Synthesis of N-[3,4-dihydro-4-(acetoxymethyl)-2,2,4-trimethyl-2H-1- benzothiopyran-6-yl]-N'-(4-nitrophenyl)thiourea and N-[3,4-dihydro-4- (hydroxymethyl)-2,2,4-trimethyl-2H-1-benzothiopyran-6-yl]-N'-(4-nitrophenyl) thiourea, a Major Metabolite of N-(3,4-d

Article abstract of DOI:10.1080/10426507.2010.534521

The compound N-(3,4-dihydro-2,2,4,4-tetramethyl-2H-1-benzothiopyran-6-yl)- N'-(4-nitrophenyl)thiourea [NSC 726189] has exhibited strong anticancer activity in several assays and cell lines and is under consideration for clinical trials for the possible tr

Full text of DOI:10.1080/10426507.2010.534521

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Phosphorus, Sulfur, and Silicon and the
Related Elements
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Synthesis of N-[3,4-Dihydro-4-
(acetoxymethyl)-2,2,4-trimethyl-2H-1-
benzothiopyran-6-yl]-N′-(4-
nitrophenyl)thiourea and N-[3,4-
dihydro-4-(hydroxymethyl)-2,2,4-
trimethyl-2H-1-benzothiopyran-6-yl]-
N′-(4-nitrophenyl)thiourea, a Major
Metabolite of N-(3,4-Dihydro-2,2,4,4-
tetramethyl-2H-1-benzothiopyran-6-YL)-
N′-(4-nitrophenyl)thiourea
Baskar Nammalwar a , Richard A. Bunce a , Doris M. Benbrook b , Tao
Lu c , Hui-Fang Li c , Ya-Dong Chen c & K. Darrell Berlin a
a Department of Chemistry , Oklahoma State University , Stillwater,
Oklahoma, USA
b University of Oklahoma, Health Sciences Center, Department of
Obstetrics/Gynecology , Oklahoma City, Oklahoma, USA
c School of Basic Sciences , China Pharmaceutical University ,
Nanjing, P. R. China
Published online: 13 Jan 2011.
To cite this article: Baskar Nammalwar , Richard A. Bunce , Doris M. Benbrook , Tao Lu , Hui-Fang
Li , Ya-Dong Chen & K. Darrell Berlin (2010) Synthesis of N-[3,4-Dihydro-4-(acetoxymethyl)-2,2,4-
trimethyl-2H-1-benzothiopyran-6-yl]-N′-(4-nitrophenyl)thiourea and N-[3,4-dihydro-4-
(hydroxymethyl)-2,2,4-trimethyl-2H-1-benzothiopyran-6-yl]-N′-(4-nitrophenyl)thiourea, a
Major Metabolite of N-(3,4-Dihydro-2,2,4,4-tetramethyl-2H-1-benzothiopyran-6-YL)-N′-(4-
nitrophenyl)thiourea, Phosphorus, Sulfur, and Silicon and the Related Elements, 186:1, 189-204, DOI:
10.1080/10426507.2010.534521
To link to this article: http://dx.doi.org/10.1080/10426507.2010.534521
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Phosphorus, Sulfur, and Silicon, 186:189–204, 2011
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2010.534521
SYNTHESIS OF N-[3,4-DIHYDRO-4-(ACETOXYMETHYL)-
2,2,4-TRIMETHYL-2H-1-BENZOTHIOPYRAN-6-YL]-Nꢀ-(4-
NITROPHENYL)THIOUREA AND N-[3,4-DIHYDRO-4-
(HYDROXYMETHYL)-2,2,4-TRIMETHYL-2H-1-
BENZOTHIOPYRAN-6-YL]-Nꢀ-(4-NITROPHENYL)-
THIOUREA, A MAJOR METABOLITE OF N-(3,4-DIHYDRO-
2,2,4,4-TETRAMETHYL-2H-1-BENZOTHIOPYRAN-6-YL)-
Nꢀ-(4-NITROPHENYL)THIOUREA
Baskar Nammalwar,1 Richard A. Bunce,1 Doris M. Benbrook,2
Tao Lu,3 Hui-Fang Li,3 Ya-Dong Chen,3 and K. Darrell Berlin1
1Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma, USA
2University of Oklahoma, Health Sciences Center, Department of
Obstetrics/Gynecology, Oklahoma City, Oklahoma, USA
3School of Basic Sciences, China Pharmaceutical University, Nanjing, P. R. China
GRAPHICAL ABSTRACT
Abstract The compound N-(3,4-dihydro-2,2,4,4-tetramethyl-2H-1-benzothiopyran-6-yl)-Nꢁ-
(4-nitrophenyl)thiourea [NSC 726189] has exhibited strong anticancer activity in several
Received 20 September 2010; accepted 21 October 2010.
We gratefully acknowledge the support by the T-BAG (Technology Business Assessment Group) program
at Oklahoma State University and the Department of Chemistry in the College of Arts and Sciences. In addition,
R.A.B. and K.D.B. are pleased to acknowledge funding for the Varian Inova 400 MHz NMR spectrometer in the
Oklahoma Statewide Shared NMR Facility by the National Science Foundation (BIR-9512269), the Oklahoma
State Regents for Higher Education, the W. M. Keck Foundation, and Conoco, Inc.
Address correspondence on the synthesis to Dr. R. A. Bunce or Dr. K. D. Berlin, Department of Chemistry,
Oklahoma State University, Stillwater, OK 74078, USA. E-mail: rab@okstate.edu; kdb@okstate.edu; Address
correspondence on the theoretical calculations to Dr. Tao Lu, School of Basic Sciences, China Pharmaceutical
University, Nanjing, P. R. China. E-mail: lutao@cpu.edu.cn
189

