Hz), 4.94 (t,
1
3
1
2
1
4
3 C
H, J = 5.62 Hz), 6.59 (s, 1H), 6.88-7.01 (m, 4H), 7.03-7.19 (m, 8 H). C NMR (100 MHz, CDCl ) δ 20.80,
5.16, 30.02, 35.48, 40.05, 49.65, 115.16, 115.36, 122.01, 129.02, 129.10, 129.88, 133.96, 135.71, 140.45,
+
40.49, 156.31, 160.10, 162.53. HRMS-ESI: (m/z) calculated for C25
H
26
F
2
N
2
O, 409.2091 [M+H] ; found
09.2127. Purity: >99%.
1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(4-nitrophenyl)urea (4d). Compound 4d was prepared in 81% yield from
1
compounds 29a and 48d using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.21-1.31 (m, 2H), 1.55
3
H
(
quin, 2H, J = 7.46 Hz), 1.93-2.03 (m, 2H), 3.22 (q, 2H, J = 6.85 Hz), 3.82 (t, 1H, J = 7.83 Hz), 5.13 (t, 1H, J =
13
5
.50 Hz), 6.87-7.00 (m, 4H), 7.04-7.17 (m, 4H), 7.22 (s, 1H), 7.45-7.53 (m, 2H), 8.07-8.19 (m, 2H). C NMR
(
100 MHz, CDCl ) δ 25.19, 29.90, 35.47, 39.99, 49.69, 115.18, 115.39, 117.47, 117.52, 125.31, 128.99,
3 C
1
4
29.07, 140.35, 140.39, 141.73, 146.10, 160.10, 162.54. HRMS-ESI: (m/z) calculated for C H F N O ,
24 23 2 3 3
+
40.1785 [M+H] ; found 440.1830. Purity: 99.70%.
1
-(4-aminophenyl)-3-(5,5-bis(4-fluorophenyl)pentyl)urea (4e). Compound 4e was prepared in 81% yield
1
from compound 4d using the general procedure B. H NMR (400 MHz, CDCl
3
) δ
H
1.12-1.27 (m, 2H), 1.48
(
quin, 2H, J = 7.30 Hz), 1.90-2.04 (m, 2H), 3.16 (q, 2H, J = 6.85 Hz) 3.69, (br. s., 2H), 3.83 (t, 1H, J = 7.83 Hz),
1
3
4
.52 (t, 1H, J = 5.50 Hz), 5.98 (s, 1H), 6.56-6.70 (m, 2H), 6.89-7.05 (m, 6H), 7.08-7.22 (m, 4H). C NMR (100
MHz, CDCl ) δ 25.13, 30.03, 35.52, 40.05, 49.65, 115.16, 115.37, 115.76, 126.69, 128.23, 129.05, 129.13,
3
C
27

1
25 2 3
40.51, 140.54, 144.74, 157.13, 160.12, 162.55. HRMS-ESI: (m/z) calculated for C24H F N O, 410.2044
+
[
M+H] ; found 410.2080. Purity: >99%.
N-(4-(3-(5,5-bis(4-fluorophenyl)pentyl)ureido)phenyl)acetamide (4f). Compound 4f was prepared in 81%
1
yield from compound 4e using the general procedure C. H NMR (400 MHz, DMSO-d ) δ 1.11-1.25 (m, 2H),
6
H
1
.43-1.48 (m, 2H), 1.92-2.07 (m, 5H), 3.02 (q, 2H, J = 6.77 Hz), 3.97 (t, 1H, J = 7.83 Hz), 6.03 (t, 1H, J = 5.75
Hz), 7.08 (dd, 4H, J = 8.90, 8.90 Hz), 7.23-7.37 (m, 6H), 7.41 (d, 2H, J = 8.80 Hz), 8.25-8.40 (m, 1H), 9.77 (s,
1
3
1
1
H). C NMR (100 MHz, DMSO-d ) δ 24.29, 25.29, 30.13, 35.15, 49.30, 115.43, 115.64, 118.35, 120.08,
6 C
29.70, 129.77, 133.44, 136.43, 141.72, 141.75, 155.73, 159.85, 162.26, 168.16. HRMS-ESI: (m/z) calculated
+
for C H F N O , 452.2149 [M+H] ; found 452.2186. Purity: >99%.
26
27
2
3
2
1
8
1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(3-(trifluoromethyl)phenyl)urea (4g). Compound 4g was prepared in
1
8% yield from compounds 29a and 48e using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.08-
3
H
.21 (m, 2H), 1.42 (quin, 2H, J = 7.40 Hz), 1.82-1.95 (m, 2H), 3.02-3.16 (m, 2H), 3.74 (t, 1H, J = 7.70 Hz), 5.53
(
t, 1H, J = 5.26 Hz), 6.84-6.97 (m, 4H), 7.00-7.12 (m, 4 H), 7.13-7.20 (m, 1H), 7.20-7.28 (m, 1H), 7.37 (d, 1H, J
13
=
8.31 Hz), 7.50 (s, 1H), 7.60 (s, 1H). C NMR (100 MHz, CDCl ) δ 25.17, 29.89, 35.43, 40.06, 49.64,
3 C
1
15.16, 115.37, 116.16, 116.20, 119.41, 122.60, 125.25, 128.95, 129.03, 129.49, 139.49, 140.35, 140.39,
+
1
56.00, 160.11, 162.54. HRMS-ESI: (m/z) calculated for C H F N O, 463.1809 [M+H] ; found 463.1845.
25
23
5
2
Purity: >99%.
1
7
1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(4-(trifluoromethyl)phenyl)urea (4h). Compound 4h was prepared in
1
3% yield from compounds 29a and 48f using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.15-
3
H
.24 (m, 2H), 1.47 (quin, 2H, J = 7.43 Hz), 1.87-1.99 (m, 2H), 3.13 (q, 2H, J = 6.85 Hz), 3.78 (t, 1H, J = 7.76
Hz), 5.18 (t, 1H, J = 5.50 Hz), 6.80-7.00 (m, 4H), 7.02-7.13 (m, 4H), 7.15 (s, 1H), 7.35 (m, 2H, J = 8.56 Hz),
1
3
7
1
.46 (m, 2H, J = 8.56 Hz). C NMR (100 MHz, CDCl ) δ 25.18, 29.93, 35.45, 40.14, 49.68, 115.19, 115.40,
3 C
18.87, 126.26, 126.30, 126.33, 128.97, 129.05, 140.32, 140.35, 142.05, 155.33, 160.14, 162.56. HRMS-ESI:
+
(
m/z) calculated for C H F N O, 463.1809 [M+H] ; found 463.1764. Purity: 99.67%.
25
23
5
2
1
-(5,5-bis(4-fluorophenyl)pentyl)-3-(3-chlorophenyl)urea (4i). Compound 4i was prepared in 66% yield from
1
compounds 29a and 48g using the general procedure A. H NMR (400 MHz, CDCl ) δ 1.14-1.27 (m, 2H) 1.49,
3
H
(
t, 2H, J = 7.09 Hz), 1.95 (q, 2H, J = 7.62 Hz), 3.07-3.22 (m, 2H), 3.80 (t, 1H, J = 7.76 Hz), 4.99 (br. s., 1H),
28