Synthesis of novel heteroarotinoids with receptor activation capabilities and tgase activity. Single crystal analysis of (E)-4-[(2,3-dihydro-2,2,4,4,- tetramethyl-2H-1-benzo-[b]thiopyran-6-Yl)-1-propenyl]-2-methylbenzoic acid

Article abstract of DOI:10.1080/104265090507678

The syntheses of ethyl (E)-4-[(2,3-dihydro-2,2,4,4-tetramethyl-2H-1- benzo[b]-thiopyran-6-yl)-1-propenyl]-2-methylbenzoate (1) and (E)-4- [(2,3-dihydro-2,2,4,4-tetramethyl-2H-1-benzo[b]thiopyran-6-yl)-1-pro- penyl]-2-methylbenzoic acid (2) have been achieved. In comparison to ester 1, acid 2 exhibited greater efficacy in activating RARα, RARβ, and RARγ as well as greater potency in activating RARα and RARβ. Interestingly, both the ester 1 and acid 2 exhibited nearly equal potency in activating RARγ. Both compounds also induced tissue transglutaminase (TGase) activity approximately 50% of the level induced by trans-retinoic acid. An X-ray diffraction analysis of (E)-2 revealed the aryl rings as nearly orthogonal [74.0(2)°] and a torsional angle of 46.5(2)° between the thiochroman group and the propenyl group. Conjugation between such groups may not be a stringent requirement for receptor activation. Copyright Taylor & Francis Inc.

Full text of DOI:10.1080/104265090507678

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Synthesis of Novel
Heteroarotinoids with Receptor
Activation Capabilities and
Tgase Activity. Single Crystal
Analysis of (E)-4-[(2,3-dihydro-
2,2,4,4,-tetramethyl-2H-1-
benzo- [b]thiopyran-6-Yl)- 1-
propenyl]-2-methylbenzoic
Acid
K. Darrell Berlin e , Shankar Subramanian a , Chad M.
Smith b , Ali Tabatabai b , Clinton D. Bryan b , Brian
Buettner b , Steve Hale b , Cynthia A. Wakefield c &
Doris M. Benbrook d
a Department of Chemistry, Oklahoma State
University, Stillwater, OK, 74078
b Department of Physical Sciences, Cameron
University, Lawton, OK
c Department of Chemistry, Oklahoma State
University, Stillwater, OK, 74078
d University of Okalahoma Health Sciences Center,
Department of Obstetrics & Gynecology, Oklahoma
City, OK
e Department of Chemistry, Oklahoma State
University, Stillwater, OK, 74078
Published online: 01 Feb 2007.

To cite this article: K. Darrell Berlin , Shankar Subramanian , Chad M. Smith , Ali
Tabatabai , Clinton D. Bryan , Brian Buettner , Steve Hale , Cynthia A. Wakefield
& Doris M. Benbrook (2005): Synthesis of Novel Heteroarotinoids with Receptor
Activation Capabilities and Tgase Activity. Single Crystal Analysis of (E)-4-[(2,3-
dihydro- 2,2,4,4,-tetramethyl-2H-1-benzo- [b]thiopyran-6-Yl)- 1-propenyl]-2-
methylbenzoic Acid, Phosphorus, Sulfur, and Silicon and the Related Elements, 180:1,
67-77
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Phosphorus, Sulfur, and Silicon, 180:67–77, 2005
Copyright © Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/104265090507678
Synthesis of Novel Heteroarotinoids with Receptor
Activation Capabilities and Tgase Activity.
Single Crystal Analysis of (E)-4-[(2,3-dihydro-
2,2,4,4,-tetramethyl-2H-1-benzo- [b]thiopyran-6-Yl)-
1-propenyl]-2-methylbenzoic Acid
Shankar Subramanian
Department of Chemistry, Oklahoma State University,
Stillwater, OK 74078
Chad M. Smith
Ali Tabatabai
Clinton D. Bryan
Brian Buettner
Steve Hale
Department of Physical Sciences, Cameron University, Lawton, OK
Cynthia A. Wakefield
Department of Chemistry, Oklahoma State University,
Stillwater, OK 74078
Doris M. Benbrook
University of Okalahoma Health Sciences Center, Department of
Obstetrics & Gynecology, Oklahoma City, OK
K. Darrell Berlin
Department of Chemistry, Oklahoma State University,
Stillwater, OK 74078
The syntheses of ethyl (E)-4-[(2,3-dihydro-2,2,4,4-tetramethyl-2H-1-benzo[b]-
thiopyran-6-yl)-1-propenyl]-2-methylbenzoate (1) and (E)-4- [(2,3-dihydro-2,2,4,4-
tetramethyl-2H-1-benzo[b]thiopyran-6-yl)-1-pro- penyl]-2-methylbenzoic acid (2)
have been achieved. In comparison to ester 1, acid 2 exhibited greater efficacy in acti-
vating RARα, RARβ, and RARγ as well as greater potency in activating RARα and
RARβ. Interestingly, both the ester 1 and acid 2 exhibited nearly equal potency in
activating RARγ . Both compounds also induced tissue transglutaminase (TGase)
Received April 18, 2004; accepted June 3, 2004.
Address correspondence to K. Darrell Berlin, Department of Chemistry, Oklahoma
State University, Stillwater Oklahoma 74078. E-mail: kdb@okstate.edu.
67