190
B. NAMMALWAR ET AL.
assays and cell lines and is under consideration for clinical trials for the possible treatment of
kidney cancer. Since the heterocycle has not shown any significant toxicity in animal studies,
it is conceivable that a metabolite could be the active agent. In a previous study, major
metabolites were tentatively identified via analyses of materials extracted from rat and human
liver microsomes, as well as extracts from rat urine, by the use of HPLC-UV and MS/MS.
However, no metabolite has been obtained in pure form. This article outlines a de novo
synthesis of a primary metabolite, namely N-[3,4-dihydro-4-(hydroxymethyl)-2,2,4-trimethyl-
2H-1-benzothiopyran-6-yl]-Nꢁ-(4-nitrophenyl)thiourea. A prodrug of the metabolite was also
prepared, namely N-[3,4-dihydro-4-(acetoxymethyl)-2,2,4-trimethyl-2H-1-benzothiopyran-6-
yl]-Nꢁ-(4-nitrophenyl)thio-urea.
Keywords
yl]-Nꢁ-(4-nitrophenyl)thiourea;
benzothiopyran-6-yl]-Nꢁ-(4-nitrophenyl)thiourea; multi-step synthesis
N-[3,4-Dihydro-4-(acetoxymethyl)-2,2,4-trimethyl-2H-1-benzothiopyran-6-
N-[3,4-dihydro-4-(hydroxymethyl)-2,2,4-trimethyl-2H-1-
INTRODUCTION
Kidney cancer is known to have a high mortality rate due to its resistance to a vari-
ety of therapies.1 The incidence of renal cancer appears to have increased over the last 50
years.2 Preliminary evidence is available that demonstrates that certain heteroarotinoids can
be effective in the treatment of such cancers.3,4 The basic structural unit 1 was originally
designed for heteroarotinoids as illustrated,4 and was based, in part, on a structural relation-
ship with trans-retinoic acid (2). Studies of the biological activity of 2 and isomers thereof
revealed them to possess high toxicity and therefore limited utility.5 Pioneering work in
OH
1
LG
O
2
3
2
X
Z
4
1
X = O, S. NR
LG = Linking
Z = CO2H, CO2R, group; amide, alkene
SO3H, NO2
ester, urea, thiourea
OH
O
OH
O
O
4
6
3
5
O
CO2H
OH
OH
O
O
OH
S
HO
H
N
H
N
H
N
H
N
S
S
S
NO2
NO2
7
8

SUBSTITUTED THIOUREA METABOLITE AND RELATED PRODRUG
191
which heteroatoms were inserted into strategic positions within the molecular framework,
as shown in 1, reduced the toxicity significantly.4,6 Since an aryl ring was also present, the
term “heteroarotinoids” was established to identify such systems.4,6 High toxicity associ-
ated with acid 2 has been presumed to arise from its metabolites, the major members of
which are shown as 3–6.7 An initial objective was to ascertain if heteroarotinoids with a
heteroatom at C-4 had reduced toxicity and could avoid oxidation at this position, which
might lead to derivatives of 2, such as 4 and 5, which were toxic. The hypothesis that het-
eratoms do reduce toxicity has been validated.4,6,8,9 Moreover, the addition of the benzene
ring fused to the cyclohexyl unit in 1 would also prevent epoxidation, which produced toxic
3 from 2. The versatility of heteroarotinoids in treating a variety of cancers is evident in that
good activity has been observed against breast, head/neck, kidney, and lung cancers.8–23
It is significant to recall that the development of useful drugs has often required an im-
provement in hydrophilicity, which increases aqueous solubility, frequently without loss of
biological activity.24,25 Examples related to our system 7 are certain diarylurea multikinase
inhibitors that inhibit tumor growth.26,27 Sorafenib (BAY 43–9006) is such a compound that
was developed by increasing the hydrophilicity of its precursor, which led to enhanced in
vivo potency.27–30 The ability of Sorafenib to prolong progression-free survival in recurrent
kidney cancer patients with manageable toxicity led to its FDA approval.31,32 However,
the toxicity of this agent remains a problem.32 Since N-(3,4-dihydro-2,2,4,4-tetramethyl-
2H-1-benzothiopyran-6-yl)-Nꢁ-(4-nitrophenyl)-thiourea (7, NSC 726189) has shown high
activity in the treatment of certain kidney cancerous cell lines without any significant toxi-
city in studies by the NCI,22 the metabolites may not be toxic. The increased polarity of the
metabolites could provide a number of pharmaceutical and pharmacokinetic advantages
with increased aqueous solubility. Higher hydrophilicity correlates well with reduced lipid
tissue uptake,33 typically resulting in higher blood or plasma concentrations available for
delivery to target tumor cells. Since 7 is a candidate for the potential treatment of kidney
cancer,22,34 the importance of meeting the Metabolites in Safety Testing (MIST) standards
for metabolites of potential drugs is well documented.35–38 One metabolite39 of 7, and
the synthetic target molecule of this article, is N-[3,4-dihydro-4-(hydroxymethyl)-2,2,4-
trimethyl-2H-1-benzothiopyran-6-yl]-Nꢁ-(4-nitrophenyl)thiourea (8). Urea derivatives as
kinase inhibitors are well recognized with many such inhibitors being marketed by Amer-
ican Custom Chemicals (P.O. Box 910574, San Diego, CA 92191–0574, USA), including
the examples Sorafenib and ABT-869, as systems with urea linkages between aryl groups
as in 7 but with the latter having a thiourea group.
N
N
H3C
CH3
H
N
H
N
O
N
H
N
O
H3C
Cl
N
H
N
H
O
CF3
F
O
Sorafenib (BAY 43 9006)
ABT-869 (KIT-3 kinase inhibitor)
[CAS Number: 796967-16-3]
CHEMISTRY
Michael addition of thiophenol (9) to 3-methyl-2-butenoic acid in standard fash-
ion gave 3-(phenylthio)-3-butenoic acid (10), which, when subjected to treatment with

