Diphenylurea derivatives for combating methicillin- and vancomycin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.ejmech.2017.02.044

A new class of diphenylurea was identified as a novel antibacterial scaffold with an antibacterial spectrum that includes highly resistant staphylococcal isolates, namely methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA & VRSA). Starting with a lead compound 3 that carries an aminoguanidine functionality from one side and a n-butyl moiety on the other ring, several analogues were prepared. Considering the pharmacokinetic parameters as a key factor in structural optimization, the structure-activity-relationships (SARs) at the lipophilic side chain were rigorously examined leading to the discovery of the cycloheptyloxyl analogue 21n as a potential drug-candidate. This compound has several notable advantages over vancomycin and linezolid including rapid killing kinetics against MRSA and the ability to target and reduce the burden of MRSA harboring inside immune cells (macrophages). Furthermore, the potent anti-MRSA activity of 21n was confirmed in?vivo using a Caenorhabditis elegans animal model. The present study provides a foundation for further development of diphenylurea compounds as potential therapeutic agents to address the burgeoning challenge of bacterial resistance to antibiotics.

Full text of DOI:10.1016/j.ejmech.2017.02.044

Accepted Manuscript
Diphenylurea derivatives for combating methicillin- and vancomycin-resistant
Staphylococcus aureus
Ibrahim E. Hassan, Haroon Mohammad, Omar A. Qassem, Waleed Younis, Tamer
M. Abdelghany, Ahmed Elshafeey, Mahmoud M. Abd Rabo Moustafa, Mohamed N.
Seleem, Abdelrahman S. Mayhoub
PII:
S0223-5234(17)30117-4
10.1016/j.ejmech.2017.02.044
EJMECH 9240
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 December 2016
Revised Date: 13 February 2017
Accepted Date: 17 February 2017
Please cite this article as: I.E. Hassan, H. Mohammad, O.A. Qassem, W. Younis, T.M. Abdelghany,
A. Elshafeey, M.M. Abd Rabo Moustafa, M.N. Seleem, A.S. Mayhoub, Diphenylurea derivatives for
combating methicillin- and vancomycin-resistant Staphylococcus aureus, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.044.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Diphenylurea Derivatives for Combating Methicillin- and Vancomycin-Resistant Staphylococcus
aureus
Ibrahim E. Hassan,a,║ Haroon Mohammad,b,║ Omar A. Qassem,a Waleed Younis,b Tamer M.
Abdelghany,c Ahmed Elshafeey,d,e Mahmoud M. Abd Rabo Moustafa,f Mohamed N. Seleem,b,g * and
Abdelrahman S. Mayhouba,h*
a.
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University,
Cairo 11884, Egypt.
b.
Department of Comparative Pathobiology, Purdue University, College of Veterinary Medicine,
West Lafayette, IN 47907, USA.
c.
Department of Pharmacology, College of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Department of Pharmaceutics, College of Pharmacy, Cairo University, Cairo 11777, Egypt.
Bioequivalence Section, Genuine Research Center, Heliopolis, Cairo 11757, Egypt.
Department of Pharmaceutical Chemistry, College of Clinical Pharmacy, University of Dammam,
d.
e.
f.
Eastern Province, KSA, P.O. Box 1982, Dammam 31441.
g.
Purdue Institute for Inflammation, Immunology, and Infectious Diseases, West Lafayette, IN
479067, USA.
h.
Biomedical Sciences, University of Science and Technology, Zewail City of Science and
Technology, Giza, Egypt
Corresponding Authors.
* ASM; e-mail, amayhoub@azhar.edu.eg
* MNS; e-mail, mseleem@purdue.edu
Abbreviations. AMR, Antimicrobial resistance; Caco-2, heterogeneous human epithelial colorectal
adenocarcinoma cells; CFUs, colony forming units; Cmax, maximum serum concentration; HaCaT, human
keratinocytes; MRT, mean residence time; Papp, apparent permeability; VISA, vancomycin-intermediate S. aureus.

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Abstract. A new class of diphenylurea was identified as a novel antibacterial scaffold with an
antibacterial spectrum that includes highly resistant staphylococcal isolates, namely methicillin- and
vancomycin-resistant Staphylococcus aureus (MRSA & VRSA). Starting with a lead compound 3 that
carries an aminoguanidine functionality from one side and a n-butyl moiety on the other ring, several
analogues were prepared. Considering the pharmacokinetic parameters as a key factor in structural
optimization, the structure-activity-relationships (SARs) at the lipophilic side chain were rigorously
examined leading to the discovery of the cycloheptyloxyl analogue 21n as a potential drug-candidate.
This compound has several notable advantages over vancomycin and linezolid including rapid killing
kinetics against MRSA and the ability to target and reduce the burden of MRSA harboring inside immune
cells (macrophages). Furthermore, the potent anti-MRSA activity of 21n was confirmed in vivo using a
Caenorhabditis elegans animal model. The present study provides a foundation for further development
of diphenylurea compounds as potential therapeutic agents to address the burgeoning challenge of
bacterial resistance to antibiotics.
Keywords: antibiotic drug resistance, Caenorhabditis elegans, Methicillin-resistant Staphylococcus
aureus, MRSA, intracellular infection, pharmacokinetics.

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1. INTRODUCTION
Antimicrobial resistance (AMR) is a major global health concern. Recent alarming estimates suggest that
deaths due to AMR may increase from the current estimate of 700,000 lives per year to ten million lives
annually by 2050, at a cost of US$100 trillion.[1, 2] Different bacterial species that were once susceptible
to several different classes of antibiotics have now acquired an array of unique resistance mechanisms.
For instance, several strains of Escherichia coli were recently found to be resistant to the 3rd and 4th
generation carbapenems[3] as well as the agent of last resort, colistin.[4] However, infections caused by
one notorious bacterial pathogen have proven especially difficult to treat. Out of more than 23,000 people
that die each year in the United States due to antibiotic-resistant bacterial infections,[5] methicillin-
resistant Staphylococcus aureus (MRSA) was found to be responsible for nearly half of these fatalities.
MRSA was first isolated in 1961,[6, 7] and became endemic in US hospitals in mid-1980, leading to the
worldwide pandemic of MRSA that continues to the present time.[8, 9]
Staphylococcus aureus is a leading source of skin, wound, and hospital-acquired infections. Successful
treatment of these infections has become a daunting challenge with the emergence of clinical isolates of
MRSA exhibiting resistance to first-line antibiotics and agents of last resort, like linezolid[10] and
vancomycin.[11] Furthermore, the effectiveness of agents of last resort (vancomycin) is limited by
prolonged, persistent or recurrent bacteraemia during therapy,[12, 13] high rates of clinical failures,[14]
severe nephrotoxicity[15] and the increasing prevalence of non-susceptible strains.[16] This highlights
the urgent need to develop new therapeutic agents with novel scaffolds to address the burden of MRSA
infections.
Our research group has been engaged extensively in developing and characterizing novel anti-
MRSA compounds with the aim of discovering promising drug candidates.[17-21] Central to our
vision of the urgent need to foster the current choices for the treatment of highly-resistant
pathogens, we continued our intensive efforts to introduce new anti-MRSA scaffold(s) to the
medicinal chemistry community. As reported earlier, the anti-MRSA phenylthiazole
1 has two

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essential structural features: a lipophilic tail and a cationic aminoguanidinyl head (Figure 1).[18]
Considering these two fundamental structural elements, an in-house library of nearly three-
hundred compounds, all with the guanidine or aminoguanidine functional group on one end and a
less lipophilic chain at the opposite end, was screened using whole-cell screening assay. The
antibacterial activity of the top promising structures in the preliminary screening, which
successfully passed the PAINS assay,[22] were selected and their antibacterial activity was
further assessed against a clinical isolate of MRSA NRS123. These efforts, collectively, furnished
compounds in Figure 2 as lead structures.
Figure 1. Chemical structure of first hit compound.