68
S. Subramanian et al.
activity approximately 50% of the level induced by trans-retinoic acid. An X-ray
diffraction analysis of (E)-2 revealed the aryl rings as nearly orthogonal [74.0(2)◦]
and a torsional angle of 46.5(2)◦ between the thiochroman group and the propenyl
group. Conjugation between such groups may not be a stringent requirement for
receptor activation.
Keywords Heteroarotinoids; NMR analyis; receptor activation; TGase activity; X-ray
data
INTRODUCTION
Retinoids have been described as key agents in the differentiation of
cells and could be important players in cancer chemotherapy.1,2 Aroti-
noids are synthetic retinoids with at least one aromatic ring in the
system,2 and heteroarotinoids have a similar structural unit but also
possess a heteroatom in a partially saturated ring usually fused to an
aromatic ring.3 We have shown that certain heteroarotinoids can acti-
vate retinoic acid receptors (RARs)3f and that selected members of this
family of retinoids have anticancer properties.3a−g We report herein
the synthesis of two novel heteroarotinoids, namely one with a ter-
minal carboxyl group and the ester precursor thereof and possessing
an adjacent methyl group. The rationale for these materials is based
on biological data obtained from lead compounds.3a−e Moreover, an as-
sessment of the methyl group on the aryl ring to alter receptor activa-
tion (since the group is near the CO2Et terminus) was envisioned. Pre-
liminary data indicate that receptor activation and ability to express
TGase activity are related to the terminus (carboxyl versus ester) on the
heteroarotinoid.3f
RESULTS AND DISCUSSION
Chemistry
Syntheses of the targets 1 and 2 (Scheme 1) were initiated from 3 which
was esterified under slightly different conditions than would be consid-
ered standard. A Dean-Stark trap was required to remove water and
a long reaction time since the carboxyl group in 3 was somewhat hin-
dered by the adjacent methyl group. Regiospecific oxidation of ester
4 was achieved with CrO3 in the presence of Ac2O which converted
the acetal formed in situ to diacetate 5. Acidification of 5 gave alde-
hyde 6 which was used immediately in the Wittig reaction with 7 to
generate ester (E)-1 (40%). Purification of (E)-1 was realized only by
chromatography and recrystallization to free the ester from several un-
known impurities. Careful saponification of (E)-1 produced acid (E)-2 in

Novel Heteroarotinoids
69
SCHEME 1
near quantitative yield. The Wittig reagent 7 was prepared as outlined
(Scheme 2) starting from 8 which was reduced to the corresponding
alcohol 9. Phosphorylation of 9 occurred with triphenylphosphine hy-
drobromide in methanol to generate salt 10. The generation of 7 had to
be accomplished at −78◦C in ether before the addition of 6. The mixture
of 7 in ether-hexane was stable only at −78◦C, and 7 was used at once.
Wittig reagent 7 was prepared (Scheme 2) starting from 8 which with
6 to obtain ester (E)-1.
SCHEME 2
Biology
In view of our previous findings that certain heteroarotinoids can be
retinoid acid receptor (RAR) active and can alter TGase induction,3f it
seemed prudent to assess in a preliminary manner such activities in
(E)-1 and (E)-2 in an effort to ascertain the influence of such action by
an ester group versus an acid group. The methodology and rationale for
the receptor activation and TGase assays have been well documented.3f
Since the potencies of the 9-cis-retinoic acid (9-c-RA) control assay vary
from 1.6-fold to 0.3-fold, any difference in this range cannot be con-
sidered significant. However, the 7-fold and 27-fold differences in the
RARγ and RARβ potencies for 1 and 2 can be considered significant.
Activity data in Table I are based on the supposition that a minimum
concentration of an agent for activation of a receptor reflects its best
potency which is clearly in favor of acid (E)-2, especially for RARα and
RARβ activation, even when compared to the standard 9-c-RA. Conse-
quently, the carboxyl group may be much more important than an ester

