Activity of oxygen-versus sulfur-containing analogs of the Flex-Het anticancer agent SHetA2

Article abstract of DOI:10.1016/j.ejmech.2018.09.036

Five series of chromans with urea and thiourea linkers connecting a chroman unit (ring A) and a 4-substituted benzene unit (ring B) have been prepared and evaluated relative to SHetA2 (NSC 721689) for activity against the human A2780 ovarian cancer cell l

Full text of DOI:10.1016/j.ejmech.2018.09.036

Accepted Manuscript
Activity of oxygen-versus sulfur-containing analogs of the Flex-Het anticancer agent
SHetA2
Field M. Watts, Jr., Tim Pouland, Richard A. Bunce, K. Darrell Berlin, Doris M.
Benbrook, Maryam Mashayekhi, Dipendra Bhandari, Donghua Zhou
PII:
S0223-5234(18)30808-0
10.1016/j.ejmech.2018.09.036
EJMECH 10747
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 June 2018
Revised Date: 3 September 2018
Accepted Date: 13 September 2018
Please cite this article as: F.M. Watts Jr., T. Pouland, R.A. Bunce, K.D. Berlin, D.M. Benbrook, M.
Mashayekhi, D. Bhandari, D. Zhou, Activity of oxygen-versus sulfur-containing analogs of the Flex-Het
anticancer agent SHetA2, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.036.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Activity of oxygen- versus sulfur-containing analogs of the
Flex-Het anticancer agent SHetA2
Field M. Watts, Jr,‡ Tim Pouland,fl Richard A. Bunce,‡* K. Darrell Berlin,‡ Doris M. Benbrook,fl ** Maryam
Mashayekhi,¶ Dipendra Bhandari,¶ and Donghua Zhou¶***
‡Department of Chemistry, Oklahoma State University, Stillwater, OK 74078
flDepartment of Obstetrics and Gynecology, Stephenson Cancer Center, University of Oklahoma
Health Sciences Center, Oklahoma City, OK 73104
¶Department of Physics, Oklahoma State University, Stillwater, OK 74078
Keywords: SHetA2; chroman-based anticancer agents; mortalin; in silico docking study; polarity and electronic
optimization
Abstract
Five series of chromans with urea and thiourea linkers connecting a chroman unit (ring A) and a 4-
substituted benzene unit (ring B) have been prepared and evaluated relative to SHetA2 (NSC 721689) for
activity against the human A2780 ovarian cancer cell line. The lead compound SHetA2 had a sulfur in place of
the oxygen in ring A and a thiourea linker to ring B. The 2-Me-4-Me series (two sets of geminal dimethyl
groups at C2 and at C4 on the ring A unit) permitted direct comparison with SHetA2. Ring B in this series was
evaluated with specific functional groups at C4 on the ring, including NO2, CO2Et, CF3, OCF3, CN and SO2NH2.
The 2-H-4-Me series (only one geminal dimethyl group at the C4 position on ring A) permitted structure-activity
relationship analysis to assess the importance of the hydrophobic geminal dimethyl groups on ring A to the
activity of SHetA2. The remaining three series 2-Et-4-Me, 2-Me-4-Et and 2-Et-4-Et (ring A methyl groups
replaced with ethyls at C2, at C4 and at both C2 and C4, respectively) offered the opportunity to modulate the
hydrophobicity of the chroman moiety. Additionally, in all these series, the influence of a urea versus a
thiourea linker was also investigated. The results of these modifications are summarized below. The exact
analog of SHetA2 with oxygen substituted for sulfur in ring A (2a) showed comparable efficacy but a
significantly lower IC50 against the ovarian cancer cell line. The urea linked analogs bearing CN, CF3 and
OCF3 at C4 of ring B (3c,d and f) showed greater efficacy than SHetA2, but also had lower IC50 values.
Removing the geminal dimethyl group at C2 (4a-c, 5a-c) caused a significant lowering of the efficacy and
percent growth inhibition, indicating that the hydrophobic geminal dimethyl group at C2 in ring A is crucial for
activity. Finally, replacing the geminal dimethyl groups with geminal diethyls on ring A in the urea derivatives
gave 6b-c, 7c-d and 8b, all of which outperformed SHetA2 with respect to efficacy and IC50. The results for
compounds 4-8 are in concurrence with modeling studies, which predicted that greater hydrophobicity in ring A
would be beneficial. Binding energies were determined for compounds docked in silico to mortalin, the protein
identified as a receptor of SHetA2. The urea linker promoted activity comparable to or, in some cases, greater
than compounds with a thiourea linker. Several compounds achieved 94% efficacy and an IC50 of 2 µM, which
were better than SHetA2 (84%, 3 µM).
*Address inquiries for the synthesis to this individual.
**Address inquiries on the biological experiments to this individual.
***Address inquires for molecular modeling and biological data analysis to this individual.
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1. Introduction
Ovarian cancer is considered the most life-threatening of all gynecologic malignancies [1] and often has
few symptoms prior to late stage diagnosis. In 2017, approximately 22,440 women were diagnosed with the
disease in the United States and 14,000 died [2]. After surgery, the use of cisplatin and taxane usually have
unacceptable side effects [3, 4]. The anti-cancer compound SHetA2 [5], a candidate for clinical trials for the
treatment/prevention of ovarian cancer, induces both intrinsic (mitochondrial mediated) and extrinsic (death
receptor mediated) apoptosis pathways in cancer cells [6]. More importantly, the agent exhibits discriminatory
activity against malignant cells versus normal cells [6-9]. An additional benefit is that the compound
demonstrates very low toxicity, with a no-observed-adverse-effect level (NOAEL) of 500 mg/kg/day in rats and
over 1500 mg/kg/day in dogs [10].
SHetA2 has been shown to interfere and interact with the activity of the heat shock protein mortalin
(PDB ID: 3N8E; HSPA9) [11]. The substrate-binding domain (SBD) of mortalin was shown to associate with
the tumor protein p53 in a concentration-dependent manner [12]. A mutation of V482F in the substrate-binding
pocket abrogated the interaction with p53 [12], consistent with a similar mutation in DnaK, a bacterial homolog
of mortalin, which also disrupts substrate binding [13]. Accordingly, pre-incubation of mortalin with a short
substrate peptide greatly reduced its binding to p53 [13]. These observations suggest that p53 binds to the
substrate-binding pocket in the substrate-binding domain of mortalin. Additional evidence showed that p53
also interacted with the nucleotide-binding domain (NBD) of mortalin [13]. Thus, one pathway for the
compounds to inhibit the growth of cancer cells in vitro is by displacing p53 from mortalin. Then, p53 migrates
to the nucleus and initiates apoptosis, and the cell dies. However, interactions of mortalin with other client
proteins, such as Bcl-2 and p66shc, may also impact cell apoptosis.
In an effort to determine if certain fragments of the lead molecule SHetA2 could be modified to enhance
aqueous solubility, targets were synthesized with an oxygen-containing chroman unit, in place of the
thiochroman, and both systems with urea and thiourea linkers were compared. The aim of this work was to
develop syntheses of oxygen counterparts (structures 2-8 in Figure 1) of SHetA2, with sufficient structural
diversity to allow a modest structure-activity relationship (SAR) analysis of this family of heterocycles. Five
series of related drug candidates with an oxygen heterocycle in ring A were prepared and evaluated relative to
SHetA2. Substituents on the chroman unit were modified to appraise the importance of hydrophobicity in this
moiety, which had been suggested by molecular modeling analyses. Preliminary data revealed that
substituting a urea linker for a thiourea linker demonstrated that the former exhibited an intramolecular
stabilizing H-bonding effect while the thiourea did not [14]. Consequently, it is not unreasonable to expect
stronger H-bonding capabilities by the urea linker compared to the thiourea linker when docked in mortalin.
Moreover, the oxygen analogs would likely be more stable to oxidative degradation both in vivo and in long-
term storage.
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Figure 1. Target compounds.
2. Results and Discussion
2.1 Chemistry
The synthesis of analogs 2 and 3 (2-Me-4-Me Series) was based, in small part, on a literature approach to
several similar compounds (Scheme 1) [15,16]. In view of the established interaction of SHetA2 with mortalin
[11], the nature of the heteroatom in ring A and the substituents on ring B were both deemed important.
Consequently, the placement of oxygen in ring A and highly polar substituents on ring B was investigated as
related to biological activity. The influence of larger alkyl groups on ring A was also examined.
Reaction of phenol (9) with sodium hydride in tetrahydrofuran (THF) gave a tan solution of sodium
phenolate, which reacted with 3-methylbut-2-enoyl chloride [0 oC ––> room temperature (23 oC)] to give phenyl
3-methylbut-2-enoate (10) in 89% yield. Treatment of 10 with aluminum chloride in dichloromethane (DCM, 0
oC ––> 23 oC) for 72 h gave the ring-closed chromanone 11 (68%). Addition of methyllithium to the carbonyl of
o
o
11 at –45 C in ether, followed by warming to 23 C overnight, afforded the ring-opened phenol-alcohol 12
o
(92%). Heating 12 for 1 h in concentrated phosphoric acid at 100 C cyclized the ether ring to give 2,2,4,4-
tetramethylchroman (13) in nearly quantitative yield. Nitration proved difficult to control, but the reaction of 13
o
with nitric acid in acetic anhydride at –5 C gave an 83% yield of a 45:55 mixture of the C6 and C8 nitrated
products 14 and 15, respectively, along with 2% of recovered starting material. Isomers 14 and 15 were
separated by column chromatography, and the C6-nitration product 14 (33%) was carried forward. Reduction
of the nitro group under 1 atmosphere of H2 over 5% Pd/C in ethanol at 23 oC gave the aniline 16 (68%), which
was immediately reacted with a series of isothiocyanates at 0 oC ––> 23 oC in THF to afford the target drug
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Scheme 1. Synthesis of oxygen analogs 2 and 3 of SHetA2 (2-Me-4-Me Series).
candidates 2a-c,e (>80%). Derivatives 3a,c,d,f were also generated in yields >80% by reacting 16 with an
appropriate isocyanate.
Additional derivatives were prepared from 11 with the geminal dimethyl groups absent from the carbon
adjacent to the oxygen in the fused heterocycle (2-H-4-Me Series) (Scheme 2). Reduction of the lactone
carbonyl of 11 with lithium aluminum hydride in THF gave diol 17. Sequential treatment of this diol with
methanesulfonyl chloride in DCM, followed by sodium hydroxide in aqueous dioxane, yielded 4,4-
dimethylchroman (18) (95%). Treatment of this chroman with nitric acid in acetic anhydride gave an 89% yield
of a 45:55 mixture of the C6 and C8 nitration products, 19 and 20, respectively. The mixture of nitro
compounds proved difficult to separate, and thus, were reduced to the corresponding anilines 21 and 22 using
o
iron/ammonium chloride in aqueous ethanol (85 C) [17]. The anilines were readily separated by column
chromatography to give 21 (42%) and 22 (52%). Once separated, 4,4-dimethylchroman-6-amine (21) was
allowed to react with a series of isothiocyanates and isocyanates in THF to give the required candidates 4a-c
and 5a-c in high yields.
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Scheme 2: Synthesis of oxygen analogs 4 and 5 of SHetA2 (2-H-4-Me Series).
In an effort to determine the influence of the position and size of hydrophobic groups on the activity,
syntheses were initiated on three additional sets of targets, 6a-c (2-Et-4-Me Series) (Scheme 3), 7a-d (2-Me-
4-Et Series) (Scheme 4) and 8a-d (4-Et-4-Et Series) (Scheme 5). All of the substituents attached to ring B in
6-8 had H-bonding capability with polar groups in the binding site of mortalin. Members of 6 retained the
geminal dimethyl group at C4, but the C2 dimethyl was replaced by a geminal diethyl group. In 7, these two
geminal groupings were reversed (di-Me at C2 and di-Et at C4). This allowed an assessment of the effects of
placing larger ethyl groups at C2 and C4, respectively, on the activity. System 8, with geminal diethyl groups
at both C2 and C4, should increase the overall capacity to interact with hydrophobic centers in mortalin.
The syntheses of 6-8 are given below. Paralleling the route to obtain 2 and 3, Scheme 3 below outlines
the conversion of 11 ––> 23 ––> 24 ––> 25 + 26 ––> 27 + 28, with 27 being the precursor to 6a-c (2-Et-4-Me
Series). Compounds 7a-d (2-Me-4-Et Series) were prepared as illustrated in Scheme 4. Phenol (9) was
treated with sodium hydride, followed by the addition of the acid chloride, prepared from 3-ethylpent-2-enoic
acid [18] and thionyl chloride, to produce ester 29. Aluminum chloride catalyzed cyclization generated 4,4-
diethylchroman-2-one (30) in 35% yield. Transformation of 30 to 2-(3-ethyl-5-hydroxy-5-methylhexan-3-
yl)phenol (31) by addition of excess methylmagnesium bromide and subsequent ring closure with concentrated
phosphoric acid, gave 4,4-diethyl-2,2-dimethylchroman (32) in 81% yield. Nitration of 32 according to the
standard protocol produced isomers 33 and 34, which could not be separated. Reduction of this mixture as
outlined above gave amines 35 (36%) and 36 (43%), which were separable by column chromatography.
Targets 7a-d were then accessed by reaction of 35 with isothiocyanates and isocyanates in the usual manner.
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Scheme 3. Synthesis of oxygen analogs 6 of SHetA2 (2-Et-4-Me Series).
Scheme 4. Synthesis of oxygen analogs 7 of SHetA2 (2-Me-4-Et Series).
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Increasing the hydrophobic properties of the systems was envisioned with compounds 8a-d (2-Et-4-Et
Series), which were prepared as outlined in Scheme 5. Treatment of 30 with ethylmagnesium bromide yielded
37, which contained an impurity that could not be removed. Thus, 37 was cyclized to 38 which also contained
the impurity. Consequently, slightly impure 38 was nitrated to give the C6 (39, 35%) and C8 (40) isomers
which could be separated. The C8 isomer (40) separated with the impurity and was discarded. Normal
reduction conditions applied to 39 generated 41 (91%), which was immediately reacted with the appropriate
isothiocyanates or isocyanates to yield 8a-d in high yields. All thiourea and urea derivatives 2-8 were fully
characterized spectroscopically and gave acceptable elemental analyses.
Scheme 5. Synthesis of oxygen analogs 8 of SHetA2 (2-Et-4-Et Series).
2.2. Biology and Docking Studies
To verify whether compounds shown in Figure 1 have the biological effects expected from the structural
changes to SHetA2, all compounds were screened against A2780 human ovarian cancer cells. The potency
(half-maximal inhibitory concentration, IC50) and efficacy, which was defined as the maximal inhibition of
cancer cell growth, have been listed in Table 1 for the compounds. The interactions of the compounds with
the mortalin substrate-binding domain were studied by virtual docking, with the resulting binding free energy
values (–∆G) also listed in Table 1. Potential correlations between binding energy and potency, between
binding energy and efficacy, and between potency and efficacy are displayed in Figures 2A, 2B, and 2C,
