Thyroid receptor agonists for the treatment of androgenetic alopecia

Article abstract of DOI:10.1016/j.bmcl.2009.10.109

A thyroid hormone receptor β subtype-selective thyromimetic 5 was found to be efficacious in both mouse and monkey hair growth models after topical applications. It penetrates the skin according to the test in human cadaver skin mounted onto Franz diffusion chambers. The serum drug level of 5 is below the limit of quantification during tests in the bald stump-tailed macaques (Macaca arctoides). It is tested negative in the 3T3 neutral red uptake (NRU) phototoxicity test, indicating a low risk for causing photo-irritation. It is also rapidly metabolized according to the PK data, thus the systemic exposure is limited.

Full text of DOI:10.1016/j.bmcl.2009.10.109

Bioorganic & Medicinal Chemistry Letters 20 (2010) 306–308
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thyroid receptor agonists for the treatment of androgenetic alopecia
a
b
Jie Jack Li a, , Lorna H. Mitchell , Robert L. Dow
*
a Department of Chemistry, Michigan Laboratories, Pfizer Global Research & Development, 2800 Plymouth Road, Ann Arbor, MI 48105, United States
b Cardiovascular and Metabolic Diseases Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A thyroid hormone receptor b subtype-selective thyromimetic 5 was found to be efficacious in both
mouse and monkey hair growth models after topical applications. It penetrates the skin according to
the test in human cadaver skin mounted onto Franz diffusion chambers. The serum drug level of 5 is
below the limit of quantification during tests in the bald stump-tailed macaques (Macaca arctoides). It
is tested negative in the 3T3 neutral red uptake (NRU) phototoxicity test, indicating a low risk for causing
photo-irritation. It is also rapidly metabolized according to the PK data, thus the systemic exposure is
limited.
Received 21 September 2009
Revised 22 October 2009
Accepted 26 October 2009
Available online 29 October 2009
Keywords:
Thyromimetics
Androgenetic alopecia
Topical drug
Ó 2009 Elsevier Ltd. All rights reserved.
Skin penetration
Photo-irritation
Androgenetic alopecia, also known male pattern baldness, oc-
curs not only in men but also in women. Current treatments such
as minoxidil (1, RogaineÒ, a potassium channel opener) and finas-
clearance as indicated by pharmacokinetic (PK) data such as
short-half-life in human liver microsome (HLM) and fast hepatic
clearance (see Fig. 2).
teride (2, PropeciaÒ, a 5
a
-reductase inhibitor) are only efficacious
Our initial drug discovery program led to a novel series of 6-
azauracil-based thyroid hormone receptor ligands as potent, TRb
subtype-selective thyromimetics.5 Although our original goal was
to search for anti-obesity drugs by taking advantage of the thermo-
genic potential of thyromimetics, preliminary tests revealed that
some of these compounds had the potential to treat hair loss as
well after topical applications.6 Herein, we report our selection,
animal model studies, discovery and improved synthesis of PF-
00277343 (5), a thyroid receptor agonist with preclinical efficacy
for hair growth after topical applications.
A group of potent, TRb subtype-selective thyromimetics were
tested in the C3H/HeN mouse model, a validated hair growth mod-
el.7a C3H/HeN mice provide a good model for studying the hair
growth in vivo because they have synchronized hair cycles, alter-
nating between periods of anagen, catagen, and telogen.7b As
shown in the Table 1, after topical applications, thyromimetics 6
in a small percentage of subjects. Novel mechanisms with robust
efficacy and long-term safety are still needed for the treatment of
androgenetic alopecia (see Fig. 1).
Thyroid hormone receptor agonists (thyromimetics) are closely
associated with hair growth. Topical triiodo-thyronine (T3, 3) stim-
ulates epidermal proliferation, dermal thickening, and hair growth
in both mice and rats.1 Some human subjects given with thyroxine
(T4, 4) to treat thyroid hormone deficiency reported hair growth as
a side effect. Thyroid hormones are also found to directly alter hu-
man hair follicle functions including anagen prolongation and
stimulation of both hair matrix keratinocyte proliferation and hair
pigmentation.2 Thyroid receptors (TR) interact with hairless gene
product (Hr), a transcription factor required for hair growth and
thyroid hormone receptor b1 is expressed in the human hair folli-
cles.3 Recently, human female hair follicles were found to be a di-
rect, nonclassical target for thyroid-stimulating hormone.4
However, use of oral thyroxine or thyroid hormones to treat andro-
genetic alopecia is impractical due to their known cardiotoxicity.