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Figure 2. Chemical structures of new hits with their minimum inhibitory concentration (MIC)
values against MRSA NRS123, All compounds were checked for pan-assay interference
compounds (PAINS) and none are PAINS.[22]
According to the chemical nature of the linker, the active hits can be classified into diarylurea
derivatives (compounds
phenylacetamides (compounds 12 and 13). The highest anti-MRSA activity was observed with
the diarylurea compounds . Additionally, after exploring the literature, we have found some
2-4), N-phenylarylamide analogues (compounds 5-9) and N-
2-4
reports that address the antimicrobial activity of diphenylurea derivatives,[23] in addition to many
others as antitumor.[24] Therefore, chemical modifications reported here involved building a

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focused library of diphenylureas with different lipophilic moieties at the phenyl para position to
define the structure−activity relationships (SARs) at this position in a rigorous manner.
Taking the metabolic stability and other key pharmacokinetic properties into account to finally
develop a drug-candidate, this work has three objectives: to investigate the antimicrobial activity
of the diphenylureas against a panel of MRSA and VRSA clinical isolates, to investigate the
capability of the most promising candidate to penetrate and eradicate intracellular MRSA, and to
confirm the potent anti-MRSA activity of the most promising analogue in a Caenorhabditis
elegans animal model.
2. RESULTS AND DISCUSSION
2.1. CHEMISTRY. The diphenylurea 16 was obtained by allowing t-butylaniline to react with the
isocyanate 15. Treatment of the obtained acetyldiphenylurea 16 with aminoguanidine, in the presence of
lithium chloride as a catalyst, afforded the final product 17 (Scheme 1). Similarly, the 1-(4-acetylphenyl)-
3-(4-hydroxyphenyl)urea (19) was prepared (Scheme 2). The latter compound was allowed to react with a
series of alkyl bromides to give the alkylated products 20a-o. These products were then treated with
aminoguanidine to afford the final products 21a-o (Scheme 2).
Scheme 1.
NH2
O
O
O
a
+
N
N
H
H
NCO
16
14
15
H
N
NH2
NH
b
O
N
N
N
H
H
17
o
Reagents and conditions:
aminoguanidine HCl, EtOH, 78 oC
a) dry THF, 78 C; b)

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2.2. BIOLOGICAL RESULTS AND DISCUSSION.
Among all tested hits, the three disubstituted urea derivatives 2-4 showed the best antibacterial effect with
minimum inhibitory concentration (MIC) values ranging between 8 and 10 µg/mL. Compound 3 was
chosen for further analysis and its stability to hepatic metabolism was briefly studied. Surprisingly,
compound 3 was found to be a good substrate for the CYP450 enzymes resulting in an ultra-short half-life
(t1/2) of 13.0 minutes and high clearance rate when incubated with human liver microsomes (HLM) (Table
2). Microsomal oxidation of 3 might occur at multiple positions; for instance, aromatic and benzylic
oxidation are highly expected. Since the active site of CYP450 is surrounded by a highly hydrophobic
seven phenylalanine cluster,[25] the benzylic carbon of 3 was initially posited to be the metabolic soft-
spot (Figure 3). This particular carbon is hypothesized to be extensively oxidized by metabolic enzymes.
Gratifyingly, literature is full of solutions for rapidly metabolized benzylic carbons that include adding

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two methyl groups or substitution with an oxygen atom.[26, 27] Therefore, two derivatives possessing a
side chain with the same number of carbon atoms were prepared; one with t-butyl (compound 17), and
one with a butoxy moiety (compound 21b). The t-butyl analogue 17 showed negligible anti-MRSA
activity, while the antibacterial effect of 21b was reasonable with MIC values ranging between 8 and 16
µg/mL (Table 1). Even more interesting, compounds 17 and 21b exhibit a marked improvement in their
stability to hepatic metabolism as noted by their increased half-lives and lower clearance rates (Table 2).
Briefly, compound 21b showed a t1/2 that was five times longer and a lower microsomal clearance rate
than the corresponding butyl hit compound 3 (Table 2). Additionally, 21b exhibited a longer half-life than
two commercially-available drugs, midazolam (t1/2 = 5.7 minutes) and verapamil (t1/2 = 15.3 minutes).
The longer half-life values of 17 and 21b support our hypothesis of the presence of metabolic soft spot at
the pended n-butyl moiety. However, this information must be taken with caution as it doesn’t completely
exclude the possibility of aromatic hydroxylation. What can be concluded from the MIC and metabolic
stability analyses of compounds 17 and 21b is that diphenylureas containing an alkoxy side chain
represent a promising novel scaffold, with suitable drug-like properties, to construct a novel class of
antimicrobial agents. Additionally, the accessibility of the synthetic protocol for ether formation and the
availability of a variety of commercial alkyl halides permit a complete study of the structure-activity and
structure-kinetics relationships at this particular position.
Figure 3. Proposed metabolic soft spot and strategies to decrease affinity to CYP450.

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Next, a series of alkoxy side chains was tethered with the core of the diphenylurea in order to
rigorously address the structure-activity and structure-kinetics relationships at this position. Thus far,
fifteen derivatives with different homologous, branched and cyclic side chains have been synthesized and
tested against a panel of methicillin-sensitive S. aureus (MSSA), MRSA and VRSA clinical isolates.
Compounds 21c, 21d, 21h, 21i, 21j and 21n are the most potent compounds against S. aureus with MIC
values in the range of 2 to 8 µg/mL (Table 1). They appear to be bactericidal given the minimum
bactericidal concentration (MBC) values match or are one-fold higher than the MIC values against most
clinical isolates. Interestingly, the newly developed diphenylureas maintained their potent antibacterial
effect when tested against strains exhibiting high-level resistance to vancomycin (VRSA4 and VRSA10).
In this regard, they have an advantage over vancomycin.
From SAR point of view, increasing the number of methylene units from 4 (as in the lead compound
21b) to five or six (compounds 21c and 21d) remarkably ameliorates the antibacterial activity of these
compounds (Table 1). The branched analogue 21i with a six carbon-unit side chain demonstrated MIC
values on par with that of linezolid, an agent of last resort for treatment of systemic MRSA infections.
Unlike the bacteriostatic nature of linezolid,[28] compound 21i appears to be bactericidal, given its MBC
value matches or is one-fold higher than its MIC value against all strains of S. aureus tested.
Table 1. The minimum inhibitory concentration (MIC in µg/mL) and the minimum bactericidal
concentration (MBC µg/mL) of diphenylurea compounds screened against S. aureus isolates.
S. aureus
NRS107
(RN4220)
MRSA
NRS123
(USA400)
MRSA
NRS382
(USA100)
MRSA
NRS383
(USA200)
MRSA
NRS384
(USA300)
VRSA4
VRSA10
MIC
MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC
MBC
MIC
MBC
64
8
16
4
128
8
16
4
17
21a
-
8
16
4
8
-
8
>64
8
8
-
4
8
4
8
16
32
32
8
-
16
-
8
16
4
4
-
16
32
4
8
-
8
16
4
8
16
16
32
4
-
16
16
4
16
16
16
32
8
-
8
16
4
8
-
8
16
4
8
-
-
8
16
4
16
32
8
32
16
16
16
32
64
16
4
21b
21c
21d
21e
21f
21g
21h
21i
4
4
8
8
8
8
16
16
32
8
16
32
32
8
8
8
8
8
16
16
32
4
16
32
32
8
16
32
4
32
64
4
16
16
4
16
32
4
16
16
4
32
16
8
2
4
2
4
4
2
4
2
4
4
4
2
4

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S. aureus
NRS107
(RN4220)
MRSA
NRS123
(USA400)
MRSA
NRS382
(USA100)
MRSA
NRS383
(USA200)
MRSA
NRS384
(USA300)
VRSA4
VRSA10
MIC
MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC
MBC
MIC
MBC
21j
21k
21l
4
-
4
-
4
-
8
-
4
8
-
4
-
8
-
8
-
8
-
4
-
4
-
4
4
128
16
-
16
-
-
32
-
16
-
16
-
16
-
32
-
16
64
16
64
32
128
16
-
16
-
16
-
16
-
21m
21n
21o
64
2
8
2
1
4
8
32
1
4
8
2
4
>64
32
2
4
2
4
>128
16
2
8
2
4
2
8
2
4
8
16
1
2
8
2
4
8
8
8
8
2
8
>64
16
16
32
0.5
Linezolid
Vanco-
mycin
0.5
0.5
<1
<1
0.5
0.5
>12 >128 >128 >128
8
Table 2. Metabolic stability analysis of compounds 3, 17, 21b, and 21n in human liver microsomes.
Tested
compound
NADPH-Dependent
CLint
NADPH-
Dependent
T1/2 (min)
5.7
NADPH-Free
CLint
(µl/min-mg)
< 9.6
NADPH-Free
T1/2 (min)
Notes
(µL/min-mg)
402
Midazolam
Verapamil
Warfarin
>240
>240
>240
High
clearance
control
High
clearance
control
Low
151
15.3
< 9.6
< 9.6
< 9.6
> 240
clearance
control
189.8
20.0
51.0
33.9
13.0
174
65.5
68.2
ND
ND
ND
42.9
ND
ND
ND
53.8
3
17
21b
21n
The two cornerstone antimicrobials clinically in use for the treatment of systemic MRSA infections are
glycopeptides such as vancomycin and oxazolidinones such as linezolid. Each of these categories has its
own drawbacks that affect their clinical efficiency. While linezolid is a bacteriostatic agent,[28]
vancomycin exhibits a very slow bactericidal mode of action[29] resulting in difficulty in clearing an
infection[30] and clinical failure in many cases.[31] In order to further investigate the observed
bactericidal activity of the new diphenylurea derivatives, the most promising compounds 21i, 21j, and
21n (at 4 × MIC) were further examined against MRSA USA400 using a standard time-kill assay. All
three compounds were able to eradicate a high inoculum of MRSA within four hours. This confirms they
possess rapid bactericidal activity. Vancomycin requires 24 hours to achieve the same effect (Figure 4).
The rapid bactericidal activity of the diphenylurea compounds may limit the ability of MRSA to rapidly