respectively. All compounds showed varying degrees of growth inhibition. In Figure 2A, a significant positive
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Table 1. Half-maximal inhibitory concentration and efficacy values derived from the cellular dose-response
data. Also shown are their standard errors of mean (SEM) and binding free energy values of compounds
docked to the mortalin substrate-binding domain (SBD).
IC50
IC50
Efficacy Efficacy
–∆G
SEM
(%)
SEM
(µM)
(kcal/mol)
Compound
SHetA2
2a
2b
2c
2e
3a
3c
3d
3f
3.17 ± 0.05
84.3
87
26
2
32
79
88.4
93
91.5
19
28
± 0.7
6
10
4
2
4
1.4
3
0.8
5
0.6
6
11
2
8.5
8.1
8.0
7.8
8.3
8.5
7.9
8.6
7.7
7.8
7.0
7.3
8.3
7.8
8.1
8.5
8.3
8.1
8.4
8.2
8.4
8.6
8.7
8.7
8.8
8.8
6.96
4.3
0.08
3.0
43.7
0.2
6.9
0.9
4.1
0.1
3.6
0.1
4.7
0.3
4.56
6.4
0.05
0.6
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
7d
8a
8b
8c
8d
5.5
5.5
4.3
0.4
16
74
47
36
6.7
0.2
5.0
0.3
3.5
0.4
3
2.9
0.1
93.9
93.2
92.4
95.7
11.8
93.7
93.3
94.0
91.4
86.2
67
0.5
0.1
0.1
0.4
0.9
0.05
0.1
1.00
0.3
0.6
3
2.17
2.45
3.69
4.7
0.04
0.04
0.04
0.5
2.05
2.43
4.6
0.02
0.05
0.2
2.09
2.0
0.02
0.1
3.00
0.06
correlation (Pearson correlation coefficient r = 0.52 with p = 0.007) between the energy and potency values
was observed, which was consistent with compounds having stronger binding affinities for mortalin (higher
–
∆
G values) and greater potency (lower IC50 values). In Figure 2B, a significant negative association (r = –
0.60, p = 0.001) between the energy and efficacy values was observed, which was consistent with compounds
having stronger binding affinities for mortalin (higher – G values) and being more effective (higher %
∆
inhibition). In Figure 2C, the potency and efficacy values for these compounds showed a significant negative
correlation (r = –0.51, p = 0.008), which was consistent with the expectation that a compound with a smaller
IC50 is more effective in competing for the receptor in mortalin versus its partner proteins. In this plot, a
sigmoid curve was sketched to capture the main trend in the data. Most compounds with IC50 below 5 µM
demonstrated efficacy comparable to or better than that of SHetA2. Since a small IC50 value necessarily
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Figure 2. Assessment of (A) binding energy-IC50, (B) binding energy-efficacy, and (C) IC50-efficacy correlation
for all compounds investigated in this study. Data for the lead compound SHetA2 is represented by “×”, the 2-
Me-4-Me series by red triangles, the 2-H-4-Me series by green squares, the 2-Et-4-Me and 2-Me-4-Et series by
blue circles, and the 2-Et-4-Et series by black diamonds. Moreover, filled symbols were used for compounds
with oxygen in both ring A and the linker, while empty symbols were used for compounds with oxygen in ring A
and sulfur in the linker. Data in (C) was fit by a sigmoid curve with a half-activity concentration of 4.9
µM.
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means a stronger affinity for receptor binding, the data seemed to indicate that these compounds needed to
have a binding affinity above a certain threshold to effectively inhibit the cancer cells. This threshold is likely
determined by interactions between the receptor and its client protein(s), which are competitively displaced by
these compounds.
The most desirable properties with the target molecules were observed via a combination of the smallest
IC50 and the highest efficacy. The group of compounds 6b, 7c, and 8b (X = NO2, Y = O) are clearly shown as
the best in Figure 2C, demonstrating excellent efficacy values of 91-94% and IC50 values of 2.0-2.4 µM, both
of which are a significant improvement over SHetA2 (84% and 3.2 µM). These three most active compounds
had three common chemical features. First, all possessed increased hydrophobicity due to the presence of
geminal diethyl groups instead of the geminal dimethyl groups in ring A. Second, they had a nitro group (X =
NO2) attached to ring B. Third, they all had a urea linker (Y = O). Docking studies showed that the NO2 group
is capable of forming two hydrogen bonds with the amino acids in mortalin, enhancing the ligand-receptor
binding affinity. The second best group of 6c, 7d, and 8c (X = CF3 and Y = O) had IC50 and efficacy values only
slightly lower than the first group. They differed from the first group only in the second chemical feature
mentioned above, the identity of the polar group attached to ring B, indicating that CF3 was slightly less
effective than NO2. The third group of 6a, 7a, and 8a (X = NO2 and Y = S) varied from the first group (6b, 7c,
and 8b) only in the third chemical feature, having a thiourea instead of a urea linker. Their efficacy values
were comparable to or even slightly better than the first groups, which might be attributed to the more flexible
thiourea linker compared to the stiff peptide bonds involved in the urea linker. However, the third group had
much lower potencies (higher IC50 values) than the first group. The importance of the first chemical feature,
increased hydrophobicity due to the larger ethyl groups in compounds 6-8 can be further appreciated by
observing the dramatically inferior performance of compounds 4 and 5 (2-H-4-Me), both of which had reduced
hydrophobicity due to the absence of the C2 geminal dimethyl.
Nevertheless, compounds 8 (2-Et-4-Et) underperformed relative to compounds 6 (2-Et-4-Me) and 7 (2-Me-
4-Et), which might indicate that the chroman unit in 8 became too large with geminal diethyl groups at both C2
and C4. Compounds 6 appeared to have slightly more consistent performance than 7, noting that 7a had a
relatively large IC50. It seemed more beneficial to place the geminal diethyl groups at C2 (as in compounds 6)
than at C4 (as in 7), a point justified by molecular docking studies (vide infra). Therefore, the appropriate
amount of hydrophobicity, the size of the chroman unit, and the position of the geminal diethyl groups in ring A
might all have influence on the receptor binding and cancer inhibition. The remaining compounds in the 6-8
groups [7b (X = CF3 with Y = S) and 8d (X = OCF3 with Y = O)] exhibited less valuable performance.
The major difference between the two 2-Me-4-Me series 2 and 3 was that the former had a thiourea linker
and the latter a urea linker. Both retained geminal dimethyl groups as in SHetA2, but the sulfur heteroatom in
ring A was replaced by oxygen. Compounds in series 2 displayed a weak performance with respect to both
efficacy and IC50 values. Compounds in series 3 demonstrated efficacy comparable to or slightly better than
SHetA2, but all had higher IC50 values. Quality performance occurred when both the linker and the chroman
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heteroatom were oxygen or sulfur (as in SHetA2), but the properties deteriorated when the heteroatoms in
these two fragments were different. This peculiar requirement might derive from the flexibility of the linker, with
the thiourea linker being more flexible than urea linker, and the ability to form hydrogen bonding with the
receptor (vide infra). Structures 2a and 5a are also somewhat unusual in the sense that they possess
respectable efficacy values, but have modest IC50 values. Of course, the possibility exists that a different
receptor might conceivably be involved.
Figures 3A to 3C depict the structure model 7c, one of the best compounds, docked to the mortalin in its
peptide/substrate-binding channel. Figures 3B and 3C clearly show urea oxygen hydrogen bonded with S473
and the nitro group hydrogen bonded with Q517. Therefore, the preference for having a urea linker and a nitro
group can be understood as they are strong hydrogen bond acceptors, while sulfur has been shown to be a
rather poor H-bond acceptor [19]. Nevertheless, this docking conformation was not static as several other
docking conformations with similar binding energies found different amino acid hydrogen-bonding partners.
This conformational diversity contributed entropically to the stabilization of the protein-ligand complex [20,21].
However, such a conformational entropic contribution is a challenge to quantify, and it was not considered in
Figure 3. Molecular docking of 7c and several other compounds with the substrate-binding domain of mortalin
(PDB ID: 3N8E). (A) The complex with compound 7c. (B)-(C) Two views of the same complex highlighting the
amino acids in contact with 7c. The two hydrogen bonds were indicated by green dotted lines. (D)-(G)
Comparison of hydrophobic ring A conformations between 7c and SHetA2, 5a, 6b, and 8b, respectively, where
7c was shown in thicker lines. These four figures were viewed from the opposite end of the tunnel in Figure 3A.
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the binding energies reported in Table 1. The chroman ring A with methyl and ethyl attachments was
accommodated in the hydrophobic pocket formed by residues V482, L450, I447, and the methyl of T449
(Figures 3B and 3C). This is consistent with a report that mutation of V482 to phenylalanine abrogated the
interaction with tumor suppressor protein p53 [12], likely because the bulky side chain of phenylalanine at that
position tends to significantly reduce the available space in the hydrophobic pocket.
In Figures 3D-G, the conformations of the hydrophobic ring A of SHetA2, 5a, 6b, and 8b were compared
with that of 7c. All compounds docked in the same hydrophobic pocket of the substrate-binding channel, yet
with subtle but important differences. In comparison to SHetA2, ring A of 7c was pushed further toward the
opening of the tunnel (closer to the viewer) by the bulky diethyl groups (Figure 3D). Therefore, 7c was
expected to displace p53 or another client protein more effectively than SHetA2. On the other hand, ring A of
5a, with only one dimethyl pair, retreated from the opening since the smaller ring posed less steric hindrance to
being drawn into the binding cavity by polar groups (including the nitro group) through interaction with the
protein (Figure 3E). Therefore, 5a was expected to be less effective in displacing the client protein. Relative
to 7c, the diethyl and dimethyl groups in compound 6b are switched. In order to pack the more hydrophobic
diethyl groups, ring A flipped by roughly 180º, taking the heteroatom oxygen from the channel opening to the
interior of the hydrophobic pocket (Figure 3F). Such a flip also caused the nitro group to switch its hydrogen
bond partner to R513. Consequently, ring A of 6b was slightly more distant from the channel opening than 7c.
The docking conformation of 8b nearly overlapped that of 7c, since in 8b diethyl groups simply replaced the
dimethyl groups next to the heteroatom. As discussed earlier, 6b and 8b had nearly the same biological
activities as 7c.
3. Conclusions
It was shown that electron-withdrawing groups (NO2, CF3) on the B ring elicited IC50 values and efficacy
responses as good or better to that of SHetA2. Moreover, the best examples also had geminal diethyl groups
replacing geminal dimethyl groups at C2, including either O or S in ring A. When geminal diethyl groups were
placed at both C2 and C4 of ring A, the efficacy values paralleled that in SHetA2 while the IC50 values varied.
Thus, the presence of two geminal diethyl groups appeared to make ring A too bulky to be accommodated in
the binding site of mortalin. Compared to SHetA2, the most exceptional agents were 6b, 7c, and 8b, exhibiting
efficacy values of 91-94% with IC50 values of 2.0-2.4 µM [SHetA2 had 84% and 3.2 µM, respectively]. In
general, the presence of a urea linker between rings A and B gave improved activity compared with a thiourea
linker. Binding of 7c in the substrate binding site of mortalin resulted in H-bonding of the urea oxygen with
amino acid S473 and H-bonding of the nitro group with Q517. All compounds docked in the hydrophobic
pocket with binding –∆G values ranging from 7.0 to 8.8 kcal/mole. These binding energies were consistent
with a common binding site for all compounds. Some fine tuning of related structures is expected to enhance
the anti-cancer activity against the human A2780 ovarian cancer cell line.
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4. Experimental Section
4.1. Chemistry
4.1.1. General Experimental Details
Commercial anhydrous solvents were used as received. Tetrahydrofuran (THF) was distilled from LiAlH4
under N2. Unless otherwise specified, all reactions were run in oven-dried glassware under dry N2. Reactions
were monitored by thin layer chromatography on silica gel GF plates (Analtech No. 21521). Column
chromatography, when necessary, was performed in quartz columns using silica gel (Davisil® grade 62, 60-200
mesh) mixed with UV-active phosphor (Sorbent Technologies No. UV-05); band elution was monitored using a
hand-held UV lamp. The saturated NaCl, saturated NH4Cl, 5% NaHCO3, and 1 M HCl used in work-up
procedures were aqueous solutions. Melting points were uncorrected. FT-IR spectra were run as thin films on
1
NaCl disks. Unless otherwise indicated, H- and 13C-NMR spectra were measured in the indicated solvents
using tetramethylsilane as the internal standard. Elemental analyses (±0.4%) were obtained by Atlantic
Microlabs, Norcross, GA.
4.2. 2,2,4,4-Tetramethyl Analogs (2-Me-4-Me Series)
4.2.1. Phenyl 3-Methylbut-2-enoate (10)
o
To an oil-free suspension of NaH (1.34 g, 56.0 mmol) in anhydrous THF (20 mL) at 0 C (ice bath) was
added over a 5-min period with stirring a solution of phenol (9, 5.0 g, 53 mmol) in THF (55 mL). The solution
was stirred for 10 min and treated with a solution of 3-methylbut-2-enoyl chloride (7.0 g, 59 mmol) in THF (25
o
mL) over a 5-min period at 0 C, and was then allowed to warm to room temperature for 3 h. The white
suspension was transferred to a separatory funnel containing water (150 mL) and acetic acid (1 mL) and was
gently shaken. The mixture was extracted with ether (150 mL), and the extract was washed with saturated
NaCl (3 × 100 mL), dried (MgSO4), filtered, and concentrated to give a light yellow oil. The product was
purified on a 40-cm × 2.5-cm silica gel column eluted with 10% ether in hexanes to give 10 (8.4 g, 89%) as a
colorless oil. IR: 1738, 1653 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.38 (t, J = 7.9 Hz, 2H, Ar-H), 7.20 (t, J = 7.8
Hz, 1H, Ar-H), 7.10 (d, J = 8.0 Hz, 2H, Ar-H), 5.91 (m, 1H, =CH), 2.22 (d, J = 1.8 Hz, 3H, CH3), 1.96 (d, J = 1.8
Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3): δ 164.9, 159.9, 150.7, 129.3, 125.5, 121.8, 115.3, 27.6, 20.5.
4.2.2. 4,4-Dimethylchroman-2-one (11)
Into a 500-mL, three-necked, round-bottomed flask equipped with a stir bar, an addition funnel and a
condenser (drying tube) was placed dichloromethane (DCM, 164 mL) to which was added AlCl3 (9.87 g, 74.0
o
mmol). The resulting suspension was stirred and cooled to 0 C (ice bath), and a solution of 10 (7.4 g, 42.0
mmol) in DCM (40 mL) was added dropwise. The reaction was gradually warmed to room temperature, and
stirring was continued for 72 h. The resulting brown solution was added to a mixture of ice and saturated NaCl,
the layers were separated, and the aqueous layer was extracted with DCM (100 mL). The combined organic
13