192
B. NAMMALWAR ET AL.
O
COOH
piperidine
PPA
HO
+
sealed tube 130 oC
70 oC
S
SH
9
10
11 (83%)
O
CN
S
TMSO CN
TMSCN
cat. ZnI2
POCl3, pyridine
benzene
S
S
12 (80%)
13 (~100%)
14 (82%)
CN
CN
NaBH4
EtOH
1) NaH, DMF
2) CH3I
50% aq H2SO4
S
S
15 (92%)
16 (90%)
OH
OAc
CO2H
LiAlH4
THF
Ac2O
DMAP
CH2Cl2
S
S
S
17 (90%)
18 (87%)
19 (98%)
OAc
OAc
O2N
H2N
HNO3
Ac2O
Fe
HOAc, 115 oC
S
S
20 (20%)
21 (95%)
OAc
H
N
H
O2N
NCS
N
21
THF
S
O2N
S
10% aq NaOH
23 (88%)
OH
OH
H
N
H
N
O2N
NCS
H2N
S
THF
S
O2N
S
22 (94%)
8 (82%)
Scheme 1
polyphosphoric acid, led to the known 2,2-dimethylthiochroman-4-one (11) in 66% overall
yield (Scheme 1).40 Conversion of 12 to the trimethylsilyl cyanohydrin 13 and treatment
directly with phosphorus oxychloride in benzene/pyridine provided the unsaturated carboni-
trile 14 in a yield of 82%.41 Saturation of the double bond in 14 with sodium borohydride

SUBSTITUTED THIOUREA METABOLITE AND RELATED PRODRUG
193
generated 15, which was smoothly alkylated to 2,2,4-trimethylthiochroman-4-carbonitrile
(16, 83% overall yield). Hydrolysis of the nitrile group in 16 gave acid 17, which was im-
mediately reduced to the corresponding key intermediate alcohol 19 (78% for two steps).
Acylation of 18 with acetic anhydride in methylene chloride in the presence of DMAP then
gave the corresponding acetate intermediate 19 in near quantitative yield.42
Although the prior steps could be optimized to give very good to excellent yields, the
nitration of 19 resulted in a modest return (∼20%) of the required nitroester 20 due to several
side reactions. This experiencehadbeennotedpreviously15 innitrationof therelated2,2,4,7-
pentamethylthiochroman system, where such reactions markedly reduced the production
of 2,2,4,4,7-pentamethyl-6-nitrothiochroman. Reduction of the nitro group in 20 using iron
and acetic acid at 115◦C afforded amine 21 in a near quantitative yield. Saponification
of 21 was easily accomplished to give amino alcohol 22. Treatment of 21 and 22 with
4-nitrophenylisothiocyanate in dry THF followed a similar pattern employed in the final
step to create 7,15 and gave 23 and 8, respectively, in high returns. Both crystalline products
had very similar melting points, but a mixture melting point determination confirmed the
different individual structures as did elemental analyses. Ester 23 can be considered as a
prodrug for alcohol 8.
RESULTS AND DISCUSSION
In view of the high activity8–23 and low toxicity9,15,22,43 of heteroarotinoids, as well
as a structural relationship to diarylureas, which interact with many kinases, a theoretical
investigation of the binding properties of 7 and 8 was initiated. Sorafenib is a well-known
multitarget kinase inhibitor, inhibiting the isoforms of Raf, KIT, VEGFR, PDGFR, and
others, which was approved for the treatment of advanced renal cell cancer (RCC) by
the FDA in 2005. Moreover, the Raf family of kinases is the main target of Sorafenib,
among which B-Raf is the most important member and an intensely studied anticancer
target. Since 7 and 8 are structurally similar to Sorafenib, a preliminary assessment of
their binding affinity and plausible binding mode was performed using molecular docking
(employing Glide as the docking program) on 7 with the crystal structure of B-Raf (PDB
code: 1UWJ). In the molecular docking study, Sorafenib was incorporated as a reference,
although the modeling showed that the Glide score44,45 of 7 (−7.7) was higher than that of
Sorafenib (−10.8) and suggested that 7 might be less potent than sorafenib.46 However, 7
was oriented in a similar manner to Sorafenib (as shown in Figure 1). Both the urea group in
Sorafenib and the thiourea group in 7 formed critical H bonds with Glu501 and Asp594.
Thus, it is predictable that 7 and its metabolite 8 would inhibit B-Raf.
It is known that B-Raf is a good therapeutic target for new agents,47 and so, it is
predictable that metabolites of 7 would inhibit kinases, especially members of the Raf
family. For experimental confirmation, 7 was submitted to Ambit Biosciences Corporation
(4215 Sorrento Valley Blvd, San Diego, CA 92121, USA) for evaluation of kinase activity
with 442 different human kinases. The technique is an active-site–dependent competition
binding assay with an immobilized, active site directed ligand.48 However, in the competitive
binding studies, the percent control for 7 with B-Raf48,49 was 87%, while that of its mutant
[B-Raf-V600E] was 77%. In contrast, agent 7 exhibited a percent control of 2.6% with
KIT-V559D, 5.2% with KIT, 11% with KIT-L576P, 17% with KIT-V559D,V6, and 12%
with the kinase PDGFRB. Consequently, binding constant Kd values were determined for
each kinase: KIT (820 nM), KIT-V559D (1300 nM), KIT-V559D,V6 (2700 nM), PDGFRB
(2900 nM), and KIT-L576P (3800 nM).46 Thus, activity at the nanomolar level, coupled with

194
B. NAMMALWAR ET AL.
Figure 1 Sorafenib and 7 were docked into the binding sites of B-Raf. Sorafenib and 7 are shown in green stick
and magnenta stick representation, respectively, and the binding pocket is shown as the white surface.
low toxicity,9,15,22 suggests that 7 has a theoretical and experimental basis for development
of an inhibitor of KIT. Sorafenib is also known to be an inhibitor of KIT (also known as
c-KIT).50
Similar experiments with metabolite 8 revealed competitive inhibition with an im-
mobilized, active-site–directed ligand in terms of percent control with values of 15% for
KIT, 15% for KIT-V559D, and 17% for PDGFRB kinases. The corresponding Kd values
were 1200 nM, 4100 nM, and 3400 nM, respectively. Thus, both 7 and 8 exhibited good
inhibitory activity against the KIT kinase. As additional support of our work, molecular
docking experiments of metabolite 8 with the crystal structure of kinase KIT (PDB code:
1t46) were conducted to explore a possible binding mode. In the complex of KIT with the
known anticancer agent Imatinib, there exist four hydrogen bonds with residues Cys673,
Thr670, Asp810, and Glu640.47 As shown in Figure 2, the docking conformation of 8 occu-
pied the same docking pocket49 and adopted a similar binding mode to Imatinib by forming
H-bond interactions with Glu640 and Asp810 and hydrophobic contacts with residues in
the hydrophobic pocket. Moreover, Imatinib is selective for BCR-ABL, inhibiting its ty-
rosine kinase activity as well as several other kinases.46 The Glide docking scores are a
measure of the binding affinities of the ligands for the kinase.44,47 The scores were −11.97
for Imatinib and −8.70 for metabolite 8.44 (The lower the score, the greater the affinity.)
Consequently, potential useful activity is predicted for the metabolite 8. It is noteworthy
that KIT kinase mutants have shown resistance to Imatinib in gastrointestinal stromal tumor
patients.47 Thus, the continued search for new agents is a worthy goal, and the identification
of 8 provides insight into the development of other KIT kinase inhibitors.