70
S. Subramanian et al.
TABLE I Potency of RAR Activation and TGase
Inducement by (E)-1 and (E)-2
Agent Efficacya
Potency (nM)b
Potency (nM) of 9-c-RA TGasec
(E)-1
33
52
76
47
68
RARα-890
RARβ-300
RARγ -420
RARα-33
RARβ-43
RARγ -640
RARα-200
RARβ-50
RARγ -53
RARα-320
RARβ-83
RARγ -17
0.52
(E)-2
0.56
141
aPercent maximal activity of the test compound/precent maximal
activity of 9-c-RA (100).
bEC50 is the concentration required to produce 50% maximal activity.
Values for 9-c-RA and 1 and 2 were calculated.
cRatio of TGase activity (in nM) of heteroarotinoid to that of t-RA.
function in this type of receptor activation.4 The TGase activity for both
compounds was approximately 50% of that of trans-retinoic acid (t-RA),
an indication of efficacy of the agents compared to the standard. The
ability to induce TGase is a well-known marker of retinoid efficacy in
leukemia cells.3d,3e
It has been recently recognized that RARγ activation requires a lig-
and which interacts in a special conformation.3f The 76% and 141% effi-
cacy of RARγ activation by 1 and 2, respectively, demonstrate that while
both compounds interact with RARγ , acid 2 has a 2-fold higher efficacy.
Since both of these efficacies were normalized to that of 9-c-RA, this
2-fold difference is significant. The potencies of concentrations 420 nM
and 640 nM for 1 and 2, respectively, however, cannot be considered
significantly different, given the variation in the receptor potencies for
the control 9-c-RA. Therefore, while 1 and 2 have similar potencies for
RARγ , 2 is significantly more activating of RARγ as expected since the
terminal carboxyl group is apparently required to form a “salt bridge”
at the terminus of the ligand.3f Consequently, it is conceivable that the
lower activation by ester 1 is due to the slow conversion by esterases to
the carboxyl group in the cells. We have previously shown that a flexible
form of a ligand for RARγ activation is likely a more accurate repre-
sentation of the ligand bound to RARγ than is a rigid form.3f Thus, the
degree of bond rotational freedom of groups attached to the double bond
in 1 or 2 is presumably sufficient to promote receptor activation. Ester
1 and acid 2 induce RARγ with similar potencies and induce TGase to
similar degrees, which is consistent with the prior observations that
RARγ mediates the effects of retinoic acid on TGase expression.8 This
study is the first where comparisons have been made regarding acti-
vation of RARs by hetero-arotinoids. The X-ray data of (E)-2 indicates

Novel Heteroarotinoids
71
that in the solid state much conjugation between the aryl rings is not
prevalent, but such a requirement in solution for RAR activation is
unknown.
X-Ray Crystallographic Analysis of the Target
Ligand (E)-2
Since the absolute structures of heteroarotinoids have rarely been
determined, an X-ray diffraction analysis of (E)-2 was performed
(Table II). Crystals were of the triclinic space group, and the struc-
ture consisted of dimers which uniquely employed hydrogen bond-
ing through the carboxyl function. Intramolecular and intermolecu-
˚
˚
lar O H bond distances of 0.905(4) A and 1.789(4) A, respectively,
were found and are typical of such hydrogen bonding. The bond an-
◦
◦
. . .
. . .
gles of O H O, C O H, and C O H were 155.6(4) , 109.8(4) , and
122.7(4)◦, respectively, and are also reasonable for such H-bonding. No
long-range bondings were noted for other groups in (E)-2. Interestingly,
the C6-C14-C15 bond angle [115.6(5)◦] was compressed by the large
cis-arranged aryl ring as revealed by the larger C14-C16-C17 bond
angle [130.8(5)◦] created presumably by ortho hydrogens at C22 (or
C18) experiencing repulsion from the methyl hydrogens of the propenyl
group.
Three torsional angles considered critical for the structure were
measured. The angle defined by the thiochroman ring system and
the phenyl ring at the end of the linker group was nearly orthog-
onal at 74.0(2)◦ (Figure 1). This angle is reminiscent of that found
for another heteroarotinoid5 but differs from that found for trans-
stilbene.6 A more exact torsional angle between the phenyl rings in
trans-stilbene was reported later to be 5.2◦.7 The angle between the
FIGURE 1 ORTEP drawing of (E)-2