Therefore, we sought thyromimetics that are active topically and
yet devoid of systemic pharmacological activities to avoid deleteri-
ous side effects. We set out to find soft drugs with high systemic
H
N
O
OH
N
H2N
NH
H
N
H
H
N
O
N
H
H
* Corresponding author at present address: Discovery Chemistry, Bristol-Myers
Squibb Company, 5 Research Parkway, Wallingford, CT 06492, USA. Tel.: +1 203 677
7225.
1, minoxidil (Rogaine®)
2, finasteride (Propecia®)
E-mail address: jack.li@bms.com (J.J. Li).
Figure 1. Current treatments for androgenetic.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.109

J. J. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 306–308
307
efficacious compound in this assay. Its MED of 0.003% represents
a 3000-fold increase in comparison to minoxidil (1) whose MED
is 2.5–5.0% qd for 4 weeks, with 5 days required for induction of
efficacy. More remarkably, single application of azauracil 5 at
1 mg/mL and 0.1 mg/mL was effective in inducing hair growth in
C3H female mice.
H
O
OH
I
OH
NH2
I
I
I
NH2
I
I
HO2C
HO2C
O
I
Ideally, topical drugs exert their desired effects locally but are
rapidly inactivated via metabolism once they reach the systemic
circulation, thereby reducing unwanted systemic effects. In vitro
analysis indicated compound 5 is rapidly metabolized in rat liver
microsomes (HLM t1/2 = 40 min).8 In rat hepatocytes, its half-life
is 45 min, clearance CLblood is 51 mL/min/kg and CLint is 149 mL/
min/kg. The hepatic extraction ratio (EH) predicted from these
in vitro data approached liver blood flow in rat, with a predicted
EH value of 1.0, indicating a high systemic clearance. Also in vitro,
compound 5 is rapidly metabolized in human liver microsomes as
well (HLM t1/2 = 39 min, CLint = 29 mL/min/kg, CLblood = 15 mL/min/
kg). The half-life in human hepatocyte is 119 min. The hepatic
extraction ratio (EH) predicted from these in vitro data in human
is 1.0 as well. Furthermore, the in vivo clearance data in rats were
consistent with the high clearance predicted in vitro in rat: Follow-
ing intravenous administration of 5 at dosage of 0.5 mg/Kg, the
mean systemic plasma clearance is 40 mL/min/kg. The mean
apparent volume of distribution at steady state was 4.88 mL/min/
kg, and the mean terminal phase half-life was 2.73 h.
3, T3
4, T4
H
O
N
O
N
OH
F
N
O
5, PF-00277343
Figure 2. Thyroid hormones T3 and T4; and thyromimetic PF00277343.
Table 1
In vivo efficacy in the C3H/HeN mouse hair growth model
Compds
TRb Kia TR
a
Ki MEDb
(nM)
(nM)
O
O
0.5
0.7
1.0
Unlike oral drugs, topical drugs are required to penetrate the
skin to exert their pharmacological effects. We evaluated skin pen-
etration of thyromimetic 5 using human cadaver skin mounted
onto Franz diffusion chambers.9 As shown in Figure 3, compound
5 did indeed penetrate the skin, with the maximal penetration rate
HO
O
N
H
OH
O
S
O
6
Cl
O
O
O
l
g/cm2/h at 28 h. After 40 h, the total skin penetration was
0.5
2.1
0.10
of 0.14
g/cm2/h, representing 6.17% of the total drug applied. Of the
HO
N
OH
2.39
l
H
7
drug that did penetrate into the skin, 0.58% was distributed in the
dermis, whereas 9.88% was in the epidermis.
Another unique requirement for topical drugs is low propensity
for photo-irritation. Thyromimetic 5 was tested negative in the 3T3
neutral red uptake (NRU) phototoxicity test,10,11 indicating a low
risk for causing photo-irritation.
Cl
N
2
126
0.10
O
O
HN
O
As preliminary in vitro and in vivo evaluation of the efficacy and
metabolic stability characteristics of 5 proved favorable, an effi-
cient synthesis was mandated to prepare enough active pharma-
ceutical ingredients (API) to support subsequent in vivo studies.