ACCEPTED MANUSCRIPT
layers were washed with saturated NaCl (2 × 75 mL), dried (MgSO4), filtered, and concentrated to give a brown
oil, which was purified on a 40 cm × 2.5 cm silica gel column eluted with 10% ether in hexanes to give 11 (5.00
1
g, 68%) as a colorless oil. IR: 1769 cm-1; H NMR (400 MHz, CDCl3): δ 7.33 (dd, J = 7.4, 1.7 Hz, 1H, Ar-H),
7.25 (td, J = 7.9, 1.7 Hz, 1H, Ar-H), 7.15 (td, J = 7.5, 1.4 Hz, 1H, Ar-H), 7.05 (dd, J = 8.0, 1.4 Hz, 1H, Ar-H),
2.62 (s, 2H, CH2), 1.36 (s, 6H, 2 CH3); 13C NMR (101 MHz, CDCl3): δ 168.1, 150.6, 131.7, 128.2, 124.8, 124.4,
117.1, 45.6, 33.2, 27.6.
4.2.3. 2-(4-Hydroxy-2,4-dimethylpentan-2-yl)phenol (12)
A solution of 11 (2.5 g, 14.2 mmol) in dry ether (40 mL) was placed in a 250-mL, three-necked, round-
o
bottomed flask equipped with a stir bar, a septum, and a condenser. The solution was cooled to –45 C (dry
ice/acetonitrile bath), and an ether solution of methyllithium (1.6 M, 22.2 mL, 35.5 mmol) was added over 20
min. The reaction was stirred for 18 h with gradual warming to room temperature. The reaction was carefully
poured into a mixture of ice and saturated NH4Cl and shaken. The layers were separated, and the aqueous
layer was washed with ether (2 × 50 mL). The combined ether layers were washed with saturated NH4Cl and
water, dried (MgSO4), filtered, and concentrated under vacuum. The resulting oil solidified on standing at room
temperature. The solid was triturated with 1% ether in pentane and filtered to give 12 (2.72 g, 92%) as a white
solid, mp 86-88 oC. IR: 3540, 3259, 1372, 753 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.29 (dd, J = 7.8, 1.7 Hz,
1H, Ar-H), 7.07 (td, J = 7.7, 1.7 Hz, 1H, Ar-H), 6.88 (td, J = 7.5, 1.4 Hz, 1H, Ar-H), 6.65 (br s, 1H, phenol OH),
6.62 (dd, J = 7.9, 1.4 Hz, 1H, Ar-H), 2.22 (s, 2H, CH2), 1.74 (br s, 1H, OH), 1.48 (s, 6H, 2CH3), 1.12 (s, 6H,
2CH3); 13C NMR (101 MHz, CDCl3): δ 154.9, 134.4, 127.8, 127.5, 120.7, 117.5, 73.2, 52.4, 37.5, 31.02, 30.95.
4.2.4. 2,2,4,4-Tetramethylchroman (13)
A 50-mL, one-necked, round-bottomed flask was charged concentrated phosphoric acid (10 mL). The acid
was heated to 100 oC, 12 (2.08 g 10 mmol) was added, and the mixture was stirred for 1 h. The crude reaction
mixture was cooled, diluted with water, and extracted with ether (3 × 25 mL). The combined organic layers
were washed with saturated NaHCO3 and saturated NaCl, dried (MgSO4), filtered, and concentrated under
vacuum. The resulting oil was passed through a 15 cm × 2.5 cm column of silica gel eluted with hexanes to
1
give 13 (1.81 g, 95%) as a colorless oil. IR: 1367, 753 cm-1; H NMR (400 MHz, CDCl3): δ 7.19 (dd, J = 7.7,
1.4 Hz, 1H, Ar-H), 6.99 (td, J = 7.9, 1.4 Hz, 1H, Ar-H), 6.83 (t, J = 7.5 Hz, 1H, Ar-H), 6.72 (d, J = 8.0 Hz, 1H,
13
Ar-H), 1.76 (s, 2H, CH2), 1.28 (s, 6H, 2CH3), 1.27 (s, 6H, 2CH3); C NMR (101 MHz, CDCl3): δ 152.5, 131.5,
127.0, 126.8, 120.6, 117.9, 74.3, 49.3, 32.8, 30.8, 28.6. The spectral data matched those previously reported
[15].
4.2.5. 2,2,4,4-Tetramethyl-6-nitrochroman (14) and 2,2,4,4-Tetramethyl-8-nitrochroman (15)
A 25-mL, three-necked, round-bottomed flask equipped with a magnetic stirrer, an addition funnel, and a
nitrogen inlet was charged with 13 (190 mg, 1.0 mmol) and freshly distilled acetic anhydride (625 µL). The
14

ACCEPTED MANUSCRIPT
o
solution was cooled to −10 C (ice/salt bath), and a cold solution of concentrated nitric acid (77 µL) in acetic
o
anhydride (625 µL) was added dropwise over 15 min. The reaction was stirred at −5 C for 30 min and then
diluted with DCM and washed with saturated NaHCO3. The NaHCO3 wash was back-extracted with 20 mL of
DCM, and the combined organic layers were washed with water and saturated NaCl, dried (MgSO4), filtered,
and concentrated under vacuum. The crude product was purified by preparative thin layer chromatography on
a silica gel plate (20 cm × 20 cm) using 1% ether in hexanes to give three bands: band 1, recovered 13 (4 mg,
2%) as a colorless oil; band 2, the 6-nitro isomer 14 (78 mg, 33%) as a white solid, mp 64-65 oC; and band 3,
the 8-amino isomer 15 (96 mg, 41%) as a white solid, mp 71-72 oC. The spectral data for 14 were: IR: 1517,
1
1339 cm-1; H NMR (400 MHz, CDCl3): δ 8.50 (d, J = 2.7 Hz, 1H, Ar-H), 8.00 (dd, J = 9.0, 2.7 Hz, 1H, Ar-H,
6.75 (d, J = 9.0 Hz, 1H, Ar-H), 1.89 (s, 2H, CH2), 1.49 (s, 6H, 2CH3), 1.48 (s, 6H, 2CH3); 13C NMR (101 MHz,
CDCl3): δ 158.5, 141.4, 132.0, 123.6, 123.2, 118.5, 76.4, 48.1, 32.7, 31.2, 28.5. The spectral data for 15 were:
1
IR: 1526, 1365 cm-1; H NMR (400 MHz, CDCl3): δ 7.60 (dd, J = 8.0, 1.6 Hz, 1H, Ar-H), 7.48 (dd, J = 7.8, 1.6
Hz, 1H, Ar-H), 6.92 (t, J = 7.9 Hz, 1H, Ar-H), 1.89 (s, 2H. CH2), 1.45 (s, 6H, 2CH3), 1.43 (s, 6H, 2CH3); 13C
NMR (101 MHz, CDCl3): δ 146.6, 140.8, 135.2, 130.9, 122.7, 119.6, 76.9, 48.4, 32.6, 31.4, 28.4.
4.2.6. 2,2,4,4-Tetramethylchroman-6-amine (16)
To a stirred solution of 14 (120 mg, 0.51 mmol) in absolute ethanol (5 mL) in a 50-mL, round-bottomed
flask was added 5% Pd/C (10 mg), and the mixture was hydrogenated under 1 atm (balloon) of H2 gas for 1.5
h. The reaction was diluted with ether, filtered through Celite® to remove the catalyst, and concentrated. The
resulting red oil was purified by passing through a plug of silica using 10-20% ether in hexanes to give 16 as a
1
yellow oil (71 mg, 68%). IR: 3435, 3358, 3221, 1627, 1498 cm-1; H NMR (400 MHz, CDCl3): δ 6.63 (s, 1H,
Ar-H), 6.61 (d, J = 7.8 Hz, 1H, Ar-H), 6.47 (dd, J = 7.8, 2.7 Hz, 1H, Ar-H), 3.50 (br s, 2H, NH2), 1.78 (s, 2H,
CH2), 1.31 (s, 6H, 2CH3), 1.29 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 145.4, 139.5, 132.3, 118.3, 114.9,
113.4, 73.8, 49.3, 32.6, 31.0, 28.4.
4.3. General Procedure for Formation of Ureas and Thioureas
o
A stirred solution of 1 mol equiv of 16 in dry THF was treated at 0 C with 1.05 mol equiv of the appropriate
isothiocyanate or isocyanate. Stirring was continued overnight with gradual warming to room temperature. The
solvent was removed under vacuum to yield an oily residue. The residue was dissolved in chloroform and
treated with a small amount of pentane until crystallization commenced. The crystals were filtered, washed
with 1:1 ether:pentane to afford the urea and thiourea products, which were dried under high vacuum at 50 oC.
4.3.1. 1-(4-Nitrophenyl)-3-(2,2,4,4-tetramethylchroman-6-yl)thiourea (2a)
o
1
Pale yellow solid (160 mg, 85%), mp 176-177 C; IR: 3307, 3190, 1542, 1335 cm-1; H NMR (400 MHz,
CDCl3): δ 8.20 (d, J = 9.1 Hz, 2H, Ar-H), 8.00 (br s, 1H, NH), 7.76 (coincident s, 1H, NH and d, J = 9.1 Hz, 2H,
Ar-H), 7.22 (d, J = 2.6 Hz, 1H, Ar-H), 7.04 (dd, J = 8.5, 2.6 Hz, 1H, Ar-H), 6.89 (d, J = 8.5 Hz, 1H, Ar-H), 1.86
15