72
S. Subramanian et al.
TABLE II Crystal and Refinement Data for (E)-2
Formula
C24H28O2S
fw
380.54
Crystal size, mm
Crystal color
Crystal mount
0.18 × 0.30 × 0.48
Colorless
On glass fiber encapsulated in epoxy
6.7122(18)
7.9440(20)
21.173(50)
˚
a, A
˚
b, A
˚
c, A
α, deg
90.670(20)
β, deg
90.040(20)
γ , deg
21.173(5)
Cell detn, refls
Cell detn, 2θ range, deg
d (calcd), g cm−3
Space group
Z
25
20–22
1.208
P-1
2
F (000)
408.45
Radiation
γ , A
MoKα, graphite monochromated
0.71073
˚
Temp, K
293
Linear abs coeff, mm−1
Diffractometer
Scan technique
Scan speed, deg min−1
Scan width, deg
2θ range, deg
16.2
Enraf-Nonius CAD-4
θ-20
4–16 (in omega)
1.0 + 0.35 tan θ
4–50
h, k, l ranges
−7,7; 0,9; −25, 25
Exposure time, hrs
Std refl indices
Drift of stds, %
Absorbtion correction
Absorbtion, range
Refl meas
25.7
0,−3,7; 3,−3,−2; 0,−3,−6
1.0
Gaussian by faces
0.96–0.97
3957
Unique refls
3665
R for merge
0.013
Data with I > 2.5 δ (I)
Solution method
Parameters refined
R(F2), Rw(F2)
1641
Direct methods
244
0.057, 0.078
GOF
1.18
0.05
0.000
P, w-1 = [δ2(I) + pI2]/4F2
Largest ꢀ/δ
Extinction correction
Final diff man, e A
Programs
Scattering factors
H atom treatment
None
−3
˚
−0.23(5), 0.24(5)
NRC386 (PC version of NRVAX)∗
Internat. Tables for Crystallography, Vol. 4
Methyl positions from maps, subsequently; idealized so that
C
˚
H = 0.95 A & U = Uc + 0.01
O atom treatment
Hydroxyl pos. from maps, subsequently; thermal Parameter U
= U◦ + 0.01
∗NRCVAX—An Interactive Program System for Structure Analysis, E. J. Gabe, Y.
LePage, J. P. Charland, F. L. Lee, and P. S. White, J. Appl. Cryst., 22, 383 (1989).

Novel Heteroarotinoids
73
aryl ring of the thiochroman unit and the propenyl linker was 46.5(2)◦
in (E)-2. In contrast, an angle of 28.9(3)◦ was present between the
propenyl linker and the single phenyl ring attached to the linker in
(E)-2. Thus, in the solid state there is little indication that conjugation
exists between the two aryl rings and the linker moiety. These data
may provide additional insight regarding future studies of activation of
RARγ by synthetic ligands in terms of orientation of groups within the
ligand.
In summary, the syntheses of two new heteroarotinoids are herein
reported. It was also been found that a difference in receptor activa-
tion capability prevails in certain heteroarotinoids with slightly dif-
fering terminal groups, that is an ester versus an acid group. This is
pres−umed to be due to the requirement for formation of a salt bridge
[CO2 ] for RARγ activation by a ligand.3f In addition, the arrange-
ment between the two aryl rings in the title acid suggests that strong
conjugation between the rings in such systems, via the linker group,
may not be absolutely critical for receptor activation as well as TGase
activity.
EXPERIMENTAL
General
Melting points were taken on a Perkin-Elmer 681 unit and were uncor-
rected. All NMR spectra were recorded on a Varian 300 MHz or Varian
400 MHz spectrometer and registered as δ or ppm, respectively, from
1
TMS. Both H and 13C spectra were taken at 299.4/399.95 MHz and
75.43/100.6 MHz, respectively. IR data were taken on a Perkin-Elmer
2000 FT-IR unit. Elemental analyses were obtained by Atlantic Inc.,
Norcross, Georgia. All chromatographic separations were effected with
˚
silica gel (Baker, 40 m, 60A, flash). Storage of all intermediates and
final products in the cold/dark minimized decomposition.
Ethyl (E)-4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H-1-
benzothiopyran-6-yl)-1-propenyl]-2-methylbenzoate (1)
A solution of n-butyllithium in hexane (0.15 mL, 1.56 mmol, 10 M)
was added dropwise (5 min, N2) to a suspension of the salt 10 (0.82 g,
1.43 mmol) in dry ether (25 mL). After stirring for 1 h at RT, the mixture
containing 7 was cooled (−78◦C, dry ice-acetone, 10 min), and a solu-
tion of ethyl 2-methyl-4-formylbenzoate (6, 0.25 g, 1.3 mmol) was added
dropwise (0.25 h). The mixture was stirred (0.75 h), and then it was al-
lowed to slowly warm to RT (1 h). The pale yellow mixture was stirred