To that end, we devised a practical and convergent synthesis that
was suitable for large-scale production of 5. As shown in the
Scheme 1, the Friedel–Crafts acylation of 1-bromo-4methoxyben-
zene with 4-fluorobenzoic acid was promoted by Eaton’s reagent12
to afford diaryl ketone 11, which was reduced using trisilylsilane in
the presence of trifluoroacetic acid (TFA) to give benzyl derivative
12. Meanwhile, a BuchwaldÀHartwig cross-coupling13 of 5-bromo-
2-methoxy-1,3-dimethylbenzene with 6-azauracil afforded adduct
N
Cl
OH
N
8
O
Cl
Cl
N
O
1.0
20
0.10
O
HN
O
N
N
Cl
OH
O
9
O
O
0.51
0.40
8.0
3.0
0.01
HN
N
N
Cl
OH
F
O
10
5
0.003
a
See note 6 of Ref. 5 for description of the binding assay method and calculation
of the Ki values.
b
MED = minimal efficacious dose, as the percentage concentration (w/v) in
mixture of 70:30 ethanol/propylene glycol.
and 7 with carboxylic acids have minimal efficacious doses (MED)
of 1.0% and 0.10% in the vehicle of 70:30 ethanol/propylene glycol,
respectively. On the other hand, 6-azauracil analogs 8–10 and 5 are
more efficacious with MEDs ranging from 0.003% to 0.10%. Azaura-
cil 5 (EC50 = 0.5 nM in Hep G-2 cell) in particular is the most
Figure 3. In vitro human cadaver skin penetration using franz diffusion chamber.

308
J. J. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 306–308
OMe
Br
OMe O
OMe
Br
Et3SiH, TFA
93%
4-fluorobenzoic acid
Eaton's reagent
quant.
F
F
11
Br
12
OH
OMe
OMe
N
CuI, K3PO4
NH
BBr3
NMP, Δ, 54%
O
N
H
O
N
O
O
CH2Cl2
97%
N
O
O
N
NH2
N
Br
NH
NH
14
NH2
13
OMe
O
CuI, Cs2CO3
NMP, 120 oC, 87%
BBr3
F
12 + 14
5
CH2Cl2
86%
O
O
O
N
N
NH
15
O
Scheme 1. Improved convergent synthesis of 5.
2. van Beek, N.; Bodo, E.; Kromminga, A.; Gaspar, E.; Meyer, K.; Zmijewski, M. A.;
Slominski, A.; Wenzel, B. E.; Paus, R. J. Clin. Endocrin. Metab. 2008, 93, 4381.
3. Billoni, N.; Buan, B.; Gautier, B.; Gaillard, O.; Mahe, Y. F.; Bernard, B. A. Br. J.
Dermatol. 2000, 142, 645.
4. Bodo, E.; Kromminga, A.; Biro, T.; Borbiro, I.; Gaspar, E.; Zmijewski, M. A.; van
Beek, N.; Langbein, L.; Slominski, A. T.; Paus, R. J. Invest. Dermatol. 2009, 12,
1126.
5. Dow, R. L.; Schneider, S. R.; Paight, E. S.; Hank, R. F.; Chiang, P.; Cornelius, P.;
Lee, E.; Newsome, W. P.; Swick, A. G.; Spitzer, J.; Hargrove, D. M.; Patterson, T.
A.; Pandit, J.; Chrunyk, B. A.; LeMotte, P. K.; Danley, D. E.; Rosner, M. H.;
Ammirati, M. J.; Simons, S. P.; Schulte, G. K.; Tate, B. F.; DaSilva-Jardine, P.
Bioorg. Med. Chem. Lett 2003, 13, 379.
13 in 54% yield, which was subsequently demethylated using BBr3
in CH2Cl2 to provide azauracil-phenol 14.
Now with fragments 12 and 14 in hand, the convergent ether
formation was achieved by Ullmann coupling using 2,2,6,6-tetra-
methylheptane-3,5-dione as a powerful ligand to accelerate the
recation.14 Routine demethylation then delivered the desired prod-
uct 5 in 86% yield. Although the yield of the cross-coupling reaction
to access 13 was moderate, the convergent nature of this route al-
lowed large-scale synthesis of 5.