SUBSTITUTED THIOUREA METABOLITE AND RELATED PRODRUG
195
Figure 2 Plausible binding mode of 8 with the crystal structure of KIT by molecular docking. Agent 8 is in green
stick and ball representation, and the magenta dashed lines represent the hydrogen bond interactions. The line
surfaces in different colors represent hydrogen bond donor (blue), hydrogen bond acceptor (red), and hydrophobic
(white) interactions, respectively.
CONCLUSIONS
The synthesis of the first metabolite of N-(3,4-dihydro-2,2,4,4-tetramethyl-2H-
1-benzothiopyran-6-yl)-Nꢁ-(4-nitrophenyl)thiourea (NSC 726189) was accomplished,
namely N-[3,4-dihydro-4-(hydroxymethyl)-2,2,4-trimethyl-2H-1-benzothiopyran-6-yl]-Nꢁ-
(4-nitrophenyl)thiourea. In addition, the acetate derivative of the metabolite, namely N-[3
,4-dihydro-4-(acetoxymethyl)-2,2,4-trimethyl-2H-1-benzothiopyran-6-yl]-Nꢁ-(4-nitrophenyl)-
thiourea, was also prepared as a possible prodrug. The compounds await biological
evaluation for anticancer activity.
EXPERIMENTAL
All reactions were run under dry nitrogen in oven-dried glassware. Reactions were
monitored by thin layer chromatography on silica gel GF plates (Analtech No. 21521)
using ultraviolet detection. Preparative separations were performed by flash column chro-
matography on silica gel (grade 62, 60–200 mesh) mixed with UV-active phosphor (Sorbent
Technologies No. UV-5); band elution was monitored using a handheld UV lamp. Melting
points were uncorrected. FT-IR spectra were taken as thin films on NaCl disks. Unless
1
otherwise indicated, H and 13C NMR spectra were measured in DCCl3 at 300 MHz and
75 MHz, respectively, and were referenced to internal TMS with coupling constants (J)

196
B. NAMMALWAR ET AL.
in Hz. Elemental analyses were provided by Atlantic Microlab (Norcross, GA). Tetrahy-
drofuran (THF) was dried over KOH pellets and distilled from LiAlH4 under nitrogen.
Commercial anhydrous N,N-dimethylformamide (DMF) was transferred by syringe into
reactions where required. Other reagents were used as received.
N-[3,4-Dihydro-4-(hydroxymethyl)-2,2,4-trimethyl-2H-1-
benzothiopyran-6-yl]-Nꢀ-(4-nitrophenyl)thiourea (8)
The alcohol (97 mg, 82%) was prepared via the general procedure employed for 23
using alcohol 22 (66 mg, 0.28 mmol) and 4-nitrophenyl isothiocyanate (54 mg, 0.30 mmol).
The product was purified by recrystallization [H3COH], mp 154–155◦C. IR: 3435 cm−1
;
1H NMR (CD3OD): δ 8.24 (d, J = 9.0 Hz, 2 H, Ar-H), 7.83 (d, J = 9.0 Hz, 2 H, Ar-H),
7.56 (d, J = 2.2 Hz, 1 H, Ar-H), 7.18 (dd, J = 8.2, 2.2 Hz, 1 H, Ar-H), 7.13 (d, J = 8.2 Hz,
1 H, Ar-H), 3.63 (d, J = 11.0 Hz, 1 H, CH2-OH), 3.57 (d, J = 11.0 Hz, 1 H, CH2OH), 2.36
(d, J = 14.3 Hz, 1 H, CH2-ring), 1.82 (d, J = 14.3 Hz, 1 H, CH2-ring), 1.44 (s, 3 H, CH3),
1.40 (s, 6 H, (CH3)2); the 2 NH and 1 OH exchanged with D3COD. 13C NMR (CD3OD):
δ 181.5, 147.2, 144.9, 141.5, 136.8, 133.2, 129.7, 125.3, 125.2, 123.7, 123.6, 72.2, 50.2,
42.9, 42.0, 31.6, 31.1, 27.7. Anal. Calcd for C20H23N3O3S2: C, 57.53; H, 5.55; N, 10.06;
S, 15.36. Found: C, 57.45; H, 5.52; N, 10.05; S, 15.23. TLC analysis, using 5% methanol
in HCCl3, showed one spot.
3-(Phenylthio)-3-methylbutanoic Acid (11)
A 200-mL, Pyrex pressure vessel (Chemglass No. CG-1880-R-112) was charged
with 3-methyl-2-butenoic acid (10, 30.0 g, 0.30 mol), piperidine (27.0 g, 0.32 mol, 1.05
eq), and thiophenol (9, 33.0 g, 0.30 mol), and then sealed and heated at 130◦C for a period
of 24 h. The resulting mixture was cooled to room temperature and diluted with 1 L of
ether. The ether layer was washed with 1 N HCl (3 × 200 mL), water (1×), and saturated
NaCl (1×). The organic solution was dried (Na2SO4) and concentrated under vacuum to
give a pale yellow solid. The compound was recrystallized (benzene:petroleum ether, 1:3)
to give 11 (52.3 g, 83%) as an off-white solid, mp 70–72◦C [lit40 mp 71◦C]. IR: 3700–2350,
1707 cm−1; 1H NMR: δ 10–11 (br s, 1 H, CO2H), 7.57 (dd, J = 7.7, 1.6 Hz, 2 H, Ar-H),
7.36 (m, 3 H, Ar-H), 2.56 (s, 2 H, CH2), 1.41 (s, 6 H, C(CH3)2); 13C NMR: δ 177.0, 137.7,
131.1, 129.2, 128.7, 46.7, 46.4, 28.4. Previous spectra were at low resolution in CCl4 and
the yield was 87%.40 Product purity was adequate to prepare 12. The TLC showed one spot
using 40% EtOAc in hexanes.
2,2-Dimethylthiochroman-4-one (12)
A 1-L, three-necked, round-bottomed flask, fitted with a mechanical stirrer, a con-
denser, and a nitrogen inlet was charged with polyphosphoric acid (400 g) and heated to
70◦C using an oil bath. To the stirred polyphosphoric acid, 3 (50 g, 0.24 mol) was added,
and the mixture was stirred at 70◦C for 45 min. The reaction mixture was cooled to 0◦C
using an ice bath and then carefully added to ice-cold water. [Caution! The reaction is
extremely exothermic.] The resulting solution was extracted with ether (3 × 600 mL). The
combined organic extracts were washed with 1 N NaOH (1×) and saturated NaCl (1×),
dried (MgSO4), and concentrated under vacuum to afford a white solid. The compound
was recrystallized [ethyl acetate:petroleum ether, 1:3] to give 36.5 g (80%) of ketone 12 as