74
S. Subramanian et al.
(48 h), and then the mixture was filtered to remove the triphenylphos-
phine oxide, the latter residue being washed with dry ether (100 mL).
This washing and the filtrate were combined, dried (Na2SO4), and
evaporated to an oil which was chromatographed (hexane:ether, 98:2).
Fractions containing crude 1 were combined and evaporated to an oil
which was treated with boiling ethanol (95%). Chilling (dry ice bath)
the final solution for 24 h induced precipitation. Filtration and recrys-
tallization (95% ethanol) of the solid gave ester (E)-1 (0.21 g, 40%),
1
mp 77–79◦C. IR (KBr) 1705 cm−1; H NMR (DCCl3) δ 1.40 [m, 3 H],
1.44 [s, 12 H], 1.98 [s, 2 H], 2.28 [s, 3 H], 2.63 [s, 3 H], 4.36 [m, 2
H], 6.77 [m, 1 H], 7.11 [m, 1 H], 7.25 [m, 3 H], 7.53 [s, H], 7.93 [m, 1
H]; 13C NMR (DCCl3) ppm 14.3, 17.6, 21.9, 31.6, 32.6, 35.6, 42.1, 54.4,
60.6, 123.7, 124.4, 125.9, 126.3, 127.9, 130.5, 130.6, 132.4, 139.1, 140.0,
140.2, 142.0, 142.4, 167.5. MS (EI) C26H32O2S (M+): 408.2123. Found:
408.2114. C26H32O2S requires: C, 76.43; H, 7.89; found: C, 76.32; H,
7.96.
(E)-4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H-1-benzothiopyran-
6-yl)-1-propenyl]-2-methylbenzoic Acid (2)
A solution of (E)-1 (0.32 g, 0.80 mmol) in water (5 mL) and ethanol
(20 mL, 95%) containing KOH (0.45 g, 8.03 mmol) was boiled (2 h),
then stirred at RT (12 h), and finally chilled to 0◦C. Dropwise addition
of HCl (∼20 mL, 2 N) resulted in the precipitation of slightly crude
(E)-2 as a white solid (0.30 g, 99%). Recrystallization (95% ethanol)
of the solid gave (E)-2 as colorless crystals (∼qt), mp 178–180◦C. IR
(KBr) 3450 (HOC O), 1690 (C O) cm−1; 1H NMR (DMSO-d6) δ 1.39 [s,
12 H], 1.95 [s, 2 H], 2.24 [s, 3 H], 2.55 [s, 3 H], 6.87 [m, 1 H], 7.06 [m,
1 H], 7.31 [m, 3 H], 7.64 [m, 1 H], 7.86 [d, 1 H]; 13C NMR (DMSO-d6)
ppm 17.1, 21.3, 31.1, 32.3, 35.1, 41.8, 53.6, 123.6, 124.3, 125.4, 126.2,
127.3, 127.8, 130.3, 138.1, 139.1, 139.5, 141.2, 142.0, 168.2. MS (EI)
C24H28O2S (M+): 380.1812. Found: 380.1810. C24H28O2S requires: C,
75.75; H, 7.42; found: C, 75.47; H, 7.49.
Ethyl 3,4-Dimethylbenzoate (4)
A solution of 3,4-dimethylbenzoic acid (3, 6.0 g, 39.9 mmol), abso-
lute ethanol (80 mL), and benzene (150 mL) with H2SO4 (1.5 mL) as
a catalyst were heated in a Dean-Stark trap (48 h). After cooling, the
solution was diluted (water, 100 mL) and extracted (ether, 3 × 40 mL).
The extracts were combined and washed with saturated NaHCO3 (3 ×
40 mL), water (2 × 50 mL), and brine. Evaporation of the solvent and
distillation (65◦C/0.25 mm) of the oil gave ester 4 (6.3 g, 88%) which
was used immediately to prepare 5. Spectral data for 4: IR 1710 cm−1
1H NMR (DCCl3) δ 1.39 [t, 3 H], 2.31 [s, 3 H], 2.58 [s, 3 H], 4.30 [m,
;