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acquire resistance to these agents. Indeed, repeated subculturing of MRSA to the diphenylurea
compounds over a two-week period did not result in the isolation of resistant mutants via a multi-step
resistance assay (data not shown).
Figure 4. Time-kill analysis of diphenylurea compounds 21i, 21n, 21j, and vancomycin against
methicillin-resistant Staphylococcus aureus (MRSA USA400) over a 24-hour incubation period at 37 °C.
DMSO served as a negative control. The error bars represent standard deviation values obtained from
triplicate samples used for each compound/antibiotic studied.
Thus far, our analysis of the antibacterial activity of the diphenylurea compounds focused exclusively
on their effect on extracellular MRSA. However, MRSA is not exclusively an extracellular pathogen.
Rather, MRSA can escape and hide intracellularly within immune cells, such as macrophages[32, 33]
inducing several life-threating diseases such as pneumonia in humans[34] and mastitis in cattle. MRSA
harbouring inside host tissues can lead to recurring infections that are very challenging for clinicians to
treat.[35] This poses a unique challenge as many antibiotics are unable to enter inside infected cells to
eradicate MRSA. For instance, the inability of vancomycin, and other glycopeptides, to penetrate and
sufficiently accumulate inside macrophages[34] has led to clinical failure in more than 40% of cases
treated with a standard vancomycin dosing regimen.[36]
Prior to examining the ability of our diphenylureas to penetrate and kill intracellular MRSA, the most
active compounds were subjected to a preliminary toxicity profiling using human keratinocytes (HaCaT).
Figure 5 indicates that the most tolerable compounds were 21c, 21n and 21i as they were not toxic up to a
concentration of 64 µg/mL (or 32 µg/mL for 21i). This represents an 8-to 16-fold difference between the

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MIC values obtained against MRSA for all three compounds. Compounds 21c and 21i are structurally
analogous; thus 21n and 21i were selected for further investigation to examine their ability to kill
intracellular MRSA harbouring inside infected macrophages.
Figure 5. Toxicity analysis of diphenylurea compounds against human keratinocytes (HaCaT). Percent
viable mammalian cells (measured as average absorbance ratio (test agent relative to DMSO)) for
cytotoxicity analysis of diphenylurea compounds 21d, 21h, 21c, 21i, 21a, 21n, and 21j (tested in
triplicate) at 16, 32, and 64 µg/mL against HaCaT cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Dimethyl sulfoxide (DMSO) was used
as a negative control to determine a baseline measurement for the cytotoxic impact of each compound.
The absorbance values represent an average of a minimum of three samples analyzed for each
compound. Error bars represent standard deviation values for the absorbance values. A one-way
ANOVA, with post hoc Dunnet’s multiple comparisons test, determined statistical difference between
the values obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05).

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Figure 6. Toxicity analysis and examination of clearance of intracellular MRSA present in murine
macrophage (J774) cells. Panel A) Percent viable mammalian cells (measured as average absorbance
ratio (test agent relative to DMSO)) for cytotoxicity analysis of diphenylurea compounds 21i and 21n
(tested in triplicate) at 8, 16, 32, and 64 µg/mL against J774 cells using the MTS 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Dimethyl sulfoxide (DMSO)
was used as a negative control to determine a baseline measurement for the cytotoxic impact of each
compound. The absorbance values represent an average of a minimum of three samples analyzed for
each compound. Error bars represent standard deviation values for the absorbance values. A one-way
ANOVA, with post hoc Dunnet’s multiple comparisons test, determined statistical difference between
the values obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05). Panel B) Percent
reduction of MRSA USA400 colony forming units inside infected murine macrophage cells after
treatment with 16 µg/mL of either compound 21n or vancomycin (tested in triplicate) for 4, 8, and 24
hours. Data were analyzed via a Student’s t-test (P < 0.05). Asterisks (*) represent significant difference
between treatment of J774 cells with 21n in comparison to vancomycin.
Initial J774 cell tolerability screening of 21i and 21n revealed that compound 21n is not toxic up to 16
µg/mL; however, compound 21i appears toxic to J774 cells even at a concentration as low as 8 µg/mL
(Figure 6A). Due to its superior toxicity profile, compound 21n was selected for further analysis for its
ability to clear MRSA harboring inside macrophage cells. As depicted in Figure 6B, after four hours, 21n
(same time required to eradicate extracellular MRSA completely via a time-kill assay) produced a 5%
reduction in MRSA CFU/mL when compared to untreated samples. The number steadily increased and
reached 35.7% reduction of MRSA after 8 hours and 69% reduction after 24 hours of treatment.
Vancomycin, as expected, was not able to reduce the presence of MRSA inside infected J774 cells, even
after 24 hours of treatment. The results collectively indicate compound 21n has the ability to gain entry
into macrophage cells (after 8 hours of treatment) at a concentration high enough to significantly reduce
the burden of MRSA inside infected macrophage cells.
After confirming this newly discovered class of compounds has potent antibacterial activity against
extracellular and intracellular MRSA with a well-tolerated safety profile against mammalian cells, we
moved to ensure that the mechanism of action of these compounds was not through physical disruption of
the bacterial cell membrane. Membrane-active agents are typically non-specific (i.e. can disrupt both
bacterial and eukaryotic cells) and thus have limited utility as therapeutic agents.

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To examine if the diphenylurea compounds exert their antibacterial effect by targeted disruption of the
bacterial cell membrane, compounds 21i, 21n, and vancomycin were incubated at a high concentration
(5.0 × MIC value) with a high-inoculum of MRSA NRS123 (USA400). Lysostaphin, a well-characterized
membrane-disruptive agent against S. aureus, was used as a positive control. The mechanism of action of
compounds 21i and 21n does not appear to be through physical disruption of the integrity of the bacterial
cell membrane (Figure 7). The compounds mimic the behavior of vancomycin, an antibiotic that inhibits
bacterial cell wall synthesis, and untreated samples (<15% leakage of 260 and 280 nm absorbing material
observed) in contrast to cells treated with lysostaphin. Subsequent investigation into the mechanism of
action of the diphenylureas indicates they interfere with bacterial cell wall synthesis (data not published).
At present, we are working to validate these findings.
Figure 7. Loss of 260 and 280 nm cellular absorbing material for compounds 21i and 21n against MRSA
NRS123. Untreated cells represent the negative control while 20 µg/mL lysostaphin (in 50 mM Tris-HCl,
pH 7.6) served as the positive control. The figure represents the ratio of the average absorbance value
obtained for each treatment against the average absorbance value obtained for the positive control. The
error bars represent standard deviation values of duplicate samples for each treatment option. A paired
t-test, P ≤ 0.05, demonstrated no statistical difference between the values obtained for vancomycin and
compounds 21i and 21n relative to untreated cells but significant difference (denoted by *) in the
absorbance values obtained for lysostaphin as compared to untreated cells.
In vivo examination of 21n and vancomycin to kill MRSA USA400 in a Caenorhabditis elegans
animal model. Thus far, promising in vitro results pertaining to the anti-MRSA activity of the
diphenylurea compounds was obtained. However, it is critical to validate in vitro results in vivo, in a

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suitable animal model of infection. To examine the efficacy of the diphenylurea compounds to treat a
MRSA infection in vivo, the Caenorhabditis elegans (C. elegans) was utilized. The C. elegans animal
model is an established system for investigating the efficacy of small molecule antibacterial agents in vivo
in early stage drug discovery[37, 38] Using this model, compound 21n (at 10 µg/mL, equal to 2.5 × MIC)
retains its potent antibacterial activity in vivo reducing the burden of MRSA USA400 by more than 50%
in infected worms (Figure 8). Vancomycin, at the same concentration, reduces the bacterial burden by
25%.
Compound 21n thus far emerged as the most promising candidate for further investigation. In order to
gauge potential therapeutic applications for 21n, it was critical to examine its pharmacokinetic profile
first. This information will prove valuable in designing future animal studies involving MRSA infections
(systemic and localized) including identifying an appropriate route of administration and frequency of
dosing.
Figure 8. Antibacterial activity of 21n and vancomycin in vivo against MRSA-infected C. elegans. In vivo
examination of antibacterial activity of diphenylurea compound 21n and vancomycin (tested at 10
µg/mL) in C. elegans AU37 infected with methicillin-resistant Staphylococcus aureus USA400.
Vancomycin served as a positive control. Worms (in L4 stage of growth) were infected with bacteria for
six hours before transferring 15-25 worms to wells of a 96-well plate. Test agents were added and
incubated with worms for 20 hours. Worms were sacrificed and the number of viable colony-forming
units of MRSA USA400 in infected worms was determined for each treatment regimen. The figure
presents the average CFU/mL of MRSA USA400 for each treatment condition. Asterisk (*) denotes
statistical significance (P < 0.05) in CFU/mL relative to untreated control using a Student’s t-test (with
Holm-Sidak correction).