ACCEPTED MANUSCRIPT
(s, 2H, CH2), 1.38 (s, 6H, 2CH3), 1.36 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 180.5, 153.9, 145.4, 144.9,
134.6, 128.4, 125.9, 125.8, 125.3, 123.6, 120.6, 75.8, 48.7, 33.0, 31.4, 28.7. Anal. Calcd for C20H23N3O3S: C,
62.32; H, 6.01; N, 10.90. Found: C, 62.51; H, 6.05; N, 10.93. Although 2a has been reported and analyzed
[15], the current purification procedure raised the mp by about 10 ˚C.
4.3.2. Ethyl 4-(3-(2,2,4,4-Tetramethylchroman-6-yl)thioureido)benzoate (2b)
White solid (70 mg, 70%), mp 140-141 oC; IR: 3349, 3284, 3195, 1709 cm-1; 1H NMR (400 MHz, CDCl3): δ
8.02 (d, J = 8.3 Hz, 2H, Ar-H), 7.91 (br s, 1H, NH), 7.72 (br s, 1H, NH), 7.57 (d, J = 8.3 Hz, 2H, Ar-H), 7.24 (d,
J = 1.9 Hz, 1H, Ar-H), 7.04 (dd, J = 8.5, 1.9 Hz, 1H, Ar-H), 6.85 (d, J = 8.5 Hz, 1H, Ar-H), 4.36 (q, J = 7.1 Hz,
2H, CO2CH2CH3), 1.86 (s, 2H), 1.38 (t, J = 7.1 Hz, 3H, CO2CH2CH3), 1.37 (s, 6H, 2CH3), 1.35 (s, 6H, 2CH3);
13C NMR (101 MHz, CDCl3): δ 179.6, 165.9, 152.5, 142.0, 133.4, 130.5, 128.2, 127.6, 125.0, 124.9, 123.0,
119.5, 75.2, 61.0, 48.5, 32.8, 31.1, 28.5, 14.3. Anal. Calcd for C23H28N2O3S: C, 66.96; H, 6.84; N, 6.79.
Found: C, 66.88; H, 6.85; N, 6.68.
4.3.3. 1-(2,2,4,4-Tetramethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)thiourea (2c)
o
1
White solid (82 mg, 81%), mp 166-168 C; IR: 3357, 3196, 1598, 1491, 1324 cm-1; H NMR (400 MHz,
CDCl3): δ 7.81 (br s, 1H, NH), 7.63 (d, J = 8.7 Hz, 2H, Ar-H), 7.61 (br s, 1H, NH), 7.59 (d, J = 8.7 Hz, 2H, Ar-H),
7.23 (d, J = 2.6 Hz, 1H, Ar-H), 7.04 (dd, J = 8.6, 2.6 Hz, 1H, Ar-H), 6.87 (d, J = 8.6 Hz, 1H, Ar-H), 1.86 (s, 2H,
CH2), 1.38 (s, 6H, 2CH3), 1.35 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 179.9, 152.6, 141.2, 133.6, 128.0,
128.6, 126.0 (q, J = 3.9 Hz), 125.1, 125.0, 123.9, 123.9 (q, J = 271.9 Hz), 119.6, 75.7, 48.8, 33.0, 31.4, 28.7.
Anal. Calcd for C21H23F3N2OS: C, 61.75; H, 5.68; N, 6.86. Found: C, 61.47; H, 5.62; N, 6.87.
4.3.4. 4-(3-(2,2,4,4-Tetramethylchroman-6-yl)thioureido)benzenesulfonamide (2e)
o
1
Tan solid (90 mg, 78%), mp 186-187 C; IR: 3351, 3258, 3179, 1339, 1162 cm-1; H NMR (400 MHz,
DMSO-d6): δ 9.92 (s, 1H, NH), 9.85 (s, 1H, NH), 7.74 (d, J = 8.0 Hz, 2H, Ar-H), 7.68 (d, J = 8.0 Hz, 2H, Ar-H),
7.39 (s, 1H, Ar-H), 7.27 (s, 2H, Ar-H), 7.14 (s, J = 7.6 Hz, 1H, Ar-H), 6.70 (d, J = 7.6 Hz, 1H, Ar-H), 1.81 (s, 2H,
CH2), 1.30 (2s, 12H, 4CH3); 13C NMR (101 MHz, DMSO-d6): δ 179.8, 149.9, 143.3, 139.3, 132.1, 131.4, 126.5,
124.0, 123.5, 122.9, 117.6, 74.7, 48.5, 33.0, 31.1, 28.6. Anal. Calcd for C20H25N3O3S2·0.5 CH3CH2OH: C,
56.99; H, 6.38; N, 9.49. Found: C, 56.67; H, 6.08; N, 9.87.
4.3.5. 1-(4-Nitrophenyl)-3-(2,2,4,4-tetramethylchroman-6-yl)urea (3a)
Compound 16 was converted to 3a using the general procedure described above. Yellow solid (175 mg,
o
1
83%), mp 219-220 C; IR: 3314, 3158, 1655, 1556, 1351 cm-1; H NMR (400 MHz, DMSO-d6): δ 9.34 (s, 1H,
NH), 8.65 (s, 1H, NH), 8.18 (d, J = 8.9 Hz, 2H, Ar-H), 7.69 (d, J = 8.9 Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-H), 7.10 (d,
J = 8.6 Hz, 1H, Ar-H), 6.67 (d, J = 8.6 Hz, 1H, Ar-H), 1.82 (s, 2H, CH2), 1.30 (s, 6H, 2CH3), 1.28 (s, 6H, 2CH3);
13C NMR (101 MHz, DMSO-d6): δ 152.6, 148.2, 147.1, 141.2, 132.3, 131.8, 125.6, 119.3, 118.2, 117.9, 117.8,
16

ACCEPTED MANUSCRIPT
74.4, 48.8, 33.0, 31.1, 28.6. Anal. Calcd for C20H23N3O4: C, 65.03; H, 6.28; N, 11.37. Found: C, 64.83; H,
6.16; N, 11.39.
4.3.6. 1-(2,2,4,4-Tetramethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)urea (3c)
o
1
White solid (93 mg, 80%), mp 245-246 C; IR: 3302, 3175, 1646, 1599, 1491, 1325 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.98 (s, 1H, NH), 8.51 (s, 1H, NH), 7.65 (d, J = 8.6 Hz, 2H, Ar-H), 7.60 (d, J = 8.6 Hz, 2H,
Ar-H), 7.43 (s, 1H), 7.09 (d, J = 8.7 Hz, 1H, Ar-H), 6.65 (d, J = 8.7 Hz, 1H, Ar-H), 1.80 (s, 2H, CH2), 1.30 (s, 6H,
13
2CH3), 1.29 (s, 6H, 2CH3); C NMR (101 MHz, DMSO-d6) δ 151.9, 146.9, 143.1, 131.5, 130.7, 125.4 (q, J =
3.8 Hz), 124.0 (q, J = 272.7 Hz), 120.9, 118.0, 117.1, 117.0, 116.8, 73.3, 47.9, 32.0, 30.1, 27.6. Anal. Calcd
for C21H23F3N2O2·0.3H2O: C, 63.40; H, 5.98; N, 7.04. Found: C, 63.71; H, 5.82; N, 7.12.
4.3.7. 1-(4-Cyanophenyl)-3-(2,2,4,4-tetramethylchroman-6-yl)urea (3d)
Tan solid (51 mg, 61%), mp 225-226 oC; IR: 3341, 3206, 2221, 1665, 1590, 1491 cm-1; 1H NMR (400 MHz,
DMSO-d6): δ 9.10 (s, 1H, NH), 8.59 (s, 1H, NH), 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 7.63 (d, J = 8.0 Hz, 2H, Ar-H),
7.43 (s, 1H, Ar-H), 7.10 (d, J = 7.9 Hz, 1H, Ar-H), 6.67 (d, J = 7.9 Hz, 1H, Ar-H), 1.80 (s, 2H, CH2), 1.29 (s, 12H,
4CH3); 13C NMR (101 MHz, DMSO-d6): δ 152.7, 148.1, 144.9, 133.7, 132.4, 131.8, 119.8, 119.2, 118.4, 118.1,
117.9, 103.4, 74.4, 48.8, 33.0, 31.1, 28.6. Anal. Calcd for C21H23N3O2: C, 72.18; H, 6.63; N, 12.03. Found: C,
72.00; H, 6.60; N, 11.76.
4.3.8. 1-(2,2,4,4-Tetramethylchroman-6-yl)-3-(4-(trifluoromethoxy)phenyl)urea (3f)
o
1
White solid (151 mg, 76%), mp 219-221 C; IR: 3313, 3206, 3154, 1646, 1256 cm-1; H NMR (400 MHz,
CDCl3): δ 7.36 (d, J = 8.1 Hz, 2H, Ar-H), 7.26 (obscured by solvent, 1H, Ar-H), 7.13 (d, J = 8.1 Hz, 2H, Ar-H),
6.97 (dd, J = 8.2, 1.5 Hz, 1H, Ar-H), 6.80 (d, J = 8.2 Hz, 1H, Ar-H), 6.69 (s, 1H, NH), 6.41 (s, 1H, NH), 1.84 (s,
2H, CH2), 1.36 (s, 6H, 2CH3), 1.34 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 153.2, 147.8, 142.9, 139.7,
132.8, 131.7, 122.1, 120.7 (q, J = 254.9 Hz), 119.7, 119.0, 117.90, 117.85, 74.3, 48.9, 33.0, 31.1, 28.6. Anal.
Calcd for C21H23F3N2O3: C, 61.76; H, 5.68; N, 6.86. Found: C, 61.91; N, 5.59; N, 6.85.
4.4. 4,4-Dimethyl Analogs (2-H-4-Me Series)
4.4.1. 2-(4-Hydroxy-2-methylbutan-2-yl)phenol (17)
A 250-mL, three-necked, round-bottomed flask equipped with a stir bar, a septum, and a condenser was
charged with a suspension of 4.18 g (110 mmol) of lithium aluminum hydride in dry THF (50 mL). The solution
was cooled to 0 oC (ice bath), and a solution of 11 (5.0 g, 28.4 mmol) in dry THF (50 mL) was added over 1 h.
The resulting suspension was stirred at room temperature for 1 h and heated at reflux for 2 h and then cooled
in ice. The excess lithium aluminum hydride was destroyed by dropwise addition of 20% ethyl acetate in THF
(8 mL). The thick suspension was carefully poured into 150 mL of 1 M H2SO4/ice. This mixture was saturated
with NaCl and extracted with ether (3 × 150 mL). The combined extracts were washed with saturated NaCl (3 ×
17