SUBSTITUTED THIOUREA METABOLITE AND RELATED PRODRUG
197
a white solid, mp 67–68◦C [lit40 66–68◦C]. IR: 1686 cm−1; 1H NMR: δ 8.10 (dd, J = 7.7,
1.1 Hz, 1 H, Ar-H), 7.39 (td, J = 8.0, 1.6 Hz, 1 H, Ar-H), 7.22 (d, J = 7.7 Hz, 1 H, Ar-H),
7.16 (td, J = 7.7, 1.1 Hz, 1 H, Ar-H), 2.87 (s, 2 H, CH2), 1.47 (s, 6 H, C(CH3)2); 13C NMR:
δ 194.8, 141.3, 133.6, 129.6, 128.6, 127.5, 124.6, 53.8, 44.6, 28.5. Only low resolution
spectra taken in CCl4 were previously reported along with a yield of 70%.40 The product
was used directly in the next reaction to prepare 14. The TLC showed one spot using 40%
EtOAc in hexanes.
2,2-Dimethyl-2H-thiochromene-4-carbonitrile (14)
A 250-mL, one-necked, round-bottomed flask, equipped with a magnetic stirrer, a
condenser, and a nitrogen inlet was charged with ketone 12 (32.0 g 0.17 mol), trimethylsilyl
cyanide (19.8 g, 25 mL, 0.20 mol, 1.2 eq), and anhydrous zinc iodide (40 mg). The
resulting solution was stirred at room temperature for 24 h under nitrogen, at which time
TLC indicated the reaction was complete. Trimethylsilyl cyanohydrin 13 was unstable to
purification and was used directly in the next step. Spectral data for only slightly impure 13
were IR: 2210 (weak) cm−1; 1H NMR: δ 7.70 (dd, J = 7.7, 1.6 Hz, 1 H, Ar-H), 7.28–7.10
(complex, 3 H, Ar-H), 2.53 (d, J = 14.0 Hz, 1 H, CH2), 2.47 (d, J = 14.0 Hz, 1 H, CH2),
1.54 (s, 3 H, CH3), 1.49 (s, 3 H, CH3), 0.22 (s, 9 H, C(CH3)3); 13C NMR: 133.4, 132.0,
129.5, 129.3, 127.8, 125.3, 121.6, 69.9, 51.5, 41.4, 31.3, 31.0, 1.2. TLC analysis, using
30% EtOAc in hexanes, indicated 13 was pure.
In a 500-mL, three-necked, round-bottomed flask, fitted with a magnetic stirrer, a
reflux condenser, an addition funnel, and a drying tube was placed the crude trimethylsilyl
cyanohydrin 13 dissolved in benzene (200 mL) containing pyridine (10 mL). To the resulting
solution, phosphorus oxychloride (33.5 g, 20 mL, 0.22 mol) was added dropwise with
stirring. After refluxing for 4 h, TLC indicated that the reaction was complete. The reaction
mixture was carefully poured onto 500 g of crushed ice, and the mixture was extracted
with ether (3 × 300 mL). The combined ether layers were washed with water (1×) and
saturated NaCl (1×), dried (MgSO4), and concentrated under vacuum. The crude product
was further purified by flash chromatography on a silica gel column (50 cm × 5 cm) using
increasing concentrations of ether (5%, 10%, 25%, 50%) in hexanes to give 27.5 g (82%)
of 14 as a white solid, mp 62–63◦C. IR: 2224 cm−1; 1H NMR: δ 7.59 (dd, J = 6.0, 2.7 Hz,
1 H, Ar-H), 7.36–7.18 (complex, 3 H, Ar-H), 6.56 (s, 1 H, CH), 1.46 (s, 6 H, C(CH3)2);
13C NMR: δ 146.8, 131.6, 129.7, 127.8, 126.9, 126.7, 126.0, 117.1, 113.5, 40.9, 28.2. TLC
analysis, using 33% EtOAc in hexanes, indicated that 14 was pure.
2,2-Dimethylthiochroman-4-carbonitrile (15)
To a 500-mL, three-necked, round-bottomed flask fitted with a magnetic stirrer, a
reflux condenser, and a nitrogen inlet, was added 14 (24.0 g, 0.12 mol) and absolute ethanol
(150 mL). The mixture was heated for 10 min to effect solution. To the warm solution,
sodium borohydride (2.25 g, 0.06 mol) was slowly added in small portions, and refluxing
was continued for 45 min. The reaction mixture was evaporated under vacuum and purified
by flash chromatography on a silica gel column (50 cm × 5 cm) using 50% ether in
1
hexanes to give 22.3 g (92%) of 15 as a white solid, mp 64–65◦C. IR: 2243 cm−1; H
NMR: δ 7.51 (d, J = 7.1 Hz, 1 H, Ar-H), 7.24–7.10 (complex, 3 H, Ar-H), 4.12 (dd,
J = 11.0, 5.2 Hz, 1 H, CH CN), 2.35 (dd, J = 13.7, 5.2 Hz, 1 H, CH2), 2.21 (dd,