Novel Heteroarotinoids
75
2 H], 7.05 [m, 2 H], 7.85 [m, 1 H]; 13C NMR (DCCl3) ppm 14.3, 21.3,
21.7, 60.4, 126.3, 126.9, 130.6, 132.4, 140.1, 142.2, 167.5.
Ethyl 2-Methyl-4-formylbenzoate (6)
A solution of ester 4 (1.6 g, 8.9 mmol), glacial acetic acid (20 mL),
and freshly distilled acetic anhydride (20 mL) with H2SO4 (1.5 mL)
was stirred at RT (0.25 h) after which time the solution was cooled
to 0◦C. The temperature was maintained <5◦C (1 h) as CrO3 (1.6 g,
16.8 mmol) was added over 0.5 h. The resulting mixture was stirred
at RT for 8 h after which the mixture was treated very cautiously
with ice water (150 mL) and then ether (40 mL). Extracts (HCCl3, 3 ×
50 mL and ether, 1 × 50 mL) were combined with the original organic
layer and washed with saturated NaHCO3 (3 × 40 mL), water (1 ×
50 mL), and brine. After drying (Na2SO4), the solvent was evaporated
to yield the diacetate 5 as an orange oil (1.9 g, 76.8%). The oil 5 was
air sensitive and thus was dissolved immediately in ethanol (15 mL) to
which was added dropwise (10 min; slightly exothermic) water (10 mL)
and conc H2SO4 (0.7 mL). Additional water (150 mL) was added to
the solution at RT, and two phases separated. Extracts (HCCl3, 5 ×
40 mL) were combined with the original organic layer and washed
with saturated NaHCO3 (4 × 35 mL), water (50 mL), and brine. The
dried (Na2SO4) solution was evaporated to a light brown oil which was
chromatographed (hexanes:ether, 8.5:1.5) to yield 6 as a pale yellow oil
(0.36 g, 21%) which solidified at 0◦C and remelted near RT.9 IR (neat)
2720 (H CO), 1725 (EtOC O), 1710 (HC O) cm−1 13C NMR (DCCl3)
;
ppm 14.1, 21.3, 61.2, 126.5, 130.8, 132.4, 135.2, 137.9, 140.5, 166.7,
191.7. The 1H NMR spectrum of 6 has been reported.7 Since 6 oxidized
rapidly, it was used at once to make (E)-1.
2,2,4,4-Tetramethythiochroman-6-(1-hydroxyethane) (9)
A solution of ketone 83c (5.8 g, 23.3 mmol) in anhydrous ether (25 mL)
was added (0.25 h, N2) to a stirred suspension of LiAlH4 (1.42 g,
37.36 mmol) in dry ether (15 mL). After stirring the grey suspension
at reflux (24 h), the mixture was cooled to RT, and EtOAc (10 mL) was
added very slowly with cooling (<5◦C). A solution of 5% HCl (50 mL)
was added slowly with stirring. Ether (50 mL) was then added, and,
after extraction (ether, 4 × mL), the extracts and original organic layer
were combined and washed with saturated NaHCO3 (3 × 40 mL), wa-
ter (50 mL), and brine. Drying (Na2SO4) and distillation gave 9 as a
thick, yellow oil (50–55◦C/0.3 mm; 5.6 g, 96%) which was used at once
without further purification to prepare 10. Data for 9 were: IR (neat)
1
3350 (O H) cm−1; H NMR (DCCl3) δ 1.41 [s, 6 H], 1.43 [s, 6 H], 1.53
[m, 3 H], 1.96 [s, 2 H], 4.84 [m, 1 H], 7.15 [m, 2 H], 7.42 [m, 1 H]; 13C