With enough quantities of 5 in hand, we carried out in vivo hair
growth experiments in the bald stump-tailed macaques (Macaca
arctoides), a validated primate model for androgenetic alopecia.15
The MED of 5 for hair growth was 0.1% (reference compound 1
has a MED of 2% for the bald stump-tailed macaques15). Therefore,
while 5 is efficacious in the monkey the MED is right-shifted rela-
tive to the C3H/HeN mouse model (MED, 0.003%). During the
course of the four-month study, there were no signs of skin irrita-
tion, erythema, flaking, edema, or skin pigmentation changes. The
serum drug level for 5 was below the lower limit of quantification
(LLQ, detection limit 2 ng/mL for the first two-month samples and
1 ng/mL for the third and fourth month samples), indicating lim-
ited systemic exposure of the drug. Although the LLQ was still
above the EC50 for these compounds, analysis of functional thyroid
endpoints indicated that there was no detectable systemic phar-
macological effects from application of 5 at efficacious dosages.
In summary, we have discovered a TRb subtype-selective thyr-
omimetic 5 that is efficacious in both mouse and monkey hair
growth models after topical applications. It is rapidly metabolized
based on pharmacokinetic data, thus the systemic exposure is lim-
ited. It penetrates the skin according to the test in human cadaver
skin mounted onto Franz diffusion chambers. The serum drug level
of 5 is below the limit of quantification during tests in the bald
stump-tailed macaques (M. arctoides). It is also tested negative in
the 3T3 neutral red uptake (NRU) phototoxicity test, indicating a
low risk for causing photo-irritation.
6. Chian, Y.-C. P.; Cornelius, P.; Doherty, N. S.; Dow, R. L. EP1262177, 2002.
7. (a) Jiang, J.; Tsuboi, R.; Kojima, Y.; Ogawa, H. J. Dermatol. 2005, 32, 243; (b)
Hamada, K.; Hirotsu, S.; Uchiwa, H.; Yamazaki, A.; Suzuki, K. J. Dermatol. Sci.
2003, 33, 195. Experimental procedure: Male C3H/HeN mice (ꢀ6 weeks old
with same date of birth) were purchased from Charles River Laboratory
(Raleigh, NC). After one week of acclimation, the mice were shaved under
isoflurane anesthesia on the lower back using an electric shaver. Only mice in
the telogen phase (pink skin) were used in studies. Twenty microliters of test
articles at various concentrations in propylene glycol/ethanol (30:70, w/v) or
the vehicle control was topically applied to the shaved lower back of the mice
to cover an area of approximately 1 cm2 (20
l
L/cm2). Ten mice were used in
each experimental group. The treatment regimen was twice daily (BID)
application for 4 weeks, 5 days/week from Monday to Friday. Local irritation
was recorded before each application and hair growth scores were recorded
every other day. After 4 weeks of treatment, mice were further observed for
one more week during which hair growth and skin irritation were scored every
other day. The scale used for scoring hair growth was: 0 = no hair growth, pink
skin; 1 = skin color changes from pink to gray or black without visible hair
growth, indicating the onset of anagen; 2 = sparse or diffuse short hair growth;
3 = dense, normal coat hair. A reference androgen receptor antagonist (RU-
58841) was included in every study.
8. Thomas, V. H.; Bhattachar, S.; Hitchingham, L.; Zocharski, P.; Naath, M.;
Surendran, N.; Stoner, C. L.; El-Kattan, A. Exp. Opin. Drug Metab. Toxicol. 2006, 2,
591.
9. See: Antille, C.; Tran, C.; Sorg, O.; Saurat, J.-H. Exp. Dermatol. 2004, 13, 558.
10. Nam, C.; An, S.; Lee, E.; Moon, S.; Kang, J.; Chang, I. ATLA, Altern. Lab. Anim. 2004,
32, 693.
11. Spielmann, H.; Alternatives to Animal Testing II, Proceedings of the International
Scientific Conference, 2nd, Brussels, 1999, pp 15–24.
12. A similar paper has recently been published: Li, Y.; Chang, M.-q.; Gao, F.; Gao,
W.-t. J. Chem. Res 2008, 640.
13. Janey, J. M. Buchwald–Hartwig Amination. In Name Reactions for Functional
Group Transformations; Li, J. J., Corey, E. J., Eds.; Wiley & Sons: Hoboken, NJ,
2007; pp 564–609.
14. see Buck, E.; Song, Z. J.; Tschaen, D.; Dormer, P. G.; Volante, R. P.; Reider, P. J.
Org. Lett. 2002, 4, 1623.
15. Uno, H.; Cappas, A.; Brigham, P. J. Am. Acad. Dermatol. 1987, 16, 657.
References and notes
1. Safer, J. D.; Fraser, L. M.; Ray, S.; Holick, M. F.. Thyroid 2001, 11, 717.