ACCEPTED MANUSCRIPT
100 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The resulting oil solidified upon standing at
room temperature. The solid was triturated with 1% ether in pentane and filtered to give 17 (4.60 g, 90%) as a
white solid, mp 108-110 oC. IR: 3531, 3251 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.21 (d, J = 8.0 Hz, 1H, Ar-H),
7.06 (t, J = 8.0 Hz, 1H, Ar-H), 6.85 (d, J = 8.0 Hz, 1H, Ar-H), 6.62 (d, J = 8.0 Hz, 1H, Ar-H), 5.85 (br s, 1H,
phenol OH), 3.53 (t, J = 7.0 Hz, 2H, CH2), 2.32 (t, J = 7.0 Hz, 2H, CH2), 1.41 (s, 6H, 2CH3), one alcohol proton
not observed; 13C NMR (101 MHz, CDCl3): δ 154.4, 133.7, 127.6, 127.4, 120.5, 116.6, 61.0, 42.9, 36.7, 28.7.
4.4.2. 4,4-Dimethychroman (18)
A solution of 4.25 g (23.6 mmol) of 17 in pyridine (50 mL) was stirred and cooled at 0 oC (ice bath) and then
o
treated with methanesulfonyl chloride (3.59 g, 31.3 mmol). The reaction was stirred at 0 C for 1 h and was
then poured into saturated NaCl (150 mL) and extracted with ether (3 × 75 mL). The combined extracts were
washed with 1 M HCl (2 × 175 mL), and dilute NaCl (2 × 75 mL), dried (MgSO4) and concentrated to give the
mesylate as a yellow oil. The crude mesylate was dissolved in dioxane (50 mL), and the solution was stirred
with 1 M NaOH (62.5 mL) at room temperature for 3 h. The two-phased mixture was diluted with saturated
NaCl (100 mL) and extracted with ether (3 × 75 mL). The combined extracts were washed with saturated NaCl
(75 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by passing
it through a plug of silica gel with 3% ether in hexanes to give 18 (3.64 g, 95%) as a colorless oil. IR: 3065,
3031, 1607, 1579, 1486 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.26 (d, J = 7.7 Hz, 1H, Ar-H), 7.06 (t, J = 7.8 Hz,
1H, Ar-H), 6.87 (t, J = 7.9 Hz, 1H, Ar-H), 6.78 (d, J = 8.2 Hz, 1H, Ar-H), 4.18 (t, J = 5.3 Hz, 2H, OCH2), 1.83 (t,
J = 5.3 Hz, 2H, CH2), 1.32 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 153.6, 131.6, 127.0, 126.9, 120.4,
116.9, 63.0, 37.7, 31.1, 30.5.
4.4.3. 4,4-Dimethyl-6-nitrochroman (19) and 4,4-Dimethyl-8-nitrochroman (20)
A 25-mL, three-necked, round-bottomed flask equipped with a magnetic stirrer, an addition funnel, and a
nitrogen inlet was charged with 18 (1.10 g, 6.8 mmol) and freshly distilled acetic anhydride (4.25 mL). The
o
solution was cooled to −10 C (ice/salt bath), and a cold solution of concentrated nitric acid (0.52 mL) in acetic
anhydride (4.25 mL) was added dropwise over 30 min. The reaction was stirred at −5 oC for 30 min and was
then diluted with DCM and washed with saturated NaHCO3. The NaHCO3 wash was back-extracted with DCM
(20 mL), and the combined organic layers were washed with water and saturated NaCl, dried (MgSO4), filtered,
and concentrated under vacuum to give 1.25 g of a yellow oil consisting of a 1:1.2 mixture of 19 and 20. As
this product mixture was difficult to purify by chromatography, it was carried on to the 6- and 8-amino
compounds, 21 and 22, respectively, before purification.
4.4.4. 4,4-Dimethylchroman-6-amine (21) and 4,4-Dimethylchroman-8-amine (22)
The following reduction procedure was found to be superior to the one outlined above for the preparation of
compound 16 [17]. A solution of the mixture of 19 and 20 (1.25 g, ca. 6.04 mmol) from above was dissolved in
18

ACCEPTED MANUSCRIPT
a mixture of 90 mL of ethanol and 20 mL of water. To the stirred solution was added ammonium chloride (0.33
g, 6.17 mmol) and iron powder (1.10 g, 19.6 mmol), and the mixture was heated at 85 oC for 2 h. The reaction
was filtered through Celite®, treated with NaHCO3 (50 mL) and extracted with ethyl acetate (2 × 50 mL). The
combined organic extracts organic were washed with saturated NaCl, dried (Na2SO4), filtered, and
concentrated under vacuum. The resulting brown oil was purified on a 30 × 2.0 cm silica gel column slurry
packed in hexane containing increasing concentrations of ether (2-30%). Elution gave two bands: band 1, the
8-amino isomer 22 (552 mg, 52%) as a yellow oil and band 2, the 6-amino isomer 21 (451 mg, 42%) as a
1
yellow oil. The spectral data for 22 were: IR: 3429, 3352, 3230, 1620, 1498 cm-1; H NMR (400 MHz,
CDCl3): δ 6.69 (m, 2H, Ar-H), 6.52 (m, 1H, Ar-H), 4.22 (t, J = 5.3 Hz, 2H, OCH2), 3.71 (br s, 2H, NH2), 1.80 (t, J
= 5.3 Hz, 2H, CH2), 1.30 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 141.4, 135.4, 131.4, 120.1, 116.3, 112.5,
1
63.1, 37.9, 31.1, 30.6. The spectral data for 21 were: IR: 3429, 3356, 3227, 1624, 1499 cm-1; H NMR (400
MHz, CDCl3): δ 6.62 (m, 2H, Ar-H), 6.53 (dd, J = 8.6, 2.7 Hz, 1H, Ar-H), 4.11 (t, J = 5.3 Hz, 2H, OCH2), 3.36 (br
s, 2H, NH2), 1.83 (t, J = 5.3 Hz, 2H, CH2), 1.31 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 146.7, 139.4,
132.3, 117.4, 115.1, 113.7, 62.9, 38.0, 31.2, 30.7. Compound 21 was used immediately in the next reaction as
it darkened rapidly on exposure to air.
4.4.5. 1-(4,4-Dimethylchroman-6-yl)-3-(4-nitrophenyl)thiourea (4a)
Compound 21 was converted to 4a using the general preparation for ureas and thioureas above. Yellow
solid (118 mg, 79%), mp 174-165 ˚C; IR: 3305, 3187,1596,1528,1495,1335 cm-1; 1H NMR (400 MHz, CDCl3): δ
8.20 (d, J = 8.8 Hz, 2H, Ar-H), 7.97 (br s, 1H, NH), 7.76 (d, J = 8.8 Hz, 2H, Ar-H), 7.71 (s, 1H, NH), 7.21 (d, J =
2.2 Hz, 1H, Ar-H), 7.03 (dd, J = 8.4, 2.2 Hz, 1H, Ar-H), 6.88 (d, J = 8.4 Hz, 1H, Ar-H), 4.24 (t, J = 5.3 Hz, 2H,
13
OCH2), 1.87 (t, J = 5.3 Hz, 2H, CH2), 1.35 (s, 6H, 2CH3); C NMR (101 MHz, CDCl3): δ 179.4, 153.9, 144.5,
144.0, 133.9, 127.4, 125.2, 125.1, 124.5, 122.8, 118.9, 63.3, 36.9, 30.94, 30.89. Anal. Calcd for C18H19N3O3S;
C, 60.49; H, 5.36; N, 11.76. Found: C, 60.44; H, 5.35; N, 11.74.
4.4.6. 1-(4,4-Dimethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)thiourea (4b)
o
1
White solid (160 mg, 75%), mp 147-149 C; IR: 3357, 3199, 1613, 1498, 1324 cm-1; H NMR (400 MHz,
CDCl3): δ 8.00 (br s, 1H, NH), 7.68 (br s, 1H, NH), 7.65 (d, J = 8.9 Hz, 2H, Ar-H), 7.61 (d, J = 8.9 Hz, 2H, Ar-H),
7.22 (s, 1H, Ar-H), 7.03 (d, J = 8.6 Hz, 1H, Ar-H), 6.86 (d, J = 8.6 Hz, 1H, Ar-H), 4.22 (t, J = 5.4 Hz, 2H, OCH2),
1.85 (t, J = 5.4 Hz, 2H, CH2), 1.34 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 179.9, 153.6, 141.5, 133.6,
127.9, 126.1 (q, J = 3.8 Hz), 125.2, 125.1, 123.9, 123.9 (q, J = 271.9 Hz), 118.6, 63.2, 36.9, 30.94, 30.86 (1
aromatic carbon not resolved). Anal. Calcd for C19H19F3N2OS: C, 59.99; H, 5.03; N, 7.36. Found: C, 60.04; H,
5.09; N, 7.37.
4.4.7. 1-(4,4-Dimethylchroman-6-yl)-3-(4-(trifluoromethoxy)phenyl)thiourea (4c)
o
1
White solid (172 mg, 90%), mp 153-154 C; IR: 3358, 3189, 1530, 1500, 1255 cm-1; H NMR (400 MHz,
19

ACCEPTED MANUSCRIPT
CDCl3): δ 7.71 (br s, 1H, NH), 7.48 (coincident d, J = 9.0 Hz, 2H, Ar-H and br s, 1H, NH), 7.26 (s, 1H, Ar-H),
7.21 (d, J = 9.0 Hz, 2H, Ar-H), 7.01 (d, J = 8.5 Hz, 1H, Ar-H), 6.85 (d, J = 8.5 Hz, 1H, Ar-H), 4.22 (t, J = 5.3 Hz,
2H, OCH2), 1.85 (t, J = 5.3 Hz, 2H, CH2), 1.34 (s, 6H, 2CH3); 13C NMR (101 MHz, CDCl3): δ 181.6, 154.5,
147.9, 137.3, 134.3, 128.8, 127.0, 126.1, 126.0, 122.8, 121.1 (q, J = 257.1 Hz), 119.3, 63.7, 37.2, 31.13, 31.05.
Anal. Calcd for C19H19F3N2O2S: C, 57.57; H, 4.83; N, 7.07. Found: C, 57.54; H, 4.76; N, 7.19.
4.4.8. 1-(4,4-Dimethylchroman-6-yl)-3-(4-nitrophenyl)urea (5a)
Compound 21 was converted to 5a using the general preparation for ureas and thioureas above. White
1
solid (94 mg, 80%), mp 240-242 oC; IR: 3281, 3189, 1640, 1556, 1333 cm-1; H NMR (400 MHz, DMSO-d6): δ
9.13 (s, 1H, NH), 8.64 (s, 1H, NH), 8.18 (d, J = 8.8 Hz, 2H, Ar-H), 7.68 (d, J = 8.8 Hz, 2H, Ar-H), 7.42 (d, J =
2.2 Hz, 1H, Ar-H), 7.10 (d, J = 8.9 Hz, 1H, Ar-H), 6.67 (d, J = 8.8, 2.2 Hz, 1H, Ar-H), 4.11 (t, J = 5.2 Hz, 2H,
CH2), 1.78 (d, J = 5.2 Hz, 2H, CH2), 1.28 (s, 6H, 2CH3); 13C NMR (101 MHz, DMSO-d6): δ 152.6, 149.4, 147.1,
141.2, 132.03, 132,02, 125.6, 119.2, 118.3, 117.8, 117.0, 62.8, 37.5, 31.3, 30.8. Anal. Calcd for C18H19N3O4:
C, 63.33; H, 5.61; N, 12.31. Found: C, 63.07; H, 5.64; N, 12.50.
4.4.9. 1-(4,4-Dimethylchroman-6-yl)-3-(4-trifluoromethyl)phenyl)urea (5b)
o
1
White solid (84 mg, 82%), mp 248-249 C; IR: 3301, 3186, 1645, 1320 cm-1; H NMR (400 MHz, DMSO-
d6): δ 8.98 (s, 1H, NH), 8.52 (s, 1H, NH), 7.65 (d, J = 8.8 Hz, 2H, Ar-H), 7.61 (d, J = 8.8 Hz, 2H, Ar-H), 7.41 (d,
J = 2.6 Hz, 1H, Ar-H), 7.11 (dd, J = 8.8, 2.6 Hz, 1H, Ar-H), 6.66 (d, J = 8.8 Hz, 1H, Ar-H), 4.10 (t, J = 5.2 Hz,
2H, CH2), 1.77 (t, J = 5.2 Hz, 2H, CH2), 1.28 (s, 6H, 2CH3); 13C NMR (101 MHz, DMSO-d6): δ 152.9, 149.2,
144.1, 132.3, 132.0, 127.1 (q, J = 3.6 Hz), 124.4 (q, J = 270.0 Hz), 124.3, 119.7, 118.7, 118.6, 117.6, 62.7,
37.5, 31.3, 30.8. Anal. Calcd for C19H19F3N2O2: C, 62.63; H, 5.26; N, 7.69. Found: C, 62.56; H, 5.32; N, 7.65.
4.4.10. 1-(4,4-Dimethylchroman-6-yl)-3-(4-(trifluoromethoxy)phenyl)urea (5c)
o
1
White solid (78 mg, 73%), mp 230-231 C; IR: 3308, 3198, 1645, 1267 cm-1; H NMR (400 MHz, DMSO-
d6): δ 8.98 (s, 1H, NH), 8.52 (s, 1H, NH), 7.65 (d, J = 8.9 Hz, 2H, Ar-H), 7.61 (d, J = 8.9 Hz, 2H, Ar-H), 7.41 (d,
J = 1.9 Hz, 1H, Ar-H), 7.09 (dd, J = 8.7, 1.9 Hz, 1H, Ar-H), 6.65 (d, J = 8.7 Hz, 1H, Ar-H), 4.10 (t, J = 5.2 Hz,
2H, CH2), 1.77 (t, J = 5.2 Hz, 2H, CH2), 1.28 (s, 6H, 2CH3); 13C NMR (101 MHz, DMSO-d6): δ 152.2, 147.9,
142.3, 138.8, 131.1, 130.5, 121.8, 120.3 (q, J = 255.4 Hz), 119.4, 118.6, 117.6, 116.6, 61.7, 36.5, 30.1, 29.7.
Anal. Calcd for C19H19F3N2O3: C, 60.00; H, 5.04; N, 7.36. Found 60.06; 5.20; 7.33.
4.5. 2,2-Diethyl-4,4-dimethyl Analogs (2-Et-4-Me Series)
4.5.1. 2-(4-Ethyl-4-hydroxy-2-methylhexan-2-yl)phenol (23)
Compound 23 was prepared on a 10.0 mmol scale from 4,4-dimethylchroman-2-one (11) and
ethylmagnesium bromide in ether (–45 oC to rt) and was used directly. The yield of 23 was 1.82 g (7.71 mmol,
77%) as a white solid, mp 109-111 oC; IR: 3532, 3206 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.29 (d, J = 7.8 Hz,
20