76
S. Subramanian et al.
NMR (DCCl3) ppm 24.9, 31.6, 32.5, 35.6, 41.9, 54.4, 70.3, 123.1, 123.9,
124.9, 128.01, 128.08, 142.7.
1-[(2,2,4,4-Tetramethylchroman-6-yl)ethyl]triphenyl-
phosphonium Bromide (10)
A solution of alcohol 9 (5.5 g, 21.96 mmol) and triphenylphosphine
hydrobromide (8.9 g, 26.4 mmol) in methanol (125 mL) was stirred at
RT (24 h, N2). Evaporation of the pale yellow solution and repeated
trituration of the clear oil obtained with dry ether induced solidification.
Stirring the suspension of the light yellow solid in dry ether (100 mL)
for 4 h gave a very light yellow solid which was filtered off and dried
(110◦C/2 mm); yield 11.1 g (88%), mp 140–150◦C. The salt 10 was used
immediately to prepare anion 7 which in turn was used to prepare 1.
1
Data for 10 were: H NMR (DCCl3) δ 1.12 [s, 3 H], 1.23 [s, 3 H], 1.37
[s, 6 H], 1.38–1.85 [m, 2 H], 1.87 [m, 3 H], 6.48–6.54 [m, 1 H], 6.60 [m,
2 H], 6.89 [m, 1 H], 7.77 [m, 15 H]; 13C NMR (DCCl3) ppm 19.6, 31.4,
31.9, 32.5, 32.7, 35.5, 54.1, 70.3, 117.2, 118.3, 126.9, 128.0, 129.8, 130.0,
130.1, 130.2, 130.4, 130.5, 132.0, 133.2, 133.3, 134.5, 134.6, 134.7, 135.1,
135.2.
Crystallographic Experimental Data for (E)-2
Suitable crystals of the acid (E)-2 were obtained by slow diffusion
of pentane into a saturated solution of the acid in absolute ethanol,
followed by slow evaporation of the ethanolic solvent. The crystals were
triclinic and of the space group P1. Data collection was accomplished
with an ENRAF-Nonius CAD-4 diffractometer with monochromated
˚
Mo Kα (λ = 0.71073 A). Crystals were mounted on a glass fiber with
silicone and then coated with epoxy to prevent decomposition. The cell
dimensions were determined by a least squares fit to 25 reflections with
2θ in the range of 20–22◦. The structure was solved by direct methods
using the program NRC386.10
The structure was refined by a full-matrix least squares method
which minimized ꢁω(ꢀF)2. A total of 3957 reflections were taken. The
hydrogen belonging to the carboxyl group was located on a difference
map and then constrained to that position. Methyl hydrogens were also
located by a difference map and then normalized to idealized positions
˚
with C H = 0.95 A. All other hydrogen atoms were generated and con-
˚
strained to idealized positions with C H = 0.95 A. All hydrogen atoms
were assigned isotropic U values of 0.01 greater than that of the at-
tached atom. The structure refined to a final R factor of 0.057.a
aTables of all X-ray data have been deposited with the Cambridge Crystallographic
Data Centre (CCDC), United Kingdom. CCDC 213322.

Novel Heteroarotinoids
77
Biological Assays
The technique for assaying for transglutaminase (TGase) activity has
been completely described.3d The methodology for determining retinoic
acid receptor activation has also been well delineated.3e
ACKNOWLEDGMENTS
We gratefully acknowledge the National Science Foundation for a De-
partmental grant (DMB-860-3864) for funds to upgrade the XL-300
NMR spectrometer and a separate grant (CHE-8718150) to purchase
the XL-400 NMR spectrometer. Thanks are extended (KDB) to the Col-
lege of Arts & Sciences. The NIH is also thanked for partial support
via the National Cancer Institute [CA-73639; CA077711 (DMB)]. We
are pleased for partial funding for a new Varian Inova 400 MHz NMR
spectrometer in the Oklahoma Statewide Shared NMR Facility by the
National Science Foundation (BIR-9512269), the Oklahoma State Re-
gents for Higher Education, the W. M. Keck Foundation, and Conoco,
Inc.
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