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Pharmacokinetic profiling
Permeability assay. We initially investigated the ability of compound 21n to permeate across the
gastrointestinal tract as modeled by the Caco-2 bidirectional permeability assay. Compound 21n is able to
permeate across the Caco-2 membrane (from the apical to basolateral direction) however at a slow rate
that is similar to talinolol (Table 3). The high efflux ratio for 21n indicates the permeability of this
compound is significantly impaired by the presence of the P-glycoprotein efflux transport system (P-gp).
This result would indicate that, for systemic application, 21n would be more suitable for administration
intravenously. As an alternative to systemic application, the compound could also be explored for topical
treatment of MRSA skin lesions. Given S. aureus is responsible for more than half of all skin and soft
tissue infections in the United States,[39] topical antibiotics are valuable allies to treat these particular
infections. This point will be further explored with 21n in a future study.
Metabolic stability analysis. As discussed earlier, the lead diphenylurea 3 that carries a n-butyl moiety at
the lipophilic side chain revealed an ultra-short half-life (t1/2 = 13 min) with a clearance rate close to 190
µL/min-mg (Table 2). Such PK data (rapid clearance and short t1/2) will significantly affect the size and
frequency of the dosing regimen. Therefore, following well-established protocols,[26, 27] the benzylic
metabolic soft spot was replaced with an oxygen atom, which cannot be oxidized under physiological
conditions. The alkoxy analogues synthesized exhibited a noticeably higher half-life value (five-fold
greater than compound 3) and lower clearance rates (3-5 times lower than for compound 3) (Table 2).
In particular, compound 21n is superior to both midazolam and verapamil as it is cleared by human
liver microsomes at a much lower rate than both control drugs (Table 2). The half-life of 68.2 minutes
indicates the compound is metabolized at a moderate rate. Interestingly, the clearance rate increases and
the half-life decreases for compound 21n in the absence of NADPH indicating this compound is not
metabolized via the typical cytochrome-P450 system in the liver.
Table 3. Caco-2 bidirectional permeability analysis for compound 21n.

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Test Article
Ranitidine
Talinolol
Test
Concentration
(µM)
Assay
Duration
(hours)
2
Mean A→B Mean B→A
Efflux
ratio (Re)
Notes
Papp (10-6
cm/sec)
0.235
Papp (10-6
cm/sec)
2.31
10
9.8
Low
permeability
control
P-glycoprotein
efflux
10
2
0.065
8.95
138
transporter
control
Warfarin
10
10
2
2
27.7
20.3
51.9
0.73
High
permeability
control
21n
0.039
>1000
In vivo Pharmacokinetics. Caco-2 results tend to over predict efflux due to P-gp overexpression. Thus,
an in vivo PK assessment was conducted. Briefly, a dose of 50 mg/kg was given to male Sprague−Dawley
rats and blood samples were collected over a 24-hour period. The aim of this preliminary assessment was
to identify the most suitable route of administration for examining the efficacy of 21n in more advanced
animal models of MRSA infection.
Thus far, the in vivo PK results confirmed the high affinity of 21n to P-gp as the maximum detected
plasma concentration (Cmax) for 21n was around 16 ng/mL (Table 4). This value is roughly two-hundred
times less than the average MIC value of 21n against MRSA. Apart from the high affinity to P-gp, the
high duration of action, indicated by the half-life (t1/2) value of approximately 16 hours (Table 4) further
supports our hypothesis that the benzylic methylene unit is the metabolic soft spot. Thus presently, 21n
would be more suitable to be examined for efficacy in suitable mouse models of infection either through
intravenous administration (for systemic MRSA infections) or topically (for MRSA skin infections).
Table 4. Oral pharmacokinetic parameters in rats after 50 mg/kg oral dose of compound 21n.
Cmax
(ng/mL)
Tmax
(h)
AUClast
((h)*(ng/mL)) ((h)*(ng/mL))
AUCtot
Lz
(1/h)
thalf
(h)
MRT
(h)
Animal
1
2
3
17.300
8.640
22.900
3
8.000
12.000
8.000
3
207.203
137.315
310.805
3
405.515
286.033
461.493
3
0.039
0.037
0.060
3
17.714
18.641
11.593
3
30.467
32.875
21.828
3
N
16.280
7.185
9.333
2.309
218.441
87.289
384.347
89.625
0.045
0.013
15.982
3.830
28.390
5.809
Mean
Stdev

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44.131
4.148
24.744
1.333
8.000
8.000
12.000
39.960
50.396
23.319
51.745
27.615
0.007
0.037
0.039
0.060
23.963
2.211
20.461
3.354
%CV
SEM
Min
8.640
137.315
207.203
310.805
286.033
405.515
461.493
11.593
17.714
18.641
21.828
30.467
32.875
17.300
22.900
Median
Max
3.CONCLUSION.
Bacterial resistance to currently available antibiotics represents a significant challenge to healthcare
providers and researchers in drug discovery. New antibacterial agents with unique chemical scaffolds and
mechanism of action are urgently needed. The present study reported a new series of synthetic
compounds bearing the diphenylurea scaffold with potent antibacterial activity against MRSA and VRSA.
Compound 21n emerged as the most promising analogue due to its superior toxicity profile, ability to kill
intracellular MRSA harboring inside infected macrophages, enhanced stability to hepatic metabolism, and
potent anti-MRSA activity in vivo in a C. elegans model. Pharmacokinetic analysis of 21n revealed, at
present, it is suitable for administration intravenously or topically (for treatment MRSA skin infections).
Future studies will aim to examine 21n in suitable mouse models of MRSA infection and to develop
novel analogues with enhanced ability to permeate across the gastrointestinal tract (to permit oral dosing).
4. EXPERIMENTAL SECTION
4.1.CHEMISTRY
4.1.1. General. 1H NMR spectra were run at 300 MHz and 13C spectra were determined at 100 MHz
in deuterated chloroform (CDCl3), or dimethyl sulfoxide (DMSO-d6) on a Varian Mercury VX-400 NMR
spectrometer. Chemical shifts are given in parts per million (ppm) on the delta (δ) scale. Chemical shifts
were calibrated relative to those of the solvents. Flash chromatography was performed on 230-400 mesh
silica. The progress of reactions was monitored with Merck silica gel IB2-F plates (0.25 mm thickness).
The infrared spectra were recorded in potassium bromide disks on pye Unicam SP 3300 and Shimadzu
FT IR 8101 PC infrared spectrophotometer. Mass spectra were recorded at 70 eV. High resolution mass
spectra for all ionization techniques were obtained from a FinniganMAT XL95. Melting points were

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determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected. HPLC analyses
were performed on an Agilent binary HPLC system (Model 1260) equipped with a multiple wavelength
absorbance UV detector set for 254 nM, and using a 5 µM C-18 reversed-phase column and
methanol:water (4:1) as a mobile phase. All yields reported refer to isolated yields
4.1.2. Preparation of Diphenylurea Derivatives 16 and 19. General Procedure. An appropriate
amine (1 equiv.) was added to 4-acetylphenyl isocyante (15, 1 equiv.) in dry THF (20 mL). The reaction
mixture was stirred at room temperature for 12 hours. The solvent was evaporated under reduced
pressure. The crude product was purified by crystallization from methanol-ethyl acetate (1:1) to afford the
desired compounds.
4.1.2.1. 1-(4-Acetylphenyl)-3-(4-tert-butylphenyl)urea (16).[40] Grayish-white solid (58% yield).
1H NMR (DMSO-d6) δ 9.14 (brs, 1 H), 8.79 (brs, 1 H), 7.88 (d, J = 8.7 Hz, 2 H), 7.61 (d, J = 8.7 Hz, 2
H), 7.41 (d, J = 8.7 Hz, 2 H), 7.13 (d, J = 8.7 Hz, 2 H), 2.56 (s, 3 H), 1.25 (s, 9 H); ESIMS m/z (rel
intensity) 310 (M+, 100); HRMS (ESI), m/z 310.1677 M+, calcd for C19H22N2O2 310.1781.
4.1.2.2. 1-(4-acetylphenyl)-3-(4-hydroxyphenyl)urea (19).[40] Off-white solid (80% yield). Rf 0.36
(50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300 MHz) δ 9.11(s, 1H), 8.97 (s, 1H), 8.46 (s, 1H), 7.90 (d,
J = 11.70 Hz, 2H), 7.57 (d, J = 11.70, 2H), 7.24 (d, J = 11.40, 2H), 6.71 (d, J = 11.07, 2H), 2.49 (s, 3H);
HRMS m/z 270.1015 (calcd for C15H14N2O3, 270.1004); Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22;
N, 10.36; found: C, 66.94; H, 5.28; N, 10.59.
4.1.3. General procedure for the nucleophilic substitution reaction. A solution of urea derivative
19 (0.17 g, 0.5 mmol), alkylhalide (1.0 mmol, 2 eq) and K2CO3 in DMF (5 mL) was stirred at 100 °C for
12 hours. After completion as indicated by TLC, 5.0 mL of EtOAc was added and the mixture was poured
into a saturated solution of NaHCO3 (15 mL). The heterogeneous mixture was separated and the aqueous
layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine, dried