198
B. NAMMALWAR ET AL.
J = 13.7, 11.0 Hz, 1 H, CH2), 1.47 (s, 3 H, CH3), 1.44 (s, 3 H, CH3); 13C NMR: δ 133.5,
128.6, 128.4, 127.7, 126.4, 125.1, 120.7, 41.9, 41.7, 30.9, 29.9, 29.4. TLC analysis, using
30% EtOAc in hexanes, indicated that 15 was pure.
2,2,4-Trimethylthiochroman-4-carbonitrile (16)
A 500-mL, three-necked, round-bottomed flask, equipped with a magnetic stirrer,
a reflux condenser, and a nitrogen inlet, was charged with of oil-free NaH (3.11 g, from
5.19 g of a 60% mineral oil suspension, 0.13 mol) in dry DMF (50 mL). To this suspension,
a solution of 7 (22.0 g, 0.11 mol) in dry DMF (50 mL) was added at room temperature.
The resulting dark brown mixture was stirred at room temperature for 30 min, and then
methyl iodide (16.9 g, 7.4 mL, 0.112 mol) was added dropwise over a period of 15 min. The
reaction mixture was stirred for 30 min and then carefully quenched by addition of 25 mL
of saturated NH4Cl followed by extraction with ether (3 × 150 mL). The combined organic
layers were washed with saturated NaCl (1×), dried (MgSO4), and concentrated under
vacuum to give a 21.1 g (90%) of 16 as a pale yellow solid, mp 71–72◦C. IR: 2231 cm−1
;
1H NMR: δ 7.56 (m, 1 H, Ar-H), 7.20–7.14 (complex, 3 H, Ar-H), 2.52 (d, J = 14.5 Hz, 1
H, CH2), 2.14 (d, J = 14.5 Hz, 1 H, CH2), 1.80 (s, 3 H, C(CH3)-CN), 1.59 (s, 3 H, CH3),
1.44 (s, 3 H, CH3); 13C NMR: δ 132.8, 131.7, 128.7, 128.5, 128.2, 125.6, 124.5, 51.3, 41.4,
34.8, 30.8, 30.6, 28.2. TLC analysis, using 30% EtOAc in hexanes, indicated that 16 was
pure.
2,2,4-Trimethylthiochroman-4-carboxylic Acid (17)
A 500-mL, one-necked, round-bottomed flask, equipped with a magnetic stirrer, a
reflux condenser, and a nitrogen inlet, was charged with 200 mL of 50% aqueous H2SO4
and 16 (20.0 g, 0.09 mol). The resulting mixture was heated at 100◦C for 2 h and then
cooled to room temperature and slowly poured onto 250 g of crushed ice. The aqueous layer
was extracted with ether:ethyl acetate (1:1; 3 × 200 mL). The combined organic layers
were washed with water (1×) and saturated NaCl (1×), dried (MgSO4), and concentrated
under vacuum to give 19.5 g (90%) of 17 as a pale yellow solid, mp 136–137◦C. The
solid was spectroscopically pure and was used in the next step without further purification.
1
IR: 3680–2360, 1699 cm−1; H NMR: δ 10.29 (br s, 1 H, CO2H), 7.41 (m, 1 H, Ar-H),
7.22–7.14 (complex, 3 H, Ar-H), 2.82 (d, J = 14.3 Hz, 1 H, CH2), 1.81 (d, J = 14.3 Hz, 1
H, CH2), 1.66 (s, 3 H, C(CH3)-CO2H), 1.43 (s, 3 H, CH3), 1.31 (s, 3 H, CH3); 13C NMR: δ
183.2, 136.6, 134.2, 128.7, 128.4, 127.3, 125.2, 50.5, 47.4, 42.6, 32.6, 29.4, 27.8. TLC of
the product, using 50% EtOAc in hexanes, revealed one spot.
(2,2,4-Trimethylthiochroman-4-yl)methanol (18)
A 500-mL, three-necked, round-bottomed flask, fitted with a magnetic stirrer, a
reflux condenser, and a nitrogen inlet, was charged with 17 (18.0 g, 0.08 mol) and dry
THF (150 mL) at room temperature. To the resulting solution, 5.78 g (0.15 mol, 2 eq) of
lithium aluminum hydride was slowly added in small portions (ca 20 mg) with stirring,
and the reaction was allowed to stir for 4 h. (Caution! The use of larger portions of lithium
aluminum hydride initially caused excessive “frothing.”) The resulting gray suspension
was slowly quenched with 20 mL of 10% NaOH solution. The reaction mixture turned to a
ꢀR
turbid, white solution, which slowly formed a white precipitate. Filtration through Celite