ACCEPTED MANUSCRIPT
1H, Ar-H), 7.20 (br s, 1H, phenol OH), 7.03 (t, J = 7.5 Hz, 1H, Ar-H), 6.86 (t, J = 7.5 Hz, 1H, Ar-H), 6.53 (d, J =
7.8 Hz, 1H, Ar-H), 2.14 (s, 2H, CH2), 1.98 (br s, 1H, OH), 1.48 (s, 6H, 2CH3), 1.42 (q, J = 7.5 Hz, 4H, 2CH2),
13
0.78 (t, J = 7.4 Hz, 6H, 2CH3); C NMR (101 MHz, CDCl3): δ 155.9, 134.6, 127.6, 127.5, 120.5, 117.5, 77.5,
48.3, 37.3, 31.4, 31.3, 8.0.
4.5.2. 2,2-Diethyl-4,4-dimethylchroman (24)
Compound 24 was prepared on a 6.00 mmol scale from 23 and purified according to the procedure
described for the preparation of 2,2,4,4-tetramethylchroman (13). The yield of 2,2-diethyl-4,4-dimethylchroman
was 1.12 g (24, 5.14 mmol, 86%) as a colorless oil; IR: 1607, 1583 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.23 (d,
J = 7.5 Hz, 1H, Ar-H), 7.04 (t, J = 8.0 Hz, 1H, Ar-H), 6.86 (t, J = 7.5 Hz, 1H, Ar-H), 6.79 (d, J = 8.0 Hz, 1H, Ar-
H), 1.78 (s, 2H, CH2), 1.70 (m, 2H, CH2CH3), 1.56 (m, 2H, CH2CH3), 1.32 (s, 6H, 2CH3), 0.88 (t, J = 7.5 Hz, 6H,
2CH2CH3); 13C NMR (101 MHz, CDCl3): δ 152.7, 132.3, 127.1, 126.5, 120.7, 118.1, 78.9, 45.6, 33.2, 30.6, 29.4,
8.0.
4.5.3. 4,4-Diethyl-2,2-dimethylchroman-6-amine (27) and 4,4-Diethyl-2,2-dimethylchroman-6-amine (28)
2,2-Diethyl-4,4-dimethylchroman (24) was nitrated in the usual manner on a 5.00 mmol scale using nitric
acid in acetic anhydride at –10 oC as described for 2,2,4,4-tetramethylchroman (13) to give a mixture of the 6-
isomer 25 and the 8-nitro isomer 26 as a yellow oil. Since the nitro derivatives were difficult to separate, the
mixture was reduced as described for the preparation of 21/22 using ammonium chloride in aqueous ethanol to
give the 6- and 8-amino compounds in a 6.2:1 ratio (86% crude yield). These were separated on a 30 cm ×
2.5 cm silica gel column eluted with increasing concentrations of ether in hexane to give two bands: band 1,
the 8-amino isomer (28, 127 mg, 0.55 mmol, 11%) eluted with 10% ether in hexanes and solidified as a light
brown oil; band 2, the 6-amino isomer (27, 792 mg, 3.40 mmol, 68%) eluted with 30% ether in hexanes as a
1
tan oil. The spectral data for 28 were: IR: 3466, 3375, 3184, 1611 cm-1; H NMR (400 MHz, CDCl3): δ 6.75-
6.67 (m, 2H, Ar-H), 6.55 (dd, J = 7.2, 2.0 Hz, 1H, Ar-H), 3.75 (br s, 2H, NH2), 1.80 (s, 2H, CH2), 1.76 (m, 2H,
13
CH2CH3), 1.60 (m, 2H, CH2CH3), 1.32 (s, 6H, 2CH3), 0.90 (t, J = 7.5 Hz, 6H, 2CH2CH3); C NMR (101 MHz,
CDCl3): δ 139.9, 136.1, 132.0, 120.2, 115.9, 112.4, 79.1, 45.2, 32.9, 30.6, 29.1, 8.0. The spectral data for 27
1
were: IR: 3429, 3355, 3221, 1624 cm-1; H NMR (400 MHz, CDCl3): δ 6.63 (d, J = 8.4 Hz, 1H, Ar-H), 6.62 (s,
1H, Ar-H), 6.46 (dd, J = 8.4, 2.8 Hz, 1H, Ar-H), 3.27 (br s, 2H, NH2), 1.74 (s, 2H, CH2), 1.70 (m, 2H, CH2CH3),
1.51 (m, 2H, CH2CH3), 1.30 (s, 6H, 2CH3), 0.87 (t, J = 7.5 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz, CDCl3):
δ 145.5, 139.5, 133.4, 118.5, 114.8, 113.3, 78.6, 45.4, 32.8, 30.8, 29.1, 7.8.
4.5.4. 1-(2,2-Diethyl-4,4-dimethylchroman-6-yl)-3-(4-nitrophenyl)thiourea (6a)
Compound 27 was converted to 6a using the general preparation for ureas and thioureas above. Yellow
solid (160 mg, 90%), mp 175-176 oC; IR: 3341, 3174, 1556, 1340, 1174 cm-1; 1H NMR (400 MHz, DMSO-d6): δ
10.2 (s, 1H, NH), 10.1 (s, 1H, NH), 8.18 (d, J = 9.0 Hz, 2H, Ar-H), 7.80 (d, J = 9.0 Hz, 2H, Ar-H), 7.38 (d, J =
21

ACCEPTED MANUSCRIPT
2.7 Hz, 1H, Ar-H), 7.14 (dd, J = 8.6, 2.7 Hz, 1H, Ar-H), 6.71 (d, J = 8.6 Hz, 1H, Ar-H), 1.77 (s, 2H, CH2), 1.63
(m, 2H, CH2CH3), 1.52 (m, 2H, CH2CH3), 1.28 (s, 6H, 2CH3), 0.85 (t, J = 7.4 Hz, 6H, 2CH2CH3); 13C NMR (101
MHz, DMSO-d6): δ 179.5, 150.0, 146.8, 142.5, 132.2, 131.9, 124.8, 123.8, 123.2, 121.8, 117.8, 79.1, 44.2,
33.2, 30.7, 29.1, 8.1. Anal. Calcd for C22H27N3O3S: C, 63.90; H, 6.58; N, 10.16. Found: C, 64.07; H, 6.64; N,
10.11.
4.5.5. 1-(2,2-Diethyl-4,4-dimethylchroman-6-yl)-3-(4-nitrophenyl)urea (6b)
Yellow solid (145 mg, 85%), mp 190-192 oC; IR: 3333, 1662, 1556, 1331 cm-1; 1H NMR (400 MHz, DMSO-
d6): δ 9.33 (s, 1H, NH), 8.64 (s, 1H, NH), 8.18 (d, J = 8.9 Hz, 2H, Ar-H), 7.69 (d, J = 8.9 Hz, 2H, Ar-H), 7.43 (d,
J = 2.7 Hz, 1H, Ar-H), 7.11 (dd, J = 8.6, 2.7 Hz, 1H, Ar-H), 6.68 (d, J = 8.6 Hz, 1H, Ar-H), 1.76 (s, 2H, CH2),
1.61 (m, 2H, CH2CH3), 1.52 (m, 2H, CH2CH3), 1.29 (s, 6H, 2CH3), 0.84 (t, J = 7.4 Hz, 6H, 2CH2CH3); 13C NMR
(101 MHz, DMSO-d6): δ 152.6, 148.1, 147.1, 141.2, 132.6, 132.3, 125.6, 119.2, 118.4, 118.0, 117.8, 78.8, 44.6,
33.1, 30.8, 29.1, 8.1. Anal. Calcd for C22H27N3O4·0.15H2O: C, 66.03; H, 6.88; N, 10.53. Found: C, 65.64; H,
6.68; N, 10.91.
4.5.6. 1-(2,2-Diethyl-4,4-dimethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)urea (6c)
o
1
White solid (166 mg, 92%), mp 208-210 C; IR: 3326, 3202, 3142, 1659, 1323 cm-1; H NMR (400 MHz,
DMSO-d6): δ 8.80 (s, 1H, NH), 8.35 (s, 1H, NH), 7.62 (d, J = 8.4 Hz, 2H, Ar-H), 7.51 (d, J = 8.4 Hz, 2H, Ar-H),
7.39 (s, 1H, Ar-H), 7.07 (d, J = 8.6 Hz, 1H, Ar-H), 6.66 (d, J = 8.6 Hz, 1H, Ar-H), 1.77 (s, 2H, CH2), 1.65 (m, 2H,
13
CH2CH3), 1.54 (m, 2H, CH2CH3), 1.32 (s, 6H, 2CH3), 0.87 (t, J = 7.3 Hz, 6H, 2CH2CH3); C NMR (101 MHz,
DMSO-d6): δ 153.0, 148.1, 143.9, 132.5, 132.3, 126 (q, J = 3.6 Hz), 124.6 (q, J = 269.8 Hz), 122.5, 122.2,
118.7, 117.7, 117.6, 78.3, 44.0, 32.2, 30.0, 28.2, 7.1. Anal. Calcd for C23H27F3N2O2: C, 65.70; H, 6.47; N, 6.66.
Found: C, 65.94; H, 6.51; N, 6.67.
4.6. 4,4-Diethyl-2,2-dimethyl Analogs (2-Me-4-Et Series)
4.6.1. Phenyl 3-Ethylpent-2-enoate (29)
Compound 29 was prepared from 9 and 3-ethylpent-2-enoyl chloride (obtained by treatment of 3-ethylpent-
2-enoic acid [18] with SOCl2) on a 53 mmol scale as described for 10 and was purified by column
chromatography on a 40 cm × 2.5 cm silica gel column eluted with 8% ether in hexane. The yield of 29 was
1
9.96 g (48.8 mmol, 92%) as a colorless oil; IR: 1738, 1640, 1593, 1200 cm-1; H NMR (400 MHz, CDCl3): δ
7.37 (t, J = 7.8 Hz, 2H, Ar-H), 7.20 (t, J = 7.4 Hz, 1H, Ar-H), 7.09 (d, J = 7.8 Hz, 2H, Ar-H), 5.85 (s, 1H, =CH),
2.67 (q, J = 7.5 Hz, 2H, CH2CH3), 2.27 (qd, J = 7.4, 1.2 Hz, 2H, CH2CH3), 1.13 (t, J = 7.4 Hz, 3H, CH2CH3),
1.10 (t, J = 7.5 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3): δ 170,8, 164.8, 150.8, 129.3, 125.7, 121.8,
112.8, 31.1, 25.8, 13.0, 12.0.
4.6.2. 4,4-Diethylchroman-2-one (30)
22