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over MgSO4, filtered through celite and concentrated under reduced pressure. Purification by flash
chromatography on silica gel using EtOAc-hexanes for elution provided the title compounds.
4.1.3.1. 1-(4-acetylphenyl)-3-(4-propoxyphenyl)urea (20a).[40] Off-white solid (70% yield, 95
mg). Rf 0.75 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ 8.99 (s, 1H), 8.57 (s, 1H), 7.90 (d,
J = 9.00, 2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 9.30 Hz, 2H), 6.88 (d, J = 9.30 Hz, 2H), 3.90 (t, J = 6.60
Hz, 2H) 2.49 (s, 3H), 1.74–1.67 (m, 2H), 0.99 (t, J = 7.50, 3H); 13C NMR (DMSO-d6) δ 196.18, 154.14,
152.27, 144.52, 132.10, 130.21, 129.57, 120.28, 116.96, 114.60, 69.09, 26.27, 22.04, 10.35; HRMS m/z
312.1466 (calcd for C18H20N2O3, 312.1474); Anal. Calcd for C18H20N2O3 (312.14): C, 69.21; H, 6.45; N,
8.97; found: C, 69.47; H, 6.51; N, 9.02.
4.1.3.2. 1-(4-Acetylphenyl)-3-(4-butoxyphenyl)urea (20b). Off-white solid (82% yield, 133 mg). Rf
0.77 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ 8.99 (s, 1H), 8.57 (s, 1H),7.90 (d, J = 8.70,
2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.00, 2H), 3.94 (t, J = 6.60, 2H) 2.49(s,
3H), 1.74–1.67 (m, 2H), 1.47–1.41 (m, 2H), 0.99 (t, J = 7.20, 3H); HRMS m/z 326.1635 (calcd for
C19H22N2O3, 326.1630); Anal. Calcd for C19H22N2O3 (326.16): C, 69.92; H, 6.79; N, 8.58; found: C,
70.21; H, 6.87; N, 8.84.
4.1.3.3. 1-(4-acetylphenyl)-3-(4-(pentyloxy)phenyl)urea (20c). Off-white solid (65% yield, 104
mg). Rf 0.79 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ9.01 (s, 1H), 8.59 (s, 1H),7.90 (d, J
= 8.70, 2H), 7.58 (d, J = 8.40, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.00, 2H), 3.94 (t, J = 6.60, 2H),
2.49 (s, 3H), 1.71–1.65 (m, 2H), 1.37–1.31 (m, 4H), 0.92–0.84 (m, 3H); HRMS m/z 340.1790 (calcd for
C20H24N2O3, 340.1787); Anal. Calcd for C20H24N2O3 (340.17): C, 70.57; H, 7.11; N, 8.23; found: C,
70.74; H, 7.18; N, 8.31.
4.1.3.4. 1-(4-acetylphenyl)-3-(4-(hexyloxy)phenyl)urea (20d). Off-white solid (80% yield, 71 mg).
1
Rf 0.81 (50% EtOAc/hexanes); H NMR (DMSO-d6, 300MHz) δ 9.04 (s, 1H), 8.62 (s, 1H),7.90 (d, J =
8.70, 2H), 7.58 (d, J = 8.40, 2H), 7.36 (d, J = 8.10, 2H), 6.88 (d, J = 8.70, 2H), 3.93 (t, J = 6.30, 2H), 2.49
(s, 3H), 1.70–1.64 (m, 2H), 1.40–1.29 (m, 6H), 0.87–0.85 (m, 3H); HRMS m/z 354.1930 (calcd for

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C21H26N2O3, 354.1943); Anal. Calcd for C21H26N2O3 (354.19): C, 71.16; H, 7.39; N, 7.90; found: C,
71.38; H, 7.46; N, 8.02.
4.1.3.5. 1-(4-Acetylphenyl)-3-(4-(heptyloxy)phenyl)urea (20e). Off-white solid (84% yield, 154
mg). Rf 0.8 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ 8.99 (s, 1H), 8.57 (s, 1H),7.90 (d, J
= 8.70, 2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.30, 2H), 3.93 (t, J = 6.60, 2H),
2.49 (s, 3H), 1.71–1.66 (m, 2H), 1.42–1.28 (m, 8H), 0.89–0.85 (m, 3H); HRMS 368.2107 (calcd for
C22H28N2O3, 368.2100); Anal. Calcd for C22H28N2O3 (368.21): C, 71.71; H, 7.66; N, 7.60; found: C,
71.88; H, 7.72; N, 7.84.
4.1.3.6. 1-(4-Acetylphenyl)-3-(4-(octyloxy)phenyl)urea (20f). Brownish solid (89% yield, 170 mg).
1
Rf 0.82 (50% EtOAc/hexanes); H NMR (DMSO-d6, 300MHz) δ 8.99 (s, 1H), 8.57 (s, 1H), 7.90 (d, J =
8.70, 2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.30, 2H), 3.93 (t, J = 6.60, 2H), 2.49
(s, 3H), 1.71–1.66 (m, 2H), 1.42–1.28 (m, 10H), 0.88–0.84 (m, 3H); HRMS m/z 382.2271 (calcd for
C23H30N2O3, 382.2256); Anal. Calcd for C23H30N2O3 (382.22): C, 72.22; H, 7.91; N, 7.32; found: C,
72.43; H, 8.00; N, 7.48.
4.1.3.7. 1-(4-Acetylphenyl)-3-(4-(nonyloxy)phenyl)urea (20g). Brownish solid (77% yield, 153
mg). Rf 0.84 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ 8.99 (s, 1H), 8.57 (s, 1H),7.90 (d, J
= 8.70, 2H), 7.58 (d, J = 8.40, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.30, 2H), 3.93 (t, J = 6.60, 2H),
2.49 (s, 3H), 1.70–1.65 (m, 2H), 1.42–1.25(m, 12H), 0.87–0.83 (m, 3H); HRMS m/z 396.2400 (calcd for
C24H32N2O3, 396.2413); Anal. Calcd for C24H32N2O3 (396.24): C, 72.70; H, 8.13; N, 7.06; found: C,
72.91; H, 8.20; N, 7.29.
4.1.3.8. 1-(4-Acetylphenyl)-3-(4-isobutoxyphenyl)urea (20h). Off-white solid (80% yield, 130 mg).
1
Rf 0.73 (50% EtOAc/hexanes); H NMR (DMSO-d6, 300MHz) δ9.05 (s, 1H), 8.63 (s, 1H),7.90 (d, J =
8.70, 2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 7.80, 2H), 6.88 (d, J = 8.70, 2H), 3.70 (d, J = 6.60, 2H),
2.49 (s, 3H), 1.99–1.95 (m, 1H), 0.98 (d, J = 6.60, 6H HRMS m/z 326.1632 (calcd for C19H22N2O3,