SUBSTITUTED THIOUREA METABOLITE AND RELATED PRODRUG
199
removed the solid, and the filtrate was concentrated under vacuum. The crude product was
purified by flash chromatography on a silica gel column (30 cm × 5 cm) using 40% ethyl
acetate in hexanes to give 14.7 g (87%) of 18 as a pale yellow oil, which was used directly
to prepare 19. IR: 3378 cm−1; 1H NMR: δ 7.36 (m, 1 H, Ar-H), 7.16–7.08 (complex, 3 H,
Ar-H), 3.60 (d, J = 10.8 Hz, 1 H, CH2OH), 3.56 (d, J = 10.8 Hz, 1 H, CH2OH), 2.38 (d,
J = 14.0 Hz, 1 H, CH2), 1.78 (d, J = 14.0 Hz, 1 H, CH2), 1.43 (s, 3 H, CH3), 1.41 (s, 3 H,
CH3), 1.40 (s, 3 H, CH3), 1.40 (br s, 1 H, CH2OH); 13C NMR: δ 138.2, 134.6, 128.6, 127.2,
126.5, 125.1, 72.0, 49.3, 41.8, 40.7, 31.3, 30.6, 27.2. TLC analysis, using 30% EtOAc in
hexanes, showed one spot.
(2,2,4-Trimethylthiochroman-4-yl)methyl Acetate (19)
A 250-mL, two-necked, round-bottomed flask, equipped with a magnetic stirrer,
a condenser, and a nitrogen inlet, was charged with alcohol 18 (14.0 g, 0.06 mol), 4-
dimethylaminopyridine (7.7 g, 0.06 mol), and dichloromethane (100 mL). A solution of
7.0 g (6.5 mL, 0.07 mol) of acetic anhydride in 5 mL of dichloromethane was added slowly
over a period of 5 min. After stirring at room temperature for 3 h, TLC confirmed the absence
of starting material. The reaction mixture was diluted with 100 mL of dichloromethane and
washed with 2 N HCl (1×), water (1×), and saturated NaCl (1×), dried (MgSO4), and
concentrated under vacuum to give 16.3 g (98%) of 19 as a dark yellow oil, which was used
1
without further purification. IR: 1742 cm−1; H NMR: δ 7.35 (m, 1 H, Ar-H), 7.17–7.07
(complex, 3 H, Ar-H), 4.18 (d, J = 11.0 Hz, 1 H, CH2OC(O)CH3), 4.14 (d, J = 11.0 Hz, 1
H, CH2OC(O)CH3), 2.23 (d, J = 14.3 Hz, 1 H, CH2-ring), 2.03 (s, 3 H, C(O)-CH3), 1.86
(d, J = 14.3 Hz, 1 H, CH2-ring), 1.42 (2s, 6 H, C(CH3)2), 1.41 (s, 3 H, CH3); 13C NMR:
δ 171.0, 138.0, 133.9, 128.5, 127.2, 126.7, 125.1, 71.9, 49.6, 41.6, 38.9, 31.3, 31.2, 27.3,
20.9. TLC analysis, using 30% EtOAC in hexanes, showed one spot.
(2,2,4-Trimethyl-6-nitrothiochroman-4-yl)methyl Acetate (20)
A 250-mL, three-necked, round-bottomed flask, equipped with a magnetic stirrer, an
addition funnel, and a nitrogen inlet, was charged with 19 (10.0 g, 0.04 mol) and freshly
distilled acetic anhydride (25 mL). The solution was cooled to −5◦C (ice/salt bath), and
a cold solution of 3.4 mL of concentrated nitric acid in 8.1 mL of acetic anhydride was
added dropwise over 10 min. The reaction was stirred at −5◦C for 90 min and then diluted
with ether and washed with saturated NaHCO3. The NaHCO3 wash was back-extracted
with 25 mL of dichloromethane (1×) and the combined organic layers were washed with
water (1×) and saturated NaCl (1×), dried (MgSO4), and concentrated under vacuum.
The crude product was purified by flash chromatography on a silica gel column (30 cm ×
2.5 cm) using increasing concentrations of ethyl acetate (5%, 10%, 25%, 50%) in hexanes
to give 2.3 g (20%) of 20 as a viscous, yellow oil, which was used directly in the next
step. IR: 1743, 1516, 1340 cm−1; 1H NMR: δ 8.29 (d, J = 2.2 Hz, 1 H, Ar-H), 7.95 (dd,
J = 8.2, 2.2 Hz, 1 H, Ar-H), 7.28 (d, J = 8.2 Hz, 1 H, Ar-H), 4.20 (d, J = 11.0 Hz, 1
H, CH2OC(O)CH3), 4.11 (d, J = 11.0 Hz, 1 H, CH2OC(O)CH3), 2.23 (d, J = 14.3 Hz,
1 H, CH2-ring), 2.05 (s, 3 H, C(O)CH3), 1.92 (d, J = 14.3 Hz, 1 H, CH2-ring), 1.52 (s, 3
H, CH3), 1.47 (s, 3 H, CH3), 1.44 (s, 3 H, CH3); 13C NMR: δ 170.7, 145.1, 144.1, 138.9,
128.6, 122.8, 121.6, 71.3, 48.9, 42.6, 39.1, 31.3, 31.2, 27.3, 20.8. TLC analysis, using 20%
EtOAc in hexanes, revealed one spot.