ACCEPTED MANUSCRIPT
Compound 30 was prepared on a 44 mmol scale as described for 4,4-dimethylchroman-2-one (13) and
was purified on a 40 cm × 2.5 cm silica gel column eluted with 8% ether in hexane. The yield of 30 was 3.12 g
1
(15.2 mmol, 35%) as a light yellow oil; IR: 1776, 1609, 1585, 1202 cm-1; H NMR (400 MHz, CDCl3): δ 7.28-
7.23 (m, 1H, Ar-H), 7.20 (dd, J = 7.8, 1.8 Hz, 1H, Ar-H), 7.15 (td, J = 7.8, 1.1 Hz, 1H, Ar-H), 7.07 (dd, J = 7.8,
1.1 Hz, 1H, Ar-H), 2.63 (s, 2H, CH2), 1.79-1.61 (m, 4H, 2CH2CH3), 0.82 (t, J = 7.4 Hz, 6H, 2CH2CH3); 13C NMR
(101 MHz, CDCl3): δ 168.9, 151.6, 128.1, 128.0, 126.2, 124.2, 117.3, 39.9, 38.5, 29.9, 8.1.
4.6.3. 2-(3-Ethyl-5-hydroxy-5-dimethylhexan-3-yl)phenol (31)
Compound 31 was prepared on a 4.90 mmol scale from 4,4-diethylchroman-2-one (30) and
o
methylmagnesium bromide in ether (–45 C ––> rt) and was used without further purification. The yield of 31
1
was 1.02 g (4.32 mmol, 88%) as a light yellow oil; IR: 3538, 3269,1601 cm-1; H NMR (400 MHz, CDCl3):
δ 7.29 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H), 7.06 (td, J = 7.9, 1.1 Hz, 1H, Ar-H), 6.89 (td, J = 7.7, 1.1 Hz, 1H, Ar-H),
6.59 (d, J = 7.8 Hz, 1H, Ar-H), 6.31 (br s, 1H, phenol OH), 2.18 (s, 2H, CH2), 2.10 (m, 2H, CH2CH3), 1.82 (m,
2H, CH2CH3), 1.63 (br s, 1H, OH), 1.10 (s, 6H, 2CH3), 0.72 (t, J = 7.4 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz,
CDCl3): δ 155.0, 132.8, 129.1, 127.3, 120.6, 117.5, 73.2, 45.9, 43.2, 31.1, 26.9, 7.9.
4.6.4. 4,4-Diethyl-2,2-dimethylchroman (32)
Compound 32 was prepared and purified on a 4.00 mmol scale according to the procedure described for
the preparation of 2,2,4,4-tetramethylchroman (13). The yield of 32 was 0.78 g (3.60 mmol, 90%) as a
colorless oil; IR: 1604, 1597, 1483, 1381, 1367, 745 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.16 (dd, J = 7.8, 1.7
Hz, 1H, Ar-H), 7.07 (ddd, J = 8.2, 7.3, 1.7 Hz, 1H, Ar-H), 6.89 (td, J = 7.3, 1.7 Hz, 1H, Ar-H), 6.79 (dd, J = 8.2,
1.7 Hz, 1H, Ar-H), 1.78 (s, 2H, CH2), 1.76-1.61 (m, 4H, 2CH2CH3), 1.34 (s, 6H, 2CH3), 0.75 (t, J = 7.4 Hz, 6H,
2CH2CH3); 13C NMR (101 MHz, CDCl3): δ 154.0, 130.3, 126.9, 126.7, 120.5, 117.9, 74.3, 40.2, 37.4, 33.5, 29.1,
8.4.
4.6.5. 4,4-Diethyl-2,2-dimethylchroman-6-amine (35) and 4,4-Diethyl-2,2-dimethylchroman-8-amine (36)
4,4-Diethyl-2,2-dimethylchroman (32) was nitrated on a 3.50 mmol scale using nitric acid in acetic
anhydride at –10 oC as described for 2,2,4,4-tetramethylchroman (13) to give a mixture of the 6-nitro (33) and
8-nitro (34) isomers as a yellow oil. Since the nitro derivatives were difficult to separate, the mixture was
reduced as described for the preparation of 21/22 using ammonium chloride in aqueous ethanol to give the 6-
and 8-amino compounds (35 and 36, respectively) in a 1:1.2 ratio (84% crude yield). These were separated on
a 30 cm × 2.5 cm silica gel column eluted with increasing concentrations of ether in hexane to give two bands:
band 1, the 8-amino isomer (34, 343 mg, 1.47 mmol, 42%) eluted with 10% ether in hexanes and solidified as
a tan solid, mp 37-38 oC; band 2, the 6-amino isomer (33, 294 mg, 1.26 mmol, 36%) eluted with 30% ether in
1
hexanes as a tan oil. The spectral data for 34 were: IR: 3471, 3374, 3194, 1612 cm-1; H NMR (400 MHz,
CDCl3): δ 6.72 (t, J = 7.7 Hz, 1H, Ar-H), 6.59 (dd, J = 7.9, 1.6 Hz, 1H, Ar-H), 6.56 (dd, J = 7.6, 1.6 Hz, 1H, Ar-
23