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326.1630); Anal. Calcd for C19H22N2O3 (326.16): C, 69.92; H, 6.79; N, 8.58; found: C, 70.13; H, 6.85; N,
8.74.
4.1.3.9. 1-(4-Acetylphenyl)-3-(4-(isopentyloxy)phenyl)urea (20i). Off-white solid (75% yield, 128
mg). Rf 0.75 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ 8.99 (s, 1H), 8.57 (s, 1H),7.90 (d, J
= 8.70, 2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.00, 2H), 3.93 (t, J = 6.60, 2H),
2.49 (s, 3H), 1.74–1.69 (m, 2H), 1.66–1.64 (m, 1H), 1.33–1.28 (m, 2H), 0.90 (d, J = 6.60, 6H); HRMS
m/z 340.1780 (calcd for C20H24N2O3, 340.1787); Anal. Calcd for C20H24N2O3 (340.17): C, 70.57; H,
7.11; N, 8.23; found: C, 70.79; H, 7.15; N, 8.42.
4.1.3.10. 1-(4-Acetylphenyl)-3-(4-((4-methylpentyl)oxy)phenyl)urea (20j). Off-white solid (92%
1
yield, 162 mg). Rf 0.75 (95% EtOAc/hexanes); H NMR (DMSO-d6, 300MHz) δ8.99 (s, 1H), 8.57 (s,
1H),7.90 (d, J = 8.70, 2H), 7.58 (d, J = 8.70, 2H), 7.36 (d, J = 9.00, 2H), 6.88 (d, J = 9.00, 2H), 3.93 (t, J
= 6.60, 2H), 2.49 (s, 3H), 1.74–1.69 (m, 2H), 1.66–1.64 (m, 1H), 1.33–1.28 (m, 2H), 0.90 (d, J = 6.60,
6H); HRMS m/z 354.1953 (calcd for C21H26N2O3, 354.1943); Anal. Calcd for C21H26N2O3 (354.19): C,
71.16; H, 7.39; N, 7.90; found: C, 71.43; H, 7.44; N, 8.06.
4.1.3.11. 1-(4-Acetylphenyl)-3-(4-(2-ethylbutoxy)phenyl)urea (20k). Off-white solid (82% yield,
145 mg). Rf 0.71 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ 9.01 (s, 1H), 8.59 (s, 1H),7.90
(d, J = 8.70, 2H), 7.56 (d, J = 8.40, 2H), 7.36 (d, J = 8.40, 2H), 6.89 (d, J = 8.40, 2H), 3.82 (d, J = 5.40,
2H), 2.49 (s, 3H), 1.62–1.56 (m, 1H), 1.66–1.64 (m, 1H), 1.47–1.31 (m, 4H), 0.92–0.85 (m, 6H); HRMS
m/z 354.1949 (calcd for C21H26N2O3, 354.1943); Anal. Calcd for C21H26N2O3 (354.19): C, 71.16; H,
7.39; N, 7.90; found: C, 71.44; H, 6.84; N, 8.03.
4.1.3.12. 1-(4-Acetylphenyl)-3-(4-(cyclobutylmethoxy)phenyl)urea (20l). Off-white solid (78%
1
yield, 132 mg). Rf 0.76 (50% EtOAc/hexanes); H NMR (DMSO-d6, 300MHz) δ8.99 (s, 1H), 8.57 (s,
1H),7.90 (d, J = 8.70, 2H), 7.58 (d, J = 9.00, 2H), 7.36 (d, J = 8.70, 2H), 6.88 (d, J = 9.40, 2H), 3.91 (d, J
= 6.60, 2H), 2.61–2.76 (m, 1H), 2.49 (s, 3H), 2.16–2.64 (m, 2H), 1.81–1.89 (m, 4H); 13C NMR (DMSO-
d6) δ 196.19, 154.27, 152.27, 144.51, 132.14, 130.21, 129.58, 120.24, 116.96, 114.66, 71.71, 34.04,

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24.35, 18.05; HRMS m/z 338.1650 (calcd for C20H22N2O3, 338.1630); Anal. Calcd for C20H22N2O3
(338.16): C, 70.99; H, 6.55; N, 8.28; found: C, 71.15; H, 6.64; N, 8.44.
4.1.3.13. 1-(4-Acetylphenyl)-3-(4-(cyclopentyloxy)phenyl)urea (20m). Off-white solid (65%
1
yield, 110 mg). Rf 0.8 (50% EtOAc/hexanes); H NMR (DMSO-d6, 300MHz) δ 9.05 (s, 1H), 8.61 (s,
1H),7.90 (d, J = 8.10, 2H), 7.58 (d, J = 8.40, 2H), 7.35 (d, J = 9.00, 2H), 6.85 (d, J = 8.40, 2H), 4.75 (br s,
1H), 2.49 (s, 3H), 1.88–1.56 (m, 8H); HRMS m/z 338.1628 (calcd for C20H22N2O3, 338.1630); Anal.
Calcd for C20H22N2O3 (338.16): C, 70.99; H, 6.55; N, 8.28; found: C, 71.24; H, 6.53; N, 8.41.
4.1.3.14. 1-(4-Acetylphenyl)-3-(4-(cycloheptyloxy)phenyl)urea (20n). Brownish white solid (68%
yield, 125 mg). Rf 0.85 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ9.6 (s, 1H), 9.16 (s, 1H),
7.89 (d, J = 8.10, 2H), 7.60 (d, J = 8.10, 2H), 7.37 (d, J = 8.40, 2H), 6.84 (d, J = 8.40, 2H), 4.41–4.39 (br
m, 1H), 2.49 (s, 3H), 1.95–1.89 (m, 2H), 1.79–1.44 (m, 10H); HRMS m/z 366.1938 (calcd for
C22H26N2O3, 366.1943); Anal. Calcd for C22H26N2O3 (366.19): C, 72.11; H, 7.15; N, 7.64; found: C,
72.40; H, 7.21; N, 7.80.
4.1.3.15. 1-(4-Acetylphenyl)-3-(4-(benzyloxy)phenyl)urea (20o).[40] Off-white solid (97% yield,
175 mg). Rf 0.91 (50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300MHz) δ9.02 (s, 1H), 8.06 (s, 1H), 7.90
(d, J = 8.70, 2H), 7.58 (d, J = 8.70, 2H), 7.45–7.32(m, 7H), 6.97 (d, J = 8.10, 2H), 5.10 (s, 2H), 2.49 (s,
3H); HRMS m/z 360.1463 (calcd for C22H20N2O3, 360.1474); Anal. Calcd for C22H20N2O3 (360.14): C,
73.32; H, 5.59; N, 7.77; found: C, 73.56; H, 5.26; N, 7.78.
4.1.4. Preparation of Carbamimidoylhydrazono Derivatives 17 and 21a-o. General
Procedures. The methyl ketones (17 and 19, 0.5 mmol) were dissolved in absolute ethanol (10 mL), and
aminoguanidine hydrochloride (111 mg, 1 mmol) and a catalytic amount of LiCl (10 mg) were added.
The reaction mixture was heated under reflux for 12-24 hours. The solvent was evaporated under reduced
pressure. The crude product was purified by crystallization from 70% methanol, then recystalization again
from ethyl acetate to afford the final products as listed below:

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4.1.4.1. 2-{1-[4-(3-(4-(tert-Butyl)phenyl)ureido)phenyl]ethylidene}hydrazine-1-
carboximidamide (17). Buff solid (76%): mp > 300 oC. 1H NMR (DMSO-d6) δ 9.70 (brs, 1 H), 8.60 (brs,
1 H), 7.72 (d, J = 8.7 Hz, 2 H), 7.41 (d, J = 8.7 Hz, 2 H), 7.37 (d, J = 8.7 Hz, 2 H), 7.27 (d, J = 8.7 Hz, 2
H), 5.83 (brs, 2 H), 5.43 (brs, 2 H), 2.19 (s, 3 H), 1.24 (s, 9 H); 13C NMR (DMSO-d6, 100MHz) δ 160.20,
153.41, 148.19, 145.02, 140.08, 137.97, 134.64, 126.82, 126.28, 119.00, 118.39, 34.78, 32.17, 14.20;
ESIMS m/z (rel intensity) 367 ([M+H]+, 100); HRMS (ESI), m/z 367.2252 M+, calcd for C20H27N6O
367.2241.
4.1.4.2. 2-(1-(4-(3-(4-Propoxyphenyl)ureido) phenyl)ethylidene) hydrazine-1-carboximidamide
1
(21a). Yellowish solid (80% yield, 147 mg). Rf 0.5 (DCM/MeOH/Et3N, 87:10:3); H NMR (DMSO-d6,
300MHz) δ11.20(s, 1H), 9.69(s, 1H), 9.48 (s, 1H), 7.91 (br s, 3H), 7.39 (d, J = 8.70, 2H), 7.54 (d, J =
8.70, 2H), 7.42 (d, J = 8.70, 2H), 6.6.77 (d, J = 8.70, 2H), 3.84 (t, J = 6.60, 2H), 2.24 (s, 3H), 1.71–1.64
(m, 2H), 0.97 (t, J = 7.50, 3H); 13C NMR (DMSO-d6, 100MHz)
δ
156.49, 154.04, 153.22, 151.96, 142.34,
133.54, 130.22, 127.63, 120.34, 117.77, 114.88, 72.56, 23.49, 14.47, 10.68; HRMS m/z 368.1980 (calcd
for C19H24N6O2, 368.1961); Anal. Calcd for C19H24N6O2 (368.19): C, 61.94; H, 6.57; N, 22.81; found: C,
62.13; H, 6.66; N, 23.06.
4.1.4.3. 2-(1-(4-(3-(4-Butoxyphenyl)ureido)phenyl)ethylidene)hydrazine-1-carboximidamide
1
(21b). Yellowish solid (82% yield, 157 g). Rf 0.51 (DCM/MeOH/Et3N, 87:10:3); H NMR (DMSO-d6,
300MHz) δ 9.90 (s, 1H), 9.56 (s, 1H),7.86 (d, J = 8.70, 2H), 7.84 (br s, 3H), 7.49 (d, J = 8.70, 2H), 7.35
(d, J = 9.00, 2H), 6.88 (d, J = 8.70, 2H), 4.68 (s, 1H), 3.89 (t, J = 6.30, 2H), 2.31(s, 3H), 1.68–1.63 (m,
2H), 1.44–1.40 (m, 2H), 0.94 (t, J = 7.20, 3H); 13C NMR (DMSO-d6, 100MHz) δ 156.42, 153,70, 152.81,
151.05, 141.63, 132.79, 129.74, 127.34, 119.47, 116.81, 114.58, 67.26, 30.80, 18.69, 14.45, 13.66;
HRMS m/z 382.2121 (calcd for C20H26N6O2, 382.2117); Anal. Calcd for C20H26N6O2 (382.21): C, 62.81;
H, 6.85; N, 21.97; found: C, 63.07; H, 6.92; N, 22.14.
4.1.4.4. 2-(1-(4-(3-(4-(Pentyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1 carboximidamide
1
(21c). Yellowish solid (75% yield, 148 mg). Rf 0.54 (DCM/MeOH/Et3N, 87:10:3); H NMR (DMSO-d6,