200
B. NAMMALWAR ET AL.
(6-Amino-2,2,4-trimethylthiochroman-4-yl)methyl Acetate (21)
A modified literature procedure was used.43 A 25-mL, three-necked, round-bottomed
flask, equipped with a magnetic stirrer, a reflux condenser, and a nitrogen inlet, was charged
with a mixture of 20 (0.50 g, 1.62 mol), acetic acid (8 mL), and 0.27 g (4.85 mmol, 3 eq)
of iron powder (>100 mesh). This mixture was heated with stirring at 115◦C (oil bath)
until TLC indicated the absence of starting material (ca. 15 min). The crude mixture was
cooled, transferred to a separatory funnel containing 50 mL of water, and extracted with
ether (3 × 25 mL). The combined ether layers were washed with water (1×), saturated
NaHCO3 (3×), and saturated NaCl (1×), dried (MgSO4), and concentrated under vacuum
to give 0.43 g (95%) of 21 as pale brown liquid, which was used at once to generate 22.
1
IR: 3451, 3365, 3224, 1734 cm−1; H NMR: δ 6.97 (d, J = 8.2 Hz, 1 H, Ar-H), 6.73 (d,
J = 2.2 Hz, 1 H, Ar-H), 6.51 (dd, J = 8.2, 2.2 Hz, 1 H, Ar-H), 4.19 (d, J = 11.0 Hz, 1
H, CH2OC(O)CH3), 4.14 (d, J = 11.0 Hz, 1 H, CH2OC(O)CH3), 3.61 (br s, 2 H, NH2),
2.19 (d, J = 14.3 Hz, 1 H, CH2-ring), 2.06 (s, 3 H, C(O)CH3), 1.80 (d, J = 14.3 Hz, 1 H,
CH2-ring), 1.39 (s, 3 H, CH3), 1.38 (s, 3 H, CH3), 1.37 (s, 3 H CH3); 13C NMR: δ 171.1,
144.1, 139.4, 129.6, 122.2, 114.4, 114.2, 71.8, 49.8, 41.6, 39.1, 31.1, 29.6, 27.1, 20.9. TLC
analysis, using 30% EtOAc in hexanes, showed one spot.
(6-Amino-2,2,4-trimethylthiochroman-4-yl)methanol (22)
A 25-mL, three-necked, round-bottomed flask, equipped with a magnetic stirrer, a
reflux condenser, and a nitrogen inlet, was charged with 2 mL of 10% NaOH and 150 mg
(0.54 mmol) of 21. The flask was heated in an oil bath at 90◦C for 30 min. The mixture
was cooled to room temperature, adjusted to pH 9 (1 N HCl), and extracted with ether (2 ×
25 mL). The combined ether layers were washed with water (1×) and saturated NaCl (1×),
dried (MgSO4), and concentrated under vacuum to give 120 mg (94%) of 22 as a viscous,
brown oil, which was used without further purification. IR: 3344 cm−1; 1H NMR: δ 6.96
(d, J = 8.2 Hz, 1 H, Ar-H), 6.73 (d, J = 2.2 Hz, 1 H, Ar-H), 6.49 (dd, J = 8.2, 2.2 Hz, 1 H,
Ar-H), 3.60 (d, J = 11.0 Hz, 1 H, CH2OH), 3.55 (d, J = 11.0 Hz, 1 H, CH2OH), 2.90 (br
s, 2 H, NH2), 2.30 (d, J = 14.3 Hz, 1 H, CH2-ring), 1.72 (d, J = 14.3 Hz, 1 H, CH2-ring),
1.39 (s, 3 H, CH3), 1.36 (s, 3 H, CH3), 1.34 (s, 3 H, CH3) 1.25 (br s, 1 H, CH2OH); 13C
NMR: δ 144.0, 139.7, 129.7, 123.1, 114.4, 114.3, 71.9, 49.9, 41.8, 41.0, 31.2, 30.4, 27.1.
TLC analysis, using 50% EtOAc in hexanes, showed one spot.
Synthesis of N-[3,4-Dihydro-4-(acetoxymethyl)-2,2,4-trimethyl-
2H-1-benzothiopyran-6-yl]-Nꢀ-(4-nitrophenyl)thiourea (23)
A 25-mL, one-necked, round-bottomed flask, equipped with a magnetic stirrer, an
addition funnel, and a nitrogen inlet, was charged with a solution of acetate 21 (79 mg,
0.28 mol) in dry THF (3 mL), and the flask was cooled to 0◦C (ice bath). To this mixture,
a solution of 54 mg (0.30 mmol) of 4-nitrophenyl isothiocyanate in 3 mL of dry THF
was added over 5 min. After the addition, the reaction mixture was allowed to warm to
room temperature, and stirring was continued for 24 h. The solvent was evaporated, and
the residue was purified using flash chromatography on a silica gel column (25 cm ×
2.5 cm) using 70% ether in hexanes to give 23 as pale yellow solid. The solid was further
recrystallized [H2CCl2:pentane, 1:1] to give 114 mg (88%) of 23 as a yellow solid, mp
156–157◦C. IR: 3291, 1727 cm−1; 1H NMR: δ 8.35 (br s, 1 H, NH), 8.20 (d, J = 9.0 Hz,

SUBSTITUTED THIOUREA METABOLITE AND RELATED PRODRUG
201
2 H, Ar-H), 7.95 (br s, 1 H, NH), 7.83 (d, J = 9.0 Hz, 2 H, Ar-H), 7.41 (d, J = 2.2 Hz, 1 H,
Ar-H), 7.18 (d, J = 8.2 Hz, 1 H, Ar-H), 6.99 (dd, J = 8.2, 2.2 Hz, 1 H, Ar-H), 4.53 (d, J =
11.0 Hz, 1 H, CH2OC(O)CH3), 3.74 (d, J = 11.0 Hz, 1 H, CH2OC(O)CH3), 2.48 (d, J =
14.3 Hz, 1 H, CH2-ring), 1.87 (s, 3 H, C(O)CH3), 1.86 (d, J = 14.3 Hz, 1 H, CH2-ring), 1.45
(s, 6 H, C(CH3)2), 1.40 (s, 3 H, CH3); 13C NMR: δ 179.6, 170.9, 144.6, 144.5, 139.3, 135.4,
132.2, 129.7, 126.0, 124.2, 124.1, 123.9, 72.6, 49.5, 42.1, 39.9, 30.9, 29.5, 27.3, 20.8. Anal.
Calcd for C22H25N3O4S2: C, 57.49; H, 5.48; N, 9.14; S, 13.95. Found: C, 57.63; H, 5.48;
N, 9.09; S, 13.83. A mixture melting point determination with 8 and 23 gave a range of
144–151◦C, confirming that the structures were different. TLC analysis, using 40% EtOAc
in hexanes, showed one spot.
Theoretical Studies
Computational docking studies were performed employing Glide (version 5.6,
Schro¨dinger suite 2010)45 using the Maestro user interface to set up the glide docking
and for visualization of the results. The Glide extra precision (XP) mode was used, and
the docked conformations were ranked by a Glide scoring function (G-score). For each
Glide run, 10,000 ligand poses, and at most 10 poses per ligand, were written out. The
crystal structures of B-Raf (1UWJ.pdb)50 and KIT (1T46.pdb) were downloaded from the
PDB and then prepared within the “Protein Preparation Wizard” workflow in Maestro.
This includes removal of the water and ligand, addition of hydrogens, and restrained en-
ergy minimization on hydrogens using the OPLS 2001 force field. After the preparation,
Glide grids were generated by “Receptor Grid Generation,” using bound ligands in the
crystal complexes to define the centers and sizes of the boxes. Compounds 7 and 8 were
first stretched in Sybyl 6.9, energy minimized with the hydrogens and Gasterger-Hu¨ckel
charges added, and then prepared by Ligprep in Maestro before docking. After the docking,
the top ranked conformations were chosen and exported with the crystal structures of the
kinases for further analysis.
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