ACCEPTED MANUSCRIPT
H), 3.73 (br s, 2H, NH2), 1.77 (s, 2H, CH2), 1.77-1.57 (m, 4H, 2CH2CH3), 1.35 (s, 6H, 2CH3), 0.75 (t, J = 7.4 Hz,
6H, 2CH2CH3); 13C NMR (101 MHz, CDCl3): δ 141.6, 136.1, 130.2, 120.2, 116.5, 112.4, 74.6, 40.4, 37.7, 33.2,
29.2, 8.5. The spectral data for 33 were: IR: 3434, 3356, 3220, 1626 cm-1; 1H NMR (400 MHz, CDCl3): δ 6.62
(d, J = 8.4 Hz, 1H, Ar-H), 6.54 (d, J = 2.7 Hz, 1H, Ar-H), 6.48 (dd, J = 8.4, 2.7 Hz, 1H, Ar-H), 3.37 (br s, 2H,
NH2), 1.73 (s, 2H, CH2), 1.72-1.56 (m, 4H, 2CH2CH3), 1.30 (s, 6H, 2CH3), 0.76 (t, J = 7.4 Hz, 6H, 2CH2CH3);
13C NMR (101 MHz, CDCl3): δ 147.1, 139.5, 131.3, 118.3, 114.6, 113.7, 73.9, 40.4, 37.7, 33.3, 28.9, 8.4.
4.6.6. 1-(4,4-Diethyl-2,2-dimethylchroman-6-yl)-3-(4-nitrophenyl)thiourea (7a)
Compound 33 was converted to 7a using the general preparation for ureas and thioureas above. Yellow solid
(155 mg, 87%), mp 174-175 oC; IR: 3344, 3183, 3115, 1546, 1333, 1257 cm-1; 1H NMR (400 MHz, DMSO-d6):
δ 10.2 (s, 1H, NH), 10.1 (s, 1H, NH), 8.19 (d, J = 9.0 Hz, 2H, Ar-H), 7.81 (d, J = 9.0 Hz, 2H, Ar-H), 7.30 (d, J =
2.5 Hz, 1H, Ar-H), 7.12 (dd, J = 8.6, 2.5 Hz, 1H, Ar-H), 6.71 (d, J = 8.6 Hz, 1H, Ar-H), 1.75 (s, 2H, CH2), 1.70-
1.54 (m, 4H, 2CH2CH3), 1.29 (s, 6H, CH3), 0.71 (t, J = 7.3 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz, DMSO-d6):
δ 179.3, 151.6, 146.9, 142.5, 131.9, 130.1, 124.9, 123.8, 123.5, 121.6, 117.7, 74.7, 40.3, 37.6, 33.5, 29.1, 8.8.
Anal. Calcd for C22H27N3O3S3: C, 63.90; H, 6.58; N, 10.16. Found: C, 63.63; H, 6.53; N, 10.06.
4.6.7. 1-(4,4-Diethyl-2,2-dimethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)thiourea (7b)
White solid (159 mg, 85%), mp 155-156 oC; IR: 3360, 3154, 1302, 1162 cm-1; 1H NMR (400 MHz, DMSO-
d6): δ 9.95 (s, 1H, NH), 9.87 (s, 1H, NH), 7.72 (d, J = 8.5 Hz, 2H, Ar-H), 7.65 (d, J = 8.5 Hz, 2H, Ar-H), 7.28 (d,
J = 2.5 Hz, 1H, Ar-H), 7.10 (dd, J = 8.5, 2.5 Hz, 1H, Ar-H), 6.69 (d, J = 8.5 Hz, 1H, Ar-H), 1.75 (s, 2H, Ar-H),
1.73-1.54 (m, 4H, 2CH2CH3), 1.29 (s, 6H, 2CH3), 0.72 (t, J = 7.2 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz,
DMSO-d6): δ 179.9, 151.4, 143.9, 132.1, 130.0, 126.0 (q, J = 3.4 Hz), 124.9 (q, J = 271.2 Hz), 124.2, 123.8,
123.6, 122.9, 117.6, 74.6, 37.5, 33.5, 29.1, 8.8 (one aliphatic carbon obscured by solvent). Anal. Calcd for
C23H27F3N2OS: C, 63.28; H, 6.23; N, 6.42. Found: C, 63.38; H, 6.21; N, 6.40.
4.6.8. 1-(4,4-Diethyl-2,2-dimethylchroman-6-yl)-3-(4-nitrophenyl)urea (7c)
o
1
Yellow solid (150 mg, 88%), mp 228-229 C; IR: 3292, 3154, 3098, 1652, 1559, 1332 cm-1; H NMR (400
MHz, DMSO-d6): δ 9.34 (s, 1H, NH), 8.67 (s, 1H, NH), 8.18 (d, J = 9.0 Hz, 2H, Ar-H), 7.68 (d, J = 9.0 Hz, 2H,
Ar-H), 7.37 (d, J = 2.5 Hz, 1H, Ar-H), 7.09 (dd, J = 8.6, 2.5 Hz, 1H, Ar-H), 6.67 (d, J = 8.6 Hz, 1H, Ar-H), 1.74
(s, 2H, CH2), 1.72-1.55 (m, 4H, 2CH2CH3), 1.28 (s, 6H, 2CH3), 0.73 (t, J = 7.3 Hz, 6H, 2CH2CH3); 13C NMR
(101 MHz, DMSO-d6) δ 152.5, 149.7, 147.1, 141.2, 132.2, 130.4, 125.6, 118.8, 118.3, 117.9, 117.8, 74.3, 40.1,
37.6, 33.6, 29.0, 8.8. Anal. Calcd for C22H27N3O4: C, 66.48; H, 6.85; N, 10.57. Found: C, 66.22; H, 6.86; N,
10.37.
4.6.9. 1-(4,4-Diethyl-2,2-dimethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)urea (7d)
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1
White solid (164 mg, 91%), mp 225-226 C; IR: 3312, 3169, 1652, 1554, 1328 cm-1; H NMR (400 MHz,
DMSO-d6): δ 8.98 (s, 1H, NH), 8.54 (s, 1H, NH), 7.65 (d, J = 8.9 Hz, 2H, Ar-H), 7.61 (d, J = 8.9 Hz, 2H, Ar-H),
7.35 (d, J = 2.7 Hz, 1H, Ar-H), 7/08 (dd, J = 8.6, 2.7 Hz, 1H, Ar-H), 6.66 (d, J = 8.6 Hz, 1H, Ar-H), 1.74 (s, 2H,
CH2), 1.68-1.54 (m, 4H, 2CH2CH3), 1.27 (s, 6H, 2CH3), 0.73 (t, J = 7.3 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz,
DMSO-d6): δ 152.9, 149.4, 144.2, 132.5, 130.3, 126.5 (q, J = 4.0 Hz), 125.1 (q, J = 270.0 Hz), 122.0, 121.7,
118.6, 118.1, 117.8, 74.3, 37.6, 33.6, 29.0, 8.8 (one aliphatic carbon obscured by solvent). Anal. Calcd for
C23H27F3N2O2: C, 65.70; H, 6.47; N, 6.66. Found: C, 65.55; H, 6.44; N, 6.61.
4.7. 2,2,4,4-Tetraethyl Analogs (2-Et-4-Et Series)
4.7.1. 2,2,4,4-Tetraethylchroman (38)
Compound 38 was prepared from lactone 30 by treatment with 2.5 equiv of ethylmagnesium bromide (–45
oC to rt) to generate 2-(3,5-diethyl-5-hydroxyheptan-3-yl)phenol
(37), followed by ring closure with H3PO4 to
promote formation of 38. The compound was purified by column chromatography to give the chroman as a
colorless oil. A small impurity was present and could not be removed. Thus, the slightly crude 38 was carried
on to the next step.
4.7.2. 2,2,4,4-Tetraethyl-6-nitrochroman (39)
Compound 39 was prepared by treatment of 38 with nitric acid in acetic anhydride at –10 oC. This reaction
gave the C6 and C8 nitrated products 39 and 40, respectively, which were difficult to separate. Purification by
preparative thin layer chromatography gave the C6 nitrated product 39 (35%) as a light yellow oil. The C8
nitrated product 40 co-eluted with the impurity cited above and was discarded. Spectral data for 39 were: IR:
1604, 1583, 1518, 1339, 1264 cm-1; 1H NMR (400 MHz, CDCl3): δ 8.10 (d, J = 2.7 Hz, 1H, Ar-H), 7.98 (dd, J =
8.9, 2.7 Hz, 1H, Ar-H), 6.86 (d, J = 8.9 Hz, 1H, Ar-H), 1.80 (s, 2H, CH2), 1.80-1.54 (m, 8H, 4CH2CH3), 0.90 (t, J
= 7.4 Hz, 6H, 2CH2CH3), 0.77 (t, J = 7.5 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz, CDCl3): δ 160.6, 141.8, 131.9,
123.9, 123.5, 118.8, 81.5, 37.9, 35.9, 34.0, 30.3, 8.7, 8.2.
4.7.3. 2,2,4,4-Tetraethylchroman-6-amine (41)
Nitrochroman 39 (1.00 g, 3.43 mmol) was reduced as described previously for 20 using iron powder and
ammonium chloride in aqueous ethanol to give the amine (41, 816 mg, 3.12 mmol, 91%) as a light brown oil.
1
IR: 3434, 3354, 3218, 1623, 1495, 1228 cm-1; H NMR (400 MHz, CDCl3): δ 6.65 (d, J = 8.4 Hz, 1H, Ar-H),
6.51 (d, J = 2.7 Hz, 1H, Ar-H), 6.48 (dd, J = 8.4, 2.7 Hz, 1H, Ar-H), 3.36 (br s, 2H, NH2), 1.69 (s, 2H, CH2),
1.76-1.44 (m, 8H, 4CH2CH3), 0.86 (t, J = 7.5 Hz, 6H, 2CH2CH3), 0.75 (t, J = 7.4 Hz, 6H, 2CH2CH3); 13C NMR
(101 MHz, CDCl3): δ 147.0, 139.5, 132.4, 118.4, 114.3, 113.7, 78.7, 37.8, 37.6, 33.3, 29.6, 8.4, 7.8.
4.7.4. 1-(4-Nitrophenyl)-3-(2,2,4,4-tetraethylchroman-6-yl)thiourea (8a)
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Compound 39 was converted to 8a using the general preparation for ureas and thioureas above. Light
o
1
yellow solid (162 mg, 85%), mp 162-164 C; IR: 3339, 3178, 1512, 1336 cm-1; H NMR (400 MHz, CDCl3):
δ 8.20 (d, J = 8.8 Hz, 2H, Ar-H), 8.05 (br s, 1H, NH), 7.75 (coincident d, J = 8.9 Hz, 2H, Ar-H and br s, 1H, NH),
7.11 (d, J = 2.1 Hz, 1H, Ar-H), 7.03 (dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 6.91 (d, J = 8.4 Hz, 1H, Ar-H), 1.78 (s, 2H,
13
CH2), 1.78-1.55 (m, 8H, 4CH2CH3), 0.91 (t, J = 7.4 Hz, 6H, 2CH2CH3), 0.77 (t, J = 7.3 Hz, 6H, 2CH2CH3); C
NMR (101 MHz, CDCl3): δ 179.4, 154.6, 144.4, 144.0, 133.4, 127.3, 125.2, 124.9, 124.5, 122.6, 119.8, 80.0,
37.6, 36.2, 33.4, 29.9, 8.5, 7.8. Anal. Calcd for C24H31N3O3S·0.2H2O: C, 64.75; H, 7.11; N, 9.44. Found: C,
64.75; H, 7.12; N, 9.36.
4.7.5. 1-(4-Nitrophenyl)-3-(2,2,4,4-tetraethylchroman-6-yl)urea (8b)
Compound 39 was converted to 8b using the general procedure described above. Light yellow solid (148
o
1
mg, 91%), mp 163-164 C; IR: 3342, 3220, 3158, 3098, 1662, 1512, 1336 cm-1; H NMR (400 MHz, DMSO-
d6): δ 9.34 (s, 1H, NH), 8.67 (s, 1H, NH), 8.18, J = 9.1 Hz, 2H, Ar-H), 7.69 (d, J = 9.1 Hz, 2H, Ar-H), 7.36 (d, J
= 2.6 Hz, 1H, Ar-H), 7.10 (dd, J = 8.6, 2.5 Hz, 1H, Ar-H), 6.69 (d, J = 8.6 Hz, 1H, Ar-H), 1.70 (s, 2H), 1.72-1.43
(m, 8H, 4CH2CH3), 0.83 (t, J = 7.3 Hz, 6H, 2CH2CH3), 0.73 (t, J = 7.3 Hz, 6H, 2CH2CH3); 13C NMR (101 MHz,
DMSO-d6): δ 152.5, 149.6, 147.1, 141.2, 132.3, 131.3, 125.6, 118.7, 118.2, 118.0, 117.7, 78.8, 37.5, 36.9, 33.5,
29.6, 8.7, 8.0. Anal. Calcd for C24H31N3O4: C, 67.74; H, 7.34; N, 9.88. Found: C, 67.59; H, 7.35; N, 9.83.
4.7.6. 1-(2,2,4,4-Tetraethylchroman-6-yl)-3-(4-(trifluoromethyl)phenyl)urea (8c)
o
1
White solid (176 mg, 91%), mp 165-166 C; IR: 3309, 3203, 3142, 3095, 1655, 1327 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.98 (s, 1H, NH), 8.54 (s, 1H, NH), 7.65 (d, J = 8.7 Hz, 2H, Ar-H), 7.61 (d, J = 8.8 Hz, 2H,
Ar-H), 7.34 (d, J = 1.3 Hz, 1H, Ar-H), 7.08 (dd, J = 8.6, 1.3 Hz, 1H, Ar-H), 6.68 (d, J = 8.6 Hz, 1H, Ar-H), 1.70
(s, 2H, CH2), 1.70-1.43 (m, 8H, 4CH2CH3), 0.83 (t, J = 7.3 Hz, 6H, 2CH2CH3), 0.72 (t, J = 7.3 Hz, 6H,
2CH2CH3); 13C NMR (101 MHz, DMSO-d6): δ 151.9, 148.3, 143.1, 131.5, 130.2, 125.4 (q, J = 3.5 Hz), 124.8 (q,
J = 270.9 Hz), 120.7 (q, J = 31.9 Hz), 117.5, 117.1, 117.0, 116.9, 77.7, 36.4, 36.0, 32.5, 28.6, 7.7, 7.0. Anal.
Calcd for C25H31F3N2O2: C, 66.95; H, 6.97; N, 6.25. Found: C, 66.94; H, 6.98; N, 6.18.
4.7.7. 1-(2,2,4,4-Tetraethylchroman-6-yl)-3-(4-(trifluoromethoxy)phenyl)urea (8d)
White solid (156 mg, 78%), mp 180-181 oC; IR: 3295, 3201, 1645, 1267 cm-1; 1H NMR (400 MHz, DMSO-
d6): δ 8.75 (s, 1H, NH), 8.46 (s, 1H, NH), 7.55 (d, J = 8.9 Hz, 2H, Ar-H), 7.32 (d, J = 2.5 Hz, 1H, Ar-H), 7.26 (d,
J = 8.8 Hz, 2H, Ar-H), 7.08 (dd, J = 8.6, 2.5 Hz, 1H, Ar-H), 6.67 (d, J = 8.6 Hz, 1H, Ar-H), 1.70 (s, 2H, CH2),
1.70-1.43 (m, 8H, 4CH2CH3), 0.83 (t, J = 7.4 Hz, 6H, 2CH2CH3), 0.72 (t, J = 7.3 Hz, 6H, 2CH2CH3); 13C NMR
(101 MHz, DMSO-d6): δ 151.2, 148.2, 141.8, 138.7, 131.8, 130.2, 121.1, 119.6 (q, J = 255.1 Hz), 118.6, 117.3,
116.83, 116.82, 77.6, 36.4, 35.9, 32.5, 28.6, 7.7, 7.0. Anal. Calcd for C25H31F3N2O3: C, 64.64; H, 6.73; N, 6.03.
Found: C, 64.38; H, 6.66; N, 5.98.
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4.8. Biology
The compounds were dissolved in DMSO at a concentration of 0.01 M. The human ovarian cancer cell line
A2780 was plated in 96-well tissue culture dishes at a concentration of 3000 cells per well in RPMI medium
supplemented with 10% fetal bovine serum and a mixture of antibiotics and antimycotics. The next day, the
cultures were treated in triplicate with compound concentrations ranging from 2 to 8 µM in 1 µM increments.
For compounds 8a-d, additional experiments were performed with concentrations of 10 µM and series two-fold
dilutions to 156 nM. Control cultures were treated with DMSO solvent only. After 72 h of incubation, the
CellTiter 96 Non-Rad Cell Proliferation Assay (Promega) was used to quantify the remaining metabolically
living cells. After subtracting blank values, the optical density (OD) readout of the assay for the treated
cultures was normalized with the average OD of the control cultures. A custom program was written in GNU
Octave, a free software compatible with Matlab, to fit the dose-response curves with four-parameter sigmoid
function, extracting the IC50 and efficacy (the maximal % growth inhibition) parameters.
4.9. Modeling Studies
Autodock 4.2 [22] was used to dock the compounds to the substrate binding domain of mortalin (Protein
Data Bank ID: 3N8E). For the individual compounds ChemSketchTM (Advanced Chemistry Development, Inc.
ADC/Labs, Toronto, Ontario, Canada) was employed to generate the SMILES notations which were
subsequently converted to PDB files with initial three-dimensional coordinates using Open Babel GUI [23].
AutoDockTool [22] was then used to prepare the protein and compound with partial charges and also for later
rotatable bonds. Only polar hydrogens were retained in the molecules. Kollman united atom partial charges
and solvation parameters were assigned. The search space of 44 Å x 47 Å x 41 Å was slightly larger than the
protein molecule, and the grid spacing was 0.375 A. Autogrid was run first to prepare the coordinate system
and the Lamarckian genetic algorithm was applied with a population size of 150 and 5 million maximum
evaluations. The minimum empirical binding free energy between each compound and receptor was recorded.
All dockings were performed on an iMac computer with a 2.4 GHz intel Core i3 processor and with 4 GB RAM.
Acknowledgments
The investigators (KDB and RAB) gratefully acknowledge the support of this research by the Stephenson
Cancer Research Center/Oklahoma Tobacco Settlement Endowment Trust (TSET). The authors also wish to
thank OSU for Niblack and Wentz Foundation Scholarships and the OSU Department of Chemistry for an
Undergraduate Research Scholarship to FMW, and for a President’s Fellows Faculty Research Award to DHZ.
The authors are grateful to the OSU College of Arts and Sciences for funds that allowed the Chemistry
Department to purchase a new A-400 MHz NMR unit for our State-Wide NMR facility. The State-Wide NMR
facility was originally established with support from the NSF (BIR-9512269), the Oklahoma State Regents for
Higher Education, the W. M. Keck Foundation, and Conoco, Inc.
27

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Competing interests
The authors declare no competing interests.
Supplemental data
Supplementary data related to this article—copies of 1H and 13C NMR spectra for all final compounds—can
be found at
References and Notes
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