ACCEPTED MANUSCRIPT
300MHz)
δ
11.01 (s, 1H), 9.45 (s, 1H), 9.21 (s, 1H), 7.80 (d, J = 8.40, 2H), 7.75 (br s, 3H), 7.53 (d, J = 8.
70, 2H), 7.39 (d, J = 8.40, 2H), 6.80 (d, J = 8.70, 2H), 3.87 (t, J = 6.30, 2H), 2.26 (s, 3H), 1.69–1.62 (m,
13
2H), 1.39–1.29 (m, 4H), 0.91–0.86 (m, 3H); C NMR (DMSO-d6, 100MHz) δ 155.88, 153,71, 152.69,
151.57, 141.83, 132.87, 129.66, 127.23, 119.92, 117.23, 114.42, 67.53, 28.43, 27.70, 21.86, 14.01, 13.85;
HRMS m/z 396.2282 (calcd for C21H28N6O2, 396.2274); Anal. Calcd for C21H28N6O2 (396.22): C, 63.62;
H, 7.12; N, 21.20; found: C, 63.80; H, 7.21; N, 21.49.
4.1.4.5. 2-(1-(4-(3-(4-(Hexyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-carboximidamide
(21d). Yellowish white solid (62% yield, 127 mg). Rf 0.56 (DCM/MeOH/Et3N, 87:10:3);1H NMR
(DMSO-d6, 300MHz) δ 9.04 (s, 1H), 8.62 (s, 1H),7.90 (d, J = 8.70, 2H), 7.58 (d, J = 8.40, 2H), 7.36 (d, J
= 8.10, 2H), 6.88 (d, J = 8.70, 2H), 3.93 (t, J = 6.30, 2H), 2.49 (s, 3H), 1.70–1.64 (m, 2H), 1.40–1.29 (m,
6H), 0.87–0.85 (m, 3H); 13C NMR (DMSO-d6, 100MHz) δ 155.89, 153,64, 152.69, 151.56, 141.90,
132.94, 129.59, 127.18, 119.88, 117.24, 114.33, 67.50, 30.98, 28.70, 25.16, 21.99, 13.81 (2C); HRMS
m/z 410.2432 (calcd for C22H30N6O2, 410.2430); Anal. Calcd for C22H30N6O2 (410.24): C, 64.37; H, 7.37;
N, 20.47; found: C, 64.51; H, 7.41; N, 20.70.
4.1.4.6. 2-(1-(4-(3-(4-(Heptyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
carboximidamide (21e). Yellowish white solid (88% yield, 187 g). Rf 0.57 (DCM/MeOH/Et3N,
87:10:3); 1H NMR (DMSO-d6, 300MHz) δ 11.15 (br s, 1H), 9.52 (s, 1H), 9.30 (s, 1H), 7.58 (d, J = 8.70,
2H), 7.71 (br s, 3H), 7.52 (d, J = 8.70, 2H), 7.40 (d, J = 8.70, 2H), 6.79 (d, J = 9.00, 2H), 3.88 (t, J = 6.30,
2H), 2.25 (s, 3H), 1.69–1.62 (m, 2H), 1.38–1.27 (m, 8H), 0.88–0.84 (m, 3H); HRMS m/z 424.2600 (calcd
for C23H32N6O2, 424.2587); Anal. Calcd for C23H32N6O2 (424.25): C, 65.07; H, 7.60; N, 19.80; found: C,
65.31; H, 7.69; N, 20.03.
4.1.4.7. 2-(1-(4-(3-(4-(octyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-carboximidamide
1
(21f). Yellowish white solid (80% yield, 175 mg). Rf 0.58 (DCM/MeOH/Et3N, 87:10:3); H NMR
(DMSO-d6, 300MHz) δ 11.02 (brs, 1H), )9.72 (s, 1H), 9.45 (s, 1H),7.77 (d, J = 9.00, 2H), 7.45 (d, J =
9.00, 2H), 7.38 (d, J = 8.70, 2H), 6.84 (d, J = 9.00, 2H), 6.76 (br s, 3H), 3.91 (t, J = 6.60 Hz, 2H), 2.25

ACCEPTED MANUSCRIPT
13
(s, 3H), 1.72–1.63 (m, 2H), 1.39–1.26 (m, 10H), 0.85–0.83 (m, 3H); C NMR (DMSO-d6, 100MHz) δ
156.44, 154.12, 153.19, 151.98, 142.32, 133.46, 130.18, 127.64, 120.37, 117.74, 114.82, 67.98, 31.70,
29.25, 29.12, 26.02, 22.53, 14.46, 14.40; HRMS m/z 438.2731 (calcd for C24H34N6O2, 438.2743); Anal.
Calcd for C24H34N6O2 (438.27): C, 65.73; H, 7.81; N, 19.16; found: C, 66.01; H, 7.89; N, 19.44.
4.1.4.8. 2-(1-(4-(3-(4-(nonyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-carboximidamide
1
(21g). Pale yellow solid (70% yield, 158 g). Rf 0.6 (DCM/MeOH/Et3N, 87:10:3); H NMR (DMSO-d6,
300MHz) δ11.10 (br s, 1H), 9.55 (s, 1H), 9.32 (s, 1H), 7.84 (br s, 3H), 7.76 (d, J = 8.40, 2H), 7.53 (d, J =
8.10, 2H), 7.40 (d, J = 8.70, 2H), 6.77 (d, J = 8.70, 2H), 3.87 (t, J = 6.60, 2H), 2.25 (s, 3H), 1.68–1.63 (m,
13
2H), 1.37–1.25(m, 12H), 0.87–0.83 (m, 3H); C NMR (DMSO-d6, 100MHz) δ 156.36, 154.08, 153.20,
152.10, 142.42, 133.52, 130.07, 127.66, 120.36, 117.75, 114.77, 67.96, 31.74, 29.44, 29.31, 29.27, 29.14,
26.02, 22.55, 14.45, 14.39; HRMS m/z 452.2893 (calcd for C25H36N6O2, 452.2900); Anal. Calcd for
C25H36N6O2 (452.29): C, 66.34; H, 8.02; N, 18.57; found: C, 66.59; H, 8.13; N, 18.81.
4.1.4.9. 2-(1-(4-(3-(4-isobutoxyphenyl)ureido)phenyl)ethylidene)hydrazine-1-carboximidamide
1
(21h). Yellow solid (65% yield, 0124 mg). Rf 0.48 (DCM/MeOH/Et3N, 87:10:3); H NMR (DMSO-d6,
300MHz) δ 11.12 (br s, 1H), 9.58 (s, 1H), 9.35 (s, 1H), 7.85 (br s, 3H), 7.76 (d, J = 7.80, 2H), 7.53 (d, J =
7.80, 2H), 7.41(d, J = 7.50, 2H), 6.79 (d, J = 8.10, 2H), 3.65 (d, J = 5.70, 2H), 2.25 (s, 3H), 1.98–1.94 (m,
13
1H), 0.96 (d, J = 6.30, 6H); C NMR (DMSO-d6, 100MHz) δ 155.92, 153.73, 152.74, 151.63, 141.95,
133.08, 129.60, 127.17, 119.90, 117.30, 114.39, 73.93, 27.71, 19.01, 13.97; HRMS m/z 382.2125 (calcd
for C20H26N6O2, 382.2117); Anal. Calcd for C20H26N6O2 (382.21): C, 62.81; H, 6.85; N, 21.97; found: C,
63.07; H, 6.92; N, 22.14.
4.1.4.10. 2-(1-(4-(3-(4-(Isopentyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
1
carboximidamide (21i). Yellow solid (60% yield, 119 mg). Rf 0.5 (DCM/MeOH/Et3N, 87:10:3); H
NMR (DMSO-d6, 300MHz) δ 11.12 (br s, 1H), 9.52 (s, 1H), 9.28 (s, 1H), 7.84 (br s, 3H), 7.79 (d, J =
8.40, 2H), 7.54 (d, J = 8.10, 2H), 7.40 (d, J = 8.40, 2H), 6.80 (d, J = 8.40, 2H), 3.92 (t, J = 6.00, 2H), 2.26
(s, 3H), 1.78–1.71 (m, 1H), 1.59–1.1.54 (m, 2H), 0.92 (d, J = 6.30, 6H); 13C NMR (DMSO-d6, 100MHz)

ACCEPTED MANUSCRIPT
δ 155.89, 153.68, 152.72, 151.61, 141.91, 132.95, 129.61, 127.22, 119.94, 117.25, 114.39, 65.96, 37.52,
24.54, 22.39, 13.99; HRMS m/z 396.2260 (calcd for C21H28N6O2, 396.2274); Anal. Calcd for
C21H28N6O2 (396.22): C, 63.62; H, 7.12; N, 21.20; found: C, 63.84; H, 7.19; N, 21.47.
4.1.4.11. 2-(1-(4-(3-(4-((4-methylpentyl)oxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
carboximid-amide (21j). Faint yellow solid (90% yield, 185 mg). Rf 0.51 (DCM/MeOH/Et3N, 87:10:3);
1H NMR (DMSO-d6, 300MHz) δ11.12 (br s, 1H), 9.50 (s, 1H), 9.24 (s, 1H), 7.80 (m, 5H), 7.53 (d, J =
8.40, 2H), 7.39 (d, J = 9.00, 2H), 6.79 (d, J = 9.00, 2H), 3.88 (t, J = 6.60, 2H), 2.26 (s, 3H), 1.69–1.63 (m,
13
2H), 1.64–1.54 (m, 1H), 1.31–1.25 (m, 2H), 0.88 (d, J = 6.60, 6H) ; C NMR (DMSO-d6, 100MHz) δ
155.92, 153.69, 152.69, 151.52, 141.82, 132.88, 129.67, 127.20, 119.90, 117.22, 114.40, 67.82, 34.68,
27.23, 26.65, 22.38, 13.99; HRMS m/z 410.2444 (calcd for C22H30N6O2, 410.2430); Anal. Calcd for
C22H30N6O2 (410.24): C, 64.37; H, 7.37; N, 20.47; found: C, 64.64; H, 7.45; N, 20.70.
4.1.4.12. 2-(1-(4-(3-(4-(2-Ethylbutoxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
carboximidamide (21k). Yellowish white solid (85% yield, 174 mg). Rf 0.52 (DCM/MeOH/Et3N,
1
87:10:3); H NMR (DMSO-d6, 300MHz) δ 11.21 (brs, 1H), 9.53 (s, 1H), 9.24 (brs, 1H), 7.95 (brs,
3H),7.81 (d, J = 8.70, 2H), 7.56 (d, J = 8.40, 2H), 7.36 (d, J = 8.40, 2H), 6.80 (d, J = 8.40, 2H), 3.82 (d, J
= 5.40, 2H), 2.38 (s, 3H), 1.62–1.56 (m, 1H), 1.47–1.31 (m, 4H), 0.92–0.85 (m, 6H); 13C NMR (DMSO-
d6, 100MHz) δ 156.39, 154.43, 153.18, 151.91, 142.31, 133.32, 130.10, 127.80, 120.28, 117.59, 114.98,
70.21, 23.26, 14.61, 11.42, 10.88; HRMS m/z 410.2439 (calcd for C22H30N6O2, 410.2430); Anal. Calcd
for C22H30N6O2 (410.24): C, 64.37; H, 7.37; N, 20.47; found: C, 64.55; H, 7.48; N, 20.71.
4.1.4.13. 2-(1-(4-(3-(4-(Cyclobutylmethoxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
1
carboximid-amide (21l). yellow solid (66% yield, 130 mg). Rf 0.49 (DCM/MeOH/Et3N, 87:10:3); H
NMR (DMSO- d6, 300MHz) δ 9.97 (s, 1H), 9.65 (s, 1H), 7.96 (brs, 3H), 7.87 (d, J = 8.10, 2H), 7.49 (d, J
= 8.40, 2H), 7.35 (d, J = 8.40, 2H), 6.85 (d, J = 8.70, 2H), 4.68 (brs, 1 H), 3.88 (d, J = 6.90, 2 H), 2.71–
2.66 (m, 1 H), 2.31 (s, 3H), 2.03–1.99 (m, 2H), 1.81–1.76 (m, 4H); HRMS m/z 394.2131 (calcd for

ACCEPTED MANUSCRIPT
C21H26N6O2, 394.2117); Anal. Calcd for C21H26N6O2 (394.21): C, 63.94; H, 6.64; N, 21.30; found: C,
64.13; H, 6.74; N, 21.56.
4.1.4.14. 2-(1-(4-(3-(4-(Cyclopentyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
1
carboximidamide (20m). Yellowish solid (58% yield, 114g). Rf 0.51 (DCM/MeOH/Et3N, 87:10:3); H
NMR (DMSO-d6, 300MHz) δ 9.82 (s, 1H), 9.49 (s, 1H), 8.91 (brs, 1H), 7.87 (d, J = 8.40 Hz, 2 H), 7.82
(brs, 1 H), 7.50 (d, J = 8.10, 2H), 7.34 (d, J = 8.70 Hz, 2 H), 7.27 (brs, 2 H), 6.82 (d, J = 9.00, 2H), 4.77
(s, 1H), 2.31 (s, 3 H), 1.86–1.56 (m, 8 H); 13C NMR (DMSO-d6, 100MHz) δ 159.79, 156.70, 153.27,
153.07, 142.21, 133.08, 130.06, 127.90, 120.05, 117.27, 116.10, 79.16, 32.68, 24.00, 14.96; HRMS m/z
394.2130 (calcd for C21H26N6O2, 394.2117); Anal. Calcd for C21H26N6O2 (394.21): C, 63.94; H, 6.64; N,
21.30; found: C, 64.12; H, 6.69; N, 21.54.
4.1.4.15. 2-(1-(4-(3-(4-(Cycloheptyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
carboximidamide (21n). Brownish yellow solid (84% yield, 177 mg). Rf 0.56 (DCM/MeOH/Et3N,
1
87:10:3); H NMR (DMSO-d6, 300MHz) δ 11.10 (br s, 1H), 9.55 (s, 1H), 9.32 (s, 1H), 7.80 (br s, 3H),
7.77 (d, J = 8.10, 2H), 7.53 (d, J = 8.40, 2H), 7.39 (d, J = 8.70, 2H), 6.76 (d, J = 8.70, 2H), 4.36–4.34 (m,
1H), 2.25 (s, 3H), 1.94–1.87 (m, 2H), 1.69–1.42 (m, 10H); 13C NMR (DMSO-d6, 100MHz) δ 156.37,
153.21, 152.68, 151.99, 142.40, 133.37, 130.07, 127.70, 120.45, 117.68, 116.47, 77.55, 33.62, 28.33,
22.80, 14.50; HRMS m/z 422.2440 (calcd for C23H30N6O2, 422.2430); Anal. Calcd for C23H30N6O2
(422.24): C, 65.38; H, 7.16; N, 19.89; found: C, 65.54; H, 7.23; N, 20.04.
4.1.4.16. 2-(1-(4-(3-(4-(Benzyloxy)phenyl)ureido)phenyl)ethylidene)hydrazine-1-
carboximidamide (21o). Yellowish brown solid (90% yield, 187 g). Rf 0.62 (DCM/MeOH/Et3N,
1
87:10:3); H NMR (DMSO-d6, 300MHz)
δ 11.12 (brs, 1H), 9.54 (s, 1H), 9.32 (s, 1H), 7.83 (br s, 3H),
7.79 (d, J = 8.40, 2H), 7.54 (d, J = 8.40, 2H), 7.36–1.24 (m, 7H), 6.90 (d, J = 8.40, 2H), 5.00 (s, 2H),
13
2.26 (s, 3H); C NMR (DMSO-d6, 100MHz) δ 155.94, 153.32, 152.71, 151.52, 141.81, 137.25, 133.29,
129.71, 128.30, 127.63, 127.56, 127.21, 119.88, 117.27, 114.82, 69.39, 13.99; HRMS m/z 416.1954